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WO2004039993A1 - Polynucleotide encoding a pyranosone dehydratase - Google Patents

Polynucleotide encoding a pyranosone dehydratase Download PDF

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Publication number
WO2004039993A1
WO2004039993A1 PCT/GB2003/004594 GB0304594W WO2004039993A1 WO 2004039993 A1 WO2004039993 A1 WO 2004039993A1 GB 0304594 W GB0304594 W GB 0304594W WO 2004039993 A1 WO2004039993 A1 WO 2004039993A1
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WO
WIPO (PCT)
Prior art keywords
polynucleotide
seq
organism
plant
nucleotide sequence
Prior art date
Application number
PCT/GB2003/004594
Other languages
French (fr)
Inventor
Shukun Yu
Egon Bech Hansen
Hans Christian Pedersen
Mark Turner
Inge Weiergang
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/283,940 external-priority patent/US20030220394A1/en
Priority claimed from PCT/GB2002/004916 external-priority patent/WO2003037918A2/en
Priority claimed from GB0226159A external-priority patent/GB0226159D0/en
Priority claimed from GB0310479A external-priority patent/GB0310479D0/en
Application filed by Danisco A/S filed Critical Danisco A/S
Priority to AU2003274375A priority Critical patent/AU2003274375A1/en
Publication of WO2004039993A1 publication Critical patent/WO2004039993A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/08—Deoxysugars; Unsaturated sugars; Osones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention relates to a novel polynucleotide encoding a pyranosone dehydratase.
  • the present invention further relates to the in situ production of a pyranosone dehydratase in plants or a part thereof, and processes for protecting plants and/or a plant part, such as a seed, from pathogens, such as pathogenic fungi, by in situ production of pyranosone dehydratase.
  • glucose can be oxidised by pyranose 2- oxidase (EC 1.1.3.10, P2O) to form glucosone (D-arabino-hexos-2-ulose), which in turn can be converted to cortalcerone by pyranosone dehydratase (PD) [Koths, K.; Halenbeck, R.; Moreland, M. (1992), Carbohydr Res. Vol. 232 No. 1, PP. 59-75; Gabriel, J.; Vole, J.; Sedmera, P.; Daniel, G.; Kubatova, E. (1993), Arch. Microbi., 160:27-34].
  • PD pyranosone dehydratase
  • P2O and PD have been purified in fungi and P2O has been cloned.
  • PD has been purified from Polyporus obtusus by Koths et al (1992), and from Phanerochaete chrysosporium by Gabriel et al (1993).
  • starch can be converted to 1,5-anhydro-D-fructose (AF) [S. Yu and J. Marcussen, Recent Advances in Carbohydrate Bioengineering; Gilbert, H. J.; Davies, G. J; Henrissat B.; Svensson, B., Eds.; Royal Society of Chemistry (RS.C) Press, 1999. 242-250].
  • AF 1,5-anhydro-D-fructose
  • Plant disease is a major cause of crop loss.
  • Various strategies have been developed to control disease, one of the most common of which is the use of chemicals. This approach is usually expensive, not always effective, and often harmful to non target organisms, including humans, and the environment.
  • a preferred approach is to develop, through breeding and/or genetic modification, genotypes resistant or tolerant to diseases.
  • Another aspect provides a construct comprising a polynucleotide sequence encoding an enzyme and a polynucleotide encoding pyranosone dehydratase selected from:
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP, operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding glucan lyase operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding pyranose-2-oxidase operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • the polynucleotide sequence according to the present invention in the expression vector may encode a pyranosone dehydratase comprising at least one amino acid sequence selected from the following: (i) KPHCEPEQPAALPLFQPQLVQGGRPDXY NEAFPFRSDSSK (SEQ ID NO: (i)
  • the polynucleotide sequence may encode for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos.2-15 or a variant, homologue or derivative thereof.
  • the polynucleotide acccording to the present invention in the expression vector may encode a pyranosone dehydratase comprising the following amino acid sequence or a variant, homologue or derivative thereof:
  • Another aspect provides an expression vector comprising a polynucleotide sequence selected from:
  • a polynucleotide comprising the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20 or the complement thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv) ; operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding an enzyme and a polynucleotide encoding a pyranosone dehydratase selected from:
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20 or the complement thereof;(ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20 or the complement thereof;(ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.20;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism; which said enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone, or APP
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding glucan lyase and a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
  • Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase and a polynucleotide selected from:
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or host organism.
  • the nucleotide sequence referred to in the present invention in particular the polynucleotide encoding a pyranosone dehydratase, such as the polynucleotide sequence present in a construct or an expression vector in accordance with the present invention, is selected from:
  • a polynucleotide which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least
  • the invention is therefore intended to encompass constructs and/or expression vectors which comprise (a) a polynucleotide sequence encoding pyranosone dehydratase; (b) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP, (c) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding glucan lyase; or (d) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding pyranose-2-oxidase.
  • the polynucleotide encoding the pyranosone dehydratase and/or the polynucleotide sequence encoding the glucan lyase and/or the polynucleotide encoding the further enzyme and/or the polynucleotide encoding pyranose-2-oxidase is fused with a polynucleotide sequence which encodes for an endogenous or heterologous targeting signal sequence for extracellular or intracellular targeting of the gene products.
  • a further aspect relates to a host cell or host organism into which has been incorporated a polynucleotide according to the present invention, a construct according to the present invention or an expression vector according to the invention.
  • Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence according to the present invention encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more antimicrobial compounds.
  • Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence according to the present invention encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP.
  • Another aspect of the invention relates to a host cell of host organism into which has been incorporated an expression vector comprising a polynucleotide selected from:
  • Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase and an expression vector comprising a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • heterologous polynucleotide sequence When it is the case that more than one heterologous polynucleotide sequence is present in a host cell or host organism, the skilled person would be a readily aware of various methods of providing two or more polynucleotide sequences to a host cell or host organism. For instance, the host cell or host organism may be transformed with a single construct or a single expression vector comprising more than one heterologous polynucleotide sequences.
  • each polynucleotide sequence may be present in a separate construct or expression vector, for example, host cells or host organisms may be transformed with (a) a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a separate construct comprising the polynucleotide sequence encoding glucan lyase, for example; or (b) a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a separate construct comprising a polynucleotide sequence encoding pyranose-2-oxidase, for example.
  • the present invention further encompasses a method whereby a first heterologous polynucleotide sequence is transformed into a first host cell or host organism, i.e. by transformation with a first construct or expression vector; and one or more further heterologous polynucleotide sequences are transformed into one or more further host cells or host organisms, i.e. by transformation with one or more further constructs or expression vectors.
  • the first host cell or host organism is crossed with the one or more further host cells or host organisms in order to provide a host cell or host organism comprising both the first heterologous polynucleotide sequence and the one or more further heterologous polynucleotide sequences.
  • a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a construct comprising the polynucleotide sequence encoding a norther enzyme, such as glucan lyase for example can be introduced into a host cell or host organism by separate transformation events, for example, by recombination or meiosis.
  • a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a construct comprising the polynucleotide sequence encoding pyranose-2-oxidase for example can be introduced into a host cell or host organism by separate transformation events.
  • the host organism may be a plant or a microorganism, such as a fungus, preferably a filamentous fungus, a yeast or a bacterium.
  • the host organism is a yeast.
  • the host organism is a transgenic plant.
  • a method of preparing pyranosone dehydratase in situ in an organism comprising culturing a host cell or host organism according to the present invention under conditions to provide for expression of pyranosone dehydratase.
  • a further aspect relates to a method of preparing one or more of microthecin or ascopyrone P (APP) in situ which comprises cultivating a host cell or host organism according to the present invention under conditions to provide for expression of the coding sequence encoding pyranosone dehydratase, whereby said pyranosone dehydratase converts constituents naturally present in the host cell or host organism, such as 1,5-D-anhydrofructose or starch dextrins for example, into microthecin and/or APP.
  • APP ascopyrone P
  • a further aspect relates to a method of preparing one or more of microthecin and/or APP in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding glucan lyase, under conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and glucan lyase, whereby said pyranosone dehydratase and glucan lyase converting starch dextrins present in the host cell or organism into one or more antimicrobial compounds, such as microthecin and or APP.
  • a further aspect relates to a method of preparing cortalcerone in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide encoding a further enzyme, under conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces an antimicrobial compounds, such as cortalcerone, whereby said pyranosone dehydratase together with said further enzyme converts constituents naturally present in the host cell or host organism, such as glucose and/or glucosone for example, into one or more antimicrobial compounds, such as cortalcerone.
  • a further aspect relates to a method of preparing cortalcerone in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding pyranose-2-oxidase, imder conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and pyranose-2- oxidase, said pyranosone dehydratase and pyranoser2-oxidase converting glucose present in the host cell or organism into a antimicrobial compound, such as cortalcerone.
  • a further aspect relates to a method of preparing one or more antimicrobial compounds, such as microthecin or a derivative thereof, in situ which method comprises:
  • the present invention yet further provides a method of preparing one or more antimicrobial compounds, such as microthecin or a derivative thereof, in situ in a host cell or in a host organism, comprising tiansforming a host cell or a host organism with a polynucleotide encoding pyranosone dehydratase according to the present invention, and culturing the transformed host cell or host organism under conditions to provide for expression of the polynucleotide such that the pyranosone dehydratase so produced converts constituents in and/or around the host cell or host organism to one or more antimicrobial compounds, such as one or more of microthecin, cortalcerone or APP.
  • one or more antimicrobial compounds such as microthecin or a derivative thereof
  • the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
  • the method may further comprise tiansforming the host cell or host organism with a further polynucleotide sequence encoding an enzyme, preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
  • an enzyme preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
  • the host cell or host organism may be cultured in a medium comprising a constituent which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase.
  • At least one of the constituents which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase may be present naturally within the host cell or host organism.
  • the host cell or host organism is a plant or a microorganism, such as a fungus, preferably a filamentous fungus, a yeast or a bacterium.
  • a further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing a microorganism in or on a transgenic organism, in particular a transgenic plant, comprising tiansforming said plant with a polynucleotide encoding pyranosone dehydratase according to the present invention; and growing said transformed plant.
  • the method may further comprise transforming the transgenic organism with a further polynucleotide sequence encoding an enzyme, preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
  • an enzyme preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
  • the method may further comprise contacting said transgenic organism with a constituent which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase.
  • At least one of the constituents which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase may be present naturally within the host cell or host organism.
  • the growth of the microorganism may be prevented and/or inhibited and/or the microorganism maybe killed due to the production of one or more antimicrobial compounds, such as microthecin or a derivative thereof, in said transgenic organism.
  • one or more antimicrobial compounds such as microthecin or a derivative thereof, in said transgenic organism.
  • the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
  • the antimicrobial compounds produced in situ in a host organism may be further modified by endogenous enzymes or heterologously introduced enzymes.
  • the present invention therefore further relates to the production of derivatives of the antimicrobial compounds according to the present invention from said antimicrobial compounds in situ in the host organism, wherein the derivatives of the microbial compounds also exhibit anti-microbial activity.
  • a further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing a microorganism in and/or on a transgenic organism, in particular a transgenic plant, comprising tiansforming said organism with a polynucleotide encoding pyranosone dehydratase according to the present invention, said method comprising contacting said organism with 1,5-D-anhydrofructose and/or glucosone.
  • a further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing microorganisms in a transgenic plant into which has been incorporated a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
  • the present invention yet further provides a method of preparing a transgenic organism, preferably a plant or part thereof, which organism or part thereof is resistant to one or more pathogens (particularly one or more fungal pathogens), comprising transforming a cell of the organism, preferably a plant cell, with a polynucleotide sequence encoding pyranosone dehydratase according to the present invention, whereby the transformed organism produces one or more antimicrobial compounds, such as microthecin or a derivative thereof, from constituents present in the cell of the organism, preferably the plant cell and/or around the cell of the organism, preferably the plant cell.
  • the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
  • the antimicrobial compound may be one or more of microthecin, cortalcerone or APP.
  • the constituents or components present in the host cell or host organism from which the antimicrobial compound, such as microthecin or a derivative thereof may be produced may be one or more of the following constituents: ⁇ -l,5-anhydrofructose, starch, starch dextrins.
  • KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No. 4);
  • VSWLENPGELR SEQ ID No. 5
  • DGVDCLWYDGAR SEQ ID No. 6
  • PAGSPTGIVRAEWTRHVLDVFGXLXXK SEQ ID No. 7
  • HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK SEQ ID No. 8
  • polynucleotide sequence may be selected from:
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20, or a fragment thereof;
  • a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No.1 or SEQ ID No.20;
  • a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No.1 or SEQ ID No.
  • a polynucleotide which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least
  • the transgenic organism for example the transgenic plant or part thereof may comprise one or more constructs and/or expression vectors as defined by the present invention.
  • the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the expression of a gene encoding a pyranosone dehydratase.
  • the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the co-expression of genes encoding (i) a pyranosone dehydratase and (ii) a glucan lyase.
  • the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the co-expression of genes encoding (i) a pyranosone dehydratase and (ii) a pyranose-2-oxidase.
  • the polynucleotide sequence(s) of the present invention may be expressed constitutively (i.e. at all times) within the organism (for example plant).
  • the polynucleotide sequence(s) of the present invention may be expressed non- constitutively (i.e. only at specific times during the life cycle of the organism (such as plant) and/or following a specific event, such as pathogen invasion, for example).
  • the gene product(s) may be produced in all or substantially all parts of the plant.
  • the gene product(s) may be produced only in specific parts, i.e. specific cells and/or tissues, of the organism (for example the plant).
  • the gene product(s) may be targeted to a specific subcellular or extracellular compartment of the organism or host cell (for example of the plant or plant cell).
  • the gene product(s) may be targeted to one or more of the following parts of the host or host cell (in particular the plant or plant cell): extracellular spaces (i.e. apoplastic space and/or cell wall); intracellular spaces; cytosol; plastid (including chloroplast or amyloplast); vacuoles (starch granules); endoplasmic reticulum (ER).
  • fungus includes any taxonomic group that is classified within one or more of the following taxons: Oomycetes, Ascomycetes, Fungi imperfecta, Deutormycetes, Basidiomycetes, Zygomycetes, or Mastigomycetes.
  • antifungal and “fungal” should be construed accordingly.
  • the present invention and particularly the organisms (such as the plants) which are resistant to fungal pathogens, may be used in combination with conventional anti-fungal strategies, such as the provision of fungicides and/or in combination with other antifungal gene traits.
  • the transgenic organism or part thereof is resistant to one or more of the following diseases: Bacterial leaf blight, Bacterial mosaic, Bacterial sheath rot, Basal glume rot, Black chaff (bacterial streak), Spike blight (gummosis), leaf spot, blights, cankers, galls.
  • Bacterial leaf blight Bacterial mosaic, Bacterial sheath rot, Basal glume rot, Black chaff (bacterial streak), Spike blight (gummosis), leaf spot, blights, cankers, galls.
  • these diseases may be caused by one or more of the following pathogens: Pseudomonas spp., Clavibacter spp., Xanthomonas spp., Rathayibacter spp.,. Corynebacterium spp., Erwinia spp. Agrobacterim spp., Xylella spp.
  • the transgentic organism or part therof (in particular where the transgenic organism is a transgenic plant) according to the present invention is resistant to one or more of the following diseases: Downey mildew, powdery mildew, grey mould, canker, black scurf, rots, soft rot, fruit rot, basal rot, crown rot, root rot, rusts, stem rusts, stripe rusts, blight, early blight, late blight, pithium blight, leaf spot, wilt, leaf blotch, glume blotch, black leg, and Black Sigatoka.
  • diseases Downey mildew, powdery mildew, grey mould, canker, black scurf, rots, soft rot, fruit rot, basal rot, crown rot, root rot, rusts, stem rusts, stripe rusts, blight, early blight, late blight, pithium blight, leaf spot, wil
  • the transgenic plant or part thereof according to the present invention may be a plantain or banana plant or part thereof and may be resistant to Mycosphaerella fijiensis (Anamorph: Paracercospora fijiensis).
  • the transgenic organism (such as the trangenic plant) or part thereof according to the present invention is resistant to one or more pathogens selected from the following genera: Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Phythium, Phytophthora, Thielaviopsis, Aspergillus, Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Eutypa, Gaeumanomyces, Helminthosporiwn, Macrophomina, Nectria,Peronospora, Phoma, Phymatotrichum, Plasmopara, Podosphaera, Puccinia, Puthium, Pyrenophora, Pyricularia, Scerotium, Sckerotinia, Sept
  • the transgenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one or more pathogens selected from the following: genera: Alternaria spp., Albugo spp., Aphanomyces spp., Amyloporia spp., Ascochyta, Aspergillus, Basidiophora, Bipolaris, Botrytis, Bremia, Cercospora, Cladosporium, Claviceps, Coniophora spp., Colletotrichum, Diplodia, Diplocarpon spp., DonUoporia spp., Drechslera, Erysiphe, Eutypa, Fibroporia spp., Fusarium, Gaeumanomyces, Geotrichum, Guignardia, Gymnosporangium, Helmintosporium, Hemileia, Kabatiella, Leptosphaeria, Macrophomina, Marssonina s
  • the transgenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one or more of the following pathogens: Alternaria brassicol; Alternaria solani, Ascochyta pisi, Botrytis cinerea, Cercospora kikuchii, Cercospora zaea-maydis; Colletotrichum lindemuthianum; Diplodia maydis; Erysiphe graminis fsp. Graminis; Erysiphe graminis f.sp.
  • Puccinia asparagi Puccinia recondita; Puccinia arachidis; Puthium aphanidenmatum; Pyrenophora tritici-repentens; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Rhizoctonia cerealis; Scerotium rolfsii; Sclerotinia sclerotiorum; Septoria Iycopersici; Septoria glycines; Septoria nodorum; Septoria tritici; Thielaviopsis basicola; Uncinula necator; Venturia inaequalis; Verticillium dahliae; Verticillium albo-atrum.
  • the transgenic organism for example the transgenic plant or part thereof according to the present invention is resistant to one or more of the following pathogens: downey mildew or powdery mildew, such as one or more of the following: Erysiphe spp.; Erysiphe cichoracearum (e.g cucumbers, endive, lettuce, melons, potato, pumpkin, squash); Erysiphe cruciferarum (e.g. broccoli, Brussels sprouts, cauliflower, and other cole crops; radicchio, radishes, turnips); Erysiphe lycopersici & Oidium lycopersicum (e.g. tomatoes); Erysiphe pisi (e.g.
  • the trangenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one ore more of the oomycete fungi selected from Pythium, Aphanomyces spp. Peronospora spp., Phytophthora spp., Albugo spp., Basidiophora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp., Peronophythora spp.
  • the oomycete fungi selected from Pythium, Aphanomyces spp. Peronospora spp., Phytophthora spp., Albugo spp., Basidiophora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp., Peronophythora spp.
  • microthecin or a derivative thereof surprisingly overcomes any instability of the microthecin or the derivative.
  • microthecin or a derivative thereof, particularly in plants or parts thereof provides effective pathogen , particularly fungal pathogen, control.
  • pathogen particularly fungal pathogen
  • the control afforded by the in situ production of microthecin is advantageously broad spectrum, i.e. targets more than one pathogen.
  • cortalcerone and or ascopyrone P particularly in plants or parts thereof, provides effective pathogen, particularly fungal pathogen, control.
  • cortalcerone is advantageously broad spectrum, i.e. target more than one pathogen.
  • the present invention may be used to advantageously control fungal pathogens on the transgenic plant during growth (cultivation) thereof.
  • the present invention may be used to control fungal pathogens on the transgenic plant or part thereof after harvest, for example during storage.
  • the transgenic plant or part thereof according to the present invention will be protected from attack by fungal pathogens both during cultivation and post-harvest, such as during storage.
  • the present invention may, therefore, be applicable to preserve stored seeds and/or other plant parts, as well as stored silage for example, and may be used to preserve timber, cotton, flax and other plant derived non-edible products.
  • the present invention may be used to control or prevent fungal infection of plant seeds after sowing, particularly prior to growth, and or during germination and initial seedling growth.
  • the transformation of a host organism with at least a polynucleotide according to the present invention may result in the production of an antimicrobial compound.
  • the derivative of microthecin may be 2-furyl-hydroxymethyl-ketone or 4-deoxy-g/ycero-hexo-2,3-diluose.
  • a typical hydrocarbyl group is a hydrocarbon group.
  • hydrocarbon means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group.
  • the term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
  • the hydrocarbyl group is an optionally substituted alkyl group.
  • the hydrocarbyl group is selected from C--C*o alkyl group, such as C C ⁇ alkyl group, and C--C 3 alkyl group.
  • Typical alkyl groups include Cj alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 7 alkyl, and C 8 alkyl.
  • the aryl group or one or more of the aryl groups may contain a hetero atom.
  • the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic.
  • Typical hetero atoms include O, N and S, in particular N.
  • the hydrocarbyl group is selected from - (CH 2 ) 1-10 -cycloalkyl, -(CH 2 ) 1 - 1 o-C 3 . 10 cycloalkyl, -(CH 2 ) 1 - 7 -C 3 . 7 cycloalkyl, -(CH ⁇ .s-C;-. 5 cycloalkyl, -(CH 2 ) ⁇ . 3 -C 3-5 cycloalkyl, and -CH 2 - C 3 cycloalkyl.
  • R 3 contains from 1 to 10 carbon atoms.
  • R 3 contains from 1 to 5 carbon atoms.
  • R 3 is a -s alkyl group.
  • R 4 is H.
  • the antimicrobial compound is a compound having Formula II
  • R_t is OH
  • R 1 is H
  • R 2 is H
  • At least one of R and R is H.
  • R 1 is H.
  • R 2 is H.
  • R 1 is H and R 2 is H.
  • R 1 may be H
  • R 2 may be H
  • R 4 may be H
  • the antimicrobial compound has the Formula
  • microthecin This compound is commonly referred to as microthecin
  • R 1 is H
  • R 2 is H
  • R 4 is OH
  • the sequence is in an isolated form.
  • isolated means that the sequence is not in its natural environment (i.e. as found in nature).
  • isolated means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.
  • the sequence may be separated from at least one other component with which it is naturally associated.
  • the sequence is in a purified form.
  • purified also means that the sequence is not in its natural environment (i.e. as found in nature).
  • purified means that the sequence is at least substantially separated from at least one other component with which the sequence is naturally associated in nature and as found in nature.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double- stranded or single-stranded whether representing the sense or antisense strand.
  • nucleotide sequence in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
  • the nucleotide sequence per se of the present invention does not cover the native nucleotide sequence according to the present invention in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment.
  • the term "non-native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • the amino acid sequence of the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
  • the amino acid sequence of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the nucleotide sequence of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).
  • the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • a nucleotide sequence encoding either an enzyme which has the specific properties as defined herein or an enzyme which is suitable for modification may be identified and/or isolated and/or purified from any cell or organism producing said enzyme.
  • Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
  • a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.
  • enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, fransforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for enzyme (i.e. maltose), thereby allowing clones expressing the enzyme to be identified.
  • an expression vector such as a plasmid, fransforming enzyme- negative bacteria
  • a substrate for enzyme i.e. maltose
  • the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805.
  • the phosphoroamidite method oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
  • the nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, pp 487-491).
  • the present invention may also encompass amino acid sequences of enzymes having the specific properties as defined herein.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.
  • the enzyme is not a native enzyme.
  • native enzyme means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • the polypeptide according to the present invention may have a molecular weight of between about 96kDa and about 102kDa, preferably of about 97kDa, as measured by SDS-Page.
  • SEQ ID No. 16 wherein X is either H or Q; or an amino acid sequence which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% homologous with SEQ ID No. 16.
  • polypeptide according to the present invention for instance the isolated polypeptide or the polypeptide encoded by the polynucleotide according to the present invention, may comprise the following amino acid sequence: MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDSSKCPNIIG
  • FKFCLTYLHFILSGHRAGGQH (SEQ ID No. 17) wherein X is either H or Q; or an amino acid sequence which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% homologous with SEQ ID No. 17.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • an homologous sequence is taken to include a nucleotide seq ⁇ ence which may be at least 40, 50, 60, 70, 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence).
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ - alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ - alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the ⁇ -carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • the nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells e.g. rat, mouse, bovine and primate cells
  • such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other ardmal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • optimised expression and/or activity in a host cell or in vitro include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in prefened environmental conditions, e.g. temperature, pH, substrate.
  • Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • the present invention also encompasses sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, imder stringent conditions (e.g. 50°C and 0.2xSSC).
  • imder stringent conditions e.g. 50°C and 0.2xSSC.
  • an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare an enzyme of the present invention.
  • Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
  • Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals.
  • Prokaryotic promoters and promoters functional in eukaryotic cells may be used.
  • Tissue specific or stimuli specific promoters may be used.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the enzyme produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the vectors of the present invention may contain one or more selectable marker genes.
  • the most suitable selection systems for industrial micro-organisms are those formed by the group of selection markers which do not require a mutation in the host organism.
  • Suitable selection markers may be the dal genes from B. subt ⁇ lis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • Alternative selection markers may be the Aspergillus selection markers such as amdS, argB, niaD and sC, or a marker giving rise to hygromycin resistance.
  • a constitutive promoter may be selected to direct the expression of the desired nucleotide sequence of the present invention.
  • Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.
  • useful promoters for transcription in a fungal host are those derived from the gene encoding A. oryzae TAKA amylase, the TPI (triose phosphate isomerase) promoter from S. cerevisiae (Alber et al (1982) J. Mol. Appl. Genet. 1, p419-434), Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ - amylase, A. niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • pCGN1761 contains fhe"double"CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has apUC-type backbone.
  • a derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRl site. This derivative is designated pCGN1761 ENX.
  • ⁇ CGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants.
  • the entire 35S promoter-coding sequence-tm/ terminator cassette of such a construction can be excised by Hindlil, Sphl, Sail, and Xbal sites 5'to the promoter and Xbal, BamHl and Bgll sites 3'to the terminator for transfer to transformation vectors such as those described below.
  • actin promoter is a good choice for a constitutive promoter.
  • the promoter from the rice Actl gene has been cloned and characterised (McElroy et al., 1990).
  • a 1. 3kb fragment of the promoter was found to contain all the regulator elements required for expression in rice protoplasts.
  • numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al, 1991). These incorporate the Actl-intron 1, Adhl 5' flanking sequence and Adhl-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter.
  • Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression.
  • the promoter expression cassettes described by McElroy et al (1991) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors.
  • the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et aL, 1993).
  • the PR-1 Promoter For Inducible Expression, the PR-1 Promoter :
  • the double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels.
  • one of the chemically regulatable promoters described in U. S. Patent No. 5, 614, 395 may replace the double 35S promoter.
  • the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector.
  • the chemically/pathogen regulatable tobacco PR1 a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of
  • EP 0 332 10 and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992).
  • pCIB1004 is cleaved with Ncol and the resultant 3 'overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
  • the fragment is then cleaved with Hindlil and the resultant PR-la promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed.
  • an Ethanol-Inducible Promoter may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U. S. Patent Nos. 5, 523, 311 and 5, 614, 395.
  • an Ethanol-Inducible Promoter may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U. S. Patent Nos. 5, 523, 311 and 5, 614, 395.
  • a promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention.
  • a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al., 1998).
  • the alcA gene encodes alcohol dehydrogenase 1, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
  • the CAT coding sequences in plasmid palcA CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et a/., 1998) are replaced by a coding sequence (polynucleotide sequence) of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.
  • the luciferase gene sequences are replaced by a gene sequence encoding a polynucleotide according to the present invention to form an expression cassette having a polynucleotide sequence according to the present invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods well known in the art.
  • the trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al., 1986) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al., 1988) fused to the hormonebinding domain of the rat glucocorticoid receptor (Picard et alirri 1988).
  • the expression of the fusion protein is controlled by any promoter suitable for expression in plants known in the art or described here.
  • This expression cassette is also comprised in the plant comprising the polynucleotide sequence according to the present invention fused to the 6xGAL4/minimal promoter.
  • tissue-or organ-specificity of the fusion protein is achieved leading to inducible tissue-or organ-specificity of the polypeptide produced.
  • a suitable root promoter is described by de Framond (1991) and also in the published patent application EP 0 452 269. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene- terminator cassette to a transformation vector of interest.
  • Patent Application WO 93/07278 describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells.
  • the gene sequence and promoter extending up to-1726 bp from the start of transcription are presented.
  • this promoter, or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner.
  • fragments containing the pith-prefened promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
  • this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a polynucleotide of the present invention in a pollen-specific manner.
  • the promoter can additionally include features to ensure or to increase expression in a suitable host.
  • the features can be conserved regions such as a Pribnow Box or a TATA box.
  • the promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
  • suitable other sequences include the Shi -intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat 1987 Gene 217, 217-225 and Dawson 1993 Plant Mol. Biol. 23: 97).
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • the above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shi -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention.
  • fused in relation to the present invention which includes direct or indirect attachment.
  • the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
  • a glucan lyase used in accordance with the present invention may have any one of the amino acid sequence shown in SEQ ID No. 21-25 (see hereinbelow).
  • a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention.
  • a nucleotide sequence is carried in a vector for the replication and expression of the nucleotide sequence.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • Gram positive bacteria from the genus Bacillus such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, Streptomyces lividans or S. murinus
  • Bacillus such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, Streptomyces lividans or S. murinus
  • Other bacteria that may be suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • Suitable filamentous fungus may be for example a strain belonging to a species of Aspergillus, such as Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp.
  • Suitable yeast organisms may be selected from the species of Kluyveromyces, Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae, or Hansenula (disclosed in UK Patent Application No. 9927801.2).
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • organism in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.
  • Suitable organisms may include a prokaryote, fungus, yeast or a plant.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis.
  • An example of an eukaryotic expression host is Hansenula polymorphia.
  • Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • Filamentous fungi cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known.
  • Aspergillus as a host microorganism is described in EP 0 238 023.
  • the host organism may be a filamentous fungus.
  • constructs used for heterologous expression preferably contain one or more of: a signal sequence which directs the amino acid sequence to be secreted, typically being of fungal origin, and a terminator (typically being active in fungi) which ends the expression system.
  • nucleotide sequence according to the present invention can be fused to a smaller or a larger part of a fungal gene encoding a stable protein.
  • This can stabilise the amino acid sequence.
  • a cleavage site recognised by a specific protease, can be introduced between the fungal protein and the amino acid sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the amino acid sequence.
  • a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli Such a fusion leads to cleavage in vivo resulting in production of the expressed product and not a larger fusion protein.
  • Host strains can generally be grown in either Vogel's or Fries minimal medium supplemented with the appropriate nutrient(s), such as, for example, any one or more of: his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol.
  • appropriate nutrient(s) such as, for example, any one or more of: his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol.
  • the transformation typically also involves a selectable gene marker which is introduced with the expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable.
  • a selectable gene marker which is introduced with the expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable.
  • Various marker/host systems are available, including the pyrG, argB and niaD genes for use with auxotrophic strains of Aspergillus nidulans; pyrG and argB genes for Aspergillus oryzae auxotrophs; pyrG, trpC and niaD genes for Penicillium chrysogenum auxotrophs; and the argB gene for Trichoderma reesei auxotrophs.
  • Dominant selectable markers including amdS, oliC, hyg and phleo are also now available for use with such filamentous fungi as A. niger, A. oryzae, A. ficuum, P. chrysogenum, Cephalosporium acremonium, Cochliobolus heterostrophus, Glomerella cingulata, Fulviafulva and Leptosphaeria maculans (for a review see Ward in Modern Microbial Genetics, 1991, Wiley-Liss, Inc., at pages 455-495).
  • a commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
  • the host organism can be of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can also be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King. et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast.
  • the constructs may contain a promoter active in yeast, such as a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells may be selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRPl, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
  • a preferred host organism suitable for the present invention is a plant.
  • the basic principle in the constraction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
  • the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
  • One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • a non-limiting example of such a Ti plasmid is pGV3850.
  • the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well- known and widely employed in the constraction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the nucleotide sequence or construct of the present invention may be first constructed in a micro-organism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful micro-organism is E. coll, but other micro-organisms having the above properties may be used.
  • a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the Ti-plasmid harbouring the nucleotide sequence or constract of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or constract of the invention, which DNA is subsequently transferred into the plant cell to be modified.
  • cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 322, the pUC series, the Ml 3 mp series, pACYC 184 etc.
  • the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is used for the transformation in E.coli.
  • the E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed.
  • the plasmid is then recovered.
  • sequence analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
  • the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al, Crit. Rev. Plant Sci., 4:1-46; and An et al, EMBO J. (1985) 4:277-284.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • transgenic plants are well known in the art.
  • either whole plants, cells or protoplasts may be transformed with a suitable nucleic acid construct encoding a target DNA (see above for examples of nucleic acid constructs).
  • a suitable nucleic acid construct encoding a target DNA
  • Suitable methods include Agrobacterium infection (see, among others, Turpen et al, 1993, J. Virol. Methods, 42: 227-239) or direct delivery of DNA such as, for example, by PEG- mediated transformation, by electroporation or by acceleration of DNA coated particles. Acceleration methods are generally preferred and include, for example, microprojectile bombardment.
  • the ballistic transformation technique (otherwise known as the particle bombardment technique) was first described by Klein et al. [1987], Sanford et al. [1987] and Klein et al. [1988] and has become widespread due to easy handling and the lack of pre-treatment of the cells or tissue in interest.
  • the principle of the particle bombardment technique is direct delivery of DNA-coated micro-projectiles into intact plant cells by a driving force (e.g. electrical discharge or compressed air).
  • a driving force e.g. electrical discharge or compressed air.
  • the micro-projectiles penetrate the cell wall and membrane, with only minor damage, and the transformed cells then express the promoter constructs.
  • PIG Particle Inflow Gun
  • the acceleration of the micro-projectiles can be controlled by a timer-relay solenoid and by regulation the provided helium pressure.
  • the use of pressurised helium as a driving force has the advantage of being inert, leaves no residues and gives reproducible acceleration.
  • the vacuum reduces the drag on the particles and lessens tissue damage by dispersion of the helium gas prior to impact [Finer et al. 1992].
  • non-biological particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • a particular advantage of microprojectile bombardment in addition to it being an effective means of reproducibly stably transforming both dicotyledons and monocotyledons, is that neither the isolation of protoplasts nor the susceptibility to Agrobacterium infection is required.
  • An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension. The screen disperses the tungsten-DNA particles so that they are not delivered to the recipient cells in large aggregates.
  • the projectiles aggregate and may be too large for attaining a high frequency of transformation. This may be due to damage inflicted on the recipient cells by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters.
  • Filters containing the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • one or more screens are also positioned between the gun and the cells to be bombarded.
  • the number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 2 to 3.
  • a preferred step is to identify the transformed cells for further culturing and plant regeneration. This step may include assaying cultures directly for a screenable trait or by exposing the bombarded cultures to a selective agent or agents.
  • An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize.
  • This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage, incubating the cells at, e.g., 18°C and greater than 180 ⁇ E m "2 s "1 , and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
  • An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • a selective agent such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
  • bombarded cells on filters are resuspended in nonselective liquid medium, cultured (e.g. for one to two weeks) and transferred to filters overlaying solid medium containing from 1-3 mg/1 bialaphos. While ranges of 1- 3 mg/1 will typically be preferred, it is proposed that ranges of 0.1-50 mg/1 will find utility in the practice of the invention.
  • the type of filter for use in bombardment is not believed to be particularly crucial, and can comprise any solid, porous, inert support.
  • Cells that survive the exposure to the selective agent may be cultured in media that supports regeneration of plants. Tissue is maintained on a basic media with hormones for about 2-4 weeks, then transferred to media with no hormones. After 2-4 weeks, shoot development will signal the time to transfer to another media.
  • Regeneration typically requires a progression of media whose composition has been modified to provide the appropriate nutrients and hormonal signals during sequential developmental stages from the transformed callus to the more mature plant.
  • Developing plantlets are transferred to soil, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO 2 , and 250 ⁇ E m "2 s "1 of light.
  • Plants are preferably matured either in a growth chamber or greenhouse. Regeneration will typically take about 3-12 weeks.
  • cells are grown on solid media in tissue culture vessels.
  • An illustrative embodiment of such a vessel is a petri dish.
  • Regenerating plants are preferably grown at about 19°C to 28°C. After the regenerating plants have reached the stage of shoot and root development, they may be transfened to a greenhouse for further growth and testing.
  • Genomic DNA may be isolated from callus cell lines and plants to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art such as PCR and/or Southern blotting.
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred.
  • Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982 ; Bevan et aL, 1983), the bargee, which confers resistance to the herbicide phosphinothricin (White et al., 1990 ; Spencer et al., 1990), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfrgene, which confers resistance to methatrexate (Bourouis et al., 1983), and the EPSPS gene, which confers resistance to glyphosate (U. S. Patent Nos. 4, 940, 935 arid 5, 188, 642).
  • T-DNA border sequence typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
  • the binary vectors pcIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner.
  • pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, 1982 ; Bevan et al., 1983 ; McBride et al., 1990).
  • Xhol linkers are ligated to the EcoRVfragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable noslnptll chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the Xhol digested fragment are cloned into Sail-digested pTJS75kan to create ⁇ CIB200 (see also EP 0 332 104, example 19).
  • pCIB200 contains the following unique polylinker restriction sites : EcoRl, Sstl, Kpnl, Bglll, Xbal, and Sail.
  • pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites.
  • Unique restriction sites in the polylinker of pCIB2001 are EcoRl, Sstl, Kpnl, Bglll, Xbal, Sail, MM, Bell, Avrll, Apal, Hpal, and Stul.
  • pCIB2001 in addition to containing these unique restriction sites also has plant and bacteria kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived M4 function for mobilization between E. coli and other hosts, and the OriTand OriVfunctions also from RK2.
  • the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulator signals.
  • pCIBlO and Hygromycin Selection Derivatives thereof are EcoRl, Sstl, Kpnl, Bglll, Xbal, Sail, MM, Bell, Avrll, Apal, Hpal, and Stul.
  • pCIB2001 in addition to containing these unique restriction sites also has
  • the binary vector pCIBlO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range . plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (1987). Various derivatives of pCIBlO are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al., 1983). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e. g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non- Agrobacterium transformation is described, a.
  • pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin).
  • the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
  • the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
  • the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
  • the resultant derivative of pCIB246 is designated pCIB3025.
  • the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
  • the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al., 1987).
  • This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlil, and BamHl.
  • This vector is suitable for the cloning of plant expression cassettes containing their own regulator signals, b.
  • pSOG19 and pSOG35 pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
  • DFR dihydrofolate reductase
  • PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGlO.
  • a 250-bp fragment encoding the E. colidihydrofolate reductase type 11 gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the opaline synthase terminator.
  • pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the opaline synthase terminator.
  • Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35.
  • pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlil, Sphl, Pstl and EcoRl sites available for the cloning of a foreign gene.
  • the gene sequence, of interest Once the gene sequence, of interest has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium.
  • Non Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., 1984 ; Potrykus et al., 1985 ; Reich et al., 1986 ; and Klein et al., 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species.
  • Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e. g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e. g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al., 1993).
  • the transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E.
  • helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
  • the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, 1988).
  • Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
  • Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4, 945, 050, 5, 036, 006, and 5, 100, 792. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e. g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i. e. cofransformation) and both these techniques are suitable for use with this invention. Cofransformation may have the advantage of avoiding complete vector constraction and of generating transgenic plants with unlined loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable.
  • a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., 1986).
  • Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, fransformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts.
  • Gordon-Kamm et al. (1990) and Fromm et al. (1990) have published techniques for transformation of A188-derived maize line using particle bombardment.
  • WO 93/07278 and Koziel et al. (1993) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1. 5-2. 5 mm length excised from a maize ear 14-15 days after pollination and a PDS-lOOOHe Biolistics device for bombardment.
  • Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
  • Protoplast-mediated fransformation has been described for Japonica-types and Indica-types (Zhang et al., 1988 ; Shimamoto et al., 1989 ; Datta et al., 1990). Both types are also routinely transformable using particle bombardment (Christou et a/., 1991).
  • WO 93/21335 describes techniques for the transformation of rice via electroporation.
  • Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the fransformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (1992) using particle bombardment into cells of type C long- term regenerable callus, and also by Vasil et al. (1993) and Weeks et al. (1993) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.
  • Each plate of embryos is shot with the DuPont Biolistics helium device using a burst pressure of- 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/1 basta in the case of pCIB3064 and 2 mg/1 methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as"GA7s "which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
  • regeneration medium MS + 1 mg/liter NAA, 5 mg/liter GA
  • the plants obtained via transformation with a gene of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra.
  • the expression of a gene of the present invention in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R. (1981) ; Wood D. R. (Ed.) (1983) ; Mayo 0. (1987) ; Singh, D. P. (1986) ; and Wricke and Weber (1986). Plant breeding can be used to produce germplasm containing frangenes inserted from different transformation events, and hence can be used to produce plants co-expressing both a pyranosone dehydratase gene and a glycan lyase gene.
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants.
  • said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
  • Specialized processes such as hydroponics or greenhouse technologies can also be applied.
  • measures are undertaken to confrol weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the present invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well- defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
  • different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means.
  • Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained, which, due to their optimized genetic"equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
  • Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD@), methalaxyl (Apron@), and pirimiphos-methyl (Actellic@). If desired, these compounds are formulated together with further carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit. It is a further aspect of the present invention to provide new agricultural methods, such as the methods examplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
  • the seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds.
  • the bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.
  • a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
  • the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type.
  • the bag, container or vessel is provided so as to. exclude or limit water and moisture from contacting the seed.
  • the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering.
  • water absorbent materials are placed between or adjacent to packaging material layers.
  • the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
  • a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.
  • a small population of tomato plants infected with powdery mildew is maintained separately and used as the source of inoculum. Conidia from these plants is used to infect six-week old healthy confrol and transgenic plants. Plant leaves infected with conidia of Erysiphe polygoni were excised and shaken above the top of the test plants to allow conidia to fall onto the healthy leaves (Fletcher & Smewin, 1988, Plant Pathol. 37: 594-598). The temperature of the greenhouse is maintained at 25 to 30 DEG C. and the humidity at above 70%. The percentage of leaf area of the inoculated plants covered by conidia or necrotic lesions is recorded.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
  • pectinolyticum P. peri ⁇ lum, P. periplocum, P. perplexum, P. polymastum, P. porphyrae, P. prolatum, P. proliferatum, P. pulchrum, P. ramificatum, P. regulare, P. rhizosaccharum, P. rostratum, P. salpingophorum, P. segnitium, P. spinosum, P. splendens, P. sulcatum, P. sylvaticum, P. terrestris, P. torulosum, P. tracheiphilum, P. ultimum, P. ultimum var. sporangiiferum, P.
  • the host (or transgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Oomycete plant fungal diseases:
  • Botrytis sp. (Attack many different plants)
  • P. infestans Late Blight, Very important pathogen of Potato/Tomato/Eggplants.
  • H. maydis (gives Helmintosporium Leaf Blight in Corn) H. carbonum (gives Helmintosporium Leaf Blight in Corn) H. turcicum ( gives Helmintosporium Leaf Blight in Corn) H. sativum (Spot Blotch, Wheat, Barley)
  • Verticillium sp. Verticillium wilt
  • V. albo-atrum Wood in alfalfa, tomato, hop
  • Gymnosporangium sp. i.e. apple rust
  • the host (or transgenic) organism or part thereof is resistant to microbial attack even post harvest.
  • the host (or transgenic) organism or part thereof is resistant to diseases which attack fruits and vegetables, such as one or more of the fungi selected from the following: Cladosporium sp, Rhizopus rot, Penicillium expansum (Blue mould) Alternaria rot, Botrytis cinerea (Grey Mould), Aspergillus niger (Black mold in many fruit and vegetable species, i.e. onion) Mon ⁇ linia sp. (Brown rot), Fusarium sp..
  • the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention is resistant to bacterial infection/contamination, suitably gram positive bacteria or gram negative bacteria.
  • the host (or fransgenic) organism or part thereof in either living or dead form, or a crude or purified extract therefrom, can be used as a antimicrobial agent (for example an antifungal or anti-bacterial agent) for use in preventing or reducing microbial contamination in and/or on a second organism or a product/composition derived therefrom, for example to reduce or eliminate microbial contamination in a food stuff.
  • a antimicrobial agent for example an antifungal or anti-bacterial agent
  • the host (or transgenic) organism or part thereof according to the present invention is a fungus which is resistant to one or more diseases, such as those selected from: bacterial or fungal diseases which occur in mushrooms for example. That is to say, those diseases caused by the following bacteria for example: Pseudomonas tolaasii, P. cepacia, Burkholderia gladioli. Also the fungus Trichoderma can cause problems in mushroom farming.
  • the host (or transgenic) organism or part thereof is resistant to one or more of the following turf grass diseases: Anthracnose and Basal Rot (Colletotrichum graminicold), Blister Smut (Entyloma dactylidis), Brown Patch (Rhizoctonia solani), Damping-off (Pythium, Fusarium, Helminthosporium and Rhizoctonia spp.), Dollar Spot (Sclerotinia homoeocarpa), Fairy Ring (Lycoperdon, Psalliota, Clitocybe species and others), Fairy Ring (Marasmius oreades), Leaf Spots, Blight, Foot Rots and Melting-out (Drechslera spp.), Powdery Mildew (Erysiphe graminis), Red Thread (Laetisaria fuciformis), Rust (Puccinia s
  • nivale and M. nivale Snow Scald (Myrioscierotinia borealis or Scierotinia borealis), Stripe Smut (Ustilago striiformis), Take-all or Ophiobolus Patch (Gaeumannomyces graminis var. avenae), Pythium blight (Pithium spp.).
  • Fungal species can grow and reproduce in most natural, domestic and industrial environments, and can cause significant loss of yield and quality to plant crops, result in considerable damage to industrial and personal property, and cause ill health, disease and allergies in humans and animals.
  • fungal mycotoxins caused by fungal mycotoxins
  • allergies to fungal spores and pathological disease caused by human infection (particularly in immune compromised individuals).
  • pathological disease caused by human infection (particularly in immune compromised individuals).
  • fungal contamination is highly unsightly, can damage the structural integrity of surfaces, furnishings, materials, carpets, wooden flows, paints and wall papers, plaster, masonry, etc. and even cause major structural damage, particularly in wood frames properties.
  • the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention can be used to prepare raw materials for application in domestic and industrial environments.
  • transgenic trees of the invention can be used to prepare timber, paper and other raw or stractural materials which are less prone to microbial contamination, and/or transgenic cotton can be prepared to produce fabric which is less prone to microbial contamination.
  • the host (or transgenic) organism or part thereof in particular a plant or part thereof) according to the present invention can be used to prevent and/or treat one or more of the following domestic or industrial problems: Wet and dry rots (also known as 'building rot'):
  • 'mildew' and 'mould' or 'house fungus' include: Aspergillus sp., Penicillium sp. Aspergillus niger, Rhizopus sp. Stachybotrys chartarum (Black mould), Alternaria, sp. and Cladosporium sp ⁇
  • the host (or transgenic) organism or part thereof in particular a plant or part thereof) according to the present invention may be used to prevent and/or confrol food and/or feed contamination by microorganisms.
  • foodstuffs as used herein means both food and feed, including raw and processed plant material and non plant material.
  • the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention can be used directly in the preparation of food and feed, or as a preservative of foodstuffs, including parts of plants, and products derived from such plants.
  • the antimicrobial compound produced by the transformed organisms in either purified or crade form, may be added to foodstuffs.
  • the host (or transgenic) organism or part thereof according to the present invention can be used as an anti-microbial either by direct application of the living or dead organisms, for example a transgenic plant, yeast, filamentous fungi or bacterial preparation, or by preparing a crude extract preparation containing the anti-microbial compound of the present invention produced by the transgenic organisms, or a purified extract of said anti-microbial compound from said fransgenic organism.
  • the anti-microbial preparations may be used in a wide range of products, for example as a preservative, to prevent microbial spoilage and contamination applications in food and feed applications, as a anti-microbial food processing agent, as an ingredients in cosmetics, personal hygiene products, oral health products, as treatment to protect plant propogative material, such as a seed treatment, as a tuber treatment.
  • Necessary formulation aids such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art, and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J., and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Kunststoff. Using these formulations, it is also possible to prepare mixtures of the present antifungal polypeptide with other pesticidally active substances, fertilizers and/or growth regulators, etc., in the form of finished formulations or tank mixes.
  • Antifungal compositions contemplated herein also include those in the form of host cells, such as bacterial and fungal cells, capable of the producing the present antifungal polypeptide, and which can colonize roots and/or leaves of plants.
  • host cells such as bacterial and fungal cells
  • Examples of bacterial cells that can be used in this manner include strains of Agrobacterium, Arthrobacter, Azospyrillum, Clavibacter, Escherichia, Pseudomonas, Rhizobacterium, and the like.
  • Numerous conventional fungal antibiotics and chemical fungicides with which the present antifungal polypeptide can be combined are known in the art and are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council.
  • the present invention provides oligonucleotide hybridization probes useful in screening genomic and other nucleic acid libraries for DNA sequences encoding peptides, polypeptides, and proteins having pyranosone dehydratase activity, which probes can be designed based on the sequences provided herein.
  • Such probes can range from about 16 to about 28 nucleotides in length, generally about 16 nucleotides in length, more typically about 20 nucleotides in length, preferably about 24 nucleotides in length, and more preferably about 28 nucleotides in length.
  • these probes specifically hybridize to genomic DNA and other DNA sequences encoding peptides, polypeptides, or proteins having the same or similar activity as that of the pyransone dehydratase enzyme.
  • Such oligonucleotide probes can be synthesised by automated synthesis, and can be conveniently labelled at the 5' end with a reporter molecule such as a radionuclide, e.g., @32 P, or biotin.
  • the library can be plated as colonies or phage, depending upon the vector employed, and the recombinant DNA is transferred to nylon or nitrocellulose membranes. Following denaturation, neutralization, and fixation of the DNA to the membrane, the membrane is hybridized with the labeled probe. Following this, the membrane is washed, and the reporter molecule detected. Colonies or phage harboring hybridizing DNA are then isolated and propagated.
  • Biologically functional equivalent genomic DNAs and cDNAs can be isolated from organisms including higher plants using degenerate oligonucleotide primers based on the sequence (SEQ ID No. 1 or SEQ ID No. 20) of pyranosone dehydratase (T. Compton (1990) In Innis et al., Eds., PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego, pp. 39-45).
  • degenerate oligonucleotide primers can be used in conjunction with PCR technology employing reverse transcriptase to amplify biologically functionally equivalent cDNAs (E. S.
  • the degenerate oligonucleotides can be used to screen genomic libraries directly, and the isolated coding sequences can be transferred into transformation/expression vectors for crop plants.
  • the degenerate oligonucleotides can be used as probes to screen cDNA libraries from plants in, for example, ⁇ -phage vectors such as ⁇ -Zapil (Stratagene, La Jolla, Calif).
  • ⁇ -phage vectors such as ⁇ -Zapil (Stratagene, La Jolla, Calif).
  • the cDNA isolated in this manner can be transferred to an appropriate transformation/expression vector for introduction into monocot or dicot plants as described below.
  • One aspect of the present invention relates to amino acid sequences that are immunologically reactive with one or more of the amino acid sequences of claim 1.
  • Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.
  • the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof.
  • fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • the sequence of the present invention or a sequence comprising an immunological epitope thereof.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunised animal is collected and treated according to known procedures.
  • polyclonal antibodies to the sequence of the present invention contains antibodies to other antigens
  • the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against the sequence of the present invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct fransformation of B lymphocytes with oncogenic DNA, or fransfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • Monoclonal antibodies to the sequence of the present invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for the substance may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulphide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD etal (1989) Science 256:1275-128 1).
  • the amino acid sequence is used for large scale applications.
  • the amino acid sequence is produced in a quantity of greater than O.lg/litre, preferably greater than 0.2g/litre, more preferably greater than 0.3 g/lifre, more preferably greater than 0.5 g/litre, even more preferably from lg per litre to about 2g/litre, and in some instances greater than 02g/litre of the total cell culture volume after cultivation of the host organism.
  • amino acid sequence is produced in a quantity of from lOOmg per litre to about 900mg per lifre of the total cell culture volume after cultivation of the host organism.
  • amino acid sequence is produced in a quantity of from 250mg per lifre to about 500mg per lifre of the total cell culture volume after cultivation of the host organism.
  • the present invention relates to a nucleotide sequence and, also to a construct comprising the same.
  • the invention also relates to new uses of a known enzyme.
  • Figure 1 shows the electrophoresis of PD1 (pyranosone dehydratase isoform 1) on gels of 8-25% gradient.
  • Figure 1 A shows SDS-PAGE: Lanes 1 and 2 (from left) protein markers from Novex and Pharmacia respectively, Lanes 3, 4 and 5, purified PD1.
  • Figure IB shows Native PAGE: Lanes 1, 2, and 3, purified PD1, Lane 4, protein markers from Pharmacia, Lane 5, partially purified. The gels were stained with PhastGel Blue R from Pharmacia.
  • Figure 2 shows partial amino acid sequences of pyranosone dehydratase.
  • Figure 3 A illustrates the use of 1,5-anhydro-D-fructose and pyranosone dehydratase for the production of microthecin.
  • the reaction mixture consisted of 1,5-Anhydro-D- fructose 5 ⁇ l (3.0%), pyranosone dehydratase preparation 5 ⁇ l, 65 ⁇ l sodium phosphate buffer (pH 6.0) and water to a final volume of 0.7 ml.
  • the reaction was monitored by scanning between 350-190 nm. Reaction time at zero min was used as blank.
  • the absorbance peak at around 230 nm indicates the formation of microthecin.
  • the absorbance at 265 nm indicate the first formation of an intermediate from AF before it converts to microthecin.
  • Figure 3B illustrates the production of microthecin and its intermediate.
  • the reaction mixture consisted of lO ⁇ l partially purified pyranosone dehydratase (a ammonium sulfate fraction between 25-50% saturation of the cell-free extract from Phanerochaete chrysosporium), 25 ⁇ l AF (3.0%, w/v), lOO ⁇ l sodium phosphate buffer (0.1M, pH6.5) and 0.84 ml water.
  • the reaction was started by the addition of the substrate AF.
  • the reaction was performed at 22 °C.
  • the formation of microthecin and its intermediate was monitored at 230 nm and 263 nm, respectively. One can see that the intermediate was first formed and levelled off after around 20 min. There was a delay for the formation of microthecin but its formation continued until nearly all the AF in the reaction mixture was consumed.
  • Figure 4 shows SEQ ID NO.l, the gene coding for pyranosone dehydratase (PD) from the fungus Phanerochaete chrysosporium including the upstream regulatory region (-1- to -288), the coding region (1-3146) and down-stream region (3147-3444).
  • the presumed starch codon is ATG (bold) and stop codons are TGA TAG(bold).
  • the purified functional pyranosone dehydratase corresponds to a N-terminal 7-amino acid truncated PD if the translation is supposed to start from the bold codon ATG.
  • Figure 5 shows the upsfream region, the coding region and the down sfream region of the pyranosone dehydratase (PD) gene from the fungus Phanerochaete chrysosporium.
  • the DNA sequence theoretically could code for three proteins with different amino acid sequences. The bold amino acids are those found by amino acid sequencing of the purified functional PD. Identified infrons are underlined.
  • Figure 6 shows the final emergence of sugar beet seeds treated in accordance with Example 3.
  • Figure 7 shows the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogen Aphanomyces cochlioides.
  • Figures 8 and 9 show the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogens Pythium ultimum and Rhizoctonia solani respectively.
  • Figure 10 shows that the Hansenula expression vector pFPMT121.
  • Figure 11 shows the specific activity of algal ⁇ -l,4-glucan lyase when cells where grown at 24°C (square), 30°C (diamond) and 37°C (triangle). Cell-free exfracts opened mechanically on a Mini Bead-Beater were used to determine the specific activity by the DNS method.
  • Figure 12 shows an ELISA-plate from activity screening by the DNS method of repressed and induced extracts prepared from cultures of transformant 2 and 8 (2 cultures of each transformant were grown).
  • A1-A12 Assay on 10, 20 and 50 ⁇ l of cell-free extracts from repressed cultures. The cells were opened mechanically on a Mini Bead-Beater.
  • C1-C12 Assay on 10, 20 and 50 ⁇ l of cell-free exfracts from induced cultures. The cells were opened mechanically on a Mini Bead-Beater.
  • Figure 13 the specific activity of algal ⁇ -l,4-glucan lyase was measured by the DNS method in cell-free extracts from repressed and induced cultures.
  • the black columns show the specific activity when the cells were repressed in YND + 2% glucose.
  • the cells were opened mechanically on a Mini-Bead Beater.
  • the pink and blue columns show the specific activity when the cells were depressed in YND + 1% glycerol and induced with 1% methanol on the second day of growth.
  • the cells were opened mechanically on a Mini Bead-Beater (white) or opened with the chemical reagent LTAB (chequered).
  • Figure 14 shows Native-PAGE on a homogenous polyacrylamide gel.: (right) shows Native-PAGE on an 8-25% gradient polyacrylamide gel (right).
  • the gels were loaded in the same order: Lane 1: Raw extract from Aspergillus Niger. Lane 2: Fraction III. Lane 3: Fraction II. Lane 4: Algal ⁇ -l,4-glucan lyase purified from Aspergillus Niger. Lane 5: Fraction I. Lane 6: Raw extract from H .polymorpha. The gels were stained with PhastGel Blue R.
  • pyranosone dehydratase may be referred to herein as PD.
  • Phanerochaete chrysosporium (white rot fungus) is a biotechnologically important fungus due to its higher growth optimum temperature (40°C) and its ability to produce a range of extracellular oxidative enzymes. Accordingly, this fungus has been used for treatment of various wastes, including explosive contaminated materials, pesticides, and toxic wastes. Furthermore, Phanerochaete chrysosporium is the first basidiomycete genome to be sequenced (University of California and Department of Energy, USA).
  • a purified a heat-stable pyranosone dehydratase was obtained from P. chrysosporium. Studies have shown that this purified PD not only uses AF as substrate, but uses it more efficiently than its natural substrate, glucosone. Furthermore, the product was shown to be microthecin, an antifungal useful in plant protection.
  • N-terminal sequence of PD and the endo-N-terminal sequences of PD after hydrolysis with two proteinases were elucidated. Together these account for 332 amino acids or 37% of the full length of the PD protein based on the assumption that it has a Mr of 97kDa.
  • the reaction mixture consisted of 25 ⁇ l of anhydrofructose solution (3.0%), 10 ⁇ l PD preparation, 93 ⁇ l 0.1 M sodium phosphate (pH 6.5), and water to a final volume of 1 ml.
  • the reaction was mixed and scanned between 190 and 320 nm at room temperature (22 °C) every 5 min or after 30 min on a Perkin Elmer Lambda 18 uv/vis specfrophotometer. Absorbance values at 265 and 230 nm were recorded.
  • One activity unit of PD is defined as the increase of 0.01 of absorbance unit at 230 nm at 22 °C per min.
  • TLC for separation of glucosone, AF and microthecin was performed as described before using a solvent system of ethylacetate, acetic acid, methanol and water (12:3:3:2) [Yu S, Ahmad T, Pedersen M, Kenne L: ⁇ -l,4-Glucan lyase, a new class of starch/glycogen degrading enzyme. III. Substrate specificity, mode of action, and cleavage mechanism, Biochim Biophys Acta 1244: 1-9 (1995)]. A Merck silica gel 60 (20x20cm) plate with a thickness of 0,15 mm was used.
  • 1,5-Anhydro-D-fructose was assayed by the DNS method [Yu S, Olsen CE, Marcussen J: Methods for the assay of 1,5-anhydro-D-fructose and ⁇ -l,4-glucan lyase, Carbohydr. Res. 305: 73-82 (1998)].
  • the purification procedure used was essentially the same as that described by Gabriel et al., (1993), Arch. Microbi., 160:27-34 except the strains used were different. In addition, an extra ammonium sulfate fractionation step was included.
  • the strain used in this application was Phanerochaete chrysosporium from American Type Culture Collection (ATCC 32629) and (ATCC 24725), while the strain used by Gabriel et al (1993) was Phanerochaete chrysosporium k-3 obtained from a Czechish collection centre.
  • the cell-free extract of Phanerochaete crysosporium was brought up to 55% ammonium sulphate saturation. It was then blended gently for 2 hours and centrifuged for 20 minutes at 4°C at lOOOOxg. The precipitate that had the PD activity was dissolved in the same volume of extraction buffer, centrifuged again and the supernatant was then used for the purification of PD using the procedure described by Gabriel et al. (1993).
  • PDl is estimated to have a molecule mass 97 kDa it had a similar migration rate as the protein marker phosphorylase b (97.4 kDa).
  • the purified PD was used for amino acid sequencing.
  • Amino acid sequencing of PD was performed as described earlier [Yu. S.; Christensen TMIE, Kragh KM, Bojsen K, Marcussen J: Efficient purification, characterization and partial amino acid sequencing of two ⁇ -l,4-gl ⁇ can lyases from fungi. Biochim Biophys Acta 1339: 311-320 (1997)].
  • PD was first partially hydrolyzed with proteinases. The generated peptide fragments were separated on HPLC. Each individual polypeptide was collected, molecule-mass determined by mass spectrometer, and sequenced on an Applied Biosystems 476A sequencer using pulsed-liquid fast cycles.
  • PD was also further characterized for its pH and temperature optimum, ion requirements for activity, stability and other kinetic properties.
  • amino acid sequences are obtained either by trypsin or endoproteinase LysC digestion .
  • Peptide purification is achieved by reverse phase HPLC and molecular weight information is generated by MALDI-TOF mass specfrometry.
  • the sequences obtained are then compared to the DNA sequences found in the White Rot Genome (Phanerochaete chrysosporium) project undertaken by The University of California. Sequence similarity alignment is done using the BLAST algorithm.
  • This peptide is found from base pair 38620 -38742. There is a start codon at base pair 38599 and at 38317 indicating a possible signal peptide. Independent confirmation that this is the N terminal of the protein is achieved by sequencing protein PD2, an isozyme.
  • Pyranosone dehydratase isozyme I (PDl) was found to be as follows:
  • V(Q) means it could be either V or Q or both (due to heterogeneity).
  • This peptide is found from base pair 38788-38963.
  • the data base sequence is interrupted by an intron from base pair 38836-38889.
  • the sequence of residues 28-41 is confirmed by trypsin peptide 8.4
  • the three X's are PNG in the data base , this fits well with the MS data.
  • Peptide 31.4a NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK This peptide is found from base pair 41293-41469 and contains an intron from base pair

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Abstract

The present invention relates to a polynucleotide sequence encoding a pyranosone dehydratase. The present invention further relates to a method for producing transgenic plants which are resistant to pathogens, particularly fungal pathogens, comprising transforming the plants or part thereof with at least a polynucleotide sequence encoding a pyranosone dehydratase. Further aspects relate to transgenic plants comprising at least a heterologous polynucleotide sequence encoding pyranosone dehydratase, which plants are resistant to pathogens, particularly fungal pathogens. The present invention further relates to the in situ production of one or more antimicrobial compounds, such as microthecin, cortalcerone and/or ascopyrone P (APP) in a host organism, such as a plant.

Description

POLYNUCLEOTIDE ENCODING A PYRANOSONE DEHYDRATASE
FIELD OF THE INVENTION
The present invention relates to a novel polynucleotide encoding a pyranosone dehydratase.
The present invention further relates to the in situ production of a pyranosone dehydratase in plants or a part thereof, and processes for protecting plants and/or a plant part, such as a seed, from pathogens, such as pathogenic fungi, by in situ production of pyranosone dehydratase.
TECHNICAL BACKGROUND AND PRIOR ART
It is well documented in the literature that glucose can be oxidised by pyranose 2- oxidase (EC 1.1.3.10, P2O) to form glucosone (D-arabino-hexos-2-ulose), which in turn can be converted to cortalcerone by pyranosone dehydratase (PD) [Koths, K.; Halenbeck, R.; Moreland, M. (1992), Carbohydr Res. Vol. 232 No. 1, PP. 59-75; Gabriel, J.; Vole, J.; Sedmera, P.; Daniel, G.; Kubatova, E. (1993), Arch. Microbi., 160:27-34]. Both P2O and PD have been purified in fungi and P2O has been cloned. PD has been purified from Polyporus obtusus by Koths et al (1992), and from Phanerochaete chrysosporium by Gabriel et al (1993).
It has been established in the art that starch can be converted to 1,5-anhydro-D-fructose (AF) [S. Yu and J. Marcussen, Recent Advances in Carbohydrate Bioengineering; Gilbert, H. J.; Davies, G. J; Henrissat B.; Svensson, B., Eds.; Royal Society of Chemistry (RS.C) Press, 1999. 242-250]. An important enzyme in the conversion of starch to AF is glucan lyase. It has further been shown that several fungal and red algal extracts can then convert AF to microthecin possibly enzymatically [Baute, M-A.; Deffieux, G.; Baute, R. (1986), Phytochemistry (Oxf) vol. 25:1472-1473; Broberg, A., Kenne, L., and Pedersen, M. (1996), Phytochemistry (oxf).41 : 151-154]. It has also been documented that ascopyrone P (APP) can be produced from AF [Baute, M-A.; Deffieux, G.; Vercauteren, J.; Baute, R.; Badoc, A. (1993), Phytochemistry (oxf) vol. 33 no. 1, 41-45],
Plant disease is a major cause of crop loss. Various strategies have been developed to control disease, one of the most common of which is the use of chemicals. This approach is usually expensive, not always effective, and often harmful to non target organisms, including humans, and the environment. A preferred approach is to develop, through breeding and/or genetic modification, genotypes resistant or tolerant to diseases.
SUMMARY OF THE INVENTION
Aspects of the present invention are presented in the claims and in the following commentary.
As used with reference to the present invention, the terms "expression", "expresses", "expressed" and "expressable" are synonymous with the respective terms "transcription", "transcribes", "transcribed" and "transcribable".
Other aspects concerning the nucleotide sequence of the present invention include: a construct comprising the sequences of the present invention; a vector comprising the sequences of the present invention; a plasmid comprising the sequences of present invention; a transformed cell comprising the sequences of the present invention; a transformed tissue comprising the sequences of the present invention; a transformed organ comprising the sequences of the present invention; a transformed host comprising the sequences of the present invention; a transformed organism comprising the sequences of the present invention. The present invention also encompasses methods of expressing the nucleotide sequence using the same, such as expression in a host cell; including methods for transferring same.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
DETAILED DISCLOSURE OF INVENTION
In one aspect, the present invention provides a polynucleotide comprising a polynucleotide sequence encoding a pyranosone dehydratase.
Suitably, the polynucleotide according to the present inveniton may comprise a nucleotide sequence which encodes a pyranosone dehydratase comprising at least one amino acid sequence selected from the following: (i) KPHCEPEQPAALPLFQPQLVQGGRPDXY VEAFPFRSDSSK (SEQ ID
No. 2 or
KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No.4);
(iii) VSWLENPGELR (SEQ ID No. 5); (iv) DGVDCLWYDGAR (SEQ ID No. 6); (v) PAGSPTGIVRAEWTRHVLDVFGXLXXK (SEQ ID No. 7); (vi) HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK (SEQ ID No. 8);
(vii) TEMEFLDVAGK (SEQ ID No. 9); (viii) KLTLVVLPPFARLDVERNVSGVK (SEQ ID No. 10); (ix) SMDELVAHNLFPAYVPDSVR (SEQ ID No. 11);
(x) NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK (SEQ ID No. 12);
(xi) TGSLVCARWPPVK (SEQ ID No. 13); (xii) NQRVAGTHSPAAMGLTSRWAVTK (SEQ ID No. 14); (xiii) GQITFRLPEAPDHGPLFLS VS AIRHQ (SEQ ID No. 15); where X is an unknown amino acid residue; or a variant, homologue or derivative thereof. Suitably, the polynucleotide may encode for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos.2- 15, or a variant, homologue or derivative thereof.
Suitably, the polynucleotide acccording to the present invention may comprise a nucleotide sequence which encodes a pyranosone dehydratase comprising the following amino acid sequence or a variant, homologue or derivative thereof:
MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDS
SKCPNIIGYGLGTYDMKSDIQMFVNPYATTNNQSSSWTPVSLAKLDF
PVAMHYADITKNGFNDGRCIFFFFCYISCFANHRTVIITDQYGSSMD
DIWAYGGRVSWLENPGELRDNWTMRTIGHSPGMHRLKAGHFTRTD
RVQVVAVPIVVASSDLTTPADVIIFTAPDDPRSEQLWQRDVVGTRHL
VHEVAIVPAAETDGEMRFDQIILAGRDGVDCLWYDGARWQKHLVG
TGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGNTVSVYT
KPAGSPTGIVRAEWTRHVLDVFGPLNGKHTGSIHQVVCADIDGDGE
DEFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRI
ATANFHSQGSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEE
VMLRVVRAGSTRFKTEMEFLDVAGKKLTLVVLPPFARLDVERNVSG
VKVMAGTVCWADENGKHERVPATRPFGCESMIVSADYLESGEEGAI
LVLYKPSSTSGRPPFRSMDELVAHNLFPAYVPDSVRAMKFPWVRCA
DRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLAHVQLWTAGIGVSA
GFHNHVEASFCEIHACIA GTGRGGMRWATVPDA FNPDSPNLEDT
ELIVVPDMHEHGPLWRTRPDGHPLLRMNDTIDYPWHAWLAGAGNP
SPQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLAL
QKNDATDGTPVLALLDLDGGPSPQAGISLMFPARTCTRSRTPRRVRL
SVLVGRPLRISVSPARTLLLPWVLRHGGPSRRTPRGRLRSVSRRRPT
MARSSLAFPLYATNREQTRFPYVIDCYPCSSFVSRIYTLSSQVIVQGD
S I E L S A W S L. (SEQ ID No.26).
In another aspect, the present invention provides a polynucleotide selected from one or more of:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No.1 or
SEQ ID No.20 or the compliment thereof; (ii) a polynucleotide compirisng a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20, (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No.1 or SEQ ID No. 20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
In a further aspect, the present invention provides a polynucleotide selected from one or more of:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 1 or
SEQ ID No. 20; (ii) a polynucleotide which hybridises to the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 20 under medium stringency, preferably high stringency, conditions;
(iii) a polynucleotide which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% homologous with SEQ ID No. 1 or SEQ ID No. 20,
(iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ΪD No. 1 or SEQ ID No. 20; and
(v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (i v) .
Preferably, the polynucleotide sequence according to the present invention encodes a polypeptide which has pyranosone dehydratase activity.
Suitably, a pyranosone dehydratase according to the present invention may be defined as an enzyme that is capable of removing water from a five-carbon sugar, preferably anhydrofructose or a derivative thereof. Suitably, the pyranosone dehydratase according to the present invention may be classified in the enzyme classification E.C. 4.2.1.x (where x can be any number).
Suitably, the polynucleotide according to the present invention may be obtainable, or obtained, from one or more of Phanerochaete chrysosporium, Polyporus obtusus or Corticium caeruleum.
Suitably, the polynucleotide may be obtainable, or obtained, from an organism in the order of Pezizales, Auricular iales, Aphyllophorales, Agaricales or Gracilariales.
Suitably, the polynucleotide may be obtainable, or obtained, from one or more of Aleuria aurantia, Peziza badia, P. succosa, Sarcophaera eximia, Morchella conica, M. costata, M. elata, M. esculenta, M. esculenta var. rotunda, M. hortensis, Gyromitra infula, Auricularia mesenterica, Pulcherricium caeruleum, Peniophora quercina, Phanerochaete sordida, Vuilleminia comedens, Stereum gausapatum, S. sanguinolentum, Lopharia spadicea, Sparassis laminosa, Boletopsis subsquamosa, Bjerkandera adusta, Trichaptum biformis, Cerrena unicolor, Pycnoporus cinnabarinus, P. sanguineus, Junghunia nitida. Ramaria flava, Clavulinopsis helvola, C. helvola var. geoglossoides, V. pulchra, Clitocybe cyathiformis, C. dicolor, C. gibba, C. odora, Lepista caespitosa, L inversa, L. luscina, L. nebularis, Mycena seynii, Pleurocybella porrigens, Marasmius oreales, Inocybe pyriodor a, Gracilaria varrucosa, Gracilaria tenuistipitata, Gracilariopsis sp, Gracilariopsis lemaneiformis. Melanosopora spp., Melanospora ornata, Microthecium spp., Microthecium compressum, Microthecium zobelii.
A further aspect of the present invention provides a construct comprising a polynucleotide sequence encoding pyranosone dehydratase.
A further aspect of the invention provides a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a further polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranose dehydratase produces one or more of microthecin, cortalcerone or ascopyrone P (APP).
A further aspect of the invention provides a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding glucan lyase.
Another aspect provides a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding pyranose-2-oxidase. The polynucleotide sequence encoding pyranosone dehydratase may suitably be one or more of the polynucleotide sequences defined herein.
Suitably, the polynucleotide sequence according to the present invention in the construct may encode for a pyranosone dehydratase comprising at least one amino acid sequence selected from the following:
(i) KPHCEPEQPAALPLFQPQLVQGGRPDXY VEAFPFRSDSSK (SEQ ID No. 2 or
KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No. 4);
(iii) VSWLENPGELR (SEQ ID No. 5); (iv) DGVDCLWYDGAR (SEQ ID No. 6); (v) PAGSPTGΓVRAE TRHVLDVFGXLXXK (SEQ ID No. 7); (vi) HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK (SEQ ID No. 8);
(vii) TEMEFLDVAGK (SEQ ID No. 9); (viii) KLTLVVLPPFARLD VERNVSGVK (SEQ ID No. 10); (ix) SMDELVAHNLFPA YVPDS VR (SEQ ID No. 11 );
(x) NDATDGTPVLALLDLDGGPSPQA NISHVPPGTDMYEIAHAK (SEQ ID No. 12);
(xi) TGSLVCARWPPVK (SEQ ID No. 13); (xii) NQRVAGTHSPAAMGLTSRWAVTK (SEQ ID No. 14); (xiii) GQITFRLPEAPDHGPLFLSVSAIRHQ (SEQ ID No. 15); where X is an unknown amino acid residue; or a variant, homologue or derivative thereof.
Suitably, the polynucleotide sequence may encode for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos.2-15, or a variant, homologue or derivative thereof.
Suitably, the polynucleotide sequence according to the present invention in the construct may encode a pyranosone dehydratase comprising the following amino acid sequence or a variant, homologue or derivative thereof:
MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDS
SKCPNIIGYGLGTYDMKSDIQMFVNPYATTNNQSSSWTPVSLAKLDF
PVAMHYADITKNGFNDGRCIFFFFCYISCFANHRTVIITDQYGSSMD
DIWAYGGRVSWLENPGELRDN TMRTIGHSPGMHRLKAGHFTRTD
RVQVVAVPIVVASSDLTTPADVIIFTAPDDPRSEQLWQRDVVGTRHL
VHEVAIVPAAETDGEMRFDQIILAGRDGVDCLWYDGARWQKHLVG
TGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGNTVSVYT
KPAGSPTGIVRAE TRHVLDVFGPLNGKHTGSIHQVVCADIDGDGE
DEFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRI
ATANFHSQGSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEE
VMLRVVRAGSTRFKTEMEFLDVAGKKLTLVVLPPFARLDVERNVSG
VKVMAGTVCWADENGKHERVPATRPFGCESMIVSADYLESGEEGAI
LVLYKPSSTSGRPPFRSMDELVAHNLFPAYVPDSVRAMKFPWVRCA
DRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLAHVQLWTAGIGVSA
GFHNHVEASFCEIHACIANGTGRGGMRWATVPDANFNPDSPNLEDT
ELIVVPDMHEHGPLWRTRPDGHPLLRMNDTIDYPWHAWLAGAGNP
SPQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLAL
QKNDATDGTPVLALLDLDGGPSPQAGISLMFPARTCTRSRTPRRVRL
SVLVGRPLRISVSPARTLLLPWVLRHGGPSRRTPRGRLRSVSRRRPT
MARSSLAFPLYATNREQTRFPYVIDCYPCSSFVSRIYTLSSQVIVQGD
S I E L S A W S L. (SEQ ID No.26):
Another aspect provides a construct comprising a polynucleotide sequence encoding a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No.1 or SEQ
ID No.20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No.1 or SEQ ID No.20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No. 20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Another aspect provides a construct comprising a polynucleotide sequence encoding an enzyme and a polynucleotide encoding pyranosone dehydratase selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof;
(ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof;
(iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
(iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv), which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP.
Another aspect provides a construct comprising a polynucleotide sequence encoding glucan lyase and a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Another aspect provides a construct comprising a polynucleotide sequence encoding pyranose-2-oxidase and a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP, operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding glucan lyase operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding pyranose-2-oxidase operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Suitably, the polynucleotide sequence according to the present invention in the expression vector may encode a pyranosone dehydratase comprising at least one amino acid sequence selected from the following: (i) KPHCEPEQPAALPLFQPQLVQGGRPDXY NEAFPFRSDSSK (SEQ ID
No. 2 or
KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFV PYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No. 4);
(iii) VSWLENPGELR (SEQ ID No. 5); (iv) DGVDCL YDGAR (SEQ ID No. 6); (v) PAGSPTGΓVRAEWTRHVLDVFGXLXXK (SEQ ID No. 7); (vi) HTGSIHQWCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK (SEQ ID No. 8);
(vii) TEMEFLDVAGK (SEQ ID No. 9); (viii) KLTLVVLPPFARLDVERNVSGVK (SEQ ID No. 10); (ix) SMDELVAHNLFPAYVPDSVR (SEQ ID No. 11);
(x) NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK (SEQ ID No. 12);
(xi) TGSLVCARWPPVK (SEQ ID No. 13); (xii) NQRVAGTHSPAAMGLTSRWAVTK (SEQ ID No. 14); (xiii) GQITFRLPEAPDHGPLFLS VS AIRHQ (SEQ ID No. 15); where X is an unknown amino acid residue; or a variant, homologue or derivative thereof. Suitably, the polynucleotide sequence may encode for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos.2-15 or a variant, homologue or derivative thereof.
Suitably, the polynucleotide acccording to the present invention in the expression vector may encode a pyranosone dehydratase comprising the following amino acid sequence or a variant, homologue or derivative thereof:
MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDS
SKCPNIIGYGLGTYDMKSDIQMFVNPYATTNNQSSSWTPVSLAKLDF
PVAMHYADITKNGFNDGRCIFFFFCYISCFANHRTVIITDQYGSSMD
DIWAYGGRVSWLENPGELRDNWTMRTIGHSPGMHRLKAGHFTRTD
RVQVVAVPIVVASSDLTTPADVIIFTAPDDPRSEQLWQRDVVGTRHL
VHEVAIVPAAETDGEMRFDQIILAGRDGVDCLWYDGARWQKHLVG
TGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGNTVSVYT
KPAGSPTGIVRAEWTRHVLDVFGPLNGKHTGSIHQVVCADIDGDGE
DEFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRI
ATANFHSQGSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEE
VMLRVVRAGSTRFKTEMEFLDVAGKKLTLVVLPPFARLDVERNVSG
VKVMAGTVCWADENGKHERVPATRPFGCESMIVSADYLESGEEGAI
LVLYKPSSTSGRPPFRSMDELVAHNLFPAYVPDSVRAMKFPWVRCA
DRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLAHVQLWTAGIGVSA
GFHNHVEASFCEIHACIANGTGRGGMRWATVPDANFNPDSPNLEDT
ELIVVPDMHEHGPL RTRPDGHPLLRMNDTIDYPWHAWLAGAGNP
SPQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLAL
QKNDATDGTPVLALLDLDGGPSPQAGISLMFPARTCTRSRTPRRVRL
SVLVGRPLRISVSPARTLLLPWVLRHGGPSRRTPRGRLRSVSRRRPT
MARSSLAFPLYATNREQTREPYVIDCYPCSSFVSRIYTLSSQVIVQGD
S I E L S A W S L. (SEQ ID No.26).
Another aspect provides an expression vector comprising a polynucleotide sequence selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv) ; operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Another aspect provides an expression vector comprising a polynucleotide sequence encoding an enzyme and a polynucleotide encoding a pyranosone dehydratase selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof;(ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID
No. 20, or a fragment thereof;
(iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20;
(iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.20; and
(v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism; which said enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone, or APP
Another aspect provides an expression vector comprising a polynucleotide sequence encoding glucan lyase and a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof;
(i) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iii) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (iv) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or organism.
Another aspect provides an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase and a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); operably linked to one or more regulatory sequences capable of directing expression of said polynucleotides in a host cell or host organism.
In a preferred embodiment, the nucleotide sequence referred to in the present invention, in particular the polynucleotide encoding a pyranosone dehydratase, such as the polynucleotide sequence present in a construct or an expression vector in accordance with the present invention, is selected from:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 1 or
SEQ ID No. 20; (ii) a polynucleotide which hybridises to the nucleotide sequence shown in SEQ ID
No. 1 or SEQ ID No. 20 under medium stringency, preferably high stringency, conditions; (iii) a polynucleotide which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least
95% homologous with SEQ ID No. 1 or SEQ ID No. 20, (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a . result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
The invention is therefore intended to encompass constructs and/or expression vectors which comprise (a) a polynucleotide sequence encoding pyranosone dehydratase; (b) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP, (c) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding glucan lyase; or (d) both a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding pyranose-2-oxidase.
Preferably, the polynucleotide encoding the pyranosone dehydratase and/or the polynucleotide sequence encoding the glucan lyase and/or the polynucleotide encoding the further enzyme and/or the polynucleotide encoding pyranose-2-oxidase, is fused with a polynucleotide sequence which encodes for an endogenous or heterologous targeting signal sequence for extracellular or intracellular targeting of the gene products. A further aspect relates to a host cell or host organism into which has been incorporated a polynucleotide according to the present invention, a construct according to the present invention or an expression vector according to the invention.
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence according to the present invention encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more antimicrobial compounds.
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence according to the present invention encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces one or more of microthecin, cortalcerone or APP.
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding glucan lyase.
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase and an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase.
Another aspect of the invention relates to a host cell of host organism into which has been incorporated an expression vector comprising a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding glucan lyase and an expression vector comprising a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No.
20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID
No. 20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Another aspect of the invention relates to a host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase and an expression vector comprising a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
When it is the case that more than one heterologous polynucleotide sequence is present in a host cell or host organism, the skilled person would be a readily aware of various methods of providing two or more polynucleotide sequences to a host cell or host organism. For instance, the host cell or host organism may be transformed with a single construct or a single expression vector comprising more than one heterologous polynucleotide sequences. Alternatively, each polynucleotide sequence may be present in a separate construct or expression vector, for example, host cells or host organisms may be transformed with (a) a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a separate construct comprising the polynucleotide sequence encoding glucan lyase, for example; or (b) a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a separate construct comprising a polynucleotide sequence encoding pyranose-2-oxidase, for example.
The present invention further encompasses a method whereby a first heterologous polynucleotide sequence is transformed into a first host cell or host organism, i.e. by transformation with a first construct or expression vector; and one or more further heterologous polynucleotide sequences are transformed into one or more further host cells or host organisms, i.e. by transformation with one or more further constructs or expression vectors. Following which, the first host cell or host organism is crossed with the one or more further host cells or host organisms in order to provide a host cell or host organism comprising both the first heterologous polynucleotide sequence and the one or more further heterologous polynucleotide sequences.
Thus in one embodiment, a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a construct comprising the polynucleotide sequence encoding a norther enzyme, such as glucan lyase for example, can be introduced into a host cell or host organism by separate transformation events, for example, by recombination or meiosis. Similarly, a construct comprising a polynucleotide sequence encoding pyranosone dehydratase and a construct comprising the polynucleotide sequence encoding pyranose-2-oxidase for example can be introduced into a host cell or host organism by separate transformation events.
Suitably, the host organism may be a plant or a microorganism, such as a fungus, preferably a filamentous fungus, a yeast or a bacterium.
In one preferred embodiment, the host organism is a yeast.
In another preferred embodiment, the host organism is a transgenic plant.
A method of preparing pyranosone dehydratase in situ in an organism, comprising culturing a host cell or host organism according to the present invention under conditions to provide for expression of pyranosone dehydratase.
A method of preparing one or more one or more antimicrobial compounds in situ in an organism, comprising culturing a host cell or host organism according to the present invention under conditions to provide for expression of pyranosone dehydratase, whereby said pyranosone dehydratase converts constituents present in the host cell or host organism, such as 1,5-anhydrofructose, starch dextrins, glucose or glucosone for example, into one or more antimicrobial compounds.
Suitably, the antimicrobial compound may be microthecin or a derivative thereof. A method of preparing one or more of microthecin or ascopyrone P (APP) or cortalcerone in situ in an organism, comprising culturing a host cell or host organism according to the present invention under conditions to provide for expression of pyranosone dehydratase, whereby said pyranosone dehydratase converts constituents present in the host cell or host organism, such as 1,5-anhydrofructose, starch dextrins, glucose or glucosone for example, into microthecin and/or APP and/or cortalcerone.
A further aspect relates to a method of preparing one or more of microthecin or ascopyrone P (APP) in situ which comprises cultivating a host cell or host organism according to the present invention under conditions to provide for expression of the coding sequence encoding pyranosone dehydratase, whereby said pyranosone dehydratase converts constituents naturally present in the host cell or host organism, such as 1,5-D-anhydrofructose or starch dextrins for example, into microthecin and/or APP.
A further aspect relates to a method of preparing one or more of microthecin or ascopyrone P (APP) in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide encoding a further enzyme, under conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces an antimicrobial compound, such as microthecin and/or APP, whereby said pyranosone dehydratase together with said further enzyme converts constituents naturally present in the host cell or host organism, such as 1,5-D-anhydrofructose or starch dextrins for example, into one or more antimicrobial compounds, such as microthecin and/or APP.
A further aspect relates to a method of preparing one or more of microthecin and/or APP in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding glucan lyase, under conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and glucan lyase, whereby said pyranosone dehydratase and glucan lyase converting starch dextrins present in the host cell or organism into one or more antimicrobial compounds, such as microthecin and or APP.
A further aspect relates to a method of preparing cortalcerone in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide encoding a further enzyme, under conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and a further enzyme, which enzyme when expressed in a host in combination with said pyranosone dehydratase produces an antimicrobial compounds, such as cortalcerone, whereby said pyranosone dehydratase together with said further enzyme converts constituents naturally present in the host cell or host organism, such as glucose and/or glucosone for example, into one or more antimicrobial compounds, such as cortalcerone.
A further aspect relates to a method of preparing cortalcerone in situ which comprises cultivating a host cell or host organism according to the invention, into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase and a polynucleotide sequence encoding pyranose-2-oxidase, imder conditions to provide for expression of the coding sequences encoding pyranosone dehydratase and pyranose-2- oxidase, said pyranosone dehydratase and pyranoser2-oxidase converting glucose present in the host cell or organism into a antimicrobial compound, such as cortalcerone.
A further aspect relates to a method of preparing one or more antimicrobial compounds, such as microthecin or a derivative thereof, in situ which method comprises:
(i) cultivating a host cell or host organism into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase according to the present invention, under conditions to provide for expression of pyranosone dehydratase; and (ii) exposing said host cell or host organism to 1,5-D-anhydrofructose and/or glucosone. A further aspect relates to a method of preparing one or more of microthecin or APP in situ which comprises
(i) cultivating a host cell or host organism into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase according to the present invention, under conditions to provide for expression of pyranosone dehydratase; and
(ii) exposing said host cell or host organism to 1,5-D-anhydrofructose.
A further aspect relates to a method of cortalcerone in situ which comprises (i) cultivating a host cell or host organism into which has been incorporated a polynucleotide sequence encoding pyranosone dehydratase according to the present invention, under conditions to provide for expression of pyranosone dehydratase; and (ii) exposing said host cell or host organism to glucosone.
The present invention yet further provides a method of preparing one or more antimicrobial compounds, such as microthecin or a derivative thereof, in situ in a host cell or in a host organism, comprising tiansforming a host cell or a host organism with a polynucleotide encoding pyranosone dehydratase according to the present invention, and culturing the transformed host cell or host organism under conditions to provide for expression of the polynucleotide such that the pyranosone dehydratase so produced converts constituents in and/or around the host cell or host organism to one or more antimicrobial compounds, such as one or more of microthecin, cortalcerone or APP.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
Suitably, the method may further comprise tiansforming the host cell or host organism with a further polynucleotide sequence encoding an enzyme, preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
Suitably, the host cell or host organism may be cultured in a medium comprising a constituent which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase.
Suitably, at least one of the constituents which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase may be present naturally within the host cell or host organism.
Preferably, the host cell or host organism is a plant or a microorganism, such as a fungus, preferably a filamentous fungus, a yeast or a bacterium.
A further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing a microorganism in or on a transgenic organism, in particular a transgenic plant, comprising tiansforming said plant with a polynucleotide encoding pyranosone dehydratase according to the present invention; and growing said transformed plant.
Suitably, the method may further comprise transforming the transgenic organism with a further polynucleotide sequence encoding an enzyme, preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
Suitably, the method may further comprise contacting said transgenic organism with a constituent which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase.
Suitably, at least one of the constituents which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase may be present naturally within the host cell or host organism.
Suitably, the growth of the microorganism may be prevented and/or inhibited and/or the microorganism maybe killed due to the production of one or more antimicrobial compounds, such as microthecin or a derivative thereof, in said transgenic organism.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
As the skilled person will readily be aware, the antimicrobial compounds produced in situ in a host organism may be further modified by endogenous enzymes or heterologously introduced enzymes. The present invention, therefore further relates to the production of derivatives of the antimicrobial compounds according to the present invention from said antimicrobial compounds in situ in the host organism, wherein the derivatives of the microbial compounds also exhibit anti-microbial activity.
A further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing a microorganism in and/or on a transgenic organism, in particular a transgenic plant, comprising tiansforming said organism with a polynucleotide encoding pyranosone dehydratase according to the present invention, said method comprising contacting said organism with 1,5-D-anhydrofructose and/or glucosone.
A further aspect relates to a method of preventing and/or inhibiting the growth of, and/or killing microorganisms in a transgenic plant into which has been incorporated a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or SEQ
ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv) said method comprising contacting said transgenic plant with 1,5-D-anhydrofructose and/or glucosone. The present invention yet further provides a method of preparing a transgenic organism, preferably a plant or part thereof, which organism or part thereof is resistant to one or more pathogens (particularly one or more fungal pathogens), comprising transforming a cell of the organism, preferably a plant cell, with a polynucleotide sequence encoding pyranosone dehydratase according to the present invention, whereby the transformed organism produces one or more antimicrobial compounds, such as microthecin or a derivative thereof, from constituents present in the cell of the organism, preferably the plant cell and/or around the cell of the organism, preferably the plant cell.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or ascopyrone P (APP).
Suitably, the method may further comprise transforming the transgenic organism or part thereof with a further polynucleotide sequence encoding an enzyme, preferably a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
Suitably, the transgenic organism or part thereof may be cultured in a medium comprising a constituent which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase.
Suitably, at least one of the constituents which is a substrate for the pyranosone dehydratase and/or the glucan lyase and/or the pyranose-2-oxidase may be present naturally within the transgenic organism or part thereof.
Preferably, the transgenic organism is a plant or a microorganism, such as a fungus (preferably a filamentous fungus), a yeast or a bacterium.
Suitably the method may further comprise the step of regenerating a the transgenic organism, preferably a transgenic plant, from the cell of the organism, preferably a plant cell, which transgenic organism, preferably transgenic plant, produces one or more antimicrobial compounds, such as microthecin or a derivative thereof in situ. Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or APP.
Suitably, the cell of the organism, preferably the plant cell, which is transformed is a regeneratable cell.
In another aspect the present invention provides a transgenic organism or part thereof, preferably a transgenic plant or part thereof (such as a plant cell or a seed for example), which is resistant to one or more pathogens (particularly one or more fungal pathogens), which organism or part thereof comprises a heterologous polynucleotide sequence encoding pyranosone dehydratase according to the present invention.
Suitably, the transgenic organism may further comprise one or more further polynucleotide sequences encoding one or more further enzymes, which when expressed in the organism in combination with the pyranosone dehydratase produces one or more antimicrobial compounds, such microthecin or a derivative thereof. Preferably at least one of the one or more further enzymes is a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or APP.
Preferably, the transgenic organism is a plant or a microorganism, such as a fungus, preferably a filamentous fungus, a yeast or a bacterium.
In another aspect the present invention provides the use of a polynucleotide sequence encoding pyranosone dehydratase according to the present invention to produce an organism, preferably a plant, or part thereof which is resistant to one or more pathogens, particularly one or more fungal pathogens. Suitably, the polynucleotide sequence encoding pyranosone dehydratase according to the present invention may be used in combination with one or more further enzymes, which further enzyme(s) when expressed in the organism in combination with the pyranosone dehydratase produces one or more antimicrobial compounds, such as microthecin or a derivative thereof. Preferably at least one of the one or more further enzymes is a glucan lyase enzyme or a pyranose-2-oxidase enzyme.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone or APP.
The term "rø situ" as used herein means that one or more antimicrobial compounds, such as microthecin or a derivative thereof, is/are produced within the host cell or host organism, in particular within the plant cell or plant. This contrasts the situation where one or more antimicrobial compounds, such as microthecin or a derivative thereof, is/are added as a formed product to the plant (or part thereof, such as seed) or plant cell. In other words, the term "in situ" as used herein means that by the transformation of the host cell or host organism, for example the plant cell or plant, with at least a polynucleotide encoding a pyranosone dehydratase enzyme according to the present invention, one or more antimicrobial compounds, such as microthecin or a derivative thereof, may be produced from components of the host cell or host organism, particularly plant cell or plant. Suitably, the components of the host cell or host organism may be the substrate(s) for the enzyme. If necessary, the components of host cell or host organism may be supplemented by addition of one or more further components which further components may be the same as those present in the host cell or host organism or may be additional to those components already present in the host cell or host organism.
Preferably, the host cell or organism, such as plant cell or plant, may be further transformed with, or comprises, one or more further polynucleotide sequences encoding one or more further enzymes, which enzyme(s) when expressed in the organism, such as the plant) in combination with the pyranosone dehydratase produces an antimicrobial compound, such as microthecin or a derivative thereof, from constituents present in the host cell.
Suitably, the antimicrobial compound may be one or more of microthecin, cortalcerone, or APP.
In one preferred aspect, the host cell or organism is transformed with, or comprises, a polynucleotide sequence encoding pyranosone dehydratase according to the present invention and a polynucleotide sequence encoding glucan lyase.
Suitably, the constituents or components present in the host cell or host organism from which the antimicrobial compound, such as microthecin or a derivative thereof may be produced, may be one or more of the following constituents: α-l,5-anhydrofructose, starch, starch dextrins.
As will be appreciated by persons skilled in the art, the level of resistance need not be absolute. Thus, the term "resistance" as used herein means that the host cell or organism (for example plant cell or plant) is capable of inhibiting or preventing the growth of one or more microbial pathogens (such as fungal pathogens) and/or of killing one or more microbial pathogens (such as fungal pathogens). The term "resistant" should be defined accordingly. For the avoidance of doubt, the term "resistance" as used herein may encompass the term "tolerance" and these terms may be used interchangeably herein.
Suitably, the methods according to the present invention may be used to prepare a transgenic organism.
Suitably, the transgenic organism according to the present invention or prepared using a method according to the present invention may produce a product (such as one or more compounds). Suitably, the trangenic organism according to the present invention and/or a product derivable (preferably derived) therefrom may be used as an antimicrobial agent. Suitably, the agent may be used to prevent and/or inhibit microbial contamination of a second organism or product derived therefrom, an inanimate object, a composition or a substance.
As will be readily appreciated by those skilled in the art, in order to have an antimicrobial effect a compound may (in addition to possible other benefits) effect one or more of the following: kill the microorganism(s), prevent and/or inhibit the spread of the microorganism(s), prevent and/or inhibit the growth of the microorganim(s), prevent and/or reduce the the detectable presence of the microorganism(s) by a human, prevent and or reduce the damage caused by the microorganism to living and nonliving materials.
Suitably, the polynucleotide sequence may encode for a fungal pyranosone dehydratase enzyme, preferably a pyranosone dehydratase enzyme from Phanerochaete chrysosporium.
Suitably, the polynucleotide sequence according to the present invention may encode for a pyranosone dehydratase comprising at least one amino acid sequence selected from the following:
(i) KPHCEPEQPAALPLFQPQLVQGGPPDXYWVEAFPFRSDSSK (SEQ ID
No. 2 or
KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No. 4);
(iii) VSWLENPGELR (SEQ ID No. 5); (iv) DGVDCLWYDGAR (SEQ ID No. 6); (v) PAGSPTGIVRAEWTRHVLDVFGXLXXK (SEQ ID No. 7); (vi) HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK (SEQ ID No. 8);
(vii) TEMEFLDVAGK (SEQ ID No. 9); (viii) KLTLVVLPPFARLDVERNVSGVK (SEQ ID No. 10); (ix) SMDELVAHNLFP AYVPDS VR (SEQ ID No. 11 ); (x) NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK (SEQ ID No.12);
(xi) TGSLVCARWPPVK(SEQIDNo.13);
(xii) NQRVAGTHSPAAMGLTSRWAVTK(SEQIDNo.14);
(xiii) GQITFRLPEAPDHGPLFLSVSAIRHQ(SEQIDNo.15); where X is an unknown amino acid residue; or a variant, homologue or derivative thereof.
Suitably, the polynucleotide sequence may encode for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos.2-15 or a variant, homologue or derivative thereof.
Suitably, the polynucleotide according to the present invention may encode for a pyranosone dehydratase comprising the following amino acid sequence or a variant, homologue or derivative thereof:
MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDS
SKCPNIIGYGLGTYDMKSDIQMFVNPYATTNNQSSSWTPVSLAKLDF
PVAMHYADITKNGFNDGRCIFFFFCYISCFANHRTVIITDQYGSSMD
DIWAYGGRVSWLENPGELRDNWTMRTIGHSPGMHRLKAGHFTRTD
RVQVVAVPIVVASSDLTTPADVIIFTAPDDPRSEQLWQRDVVGTRHL
VHEVAIVPAAETDGEMRFDQIILAGRDGVDCLWYDGARWQKHLVG
TGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGNTVSVYT
KPAGSPTGIVRAEWTRHVLDVFGPLNGKHTGSIHQVVCADIDGDGE
DEFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRI
ATANFHSQGSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEE
VMLRVVRAGSTRFKTEMEFLDVAGKKLTLVVLPPFARLDVERNVSG
VKVMAGTVCWADENGKHERVPATRPFGCESMIVSADYLESGEEGAI
LVLYKPSSTSGRPPFRSMDELVAHNLFPAYVPDSVRAMKFPWVRCA
DRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLAHVQLWTAGIGVSA
GFHNHVEASFCEIHACIANGTGRGGMRWATVPDANFNPDSPNLEDT
ELIVVPDMHEHGPLWRTRPDGHPLLRMNDTIDYPWHAWLAGAGNP
SPQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLAL
QKNDATDGTPVLALLDLDGGPSPQAGISLMFPARTCTRSRTPRRVRL
SVLVGRPLRISVSPARTLLLPWVLRHGGPSRRTPRGRLRSVSRRRP MARSSLAFPLYATNREQTRFPYVIDCYPCSSFVSRIYTLSSQVIVQGD SIELSAWSL.(SEQIDNo.26).
Suitably, the polynucleotide sequence may be selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No.1 or SEQ
ID No.20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No.1 or SEQ ID No.20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No.1 or SEQ ID No.20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No.1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
In a preferred embodiment, the nucleotide sequence referred to in the present invention, such as the polynucleotide sequence used in the transformation of a host cell or organism
(for example a plant cell or plant) in accordance with the present invention, is selected from:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No.1 or
SEQ ID No.20; (ii) a polynucleotide which hybridises to the nucleotide sequence shown in SEQ ID
No.1 or SEQ ID No.20 under medium stringency, preferably high stringency, conditions; (iii) a polynucleotide which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least
95% homologous with SEQ ID No.1 or SEQ ID No.20, (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No.1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
Suitably, the transgenic organism (for example the transgenic plant) or part thereof may comprise one or more constructs and/or expression vectors as defined by the present invention.
In one aspect, the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the expression of a gene encoding a pyranosone dehydratase.
In a further aspect, the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the co-expression of genes encoding (i) a pyranosone dehydratase and (ii) a glucan lyase.
In a further aspect, the present invention may provide a transgenic organism (such as a transgenic plant) with effective protection against fungal pathogen attack, which protection is afforded by the co-expression of genes encoding (i) a pyranosone dehydratase and (ii) a pyranose-2-oxidase.
Suitably, the polynucleotide sequence(s) according to the present invention may be placed under the control of promoters and/or other regulatory sequences which allows suitably strong expression in one or more of the organisms tissues, for example in one or more of the plant tissues.
Suitably, the polynucleotide sequence(s) of the present invention may be expressed constitutively (i.e. at all times) within the organism (for example plant). Alternatively, the polynucleotide sequence(s) of the present invention may be expressed non- constitutively (i.e. only at specific times during the life cycle of the organism (such as plant) and/or following a specific event, such as pathogen invasion, for example). Preferably, the gene product(s) may be produced in all or substantially all parts of the plant. Alternatively, the gene product(s) may be produced only in specific parts, i.e. specific cells and/or tissues, of the organism (for example the plant).
Preferably, the gene product(s) may be targeted to a specific subcellular or extracellular compartment of the organism or host cell (for example of the plant or plant cell). For example, the gene product(s) may be targeted to one or more of the following parts of the host or host cell (in particular the plant or plant cell): extracellular spaces (i.e. apoplastic space and/or cell wall); intracellular spaces; cytosol; plastid (including chloroplast or amyloplast); vacuoles (starch granules); endoplasmic reticulum (ER).
The term "fungus"as used herein includes any taxonomic group that is classified within one or more of the following taxons: Oomycetes, Ascomycetes, Fungi imperfecta, Deutormycetes, Basidiomycetes, Zygomycetes, or Mastigomycetes. The terms "antifungal" and "fungal" should be construed accordingly.
The present invention and particularly the organisms (such as the plants) which are resistant to fungal pathogens, may be used in combination with conventional anti-fungal strategies, such as the provision of fungicides and/or in combination with other antifungal gene traits.
Preferably, the transgenic organism or part thereof (in particular where the transgenic organism is a transgenic plant) according to the present invention is resistant to one or more of the following diseases: Bacterial leaf blight, Bacterial mosaic, Bacterial sheath rot, Basal glume rot, Black chaff (bacterial streak), Spike blight (gummosis), leaf spot, blights, cankers, galls.
Suitably, these diseases may be caused by one or more of the following pathogens: Pseudomonas spp., Clavibacter spp., Xanthomonas spp., Rathayibacter spp.,. Corynebacterium spp., Erwinia spp. Agrobacterim spp., Xylella spp. Preferably, the transgentic organism or part therof (in particular where the transgenic organism is a transgenic plant) according to the present invention is resistant to one or more of the following diseases: Downey mildew, powdery mildew, grey mould, canker, black scurf, rots, soft rot, fruit rot, basal rot, crown rot, root rot, rusts, stem rusts, stripe rusts, blight, early blight, late blight, pithium blight, leaf spot, wilt, leaf blotch, glume blotch, black leg, and Black Sigatoka.
Black Sigatoka often affects plantain and banana, and is caused by the ascomycete Mycosphaerelϊa fijiensis (Anamorph: Paracercospora fijiensis). Thus, the transgenic plant or part thereof according to the present invention may be a plantain or banana plant or part thereof and may be resistant to Mycosphaerella fijiensis (Anamorph: Paracercospora fijiensis).
Preferably, the transgenic organism (such as the trangenic plant) or part thereof according to the present invention is resistant to one or more pathogens selected from the following genera: Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Phythium, Phytophthora, Thielaviopsis, Aspergillus, Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Eutypa, Gaeumanomyces, Helminthosporiwn, Macrophomina, Nectria,Peronospora, Phoma, Phymatotrichum, Plasmopara, Podosphaera, Puccinia, Puthium, Pyrenophora, Pyricularia, Scerotium, Sckerotinia, Septoria, Uncinula, Venturia, Verticillium or Penicillium, including both the anamorph and/or teleomorph stage of any one thereof.
Even more preferably, the transgenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one or more pathogens selected from the following: genera: Alternaria spp., Albugo spp., Aphanomyces spp., Amyloporia spp., Ascochyta, Aspergillus, Basidiophora, Bipolaris, Botrytis, Bremia, Cercospora, Cladosporium, Claviceps, Coniophora spp., Colletotrichum, Diplodia, Diplocarpon spp., DonUoporia spp., Drechslera, Erysiphe, Eutypa, Fibroporia spp., Fusarium, Gaeumanomyces, Geotrichum, Guignardia, Gymnosporangium, Helmintosporium, Hemileia, Kabatiella, Leptosphaeria, Macrophomina, Marssonina spp., Monilinea, Merulus, Mycosphaerella, Nectria, Paracercospora, Penicittium, Peronophythora, Peronospora, Phelleήas spp., Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmophora, Podosphaera, Porai spp., Pseudocercosporella, Pseudoperonospora, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Rhizopus, Sclerophthora, Sclerotinia, Sclerotium, Septoria, Serpula spp., Sphaerotheca, Stagonospora, Taphrina, Thielaviopsis, Tϊlletia, Trichoderma, Uncinula, Ustilago, Venturia, Verticϊllium, including both the anamorph and/or teleomorph stage of any one thereof.
Preferably, the transgenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one or more of the following pathogens: Alternaria brassicol; Alternaria solani, Ascochyta pisi, Botrytis cinerea, Cercospora kikuchii, Cercospora zaea-maydis; Colletotrichum lindemuthianum; Diplodia maydis; Erysiphe graminis fsp. Graminis; Erysiphe graminis f.sp. hordei; Eutypa lata; Fusarium nivale; Fusarium oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusarium moniliforme; Fusarium roseum; Gaeumanomyces graminis f.sp. tritici;Helminthosporium turcicum; Helminthosporium carbonum; Helminthosporium maydis; Macrophomina phaseolina; Maganaporthe grisea; Nectria heamatococca; Peronospora manshurica; Peronospora tabacina; Phoma betae; Phymatotrichum omnivorum; Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthόra parasitica; Phytophthora citrophthora; Phytophthora megasperma fsp. sojae; Phytophthora infestans; Plasmopara viticola; Podosphaera leucotricha; Puccinia sorghi; Puccinia striiformis; Puccinia graminis fsp. tritici; Puccinia asparagi; Puccinia recondita; Puccinia arachidis; Puthium aphanidenmatum; Pyrenophora tritici-repentens; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Rhizoctonia cerealis; Scerotium rolfsii; Sclerotinia sclerotiorum; Septoria Iycopersici; Septoria glycines; Septoria nodorum; Septoria tritici; Thielaviopsis basicola; Uncinula necator; Venturia inaequalis; Verticillium dahliae; Verticillium albo-atrum.
Preferably, the transgenic organism (for example the transgenic plant) or part thereof according to the present invention is resistant to one or more of the following pathogens: downey mildew or powdery mildew, such as one or more of the following: Erysiphe spp.; Erysiphe cichoracearum (e.g cucumbers, endive, lettuce, melons, potato, pumpkin, squash); Erysiphe cruciferarum (e.g. broccoli, Brussels sprouts, cauliflower, and other cole crops; radicchio, radishes, turnips); Erysiphe lycopersici & Oidium lycopersicum (e.g. tomatoes); Erysiphe pisi (e.g. peas); Erysiphe heraclei (e.g. carrots, parsley, parsnips); Erysiphe polygoni (e.g. beets); Leveillula taurica (e.g. artichoke, eggplant, peppers, tomatillo, tomatoes); Sphaerotheca spp.; Sphaerothecafuliginea (e.g. beans, black-eyed peas, cucurbits, okra).
More preferably still, the transgenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one or more plant pathogens selected from the following: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Rhizoctonia, Pythium, Aphanomyces, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), Mycosphaerella fijiensis, Paracercospora fijiensis, Ascomycetes spp, Leptosphaeria maculans and Eutypa lota.
In a preferred aspect, the trangenic organism (such as the transgenic plant) or part thereof according to the present invention is resistant to one ore more of the oomycete fungi selected from Pythium, Aphanomyces spp. Peronospora spp., Phytophthora spp., Albugo spp., Basidiophora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp., Peronophythora spp.
Preferably, the transgenic plant according to the present invention is one or more of the following: a crop plant, a monocotyledonous crop plant, a dicotyledonous crop plant, a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, fruit crops, vegetables, apple, pear, strawberry, pea, tomato, grape (vine), Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, agricultural crops, livestock nutritional plants, oilseed rape, sunflowers, soybean, peanuts, broccoli, cabbage, carrot, citrus, garlic, onion, pepper (Capsicum), potato, and strawberry. Suitably, the present invention may encompass a potato seed piece produced by potato plants.
Suitably, the plant or part thereof may be a foodstuff.
ADVANTAGES
The present invention teaches the in situ production of an antimicrobial compound, such as microthecin (or a derivative thereof) and/or cortalcerone and/or ascopyrone P (APP).
The in situ production of microthecin or a derivative thereof according to the present invention surprisingly overcomes any instability of the microthecin or the derivative.
The in situ production of microthecin or a derivative thereof, particularly in plants or parts thereof, provides effective pathogen , particularly fungal pathogen, control. The control afforded by the in situ production of microthecin is advantageously broad spectrum, i.e. targets more than one pathogen.
The in situ production of cortalcerone and or ascopyrone P (APP), particularly in plants or parts thereof, provides effective pathogen, particularly fungal pathogen, control.
The control afforded by the in situ production of cortalcerone is advantageously broad spectrum, i.e. target more than one pathogen.
The present invention may be used to advantageously control fungal pathogens on the transgenic plant during growth (cultivation) thereof. Suitably, however, the present invention may be used to control fungal pathogens on the transgenic plant or part thereof after harvest, for example during storage. Advantageously, the transgenic plant or part thereof according to the present invention will be protected from attack by fungal pathogens both during cultivation and post-harvest, such as during storage. As will be readily appreciated by the skilled person, the present invention may, therefore, be applicable to preserve stored seeds and/or other plant parts, as well as stored silage for example, and may be used to preserve timber, cotton, flax and other plant derived non-edible products.
The present invention may be used to control or prevent fungal infection of plant seeds after sowing, particularly prior to growth, and or during germination and initial seedling growth.
ANTIMICROBIAL COMPOUND
In one aspect of the present invention the transformation of a host organism with at least a polynucleotide according to the present invention may result in the production of an antimicrobial compound.
In one aspect, the antimicrobial compound is selected from microthecin or a derivative thereof.
In one embodiment, the derivative of microthecin may be 2-furyl-hydroxymethyl-ketone or 4-deoxy-g/ycero-hexo-2,3-diluose.
In another embodiment, the derivative of microthecin may be 2-furylglyoxal.
In one aspect the antimicrobial compound is a compound having Formula I, Formula I
Figure imgf000039_0001
wherein R* and R2 are independently selected from H and C(=O)R3 wherein R3 is a hydrocarbyl group, and R is from H and OH . The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.
A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
In some aspects of the present invention, the hydrocarbyl group is selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.
In some aspects of the present invention, the hydrocarbyl group is an optionally substituted alkyl group.
In some aspects of the present invention, the hydrocarbyl group is selected from C--C*o alkyl group, such as C Cβ alkyl group, and C--C3 alkyl group. Typical alkyl groups include Cj alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C7 alkyl, and C8 alkyl.
In some aspects of the present invention, the hydrocarbyl group is selected from C--C10 haloalkyl group, Cj--C6 haloalkyl group, C--C3 haloalkyl group, C--C10 bromoalkyl group, C Cβ bromoalkyl group, and C--C3 bromoalkyl group. Typical haloalkyl groups include C\ haloalkyl, C2 haloalkyl, C3 haloalkyl, C haloalkyl, C5 haloalkyl, C7 haloalkyl, C8 haloalkyl, C- bromoalkyl, C2 bromoalkyl, C3 bromoalkyl, C4 bromoalkyl, C5 bromoalkyl, C7 bromoalkyl, and Cs bromoalkyl.
In some aspects of the present invention, the hydrocarbyl group is selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH^-.-o-aryl, -(CH2)1-10-Ph, (CH2)ι-ιo- Ph-C-.10 alkyl, -(CH2)1-5-Ph, (CH^-.s-Ph- .s alkyl, -(CH2)1-3-Ph, (CH2)1-3-Ph-C-.3 alkyl, -CH2-Ph, and -CH2-Ph-C(CH3)3.
When the hydrocarbyl group is or contains an aryl group, the aryl group or one or more of the aryl groups may contain a hetero atom. Thus the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N.
In some aspects of the present invention, the hydrocarbyl group is selected from - (CH2)1-10-cycloalkyl, -(CH2)1-1o-C3.10cycloalkyl, -(CH2)1-7-C3.7cycloalkyl, -(CH^.s-C;-. 5cycloalkyl, -(CH2)ι.3-C3-5cycloalkyl, and -CH2- C3cycloalkyl.
In some aspects of the present invention, the hydrocarbyl group is an alkene group. Typical alkene groups include C-- 0 alkene group, C!-C6 alkene group, Cι-C3 alkene group, such as Cls C2, C3, C , C5, C6, or C7 alkene group. In a preferred aspect the alkene group contains 1, 2 or 3 C=C bonds. In a prefened aspect the alkene group contains 1 C=C bond. In some preferred aspect at least one C=C bond or the only C=-C bond is to the terminal C of the alkene chain, that is the bond is at the distal end of the chain to the ring system.
In one aspect R3 contains from 1 to 10 carbon atoms.
In one aspect R3 contains from 1 to 5 carbon atoms.
In one aspect R3 is a -s alkyl group. In one aspect R4 is H. Thus, in this aspect the antimicrobial compound is a compound having Formula II
Formula II
Figure imgf000042_0001
wherein Rι and R2 are independently selected from H and C(=O)R3 wherein R3 is a hydrocarbyl group.
In one aspect of the present invention when R_t is OH, R1 is H and R2 is H.
In one aspect preferably at least one of R and R is H.
Preferably R1 is H.
Preferably R2 is H.
In one aspect preferably R1 is H and R2 is H.
In one aspect R1 may be H, R2 may be H and R4 may be H. Thus in this aspect the antimicrobial compound has the Formula
Figure imgf000042_0002
This compound is commonly referred to as microthecin
In one highly preferred aspect R1 is H, R2 is H and R4 is OH. Thus in this aspect the antimicrobial compound has the Formula
Figure imgf000043_0001
This compound is commonly referred to as cortalcerone.
ISOLATED
In one aspect, preferably the sequence is in an isolated form. The term "isolated" means that the sequence is not in its natural environment (i.e. as found in nature). Typically the term "isolated" means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature. Here, the sequence may be separated from at least one other component with which it is naturally associated.
PURIFIED
In one aspect, preferably the sequence is in a purified form. The term "purified" also means that the sequence is not in its natural environment (i.e. as found in nature). Typically the term "purified" means that the sequence is at least substantially separated from at least one other component with which the sequence is naturally associated in nature and as found in nature.
NUCLEOTIDE SEQUENCE
The present invention encompasses nucleotide sequences encoding enzymes having the specific properties as defined herein. The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double- stranded or single-stranded whether representing the sense or antisense strand. The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
In a preferred embodiment, the nucleotide sequence per se of the present invention does not cover the native nucleotide sequence according to the present invention in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence of the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
Typically, the nucleotide sequence of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
PREPARATION OF THE NUCLEOTIDE SEQUENCE
A nucleotide sequence encoding either an enzyme which has the specific properties as defined herein or an enzyme which is suitable for modification may be identified and/or isolated and/or purified from any cell or organism producing said enzyme. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.
Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, fransforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for enzyme (i.e. maltose), thereby allowing clones expressing the enzyme to be identified.
In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.
The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al (Science (1988) 239, pp 487-491).
AMINO ACID SEQUENCES
The present invention may also encompass amino acid sequences of enzymes having the specific properties as defined herein.
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".
The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
The enzyme of the. present invention may be used in conjunction with other enzymes. Thus the present invention also covers a combination of enzymes wherein the combination comprises the enzyme of the present invention and another enzyme, which may be another enzyme according to the present invention. This aspect is discussed in a later section.
Preferably the enzyme is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
Suitably, the polypeptide according to the present invention, for instance the isolated polypeptide or the polypeptide encoded by the polynucleotide according to the present invention, may have a molecular weight of between about 96kDa and about 102kDa, preferably of about 97kDa, as measured by SDS-Page.
Suitably, the polypeptide according to the present invention, for instance the isolated polypeptide or the polypeptide encoded by the polynucleotide according to the present invention, may comprise the following amino acid sequence:
MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDSSKCPNIIG
YGLGTYDMKSDIQMFVNPYATTNNXSSSWTPVSLAKLDFPVAMHYADITKNG
FNDGRCIFFFFCYISCFANHRTVIITDQYGSSMDDIWAYGGRVSWLENPGELRD
NWTMRTIGHSPGMHRLKAGHFTRTDRVQVVAVPIVVASSDLTTPADVIIFTAP
DDPRSEQLWQRDVVGTRHLVHEVAIVPAAETDGEMRFDQIILAGRDGVDCLW
YDGARWQKHLVGTGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGN
TVSVYTKPAGSPTGIVRAEWTRHVLDVFGPLNGKHTGSIHQVVCADIDGDGED
EFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRIATANFHSQ
GSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEEVMLRWRAGSTRFKTE
MEFLDVAGKKLTLVVLPPFARLDVERNVSGVKVMAGTVCWADENGKHERVP
ATPJ>FGCESMIVSAX)YLESGEEGAILVLYKPSSTSGRPPFRSMDELVAHNLFPA
YVPDSVRAMKFPWVRCADRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLAH
VQLWTAGIGVSAGFHNHVEASFCEIHACIANGTGRGGMRWATVPDANFNPDS
PNLEDTELIVVPDMHEHGPLWRTRPDGHPLLRMNDTIDYPWHAWLAGAGNPS
PQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLALQKNDATD
GTPVLALLDLDGGPSPQAWNISHVPGTDMYEIAHAKTGSLRVAGTHSPAAMG
LTSRWAVTKNTKGQITNKAMCYRQWSSNDSTCLKRMRGYVT
(SEQ ID No. 16) wherein X is either H or Q; or an amino acid sequence which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% homologous with SEQ ID No. 16.
Suitably, the polypeptide according to the present invention, for instance the isolated polypeptide or the polypeptide encoded by the polynucleotide according to the present invention, may comprise the following amino acid sequence: MYSKVFLKPHCEPEQPAALPLFQPQLVQGGRPDGYWVEAFPFRSDSSKCPNIIG
YGLGTYDMKSDIQMFVNPYATTNNXSSSWTPVSLAKLDFPVAMHYADITKNG
FNDGRCIFFFFCYISCFANHRTVIITDQYGSSMDDIWAYGGRVSWLENPGELRD
NWTMRTIGHSPGMHRLKAGHFTRTDRVQVVAVPIVVASSDLTTPADVIIFTAP
DDPRSEQLWQRDVVGTRHLVHEVAIVPAAETDGEMRFDQIILAGRDGVDCLW
YDGARWQKHLVGTGLPEERGDPYWGAGSAAVGRVGDDYAGYICSAEAFHGN
TVSVYTKPAGSPTGIVRAEWTRHVLDVFGPLNGKHTGSIHQVVCADIDGDGED
EFLVAMMGADPPDFQRTGVWCYKVDRTNMKFSKTKVSSVSAGRIATANFHS
QGSEVDIATISYSVPGYFESPNPSINVFLSTGILAERLDEEVMLRVVRAGSTRFK
TEMEFLDVAGKKLTLVVLPPFARLDVERNVSGVKVMAGTVCWADENGKHER
VPATPvPFGCESMIVSADYLESGEEGAILVLYKPSSTSGRPPFRSMDELVAHNLFP
AYVPDSVRAMKFPWVRCADRPWAHGRFKDLDFFNLIGFHVNFADDSAAVLA
HVQLWTAGIGVSAGFHNHVEASFCEIHACIANGTGRGGMRWATVPDANFNPD
SPNLEDTELIVVPDMHEHGPLWRTRPDGHPLLRMNDTIDYPWHAWLAGAGNP
SPQAFDVWVAFEFPGFETFSTPPPPRVLEPGRYAIRFGDPHQTASLALQKNDAT
DGTPVLALLDLDGGPSPQAWNISHVPGTDMYEIAHAKTGSLRVAGTHSPAAM
GLTSRWAVTKNTKGQITFRLPEAPDHGPLFLSVSAIRHQQGADAIPVRDRLLSL
FKFCLTYLHFILSGHRAGGQH (SEQ ID No. 17) wherein X is either H or Q; or an amino acid sequence which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% homologous with SEQ ID No. 17.
VARIANTS/HOMOLOGUES/DERTVATIVES
The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme of the present invention or of any nucleotide sequence encoding such an enzyme. Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".
In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
In the present context, an homologous sequence is taken to include a nucleotide seqμence which may be at least 40, 50, 60, 70, 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% Homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000052_0001
The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone**, L-norleucine*, L-norvaline*, p-nitro- L-phenylalanine*, L-hydroxyproline , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) , L- Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tefrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid * and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non- homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β- alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other ardmal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
The present invention also encompasses polynucleotides which have undergone molecular evolution via random processes, selection mutagenesis or in vitro recombination. As a non-limiting example, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, arid to subsequently screen for improved functionality of the encoded polypeptide by various means. In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wildtype or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide. The production of new preferred variants can be achieved by various methods well established in the art, for example the Error Threshold Mutagenesis (WO 92/18645), oligonucleotide mediated random mutagenesis (US 5,723,323), DNA shuffling (US 5,605,793), exo-mediated gene assembly WO 00/58517. The application of these and similar random directed molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in prefened environmental conditions, e.g. temperature, pH, substrate.
Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
BIOLOGICALLY ACTIVE
Preferably, the variant sequences etc. are at least as biologically active as the sequences presented herein.
As used herein "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.
ISOZYMES
The polypeptide of the present invention may exist in the form of one or more different isozymes. As used herein, the term "isozyme" encompasses variants of the polypeptide that catalyse the same reaction, but differ from each other in properties such as substrate affinity and maximum rates of enzyme-substrate reaction. Owing to differences in amino acid sequence, isozymes can be distinguished by techniques such as electrophoresis or isoelectric focusing. Different tissues often have different isoenzymes. The sequence differences generally confer different enzyme kinetic parameters that can sometimes be interpreted as fine tuning to the specific requirements of the cell types in which a particular isoenzyme is found.
ISOFORMS
The present invention also encompasses different isoforms of the polypeptide described herein. The term "isoform" refers to a protein having the same function (namely pyranosone dehydratase activity), which has a similar or identical amino acid sequence, but which is the product of a different gene.
HYBRIDISATION
The present invention also encompasses sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein. Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g. 50°C and 0.2xSSC {lxSSC = 0.15 M NaCI, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e.g. 65°C and O.lxSSC {lxSSC = 0.15 M NaCI, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, imder stringent conditions (e.g. 50°C and 0.2xSSC).
In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringency conditions (e.g. 65°C and O.lxSSC). SITE-DIRECTED MUTAGENESIS
Once an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare an enzyme of the present invention.
Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
A suitable method is disclosed in Morinaga et al (Biotechnology (1984) 2, p646-649), wherein a single-stranded gap of DNA, the enzyme-encoding sequence, is created in a vector carrying the enzyme gene. The synthetic nucleotide, bearing the desired mutation, is then annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase.
US 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the above mentioned Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.
Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151). This method involves the 3 -step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesised DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
By way of example, Sierks et al (Protein Eng (1989) 2, 621-625 and Protein Eng (1990) 3, 193-198) describes site-directed mutagenesis in Aspergillus glucoamylase. . RECOMBINANT
In one aspect of the present invention the sequence is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.
SYNTHETIC
In one aspect of the present invention the sequence is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes but is not limited to sequences made with optimal codon usage for host organisms, such as the methylotrophic yeasts Pichia and Hansenula.
EXPRESSION OF ENZYMES
The nucleotide sequence for use in the present invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in enzyme form, in and/or from a compatible host cell. Both homologous and heterologous expression is contemplated.
For heterologous expression, preferably the gene of interest or nucleotide sequence of interest is not in its naturally occurring genetic context. In the case where the gene of interest or nucleotide sequence of interest is in its naturally occurring genetic context, preferably expression is driven by means other than or in addition to its naturally occurring expression mechanism; for example, by overexpressing the gene of interest by genetic intervention
Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. The enzyme produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
EXPRESSION VECTOR
The term "expression vector" means a construct capable of resulting in in vivo of in vitro expression when introduced into a host cell or other system capable of expression.
Preferably, the expression vector is incorporated in the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.
The host organism can be the same or different to the gene of interest source organism, giving rise to homologous and heterologous expression respectively.
Preferably, the vector of the present invention comprises a construct according to the present invention. Alternatively expressed, preferably the nucleotide sequence of the present invention is present in a vector and wherein the nucleotide sequence is operably linked to regulatory sequences such that the regulatory sequences are capable of providing the expression of the nucleotide sequence by a suitable host organism, i.e. the vector is an expression vector.
The vectors of the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention. Thus, in a further aspect the invention provides a process for preparing polypeptides for subsequent use according to the present invention which comprises cultivating a host cell transformed or transfected with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The choice of vector will often depend on the host cell into which it is to be introduced.
In one aspect, Hansenula polymorpha may be used as the host for heterologous protein production.
The vectors of the present invention may contain one or more selectable marker genes. The most suitable selection systems for industrial micro-organisms are those formed by the group of selection markers which do not require a mutation in the host organism. Suitable selection markers may be the dal genes from B. subtϊlis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Alternative selection markers may be the Aspergillus selection markers such as amdS, argB, niaD and sC, or a marker giving rise to hygromycin resistance. Examples of other fungal selection markers are the genes for ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate-decarboxylase (pvrA), phleomycin and benomyl resistance (benA). Examples of non-fungal selection markers are the bacterial G418 resistance gene (this may also be used in yeast, but not in filamentous fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for β-glucuronidase (GUS). Further suitable selection markers include the dal genes from B subtilis or B. licheniformis. Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243).
Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
Thus, nucleotide sequences for use according to the present invention can be incorporated into a recombinant vector (typically a replicable vector), for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.
The procedures used to ligate a DNA construct of the invention encoding an enzyme which has the specific properties as defined herein, and the regulatory sequences, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (for instance see Sambrook et al Molecular Cloning: A laboratory Manual, 2nd Ed. (1989)).
The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences encoding a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the amino acid sequence to a host cell organelle such as a peroxisome or to a particular host cell compartment. In the present context, the term 'expression signal" includes any of the above control sequences, repressor or activator sequences. For expression under the direction of control sequences, the nucleotide sequence is operably linked to the control sequences in proper manner with respect to expression. CONSTRUCTION OF PLANT EXPRESSION CASSETTES
Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors as described herein. A description of various components of typical expression cassettes is also provided herein.
REGULATORY SEQUENCES
In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.
The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site. Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the enzyme of the present invention. In eukaryotes, polyadenylation sequences may be operably connected to the nucleotide sequence encoding the enzyme.
PROMOTERS
Preferably, the nucleotide sequence of the present invention may be operably linked to at least a promoter.
Aside from the promoter native to the gene encoding the nucleotide sequence of the present invention, other promoters may be used to direct expression of the polypeptide of the present invention. The promoter may be selected for its efficiency in directing the expression of the nucleotide sequence of the present invention in the desired expression host.
The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i. e., ability to promote transcription.
In another embodiment, a constitutive promoter may be selected to direct the expression of the desired nucleotide sequence of the present invention. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.
Depending upon the host cell system utilised, any one of a number of suitable promoters can be used, including the gene's native promoter.
Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial host include the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a- amylase gene (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes and a promoter derived from a Lactococcus sp.-derived promoter including the P170 promoter. When the nucleotide sequence is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
For transcription in a fungal species, examples of useful promoters are those derived from the genes encoding the, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, A. niger acid stable α- amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.
Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3 -phosphate dehydrogenase (gpd) promoters. Other examples of useful promoters for transcription in a fungal host are those derived from the gene encoding A. oryzae TAKA amylase, the TPI (triose phosphate isomerase) promoter from S. cerevisiae (Alber et al (1982) J. Mol. Appl. Genet. 1, p419-434), Rhizomucor miehei aspartic proteinase, A. niger neutral α- amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A oryzae triose phosphate isomerase or A. nidulans acetamidase.
Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.
Hybrid promoters may also be used to improve inducible regulation of the expression construct.
The following are non-limiting examples of promoters that may be used in expression cassettes, particularly for use with a plant host.
For Constitutive Expression, the Ubiquitin Promoter:
Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e. g. sunflower-Binet et aL, 1991 ; maize-Christensen et aL, 1989 ; and Arabidopsis-Norris et al., 1993). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol). Taylor et al. (1993) describe a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons..
For Constitutive Expression, the CaMV 35 S Promoter :
Construction of the plasmid pCGN1761 is described in the published patent application EP 0 392 225 (Example 23). pCGN1761 contains fhe"double"CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has apUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRl site. This derivative is designated pCGN1761 ENX. ρCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence-tm/ terminator cassette of such a construction can be excised by Hindlil, Sphl, Sail, and Xbal sites 5'to the promoter and Xbal, BamHl and Bgll sites 3'to the terminator for transfer to transformation vectors such as those described below. Furthermore, the double 35S promoter fragment can be removed by 5 'excision with Hindlil, Sphl, Sail, Xbal, or Pstl, and 3 'excision with any of the polylinker restriction sites (EcoRl, Notl or Xhon for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U. S. Patent No. 5, 639, 949.
For Constitutive Expression, the Actin Promoter :
Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Actl gene has been cloned and characterised (McElroy et al., 1990). A 1. 3kb fragment of the promoter was found to contain all the regulator elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al, 1991). These incorporate the Actl-intron 1, Adhl 5' flanking sequence and Adhl-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al (1991) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et aL, 1993).
For Inducible Expression, the PR-1 Promoter :
The double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, one of the chemically regulatable promoters described in U. S. Patent No. 5, 614, 395 may replace the double 35S promoter. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR1 a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of
EP 0 332 104) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992). pCIB1004 is cleaved with Ncol and the resultant 3 'overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with Hindlil and the resultant PR-la promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlil and isolation of the larger vector-terminator containing fragment into which the pCIB 1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and an intervening polylinker with unique EcoRl and Notl sites. The selected coding sequence can be inserted into this vector, and the fusion products (i. e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described infra. Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U. S. Patent Nos. 5, 523, 311 and 5, 614, 395. For Inducible Expression, an Ethanol-Inducible Promoter :
A promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention. Such a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al., 1998). In A. nidulans, the alcA gene encodes alcohol dehydrogenase 1, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
For the purposes of the present invention, the CAT coding sequences in plasmid palcA: CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et a/., 1998) are replaced by a coding sequence (polynucleotide sequence) of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.
For Inducible Expression, a Glucocorticoid-Inducible Promoter: Induction of expression of a polynucleotide of the present invention using systems based on steroid hormones is also contemplated. For example, a glucocorticoid- mediated induction system is used (Aoyama and Chua, 1997) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0. 1 mM to lmM, more preferably from 1 OmM to 1 OOmM. For the purposes of the present invention, the luciferase gene sequences are replaced by a gene sequence encoding a polynucleotide according to the present invention to form an expression cassette having a polynucleotide sequence according to the present invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods well known in the art. The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al., 1986) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al., 1988) fused to the hormonebinding domain of the rat glucocorticoid receptor (Picard et al„ 1988). The expression of the fusion protein is controlled by any promoter suitable for expression in plants known in the art or described here. This expression cassette is also comprised in the plant comprising the polynucleotide sequence according to the present invention fused to the 6xGAL4/minimal promoter. Thus, tissue-or organ-specificity of the fusion protein is achieved leading to inducible tissue-or organ-specificity of the polypeptide produced.
Root Specific Expression:
Another pattern of gene expression is root expression. A suitable root promoter is described by de Framond (1991) and also in the published patent application EP 0 452 269. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene- terminator cassette to a transformation vector of interest.
Wound-lnducible Promoters:
Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e. g. Xu et, al., 1993) ; Logemann et al., 1989 ; Rohrmeier & Lehle, 1993 ; Firek et al., 1993 ; Warner et al., 1993) and all are suitable for use with the instant invention. Logemann et al. describe the 5'upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et aL and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.
Pith-Preferred Expression :
Patent Application WO 93/07278 describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to-1726 bp from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-prefened promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
Leaf-Specific Expression :
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (1989). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants. k. Pollen-Specific Expression : WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells. The gene sequence and promoter extend up to 1400 bp from the start of transcription. Using standard molecular biological techniques, this promoter or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a polynucleotide of the present invention in a pollen-specific manner.
The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable other sequences include the Shi -intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat 1987 Gene 217, 217-225 and Dawson 1993 Plant Mol. Biol. 23: 97).
TRANSCRIPTIONAL TERMINATORS
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tm/terminator, the opaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used.
ENHANCER SEQUENCES
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis etal., 1987). In the same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression, intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the"W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e. g. Gallie et al., 1987 ; Skuzeski et al, 1990). TARGETING OF A GENE PRODUCT (POLYPEPTIDE) WITHIN THE CELL
Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterised in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e. g. Comai et aL, 1988). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al., 1985). DNA encoding for appropriate signal sequences can be isolated from the 5 'end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting"in Example 37 of U. S. Patent No. 5, 639, 949.
Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e. g. Unger et al., 1989). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles.
Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (1985).
In addition, sequences have been characterised which cause the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, 1990). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., 1990).
By the fusion of the appropriate targeting sequences described above to polynucleotide sequences of interest it is possible to direct the polypeptide product(s) to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. (1982) and Wasmann et al. (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.
CONSTRUCTS
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shi -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment. The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred. Various markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin, or other selectable markers such as the galactose selection marker (see WO00/09705).
For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.
GLUCAN LYASES
Suitable glucan lyases and polynucleotides encoding same which can be used in accordance with the present invention are known. Suitable glucan lyases are taught in WO96/12026, WO95/10618 and WO95/10617 for example.
By way of example only, a glucan lyase used in accordance with the present invention may have any one of the amino acid sequence shown in SEQ ID No. 21-25 (see hereinbelow).
SEQ ID No. 21:
MAGFSDPLNF CKAEDYYSVA LDWKGPQKII GVDTTPPKST KFPKNWHGVN LRFDDGTLGV VQFIRPCVWR VRYDPGFKTS DEYGDENTRT IVQDYMSTLS NKLDTYRGLT WETKCEDSGD FFTFSSKVTA VEKSERTRNK VGDGLRIHLW KSPFRIQWR TLTPLKDPYP IPNVAAAEAR VSDKWWQTS PKTFRKNLHP QHKMLKDTVL DIVKPGHGEY VGWGEMGGIQ FMKEPTFMNY FNFDNMQYQQ VYAQGALDSR EPLYHSDPFY LDVNSNPEHK NITATFIDNY SQIAIDFGKT NSGYTKLGTR YGGIDCYGIS ADTVPEIVRL YTGLVGRSKL KPRYTLGAHQ ACYGYQQESD LYSVVQQYRD CKFPLDGIHV DVDVQDGFRT FTTNPHTFPN PKEMFTNLRN NGIKCSTNIT PVISINNREG GYSTLLEGVD KKYFIMDDRY TEGTSGNAKD VRYMYYGGGN KVEVDPNDVN GRPDFKDNYD FPANFNSKQY PYHGGVSYGY GNGSAGFYPD LNRKEVRIWW GMQYKYLFDM GLEFVWQDMT TPAIHTSYGD MKGLPTRLLV TSDSVTNASE KKLAIETWAL YSYNLHKATW HGLSRLESRK NKRNFILGRG SYAGAYRFAG LWTGDNASNW EFWKISVSQV LSLGLNGVCI AGSDTGGFEP YRDANGVEEK YCSPELLIRW YTGSFLLPWL RNHYVKKDRK WFQEPYSYPK HLETHPELAD QAWLYKSVLE ICRYYVELRY SLIQLLYDCM FQNWDGMPI TRSMLLTDTE DTTFFNESQK FLDNQYMAGD DILVAPILHS RKEIPGENRD VYLPLYHTWY PSNLRPWDDQ GVALGNPVEG GSVINYTARI VAPEDYNLFH SVVPVYVREG AIIPQIEVRQ WTGQGGANRI KFNIYPGKDK EYCTYLDDGV SRDSAPEDLP QYKETHEQSK VEGAEIAKQI GKKTGYNISG TDPEAKGYHR KVAVTQTSKD KTRTVTIEPK HNGYDPSKEV GDYYTIILWY APGFDGSIVD VSKTTVNVEG GVEHQVYKNS DLHTWIDVK EVIGTTKSVK ITCTAA
SEQ ID No.22:
MAGLSDPLNF RKAEDYYAAA KGWSGPQKII RYDQTPPQGT KDPKSWHAVN LPFDDGTMCV VQFVRPCVWR VRYDPSVKTS DEYGDENTRT IVQDYMTTLV GNLDIFRGLT WVSTLEDSGE YYTFKSEVTA VDETERTRNK VGDGLKIYLW KNPFRIQVVR LLTPLVDPFP IPNVANATAR VADKVVWQTS PKTFRKNLHP QHKMLKDTVL DIIKPGHGEY VGWGEMGGIE FMKEPTFMNY FNFDNMQYQQ VYAQGALDSR EPLYHSDPFY LDVNSNPEHK NITATFIDNY SQIAIDFGKT NSGYIKLGTR YGGIDCYGIS ADTVPEIVRL YTGLVGRSKL KPRYILGAHQ ACYGYQQESD LHAVVQQYRD TKFPLDGLHV DVDFQDNFRT FTTNPITFPN PKEMFTNLRN NGIKCSTNIT PVISIRDRPN GYSTLNEGYD KKYFIMDDRY TEGTSGDPQN VRYSFYGGGN PVEVNPNDVW ARPDFGDNYD FPTNFNCKDY PYHGGVSYGY GNGTPGYYPD LNREEVRIWW GLQYEYLFNM GLEFVWQDMT TPAIHSSYGD MKGLPTRLLV TADSVTNASE KKLAIES AL YSYNLHKATF HGLGRLESRK NKRNFILGRG SYAGAYRFAG LWTGDNASTW EFWKISVSQV LSLGLNGVCI AGSDTGGFEP ARTEIGEEKY CSPELLIRWY TGSFLLPWLR NHYVKKDRKW FQEPYAYPKH LETHPELADQ AWLYKSVLEI CRYWVELRYS LIQLLYDCMF QNWDGMPLA RSMLLTDTED TTFFNESQKF LDNQYMAGDD ILVAPILHSR NEVPGENRDV YLPLFHTWYP SNLRPWDDQG VALGNPVEGG SVTNYTARIV APEDYNLFHN WPVYIREGA IIPQIQVRQW IGEGGPNPIK FNIYPGKDKE YVTYLDDGVS RDSAPDDLPQ YREAYEQAKV EGKDVQKQLA VIQGNKTNDF SASGIDKEAK GYHRKVSIKQ ESKDKTRTVT IEPKHNGYDP SKEVGNYYTI E WYAPGFDG SΓVDVSQATV NIEGGVECEI FKNTGLHTW VNVKEVIGTT KSVKITCTTA
SEQ ID No.23:
Met Phe Pro Thr Leu Thr Phe He Ala Pro Ser Ala Leu Ala Ala Ser Thr Phe Val Gly Ala Asp lie Arg Ser Gly He Arg He Gin Ser Ala Leu Pro Ala Val Arg Asn Ala Val Arg Arg Ser Lys His Tyr Asn Val Ser Met Thr Ala Leu Ser Asp Lys Gin Thr Ala He Ser He Gly Pro Asp Asn Pro Asp Gly He Asn Tyr Gin Asn Tyr Asp Tyr lie Pro Val Ala Gly Phe Thr Pro Leu Ser Asn Thr Asn Trp Tyr Ala Ala Gly Ser Ser Thr Pro Gly Gly He Thr Asp Trp Thr Ala Thr Met Asn Val Lys Phe Asp Arg He Asp Asn Pro Ser Tyr Ser Asn Asn His Pro Val Gin He Gin Val Thr Ser Tyr Asn Asn Asn Ser Phe Arg He Arg Phe Asn Pro Asp Gly Pro He Arg Asp Val Ser Arg Gly Pro He Leu Lys Gin Gin Leu Thr Trp He Arg Asn Gin Glu Leu Ala Gin Gly Cys Asn Pro Asn Met Ser Phe Ser Pro Glu Gly Phe Leu Ser Phe Glu Thr Lys Asp Leu Asn Val He He Tyr Gly Asn Cys Lys Met Arg Val Thr Lys Lys Asp Gly Tyr Leu Val Met Glu Asn Asp Glu Cys Asn Ser Gta Ser Asp Gly Asn Lys Cys Arg Gly Leu Met Tyr Val Asp Arg Leu Tyr Gly Asn Ala He Ala Ser Val Gta Thr Asn Phe His Lys Asp Thr Ser Arg Asn Glu Lys Phe Tyr Gly Ala Gly Glu Val Asn Cys Arg Tyr Glu Glu Gta Gly Lys Ala Pro Thr Tyr Val Leu Glu Arg Ser Gly Leu Ala Met Thr Asn Tyr Asn Tyr Asp Asn Leu Asn Tyr Asn Gta Pro Asp Val Val Pro Pro Gly Tyr Pro Asp His Pro Asn Tyr Tyr He Pro Met Tyr Tyr Ala Ala Pro Trp Leu Val Val Gin Gly Cys Ala Gly Thr Ser Lys Gta Tyr Ser Tyr Gly Trp Phe Met Asp Asn Val Ser Gta Ser Tyr Met Asn Thr Gly Asp Thr Ala Trp Asn Cys Gly Gin Glu Asn Leu Ala Tyr Met Gly Ala Gta Tyr Gly Pro Phe Asp Gin His Phe Val Tyr Gly Asp Gly Asp Gly Leu Glu Asp Val Val Lys Ala Phe Ser Phe Leu Gta Gly Lys Glu Phe Glu Asp Lys Lys Leu Asn Lys Arg Ser Val Met Pro Pro Lys Tyr Val Phe Gly Phe Phe Gin Gly Val Phe Gly Ala Leu Ser Leu Leu Lys Gta Asn Leu Pro Ala Gly Glu Asn Asn He Ser Val Gta Glu He Val Glu Gly Tyr Gta Asp Asn Asp Tyr Pro Phe Glu Gly Leu Ala Val Asp Val Asp Met Gin Asp Asp Leu Arg Val Phe Thr. Thr Lys Pro Glu Tyr Trp Ser Ala Asn Met Val Gly Glu Gly Gly Asp Pro Asn Asn Arg Ser Val Phe Glu Trp Ala His Asp Arg Gly Leu Val Cys Gta Thr Asn Val Thr Cys Phe Leu Arg Asn Asp Asn Ser Gly Lys Pro Tyr Glu Val Asn Gin Thr Leu Arg Glu Lys Gta Leu Tyr Thr Lys Asn Asp Ser Leu Asn Asn Thr Asp Phe Gly Thr Thr Ser Asp Gly Pro Gly Asp Ala Tyr He Gly His Leu Asp Tyr Gly Gly Gly Val Glu Cys Asp Ala lie Phe Pro Asp Trp Gly Arg Pro Asp Val Ala Gta Trp Trp Gly Glu Asn Tyr Lys Lys Leu Phe Ser He Gly Leu Asp Phe Val Trp Gta Asp Met Thr Val Pro Ala Met Met Pro His Arg Leu Gly Asp Ala Val Asn Lys Asn Ser Gly Ser Ser Ala Pro Gly Trp Pro Asn Glu Asn Asp Pro Ser Asn Gly Arg Tyr Asn Trp Lys Ser Tyr His Pro Gta Val Leu Val Thr Asp Met Arg Tyr Gly Ala Glu Tyr Gly Arg Glu Pro Met Val Ser Gin Arg Asn He His Ala Tyr Thr Leu Cys Glu Ser Thr Arg Arg Glu Gly He Val Gly Asn Ala Asp Ser Leu Thr Lys Phe Arg Arg Ser Tyr He He Ser Arg Gly Gly Tyr He Gly Asn Gta His Phe Gly Gly Met Trp Val Gly Asp Asn Ser Ala Thr Glu Ser Tyr Leu Gta Met Met Leu Ala Asn He He Asn Met Asn Met Ser Cys Leu Pro Leu Val Gly Ser Asp He Gly Gly Phe Thr Gin Tyr Asn Asp Ala Gly Asp Pro Thr Pro Glu Asp Leu Met Val Arg Phe Val Gin Ala Gly Cys Leu Leu Pro Trp Phe Arg Asn His Tyr Asp Arg Trp He Glu Ser Lys Lys His Gly Lys Lys Tyr Gta Glu Leu Tyr Met Tyr Pro Gly Gta Lys Asp Thr Leu Lys Lys Phe Val Glu Phe Arg Tyr Arg Trp Gin Glu Val Leu Tyr Thr Ala Met Tyr Gin Asn Ala Thr Thr Gly Glu Pro He He Lys Ala Ala Pro Met Tyr Asn Asn Asp Val Asn Val Tyr Lys Ser Gta Asn Asp His Phe Leu Leu Gly Gly His Asp Gly Tyr Arg He Leu Cys Ala Pro Val Val Arg Glu Asn Ala Thr Ser Arg Glu Val Tyr Leu Pro Val Tyr Ser Lys Trp Phe Lys Phe Gly Pro Asp Phe Asp Thr Lys Pro Leu Glu Asn Glu He Gta Gly Gly Gta Thr Leu Tyr Asn Tyr Ala Ala Pro Leu Asn Asp Ser Pro He Phe Val Arg Glu Gly Thr He Leu Pro Thr Arg Tyr Thr Leu Asp Gly Val Asn Lys Ser He Asn Thr Tyr Thr Asp Asn Asp Pro Leu Val Phe Glu Leu Phe Pro Leu Glu Asn Asn Gin Ala His Gly Leu Phe Tyr His Asp Asp Gly Gly Val Thr Thr Asn Ala Glu Asp Phe Gly Lys Tyr Ser Val He Ser Val Lys Ala Ala Gin Glu Gly Ser Gin Met Ser Val Lys Phe Asp Asn Glu Val Tyr Glu His Gin Trp Gly Ala Ser Phe Tyr Val Arg Val Arg Asn Met Gly Ala Pro Ser Asn He Asn Val Ser Ser Gin He Gly Gin Gta Asp Met Gin Gta Ser Ser Val Ser Ser Arg Ala Gta Met Phe Thr Ser Ala Asn Asp Gly Glu Tyr Trp Val Asp Gin Ser Thr Asn Ser Leu Trp Leu Lys Leu Pro Gly Ala Val He Gta Asp Ala Ala He Thr Val Arg
SEQ ID No. 24:
Met Thr Asn Tyr Asn Tyr Asp Asn Leu Asn Tyr Asn Gta Pro Asp Leu He Pro Pro Gly His Asp Ser Asp Pro Asp Tyr Tyr He Pro Met Tyr Phe Ala Ala Pro Trp Val He Ala His Gly Tyr Arg Gly Thr Ser Asp Gin Tyr Ser Tyr Gly Trp Phe Leu Asp Asn Val Ser Gin Ser Tyr Thr Asn Thr Gly Asp Asp Ala Trp Ala Gly Gin Lys Asp Leu Ala Tyr Met Gly Ala Gin Cys Gly Pro Phe Asp Gta His Phe Val Tyr Glu Ala Gly Asp Gly Leu Glu Asp Val Val Thr Ala Phe Ser Tyr Leu Gta Gly Lys Glu Tyr Glu Asn Gin Gly Leu Asn He Arg Ser Ala Met Pro Pro Lys Tyr Val Phe Gly Phe Phe Gin Gly Val Phe Gly Ala Thr Ser Leu Leu Arg Asp Asn Leu Pro Ala Gly Glu Asn Asn Val Ser Leu Glu Glu He Val Glu Gly Tyr Gta Asn Gta Asn Val Pro Phe Glu Gly Leu Ala Val Asp Val Asp Met Gin Asp Asp Leu Arg Val Phe Thr Thr Arg Pro Ala Phe Trp Thr Ala Asn Lys Val Gly Glu Gly Gly Asp Pro Asn Asn Lys Ser Val Phe Glu Trp Ala His Asp Arg Gly Leu Val Cys Gin Thr Asn Val Thr Cys Phe Leu Lys Asn Glu Lys Asn Pro Tyr Glu Val Asn Gin Ser Leu Arg Glu Lys Gta Leu Tyr Thr Lys Ser Asp Ser Leu Asp Asn He Asp Phe Gly Thr Thr Pro Asp Gly Pro Ser Asp Ala Tyr He Gly His Leu Asp Tyr Gly Gly Gly Val Glu Cys Asp Ala Leu Phe Pro Asp Trp Gly Arg Pro Asp Val Ala Gin Trp Trp Gly Asp Asn Tyr Lys Lys Leu Phe Ser He Gly Leu Asp Phe Val Trp Gin Asp Met Thr Val Pro Ala Met Met Pro His Arg Leu Gly Asp Pro Val Gly Thr Asn Ser Gly Glu Thr Ala Pro Gly Trp Pro Asn Asp Lys Asp Pro Ser Asn Gly Arg Tyr Asn Trp Lys Ser Tyr His Pro Gin Val Leu Val Thr Asp Met Arg Tyr Asp Asp Tyr Gly Arg Asp Pro He Val Thr Gta Arg Asn Leu His Ala Tyr Thr Leu Cys Glu Ser Thr Arg Arg Glu Gly He Val Gly Asn Ala Asp Ser Leu Thr Lys Phe Arg Arg Ser Tyr He He Ser Arg Gly Gly Tyr He Gly Asn Gin His Phe Gly Gly Met Trp Val Gly Asp Asn Ser Ser Thr Glu Asp Tyr Leu Ala Met Met Val He Asn Val He Asn Met Asn Met Ser Gly Val Pro Leu Val Gly Ser Asp He Gly Gly Phe Thr Glu His Asp Lys Arg Asn Pro Cys Thr Pro Asp Leu Met Met Arg Phe Val Gin Ala Gly Cys Leu Leu Pro Trp Phe Arg Asn His Tyr Asp Arg Trp He Glu Ser Lys Lys His Gly Lys Asn Tyr Gin Glu Leu Tyr Met Tyr Arg Asp His Leu Asp Ala Leu Arg Ser Phe Val Glu Leu Arg Tyr Arg Trp Gin Glu Val Leu Tyr Thr Ala Met Tyr Gin Asn Ala Leu Asn Gly Lys Pro He He Lys Thr Val Ser Met Tyr Asn Asn Asp Met Asn Val Lys Asp Ala Gta Asn Asp His Phe
SEQ ID No. 25:
MFSTLAFVAP SALGASTFVG AEVRSNVRIH SAFPAVHTAT RKTNRLNVSM TALSDKQTAT AGSTDNPDGI DYKTYDYVGV WGFSPLSNTN WFAAGSSTPG GITDWTATMN VNFDRIDNPS ITVQHPVQVQ VTSYNNNSYR VRFNPDGPIR DVTRGPILKQ QLDWIRTQEL SEGCDPGMTF TSEGFLTFET KDLSVΠYGN FKTRVTRKSD GKVIMENDEV GTASSGNKCR GLMFVDRLYG NAIASVNKNF RNDAVKQEGF YGAGEVNCKY QDTYILERTG IAMTNYNYDN LNYNQWDLRP PHHDGALNPD YYTPMYYAAP WLIVNGCAGT SEQYSYGWFM DNVSQSYMNT GDTTWNSGQE DLAYMGAQYG PFDQHFVYGA GGGMECVVTA FSLLQGKEFE NQVLNKRSVM PPKYVFGFFQ GVFGTSSLLR AHMPAGENNI SVEEIVEGYQ NNNFPFEGLA VDVDMQDNLR VFTTKGEFWT ANRVGTGGDP NNRSVFEWAH DKGLVCQTNI TCFLRNDNEG QDYEVNQTLR ERQLYTKNDS LTGTDFGMTD DGPSDAYIGH LDYGGGVECD ALFPDWGRPD VAEWWGNNYK KLFSIGLDFV WQDMTVPAMM PHKIGDDINV KPDGNWPNAD DPSNGQYNWK TYHPQVLVTD MRYENHGREP MVTQRNIHAY TLCESTRKEG IVENADTLTK FRRSYIISRG GYIGNQHFGG MWVGDNSTTS NYIQMMIANN INMNMSCLPL VGSDIGGFTS YDNENQRTPC TGDLMVRYVQ AGCLLPWFRN HYDRWIESKD HGKDYQELYM YPNEMDTLRK FVEFRYRWQE VLYTAMYQNA AFGKPIIKAA SMYNNDSNVR RAQNDHFLLG GHDGYRILCA PWWENSTER ELYLPVLTQW YKFGPDFDTK PLEGAMNGGD RIYNYPVPQS ESPFVREGA ILPTRYTLNG ENKSLNTYTD EDPLVFEVFP LGNNRADGMC YLDDGGVTTN AEDNGKFSVV KVAAEQDGGT ETITFTNDCY EYVFGGPFYV RVRGAQSPSN IHVSSGAGSQ DMKVSSATSR AALFNDGENG DFWVDQETDS LWLKLPNWL PDAVITIT
Suitable glucan lyases can be obtained from a range of organisms using degenerative primers based on the flowing conserved amino acid sequences FVWQDMT and PWLRHNY, for example glucan lyase can be obtained from Anthracobia melaloma using the following PCR primers: TA(GA)TGGTT(TC)CT(GA)A(GA)CCA(CT) TTT(TC)GT(GATC)TGGCA(GA)GA(TC)ATGAC
PYRANOSE -2-OXIDASE
Suitable pyranose-2-oxidase genes and enzymes are disclosed in US6, 146,865 and US
5,712,139.
By way of example only the pyranose-2-oxidase may be encoded by the following sequences: gi I 34452036 | gb|AY370876.11 Peniσphora gigantea pyranose oxidase (poxB) mRNA, complete eds (GENBANK)
GCTACCTTCTTAACGGACAGGCATCCCAGCTTTTCCGCCCTACAACACCCACCCCGCGCACAAATGTCGG
CCAGCTCGAGTGACCCCTTCCACAGCTTCGCGAAGACGAGCTTCACGAGCAAGGCGGCGAAGAGGGCCAC
TGCGCACTCTCTCCCGCCGCTGCCTGGTCCCGGCGACCTGCCGCCTGGTATGAATGTTGAGTACGACGTT
GCCATCGTCGGCTCGGGGCCAATTGGCTCCACATATGCGCGCGAGCTCGTTGAGGCCGGCTTCAACGTCG
CCATGTTCGAGATTGGAGAGATCGACTCCGGCTTGAAGATCGGCTCACACAAGAAGAACACCGTCGAGTA
CCAGAAGAACATCGACAAATTCGTAAATGTTATACAAGGGCAACTTATGCCCGTCTCGGTGCCCGTCAAC.
ACGATGGTCGTTGACACGCTAAGCCCGGCGTCATGGCAAGCTTCGACGTTCTTCGTCCGCAACGGGGCGA
ATCCAGAGCAAGACCCGCTGCGCAACCTTAGTGGCCAGGCGGTCACCCGCGTCGTCGGCGGCATGTCTAC
GCACTGGACGTGCGCGACGCCGCGCTTCGAGAAGTTGCAGCGCCCGCTACTCGTGAAGAACGACCCCGTA
GCGGACGACGCCGAGTGGGACAGGCTCTACAAGAAGGCCGAGTCGTACTTCAAGACCGGCACGACCCAGT
TCGCCGAGTCGATCCGCCACAACCTCGTGCTCAAGAAGCTGCAGGAGGAGTACAAGGGCGTGCGCGACTT
CCAGCAGATCCCGCTCGCGGCGACGCGCCAGAGCCCGACGTTCGTCGAGTGGAGCTCGGCGCACACCGTG TTCGATCTCGAGAACCGGCCGAACAAGGACGCGCCGAAGCAGCGCTTCAATCTCTTCCCCGCCGTCGCGT
GCACGAGCGTGAGGCGCAATGACGCGAACTCGGAGATCATAGGCCTGGATGTCCGCGACCTCCACGGGGG
CAAGAGCATCACCATCAAGGCCAAGGTGTACATCCTCACCGCCGGCGCGGTCCACAACGCGCAGCTCCTC
GCGGCCTCTGGATTCGGGCAGCTGGGTCGTCCCGACCCCGCCAAGCCGCTGCCGTCTCTGCTGCCGTACC
TGGGGACCCACATCACCGAGCAGACGCTCGTCTTCTGCCAGACCGTCATGAGCACGGAGCTCATCAACAG
CGTCACGGCGGATATGACCATTGTCGGGAAGCCGGGCGACCCGGACTATAGCGTCACGTACACCTCGGGC
AGCCCGAACAACAAGCACCCGGACTGGTGGAACGAGAAGGTGAAGAAGCACATGATGGACCACCAGGAGG
ACCCGCTCCCGATCCCGTTCGAGGACCCCGAGCCGCAGGTCACCACGCTCTTCAAGGCATCGCACCCG.TG
GCACACTCAGATCCACC'GCGACGCCTTCAGCTACGGCGCCGTGCAGCAGAGCATCGACTCGCGGCTCATC
GTCGACTGGCGGTTCTTCGGACGCACCGAGCCCAAGGAAGAGAACAAGCTATGGTTCTCGGACAAGATCA
CGGACGCGTACAACCTGCCACAGCCGACGTTCGACTTCCGCTTCCCCGGGGGTCGCGAAGCGGAGGACAT
GATGACCGACATGTGCGTCATGTCGGCGAAGATCGGTGGATTCCTGCCTGGGTCCTACCCACAGTTCATG
GAGCCCGGTCTTGTCCTGCACCTTGGTGGGACGCACCGCATGGGCTTCGACGAGAAGGCGGACAAGTGCT
GCGTCGACACCGACTCACGCGTCTTCGGCTTCAAGAACCTCTTCCTCGGCGGCTGCGGGAACATCCCCAC'
CGCGTACGCCGCGAACCCGACGCTCACCGCAATGTCGCTTGCGATCAAGAGCTGCGAGTACATCAAGAAG
AACTTCGAGCCGAGCCCGAACCCCGTGAAGCACCACAACTGATGCGTTGCTTCCGATGTCATTGCCTGTC
CGATCTCAACTTGAAGCCTCGCTGGCCCTGAΆGCATCCTAGGGTGTTGTACGAGTATCCGGTAGCATCCG
CCTGACAGCGTTTATGTGTACGAGTAGTCGCAΆTCCTCATATCCGACGTTTGAGCAAAAAAAAAAAAAAA
A (SEQ ID No . 27 )
gi|31044223|gb|AY291124.1| Trametes ochracea strain MB49 pyranose oxidase (p2o) mRNA, complete eds
ACTTAAGGAGCCCCTGCGACTCTACTGTAGCCCTCAAΆCTACACCAGCATGTCTACCAGCTCGAGCGACC CGTTCTTCAACTTCGCGAAGTCGAGCTTCAGGAGCGCGGCGGCGCAGAAGGCCTCGGCGAGTTCTCTGCC GCCGCTGCCGGGTCCCGACAAGAAAGTGCCCGGAATGGACATCAAGTACGACGTTGTCATAGTAGGCTCC GGACCGATTGGATGCACGTATGCCCGTGAGCTCGTCGGCGCAGGTTACAAGGTCGCCATGTTCGACATCG GAGAGATCGACTCTGGCCTGAAGATTGGTGCCCACAAGAAGAACACCGTCGAGTACCAGAAGAACATCGA CAAGTTTGTGAACGTCATTCAGGGACAACTGATGTCTGTTTCCGTTCCCGTCAATACCCTCGTGGTCGAC ACGCTCAGCCCGACGTCTTGGCAAGCTTCGACGTTCTTCGTCCGCAACGGCTCGAACCCAGAGCAGGACC CACTTCGTAACCTCAGTGGTCAGGCGGTCACGCGCGTCGTCGGAGGCATGTCTACGCACTGGACATGCGC CACACCCCGTTTCGACCGCGAGCAACGCCCGCTGCTTGTGAAGGACGACGCGGACGCTGACGACGCTGAG TGGGACCGGCTCTACACCAAGGCCGAGTCGTACTTCCAGACTGGGACGGACCAGTTCAΆGGAGTCGATCC GCCACAATCTCGTGCTCAACAAGCTCACGGAGGAATACAAGGGCCAACGCGACTTCCAGCAGATTCCACT CGCGGCAACCCGCCGGAGCCCGACCTTCGTCGAATGGAGCTCGGCGAΆCACTGTTTTCGACCTCCAGAAC AGGCCGAACACGGACGCGCCGGAGGAGCGCTTCAACCTCTTCCCCGCGGTCGCGTGTGAGCGCGTCGTGC GCAACGCGTTGAACTCGGAGATCGAGAGTCTGCACATCCACGACCTCATCTCGGGCGACCGCTTCGAAAT CAAGGCTGACGTGTACGTCCTCACCGCCGGGGCGGTCCACAACACGCAGCTTCTCGTGAACTCTGGCTTT GGACAGCTGGGCCGACCGAACCCCGCAΆACCCACCGGAGCTGCTGCCGTCCCTGGGGAGCTACATCACCG AGCAGTCGCTCGTCTTCTGCCAGACCGTGATGAGCACCGAGCTCATCGACAGCGTCAAGTCCGACATGAC CATCAGGGGAACCCCTGGCGAGCTGACGTACAGCGTGACGTACACGCCGGGCGCGTCGACCAACAAGCAC CCGGACTGGTGGAACGAGAAGGTGAAAAACCACATGATGCAGCACCAGGAGGACCCGCTCCCGATCCCGT TCGAGGACCCCGAGCCGCAGGTTACCACTCTGTTTCAΆCCGTCGCACCCGTGGCACACTCAGATCCACCG CGATGCTTTCAGTTACGGCGCAGTGCAGCAAAGCATCGACTCGCGTCTCATCGTGGACTGGCGCTTCTTC GGCCGGACGGAGCCCAAGGAGGAGAACAAGCTCTGGTTCTCAGACAAGATCACGGACGCGTACAACAT-GC CGCAGCCCACGTTCGACTTCCGCTTCCCGGCCGGCCGCACGAGCAAGGAGGCGGAGGACATGATGACCGA CATGTGCGTTATGTCGGCGAAGATCGGTGGCTTCCTACCCGGCTCCCTCCCGCAATTCATGGAGCCTGGT CTTGTCCTTCACCTTGGTGGTACGCACCGCATGGGCTTCGACGAGAAGGAGGACAACTGCTGCGTCAACA CGGACTCGCGCGTGTTCGGCTTCAAGAACCTCTTCCTCGGTGGCTGCGGAAACATTCCTACCGCGTACGG CGCGAACCCGACGCTCACCGCAATGTCGCTCGCGATCAAGAGTTGCGAGTACATCAAGCAGAACTTCACG CCGAGCCCATTCACGTCGGAGGCTCAGTGAGTGGTCGCTCGCCGACCTAGCTTAGTGTGGATGCGAAGCC CCTTACGTGGCGTGGAAACTCGCTGAGTAGTTTGTACGATCAGAATGAGCGAAΆTCGG'CGCGACTGTCAT
TTGTACCATTAGTATCAATGTGTATGGCTATGACCACCC (SEQ ID No. 28).
>gi | 1845548 | dbj I D73369. 1 I Coriolus versicolor mRNA for pyranose oxidase, complete eds ACTGTAGCTCTCAAACAACGCCAACATGTCTACTAGCTCGAGCGACCCGTTCTTCAACTTCACGAAGTCG AGCTTTAGGAGCGCGGCGGCGCAGAAGGCCTCGGCGACTTCTCTGCCGCCGCTGCCTGGTCCCGACAAGA AAGTCCCTGGAATGGACATCAAGTACGACGTTGTCATAGTAGGCTCCGGACCGATTGGATGCACGTATGC CCGTGAGCTCGTCGAAGCCGGTTACAAGGTCGCCATGTTCGACATCGGGGAAATTGACTCTGGCCTGAAG ATCGGTGCCCACAAGAAGAACACCGTCGAATACCAGAAGAACATTGACAAGTTTGTGAΆCGTCATTCAGG GCCAATTGATGTCTGTTTCCGTTCCCGTCAATACCCTCGTGATCGACACGCTCAGCCCGACGTCTTGGCA AGCTTCATCGTTCTTCGTCCGCAATGGCTCGAACCCAGAGCAGGACCCGCTTCGTAACCTCAGTGGTCAG GCGGTCACGCGTGTCGTCGGAGGCATGTCCACGCACTGGACATGCGCGACACCGCGCTTTGACCGCGAGC AGCGCCCGTTGCTCGTGAAGGACGACCAGGACGCTGACGACGCCGAGTGGGACCGGCTGTACACCAAGGC CGAGTCATACTTCAAGACCGGGACGGACCAGTTCAAGGAGTCGATCCGCGACAACCTCGTGCTCAACAAG CTCGCGGAGGAATACAAAGGTCAGCGCGACTTCCAGCAGATCCCGCTCGCGGCAACGCGTCGCAGTCCGA CCTTCGTCGAGTGGAGCTCGGCGAACACCGTGTTCGACCTCCAGAACAGGCCGAACACGGACGCGCCGAA TGAGCGCTTCAACCTCTTCCCCGCGGTTGCATGTGAGCGCGTCGTGCGCAACACGTCGAACTCCGAGATC GAGAGTCTGCACATCCACGACCTCATCTCGGGCGACCGCTTCGAAATCAAAGCAGACGTGTTCGTTCTTA CAGCCGGGGCGGTCCACAACGCGCAGCTTCTCGTGAACTCTGGCTTTGGACAGCTGGGCCGGCCGGACCC CGCGAACCCGCCGCAGTTGCTGCCGTCCCTGGGAAGCTACATCACCGAGCAGTCGCTCGTCTTCTGCCAG ACCGTGATGAGCACCGAGCTCATCGACAGCGTCAAGTCCGACATGATCATCAGGGGCAACCCTGGCGATC TGGGGTACAGCGTCACGTACACGCCCGGCGCGGAGACCAACAAGCACCCGGACTGGTGGAACGAAAAGGT GAAGAACCACATGATGCAGCACCAGGAGGACCCGCTTCCAATCCCGTTCGAGGACCCCGAGGCGCAGGTC ACCACCTTGTTCCAGCCATCGCACCCGTGGCACACTCAGATTCACCGCGATGCGTTCAGTTACGGCGCGG TGCAGCAAAGCATCGACTCACGTCTCATCGTCGACTGGCGCTTCTTCGGCCGGACGGAGCCAAAGGAGGA GAACAAGCTCTGGTTCTCGGACAAAATTACGGACACGTACAACATGCCGCAGCCGACGTTCGACTTCCGC TTCCCGGCGGGCCGCACGAGCAAGGAGGCGGAGGACATGATGACCGATATGTGCGTTATGTCGGCGAAGA TTGGTGGCTTCGTGCCCGGCTCCCTCCCGCAATTCATGGAGCCCGGTCTTGTCCTTCACCTCGGTGGTAC GCACCGCATGGGCTTCGACGAGCAGGAGGACAAGTGCTGCGTCAACACGGACTCGCGCGTGTTTGGCTTC AAGAACCTGTTCCTCGGTGGCTGCGGAAACATTCCCACCGCGTACGGCGCGAACCCGACGCTCACCGCAA TGTCGCTCGCGATCAAGAGTTGCGAGTACATCAAGAACAACTTCACACCGAGCCCTTTCACAGATCAGGC TGAGTGAGTGGTCGCTCGCTGACCTTGCTTAGTATGGATGCGAAGCGTTTTACGTGGCGTGAAAACTCGC TGAGTAGTTTGTACGATCAGAATGAGCGAAATCGGCGCGACTGTCGTTTGTACCATTAGTATCAATGCGT ATGGCTGTTACCACCC (SEQ ID NO. 29) .
HOST CELLS
The term "host cell" - in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein. The nucleotide of interest may be homologous or heterologous to the host cell.
Thus, a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the enzyme of the present invention. Preferably said nucleotide sequence is carried in a vector for the replication and expression of the nucleotide sequence. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells. Examples of suitable bacterial host organisms are gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentύs, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacϊllus spp. including Lactobacϊllus reuteri, Leuconostoc spp., Pediococcus spp. and Streptococcus spp. Alternatively, strains of a gram-negative bacterial species belonging to Enterobacteriaccae including E. coli, or to Pseudomonαdαceαe can be selected as the host organism.
The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
In contrast to E. coli, Gram positive bacteria from the genus Bacillus, such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, Streptomyces lividans or S. murinus, may be very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria that may be suitable as hosts are those from the genera Streptomyces and Pseudomonas.
Depending on the nature of the nucleotide sequence encoding the enzyme of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are prefened over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected. Typical fungal expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.
Suitable filamentous fungus may be for example a strain belonging to a species of Aspergillus, such as Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp. Cereάlis), or Fusarium sulphur eum (in the perfect state named Gibberella puricaris, synonym with Fusarium trichothercioides, Fusarium bactridioides, Fusarium sambucium, Fusarium roseum and Fusarium roseum var. graminearum), Fusarium cerealis (synonym with Fusarium crokkwellnse) or Fusarium venenatum.
Suitable yeast organisms may be selected from the species of Kluyveromyces, Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae, or Hansenula (disclosed in UK Patent Application No. 9927801.2).
The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
The host cell may be a protease deficient or protease minus strain. This may for example be the protease deficient strain Aspergillus oryzae JaL 125 having the alkaline protease gene named "alp" deleted. This strain is described in WO97/35956.
ORGANISM
The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.
Suitable organisms may include a prokaryote, fungus, yeast or a plant.
The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.
The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof. For example the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter.
TRANSFORMATION OF HOST CELLS/ORGANISM
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. An example of an eukaryotic expression host is Hansenula polymorphia. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
Filamentous fungi cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known. The use of Aspergillus as a host microorganism is described in EP 0 238 023.
Another host organism can be a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food- Industry Hi-Tech March/ April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
General teachings on the transformation of fungi, yeasts and plants are presented in following sections.
TRANSFORMED FUNGUS
A host organism may be a fungus - such as a mold. Examples of suitable such hosts include any member belonging to the genera Phanerochaete, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like - such as Thermomyces lanuginosis, Acremonium chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillinum chrysogenem, Mucor javanious, Neurospora crassa, Trichoderma viridae, Phanerochaete chrysosporium, and the like.
In one embodiment, the host organism may be a filamentous fungus.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting the nucleotide sequence according to the present invention into a construct designed for expression in filamentous fungi.
Several types of constructs used for heterologous expression have been developed. These constructs preferably contain one or more of: a signal sequence which directs the amino acid sequence to be secreted, typically being of fungal origin, and a terminator (typically being active in fungi) which ends the expression system.
Another type of expression system has been developed in fungi where the nucleotide sequence according to the present invention can be fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilise the amino acid sequence. In such a system a cleavage site, recognised by a specific protease, can be introduced between the fungal protein and the amino acid sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the amino acid sequence. By way of example, one can introduce a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli. Such a fusion leads to cleavage in vivo resulting in production of the expressed product and not a larger fusion protein.
Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the nucleotide sequence according to the present invention is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention is equipped with a signal sequence the protein will accumulate extracellularly. With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
Teachings on tiansforming filamentous fungi are reviewed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N. crassa is found, for example in Davis and de Serres, Methods Enzymol (1971) 17A:79-143. Standard procedures are generally used for the maintenance of strains and the preparation of conidia. Mycelia are typically grown in liquid cultures for about 14 hours (25°C), as described in Lambowitz et al, J Cell Biol (1979) 82:17-31. Host strains can generally be grown in either Vogel's or Fries minimal medium supplemented with the appropriate nutrient(s), such as, for example, any one or more of: his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol.
Further teachings on fransforming filamentous fungi are reviewed in US-A-5674707 which states that once a construct has been obtained, it can be introduced either in linear form or in plasmid form, e.g., in a pUC-based or other vector, into a selected filamentous fungal host using a technique such as DΝA-mediated transformation, electroporation, particle gun bombardment, protoplast fusion and the like. In addition, Ballance 1991 (ibid) states that transformation protocols for preparing transformed fungi are based on preparation of protoplasts and introduction of DΝA into the protoplasts using PEG and Ca2+ ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers.
To allow for selection of the resulting transformants, the transformation typically also involves a selectable gene marker which is introduced with the expression cassette, either on the same vector or by co-transformation, into a host strain in which the gene marker is selectable. Various marker/host systems are available, including the pyrG, argB and niaD genes for use with auxotrophic strains of Aspergillus nidulans; pyrG and argB genes for Aspergillus oryzae auxotrophs; pyrG, trpC and niaD genes for Penicillium chrysogenum auxotrophs; and the argB gene for Trichoderma reesei auxotrophs. Dominant selectable markers including amdS, oliC, hyg and phleo are also now available for use with such filamentous fungi as A. niger, A. oryzae, A. ficuum, P. chrysogenum, Cephalosporium acremonium, Cochliobolus heterostrophus, Glomerella cingulata, Fulviafulva and Leptosphaeria maculans (for a review see Ward in Modern Microbial Genetics, 1991, Wiley-Liss, Inc., at pages 455-495). A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
In one aspect, the host organism can be of the genus Aspergillus, such as Aspergillus niger.
A transgenic Aspergillus according to the present invention can also be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong, S.A., Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D., Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). TRANSFORMED YEAST
In another embodiment the transgenic organism can be a yeast.
In this regard, yeast have also been widely used as a vehicle for heterologous gene expression.
By way of example, the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King. et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs may contain a promoter active in yeast, such as a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells may be selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRPl, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
TRANSFORMED PLANTS/PLANT CELLS
A preferred host organism suitable for the present invention is a plant.
In this respect, the basic principle in the constraction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March April 1994 17-27). EP-B-0470145 and CA-A-2006454 provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
The vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.
The nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct. Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well- known and widely employed in the constraction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
In the constraction of a transgenic plant the nucleotide sequence or construct of the present invention may be first constructed in a micro-organism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful micro-organism is E. coll, but other micro-organisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the nucleotide sequence or constract of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or constract of the invention, which DNA is subsequently transferred into the plant cell to be modified.
As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, the pUC series, the Ml 3 mp series, pACYC 184 etc.
In this way, the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E.coli. The E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
After each introduction method of the desired promoter or constract or nucleotide sequence according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al, Crit. Rev. Plant Sci., 4:1-46; and An et al, EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). With this technique, infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the promoter and or the GOI, a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
Other techniques for transforming plants include ballistic transformation, the silicon whisker carbide technique (see Frame BR, Drayton PR, Bagnaall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA & Wang K (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation, The Plant Journal 6: 941-948) and viral transformation techniques (e.g. see Meyer P, Heidmann I & Niedenhof I (1992) The use of cassava mosaic virus as a vector system for plants, Gene 110: 213-217). Teachings on ballistic transformation are presented in following section.
Further teachings on plant transformation may be found in EP-A-0449375.
BALLISTIC TRANSFORMATION OF PLANTS AND PLANT TISSUE
As indicated, techniques for producing transgenic plants are well known in the art. Typically, either whole plants, cells or protoplasts may be transformed with a suitable nucleic acid construct encoding a target DNA (see above for examples of nucleic acid constructs). There are many methods for introducing transforming DNA constructs into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods include Agrobacterium infection (see, among others, Turpen et al, 1993, J. Virol. Methods, 42: 227-239) or direct delivery of DNA such as, for example, by PEG- mediated transformation, by electroporation or by acceleration of DNA coated particles. Acceleration methods are generally preferred and include, for example, microprojectile bombardment.
Originally developed to produce stable transformants of plant species which were recalcitrant to transformation by Agrobacterium tumefaciens, ballistic transformation of plant tissue, which introduces DNA into cells on the surface of metal particles, has found utility in testing the performance of genetic constructs during transient expression. In this way, gene expression can be studied in transiently transformed cells, without stable integration of the gene in interest, and thereby without time-consuming generation of stable transformants.
In more detail, the ballistic transformation technique (otherwise known as the particle bombardment technique) was first described by Klein et al. [1987], Sanford et al. [1987] and Klein et al. [1988] and has become widespread due to easy handling and the lack of pre-treatment of the cells or tissue in interest.
The principle of the particle bombardment technique is direct delivery of DNA-coated micro-projectiles into intact plant cells by a driving force (e.g. electrical discharge or compressed air). The micro-projectiles penetrate the cell wall and membrane, with only minor damage, and the transformed cells then express the promoter constructs.
One particle bombardment technique that can be performed uses the Particle Inflow Gun (PIG), which was developed and described by Finer et al. [1992] and Vain et al [1993]. The PIG accelerates the micro-projectiles in a stream of flowing helium, through a partial vacuum, into the plant cells.
One of advantages of the PIG is that the acceleration of the micro-projectiles can be controlled by a timer-relay solenoid and by regulation the provided helium pressure. The use of pressurised helium as a driving force has the advantage of being inert, leaves no residues and gives reproducible acceleration. The vacuum reduces the drag on the particles and lessens tissue damage by dispersion of the helium gas prior to impact [Finer et al. 1992].
In some cases, the effectiveness and ease of the PIG system makes it a good choice for the generation of transient transformed guar tissue, which were tested for transient expression of promoter/reporter gene fusions. A typical protocol for producing transgenic plants (in particular moncotyledons), taken from U.S. Patent No. 5, 874, 265, is described below.
An example of a method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, non-biological particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming both dicotyledons and monocotyledons, is that neither the isolation of protoplasts nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension. The screen disperses the tungsten-DNA particles so that they are not delivered to the recipient cells in large aggregates. It is believed that without a screen intervening between the projectile apparatus and the cells to be bombarded, the projectiles aggregate and may be too large for attaining a high frequency of transformation. This may be due to damage inflicted on the recipient cells by projectiles that are too large.
For the bombardment, cells in suspension are preferably concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more clusters of cells transiently expressing a marker gene ("foci") on the bombarded filter. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 2 to 3. After effecting delivery of exogenous DNA to recipient cells by any of the methods discussed above, a preferred step is to identify the transformed cells for further culturing and plant regeneration. This step may include assaying cultures directly for a screenable trait or by exposing the bombarded cultures to a selective agent or agents.
An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage, incubating the cells at, e.g., 18°C and greater than 180 μE m"2 s"1, and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
To use the bar-bialaphos selective system, bombarded cells on filters are resuspended in nonselective liquid medium, cultured (e.g. for one to two weeks) and transferred to filters overlaying solid medium containing from 1-3 mg/1 bialaphos. While ranges of 1- 3 mg/1 will typically be preferred, it is proposed that ranges of 0.1-50 mg/1 will find utility in the practice of the invention. The type of filter for use in bombardment is not believed to be particularly crucial, and can comprise any solid, porous, inert support.
Cells that survive the exposure to the selective agent may be cultured in media that supports regeneration of plants. Tissue is maintained on a basic media with hormones for about 2-4 weeks, then transferred to media with no hormones. After 2-4 weeks, shoot development will signal the time to transfer to another media.
Regeneration typically requires a progression of media whose composition has been modified to provide the appropriate nutrients and hormonal signals during sequential developmental stages from the transformed callus to the more mature plant. Developing plantlets are transferred to soil, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO2, and 250 μE m"2 s"1 of light. Plants are preferably matured either in a growth chamber or greenhouse. Regeneration will typically take about 3-12 weeks. During regeneration, cells are grown on solid media in tissue culture vessels. An illustrative embodiment of such a vessel is a petri dish. Regenerating plants are preferably grown at about 19°C to 28°C. After the regenerating plants have reached the stage of shoot and root development, they may be transfened to a greenhouse for further growth and testing.
Genomic DNA may be isolated from callus cell lines and plants to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art such as PCR and/or Southern blotting.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205- 225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
CONSTRUCTION OF PLANT TRANSFORMATION VECTORS:
Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982 ; Bevan et aL, 1983), the bargee, which confers resistance to the herbicide phosphinothricin (White et al., 1990 ; Spencer et al., 1990), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfrgene, which confers resistance to methatrexate (Bourouis et al., 1983), and the EPSPS gene, which confers resistance to glyphosate (U. S. Patent Nos. 4, 940, 935 arid 5, 188, 642).
VECTORS SUITABLE FOR AGROBACTERIUM TRANSFORMATION
Many vectors are available for transformation using Agrobacterium tumefaciens.
These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
pCIB200 and pCIB2001:
The binary vectors pcIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, 1982 ; Bevan et al., 1983 ; McBride et al., 1990). Xhol linkers are ligated to the EcoRVfragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable noslnptll chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the Xhol digested fragment are cloned into Sail-digested pTJS75kan to create ρCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites : EcoRl, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRl, Sstl, Kpnl, Bglll, Xbal, Sail, MM, Bell, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacteria kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived M4 function for mobilization between E. coli and other hosts, and the OriTand OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulator signals. pCIBlO and Hygromycin Selection Derivatives thereof :
The binary vector pCIBlO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range . plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (1987). Various derivatives of pCIBlO are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al., 1983). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
VECTORS SUITABLE FOR NON-AGROBACTERIUM TRANSFORMATION
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e. g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non- Agrobacterium transformation is described, a. pCIB3064 : pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al., 1987). This generated pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlil, and BamHl. This vector is suitable for the cloning of plant expression cassettes containing their own regulator signals, b. pSOG19 and pSOG35 : pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGlO. A 250-bp fragment encoding the E. colidihydrofolate reductase type 11 gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the opaline synthase terminator.
Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the opaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlil, Sphl, Pstl and EcoRl sites available for the cloning of a foreign gene.
TRANSFORMATION
Once the gene sequence, of interest has been cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.
TRANSFORMATION OF DICOTYLEDONOUS PLANTS
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., 1984 ; Potrykus et al., 1985 ; Reich et al., 1986 ; and Klein et al., 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e. g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e. g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al., 1993). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, 1988).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4, 945, 050, 5, 036, 006, and 5, 100, 792. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e. g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
TRANSFORMATION OF MONOCOTYLEDONOUS PLANTS
Transformation of most monocotyledon species has now also become routine.
Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i. e. cofransformation) and both these techniques are suitable for use with this invention. Cofransformation may have the advantage of avoiding complete vector constraction and of generating transgenic plants with unlined loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., 1986). Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, fransformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (1990) and Fromm et al. (1990) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (1993) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1. 5-2. 5 mm length excised from a maize ear 14-15 days after pollination and a PDS-lOOOHe Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated fransformation has been described for Japonica-types and Indica-types (Zhang et al., 1988 ; Shimamoto et al., 1989 ; Datta et al., 1990). Both types are also routinely transformable using particle bombardment (Christou et a/., 1991). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the fransformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (1992) using particle bombardment into cells of type C long- term regenerable callus, and also by Vasil et al. (1993) and Weeks et al. (1993) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0. 75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, 1962) and 3 mg/1 2, 4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i. e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene- carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics helium device using a burst pressure of- 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/1 basta in the case of pCIB3064 and 2 mg/1 methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as"GA7s "which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
Transformation of banana is taught in PCT/US98/14661. Transformation of monocotyledons using Agrobacterium has also been described, see for example WO 94/00977 and U. S. Patent No. 5, 591, 616.
BREEDING
The plants obtained via transformation with a gene of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R. (1981) ; Wood D. R. (Ed.) (1983) ; Mayo 0. (1987) ; Singh, D. P. (1986) ; and Wricke and Weber (1986). Plant breeding can be used to produce germplasm containing frangenes inserted from different transformation events, and hence can be used to produce plants co-expressing both a pyranosone dehydratase gene and a glycan lyase gene.
The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to confrol weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.
Use of the advantageous genetic properties of the transgenic plants and seeds according to the present invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well- defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic"equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
SEED PRODUCTION
In seeds production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD@), methalaxyl (Apron@), and pirimiphos-methyl (Actellic@). If desired, these compounds are formulated together with further carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit. It is a further aspect of the present invention to provide new agricultural methods, such as the methods examplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to. exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.
DISEASE RESISTANCE EVALUATION
Disease resistance evaluation is performed by methods known in the art. See, Uknes et al. (1993) ; Grlach et al. (1996) ; Alexander et al. (1993). For example, a disease resistance assay for powdery mildew is described below. Powdery Mildew Inoculation and Assessment
A small population of tomato plants infected with powdery mildew is maintained separately and used as the source of inoculum. Conidia from these plants is used to infect six-week old healthy confrol and transgenic plants. Plant leaves infected with conidia of Erysiphe polygoni were excised and shaken above the top of the test plants to allow conidia to fall onto the healthy leaves (Fletcher & Smewin, 1988, Plant Pathol. 37: 594-598). The temperature of the greenhouse is maintained at 25 to 30 DEG C. and the humidity at above 70%. The percentage of leaf area of the inoculated plants covered by conidia or necrotic lesions is recorded.
Germination of Spores of Powdery Mildew
The effect of fatty acids on the germination of E. polygoni spores and germ tube growth is evaluated by germinating the spores in the presence of various fatty acids. Recently colonized leaves from control plants is flicked over petri dishes containing 0.7% agar and selective fatty acid. Attempts are made to allow conidia to distribute evenly on the agar surface. The petri dishes is then incubated at 25 DEG C. in the dark for 12 hr. Rates of germination is determined with the aid of a microscope and recorded at the end of 12 h.
Resistance to Powdery Mildew
The presence and number of whitish powdery spots, an indication of powdery mildew, present on the older leaves of both the confrol and fransgenic plants is recorded one week after the inoculation. The colonies are found mostly on the upper surface of the leaves but a few also appeared on the lower surface and on the petioles and stems.
CULTURING AND PRODUCTION
Host cells transformed with the nucleotide sequence may be cultured under conditions conducive to the production of the encoded enzvme and which facilitate recovery of the enzyme from the cells and/or culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
The enzyme may be secreted from the host cells and may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
SECRETION
Often, it is desirable for the enzyme to be secreted from the expression host into the culture medium from where the enzyme may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.
Typical examples of heterologous secretion leader sequences are those originating from ill
the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the α-amylase gene (Bacillus).
DETECTION
A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the POI may be used or a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1).
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting the amino acid sequence include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled nucleotide. Alternatively, the NOI, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WT), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3, 850,752; US-A-3,939,350; US-A- 3,996,345; US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241. Also, recombinant immunoglobulins may be produced as shown in US-A-4,816,567. Additional methods to quantitate the expression of the amino acid sequence include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a confrol nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a specfrophotomefric or calorimetric response gives rapid quantitation.
Although the presence/absence of marker gene expression suggests that the nucleotide sequence is also present, its presence and expression should be confirmed. For example, if the nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing nucleotide sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a nucleotide sequence under the confrol of the promoter of the present invention or an alternative promoter (preferably the same promoter of the present invention). Expression of the marker gene in response to induction or selection usually indicates expression of the amino acid sequence as well.
Alternatively, host cells which contain the nucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip- based technologies for the detection and/or quantification of the nucleic acid or protein.
FUSION PROTEINS
The amino acid sequence of the present invention may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-fransferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.
The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
ADDITIONAL POIs
The sequences of the present invention may be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NOIs).
Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, α-galactosidases, β-galactosidases, α-glucanases, glucan lysases, endo-β- glucanases, gluc amylases, glucose oxidases, α-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, upases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno- galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: O2-oxidoreductase, EC 1.1.3.5) or combinations thereof or contain sense and antisense sequences. The NOI may even be an antisense sequence for any of those sequences.
The POI may even be a fusion protein, for example to aid in extraction and purification.
Examples of fusion protein partners include the maltose binding protein, glutathione-S- fransferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion components.
The POI may even be fused to a secretion sequence. Examples of secretion leader sequences are those originating from the amyloglucosidase gene, the α-factor gene, the α-amylase gene, the lipase A gene, the xylanase A gene.
Other sequences can also facilitate secretion or increase the yield of secreted POI. Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198.0.
The NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference. By way of further example, the NOI may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
The NOI may include within it synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the NOI may be modified by any method available in the art. Such modifications may be carried out in to enhance the in vivo activity or life span of the NOI.
The NOI may be modified to increase infracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
In one aspect, the present invention provides transformed plants that co-express the polypeptide according to the present invention and with other antifungal, antibacterial, or antiviral pathogenesis-related peptides, polypeptides, or proteins; insecticidal proteins, e.g., Bacillus thuringiensis (B.t.) proteins; or proteins involved in improving the quality of plant products or agronomic performance of plants. Simultaneous co-expression of multiple antifungal proteins in plants is advantageous in that it exploits more than one mode of action to control fungal damage. This can minimize the possibility of developing resistant fungal strains, broaden the scope of resistance, and potentially result in a syngergistic antifungal effect, thereby enhancing the level of resistance (see WO 92/17591, for example).
Non-limiting examples of DNAs that can be co-expressed along with DNAs encoding the polypeptides of the present invention include 1) DNAs encoding enzymes such as: glucose oxidase (which converts glucose to gluconic acid, concomitantly producing hydrogen peroxide which confers broad spectrum resistance to plant pathogens); pyruvate oxidase; oxylate oxidase; cholesterol oxidase; amino acid oxidases; and other oxidases that use molecular oxygen as a primary or secondary substrate to produce peroxides, including hydrogen peroxide; 2) pathogenesis related proteins such as SAR8.2a and SARB.2b proteins; the acidic and basic forms of tobacco PR-la, PR-lb, PR-lc, PR-1', PR-2, PR-3, PR-4, PR-5, PR-N, PR-O, PR-O', PR-P, PR-Q, PR-S, and PR-R proteins; chitinases such as tobacco basic cbitinase and cucumber cbitinase/lysozyme; peroxidases such as cucumber basic peroxidase; glucanases such as tobacco basic glucanase; osmotin-like proteins; 3) viral capsid proteins and replicases of plant viruses; 4) plant R-genes (resistance genes), such as Arabidopsis RPS2 (Bent et al. (1994) Science 265:1856-1860), Arabidopsis RPM1 (Grant et al. (1995) Science 269:843-846), tobacco N-gene and N-gene (Whitham et al. (1994) Cell 78:1101-1115), tomato Cf-9 (Jones et al. (1994) Science 266:789-793), flax L@6 (Ellis et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 4185), and rice Xa21 (Song et al. (1995) Science 270: 1804-1806). These genes can be expressed using constitutive promoters, tissue-specific promoters, or promoters inducible by fungal pathogens or other biological or chemical inducers; 5) pathogen Avr genes, such as Cladosporium fulvum Avr9 (Van Den Ackerveken et al. (1992) Plant J. 2:359), that can be expressed using pathogen- or chemical-inducible promoters; and 6) genes that are involved in the biosynthesis of salicylic acid, such as benzoic acid 2-hydroxylase (Leon et al. (1995) Proc. Natl. Acad. Sci. USA 92:10413-10417).
A number of publications have discussed the use of plant and bacterial glucanases, chitinases, and lysozymes to produce transgenic plants exhibiting increased resistance to various microorganisms such as fungi. These include EP 0 292 435, EP 0 290 123, WO 88/00976, U.S. Pat. No. 4,940,840, WO 90/07001, EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304, EP 0 418 695, EP 0 448 511, and WO 91/06312. The use of osmotin-like proteins is discussed in WO 91/18984.
PATHOGENS AND DISEASES
Suitably, the host (or transgenic) organism or part thereof according to the present invention is resistant to one or more pathogens, which pathogens are capable of causing one or more diseases in or on the host organism or part thereof.
When the host (or transgenic) organism or part thereof according to the present invention is a plant or part thereof, the plant or part thereof may be resistant to one or more of the following diseases: Bacterial leaf blight, Bacterial mosaic, Bacterial sheath rot, Basal glume rot, Black chaff (bacterial streak), Spike blight (gummosis), leaf spot, blights, cankers, galls. Suitably, when the host (or transgenic) organism or part thereof according to the present invention is a plant or part thereof, the plant or part thereof may be resistant to one or more of the following: Pseudomonas spp., Clavibacter spp., Xanthomonas spp., Rathayibacter spp., Corynebacterium spp., Erwinia spp. Agrobacterim spp., Xylella spp.
In one embodiment, when the host (or transgenic) organism or part thereof according to the present invention is a plant or part thereof, preferably the plant or part thereof produces a fungicidal or fungistatic compound which is effective against one or more of the fungal plant diseases: Downey mildew, powdery mildew, grey mould, canker, black scurf, rots, soft rot, fruit rot, basal, rot, crown rot, root rot, rusts, stem rusts, stripe rusts, blight, early blight, late blight, pithium blight, leaf spot, wilt, leaf blotch, glume blotch, black leg and black sigitoka.
Suitably, the host (or fransgenic) organism or part thereof according to the present invention produces a fungicidal or fungistatic compound which is effective against a fungus from one or more of the following fungal genera: Alternaria spp , Albugo spp. Aphanomyces spp , Amyloporia spp., Ascochyta, Aspergillus, Basidiophora, Bipolaris, Botrytis, Bremia, Cercospora, Cladosporium, Claviceps, Coniophora spp. Colletotrichum, Diplodia, Diplocarpon spp., Donkioporia spp., Drechslera, Erysiphe, Eutypa, Fϊbroporia spp., Fusarium, Gaeumanomyces, Geotrichum, Guignardia, Gymnosporangium, Helmintosporium, Hemileia, Kabatiella, Leptosphaeria, Macrophomina, Marssonina spp., Monilinea, Merulus, Mycosphaerella, Nectria, Paracercospora, Penicillium, Peronophythora, Peronospora, Phellerias spp., Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmophora, Podosphaera, Porai spp. Pseudocercosporella, Pseudoperonospora, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Rhizopus, Sclerophthora, Sclerotinia, Sclerotium, Septoria, Serpula spp., Sphaerotheca, Stagonospora, Taphrina, Thielaviopsis, Tilletia, Trichoderma, Uncinula, Ustilago, Venturia and Verticillium.
In one aspect the host (or transgemc) organism or part thereof (in particular a plant or part thereof) according to the present invention is resitant to one or more oomycete fungi selected from Pythium, Aphanomyces spp. Peronospora spp., Phytophthora spp., Albugo spp., Basidiophora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp. and Peronophythorα spp..
In one aspect the host (or fransgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Pythium αcαnthicum, P. αcαnthophoron, P. αphαnidermαtum, P. αquαtile, P. αristosporum, P. αrrhenomαnes, P. bifurcαtum, P. buismαniαe, P. butleri , P. cαmpαnulαtum, P. cαnαriense, P. cαrbonicum, P. cαroliniαnum, P. cαtenulαtum, P. chαmαehyphon , P. citrinum, P. colorαtum, P. contiguαnum, P. cylindrosporum, P. Debαryαnum, P. Deliense, P. destruens, P. diclinum, P. dimorphum, P. Dissotocum, P. echinulαtum, P. erinαceum, P. glomerαtum, P. grαminicolα, P. grαndisporαngium, P. helicoides, P. heterothαllicum, P. hydnosporum, P. inflαtum, P. insidiosum, P. intermedium, P. irregular , P. iwagamai, P. jasmonium, P. longandrum, P. macrosporum, P. mamillatum, P. mastophorum, P. megacarpum, P. megalacanthum, P. middletonii, P. monospermum, P. montanum, P. myriotylum, P. nodosum, P. nunn, P. oedochilum, P. okanoganense, P. oligandrum, P. ornacarpum, P: prthogonon, P. ostracodes, P. pachycaule, P. pachycaule var. ramificatum, P. paddicum, P. paroecandrum, P. pectinolyticum, P. periϊlum, P. periplocum, P. perplexum, P. polymastum, P. porphyrae, P. prolatum, P. proliferatum, P. pulchrum, P. ramificatum, P. regulare, P. rhizosaccharum, P. rostratum, P. salpingophorum, P. segnitium, P. spinosum, P. splendens, P. sulcatum, P. sylvaticum, P. terrestris, P. torulosum, P. tracheiphilum, P. ultimum, P. ultimum var. sporangiiferum, P. ultimum var. ultimum, P. uncinulatum, P. undulatum, P. vanterpoolii, P. aff. vanterpoolii, P. vexans, P. violae, P. volutum, P. zingiberum, and P. sp. Abappressorium.
In one aspect the host (or fransgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Aphanomyces astaci, A. cochlioides, A. euteiches, A.. helicoides, A. invadans, A. laevis, A. piscicid , A. stellatus, A. sp. 84-1240, A. sp. AR_11, A. sp. TF33 and A. sp. TF5. A. euteiches, which cause Aphanomyces root rot in pea and alfalfa In one aspect the host (or transgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Rhizoctonia cerealis, R. oryzae-sativae, R. solani, Rhizina undulata, Rhizoctonia leguminicola, Rhizoctonia zeae, Rhizoctonia repens, Rhizoctonia crocorum, Rhizoctonia muneratii and Rhizoctonia stahlii.
In one aspect the host (or transgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Cercospora apii, C. asparagi, C. beticola, C. canescens, C. caricis, C. cqffeicola, C. hayi, C. kalmiae, C. kikuchii, C. nicotianae, C. piaropi, C. sojina, C. violae, C. zeae-maydis, C. zebrina and C. sp. PoGDS8-2.
In one aspect the host (or transgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to a fungi selected from one or more of Oomycete plant fungal diseases:
Genera Example of disease
Pythium Root rot and damping off in many important crop plants. P. sulcatum causes cavity spot in carrots Aphanomyces A. euteiches causes Aphanomyces root rot in pea and alfalfa. A cochlioides in sugar beet Peronospora Downy mildew in many important crops
Phytophthora Late Blight in potato, tomato, eggplant
Albugo White rust on beet, cabbage, cauliflower, horseradish, kale and the like. Basidiophora Downy mildew
Bremia Downy mildew in lettuce
Plasmopara Downy mildew in grapes
Pseudoperonospora Hop Downy mildew Peronophythora P. litchii in Litchi In one aspect the host (or fransgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resitant to one or more of the following seed and seedling diseases:
Pythium sp. Widespread: Root rot diseases (i.e. Root rot in soybean, Rice Seedling Blight etc.)
P. ultimum in beet (Causing Damping off and root rot) P. ultimum (other crops) P. aphanidermatum (Pythium blight in turfs) P. graminicola (Pythium blight in turf)
Rhizoctonia sp. Widespread: Root rot diseases
R. solani in beet (Damping off, root rot and violet disease) R. solani (i.e., Black scurf in potatoes; Sheath Blight of Rice)
Aphanomyces sp. Widespread: Root rot diseases
A. cochlioides in beet (Damping off and root rot) A. euteiches (pea, alfalfa)
Fusarium sp. (Widespread: Root rot diseases, Causing fusariose (Fusarium wilt) in many different crops)
F. oxysporum (i.e., Fusarium root rot in soybeans, conifers, Spinach
Fusarium wilt)
F. solani (Sudden Death Syndrome, soybean)
Sclerotinia sp.
S. sclerotiorium (i.e., white mould (stem rot) in soybean)
Sclerotium sp.
S. rolfsii (Sclerotium rot, sugar beet)
Phytophthora sp. P. megasperma (Stem rot in soybean)
Cercospora sp.
C. kikuchii (Purple seed stain in soybean) C. beticola (Leaf spot, sugarbeet)
Phoma sp.
P. betae in beet,
P. lingam (oil seed rape)
Alternaria sp.
A. solani (Early Blight, tomato)
A. dauci (Early Blight, tomato, carrot)
A. radicina (Early Blight, carrots)
A. triticina (Alternaria Leaf Blight, Wheat)
Thielaviopsis sp.
T. basicola (Black Root Rot in tobacco and many other crops: Vinca)
U. tritici (Loose Smut of Wheat) Gaeumannomyces graminis (Take All in Wheat) Albugo Candida (White Rust, radish)
Bipolaris sp.
B. sorokiniana (Spot Blotch in Barley; Root Rot in wheat)
In one aspect the host (or transgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention may be a transgenic potato which is resistant to Black scurf (stem canker) caused by Rhizoctonia solani. In one aspect the host (or transgenic) organism or part theroef (in particular a plant or part thereof) according to the present invention is resistant to one or more of the following foliar and other diseases:
Botrytis sp. (Attack many different plants)
B. cinerea (i.e. Botrytis Blight, Bunch Rot and Grey Mould of grape, Kiwifruit Grey Mould, Douglas fir grey mould)
B. squamosa (Leaf Blight in onion)
Colletrotrichum sp.
C. sublineolum (formerly C. graminicola; Anthracnose Leaf Blight, corn, sorghum)
Kabatiella zeae (Eyespot, corn)
Puccinia sp. (Rust diseases, especially important in cereals) P. sorghi (Common Rust, corn) P. hordei (Brown rust/Leaf Rust, Barley) P. poae-nemoralis and P. poarum (Rust) P. striiformis (Yellow rust/Stripe Rust, Wheat) P. horiana (White Rust of chysanfhemum) P. graminis f.sp. triticale (Stem rust, Wheat)
Drechslera sp.
D. teres (Net Blotch, barley)
D. graminea (Leaf Stripe, Barley)
Erysiphe sp. (Powdery Mildew)
E. graminis f. sp. hordei (Powdery Mildew, Barley/Wheat) = Blumeria graminis
E. betae (Powdery Mildew, Beet) Ustilago sp. (Smut fungi - very widespread) U. maydis (Corn Smut) U. nuda (Loose Smut, Barley) U. tritici (Loose Smut of Wheat) U. scitaminea (Sugar Cane Smut)
Claviceps purpurea (Ergot, cereals, grasses)
Tilletia sp.
T. caries (Bunt of Wheat)
Septoria sp. and Staganospora sp.
S. nodorum (Leaf Blotch, wheat)
S. tritici (Leaf Blotch, wheat)
S. avenae f.sp. triticea (Leaf Blotch, wheat)
Phytophthora sp.
P. infestans (Late Blight, Very important pathogen of Potato/Tomato/Eggplants).
Pseudocercosporella herpotrichoides (Eyespot, Cereals)
Pseudoperonospora sp.
P. cubensis (Downy Mildew, cucumber)
Sphaerotheca sp.
S.fuliginea (Powdery Mildew, cucumber) S. pannosa (Powdery Mildew, Roses)
Cercospora sp.
C. maydis (Gray leaf spot, Corn) Helmintosporium sp.
H. maydis (gives Helmintosporium Leaf Blight in Corn) H. carbonum (gives Helmintosporium Leaf Blight in Corn) H. turcicum ( gives Helmintosporium Leaf Blight in Corn) H. sativum (Spot Blotch, Wheat, Barley)
Pyrenophora
P. triticirepensis (Tan spot, Wheat)
Fusarium sp.
F. oxysporum (Fusarium Wilt in Banana and many other species)
Mycosphaerella sp.
M. musicola (Banana Leaf spot, Sigatoka disease)
M. brassicicola (Black Blight in cabbage, cauliflower, Brassels sprouts, broccoli)
Phomopsis sp.
P. viticola (Cane and Leaf Spot in grape)
Sphaerotheca sp.
S. macularis (Powdery Mildew in Strawberry, Raspberry, Blackberry)
Verticillium sp. (Verticillium wilt)
V. albo-atrum (Wilt in alfalfa, tomato, hop)
Gymnosporangium sp. (i.e. apple rust)
Hemileia vastatrix (Coffee Rust)
Guignardia bidwellii (Black Rot, Grape) Taphrina sp. (Leaf curl diseases)
Pyricularia oryzae (Rice blast)
In one aspect the host (or transgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention is resistant to microbial attack even post harvest. Preferably, the host (or transgenic) organism or part thereof is resistant to diseases which attack fruits and vegetables, such as one or more of the fungi selected from the following: Cladosporium sp, Rhizopus rot, Penicillium expansum (Blue mould) Alternaria rot, Botrytis cinerea (Grey Mould), Aspergillus niger (Black mold in many fruit and vegetable species, i.e. onion) Monϊlinia sp. (Brown rot), Fusarium sp.. (Pink or Yellow Molds), Geotrichum (sour rots) or one or more of the bacteria selected from Pseudomonas syringae (responsible for a number of economically important diseases in a wide variety of fruits and vegetables, and in ornamental plants - Bacterial Blight, Flower Blast, Necrotic Leaf Spots, Spots and blisters on fruits), Erwinia carotovora (Erwinia Soft Rot in many vegetables), Xanthomonas sp. (Bacterial Leaf spots in many vegetables), X. campestris ^causes Walnut Blight).
In one aspect the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention is resistant to bacterial infection/contamination, suitably gram positive bacteria or gram negative bacteria.
In one aspect the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention, in either living or dead form, or a crude or purified extract therefrom, can be used as a antimicrobial agent (for example an antifungal or anti-bacterial agent) for use in preventing or reducing microbial contamination in and/or on a second organism or a product/composition derived therefrom, for example to reduce or eliminate microbial contamination in a food stuff.
In one aspect, the host (or transgenic) organism or part thereof according to the present invention is a fungus which is resistant to one or more diseases, such as those selected from: bacterial or fungal diseases which occur in mushrooms for example. That is to say, those diseases caused by the following bacteria for example: Pseudomonas tolaasii, P. cepacia, Burkholderia gladioli. Also the fungus Trichoderma can cause problems in mushroom farming.
In one aspect the host (or transgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention is resistant to one or more of the following turf grass diseases: Anthracnose and Basal Rot (Colletotrichum graminicold), Blister Smut (Entyloma dactylidis), Brown Patch (Rhizoctonia solani), Damping-off (Pythium, Fusarium, Helminthosporium and Rhizoctonia spp.), Dollar Spot (Sclerotinia homoeocarpa), Fairy Ring (Lycoperdon, Psalliota, Clitocybe species and others), Fairy Ring (Marasmius oreades), Leaf Spots, Blight, Foot Rots and Melting-out (Drechslera spp.), Powdery Mildew (Erysiphe graminis), Red Thread (Laetisaria fuciformis), Rust (Puccinia spp.), Slime Molds (usually Physarum cinereum), Snow Mold, Cottony or Ltb/Sltb Snow Molds (Coprinus psychromorbidus), Snow Mold, Gray or Speckled (Typhula spp.), Snow Mold, Pink or Fusarium and Microdochium Patch (F. nivale and M. nivale), Snow Scald (Myrioscierotinia borealis or Scierotinia borealis), Stripe Smut (Ustilago striiformis), Take-all or Ophiobolus Patch (Gaeumannomyces graminis var. avenae), Pythium blight (Pithium spp.).
Fungal species can grow and reproduce in most natural, domestic and industrial environments, and can cause significant loss of yield and quality to plant crops, result in considerable damage to industrial and personal property, and cause ill health, disease and allergies in humans and animals.
The control of fungal contamination can be achieved via use of fungicides in addition to other fungal management measures. For example, in the domestic and industrial environments, fungal contaminations can, in many instances be reduced by ensuring good ventilation to avoid the build up of fungal spores and ensure appropriate control of moisture. However, in modern domestic and industrial property, the. widespread use of heating, and poor ventilation, results in a environment that is ideal for fungal contamination and growth. Therefore the indoor environment is a creation of the modern era. Previously, buildings were notable for the extent to which they were reall open to the outside air, a system that could be referred to as natural ventilation. But, technological advances have permitted us to seal buildings tightly, recirculate the air within them, and fill them with a variety of particle- and chemical-emitting materials and objects. Shelton, B.G. Kirkland, K.H. Flanders, W.D. Morris, G.K. Profiles of airborne fungi in buildings and outdoor environments in the United States. Applied and Environmental Microbiology 68, 1743 - 1753 (2002).
Therefore, there is increasing awareness and concern over the rise in fungal related disorders caused by fungal mycotoxins, allergies to fungal spores, and pathological disease caused by human infection (particularly in immune compromised individuals). In addition fungal contamination is highly unsightly, can damage the structural integrity of surfaces, furnishings, materials, carpets, wooden flows, paints and wall papers, plaster, masonry, etc. and even cause major structural damage, particularly in wood frames properties.
In one aspect the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention can be used to prepare raw materials for application in domestic and industrial environments. By way of example only, transgenic trees of the invention can be used to prepare timber, paper and other raw or stractural materials which are less prone to microbial contamination, and/or transgenic cotton can be prepared to produce fabric which is less prone to microbial contamination.
In one aspect the host (or transgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention can be used to prevent and/or treat one or more of the following domestic or industrial problems: Wet and dry rots (also known as 'building rot'):
Merulius lacrymans (Serpula lacrymans) - dry rot; Coniophporan cerebella arid C. puteanaw (brown rot), Poria incrassata - Wet rot; Amyloponia xantha, Fϊbroporia vaillanti, Poria placenta, Phellinas contignus, Donkioporia expansa, Asterostrama sp., Paxillus pansuides, white rot. Mildew and Moulds:
Many fungal species are encompassed by the common terms, 'mildew' and 'mould' or 'house fungus' and include: Aspergillus sp., Penicillium sp. Aspergillus niger, Rhizopus sp. Stachybotrys chartarum (Black mould), Alternaria, sp. and Cladosporium sp¬
in one aspect the host (or transgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention may be used to prevent and/or confrol food and/or feed contamination by microorganisms.
The term "foodstuffs" as used herein means both food and feed, including raw and processed plant material and non plant material.
In one aspect the host (or fransgenic) organism or part thereof (in particular a plant or part thereof) according to the present invention can be used directly in the preparation of food and feed, or as a preservative of foodstuffs, including parts of plants, and products derived from such plants. Alternatively, the antimicrobial compound produced by the transformed organisms, in either purified or crade form, may be added to foodstuffs.
In one aspect the host (or transgenic) organism or part thereof according to the present invention can be used as an anti-microbial either by direct application of the living or dead organisms, for example a transgenic plant, yeast, filamentous fungi or bacterial preparation, or by preparing a crude extract preparation containing the anti-microbial compound of the present invention produced by the transgenic organisms, or a purified extract of said anti-microbial compound from said fransgenic organism. The anti-microbial preparations may be used in a wide range of products, for example as a preservative, to prevent microbial spoilage and contamination applications in food and feed applications, as a anti-microbial food processing agent, as an ingredients in cosmetics, personal hygiene products, oral health products, as treatment to protect plant propogative material, such as a seed treatment, as a tuber treatment.
ANTIFUNGAL COMPOSITIONS
The present invention further provides an antifungal composition, comprising an antifungal effective amount of an isolated polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NO:2-17, and an acceptable carrier. The antifungal composition can be used for inhibiting the growth of, or killing, pathogenic fungi. These compositions can be formulated by conventional methods such as those described in, for example, Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, Second Edition, Marcel Dekker, N.Y.; and K Martens (1979) Spray Drying Handbook, Third Edition, G. Goodwin, Ltd., London. Necessary formulation aids, such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art, and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J., and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich. Using these formulations, it is also possible to prepare mixtures of the present antifungal polypeptide with other pesticidally active substances, fertilizers and/or growth regulators, etc., in the form of finished formulations or tank mixes.
Antifungal compositions contemplated herein also include those in the form of host cells, such as bacterial and fungal cells, capable of the producing the present antifungal polypeptide, and which can colonize roots and/or leaves of plants. Examples of bacterial cells that can be used in this manner include strains of Agrobacterium, Arthrobacter, Azospyrillum, Clavibacter, Escherichia, Pseudomonas, Rhizobacterium, and the like. Numerous conventional fungal antibiotics and chemical fungicides with which the present antifungal polypeptide can be combined are known in the art and are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council. These include, for example, polyoxines, nikkomycines, carboxyamides, aromatic carbohydrates, carboxines, morpholines, inhibitors of sterol biosynthesis, and organophosphoras compounds. Other active ingredients which can be formulated in combination with the present antifungal polypeptide include, for example, insecticides, attractants, sterilizing agents, acaricides, nematocides, and herbicides. U.S. Pat. No. 5,421,839 contains a comprehensive summary of the many active agents with which substances such as the present antifungal polypeptide can be formulated.
GENOMIC PROBES
In another aspect, the present invention provides oligonucleotide hybridization probes useful in screening genomic and other nucleic acid libraries for DNA sequences encoding peptides, polypeptides, and proteins having pyranosone dehydratase activity, which probes can be designed based on the sequences provided herein. Such probes can range from about 16 to about 28 nucleotides in length, generally about 16 nucleotides in length, more typically about 20 nucleotides in length, preferably about 24 nucleotides in length, and more preferably about 28 nucleotides in length. Preferably, these probes specifically hybridize to genomic DNA and other DNA sequences encoding peptides, polypeptides, or proteins having the same or similar activity as that of the pyransone dehydratase enzyme.
Such oligonucleotide probes can be synthesised by automated synthesis, and can be conveniently labelled at the 5' end with a reporter molecule such as a radionuclide, e.g., @32 P, or biotin. The library can be plated as colonies or phage, depending upon the vector employed, and the recombinant DNA is transferred to nylon or nitrocellulose membranes. Following denaturation, neutralization, and fixation of the DNA to the membrane, the membrane is hybridized with the labeled probe. Following this, the membrane is washed, and the reporter molecule detected. Colonies or phage harboring hybridizing DNA are then isolated and propagated. Candidate clones or PCR-amplified fragments can be verified as comprising DNA encoding pyranosone dehydratase or related peptides, polypeptides, or proteins having antifungal activity the same as or similar to that of pyranosone dehydratase by a variety of means. For example, the candidate clones can be hybridized with a second, non-overlapping probe, or subjected to DNA sequence analysis. The pyranosone dehydratase of the peptide, polypeptide, or protein encoded thereby can be assessed by cloning and expression of the DNA in an appropriate host such as yeast or E. coli, followed by isolation of the peptide, polypeptide, or protein, and assay of the pyranosone dehydratase activity thereof by the method described in Example 8. By such means, plant nucleic acids encoding pyranosone dehydratase, or peptides, polypeptides, or proteins biologically functionally equivalent thereto, useful in controlling undesired fungi and protecting plants against fungal pathogens can be isolated.
PCR PRIMERS
Biologically functional equivalent genomic DNAs and cDNAs can be isolated from organisms including higher plants using degenerate oligonucleotide primers based on the sequence (SEQ ID No. 1 or SEQ ID No. 20) of pyranosone dehydratase (T. Compton (1990) In Innis et al., Eds., PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego, pp. 39-45). Such degenerate oligonucleotide primers can be used in conjunction with PCR technology employing reverse transcriptase to amplify biologically functionally equivalent cDNAs (E. S. Kawasaki (1990) In Innis et al., Eds., PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego, pp. 21-27). These cDNAs can then be easily cloned in appropriate transformation/expression vectors and introduced into monocots and dicots, and transformed plants expressing the DNAs in these vectors can be isolated using established procedures as discussed below.
The degenerate oligonucleotides can be used to screen genomic libraries directly, and the isolated coding sequences can be transferred into transformation/expression vectors for crop plants.
Alternatively, the degenerate oligonucleotides can be used as probes to screen cDNA libraries from plants in, for example, λ-phage vectors such as λ-Zapil (Stratagene, La Jolla, Calif). The cDNA isolated in this manner can be transferred to an appropriate transformation/expression vector for introduction into monocot or dicot plants as described below.
ANTIBODIES
One aspect of the present invention relates to amino acid sequences that are immunologically reactive with one or more of the amino acid sequences of claim 1.
Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with the sequence of the present invention (or a sequence comprising an immunological epitope thereof). Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the sequence of the present invention (or a sequence comprising an immunological epitope thereof) contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
Monoclonal antibodies directed against the sequence of the present invention (or a . sequence comprising an immunological epitope thereof) can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct fransformation of B lymphocytes with oncogenic DNA, or fransfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
Monoclonal antibodies to the sequence of the present invention (or a sequence comprising an immunological epitope thereof) may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the substance specific single chain antibodies.
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
Antibody fragments which contain specific binding sites for the substance may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulphide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD etal (1989) Science 256:1275-128 1).
LARGE SCALE APPLICATION
In one preferred embodiment of the present invention, the amino acid sequence is used for large scale applications.
Preferably the amino acid sequence is produced in a quantity of greater than O.lg/litre, preferably greater than 0.2g/litre, more preferably greater than 0.3 g/lifre, more preferably greater than 0.5 g/litre, even more preferably from lg per litre to about 2g/litre, and in some instances greater than 02g/litre of the total cell culture volume after cultivation of the host organism.
Preferably the amino acid sequence is produced in a quantity of from lOOmg per litre to about 900mg per lifre of the total cell culture volume after cultivation of the host organism.
Preferably the amino acid sequence is produced in a quantity of from 250mg per lifre to about 500mg per lifre of the total cell culture volume after cultivation of the host organism.
SUMMARY
In summation, the present invention relates to a nucleotide sequence and, also to a construct comprising the same. The invention also relates to new uses of a known enzyme.
The invention is further illustrated in the following non-limiting examples, and with reference to the following figures wherein:
Figure 1 shows the electrophoresis of PD1 (pyranosone dehydratase isoform 1) on gels of 8-25% gradient. In more detail, Figure 1 A, shows SDS-PAGE: Lanes 1 and 2 (from left) protein markers from Novex and Pharmacia respectively, Lanes 3, 4 and 5, purified PD1. Figure IB, shows Native PAGE: Lanes 1, 2, and 3, purified PD1, Lane 4, protein markers from Pharmacia, Lane 5, partially purified. The gels were stained with PhastGel Blue R from Pharmacia.
Figure 2 shows partial amino acid sequences of pyranosone dehydratase.
Figure 3 A illustrates the use of 1,5-anhydro-D-fructose and pyranosone dehydratase for the production of microthecin. The reaction mixture consisted of 1,5-Anhydro-D- fructose 5μl (3.0%), pyranosone dehydratase preparation 5μl, 65μl sodium phosphate buffer (pH 6.0) and water to a final volume of 0.7 ml. The reaction was monitored by scanning between 350-190 nm. Reaction time at zero min was used as blank. The absorbance peak at around 230 nm indicates the formation of microthecin. The absorbance at 265 nm indicate the first formation of an intermediate from AF before it converts to microthecin.
Figure 3B illustrates the production of microthecin and its intermediate. The reaction mixture consisted of lOμl partially purified pyranosone dehydratase (a ammonium sulfate fraction between 25-50% saturation of the cell-free extract from Phanerochaete chrysosporium), 25μl AF (3.0%, w/v), lOOμl sodium phosphate buffer (0.1M, pH6.5) and 0.84 ml water. The reaction was started by the addition of the substrate AF. The reaction was performed at 22 °C. The formation of microthecin and its intermediate was monitored at 230 nm and 263 nm, respectively. One can see that the intermediate was first formed and levelled off after around 20 min. There was a delay for the formation of microthecin but its formation continued until nearly all the AF in the reaction mixture was consumed.
Figure 4 shows SEQ ID NO.l, the gene coding for pyranosone dehydratase (PD) from the fungus Phanerochaete chrysosporium including the upstream regulatory region (-1- to -288), the coding region (1-3146) and down-stream region (3147-3444). The presumed starch codon is ATG (bold) and stop codons are TGA TAG(bold). The purified functional pyranosone dehydratase corresponds to a N-terminal 7-amino acid truncated PD if the translation is supposed to start from the bold codon ATG.
Figure 5 shows the upsfream region, the coding region and the down sfream region of the pyranosone dehydratase (PD) gene from the fungus Phanerochaete chrysosporium. The DNA sequence theoretically could code for three proteins with different amino acid sequences. The bold amino acids are those found by amino acid sequencing of the purified functional PD. Identified infrons are underlined.
Figure 6 shows the final emergence of sugar beet seeds treated in accordance with Example 3.
Figure 7 shows the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogen Aphanomyces cochlioides.
Figures 8 and 9 show the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogens Pythium ultimum and Rhizoctonia solani respectively.
Figure 10 shows that the Hansenula expression vector pFPMT121. Figure 11 shows the specific activity of algal α-l,4-glucan lyase when cells where grown at 24°C (square), 30°C (diamond) and 37°C (triangle). Cell-free exfracts opened mechanically on a Mini Bead-Beater were used to determine the specific activity by the DNS method.
Figure 12 shows an ELISA-plate from activity screening by the DNS method of repressed and induced extracts prepared from cultures of transformant 2 and 8 (2 cultures of each transformant were grown). In columns 1-3 and 4-6 extracts from culture 1 and 2 of transformant 2 were assayed. In column 7-9 and 10-12 extracts from culture 1 and 2 of transformant 8 were assayed. A1-A12: Assay on 10, 20 and 50 μl of cell-free extracts from repressed cultures. The cells were opened mechanically on a Mini Bead-Beater. C1-C12: Assay on 10, 20 and 50 μl of cell-free exfracts from induced cultures. The cells were opened mechanically on a Mini Bead-Beater. E1-E12: Assay on 1, 5 and 10 μl of cell-free extract from induced cultures. The cells were opened with the chemical reagent LTAB and the supernatant was used for the assay. G1-G12: The pellet from the LTAB opening was resuspended in 0.1 M MOPS-NaOH pH=6.2 and 1, 5 and 10 μl was used for the assay. The respective blanks are shown in B1-B12, D1-D12, F1-F12 and H1-H12.
Figure 13 the specific activity of algal α-l,4-glucan lyase was measured by the DNS method in cell-free extracts from repressed and induced cultures. The black columns show the specific activity when the cells were repressed in YND + 2% glucose. The cells were opened mechanically on a Mini-Bead Beater. The pink and blue columns show the specific activity when the cells were depressed in YND + 1% glycerol and induced with 1% methanol on the second day of growth. The cells were opened mechanically on a Mini Bead-Beater (white) or opened with the chemical reagent LTAB (chequered).
Figure 14 (left) shows Native-PAGE on a homogenous polyacrylamide gel.: (right) shows Native-PAGE on an 8-25% gradient polyacrylamide gel (right). The gels were loaded in the same order: Lane 1: Raw extract from Aspergillus Niger. Lane 2: Fraction III. Lane 3: Fraction II. Lane 4: Algal α-l,4-glucan lyase purified from Aspergillus Niger. Lane 5: Fraction I. Lane 6: Raw extract from H .polymorpha. The gels were stained with PhastGel Blue R.
EXAMPLES
The term pyranosone dehydratase may be referred to herein as PD.
Pyranosome Dehydratase purified from the fungus Phanerochaete chrysosporium
Phanerochaete chrysosporium (white rot fungus) is a biotechnologically important fungus due to its higher growth optimum temperature (40°C) and its ability to produce a range of extracellular oxidative enzymes. Accordingly, this fungus has been used for treatment of various wastes, including explosive contaminated materials, pesticides, and toxic wastes. Furthermore, Phanerochaete chrysosporium is the first basidiomycete genome to be sequenced (University of California and Department of Energy, USA).
In the search for enzymes that metabolise anhydrofructose (AF), a purified a heat-stable pyranosone dehydratase (PD) was obtained from P. chrysosporium. Studies have shown that this purified PD not only uses AF as substrate, but uses it more efficiently than its natural substrate, glucosone. Furthermore, the product was shown to be microthecin, an antifungal useful in plant protection.
The N-terminal sequence of PD, and the endo-N-terminal sequences of PD after hydrolysis with two proteinases were elucidated. Together these account for 332 amino acids or 37% of the full length of the PD protein based on the assumption that it has a Mr of 97kDa.
Through database search using the above partial amino acid sequences on the fungal genome, the full length PD gene was identified in Scaffold 62 (Figure 4). The transcription start and stop codens together with 3 introns were identified (Figure 4). It appears that the purified PD is N-terminal 7-amino acid truncated, but still functional.. Since the enzyme PD has not been found in culture medium, it may not have a signal peptide.
Assay methods
Measuring of the pyranosone dehydratase (PD) activity
The reaction mixture consisted of 25 μl of anhydrofructose solution (3.0%), 10 μl PD preparation, 93 μl 0.1 M sodium phosphate (pH 6.5), and water to a final volume of 1 ml. The reaction was mixed and scanned between 190 and 320 nm at room temperature (22 °C) every 5 min or after 30 min on a Perkin Elmer Lambda 18 uv/vis specfrophotometer. Absorbance values at 265 and 230 nm were recorded. One activity unit of PD is defined as the increase of 0.01 of absorbance unit at 230 nm at 22 °C per min.
A protein assay was carried out using the Bio-Rad Method (Bradford method) using the reagent and instractions form Bio-Rad laboratories [Peterson, GL: Determination of total protein, Methods Enzymol. 91, 95-119 (1983)]
TLC for separation of glucosone, AF and microthecin was performed as described before using a solvent system of ethylacetate, acetic acid, methanol and water (12:3:3:2) [Yu S, Ahmad T, Pedersen M, Kenne L: α-l,4-Glucan lyase, a new class of starch/glycogen degrading enzyme. III. Substrate specificity, mode of action, and cleavage mechanism, Biochim Biophys Acta 1244: 1-9 (1995)]. A Merck silica gel 60 (20x20cm) plate with a thickness of 0,15 mm was used. 1,5-Anhydro-D-fructose was assayed by the DNS method [Yu S, Olsen CE, Marcussen J: Methods for the assay of 1,5-anhydro-D-fructose and α-l,4-glucan lyase, Carbohydr. Res. 305: 73-82 (1998)].
Purification of PD
The purification procedure used was essentially the same as that described by Gabriel et al., (1993), Arch. Microbi., 160:27-34 except the strains used were different. In addition, an extra ammonium sulfate fractionation step was included. The strain used in this application was Phanerochaete chrysosporium from American Type Culture Collection (ATCC 32629) and (ATCC 24725), while the strain used by Gabriel et al (1993) was Phanerochaete chrysosporium k-3 obtained from a Czechish collection centre.
The cell-free extract of Phanerochaete crysosporium was brought up to 55% ammonium sulphate saturation. It was then blended gently for 2 hours and centrifuged for 20 minutes at 4°C at lOOOOxg. The precipitate that had the PD activity was dissolved in the same volume of extraction buffer, centrifuged again and the supernatant was then used for the purification of PD using the procedure described by Gabriel et al. (1993).
The purification of PD procedure was followed by SDS-PAGE, and native-PAGE using PhastSystem (Pharmacia) using 8-25 % gradient gels according to the manufacturer's instractions. Visualization of protein bands on the gels was made with Coomassie brilliant blue staining (PhastGel Blue R). From Fig. 1A, PDl is estimated to have a molecule mass 97 kDa it had a similar migration rate as the protein marker phosphorylase b (97.4 kDa).
Amino acid sequencing
The purified PD was used for amino acid sequencing. Amino acid sequencing of PD was performed as described earlier [Yu. S.; Christensen TMIE, Kragh KM, Bojsen K, Marcussen J: Efficient purification, characterization and partial amino acid sequencing of two α-l,4-glμcan lyases from fungi. Biochim Biophys Acta 1339: 311-320 (1997)]. PD was first partially hydrolyzed with proteinases. The generated peptide fragments were separated on HPLC. Each individual polypeptide was collected, molecule-mass determined by mass spectrometer, and sequenced on an Applied Biosystems 476A sequencer using pulsed-liquid fast cycles. PD was also further characterized for its pH and temperature optimum, ion requirements for activity, stability and other kinetic properties. Amino acid sequences obtained from Pyranosome Dehydratase purified from the fungus Phanerochaete chrysosporium.
The following amino acid sequences are obtained either by trypsin or endoproteinase LysC digestion . Peptide purification is achieved by reverse phase HPLC and molecular weight information is generated by MALDI-TOF mass specfrometry. The sequences obtained are then compared to the DNA sequences found in the White Rot Genome (Phanerochaete chrysosporium) project undertaken by The University of California. Sequence similarity alignment is done using the BLAST algorithm.
All peptides producing significant alignments are found in Scaffold 62
LysC peptides
Peptide 27.3 (N terminal) KPHCEPEQPAALPLFQPQLVQGGRPDXYWVEAFPFRSDSSK
V possible heterogeneity
This peptide is found from base pair 38620 -38742. There is a start codon at base pair 38599 and at 38317 indicating a possible signal peptide. Independent confirmation that this is the N terminal of the protein is achieved by sequencing protein PD2, an isozyme.
The X at residue 27 is G in the data base, this fits well with the MS data. . MSc + = 4669.10 MSo+ = 4668.01 -0.023%
N-terminal
The N-terminal of Pyranosone dehydratase isozyme I (PDl) was found to be as follows:
KPHXEPEQPAALPLFQPQLW(Q)GGRPDXY
X is unknown. V(Q) means it could be either V or Q or both (due to heterogeneity).
The N-terminal sequence above (Peptide 27.3) was isozyme II (PDII). The N-terminals of PDl and PDII are very similar or the same. Peptide 31.4 b SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK
D possible heterogenity
This peptide is found from base pair 38788-38963. The data base sequence is interrupted by an intron from base pair 38836-38889. The sequence of residues 28-41 is confirmed by trypsin peptide 8.4
The X at residue 19 is S in the data base sequence, this fits with the MS data. MSc+ = 4591.22 MSo+ = 4591.55 + 0.007%
Trypsin peptides
Peptide 6a VSWLENPGELR
This peptide is found from base pair 39096-39128. MSc+ =1300.44 MSo+ = 1300.45 + 0.001%
Peptide 5 DGVDCLWYDGAR
This peptide is found from base pair 39426-39461 MSc+ = 1427.48 MSo+ = 1427.48
LysC peptides
Peptide 27.4a
PAGSPTGΓVRAEWTRHVLDVFGXLXXK
This peptide is found from base pair 39673-39753
The three X's are PNG in the data base , this fits well with the MS data.
MSc+ = 2876.27 MSo+ = 2876.80 + 0.021% Peptide 29.4.8 HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK
This peptide is found from base pair 39754-39879 MSc+ = 4727.13 MSo+ = 4727.70 +0.012%
Peptide 13.11 TEMEFLDVAGK
This peptide is found from base pair 40244-40276 MSc+ = 1240.42 MSo+ = 1240.53 + 0.009%
Peptide 14.2 KLTLVVLPPFARLDVERNVSGVK
This peptide is found from base pair 40277-40345 MSc+ = 2552.08 MSo+ = 25551.35 - 0.029%
Trypsin peptide
Peptide 10.5 SMDELVAHNLFPAYVPDSVR
This peptide is found from base pair 40526-40585 MSc+ = 2259.55 MSo+ = 2259.77 + 0.009%
LysC peptide
Peptide 31.4a NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK This peptide is found from base pair 41293-41469 and contains an intron from base pair
41362-41416
MSc+ = 4289.73 MSo+ = 4289.45 - 0.007%
Peptide 2b TGSLVCARWPPVK
This peptide is found from base pair 41470-41508 MSc+ = 1471.71 MSo+ = 1472.62 +0.062%
Peptide 2a
NQRVAGTHSPAAMGLTSRWAVTK
This peptide is found from base pair 41509-41577
MSc+ = 2440.71 MSo+ = 2441.58 + 0.036%
Peptide 11.3 GQITFRLPEAPDHGPLFLSVSAJRHQ
This peptide is found from base pair 41641-41718
MSc+ = 2888.34 MSo+ = 2888.25 - 0.031%
This peptide does not end with K which is an indication of the C terminal. The sequence is also followed by a stop codon.
The molecular weight of this protein is approximately 97 KD. Based on the assumption that the average molecular weight of an amino acid is 110, the expected number of residues would be 880, which would give a total number of base pairs of 2640.
The number of base pairs calculated from the data base sequence is 3100. The two known infrons comprise of 53 and 54 base pairs so if it is assumed that this figure is normal then the data base sequence is expected to contain about 8 infrons. The total number of residues sequenced here is 332 amino acids, which accounts for 37% of the protein.
Example 1: Use of 1,5-anhydro-D-fructose and PD for the production of microthecin
The reaction mixture consisted of l,5-Anhydro-E)-fructose 5μl (3.0%), PD preparation 5μl, 65μl sodium phosphate buffer (pH 6.0) and water to a final volume of 0.7 ml. The reaction was monitored by scanning between 350-190 nm. Reaction time at zero min was used as blank. The absorbance peak at around 230 nm indicates the formation of microthecin. The absorbance at 265 nm indicate the first formation of an intermediate from AF before it converts to microthecin.
The microthecin formed was further confirmed by relative migration rate on TLC and its conversion of 2-furyhydoroxymethylketone that exhibits a typical absorbance peak at 275 nm [Baute M.-A. et al., 1986].
In larger scale production of microthecin, AF used was from 0.4% to 20%. The reaction was followed by AF disappearing from the reaction mixture using the DNS method [Yu. S.; Christensen TMIE, Kragh KM, Bojsen K, Marcussen J, Biochim Biophys Acta 1339: 311-320 (1997)]. The formation of microthecin was monitored at 265nm and its shift to 230nm, and was further monitored by TLC method.
1.5-Anhydro-D-fructose is found to be a much better substrate for the pyranosone dehydratase (PD) than for its natural substrate glucosone. The Vmax is around 4.7 times higher with AF than with glucosone (Table 1).
Table 1
Figure imgf000146_0001
Figure imgf000147_0001
The reaction system consisted of AF or glucosone 1-15μl, 25 μl sodium phosphate buffer (6.5. 0.1M), water, 1.4μl PD to a final volume of 200μl. The reaction was performed at 22°C for 5.5 hours. The formation of microthecin from AF and cortalcerone from glucosone were monitored at 226nm.
Example 2: Production of Cortalcerone
Cortalcerone may be produced in one step by incubating a starch-type substrate, such starch, waxy starch, dextrins, with starch hydrolases, such amyloglucosidase and a debranching enzyme or cyclodextrin fransferase, pyranose 2-oxidase, and PD. After incubation Cortalcerone can be separated from the reaction mixture by ultrafiltation using membrane cut-off of 300-30,000, preferably 10,000.
Example 3: Use of 1,5-anhydro-D-fructose, PD and ascopyrone P synthase for the production of APP
The reaction mixture consisted of 1,5-Anhydro-D-fructose 50μl (3.0%), PD preparation 5μl, ascopyrone P synthase 5 μl, 0.1 ml sodium phosphate buffer (pH 6.0) and water to a final volume of 0.8 ml. The reaction was monitored by the formation of APP at 289 nm spectrophotometrically. The reaction temperature was 22 °C and reaction time was 24 hours. At the end of 90% of AF had been converted to APP. The structure of APP was corifirmed using NMR as described earlier [WO 00/56838 filed 16/3/00, claiming priority from GB9906457.8, filed 19/3/99].
Expression of PD gene The PD gene may be expressed in a production organism such as Pichia pastoris, Aspergillus niger, and Hansenulla polymorph by techniques well known in the art and referenced hereinbefore in the description.
Antibody production
Antibodies were raised against the amino acid of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild ("Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre" In A Manual of Quantitative. Immunoelectrophoresis, Methods and Applications, N H Axelsen, et al (eds.), Universitetsforlaget, Oslo, 1973) and by T G Cooper ("The Tools of Biochemistry", John Wiley & Sons, New York, 1977).
Microthecin as an Anti-Fungal
Fungal growth in plant causes enormous economical damages. Examples are their damage to sugar beet seedlings and their leaves. As soon as the sugar beet seed is germinated in the soil it is immediately exposed to fungal attack by the species such as Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides. In the present invention, it was found microthecin was able to inhibit the growth of these disease- causing fungi. Hence, the seeds of economical crops, sugar as sugar beet seeds are coated with a paste containing microthecin at 50-2000 ppm and dried before use for planting. Alternatively, aqueous solution of microthecin may be directly sprayed on the plant and its leaves.
Experimental
Basic microthecin solution: 24 mg/ml Batch no. Mic20011016
Dilutions used:
Figure imgf000148_0001
Figure imgf000149_0001
All solutions were filtered through a 0.22 μm filter for sterilisation. The solutions were tested against the following fungi:
Figure imgf000149_0002
A circular plug (diameter 10 mm) of fresh mycelium was placed at the centre on a petri-dish (diameter 9 cm) containing PDA medium. (PDA= Potato dextrose agar Difco no. 213400). Wells with a diameter of 5 mm were cut along the periphery of the agar plate. In each well were placed 50 μl of a test solution. Alternatively, 20 μl of each test solution were placed directly oil the agar along the periphery of the plate. Also, 50μl of each test solution were placed directly on top of the fungal mycelium plug.
The agar plates were placed at room temperature in daylight, but protected from direct sunlight.
The reaction (inhibition zones) of the fungi to the test substance was judged as follows:
Rhizoctonia solani: after 2-3 days of growth Pythium ultimum: after 1-2 days of growth Aphanomyces cochlioides: after 3-4 days of growth Cercospora beticola after 3-4 weeks of growth Results
Microthecin as a fungal growth regulator was inhibitory against Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides and Cercospora beticola. The minimum inhibition concentration (mic) of microthecin against these fungi were 240, 480, 1200 and 2400 ppm, respectively.
Example 4: Effect of Microthecin on pelleted sugar beet seeds
The effect of microthecin on the plant pathogenic fungi Pythium ultimum, Rhizoctonia solani and Aphanomyces cochlioides in vitro was investigated by screening for growth inhibition of the pathogens on agar-plates (Figures 6, 7, 8).
Figure 7 shows the screening effect of microthecin in different concentrations against Aphanomyces cochlioides, whereas Figures 8 and 9 show the screening effect against Pythium ultimum and Rhizoctonia solani respectively. In each case, microthecin was dissolved in water and placed in wells in the periphery. An agar block containing the pathogen was placed in the centre. The pathogens were allowed to grow out on the PDA-agar plates for 3-5 days. These investigations showed that microthecin in very low concentrations was able to reduce the growth of Aphanomyces.
Similar tests with other microorganisms showed that Microthecin has no effect on Cercospora. Pseudomonads (P. fluorescens DS96.578, P. mendocina DS98.124) are slightly affected, whereas it has no effect on the growth of Bacillus (B. Pumilus DS96.734, B. megaterium DS98.124).
Based on these findings, the efficiency of microthecin was further investigated in a field emergence trial. The trial was sown relatively late giving it a higher chance for presence of the pathogen Aphanomyces in the trial field.
Materials and methods
Pellet TKW
1. Manhattan CAC-7-2306 kb5, 3,0-4,25mm, 19,2 (7) 19,1 (1) 2. Tower MIT-1-0290 kb5, 3,0-4,25mm. 17,3 (8) 17,8 (2)
Seeds were pelleted with standard PI pelleting mass with (1,2) or without (7,8)Thirani.
Standard seed coating:
Inner coating: 0,3gai/U microthecin as a 0,5% solution in water or
14,7gai/U Hymexazol.
60gai/U Imidacloprid. Standard metallic green seed cover film.
The following combinations were included in the trial:
R F0 Without fungicides
R FT With Thiram (in pellet)
R FH With Hymexazol
R FM With Microthecin
R P1 STD With Thiram (in pellet) + Hymexazol
R FTM With Thiram (in pellet) + Microthecin
Trial place Bukkehave, DK. (4 reps, 200 seeds/plot)
Trial sown 21.05.2002
1. Count 28.05.2002 (speed)
2. Count 29.05.2002 (speed)
3. Count 24.06.2002 (final)
Results
Lab and field emergence figures can be fotmd in Table 2 (Trial FEHCP034
Aphanomyces). The final emergence is shown in Figure 6. Table 2
Figure imgf000152_0001
The results of the lab studies indicate that the inclusion of microthecin decreases the speed of laboratory germination (4d), but this is not reflected in the 4d>15mm figures. This is the opposite effect of Hymexazol that has a low 4d>15mm germination.
With regard to the speed of germination, the field emergence trials indicate that pellets containing Hymexazol - either alone, or in combination with Thiram - germinate relatively slow (as expected from the 4d>15mm lab germination). Pellets containing microthecin show a speed of germination comparable with pellets only containing Thiram. In contrast to the fast germinating Thiram containing pellets the pellets containing Microthecin show a high final germination (comparable with Hymexazol containing pellets).
Although the actual attack by root rot causing pathogens was rather limited, the 4% (approximately) missing plantlets in the FT-plots arise from attack of plantlets by pathogens that can be controlled by Hymexazol (most probably Aphanomyces). The final number of plantlets in the FT-plots are lower than the number of plantlets in the F0 (no fungicides) plots. This can be explained by the action of Thiram, that controls other microbes, but not Aphanomyces, thereby allowing easier access of Aphanomyces to the plantlets.
The microthecin containing pellets are the only pellets that both show a fast germination and a high final germination. It is believed that microthecin might therefore be an alternative to the rather expensive chemical Hymexazol.
Example 5: Test of microthecin and selected compositions against powdery mildew
Method Reference:
Lyngs Jørgensen, H.J., Andresen, H. and Smedegaard-Petersen, V. (1996).
Confrol of Drechslera teres and Other Barley Pathogens by Preinoculation with
Bipolaris maydis and Septoria nodorum. Phytopathology 86, 602-607.)
Barley plants, isoline P01 of the cultivar Pallas, were grown in a growth chamber, at approximately 200 μE m"2 s (16 h of light at 19°C with 50 to 60 % relative humidity and 8 h of darkness at 16°C with 80 to 90 % relative humidity) in plastic pots (12 by 13.5 cm) containing the soil mix 'Weibulls Enhetsjoπf (K jord, Svalδf Weibull AB, Hammenhδg, Sweden).
11 days after sowing the first leaf of each of 10 plants per pot was fixed in a horizontal position, adaxial side upwards, on bent plastic plates using unbleached cotton strings. 2.5 ml of each test solution was sprayed on the fixed leaves of a total of 4 pots. After 1 hour the treatment was repeated with extra 2.5 ml of the same test solution.
The experimental set-up was as shown in the Table 3 below.
Table 3
Figure imgf000154_0001
Test solutions
Pyranosone dehydratase
Stock solution 46.4 units/ml in 25 mM phosphate buffer (pH 6.5).
Used solution: 50μl stock solution in 10 ml 20 mM sodium phosphate buffer (pH 6.5)
=> 0.23 units/ml.
Anhydrofructose:
Stock solution: 49.5 mg/ml of water.
Used solution: 436 μl stock solution was diluted with 20 mM sodium phosphate buffer
(pH 6.5) to a total of 10.00 ml => 2.1 mg anhydrofructose/ml.
Anhydrofructose + pyrasonone dehydratase:
436 μl stock solution, 50 μl pyrasonone dehydratase stock solution and 9514 μl sodium phosphate buffer (pH 6.5) were mixed V/2 hours before use. Microthecin:
Stocksolution: Approximately 44mg/ml 20 mM sodium phosphate buffer pH=6.5 Used solution: 460 μl stock solution was diluted with 20 mM sodium phosphate buffer (pH 6.5) to a total of 10.00 ml => 2.0 mg microthecin/ml.
Inoculum of Powdery Mildew, Blumeria graminis f. sp. hordei was produced on barley plants, isoline P01 of the cultivar Pallas. After 7 days of incubation (16 h of light at 18 to 20°C and 8 h of darkness at 15 to 16°C) the powdery mildew fungus sporalated abundantly and was used for inoculation of the treated barley leaves. An inoculum concentration of 3 conidia/mm2 leaf area was applied. The pots were randomly placed in the growth chamber (200 μE m"2 s (16 h of light at 19°C with 50 to 60 % relative humidity and 8 h of darkness at 16°C with 80 to 90 % relative humidity) for 7 days before disease assessment.
Disease assessment
Seven days after treatment the disease development was assessed by judging percentage leaf coverage with mildew colonies on the ten fixed leaves on each pot.
Results Table 4
Figure imgf000155_0001
As can be seen from Table 4, freatments with microthecin as well as the combined anhydrofructose + pyranosone dehydratase (AF + PD) significantly reduces the mildew colony formation.
Example 6: Testing of microthecin against various plant pathogens
The effect of microthecin against a range of different plant pathogens was investigated, including those listed below in Table 5.
Table 5
Figure imgf000156_0001
* Isolate received from Section for Plantpathology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
The results are shown below in Table 6, where "+" indicates a positive effect against a particular pathogen.
Table 6
Figure imgf000157_0001
Example 7: Testing microthecin and other products on agar plates
The effect of microthecin and the products for the enzymatic production of microthecin on different plant pathogens were tested on agar plates. The tests were performed on 9- cm agar plates, pH 6,1-pH 6,5. On each plate a 9mm PDA-block of an actively growing pathogen was placed in the cenfre and allowed to grow out. Different products and enzymes were placed in 5mm diameter cut holes in the periphery of the agar plates, in total about 45-50μl/hole. The growth and growth inhibition of the pathogenic fungi in the vicinity of the holes were monitored:
+++: pronounced growth inhibition, ++ : good inhibition, + : some inhibition, : no growth inhibition.
Test of Anhydrofructose (AF) + Pyranosone dehydratase (PD) Growth medium: Potato Dextrose Agar (PDA) plates, pH 6,1.
Table 7
Figure imgf000158_0001
lx PD corresponds to 0,02U/mg AF.
Table 7 shows that Aphanomyces and Pythium are sensitive to AF+PD, whereas Rhizoctonia is less sensitive and finally will overgrow the holes.
Time-effect study of Anhydrofructose (AF) + Pyranosone dehydratase (PD)
The AF and PD were mixed and at given times filtered through a MW 10.000 sieve to stop the enzymatic reaction. The starting mixture contained AF 44,6mg/ml and PD
40U/ml.
The set-up of the experiment was the same as in Example 1. 15μl probe + 30μl 20mM sodium phosphate buffer, pH 6,5 were added to each hole. Controls were sodium phosphate buffer pH 6,5 and concentrated pyranosone dehydratase ( 46U/ml). The results are shown below in Table 8.
Table 8
Figure imgf000159_0001
As can be seen from Table 8 above, the effect of the mixture is most pronounced on Aphanomyces cochlioides in the time span from 1 hr to 2 days. After 2 days the inhibitory effect decreases. There is no inhibitory effect on Rhizoctonia solani and only weak inhibitory effect on Pythium ultimum.
The time restricted inhibitory effect corresponds to the peak of microthecin in the mixture. Microthecin starts to decompose to other compounds after short time and after 7 days the microthecin in the mixture is halved compared to the maximum amount present in the mixture.
Pyranosone dehydratase in very high concentrations (PD control) is in itself controlling the growth of the tested plant pathogens. Test of inhibitory effect of dextrin and amylopectin-derived products formed directly in the holes on the agar plates
To rale out if it was possible to produce inhibitory compounds directly from the starch derivatives dextrin and amylopectin using the combined enzyme reaction Glucan Lyase (GL) plus Pyranosone dehydratase (PD), an experiment was set up where the mixtures were added to the holes immediately after blending. The inhibitory effect is shown in Table 9 below.
Dextrin 10 (Fluka 31410, CASno 9050-36-6) 1,25% in 20mM sodium phosphate buffer, pH 6,5. Amylopectin (Sigma A8515) 1,25% in 20mM sodium phosphate buffer, pH 6,5. Dextrin and amylopectin was boiled for 20min and cooled before adding to the holes (approx. 45μl/hole).
Glucan Lyase (GL) 1U per hole, Pyranosone dehydratase (PD) 0,25U per hole. Growth medium: Potato Dextrose Agar (PDA) plates, pH 6,1.
Table 9
Figure imgf000160_0001
As can be seen from Table 9 above, the starch derivative plus Glucan lyase has no effect on fungal growth. Addition of Pyranosone dehydratase to the starch derivatives has a slight inhibitory effect, whereas the best inhibitory effect was obtained by adding both Glucan lyase and Pyranosone dehydratase to the starch derivatives.
Water Agar
To clarify if the inhibitory effect of the Pyranosone dehydratase was due to assessable substrate present in the potato dextrose agar plates, an experiment was carried out using water agar as the growth medium. The pH in the water agar was adjusted to pH 6,2 with 20mM sodium phosphate buffer.
Also tested was the effect of boiling versus sterile filtration of the dextrin 10 used as the starch derivative (1,25%) in this experiment. No difference in inhibitory effect between boiling and sterile filtration was found. The experiment was performed using actively growing Aphanomyces cochlioides and Pythium ultimum on PDA-block placed in the middle of the test plates. Growth inhibition was monitored after 5days (for Pythium also after 2 days). The results are shown below in Table 10.
Table 10
Figure imgf000161_0001
The inhibitory effect on Pythium seems to be transient as the PDA plates are completely overgrown after 5 days. In Aphanomyces, the combined Dextrin + Glucan lyase + Pyranosone dehydratase has a pronounced effect also after 5 days on PDA- plates.
Test of seeds coated with microthecin or anhydrofructose + Pyranosone dehydratase complex
Pelleted sugar beet (Beta vulgaris L.) seeds (lUnit = 100.000 seeds) were coated with
1) 0,3g/Unit Microthecin (FM, FM2)
2) 0,9g/Unit Anhydrofructose (FA)
3) 0,9g/Unit Anhydrofructose + 4 enzymeU/Unit Pyranosone dehydratase (FAD) in the underfilm. The underfilm also contained the insecticide Imidacloprid (60gai/Unit). The coating was finished with a standard green coverfilm. Treatments FT (Thiram) and FH (Hymexazol) are standard fungicide treatments for sugar beet seeds. Treatment FM differs from FM2 in being prepared 6 months before agar plate testing.
After coating, the seeds were tested on PDA-agar plates by placing seeds in the periphery with actively growing pathogenic fungi in the middle of the agar plate. Inhibitory effect was monitored after 2 days. The results are shown below in Table 11.
Table 11
Figure imgf000162_0001
(1) No overgrowth of seed with pathogen.
Treatment of seeds with low levels of Microthecin act at a level comparable with the standard sugar beet fungicides Thiram and Hymexazol against the pathogen Aphanomyces. The level of Microthecin is too low to act against the other two tested pathogens in this experiment.
No phytotoxic effects were observed on seed germination (The pelleted seeds germinated after a few days on the agar-plates).
PDA plates. Action against other plant pathogenic diseases
A number of other plant pathogenic fungi were tested on agar plates. Test system consisted of actively growing fungi placed in the cenfre of the 9-cm agar plate (pH.6.2) and 9mm holes were cut in the periphery of the agar plate. The 7 holes per plate contained the following:
Figure imgf000163_0001
GL: Glucan lyase
PD: Pyranosone dehydratase
The results are shown below in Table 12. Table 12
Figure imgf000163_0002
Septoria tritici was tested after plating of spores on PDA-plates. The spores seem not able to germinate in presence of media containing Pyranosone dehydratase. Substitution of dextrin with amylopectin resulted in the same pattern of fungal growth inhibition. In general treatment 3 (Substrate+GL+PD) seems to control the growth of a variety of fungal species.
EXAMPLE 8: Method to identify enzyme activities of biological samples containing pyranosone dehydratase activity.
Candidate pyranosone dehydratase genes are identified based on their hybridisation or homology to SEQ ID No 1, or by the identification of homology between the amino acid sequence encoded by the candidate sequence and sequences SEQ ID Nos 2-17, Candidate pyranosone dehydratase genes are also identified using antibodies raised against a known PD enzyme (such as an enzyme comprising the amino acid sequence shown as SEQ ID No. 26), or fragment thereof.
Candidate pyranosone dehydratase genes are identified by expression cloning- Selected candidate pyranosone dehydratase cDNA genes identified by basis of homology, and random cDNA clones or libraries are cloned into E.coli or yeast expression vectors and heterologously expressed.
Pyranosone dehydratase (PD) clones may also be obtained by PCR or RT-PCR using degenerate PCR primers. Suitable protocols for designing and performing such protocols are: i) PCR Protocols: A guide to methods and Applications, Academic Press Inc. Chapters 5 & 6; and ii) Rose et al Nucleic Acid Research 1998, 26(7) ppl628- 1635.
It will be recognised that once a skilled person has been taught that pyranosone dehydratase enzymes can be used to produce microthecin, and is provided with information relating to the pyranosone dehydratase gene and/or protein, that other pyranosone dehydratase enzymes can be identified and used for the purpose of performing the present invention. The genes encoding other pyranosone dehydratase enzymes may be obtained from Polyporus obtusus, and other fungal and algal sources mentioned hereinabove. It is also envisaged that pyranosone dehydratase can be obtained from numerous other sources, including plants, animals, bacteria, and cyanobacteria. In order to determine whether an organism contains pyranosone dehydratase activity crude native protein extracts are obtained and screened using the pyranosone dehydratase assay disclosed hereinbelow.
Crude Native protein exfracts are obtained by freeze thawing cells in liquid nitrogen, optionally with mechaniscal disruption (e.g. use of pestle/motar or French press) and extracting the protein in protein extraction buffer (Gabriel et al 1993) Arch. Microbi., 160:27-34. Pyranosone Dehydratase Assay
The reaction mixture consists of sodium phosphate (0.1M pH 6.5, 50 μl), 1,5-Anhydro- D-fructose (AF) (final concenfration 1-100 mM), candidate pyranosone dehydratase sample and water to a final volume of 0.2 ml. The reaction was followed at 26°C using an uv/vis microplate scanning spectrophotometer (model PowerWavex) (Bio-TEK Instruments, Inc., Vermont, USA) in either the scanning mode (350-200 nm) or time course mode (226nn and 263 nm). Pyranosone dehydratase activity is indicated by the increase in absorbance at 226nm.
cDNA libraries are constructed from organisms containing pyranosone dehydratase activity. Total RNA is extracted using RNAeasy standard protocols (Qiagen, http://www.qiagen.com). cDNA is produced using RT-PCR (Gene-Racer, Invifrogen). cDNA libraries are created using standard protocols
cDNA libraries based in Gateway™ vectors are obtained from Invifrogen. cDNA libraries are constructed using standard protocols supplied by Invifrogen. The Gateway™ system is used to convert the cDNA libraries into the appropriate E.coli or yeasts expression vectors.
cDNAs or synthetic genes may be cloned into a suitable E.coli expression vector and the putative enzyme may be produced using the manufacturers standard protocols (TOPO Cloning Kit - pBAD/D-TOPO Expression kits (Invifrogen) http://www.invitrogen.coni/content/sfs/manuals/pbad dtopo man.pdf.
For yeast expression, candidate cDNAs may be cloned into the pPICZalpha (Invifrogen Inc) and expressed as per standard Pichia pastoris Expression System protocols (Invifrogen Inc). pPICZalpha contains the alpha factor secretion signal for targeting of the protein product to the media.
Individual colonies expressing random or pyranosone dehydratase candidate cDNAs are picked into microtitre plates and pyranosone dehydratase activity is identified. Candidate pyranosone dehydratase genes are identified using antibodies raised against one or more of the animo acid sequences shown as SEQ ID Nos. 2-17.
Candidate pyranosone dehydratase genes are identified by assessing the presence of pyranosone dehydratase activity in crude exfracts or in the media or in cells expressing the candidate pyranosone dehydratase genes, using the pyranosone dehydratase assay.
EXAMPLE 9: Cloning Strategy from Phanerochaete chtysosoporium pyranosone dehydratase gene
A cDNA of the Phanerochaete chrysosporium pyranosone dehydratase gene is. obtained by RT-PCR (see below).
Source of Phanerochaete chrysosporium :
Phanerochaete chrysosporium BKM-F-1767 - ATCC 24725 - publically available from the ATCC Collection.
Phanerochaete chrysosporium (Filamentous Fungi) publically obtainable from DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, Strain ID: DSM 1556. The strain can be provided on agar or in liquid culture.
Culture of Phanerochaete chrysosporium - is cultured using the methods described in Kullman and
Figure imgf000166_0001
Environ. Microbiol 63 (7): 2741.
Total RNA extraction from Phanerochaete chrysosporium is performed using the RNeasy Plant Total RNA kit (Qiagen, http://www.qiagen.com)
RT-PCR Reverse transcriptase of total Phanerochaete chrysosporium RNA may be performed as described in Kullman and Matsumura4pP-r- Environ. Microbiol 63 (7): 2741.
Second strand synthesis and subsequent PCR amplification may be performed as described in Kullman and MatsυmvaεiAppl Environ. Microbiol 63 (7): 2741, however primers designed to the 5' and 3' of the ATG of the Phanerochaete chrysosporium pyranosone dehydratase gene are used, such as:
5'PRIMER 1 CGCGACGATGTACAGCAAAGTC (SEQIDNo.18)
3' PRIMER 1 CACGCCGAAAGCTCAATGCTGTC (SEQ IDNo.19)
Alternative primer pairs would be easily identified by a person skilled in the art or could be predicted using suitable primer design software such as Informax VectorNTI. Such programs can also predict the optimal PCR conditions for amplification of such primer combination pairs
PCR Conditions may be performed as described in Kullman and Matsumura AppL Environ. Microbiol 63 (7): 2741 (sequence specific primer conditions) although annealing temperature is performed at 55°C for 1 minute.
Alternatively, PCR may be performed using Invifrogen™ One Step RT-PCR for long templates as described at: http://www.alab.com.pl/pdf/life/11922010%20superscript%20one%20step%20rt- pcr%20for%201ong%20temρlate.pdf
Alternatively a synthetic codon optimised Phanerochaete chrysosporium pyranosone dehydratase gene is obtained by designing a codon optimised gene for the targeted plant species. The genomic sequence of the Phanerochaete chrysosporium pyranosone dehydratase gene is disclosed in Figures 4 and 5.
The cDNA sequence is: TCGCGACGATGTACAGCAAAGTCTTCCTCAAGCCGCACTGTGAGCCCGAGC
AGCCTGCCGCTCTCCCTCTCTTCCAGCCCCAACTCGTGCAGGGAGGACGTCC
TGATGGCTACTGGGTCGAGGCATTCCCCTTTCGCTCAGACTCCAGCAAATG
CCCCAACATCATTGGCTATGGACTCGGCACGTACGACATGAAGAGCGACAT
CCAGATGTTTGTCAACCCATACGCAACTACCAACAATCAGAGCTCGTCTTG
GACCCCTGTCTCACTGGCAAAACTCGATTTCCCGGTCGCAATGCACTATGCC
GACATCACGAAGAATGGTTTTAATGATGGTCGGTGTATTTTTTTTTTTTTTTG
CTATATCTCATGCTTTGCTAACCATCGCACAGTTATCATCACGGACCAATAC
GGCTCCTCGATGGACGACATCTGGGCCTATGGTGGACGCGTCAGCTGGCTC
GAGAATCCCGGCGAGCTGCGCGACAATTGGACGATGCGCACGATTGGGCA
CAGCCCGGGCATGCACCGGCTCAAGGCGGGGCACTTCACGCGCACGGACC
GTGTGCAGGTCGTCGCAGTGCCGATCGTCGTTGCGTCCAGCGACCTCACGA
CGCCGGCGGACGTCATCATCTTCACTGCCCCCGACGATCCTCGCTCAGAGC
AGCTCTGGCAGCGTGACGTCGTCGGCACGCGCCACCTCGTCCATGAGGTCG
CCATCGTCCCCGCCGCCGAAACTGATGGCGAAATGCGCTTCGACCAGATCA
TCCTTGCGGGACGCGACGGTGTCGACTGCCTGTGGTATGACGGCGCCAGGT
GGCAGAAGCATCTCGTCGGCACGGGCCTTCCGGAAGAGCGCGGAGACCCC
TATTGGGGTGCGGGCTCCGCTGCGGTTGGACGCGTAGGCGACGACTATGCG
GGATACATCTGCTCTGCCGAGGCATTCCACGGCAATACCGTCTCGGTCTAT
ACAAAGCCCGCTGGCTCACCGACGGGCATCGTCCGCGCAGAGTGGACGAG
ACATGTGCTCGACGTCTTCGGGCCACTCAACGGGAAGCACACCGGGAGCAT
TCACCAGGTCGTCTGCGCGGACATCGATGGAGACGGGGAAGACGAATTTCT
CGTAGCCATGATGGGCGCAGATCCTCCGGACTTCCAGAGGACAGGCGTTTG
GTGCTATAAGGTCGACAGGACAAACATGAAGTTCTCCAAGACCAAAGTCA
GTAGTGTTTCTGCCGGGCGCATCGCAACAGCGAACTTCCACTCGCAGGGCT
CCGAAGTGGACATTGCCACCATCTCTTACTCTGTTCCTGGATATTTTGAGTC
CCCCAACCCGTCCATCAACGTCTTCCTCTCCACCGGCATTCTTGCCGAGCGG
CTTGACGAAGAGGTGATGCTCAGGGTGGTCCGCGCAGGATCGACGCGCTTC
AAGACCGAGATGGAGTTCCTTGACGTCGCGGGAAAGAAGCTTACGCTTGTC
GTGCTGCCGCCCTTCGCACGCCTCGATGTCGAACGCAATGTGTCCGGTGTG
AAGGTCATGGCCGGGACAGTCTGTTGGGCCGACGAGAACGGGAAGCATGA . ACGCGTGCCTGCAACGCGCCCATTCGGCTGCGAGAGCATGATCGTCTCCGC
AGACTATCTCGAGAGCGGGGAAGAGGGCGCGATCCTCGTCCTCTACAAGCC
CTCGAGCACCTCAGGCCGGCCGCCGTTCCGTTCTATGGACGAACTTGTGGC
GCACAACCTGTTCCCCGCGTACGTCCCCGATAGTGTTCGCGCGATGAAGTT
CCCCTGGGTACGCTGCGCAGATCGCCCGTGGGCGCATGGCCGCTTCAAGGA
CCTTGACTTCTTCAACCTCATCGGCTTCCACGTCAACTTTGCGGATGATTCC
GCGGCTGTGCTCGCGCACGTTCAGCTCTGGACGGCGGGCATTGGCGTCTCC
GCTGGGTTCCACAACCACGTCGAAGCGTCGTTCTGCGAGATCCATGCCTGC
ATCGCGAACGGCACCGGTCGCGGCGGGATGCGCTGGGCAACCGTTCCCGAT
GCCAATTTCAACCCAGACAGCCCGAACCTCGAGGACACGGAGCTGATTGTC
GTGCCTGACATGCACGAGCACGGCCCACTCTGGCGCACGCGTCCTGATGGA
CACCCGCTCCTGCGCATGAATGACACCATCGACTACCCATGGCATGCTTGG
CTGGCGGGCGCCGGCAACCCCAGCCCGCAGGCGTTCGACGTCTGGGTTGCG
TTCGAGTTCCCCGGGTTCGAAACGTTCTCGACTCCTCCGCCTCCGCGCGTAC
TCGAGCCCGGGAGGTACGCAATCCGGTTTGGAGACCCTCACCAGACCGCAT
CGCTTGCCCTTCAGAAGAACGATGCCACAGACGGCACCCCCGTTCTCGCGC
TCCTCGACCTCGATGGCGGCCCGTCGCCGCAGGCTGGAATATCTCTCATGTT
CCCGGCACGGACATGTACGAGATCGCGCACGCCAAGACGGGTTCGCTTGTC
TGTGCTCGTTGGCCGCCCGTTAAGAATCAGCGTGTCGCCGGCACGCACTCT
CCTGCTGCCATGGGTCTTACGTCACGGTGGGCCGTCACGAAGAACACCAAG
GGGCAGATTACGTTCCGTCTCCCGGAGGCGCCCGACCATGGCCCGCTCTTC
CTTAGCGTTTCCGCTATACGCCACCAACAGGGAGCAGACGCGATTCCCGTA
CGTGATAGACTGCTATCCCTGTTCAAGTTTTGTCTCACGTATTTACACTTTAT
CCTCTCAGGTCATCGTGCAGGGGGACAGCATTGAGCTTTCGGCGTGGTCTC
TTGT (SEQ ID No. 20)
cDNA sequences may be deduced from genomic sequences using infron prediction, exon prediction, open reading frame and gene structure prediction prediction bioinformatics software or by manual identification of the intron cleavage sites, for. example the Webgene-Gene builder available through http://125.itba.mi.cnr.it ~webgene/genebuilder.html, combined with the peptide sequence information relating to the pyranosone dehydratase gene. The Numerous bioinformatics programs have been developed to provide an optimised codon sequence for a large range of organisms. Prefened codon usage tables are available at http://www.kazusa.or.ip/codon/T.html. Both the design and the constraction of the synthetic pyranosone dehydratase gene can be undertaken by one of numerous technology service providers, for example www.entelechon.com. Synthetic genes can be modified to remove cryptic intron splice sites that can be a problem in heterologous expression, or to overcome other transcriptional or post-transciptional or translation or protein stability problems. In addition unwanted restriction sites can be removes to facilitate down-stream cloning into desired expression constructs, or for the inclusion of protein targeting signals. The pyranosone dehydratase gene is made synthetically for expression in both monocotyledonous plants and dicolyledenous plants (codon optimised for expression in Triticum aesitivum), and any unwanted restriction sites are removed.
EXAMPLE 10: Transformation of a plant with a polynucleotide sequence encoding pyranosone dehydratase and/or a polynucleotide sequence encoding glucan lyase
The cDNA clone of the Phanerochaete chrysosporium pyranosone dehydratase (SEQ ID 20) is obtained by RT-PCR using Invifrogen's Superscrpt One-Step RT-PCR for long templates, standard protocols (US 6,063,608). The RT-PCR product is cloned into pTOPO (Invifrogen). EcoRl is used to excise the inserted fragment.
A codon optimised cDNA clone of the Phanerochaete chrysosporium pyranosone dehydratase gene is obtained synthetically. Suitable restriction unique restriction sites (e.g. EcoRl) sites are engineered at the 5' and 3' flanking regions and the optimised gene, and the optimised sequence is designed to contain Kpnl or EcoRV sites.
Several fungal and algal glucan lyase sequences have been isolated and characterised and are suitable for use in the present invention (see for example WO 95/10616, WO 95/10617, WO 95/10618, WO 96/12026, and Yu S, Bojsen K, Svensson B, Marcussen J.Biochim Biophys Acta 1999 Aug 17;1433(1-2):1-15).
The cDNA clone (Agl l l, Genbank Accession Number Y18737) from the red alga Gracilariopsis lemaneiformis has been cloned (see Bojsen,K., Yu,S., Kragh,K.M. and Marcussen, J. Biochim. Biophys. Acta 1430 (2), 396-402 (1999). The Agl l l clone is modified to remove unwanted restriction sites using Sfratagene QuickChangeTM Kit (three EcoRl sites, 1184 2556 2744, and a single EcoRV site at 2712 are removed by altering the third positions of the relevant codons). The Agl 11 clone is ligated into pJIT 163. The Agl 11 clone insert is prepared as a Notl-Hindlll fragment, blunted and cloned into the Smal site of JIT 163 and ligation products with the correct orientation are determined by restriction analysis. A codon optimised glucan lyase (GL) gene is also constructed using the appropriate codon preference for the host species. The codon optimised gene can be used in place of the native cDNA clone in the following experiments.
Linear restriction fragments fragments containing either the PD cDNA or the glucan lyase are cloned into the expression vector pJIT 163 (available from the John Junes Center, Norwich, UK http://www.pgreen.ac.uk. (Roger P. Hellens, E. Anne Edwards, Nicola R. Leyland, Samantha Bean and Philip M. Mullineaux (2000) "pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation", Plant Mol. Bio. 42: 819-832.).
The expression cassette is excised using Kpnl and EcoRV and cloned into the equivalent sites in the Agrobacterium fransformation vector pGreen to make pGreen- PD (native pyranosone dehydratase), and pGreen-PDco (codon optimized pyranosone dehydratase) and pGreen-GL (glucan lyase) Elecfrocompetent Agrobacteria cells (LBA4404) are prepared as per the JIC protocol, and the prepared transformation vectors are introduced into Agrobacteria strain LBA4404 (widely available standard plant transformation Agrobacterium tumafaciens strain). As will be readily appreciated by those skilled in the art, alternative expression cloning strategies, expression vectors and/or fransformation vectors or other Agrobacterium strains may be used to produce fransgenic plants.
Plant fransformation is carried out using Agrobacteria contaimng pGreen-PD (or PGreen-PDco). Plant fransformation is carried out using a mixture of Agrobacteria containing pGreen-PD (or PGreen-PDco). Plant fransformation is carried out using Agrobacteria containing an empty pGreen vector. Plant fransformation is also carried out using Agrobacteria containing pGreen.GL (or a codon optimised glucan lyase [GL]).
Tomato transformation may be carried out using standard fransformation protocols (Bird et al, 1988, Plant Molecular Biology, 11:651-662.
The polynucleotides of the present inventino can be introduced using plant transformation techniques into timber plants, such as conifer and hardwood trees, including Poplar spp. and Larix spp. (such as poplar and larch aspen) and Pinus spp. Protocols for the fransformation of such free species are detailed in US2003038919 and WO01/66777. Protocols for the fransformation of Pinus spp. can be found in US 6,518,485.
Transformed plants are identified using PCR to detect the presence of the CaMV35S promoter, the introduced pyranosone anhydratase (PD), PDco and glucan lyase (GL), using appropriate primer pairs under standard PCR reaction conditions. The following populations of plants are identified:
Confrol plants - contain empty pGreen T-DNA insert
PD only - contain native pyranosone hydratase (PD) containing pGreen T-DNA
PD & GL - contains both PD and glucan lyase (GL) T-DNA inserts
PD-co - contains codon optimized pyranosone hydratase (PD-co) containing pGreen T-
DNA
PD-co & GL - contains both PD-co and GL T-DNA inserts Selected lines expressing the respective fransgene inserts are identified by RT-PCR arii Northern blotting techniques. Expressing lines are used in fungal resistance analysis.
EXAMPLE 11: Resistance to Fungal pathogens - Transgenic plants transformei with a polynucleotide sequence encoding pyranosone dehydratase and/or polynucleotide sequence encoding glucan lyase.
Powdery Mildew Inoculation and Assessment:
A small population of tomato plants infected with powdery mildew is maintaine separately and used as the source of inoculum. Conidia from these plants is used t infect six-week old healthy control and transgenic plants. Plant leaves infected wit conidia of Erysiphe polygoni were excised and shaken above the top of the test plant to allow conidia to fall onto the healthy leaves (Fletcher & Smewiή, 1988, Plant Patho 37: 594-598). The temperature of the greenhouse is maintained at 25 to 30 DEG C. an the humidity at above 70%. The percentage of leaf area of the inoculated plants covere by conidia or necrotic lesions is recorded.
Germination of Spores of Powdery Mildew:
The effect of expression of the pyranosone dehydratase (PD) gene, either alone or i combination with expression of a glucan lyase gene on the germination of E. polygot spores and germ tube growth is evaluated by germinating the spores on selecte fransgenic plants known to express the appropriate fransgene, or contain just the empt pGreen T-DNA insert. Recently colonized leaves from control plants is flicked ov< petri dishes containing 0.7% agar. Attempts are made to allow conidia to distribul evenly on the agar surface. The petri dishes is then incubated at 25 DEG C. in the dar for 12 hr. Rates of germination is determined with the aid of a microscope and recorde at the end of 12 h. Resistance to Powdery Mildew:
The presence and number of whitish powdery spots, an indication of powdery mildew, present on the older leaves of both the confrol and fransgenic plants is recorded one week after the inoculation. The colonies are found mostly on the upper surface of the leaves but a few also appear on the lower surface and on the petioles and stems.
Preliminary results indicate that plants fransfected with PD or PD together with GL or PD-co would have a lower degree of infection compared with confrol plants.
EXAMPLE: 12 HANSENULA POLYMORPHA AS HOST FOR HETEROLOGOUS PROTEIN PRODUCTION:
H polymorpha possesses several strong promoters, for example the promoters of the alcohol oxidase (MOX), dihydroxyacetone synthase (DHAS) and formate dehydrogenase (FMD) genes. These three promoters are most often used to confrol heterologous protein expression. The promoters are fully repressed by glucose and derepressed by glycerol. They are strongly induced during growth on methanol. When grown on glycerol the MOX and DHAS promoters can reach 10-30% of the induced level while the FMD promoter can reach up to 60% of the induced level (2,5).
In this study the strain RBll (urd) and the shuttle vector pFPMT121 of H polymorpha has been used. RBll is an auxofrophic mutant that is deficient in orotidiene-5' -phosphate decarboxylase (URA3). The pFPMT 121 expression vector (see Figure 10) contains the URA3 gene of Saccharomyces cerevisie as a selection marker, an ampicillin resistance gene, an origin of replication of pBR322 for propagation in E. coli and an autonomously replicating sequence (HARS) that leads to amplification of the plasmid within the yeast cell. In addition the vector contains an expression cassette with the FMD promoter, linker sequences EcόRl-BglE-BamHl for insertion of the foreign gene and the MOX terminator. In H. polymorpha the plasmid integrates spontaneously into the genome in a tandem head-to-tail arrangement. When the FMD promoter is used up to 50 copies of the expression vector are integrated into the genome. This makes H. Polymorpha a very effective organism for production of heterologous proteins since a high-scale production can be obtained (see Gatzke R, Weydemann U, Janowicz ZA and Ηollenberg CP (1995). Stable multicopy integration of vector sequences in Hansenula polymorpha. Appl Microbiol Biotechnol 43 p.844- 849; Gellissen G (2002). Hansenula polymorpha - Biology and Applications. WILEY- VCH Chapter 8 and 17; Gellissen G (2000). Heterologous protein production in methylofrophic yeasts. Review. Appl Microbiol Biotechnol 54 p.741-750). The proteins can either be expressed infracellular or secreted into the culture media. With intracellular expression the proteins can be isolated from extracts after breakage of the cells. When proteins are secreted into the media the proteins are expressed as a pre- protein with a signal sequence that targets the protein into the secretory pathway. During the secretion process, the signal sequence is removed and the protein is secreted into the medium facilitating an easy purification of the protein (Gellissen G (2002). As above, Veenhuis M og van der Klei I (2000). The methylofrophic yeast Hansenula Polymorpha; a versatile cell factory. Enzyme and Microbial Technology 26 p.793-800).
EXAMPLE 13: CONSTRUCTION OF A GLUCAN LYASE EXPRESSION VECTOR
The Hansenula expression vector pFPMT121 was used to construct the glucan lyase expression vector (see Figure 10).
The glucan lyase gene was assembled using PCR using the following primers
US-agll: GAATTC ATGACC GCATTGTCC GAC AAA CAAACGGCT
LS-agl2: ACC CGGGGTAGAAGAGCC GGC AGCAAACCAGTT
US-agl5: GGGTGA GCTCTG CCACTT CCA GGGCTGCGC TGTTC
LS-agl6: GGA GAT CTT TAT TAA TGG TGA TGG TGA TGG TGG GTA ATT
GTGATCACA
GCGTCC GG The PCR protocol used is as follows: The 3' end of the glucan lyase gene was amplified using primers US-agl5 and LS-agl6, and the 5' end was amplified using US- agll and LS-agl2, and the respective PCR products were ligated into pCR-Blunt II- TOPO and transformed into TOP 10 E.coli cells using standard protocols (Strategene/Invitrogen). The 3' end product was excised from pCR Blunt using EcoRI and Bglll and ligated into pFPMT121 and transformed into TOP 10 E.coli cells, the resultant plasmid was cut with EcoRI to produce vector fragment 1. The 5 'end product was excised using EcoRI and Xmal to make insert fragment 1. Insert fragment 1. and vector fragment 1 were ligated using standard protocols to prepare the pFPMT121- glucan lyase expression vector. The PCR products were sequenced to ensure no errors had been introduced during the cloning strategy.
Preparation of Hansenula polymorpha competent cells:
The strain RBll (urd) was grown in 5 ml of YPD containing 2% peptone, 1% yeast extract and 2% glucose at 37°C with shaking over night. The culture was diluted 50- fold in 200 ml of prewarmed YPD and the culture was grown at 37°C to an OD66onm =l-0-1.3. The culture was transferred to a centrifuge tube and the cells were harvested by centrifugation at 3000 rpm for 5 minutes at room temperature. The cells were resuspended in 20 ml of PPD buffer (prewarmed to 37°C) (See Appendix 2 for used buffers) and incubated for 15 minutes at 37°C. Cells were harvested by centrifugation at 3000 rpm for 5 minutes at room temperature. The cells were washed three times with 50 ml of STM buffer. After last wash and centrifugation the cells were put on ice and resuspended in 1 ml of ice-cold STM buffer. Batches of 60 μl cell suspensions were transferred to storage tubes and directly frozen in liquid N2 and kept at -80°C. Transformation of gene constructs in Hansenula polymorpha by electroporation:
The constructs were transformed into H. polymorpha by electroporation in which the cells get an electric pulse that perforates their cell walls and facilitates the uptake of foreign DNA. 1 μg DNA of each gene construct was used for the transformations. DNA of pFPMT121 without insert and sterile distilled water were transformed as positive and negative control, respectively. The DNA was added to 60 μl of RBll competent cells and the mixture was transferred to a prechilled 2-mm electroporation cuvet that was kept on ice until electroporation. The genepulser was adjusted to 1.5 kV, 25 μF, 200 Ω so it was ready to fire. Immediately after the pulse 1 ml of YPD medium was added to the cuvet. The cell suspension was incubated at 37°C for one hour and transferred to eppendorf tubes. The cells were harvested by centrifugation at 3600 rpm for 5 minutes. The cells were washed twice with YND medium (0.14 % yeast nitrogen base without amino acids and ammonium sulfate, 2% ammonium sulfate, 2% glucose (2% agarose was added for plates)) and resuspended in 0.5 ml of YND. The samples were plated on YND plates and incubated at 37°C. Transformants appeared on the plates after 3-5 days.
Integration of the construct into the genome of H. polymorpha requires time and proper conditions. From the YND-plates transformants were inoculated in 3 ml YND and grown at 37°C with shaking for two days. As a confrol 5 transformants of vector DNA were also picked. Every second day 50 μl of cells were fransferred to 3 ml fresh YND (repeated 7 times). After the seventh passage 50 μl of cells were transferred to 3 ml YPD and grown over night (repeated once). 20 μl of cells were transferred to 3 ml YND and streaked on YND-plates. The plates were incubated at 37°C until transformants appeared. This passaging of transformants allows stabilization of the construct that initially exists as a free replicating plasmid and results in forced integration into the chromosomal DNA (1,2). One colony from each YND-plate was inoculated in 3 ml YPD and grown overnight at 37°C with shaking. To induce the expression of the integrated constructs 100 μl of cells were transferred to 3 ml YND containing 1% glycerol and grown for two days at 37°C. Transformants were screened using PCR using primers US3-alcore and LS4.
1.0 ml of cell culture was pelleted by centrifugation in a microcentrifuge tube. The supernatant was decanted. One third of the tube was filled with acid washed glass beads (425-600 microns) and 400 μl of 0.1 M MOPS-NaOH (ρH-6.2) was added. The cells were opened by shaking in a Mini Bead-Beater (Biospec Products, Bartlesville, OK) 4 times 20 seconds at maximum speed. With a hot glowing needle a hole was made in the bottom of each microcentrifuge tube and the tubes were placed in eppendorf tubes. The tubes were centrifuged at low speed so the cell-free exfracts were transferred to the eppendorf tubes and the glass beads were retained in the microcentrifuge tubes.
In a PCR tube 10 μl of cell-free exfract was mixed with 50 pmole of primers, 1 μl of each dNTP, 10 μl of AmpliTaq DNA Polymerase Buffer, 1 U of AmpliTaq DNA Polymerase and water to a final volume of 50 μl. After preheating for 30 seconds at 95°C the PCR-program consisted in 30 cycles of 95°C for 30 seconds, 55°C for 1 minute and 68°C for 2 minutes and 5 minutes at 72°C extension at the end. The PCR products were loaded on 2% agarose gels to check the size of the products. The two primers US3-aglcore and LS4-aglcore were used for the PCR-screening
US3-aglcore: GGA GAT ACT ACC TGG AAC TCT GGA CAA GAG GAC LS4-aglcore: GTT TGG ATC CCC GCC AGT ACC CAC
Intracelluar protein expression was determined using western blot analysis using polyclonal antibodies raised against the glucan lyase protein and raised in rabbit, and conjugated swine anti-rabbit immunoglobulin (DAKA A/S) using standard techniques.
Hansenula colonies which were positive by both PCR and western blot analysis were further analysed to determine their glucan lyase activity using the DNA glucan lyase assay and standard Biorad™ protein Assay to determine the total protein concenfration (Yu S et al (1998) Carbohyrdate Research 305, pρ73-82). Transformant Specific activity P Prrootteeiinn concentration
(μmol 1,5-anhydrofrucose/min ml) (mg/ml)
Figure imgf000179_0001
Table: Eight fransformants from transformation of the full-length glucan lyase gene showed a high glucan lyase activity when assayed by the DNS method. The protein concenfration was determined by the BioRad protein assay. The specific activity and the protein concenfration is the average of four independent measurements.
The specific activity of glucan lyase is defined as mol 1,5-anhydrofructose produced per minute per ml enzyme solution or as mol 1,5-anhydrofructose produced per minute per mg protein.
The specific activity can be determined by using the absorbance measured at 550 nanometers by the DNS method: 1,5-anhydrofructose (mol) = 0.69577 (OD550nm - Blank) + 0.05978. The specific activity of glucan lyase = 1,5-anhydrofructose (mol)/(time (min) x ml cell-free exfract used). 1 activity unit is defined as the amount of enzyme needed to produce 1 mol 1,5-anhydrofructose per minute under optimal assay conditions (pH=4 and a temperature of 45C and glycogen as substrate (final 2%). The protein concentration is determined by using the protein standard curve. Protein concentration (mg/ml) = (15.558 OD595nm - 0.0445)/x 1 cell-free extract.
Growth experiment: To investigate if expression of glucan lyase could be optimised at other temperatures two transformants from transformation of the full-length glucan lyase gene were grown at three different temperatures: 24°C, 30°C and 37°C. In 500 ml shake flasks with baffles 100 ml of YND with 1% glycerol were inoculated with YPD culture so an OD=l was obtained. The shake flasks were incubated with shaking for five days. As a confrol one transformant from transformation of vector DNA was also grown under the same conditions.
Transformant number 2 and 8 that expressed a high level of glucan lyase were grown for 5 days at 24°C, 30° C and 37°C in order to examine if the expression of glucan lyase could be optimised at other temperatures. Each day the growth was followed by measuring OD600nm and activity screening and BioRad protein assay were done. Figure 11 shows the specific activity for fransformant number 2 during the five days of growth. The curves clearly indicate that the expression of glucan lyase is temperature dependent and can be optimised when the temperature is lowered to 24°C or 30°C. After only two days of growth at 24°C or 30°C an almost 10-fold increase in the specific activity was observed compared with growth at 37°C. Similar results were obtained when growing fransformant number 8 under the same conditions and temperatures.
Purification of recombinant algal a-l,4-glucan lyase:
The recombinant algal α-l,4-glucan lyase expressed in H. polymorpha was purified by affinity chromatography on a starch column connected to a FPLC. Two transformants from fransformation of the full-length glucan lyase gene were grown in 250 ml of YND+1% glycerol in 2 L shakeflaks with baffles at 24°C with shaking. The cultures were inoculated with cells grown in YND+2% glucose so an OD=l was obtained in the new media. On the second day of growth the cultures were induced with 1% methanol. After three days of growth the cells were harvested by centrifuging for 10 minutes at 4000 rpm. The cells were resuspended in 5 mM sodium acetate pΗ=5.5 with 0.2% LTAB at 30% wet biomass in order to lyse the cells and release the intraceUular glucan lyase. The tubes were mcubated over mght with shaking at 7"C The cells were harvested by centrifuging at 4000 rpm in 10 minutes and the glucan lyase activity was determined in the cell-free exfract and in the pellet. This was done to check if LTAB had opened the cells successfully before starting the purification of glucan lyase.
Glucan lyase was purified by affinity chromatography on a starch column connected to a Fast Protein Liquid Chromatography system (FPLC). An AKTA explorer 10S from Pharmacia Biotech was used and it measured the absorbance at 260 nm and 280 nm. 1.5 g of starch/mg glucan lyase resuspended in 5 mM potassium acetate pH=4 was used to pack a column with a diameter of 1.6 cm and a volume of 23 ml. The column was equilibrated with 5 mM potassium acetate pH=4. The cell-free extract was adjusted to pH=4 and loaded on another column. Both columns were connected to the AKTA. Before starting the purification the system was washed with sterile distilled water and the pumps were washed with 5 mM potassium acetate pH=4 and 20 mM Bis-Tris-HCl pH= 6.6 + 2% dextrinlO (elusion buffer). The starch column was equilibrated with 5 column volumes of 5 mM potassium acetate pH=4. Then the cell-free exfract was loaded automatically on the starch column and the column was washed with 5 column volumes of 5 mM potassium acetate pH=4. Glucan lyase was eluted with 20 mM Bis- Tris-HCl pH=6.6 with 2% dextrinlO in fractions of 1 ml. The fractions with a high absorbance at 260 and 280 nanometers were tested for glucan lyase activity and the fractions with highest activity were collected into three large fractions. The three fractions were separately concentrated with a Centriprep YM-30 from Millipore by centrifuging at 1500 rpm at 4°C so molecules smaller than 30 kDa were removed. The three fractions were mixed and filtrated, and the glucan lyase was purified by ion- exchange chromatography on a MonoQ column (an anion exchange column from Pharmacia Biotech) connected to the same FLPC system as used above. The column was equilibrated with 10 mM Bis-Tris-HCl pH=7 and the glucan lyase fractions were injected into the system with a needle. The column was washed with 10 mM Bis-Tris- HCl pH=7 and the glucan lyase was eluted with 10 mM Bis-Tris-HCl pH=7 + 1 M NaCI. The fractions with a high absorbance at 260 nm and 280 nm were tested for glucan lyase activity. Two fractions with high glucan lyase activity were concentrated separately with a Cenfriprep YM-10 from Millipore by centrifuging at 3000 rpm at 4°C where the molecule cutoff was 10 kDa.
LTAB Greatly increases recoverable yield of glucan lyase from Hansenula polymorpha
Two fransformants that expressed a high level of glucan lyase (fransformant number 2 and 8) were grown at 24°C (2 cultures of each fransformant were started). To compare the specific activity under repressed and induced conditions samples were collected when the cells were grown in YND+2% glucose (repressed) and in YND+1% glycerol with 1% methanol added on the second day of growth (induced). So far activity screening of all fransformants and in the growth experiment has only been determined in cell-free exfracts prepared by opening the cells mechanically on a Mini Bead-Beater. This is very time-consuming and a very slowprocess especially when you have many samples. The chemical reagent LTAB (Lauroyl Trimethyl Ammonium Bromide) was used to open the cells efficiently. Figure 12 shows the ELISA-plate from the activity screening by the DNS method of the repressed and induced extracts. The glucan lyase activity was much stronger when the induced cells were opened with LTAB compared with opening of the cells on a Mini Bead-Beater (E1-E12 compared with C1-C12). The specific activity was almost 60-fold higher in the case of LTAB-treated cellsindicating that this is a much more effective way of releasing intracellular proteins in H polymorpha (See figure 7). When the cells were grown in YND + 2% glucose a very low specific activity was Observed as expected since the FMD promoter is repressed in this media. The pellet from the LTAB opening was resuspended in 0.1 M MOPS- NaOH pH=6.2 and also assayed to check if some glucan lyase was still bound in the pellet. The assay detected a high glucan lyase activity in the pellet. A second round of LTAB incubation of the pellet did not release the glucan lyase so it is possible that the protein is bound to membranes.
The cell-free exfract from the LTAB treated cells was used to purify the recombinant algal α-1,4- glucan lyase by FPLC on a starch column. The glucan lyase was eluted with 20 mM Bis-Tris-HCl pH=6.6 + 2% dextrinlO and a broad peak in the absorbance at 260 nm and at 280 was observed (See Appendix 8). Fraction 21-40 was tested tor glucan lyase activity and the fractions with highest specific activity were collected into three larger fractions: Fraction I (fractions 21-26), fraction II (fraction 27-32) and fraction III (fraction 32-38) with fraction II having the highest specific activity. The purification of glucan lyase resulted in a yield of 61% and a fold of purification of 1.43 (see the table below).
Figure imgf000183_0001
on a starch column.
Calculations in table 2:
Cell-free-exfract: Total activity = 21 ml 58.95 mol 1,5-anhydrofructose/min ml = 1237.95 mol 1,5-anhydrofructose/min = 1237.95 U. Total protein = 21 ml 0.56 mg protein/ml = 11.76 mg protein. Specific activity = 1237 U/11.76 mg protein = 105.26 U/mg
Starch column: The total activity is calculated as the sum of the activity in fraction I, II and III:
Total activity fraction I = 6 ml 12.57 mol 1,5-anhydrofructose/min ml = 75.42 U Total activity fraction II = 6 ml 77.80 mol 1,5-anhydrofructose/min ml = 466.8 U Total activity fraction III = 6 ml 35.94 mol 1,5-anhydrofructose/min ml = 215.64 U
Total activity (average) = 757.86 U
Total protein is calculated as the sum of amount of protein in fraction I, II and III: Total protein fraction I = 6 ml 0.07 = 0.42 mg Total protein fraction 11 = 6 ml 0.54 = 3.24 mg Total protein fraction III = 6 ml 0.23 = 1.38 mg
Total protein (average) = 5.04 mg
Specific activity = 757.86 U/5.04 mg = 150.37 U/mg. Fold = Specific activity (step x)/specific activity (step 1). Fold = (150.37 U/mg)/(105.26 U/mg) = 1.43. Yield = (Total activity (step x)/total activity (step 1)) 100%. Yield = (757.86 U/1237.95 U). 100% = 61%
The three fractions were concentrated with a Centriprep YM-30 and the purity of the glucan lyase was analysed by native PAGE (Figure 14).
MALDTI-TOF Analysis
Prior to MALDI-TOF mass specfrometry the glucan lyase in the three fractions was further purified by FPLC on an ion-exchange column in order to remove dextrinlO from the elution buffer. The glucan lyase was eluted from the ion-exchange column with 10 mM Bis-Tris-HCl pH=7 + 1 M NaCI and activity screening of fraction B2-B6 revealed that all glucan lyase had been eluted in fraction B4 and B5. These two fractions were concentrated with a Centriprep YM-10 and the buffer was changed to 10 mM Bis-Tris-HCl pH=7 to remove the salts. A few microliter of fraction B5 was further desalted prior to the mass specfrometry analysis. The molecular weight of the purified glucan lyase was determined as 115722±57Da. This purified algal α-1,4- glucan lyase from H polymorpha has a smaller molecular weight than the algal α-1,4- glucan lyase purified from Aspergillus niger which has a molecular weight of 117030 Da as determined by MALDI-TOF mass specfrometry.
N-terminal Sequencing
The purified glucan lyase was analysed by N-terminal sequencing on an Applied Biosystems 476 A Protein Sequencer. The N-terminal sequencing of the purified glucan lyase resulted in a sequence of 20 amino acids (GSTDNPDGIDYKTYDYV GVW) that was 100% identical with the wild type algal glucan lyase. Surprisingly the glucan lyase irom tl. polymorpha is very active even though the N-terminal is 11 amino acids shorter than the wild type protein.
N-terminal sequence
Figure imgf000185_0001
Table. The N-terminal sequence of the wildtype algal α-l,4-glucan lyase and of the algal α-l,4-glucan lyase from H. polymorpha. An Applied Biosystems 476A Protein Sequencer was used for the N-terminal sequencing.
The shorter N-terminal observed in the glucan lyase from H. polymorpha can explain the lower molecular weight determined by MALDI-TOF mass specfrometry. The molecular weight of the 11 amino acids in the N-terminal is 1088 Da. Thus, the molecular weight of the glucan lyase from H. polymorpha is expected to be 115942 Da (117030 Da - 1088 Da) which is consistent with the molecular weight of 115722 Da determined by MALDI-TOF mass specfrometry.
EXAMPLE 14: THE HANSENULA EXPRESSION VECTOR PFPMT121 IS USED TO CONSTRUCT A PYRANOSONE DEHYDRATASE (PD) EXPRESSION VECTOR.
The PD cDNA gene is obtained by the methodology listed in Example 9 assembled and is inserted into pFPMT121 using the protocols described in Example 13.
Hansenula colonies containing PD expression constructs are identified using standard PCR protocols.
Active PD enzyme is obtained using LTAB to break the cells as described in the glucan lyase example above. EXAMPLE 15: CO-EXPRESSION OF GLUCAN LYASE AND PYRANOSONE DEHYDRATASE (PD) IN HANSENULA POLYMORPHA.
The pFPMT121 expression vectors for both glucan lyase and PD are co*-transformed using the electroporation protocol for Hansenula (see Example 13 above). Alternatively a single double expression vector containing both a glucan lyase expression cassette and a PD expression cassette are made using standard cloning techniques.
Hansenula colonies containing both PD and GL expression constructs are identified using standard PCR protocols.
Active PD and GL enzyme is obtained using LTAB to break the cells as described in the glucan lyase example above.
Mention is made of U.S. applications Serial Nos. 10/283,940 (attorney ref.
P11937US), 10/283,988 (attorney ref. P12627US), 10/283,987 (attorney ref.
P11933US), 10/283,936 (attorney ref. P11934US), and 10/283,963 (attorney ref. PI 1938US) each filed on 30 October 2002.
Mention is also made of U.S. provisional applications Serial Nos. 60/343,485 (attorney ref. P11937USO), 60/343,313 (attorney ref. P12627USO), 60/343,447 (attorney ref. P11933USO), 60/343,368 (attorney ref. P11934USO), and 60/343,316 (attorney ref. PI 1938USO) each filed 21 December 2001.
Mention is also made of UK patent application Nos. 0126164.3 (attorney ref. PI 1937GB), 0126163.5 (attorney ref. P12627GB), 0126165.0 (attorney ref. PI 1933 GB), 0126186.6 (attorney ref. PI 1934 GB), and 0126162.7 (attorney ref. PI 1938 GB), each filed on 31 October 2001.
Mention is also made of UK patent application Nos. 0226159.2 (attorney ref. PI 5628GB) filed on 8 November 2002, 0310479.1 (attorney ref. P15628GBR) filed on 7 May 2003, 0306315.3 (attorney ref. P16678GB) filed on 19 March 2003, and 0310480.9 (attorney ref. P16679GB) filed on 7 May 2003.
Mention is also made of U.S. provisional applications Serial No. 60/468,954 (attorney ref. P15628USO) filed on 7 May 2003.
Each of these applications, together with any document cited or referenced in each of these applications, is hereby incorporated herein by reference. All documents cited herein and all documents cited or referenced in herein cited documents (including any manufacturer's specifications, instructions, etc. as to products mentioned herein or in documents cited or referenced in herein cited documents) are hereby incorporated herein by reference.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following.

Claims

1. A polynucleotide comprising a polynucleotide sequence encoding a polypeptide with pyranosone dehydratase activity.
2. A polynucleotide according to claim 1 wherein said polypeptide comprises at least one amino acid sequence selected from the following:
(i) KPHCEPEQPAALPLFQPQLVQGGRPDXYWVEAFPFRSDSSK (SEQ ID No. 2 or
KPHXEPEQPAALPLFQPQLVV(Q)GGRPDXY (SEQ ID No. 3); (ii) SDIQMFVNPYATTNNQSSXWTPVSLAKLDFPVAMHYADITK (SEQ ID No. 4);
(iii) VSWLENPGELR (SEQ ID No. 5); (iv) DGVDCLWYDGAR (SEQ ID No. 6); (v) PAGSPTGIVRAEWTRHVLDVFGXLXXK (SEQ ID No. 7); (vi) HTGSIHQVVCADIDGDGEDEFLVAMMGADPPDFQRTGVWCYK (SEQ ID No. 8);
(vii) TEMEFLDVAGK (SEQ ID No. 9); (viii) KLTLVVLPPFARLDVERNVSGVK (SEQ ID No. 10); (ix) SMDELVAHNLFPAYVPDS VR (SEQ ID No. 11 );
(x) NDATDGTPVLALLDLDGGPSPQAWNISHVPPGTDMYEIAHAK (SEQ ID No. 12);
(xi) TGSLVCARWPPVK (SEQ ID No. 13); (xii) NQRVAGTHSPAAMGLTSRWAVTK (SEQ ID No. 14); (xiii) GQITFRLPEAPDHGPLFLSVSAIRHQ (SEQ ID No. 15); where X is an unknown amino acid residue; or a variant, homologue or derivative thereof.
3. A polynucleotide according to claim 2, wherein said polynucleotide encodes for an amino acid comprising at least two, suitably at least three, suitably at least four, suitably at least five, suitably at least six, suitably at least seven, suitably at least eight, suitably at least nine, suitably at least ten, suitably at least eleven, suitably at least twelve, or suitably at least thirteen, of the amino acids shown as SEQ ID Nos. 2-15, or a variant, homologue or derivative thereof.
4. A polynucleotide according to any one of the preceding claims selected from one or more of:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 1 or
SEQ ID No. 20 or the compliment thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No,
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
5. A polynucleotide according to any one of the preceding claims selected from one or more of:
(i) a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 1 or
SEQ ID No. 20; (ii) a polynucleotide which hybridises to the nucleotide sequence shown in SEQ ID
No. 1 or SEQ ID No. 20 under medium stringency, preferably high stringency, conditions; (iii) a polynucleotide which is at least 70%, preferably at least 75%, preferably at least
80%, preferably at least 85%, preferably at least 90%, preferably at least 95% homologous with SEQ ID No. 1 or SEQ ID No. 20, (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No.
20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv).
6. A polynucleotide according to any one of the preceding claims wherein said polynucleotide is obtainable from one or more of Phanerochaete chrysosporium, Polyporus obtusus or Corticium caeruleum.
7. A polynucleotide according to any one of the preceding claims wherein said polynucleotide is obtainable from an organism in the order of Pezizales, Auriculariales, Aphyllophorales, Agaricales or Gracilariales.
8. A polynucleotide according to the present invention wherein said polynucleotide is obtainable from one or more of Aleuria aurantia, Peziza badia, P. succosa, Sarcophaera eximia, Morchella conica, M. costata, M. elata, M. esculenta, M. esculenta var. rotunda, M. hortensis, Gyromitra infula, Auricularia mesenterica, Pulcherricium caeruleum, Peniophora quercina, Phanerochaete sordida, Vuilleminia comedens, Stereum gausapatum, S. sanguinolentum, Lopharia spadicea, Sparassis laminosa, Boletopsis subsquamosa, Bjerkandera adusta, Trichaptum biformis, Cerrena unicolor, Pycnoporus cinnabarinus, P. sanguineus, Junghunia nitida. Ramariafiava, Clavulinopsis helvola, C. helvola var. geoglossoides, V. pulchra, Clitocybe cyathiformis, C. dicolor, C. gibba, C. odora, Lepista caespitosa, L inversa, L. luscina, L. nebularis, Mycena seynii, Pleurocybella porrigens, Marasmius oreales, Inocybe pyriodora, Gracilaria varrucosa, Gracilaria tenuistipitata, Gracilariopsis sp, Gracilariopsis lemaneiformis. Melanosopora spp., Melanospora ornata, Microthecium spp., Microthecium compressum, Microthecium zobelii.
9. A constract comprising a polynucleotide encoding pyranosone dehydratase according to any one of the preceding claims.
10. A constract according to claim 9 wherein said constract comprises a polynucleotide sequence encoding a further enzyme.
11. A construct according to claim 10 wherein said further enzyme is glucan lyase..
12 A constract according to claims 9 wherein said further enzyme is pyranose-2- oxidase.
13. . An expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to any one of claims 1-8 operably linked to one or more regulatory sequences capable of directing expression of said polynucleotide in a host cell or organism.
14. An expression vector according to claim 13 wherein said expression vector further comprises a polynucleotide sequence encoding a further enzyme, wherein said polynucleotide sequence encoding said further enzyme is operably linked to one or more regulatory sequences capable of directing expression of said polynucleotide in a host cell or organism.
15. An expression vector according to claim 14, wherein said polynucleotide sequence encoding pyranosone dehydratase and said polynucleotide sequence encoding said further enzyme are both operably linked to the same one or more regulatory sequences.
16. An expression vector according to claim 14 or claim 15 wherein said further enzyme is glucan lyase.
17. An expression vector according to claim 14 or claim 15 wherein said further enzyme is pyranose-2-oxidase.
18. A host cell or host organism into which has been incorporated any one of the polynucleotide according to any one of claims 1-8 or the constract according to any one of claims 9-12 or the expression vector according to any one of claims 13 to 17.
19. A host organism according to claim 19 which is a yeast.
20. A host organism according to claim 19 which is a plant.
21. A host organism according to claim 20 which is a fransgenic plant.
22. A host organism according to claim 21 which further comprises a heterologous secretion sequence.
23. A host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to any one of claim 1-8 and an expression vector comprising a polynucleotide sequence encoding glucan lyase.
24. A host cell or host organism into which has been incorporated an expression vector comprising a polynucleotide sequence encoding pyranosone dehydratase according to any one of claims 1-8 and an expression vector comprising a polynucleotide sequence encoding pyranose-2-oxidase.
25. A method of preparing one or more antimicrobial compound in situ in an organism which comprises cultivating a host cell or host organism according to any one of claims 18-24, under conditions to provide for expression of at least pyranosone dehydratase; whereby said pyranosone dehydratase converts constituents present in the host cell or host organism, such as 1,5-anhydrofructose, starch dextrins, glucose or glucosone for example, into one or more antimicrobial compounds.
26. A method according to claim 25, wherein the antimicrobial compound is one or more of microthecin and/or APP and/or cortalcerone.
27. A method according to claim 25 or claim 26, wherein the method further comprises exposing said host cell or host organism to 1,5-D-anhydrofructose.
28. A method according to claim 25 or claim 26 wherein the method fiirther comprises exposing said host cell or host organism to glucosone.
29. A method of preparing microthecin in situ which comprises
(a) cultivating a host cell or host organism into which has been incorporated a polynucleotide selected from: (i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or
SEQ ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID
No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID
No. 1 or SEQ ID No. 20; and (v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv) under conditions to provide for expression of pyranosone dehydratase; and (b) exposing said host cell or host organism to 1 ,5-D-anhydrofructose.
30. A method of preparing cortalcerone in situ which comprises
(a) cultivating a host cell or host organism into which has been incorporated a polynucleotide selected from:
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1 or
SEQ ID No. 20 or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 20, or a fragment thereof ; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridising to the complement of the nucleotide sequence of SEQ ID. No. 1 or SEQ ID No. 20; (iv) a polynucleotide comprising a polynucleotide sequence which is degenerate as a result of the genetic code to the polynucleotide of SEQ ID No. 1 or SEQ ID No. 20; and
(v) a polynucleotide comprising a nucleotide sequence capable of hybridising to a polynucleotide of (iv); under conditions to provide for expression of pyranosone dehydratase; and
(b) exposing said host cell or host organism to glucosone.
31. A method according to any one of claims 25-23 wherein the host cell is a plant or a yeast.
32. A method of preparing one or more antimicrobial compounds in situ in a host cell or in a host organism, comprising fransforming a host cell or host organism with a polynucleotide encoding pyranosone dehydratase according to any one of claims 1-8, and culturing the fransformed host cell or host organism under conditions to provide for expression of the polynucleotide such that the pyranosone dehydratase so produced converts constituents in and/or around the host cell or host organism to one or more antimicrobial compounds.
33. A method according to claim 32 wherein the antimicrobial compound is one or more of microthecin, cortalcerone or ascopyrone P (APP).
34. A method according to claim 32 or claim 33 comprising fransforming the host cell or host organism with a further polynucleotide sequence encoding an enzyme.
35. A method according to claim 34, wherein the enzyme is one or more of glucan lyase or pyranosone-2-oxidase.
36. A method according to claim 32 or claim 33 comprising culturing the host cell or host organism in a medium comprising a constituent which is a subsfrate for the pyranosone dehydratase.
37. A method according to claim 35 comprising culturing the host cell or host organism in a medium comprising one or more constituents which is/are a subsfrate for the pyranosone dehydratase and/or glucan lyase and/or pyranose-2-oxidase.
38. A method according to any one of claims 32-37 wherein the host cell or host organism is a plant cell or a plant.
39. A method of preventing and/or inhibiting the growth of, and/or killing a microorganism in or on a fransgenic organism, comprising fransforming said orgamsm with a polynucleotide encoding pyranosone dehydratase according to any one of claims 1- 8, and growing said fransformed plant.
40. A method according to claim 39 comprising transforming the organism with a further polynucleotide sequence encoding an enzyme.
41. A method according to claim 40, wherein the enzyme is one or more of glucan lyase or pyranosone-2-oxidase.
42. A method according to any one of claims 39-41 comprising contacting the fransgenic organism with a constituent which is a subsfrate for the pyranose dehydratase.
43. A method according to claim 41 comprising contacting the fransgenic organism with a constituent which is a subsfrate for the pyranose dehydratase and/or the glucan lyase and/or the pyranosoe-2-oxidase.
44. A method according to any one of claims 39-43 wherein the host cell or host organism is a plant cell or a plant.
45. A method according to any one of claims 39-43 comprising contacting said plant with 1,5-D-anhydrofructose and/or glucosone.
46. A method according to any one of claims 39-43 wherein the microorganism is pathogen.
47. A method of preparing a fransgenic organism or part thereof, which organism or part thereof is resistant to one or more pathogens, comprising fransforming a cell of the organism with a polynucleotide sequence encoding pyranosone dehydratase according to any one of claims 1-8 or with a construct according to any one of claims 9-12 or with a expression vector according to any one of claims 13-17, whereby the fransformed organism produces one or more antimicrobial compounds from constituents present in the cell of the organism.
48. A method according to claim 47, wherein the the antimicrobial agent is one or more of of microthecin, cortalcerone or ascopyrone P (APP).
49. A method according to claim 47 or claim 48 comprising fransforming the organism or part thereof with a further polynucleotide sequence encoding an enzyme.
50. A method according to claim 49, wherein the enzyme is one or more of glucan lyase or pyranosone-2-oxidase.
51. A method according to claim 47 or claim 48 comprising culturing the organism or part thereof in a medium comprising a constitaent which is a subsfrate for the pyranosone dehydratase.
52. A method according to claim 50 comprising culturing the organism or part thereof in a medium comprising one or more constituents which is/are a substrate for the pyranosone dehydratase and/or glucan lyase and/or pyranose-2-oxidase.
53. A method according to any one of claims 47-52 wherein the organism or part thereof is a plant or part thereof.
54. A method according to any one of claims 47-53 wherein said constitaent is selected from one or more of the following constituents: α-l,5-anhydrofructose, starch, starch dextrins, glucose, glucosone.
55. A method according to any one of claims 47-54, wherein said pathogen is one or more of the fungal pathogens selected from the following: downey mildew, powdery mildew, Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora. Thielaviopsis, Aspergillus, Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum Diplodia; Erysiphe; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria, Peronospora; Phoma; Phymatotrichum; Plasmopara; Podosphaera; Puccinia; Puthium. Pyrenophora; Eutypa; Pyricularia; Scerotium; Sclerotinia; Septoria; Uncinula, Venturia; Verticillium, Alternaria spp , Albugo spp., Aphanomyces spp , Amyloporia spp. Ascochyta, Aspergillus, Basidiophora, Bipolaris, Botrytis, Bremia, Cladosporium, Claviceps, Coniophora spp., Diplocarpon spp., Donkioporia spp., Drechslera, Eutypa, Fibroporia spp., Fusarium, Geotrichum, Guignardia, Gymnosporangium, Hemileia, Kabatiella, Leptosphaeria, Marssonina spp., Monilinea, Merulus, Mycosphaerella, Paracercospora, Penicillium, Peronophythora, Peronospora, Phellerias spp., Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmophora, Podosphaera, Porai spp., Pseudocercosporella, Pseudoperonospora, Puccinia, Pyrenophora, Pythium, Rhizoctonia, Rhizopus, Sclerophthora, Serpula spp., Sphaerotheca, Stagonospora, Taphrina, Thielaviopsis, Tilletia, Trichoderma andUstilago.
56. A method according to any one of claims 47-55, wherein said pathogen is one or more of the fungal pathogens selected from the following: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Rhizoctonia, Pythium, Aphanomyces, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), Mycosphaerella fijiensis, Paracercospora fijiensis, Ascomycetes spp, Leptosphaeria maculans and Eutypa lata.
57. A method according to any one of claims 47-56 wherein the fransgenic plant is one of the following: a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, apple, pear, sfrawberry, pea, tomato, grape, Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, oilseed rape, sunflower, soybean, peanut, carrot, and timber trees such as poplar.
58. A fransgenic organism or part thereof which is resistant to one or more pathogens, which organism or part thereof comprises a heterologous polynucleotide sequence encoding pyranosone dehydratase according to any one of claims 1-8.
59. A fransgenic organism or part thereof according to claim 58 wherein said plant or part thereof comprises one or more further polynucleotide sequences encoding one or more further enzymes, which enzyme(s) when expressed in the plant in combination with the pyranosone dehydratase produces one or more antimicrobial compounds, such as microthecin, cortalcerone and/or APP, from constituents present in the plant cell.
60. A fransgenic organism or part thereof according to claim 59 wherein said further nucleotide sequence encodes a glucan lyase enzyme and/or a pyranose-2-oxidase enzyme.
61. A fransgenic organism or part thereof according to any one of claims 58-60, wherein said pathogen is one or more of the following fungal pathogens: downey mildew, powdery mildew, Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora, Thielaviopsis, Aspergillus, Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria; Peronospora; Phoma; Phymatotrichum; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Eutypa; Pyricularia; Scerotium; Sclerotinia; Septoria; Uncinula; Venturia; Verticillium, Alternaria spp , Albugo spp., Aphanomyces spp , Amyloporia spp., Ascochyta, Aspergillus, Basidiophora, Bipolaris, Botrytis, Bremia, Cladosporium, Claviceps, Coniophora spp., Diplocarpon spp., Donkioporia spp., Drechslerd, Eutypa, Fibroporia spp., Fusarium, Geotrichum, Guignardia, Gymnosporangium, Hemileia, Kabatiella, Leptosphaeria, Marssonina spp., Monilinea, Merulus, Mycosphaerella, Paracercospora, Penicillium, Peronophythora, Peronospora, Phellerias spp., Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmophora, Podosphaera, Porai spp., Pseudocercosporella, Pseudoperonospora, Puccinia, Pyrenophora, Pythium, Rhizoctonia, Rhizopus, Sclerophthora, Serpula spp., Sphaerotheca, Stagonospora, Taphrina, Thielaviopsis, Tilletia, Trichoderma andUstilago.
62. A transgenic organism or part thereof according to any one of claims 58-61, wherein said pathogen is one or more of the following fungal pathogens: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Rhizoctonia, Pythium, Aphanomyces, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), Mycosphaerella fijiensis, Paracercospora fijiensis, Ascomycetes spp, Leptosphaeria maculans and Eutypa lata.
63. A fransgenic organism or part thereof according to any one of claims 58-62 wherein said organism is a plant.
64. A fransgenic organism or part thereof according to claim 63 wherein the plant is one of the following: a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, apple, pear, strawberry, pea, tomato, grape, Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, oilseed rape, sunflower, soybean, peanut.
65. Use of a polynucleotide sequence according to any one of claims 1-8 to produce an organism or part thereof which is resistant to one or more pathogens, particularly one or more fungal pathogens.
66. Use according to claim 65 wherein the polynucleotide sequence encoding pyranosone dehydratase is used in combination with a polynucleotide sequence encoding one or more of a glucan lyase or a pyranose-2-oxidase.
67. Use according to claim 65 or claim 66 wherein the organism is a plant.
68. A polynucleotide, constract, expression vector, host cell or. host organism substantially as described herein and with reference to the accompanying Examples.
69. A method of preventing and/or inhibiting the growth of, and/or killing a microorganism in or on an organism substantially as described herein with reference to the accompanying Examples.
70. A fransgenic organism substantially as described herein with reference to the accompanying Examples.
71. A method of preparing one or more of microthecin, cortalcerone or ascopyrone P (APP) in situ in a host cell or host organism substantially as described herein and with reference to the accompanying Examples.
PCT/GB2003/004594 2002-10-30 2003-10-24 Polynucleotide encoding a pyranosone dehydratase WO2004039993A1 (en)

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CN113234766A (en) * 2021-06-03 2021-08-10 淮北师范大学 Biosynthesis method of epsilon-polylysine by adding microbial elicitor
CN113943670A (en) * 2021-10-13 2022-01-18 中国农业科学院农业资源与农业区划研究所 Pseudomonas toralis for preventing diseases and promoting growth and application thereof
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CN114774580A (en) * 2022-06-02 2022-07-22 吉林农业大学 A kind of Molecular ID card of Ochre deer flower fungus and its amplification primer, detection kit and species identification method
CN115820686A (en) * 2022-08-22 2023-03-21 西南大学 A kind of citrus CsGSTU18 gene and its application

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CN105255953A (en) * 2015-10-09 2016-01-20 淮阴工学院 Method for pre-processing corn stalks through physical-chemical-biological method
CN105255953B (en) * 2015-10-09 2018-12-25 淮阴工学院 Physical-chemical-Biological Pretreatment corn stover method
US11319553B2 (en) 2017-11-29 2022-05-03 Rahan Meristem (1998) Ltd. Compositions and methods conferring resistance to fungal diseases
CN113234766A (en) * 2021-06-03 2021-08-10 淮北师范大学 Biosynthesis method of epsilon-polylysine by adding microbial elicitor
CN113234766B (en) * 2021-06-03 2022-04-05 淮北师范大学 Biosynthesis method of epsilon-polylysine by adding microbial elicitor
CN113943670A (en) * 2021-10-13 2022-01-18 中国农业科学院农业资源与农业区划研究所 Pseudomonas toralis for preventing diseases and promoting growth and application thereof
CN114774580A (en) * 2022-06-02 2022-07-22 吉林农业大学 A kind of Molecular ID card of Ochre deer flower fungus and its amplification primer, detection kit and species identification method
CN115820686A (en) * 2022-08-22 2023-03-21 西南大学 A kind of citrus CsGSTU18 gene and its application
CN115820686B (en) * 2022-08-22 2023-09-12 西南大学 Citrus CsGSTU18 gene and application thereof

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