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WO2017011879A1 - Mono-oxygénases et procédé de production de cinéol hydroxylé - Google Patents

Mono-oxygénases et procédé de production de cinéol hydroxylé Download PDF

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WO2017011879A1
WO2017011879A1 PCT/AU2016/050654 AU2016050654W WO2017011879A1 WO 2017011879 A1 WO2017011879 A1 WO 2017011879A1 AU 2016050654 W AU2016050654 W AU 2016050654W WO 2017011879 A1 WO2017011879 A1 WO 2017011879A1
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seq
cineole
ferredoxin
similar
polypeptide
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PCT/AU2016/050654
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English (en)
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Geoff DUMSDAY
Birgit UNTERWEGER
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Commonwealth Scientific And Industrial Research Organisation
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Priority claimed from AU2015902940A external-priority patent/AU2015902940A0/en
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Publication of WO2017011879A1 publication Critical patent/WO2017011879A1/fr

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • 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
    • 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 the field of biocatalysts and more specifically to the field of monooxygenases. More specifically, the present invention relates to methods for functionalizing 1 ,8-cineole and polynucleotides, polypeptides, host cells and vectors for doing the same. The invention also relates to biohydroxylated 1 ,8-cineole, which gives rise to 2-hydroxycineole.
  • 1 ,8-cineole (1 ,3,3-Trimethyl-2-oxabicyclo[2,2,2]octane) is a chemically stable saturated ditertiary ether. It is a bicyclic monoterpenoid and a major component of many essential oils, including eucalyptus oil. It possesses antimicrobial properties and a camphor-like scent. 1 ,8-cineole is an abundant natural resource, especially from the essential oil of several species of the genus Eucalyptus and from turpentine as a by-product of commercial pine oil production.
  • 1 ,8-cineole is relatively chemically inert as there are no activated C-H bonds in the molecule, it is a compound of small economic significance. It requires to be functionalised before it is suitable for further modification such as esterification, etc.
  • the product of functionalizing 1 ,8-cineole such as oxidised or hydroxylated-cineole derivatives are chiral compounds that can serve for example, as building blocks and precursors for uses in organic chemistry.
  • 1 ,8-cineole and its functionalised forms have applications for example in the manufacture of pharmaceuticals, insecticides, insect repellents, fragrances, cleaning products, and as flavouring in the food industry.
  • Examples of processes for producing functionalised 1 ,8-cineole using organic chemistry processes include multi-step syntheses using hot aqueous acids, multiple oxidative treatments, bromination, peracid treatments and other harsh or even hazardous chemical techniques. Free-radical reactions such as photochlorination yield a complex mixture of products. These chemical processes generally require the use of harsh reaction conditions and there is little to no control over the stereoisomers created during the chemical reaction. Furthermore, these chemical processes are usually complex and multi-stepped.
  • Biotransformation of 1 ,8-cineole can also occur within bacterial cells.
  • Bacterial 1 ,8-cineole metabolism was first discovered in the late 1970s when the isolation of Pseudomonas flava capable of growing on 1 ,8-cineole as a sole source of carbon was reported. Subsequently, 1 ,8-cineole biohydroxylation has also been observed in several other bacterial species including Rhodococcus C1 , Bacillus cereus, Citrobacter braakii, Novosphingobium subterranea and Sphingomonas capsulata.
  • One aspect of the invention provides an isolated polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof selected from the group comprising:
  • sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 2 or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 35 or SEQ ID NO: 36 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 32, or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 37 or SEQ ID NO: 38 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 34, or a biologically active fragment or equivalent thereof; and
  • the present invention is based in part on the identification and characterisation of Sphingobium yanoikuyae polypeptides that are capable of functionalizing 1 ,8-cineole.
  • the P450 monooxygenases of the present invention demonstrate stereoselectivity in that they functionalize 1 ,8-cineole preferably in the 2-position to produce the product 2-hydroxy cineole. Accordingly, in one embodiment the main product of 1 ,8-cineole hydroxylation is 2-hydroxy cineole.
  • substantially purified and/or recombinant polypeptide is selected from the group comprising:
  • the invention provides a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.
  • the invention provides a host cell comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described, and/or at least one plasm id comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.
  • the host cell expresses any one of the P450 monooxygenases of the present invention and facilitates the whole-cell biotransformation or functionalization of 1 ,8-cineole to 2-hydroxy-cineole or oxidises a hydroxy-1 ,8-cineole such as 6-hydroxy-1 ,8-cineole in the 2-position to form oxo- hydroxy-1 ,8-cineole.
  • the host cell may comprise one, two or three of the P450 monooxygenases of the present invention and expresses one, two or three of the P450 monooxygenases.
  • the host cell of the present invention is capable of metabolising further cineole-derivatives including but not limited to oxo-hydroxy-1 ,8-cineole, hydroxy-2,6-oxo-1 ,8-cineole from a hydroxyl 1 ,8 cineole such as (1 R)-6p-hydroxy-1 ,8-cineole.
  • the invention provides a host cell comprising a polynucleotide or vector as hereinbefore described and further comprising an electron transport partner such as but not limited to a ferredoxin (FdX) and/or a ferredoxin reductase (FdR).
  • the electron transport partners such as FdX and/or the FdR may be expressed on separate vectors.
  • a method of expressing a monooxygenase which functionalizes 1 ,8-cineole to a 2 hydroxy cineole comprising:
  • a method of functionalizing 1 ,8 cineole comprising contacting 1 ,8 cineole with an effective amount of a monooxygenase polypeptide as hereinbefore described to produce 2-hydroxycineole.
  • the monooxygenase polypeptide which contacts 1 ,8- cineole may be any one of the polypeptides of P450 B ui , P450 B u2 or P450 B u3 as hereinbefore described.
  • the invention provides a method of functionalizing 1 ,8 cineole said method comprising
  • a further aspect of the invention provides an isolated polynucleotide which encodes a ferredoxin or a biologically active fragment or equivalent thereof selected from the group comprising:
  • the invention provides an isolated polynucleotide which encodes a ferredoxin reductase or a biologically active fragment or equivalent thereof selected from the group comprising:
  • the invention provides a vector comprising at least one polynucleotide encoding a ferredoxin reductase as hereinbefore described.
  • Figure 1 shows a Coomassie-stained SDS-PAGE (4-12% Bis-Tris, MES running buffer) of PF (purified fraction) 1 and 2 after IEX (ion-exchange) and GF (gel filtration).
  • Lane 1 Molecular weight marker.
  • Lane 2 PF 1 after IEX.
  • Lane 3 and 4 PF 1 after GF.
  • Lane 5 PF 2 after IEX.
  • Lane 3 and 4 PF 2 after GF.
  • Figure 2 shows SDS-PAGE analysis of purified P450 proteins.
  • Figure 2A lane 1 molecular weight markers
  • lane 2 purified P450 B ui protein
  • lane 2 P450 B u3 protein
  • Figure 2B lane 1 molecular weight markers
  • lane 2 purified P450 B u2 protein.
  • Figure 3 shows absorbance spectra of (A) P450 BU i , (B) P450 BU2 and (C) P450 B u3 in 50 mM Tris, pH 7.4, of the oxidised (full black line), oxidised in the presence of 1 ,8-cineole (full gray line), reduced by the addition of sodium dithionite in the presence of 1 ,8-cineole (broken black line) and reduced and in complex with CO in the presence of 1 ,8-cineole (broken grey line) forms. All three P450s show the characteristic absorbance peaks and shifts.
  • Figure 4A shows a schematic representing the combination of the three expression vectors transformed into E. coli. Conversion of 1 ,8-cineole to 2- hydroxycineole using the various combinations of P450 BU i and ferredoxins found in the new strain of S. yanoikuyae.
  • Figure 4B shows a bar graph representing the efficiency of hydroxylating 1 ,8-cineole by E. coli transformed with each combination of P450 B ui with S. yanoikuyae ferredoxin FdX1 , FdX2, FdX3, FdX4, FdX5, FdX6 or FdX7.
  • the negative control is E. coli with "empty vectors".
  • the error bars represent one standard deviation from the mean.
  • Figure 5A shows a schematic representation of the plasm ids transformed into E. coli.
  • B Conversion of 1 ,8-cineole to 2-hydroxycineole using the various combinations of P450 B ui and ferredoxin reductases found in the S. yanoikuyae of the present invention.
  • Figure 5B shows a bar graph representing the efficiency of hydroxylating 1 ,8-cineole by E. coli transformed with each combination of P450 B ui with S. yanoikuyae ferredoxin reductase FdR1 , FdR2, FdR3, FdR4 or FdR4s.
  • the negative control is E coli with all three "empty vectors”.
  • the error bars represent standard deviation from the mean.
  • Figure 6 shows a bar graph representing the efficiency of each combination of P450 B ui with any one of S. yanoikuyae ferredoxin FdX1 , FdX2, FdX3, FdX4, FdX5, FdX6 and FdX7 and any one of S. yanoikuyae ferredoxin reductase FdR1 , FdR2, FdR3, FdR4 and FdR4s and its efficiency at hydroxylating 1 ,8-cineole to 2-hydroxycineole.
  • the negative control is E coli with all three "empty vectors".
  • the error bars represent one standard deviation from the mean.
  • Figure 7A shows the rates of hydroxy-cineole production by various combinations of P450 BU i with S. yanoikuyae ferredoxin reductases and ferredoxins. 1 ,8-cineole was added to the cell at 0 hours, 3 hours and 6 hours. The error bars represent one standard deviation from the mean.
  • Figure 8 show the rate of production of 2-hydroxycineole using recombinant P450 BU i in combination with FdR3 and FdX2 in a bioreactor.
  • Figure 9 shows the rate of production of 2-hydroxycineole using recombinant P450 BU2 in combination with FdR3 and FdX2 in a bioreactor when cultured at 30°C.
  • Figure 10 shows the rate of production of 2-hydroxycineole using recombinant P450 B u3 in combination with FdR3 and FdX2 in a bioreactor when cultured at 30°C.
  • Figure 1 1 shows the rate of production of 2-hydroxycineole using recombinant P450 B u3 in combination with FdR3 and FdX2 in a bioreactor when cultured at 20°C.
  • Figure 12 shows production of cineole derivatives in a bioreactor using P450 B ui-FdR3-FdX2 at 30°C using Terrific broth as the growth medium.
  • the substrate used was (1 R)-6B-hydroxy-1 ,8-cineole. Samples of the culture were taken just prior to and just after addition of the substrate and when the culture was ended.
  • Figure 14 shows the polynucleotide and polypeptide sequences of P450 B ui , P450 B u2 and P450 BU 3, as well as the T7 sequencing primers suitable amplifying the cloned sequences from pCDFDuet and the oligonucleotide primers for PCR amplifying P450 BU i , P450 BU 2 and P450 BU3 from messenger RNA.
  • the invention provides an isolated polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof selected from the group comprising:
  • sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 2 or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 35 or SEQ ID NO: 36 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 32, or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 37 or SEQ ID NO: 38 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 34, or a biologically active fragment or equivalent thereof; and (v) a sequence complementary to any one of (i) to (iv).
  • Cytochrome P450 proteins are a diverse family of proteins containing a heme cofactor and therefore are classified as hemoproteins. P450 proteins use a variety of small and large molecules as substrates in enzymatic reactions. P450 proteins have been identified in all domains of life, such as but not limited to animals, plants, fungi, protists, bacteria, archaea, and even in viruses.
  • Monooxygenases are enzymes that incorporate one hydroxyl group into its substrate.
  • the phrase "monooxygenase activity" refers to an enzyme's ability to incorporate a hydroxyl group into its substrate.
  • the present invention is based in part on the identification and characterisation of Sphingobium yanoikuyae polypeptides that are capable of functionalizing 1 ,8-cineole.
  • these isolated polypeptides were N-terminally sequenced using an Applied Biosystems Procise sequencer using solid support (glass fibre disc) and following traditional Edman degradation techniques.
  • the amino acid sequences of these polypeptides were then used to infer the possible encoding polynucleotide sequences based on the sequenced genome of S. yanoikuyae.
  • Sphingobium sp. is a relatively common soil bacterium although S. yanoikuyae was originally isolated from a clinical sample.
  • the Sphingobium yanoikuyae of the present invention was isolated from activated sludge and in part selected on the basis of its ability to metabolise 1 ,8-cineole as the sole source of carbon. The skilled addressee would appreciate that activated sludge is a diverse microbial culture.
  • Sphingobium species are capable of degrading a variety of chemicals in the environment such as aromatic and chloroaromatic compounds, phenols like nonylphenol and pentachlorophenol, herbicides such as (RS)-2-(4-chloro-2-methylphenoxy) propionic acid and hexachlorocyclohexane, and polycyclic aromatic hydrocarbons.
  • herbicides such as (RS)-2-(4-chloro-2-methylphenoxy) propionic acid and hexachlorocyclohexane
  • polycyclic aromatic hydrocarbons e.g.
  • strain XLDN2-5 is able to metabolise carbazole and strain B1 is able to metabolise biphenyl, naphthalene, phenanthrene, toluene, and m-/p-xylene as sole sources of carbon energy. Therefore the selection of S. yanoikuyae as a source of a monooxygenase which is capable of hydroxylating 1 ,8-cineole is surprising.
  • polynucleotides were found to encode proteins which presented with the typical Soret absorbance of a P450 protein, that is an absorbance maxima of 416 - 417 nm in the absence of its substrate. The absorbance maxima shift to a lower range of wavelengths of 392 - 396 nm upon binding to their substrates 1 ,8-cineole. Accordingly, the polynucleotides encode a P450 protein, preferably a P450 monooxygenase.
  • the P450 proteins of the present invention have been further demonstrated to preferentially bind 1 ,8-cineole.
  • Four other related substrates, 2- adamantanone, ⁇ -ionone, (1 S)-(-)-camphor and (1 R)-(+)-camphor were also tested for binding to the P450 monooxygenases of the present invention and none of them bound to any significant extent, except for weak binding by ⁇ -ionone.
  • 1 ,8-Cineole has low chemical reactivity since there are no activated C-H bonds in the molecule.
  • the regioselective hydroxylation of such structure remains a challenge in organic synthesis and studies on the chemistry of this monoterpenoid is mostly related to the cleavage of the ether bridge to obtain p-menthane derivatives (Boggiato et al., 1987; Liu and Rosazza, 1990).
  • Known chemical processes for functionalising 1 ,8-cineole such as by chemical techniques, generally require the use of harsh reaction conditions and there is little or no control over the stereoisomer created during the chemical reaction. As a result, significant time and costs are required downstream to purify the desired isomer from the reaction product mixture.
  • Microbial transformation of 1 ,8-cineole has a number of advantages including mild reaction conditions, biodegradable reagents, etc.
  • Another main advantage of biotransformation is the stereoselectivity or stereospecificity of enzymes.
  • Stereoselectivity of an enzyme refers to a property of the enzyme wherein during biotransformation, a single reactant is transformed into an unequal mixture of stereoisomers during the non-stereospecific transformation of the reactant.
  • Stereospecificity of an enzyme refers to a property of an enzyme wherein during biotransformation, a single reactant is transformed to a single stereoisomer. This means that biotransformation produces only specific stereoisomers and the process is predictable.
  • the isomerism of the functionalised groups is important for further processing of the product. For example, in certain circumstances, a specific stereoisomer is desired.
  • the P450 monooxygenases of the present invention demonstrate stereoselectivity in that they functionalize 1 ,8-cineole preferably in the 2-position to produce the product 2-hydroxy cineole. Accordingly, in one embodiment the main product of 1 ,8-cineole hydroxylation is 2-hydroxy cineole.
  • the P450 monooxygenases of the present invention are also able to utilise (1 R)-6B-hydroxy- 1 ,8-cineole as its substrate and functionalize (1 R)-6B-hydroxy-1 ,8-cineole to oxo- hydroxy-1 ,8-cineole thereby producing a difunctionalized 1 ,8 cineole.
  • the invention allows for functionalization at additional positions of the compound for any hydroxylated cineole.
  • any 1 ,8 cineole may be used as a substrate that can be further functionalized.
  • any one of 2-hydroxy-1 ,8 cineole, 3-hydroxy-1 ,8 cineole, 5-hydroxy-1 ,8 cineole, 6-hydroxy-1 ,8 cineole, 7-hydroxy-1 ,8 cineole, 9-hydroxy-1 ,8 cineole, or 10-hydroxy-1 ,8 cineole may be further functionalized to provide a multifunctionalized 1 ,8 cineole being functionalized at least at one additional position of the compound.
  • the polynucleotides of the present invention are not required to encode for the full length P450 monooxygenase polypeptides, rather, they may encode biologically active fragments or equivalents thereof.
  • the polynucleotides of the present invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • Such polynucleotides encoding P450 monooxygenases may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al.), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • biologically active fragment when used to refer to a polypeptide refers to a portion of a larger molecule, which retains the same activity as the polypeptides of the present invention.
  • polypeptides when used herein refers to a molecule which is different but retains the same activity as the polypeptides of the present invention.
  • two polypeptides may have different polypeptide sequences but both polypeptides are capable of functionalizing or biohydroxylating 1 ,8-cineole to 2- hydroxycineole or functionalizing or biohydroxylating (1 R)-6B-hydroxy-1 ,8-cineole to oxo-hydroxy-1 ,8-cineole.
  • polynucleotides may have different sequences but encode for polypeptides having the same activity as the polypeptides of the present invention and hence provide equivalents.
  • polypeptides encoded by these polynucleotides are therefore capable of functionalizing or biohydroxylating 1 ,8-cineole to 2-hydroxycineole or functionalizing or biohydroxylating (1 R)-6B- hydroxy-1 ,8-cineole to oxo-hydroxy-1 ,8-cineole.
  • a substrate-binding assay based on observing the spectral absorbance shift when a P450 monooxygenase binds to its substrate may be used.
  • the polypeptide may be co-expressed with the P450 monooxygenase of the present invention, and the rate of functionalization or biohydroxylation of 1 ,8-cineole may be measured to determine if there is an increase in the rate of functionalization or biohydroxylation.
  • variant when used to refer to a polynucleotide refers to a sequence of a polynucleotide that still comprises the information for translation into the same protein. Degeneracy in the genetic code or codons is one way in which a variant of the polynucleotides of the present invention still comprises the information for translation into the same polypeptide.
  • the polynucleotide can be translated into a polypeptide in silico by reference to a codon degeneracy table, then comparing the sequence of the resultant polypeptide to the sequences of the polypeptides of the present invention.
  • a polynucleotide can be cloned into an expression vector and transformed into a host cell. The host cell can then be cultured in the presence of 1 ,8-cineole and assessed for its ability to functionalize 1 ,8-cineole to 2a- hydroxycineole.
  • the term “functionalize”, “functionalizing” or “functionalization” as used herein refers to modifying a compound such as by hydroxylation, biohydroxylation, or oxidation to add a functional group to the compound.
  • the polynucleotides of the present invention encode for P450 monooxygenases that display stereoselectivity when they functionalize 1 ,8-cineole to 2-hydroxy cineole, and were further characterised to be (1 S)-2a-hydroxy-1 ,8-cineole as determined preferably by chiral gas chromatography (GC), GC-MS, nuclear magnetic resonance (NMR) and optical rotation measurements. The analysis also determined that the hydroxy-cineole is not (1 R)-6p-hydroxy-1 ,8-cineole or its enantiomer (1 S)-2p-hydroxy-1 ,8-cineole.
  • GC chiral gas chromatography
  • NMR nuclear magnetic resonance
  • the polynucleotides of the P450 monooxygenases of the present invention are defined in SEQ ID NO: 2, 32 or 34.
  • the polynucleotides may comprise polynucleotides that encode polypeptides according to SEQ ID NO: 1 , 31 or 33.
  • the P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 2 and the polypeptide of SEQ ID NO: 1 is herein referred to as P450 B ui -
  • P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 32 and the polypeptide of SEQ ID NO: 31 is herein referred to as P450 BU2 .
  • the P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 34 and the polypeptide of SEQ ID NO: 33 is herein referred to as P450 B u3-
  • P450 monooxygenases of the present invention will be referred to as P450 B ui , P450 BU 2 or P450 BU 3- Where P450 BU i , P450 BU 2 or P450 BU 3 are referred to, these include the biologically active fragments and equivalents of their polynucleotide sequences and the variants of their polypeptide sequences.
  • nucleotides encoding the STOP codon of the P450 monooxygenases of the present invention have been substituted from TGA to TAA to optimise protein translation in E. coli. Further codon optimisation is possible.
  • the polynucleotide may encode a polypeptide that is at least 75% similar to the polypeptide of SEQ ID NO: 1 , 31 or 33. More preferably, the polynucleotide encodes a polypeptide that is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of SEQ ID NO: 1 , 31 or 33.
  • This aspect of the invention further provides oligonucleotide primers according to SEQ ID NOs: 27, 28, 35, 36, 37 or 38, which may be used to obtain polynucleotides that encode polypeptides comprising polypeptide sequences according to SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof.
  • oligonucleotides may be used to obtain polynucleotides as hereinbefore described using a method such as but not limited to polymerase chain reaction (PCR).
  • the oligonucleotide primers may also comprise restriction enzyme sites for Ndel (5 ATATG-3') and Xhol (5'-CTCGAG-3') to facilitate molecular cloning of the polynucleotides into a vector.
  • restriction enzyme sites are not always required for cloning into a vector as the polynucleotides may be cloned first into a cloning vector without the use of any restriction enzymes, e.g. using a TA cloning technique, before transferring into the final vector.
  • the restriction enzyme sites may be substituted for any other appropriate restriction enzyme sites and the choice of restriction enzyme is in part dependent on the vector to be cloned into.
  • This aspect of the invention further provides polynucleotide sequences that are complementary to the polynucleotide sequences of the present invention as hereinbefore described.
  • the present aspect of the invention also provides variants of the polynucleotides of the present invention as hereinbefore described.
  • the polynucleotides of the present invention may be recombinantly expressed to produce the P450 monooxygenase they respectively encode. These enzymes can then be used for biotransforming or biohydroxylating 1 ,8-cineole to 2- hydroxycineole, preferably (1 S)-2a-hydroxy-1 ,8-cineole.
  • isolated and substantially purified refers to a molecule that is substantially free of its natural environment.
  • an isolated polynucleotide is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived; or at least 45-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
  • polynucleotide refers to a nucleic acid fragment that encodes a specific protein.
  • the polynucleotide may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • encodes refers to a nucleic acid or polynucleotide which comprises the information for translation into a specific protein.
  • hydroxylates refers to the addition of a chemical process that introduces a hydroxyl group (-OH) into an organic compound.
  • ,8-cineole refers to the chemical compound which has the lUPAC name 1 ,3,3-trimethyl-2-oxabicyclo[2,2,2]octane.
  • the naming of the compound and numbering of the structure used herein is the same as those of Azerad et al., 2014.
  • the numbering of the carbon atoms used herein and according to Azerad et al. is as follows:
  • the invention provides isolated polynucleotides as hereinbefore described, wherein the polynucleotides encode P450 monooxygenases or biologically active fragments or equivalents thereof, wherein said P450 monooxygenases hydroxylate 1 ,8-cineole to form 2-hydroxycineole.
  • the isolated polynucleotides of the present invention encode P450 monooxygenases or biologically active fragments or equivalents thereof that are able to functionalize 1 ,8-cineole to produce 2-hydroxycineole.
  • Analysis of the biotransformation product of the P450 monooxygenase of the present invention using NMR, GC-MS and optical rotation measurements demonstrated that the major product is (1 S)-2a-hydroxy-1 ,8-cineole or oxidises 6-hydroxy-1 ,8-cineole to form oxo- hydroxy-1 ,8-cineole.
  • amino acid will refer to the basic chemical structural unit of a protein or polypeptide.
  • polynucleotides or polypeptides refers to polynucleotides or polypeptides formed by laboratory methods of genetic recombination (such as molecular cloning).
  • Recombinant expression of a polynucleotide refers to the laboratory manipulation of the polynucleotide that allows the expression of the polynucleotide, for example in a host cell.
  • sequence identity refers to the amount of nucleotides/amino acids which match exactly between two different sequences. In calculating sequence identity, gaps are not counted and the measurement is relational to the shorter of the two sequences.
  • nucleotide bases that are capable to hybridizing to one another.
  • adenosine is complementary to thymine and cytosine is complementary to guanine.
  • polynucleotides it is referring to the percentage of the number of nucleotide bases between the two polynucleotide sequences that are complementary.
  • two polynucleotide sequences are complementary, preferably at least 50% of their nucleotide bases are complementary. More preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of their nucleotide bases are complementary.
  • polypeptides or biologically active fragments or equivalents thereof that are able to functionalize or hydroxylate 1 ,8- cineole in the 2-position are any one of P450 B ui or P450 B u2 and the polypeptide sequences are at least 75% similar to the polypeptide sequences according to SEQ ID NO: 1 or 31 , respectively.
  • the polypeptide is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of SEQ ID NO: 1 or 31 .
  • substantially purified and/or recombinant polypeptide is selected from the group comprising:
  • P450 monooxygenase as hereinbefore described; and biologically active fragment or equivalent thereof.
  • the polypeptide comprises the polypeptide sequence of P450 B ui according to SEQ ID NO: 1 , or the polypeptide sequence of P450 B u2 according to SEQ ID NO: 31.
  • the polypeptides may be encoded by any of the polynucleotides or variants which encode P450 monooxygenases or biologically active fragments or equivalents thereof as hereinbefore described.
  • the P450 monooxygenases P450 B ui , P450 B u2 and P450 B u3 are able to functionalize or biohydroxylate 1 ,8-cineole in relatively mild conditions, i.e. conditions that are suitable for the growth of microorganisms such as E. coli.
  • the recombinant expression of P450 B ui , P450 BU 2 or P450 BU 3 does not negatively impact the biological function and growth of the host cell, which may be E. coli in this instance.
  • P450 BU i , P450 BU 2 and P450 BU 3 have the typical Soret absorbance of a P450 protein, that is an absorbance maxima of 416 - 417 nm in the absence of its substrate. This absorbance maxima shift to a lower range of wavelengths of 392 - 396 nm upon binding to their substrate 1 ,8-cineole.
  • P450 B ui and P450 BU 2 were further demonstrated to preferentially bind 1 ,8- cineole.
  • Five other related substrates, 2-adamantanone, ⁇ -ionone, (1 S)-(-)-camphor, toluene and (1 R)-(+)-camphor were also tested for binding to the P450 monooxygenases of the present invention and none of these bound to any significant extent, except for weak binding by ⁇ -ionone.
  • the binding affinity of P450 BU i and P450 BU 2 for 1 ,8-cineole was found to be about 20 ⁇ , which increased by a factor of two in the presence of 0.2 M potassium chloride.
  • the binding affinity of P450 BU 3 for 1 ,8-cineole was found to be about 8 ⁇ , which increased by a factor of 1 .5 in the presence of 0.2 M potassium chloride.
  • the invention provides a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.
  • the vector may be an expression vector, which comprises promoter elements that enhance the expression of the polynucleotide.
  • the vector may be a cloning vector, which contains features that allow for the convenient insertion or removal of a DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use, e.g. an expression vector. It should be appreciated that the choice of vector is dependent on the intended purpose.
  • Classes of cloning vectors include plasm ids, cosmids, bacteriophages, bacterial artificial chromosomes, yeast artificial chromosomes, human artificial chromosomes etc.
  • Non-limiting specific examples of cloning vectors include Promega pGEM-T Easy, Life Technology TOPO- TA, pUC-19, pCR2.1 , etc.
  • plasm id and “vector” are used interchangeably and refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • the invention provides a vector as hereinbefore described wherein the vector is an expression vector.
  • An expression vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene.
  • the vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
  • the expression vectors may be part of the Duet Vectors from Novagen. This vector system is preferred based on compatibility of the origins of replication and antibiotics resistance when multiple vectors are transformed into a single Escherichia coli cell.
  • the pETDuet vector carries the ColE1 replicon with ampicillin resistance
  • the pRSFDuet vector carries the RSF1030 replicon with kanamycin resistance
  • the pCDFDuet vector carries the CloDF13 replicon with streptomycin resistance.
  • this is particularly useful for E. coli. Should other host cells be used, any alternative vectors can be adopted which complement the host cell selected. Accordingly, co-transformation of all three vectors into a single E.
  • coli cell results in the expression of all three genes encoded by each vector as well as resistance to ampicillin, kanamycin and streptomycin. This allows the selection of transformed E. coli cells which express all three vectors. This also means that the transformation and antibiotics selection can be carried out for all three vectors simultaneously rather than in a step-wise fashion, i.e. there is no need to transform E. coli with a pETDuet vector and selecting for ampicillin resistance, followed by transforming with a pRSFDuet vector and selecting for kanamycin resistance and then transforming with a pCDFDuet vector and selecting for streptomycin resistance.
  • all three specific pRSFDuet, PETDuet and pCDFDuet vectors may be used and contain a coding sequence for a six (6) histidine tag 5' to the multi-cloning site.
  • the expressed protein comprises an N-terminal 6x histidine tag that facilitates purification of the expressed protein using an affinity purification column such as a nickel column. It should be readily appreciated that a his-tag is not required if purification of the expressed proteins is not required.
  • the expression vector may also be pET28a.
  • Other non-limiting examples of expression vectors include pcDNA3.1 , pDEST vector systems, pET vector systems, etc. The skilled addressee would readily appreciate that any expression vector that is suitable for co-expressing multiple proteins in a host cell is suitable.
  • the invention provides a host cell comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described, and/or at least one plasm id comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.
  • the host cell expresses any one of the P450 monooxygenases of the present invention and facilitates the whole-cell biotransformation or functionalization of 1 ,8-cineole to 2-hydroxy-cineole.
  • the host cell may comprise one, two or three of the P450 monooxygenases of the present invention and expresses one, two or three of the P450 monooxygenases.
  • the term "host cell” refers to those transformable cells capable of growth in culture and expressing a desired polynucleotide and/or polypeptide. While the preferred host cells of this invention are bacterial cells such as E. coli, other microorganism cells may be used, for example, other bacterial cells, yeast cells, fungal cells, insect cells, vertebrate cells, etc.
  • the host cell of the present invention is not capable of metabolising 1 ,8-cineole without the polypeptides or polynucleotides of the present invention. Accordingly, the host cell may be unable to further metabolise the 2-hydroxycineole eventually into water and carbon and therefore the functionalised intermediate products of the biotransformation may be purified.
  • the host cell of the present invention is capable of metabolising further cineole-derivatives from (1 R)-6p-hydroxy-1 ,8-cineole including but not limited to oxo-hydroxy-1 ,8-cineole, hydroxy-2,6-oxo-1 ,8-cineole or 6-oxo-2- hydroxy-1 ,8-cineole.
  • the monooxygenases of the present invention are capable of further functionalising at other positions of the substrate. Accordingly, any hydroxylated 1 ,8 cineole may be used and functionalized to provide a multifunctionalized 1 ,8 cineole.
  • the invention provides a host cell comprising a polynucleotide or vector as hereinbefore described and further comprising an electron transport partner such as but not limited to a ferredoxin (FdX) and/or a ferredoxin reductase (FdR).
  • the electron transport partners such as FdX and/or the FdR may be expressed on separate vectors.
  • a number of other enzymes may act as electron transport partners. In some bacterial systems, the most commonly used electron transport partners are FdX/FdR.
  • Flavodoxin reductase and a Flavodoxin may be used instead.
  • a review of other electron transport partners may be found in Hannemann F et al (2007) Biochimica et Biophysica Acta 1770 330-344.
  • P450 proteins are usually associated with one or more electron transport partners such as but not limited to ferredoxins and ferredoxin reductases in a biochemical pathway.
  • the ferredoxins serve as electron carriers between the corresponding NADH or NADPH-dependent ferredoxin reductases and the P450 protein.
  • the host cell of the present invention may further comprise an electron transport partner such as a ferredoxin (Fdx) and/or a ferredoxin reductase (FdR), wherein the electron transport partner such as a FdR and/or the FdX may be derived from the host cell or may be introduced into the host cell.
  • Fdx ferredoxin
  • FdR ferredoxin reductase
  • the FdX and/or FdR may additionally be recombinantly over-expressed.
  • P450 B ui , P450 B u2 or P450 B u3 are capable of functionalizing 1 ,8-cineole when coupled with electron transport partners. Accordingly, whole-cell biotransformation of 1 ,8-cineole with P450 B ui , P450 B U2, or P450 B u3 is possible with surrogate electron transport partners.
  • the surrogate electron transport partners are derived from E. coli.
  • the invention provides a host cell as hereinbefore described, further comprising a heterologous electron transport partner such as a ferredoxin and/or a heterologous ferredoxin reductase.
  • a heterologous electron transport partner such as a ferredoxin and/or a heterologous ferredoxin reductase.
  • the ferredoxin and/or ferredoxin reductases may also be heterologous to the host cell.
  • the ferredoxin and/or ferredoxin reductases may be recombinantly introduced into a host organism that does not normally express that electron transport partner such as a ferredoxin and/or ferredoxin reductases.
  • the invention provides a host cell as hereinbefore described wherein the electron transport partner such as a ferredoxin and/or the ferredoxin reductase are from Sphingobium and more preferably from Sphingobium yanoikuyae.
  • the electron transport partner such as a ferredoxin and/or the ferredoxin reductase are from Sphingobium and more preferably from Sphingobium yanoikuyae.
  • the ferredoxin and/or the ferredoxin reductase may be derived from Sphingobium yanoikuyae. It is demonstrated that P450 B ui , P450 B u2 or P450 BU 3's efficiency at functionalizing 1 ,8-cineole may be improved when P450 BU i is co-expressed with a ferredoxin and/or ferredoxin reductase from S. yanoikuyae. Furthermore, it is likely that ferredoxin and/or ferredoxin reductases from other Sphingobium species may also be used. The ferredoxins and ferredoxin reductases are most preferably derived from S.
  • a heterologous electron transport partner such as a ferredoxin and/or ferredoxin reductase
  • the polynucleotide encoding FdR and/or FdX may be cloned into a vector or expression vector then recombinantly introduced into the host cell separately to the vector, which expresses the monooxygenases, such as but not limited to, P450 B ui , P450 B u2 or P450 B u3-
  • the electron transport partners are FdX and/or FdR.
  • the Applicants have found advantages and greater efficiencies and effectiveness in expressing these components separately to provide a monooxygenase which can hydroxylate or functionalize 1 ,8-cineole.
  • the second electron transport partner is expressed on the third vector.
  • all three vectors are inserted into the host cell and express the monooxygenase and electron partners individually and separately from three separate vectors.
  • the monooxygenase, and electron transport partners such as FdX and/or FdR are as hereinbefore described.
  • the method of separately expressing a monooxygenase, and the electron transport partners such as a FdX and FdR on expression vectors may be applied to any monooxygenase system.
  • the introduction of the vectors expressing the monooxygenase, and the electron transport partners such as FdX and/or FdR into a host cell may be separately or simultaneously introduced by methods available to the skilled addressee.
  • the electron transport partners are FdX and FdR.
  • the invention provides a host cell as hereinbefore described or a vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin (Fdx) is encoded by a polynucleotide selected from the group comprising:
  • the host cell or the vector or a method of expressing a monooxygenase comprises a heterologous ferredoxin polynucleotide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), ferredoxin 6(FdX6) or ferredoxin 7(FdX7), as defined by SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16, respectively.
  • ferredoxin 1 ferredoxin 1
  • ferredoxin 2(FdX2) ferredoxin 3(FdX3)
  • ferredoxin 4(FdX4) ferredoxin 5(FdX5)
  • ferredoxin 6(FdX6) or ferredoxin 7(FdX7) as defined by SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16, respectively.
  • the host cell or the vector may also comprise a heterologous ferredoxin polypeptide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), ferredoxin 6(FdX6) or ferredoxin 7(FdX7), as defined by SEQ ID NO: 3, 5, 7, 9, 1 1 , 13 and 15, respectively.
  • the polynucleotide encoding the ferredoxin 1 may be at least 50% similar to SEQ ID NO: 4. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 4. More preferably, the polynu
  • the polynucleotide encoding the ferredoxin 2 may be at least 59% similar to SEQ ID NO: 6. More preferably, the polynucleotide encodes a polypeptide that is at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 6.
  • the polynucleotide encoding the ferredoxin 3 may be at least 50% similar to SEQ ID NO: 8. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 8.
  • the polynucleotide encoding the ferredoxin 4 may be at least 50% similar to SEQ ID NO: 10. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 10.
  • the polynucleotide encoding the ferredoxin 5 may be at least 46% similar to SEQ ID NO: 12. More preferably, the polynucleotide encodes a polypeptide that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 12. More preferably
  • the polynucleotide encoding the ferredoxin 6 may be at least 84% similar to SEQ ID NO: 14. More preferably, the polynucleotide encodes a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 14.
  • the polynucleotide encoding the ferredoxin 7 may be at least 98% similar to SEQ ID NO: 16. More preferably, the polynucleotide encodes a polypeptide that is at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 16.
  • the polynucleotides encoding the ferredoxin 1 (FdX1 ) polypeptide of the present invention may encode a polypeptide that is at least 71 % similar to SEQ ID NO: 3. More preferably, the polynucleotide encodes a polypeptide that is at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 3.
  • the polynucleotides encoding the ferredoxin 5 (FdX5) polypeptide of the present invention may encode a polypeptide that is at least 67% similar to SEQ ID NO: 1 1. More preferably, the polynucleotide encodes a polypeptide that is at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides
  • the heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin as hereinbefore described.
  • the heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin polypeptide as hereinbefore described.
  • the polynucleotide may not encode for the entire ferredoxin polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.
  • the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin is encoded by a polynucleotide selected from the group comprising:
  • FdX6 at least 84% similar to FdX6 (SEQ ID NO: 14); a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), and FdX6 (SEQ ID NO: 13), or a biologically active fragment or equivalent thereof; a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence,
  • the host cell or vector or a method of expressing a monooxygenase comprises a heterologous ferredoxin polynucleotide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), or ferredoxin 6(FdX6) , as defined by SEQ ID NO: 4, 6, 8, 10, 12 or 14, respectively, or a variant thereof.
  • ferredoxin 1 ferredoxin 1
  • ferredoxin 2(FdX2) ferredoxin 3(FdX3)
  • ferredoxin 4(FdX4) ferredoxin 5(FdX5)
  • ferredoxin 6(FdX6) as defined by SEQ ID NO: 4, 6, 8, 10, 12 or 14, respectively, or a variant thereof.
  • the host cell or vector or a method of expressing a monooxygenase may also comprise a heterologous ferredoxin polypeptide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 , ferredoxin 2, ferredoxin 3, ferredoxin 4, ferredoxin 5 or ferredoxin 6, as defined by SEQ ID NO: 3, 5, 7, 9, 1 1 and 13, respectively, or a biologically active fragment or equivalent thereof.
  • the heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin as hereinbefore described.
  • the heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin polypeptide as hereinbefore described.
  • the polynucleotide may not encode for the entire ferredoxin polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.
  • the invention provides a host cell or a vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin is encoded by a polynucleotide encoding an amino acid sequence provided in FdX2 (SEQ ID NO: 5).
  • the polynucleotide is preferably SEQ ID NO: 6.
  • FdX2 facilitates an effective functionalization by P450 B ui out of the seven ferredoxins hereinbefore described. FdX2 has also demonstrated effective functionalization of 1 ,8-cineole when in combination with
  • the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase (FdR) as hereinbefore described is encoded by a polynucleotide selected from the group comprising:
  • the host cell or vector or a method of expressing a monooxygenase as hereinbefore described may further comprise a heterologous ferredoxin reductase from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 1 (FdR1 ), ferredoxin reductase 2 (FdR2), ferredoxin reductase 3 (FdR3), ferredoxin reductase 4 (FdR4) or ferredoxin reductase 4s (FdR4s), as defined by SEQ ID NO: 18, 20, 22, 24 and 26, respectively.
  • FdR1 ferredoxin reductase 1
  • FdR2 ferredoxin reductase 2
  • FdR3 ferredoxin reductase 3
  • FdR4 ferredoxin reductase 4s
  • the host cell or vector or a method of expressing a monooxygenase may also comprise a heterologous ferredoxin reductase polypeptide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 1 (FdR1 ), ferredoxin reductase 2 (FdR2), ferredoxin reductase 3 (FdR3), ferredoxin reductase 4 (FdR4) or ferredoxin reductase 4s (FdR4s), as defined by SEQ ID NO: 17, 19, 21 , 23 and 25, respectively.
  • FdR1 ferredoxin reductase 1
  • FdR2 ferredoxin reductase 2
  • FdR3 ferredoxin reductase 3
  • FdR4 ferredoxin reductase 4s
  • the polynucleotide encoding the ferredoxin reductase 1 may be at least 86% similar to SEQ ID NO: 18. More preferably, the polynucleotide encodes a polypeptide that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 18.
  • the polynucleotide encoding the ferredoxin reductase 3 may be at least 71 % similar to SEQ ID NO: 22. More preferably, the polynucleotide encodes a polypeptide that is at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 22.
  • the polynucleotide encoding the ferredoxin reductase 4 may be at least 59% similar to SEQ ID NO: 24. More preferably, the polynucleotide encodes a polypeptide that is at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 24. More preferably, the
  • the polynucleotide encoding the ferredoxin reductase 4s may be at least 67% similar to SEQ ID NO: 26. More preferably, the polynucleotide encodes a polypeptide that is at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to
  • the polynucleotides encoding the ferredoxin reductase 4 (FdR4) polypeptide of the present invention may encode a polypeptide that is at least 98% similar to SEQ ID NO: 23. More preferably, the polynucleotide encodes a polypeptide that is at least 99% similar to the polypeptide according to SEQ ID NO: 23.
  • the polynucleotides encoding the ferredoxin reductase 4s (FdR4s) polypeptide of the present invention may encode a polypeptide that is at least 99% similar to SEQ ID NO: 25.
  • the heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin reductase as hereinbefore described.
  • the heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin reductase polypeptide as hereinbefore described.
  • the polynucleotide may not encode for the entire ferredoxin reductase polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.
  • the host cell or vector or a method of expressing a monooxygenase as hereinbefore described may comprise a heterologous ferredoxin, a heterologous ferredoxin reductase or a combination thereof.
  • the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide selected from the group comprising:
  • the host cell or vector or the method of expressing a monooxygenase as hereinbefore described may further comprise a heterologous ferredoxin reductase polynucleotide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 2, ferredoxin reductase 3 or ferredoxin reductase 4s, as defined by SEQ ID NO: 20, 22 and 26, respectively.
  • the host cell or vector or the method of expressing a monooxygenase may also comprise heterologous ferredoxin reductase polypeptide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 2, ferredoxin reductase 3 or ferredoxin reductase 4s, as defined by SEQ ID NO: 19, 21 and 25, respectively.
  • the heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin reductase as hereinbefore described.
  • the heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin reductase polypeptide as hereinbefore described.
  • the polynucleotide may not encode for the entire ferredoxin reductase polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.
  • the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide which encodes an amino acid sequence provided in FdR3 (SEQ ID NO: 21 ).
  • the polynucleotide is preferably SEQ ID NO: 22.
  • the electron transport partners such as the FdXs and FdRs of the present invention may be used in combination to facilitate and improve the biohydroxylation or functionalization of 1 ,8-cineole by the monooxygenases of the present invention.
  • the electron transport partners such as but not limited to FdXs and the FdRs are ideally expressed by separate vectors in a host cell.
  • the invention provides a host cell or vector or method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide provided in FdR3 (SEQ ID NO: 22) and the ferredoxin is encoded by a polynucleotide provided in FdX2 (SEQ ID NO: 6).
  • the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin having an amino acid sequence selected from the group comprising FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent or variant thereof.
  • a ferredoxin having an amino acid sequence selected from the group comprising FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent or variant thereof.
  • the host cell or vector of the present invention expresses a heterologous ferredoxin polypeptide from S. yanoikuyae.
  • the ferredoxin may be one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent or variant thereof.
  • the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin reductase polypeptide having a sequence selected from the group comprising FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent or variant thereof.
  • the host cell or vector expresses a heterologous ferredoxin reductase from S. yanoikuyae.
  • the ferredoxin reductase may be one of FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent or variant thereof.
  • the invention provides a host cell as hereinbefore described which expresses a ferredoxin having a polypeptide sequence selected from the group comprising FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent thereof and a ferredoxin reductase having a polypeptide sequence selected from the group comprising FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof, or a combination of a FdR and a FdX thereof
  • the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin having a polypeptide sequence provided in FdX2 (SEQ ID NO: 5) and a ferredoxin reductase having a polypeptide sequence provided in FdR3 (SEQ ID NO: 21 ).
  • the polynucleotide encoding the FdX2 is preferably SEQ ID NO: 6 and the polynucleotide encoding the FdR3 is preferably SEQ ID NO: 22.
  • the invention provides a host cell that is an E. coli cell.
  • E. coli is not normally capable of metabolising or functionalizing 1 ,8-cineole. Accordingly, it also lacks the metabolic pathways that further metabolise 2- hydroxycineole eventually to water and carbon dioxide. This allows the functionalised intermediate products to be isolated.
  • a method of functionalizing 1 ,8 cineole comprising contacting 1 ,8 cineole with an effective amount of a monooxygenase polypeptide as hereinbefore described to produce 2-hydroxycineole.
  • the monooxygenase polypeptide which contacts 1 ,8- cineole may be any one of the polypeptides of P450 B ui , P450 B u2 or P450 B u3 as hereinbefore described.
  • the term "effective amount" when used to refer to the polypeptides of the present invention refers to a quantity of the polypeptides such that measurable amounts of 1 ,8-cineole hydroxylation or functionalization can be observed.
  • the functionalization may be observed as hydroxylation or oxidation.
  • the present invention provides a method of producing 2- hydroxycineole said method comprising hydroxylating or functionalizing 1 ,8-cineole in the presence of the host cell as hereinbefore described.
  • This embodiment of the invention provides a method of whole-cell functionalization or biohydroxylation of 1 ,8-cineole using the host cell as hereinbefore described. [0174] In one embodiment the invention provides a method of functionalizing 1 ,8 cineole said method comprising
  • transforming bacterial host cells include electroporation, cationic liposome formulations such as Lipofectamine®, calcium phosphate transfection, the use of chemically competent cells, etc.
  • chemically competent E. coli BL21 (DE3) can be transformed with the P450 B ui -expressing vector P450 B ui- pET28a(+), then cultured in a 500 ml of TB media at 30°C and agitated at 180 rpm until the OD 6 oo reached 0.6 to 0.8.
  • the culture can then be induced with 1 mM IPTG and at the same time 1 ,8-cineole is added to the culture.
  • the recombinant host cell may then biohydroxylate 1 ,8-cineole to 2-hydroxycineole, which can then be isolated from the culture supernatant using ethyl acetate.
  • the substrate of 1 ,8 cineole may be a non-hydroxylated or hydroxylated form of 1 ,8 cineole.
  • the substrate may be further functionalized using the monooxygenases of the present invention selected from any one of P450 B ui , P450 B u2 or P450 BU 3 as hereinbefore described.
  • the substrate may be 6 - hydroxy-1 ,8-cineole and functionalizing using the monooxygenases of the present invention may result in a difunctionalized cineole preferably to produce oxo-hydroxy cineole.
  • the invention provides a method of functionalizing 1 ,8 cineole wherein the host cell expresses a polypeptide having: (i) an amino acid sequence as provided in SEQ ID NO: 1 , 31 or 33; or
  • the method of functionalizing or biohydroxylating 1 ,8- cineole uses a host cell that expresses any one of the P450 monooxygenases of the present invention as hereinbefore described according to the polypeptide of SEQ ID NO: 1 , 31 or 33 or a polypeptide encoded by any of the P450 monooxygenase polynucleotides as hereinbefore described or biologically active fragments or equivalents or variant thereof.
  • the method monofunctionalizes a substrate of 1 ,8 cineole to produce 2 hydroxycineole.
  • Another aspect of the invention provides a method of functionalizing 1 ,8 cineole further including conducting the functionalization or hydroxylation in the presence of an electron transport partner.
  • a suitable electron transport partner protein is a protein that can interact with the monooxygenase of the present invention and act as an electron carrier to the monooxygenase.
  • the method of the present invention may be performed as a whole-cell biotransformation, wherein the host cell expresses at least one of P450 B ui , P450 B u2 or P450 B u3-
  • the host cell is transformed to express the monooxygenases, and the electron transport partners of the present invention together.
  • the monooxygenases, and the electron transport partners of the present invention may be expressed from a single vector or from separate vectors.
  • the method may be carried out in the absence of a host cell.
  • the electron transport partners are Fdx or FdR. More preferably, they are selected from the FdX and the FdR as hereinbefore described.
  • the invention provides a method of producing 2-hydroxycineole wherein the electron transport partner is a ferredoxin (FdX) and/or a ferredoxin reductase (FdR).
  • the invention provides a method of producing 2-hydroxycineole wherein the electron transport partner is a heterologous ferredoxin (FdX) and/or a heterologous ferredoxin reductase (FdR).
  • FdX heterologous ferredoxin
  • FdR heterologous ferredoxin reductase
  • ferredoxin and/or ferredoxin reductase is heterologous as compared to the P450 monooxygenase of the present invention.
  • the invention provides a method of producing 2-hydroxycineole wherein the ferredoxin (FdX) comprises an amino acid sequence according to any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13), FdX7 (SEQ ID NO: 15) and a biologically active fragment or equivalent or variant thereof.
  • FdX ferredoxin
  • the ferredoxin or a biologically active fragment or equivalent thereof is a S. yanoikuyae ferredoxin.
  • the invention provides a method of producing 2-hydroxycineole wherein the ferredoxin (FdX) comprises an amino acid sequence as provided in any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), and FdX6 (SEQ ID NO: 15) and a biologically active fragment or equivalent thereof.
  • FdX ferredoxin
  • the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin (FdX) comprises an amino acid sequence according to FdX2 (SEQ ID NO: 5).
  • the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to any one of FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23), FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof as hereinbefore described.
  • FdR ferredoxin reductase
  • the ferredoxin reductase or a biologically active fragment or equivalent thereof is a S. yanoikuyae ferredoxin reductase.
  • the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to any one of FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof as hereinbefore described.
  • FdR ferredoxin reductase
  • the invention provides a method as hereinbefore described wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to FdR3 (SEQ ID NO: 21 ) or a biologically active fragment or equivalent thereof as hereinbefore described.
  • FdR ferredoxin reductase
  • the method of functionalizing 1 ,8 cineole proves a means to prepare 2- hydroxycineole from 1 ,8 cineole or stereospecifically target functionalization at the 2 position of 1 ,8 cineole.
  • the invention provides a 2-hydroxycineole prepared by the methods as hereinbefore described.
  • the method of the present invention may be carried out at an experimental or research scale. Alternatively, it may also be scaled up, e.g. in a bioreactor.
  • the bioreactor may be static or agitated, it may also employ permanent or semipermanent growth chambers. Alternatively, the bioreactor may be disposable. It should be readily appreciated that the choice of bioreactor and the requirements for up-scaling is dependent in part on the host cell used for biotransformation. Alternatively, if the biotransformation is performed in the absence of a host cell, the up-scaling may be performed in a bioreactor suitable for such a reaction.
  • a further aspect of the invention provides an isolated polynucleotide which encodes a ferredoxin or a biologically active fragment or equivalent thereof selected from the group comprising:
  • the polynucleotide encodes a S. yanoikuyae ferredoxin or a biologically active fragment or equivalent thereof.
  • ferredoxin 1 to 7 seven different ferredoxins were identified and named ferredoxin 1 to 7.
  • the ability of each of these ferredoxins to function as an electron carrier protein for the P450 monooxygenases of the present invention was verified by co-expressing the ferredoxin with the P450 B ui , P450 B u2 or P450 B u3 as hereinbefore described, in a host cell and measuring the ability of the host cell to hydroxylate 1 ,8- cineole.
  • the other electron transport partners were provided by the host cell, E. coli.
  • Six of the seven ferredoxins were able to function as electron carrier proteins for the P450 B ui , P450 BU 2 or P450 BU 3.
  • the invention provides a substantially purified and/or recombinant polypeptide having electron carrier activity or a biologically active fragment or equivalent thereof, wherein the polypeptide comprises an amino acid sequence, which is: at least 71 % similar to FdX1 (SEQ ID NO: 3);
  • the invention provides a vector as hereinbefore described comprising at least one polynucleotide encoding a ferredoxin as hereinbefore described.
  • the invention provides that the vector as hereinbefore described comprising a polynucleotide encoding a ferredoxin is an expression vector.
  • the polynucleotides encoding the ferredoxins of the present invention were cloned into expression vectors to allow recombinant expression of the ferredoxin within a host cell.
  • the expression vector used was the pRSFDuet from Novagen, however any other expression vectors as hereinbefore described may be suitable.
  • the invention provides an isolated polynucleotide which encodes a ferredoxin reductase or a biologically active fragment or equivalent thereof selected from the group comprising:
  • the START and STOP codons within the polynucleotides of the ferredoxin reductases of the present invention were codon modified for better translation in E. coli. Specifically, the following codon changes were made to the ferredoxin reductases polynucleotides: Gene Name START Codon STOP Codon
  • the invention provides a substantially purified and/or recombinant polypeptide having electron carrier activity or a biologically active fragment or equivalent thereof, wherein the polypeptide comprises an amino acid sequence, which is:
  • the invention provides a vector comprising at least one polynucleotide encoding a ferredoxin reductase as hereinbefore described.
  • the invention provides that the vector comprising a polynucleotide encoding a ferredoxin reductase is an expression vector.
  • the polynucleotides encoding the ferredoxin reductases of the present invention were cloned into expression vectors to allow recombinant expression of the ferredoxin reductase within a host cell.
  • the expression vector used is the pETDuet expression vector from Novagen, however any other expression vectors may be suitable.
  • the pH of the medium was adjusted to pH 7.0 using 10% (v/v) ammonia solution and the bioreactor pH controller.
  • Example 1 Isolation of a Cineole-Metabolising Microorganism
  • Microorganisms capable of metabolising 1 ,8-cineole were isolated from activated sludge (obtained from Lilydale Sewage Treatment Plant, Victoria, Australia) using a modified continuous culture method in combination with selection based on growth with 1 ,8-cineole as the sole source of carbon and energy.
  • the medium used for the enrichment process was a fully defined mineral salt medium (DM2).
  • DM2 mineral salt medium
  • One isolate was chosen because of its ability to grow in relatively high concentrations of 1 ,8-cineole (Dumsday et al., 2007). Once established as a pure culture the strain was shown to be able to utilise 1 ,8-cineole as a sole carbon source and could grow in the presence of 1 ,8-cineole concentrations up to 2 g L 1 .
  • Table 1 16S rRNA identification of the new isolate from activated sludge using the blastn algorithm. The best 3 hits are shown when the 1492 bp 16S rRNA gene sequence from the isolate was blasted against the " 16S ribosomal sequences (Bacteria and Archae)" database from NCBI. The new isolate was identified as Sphingobium yanoikuyae.
  • Example 2 Production, Purification and Structural Analysis of 1,8-cineole Metabolites Produced by S. yanoikuyae
  • yanoikuyae was grown in 500 mL of DM medium in a 2 L Erlenmeyer flask) and once purified the structure of each compound was partially elucidated using Nuclear Magnetic Resonance (NMR) and Gas Chromatography Mass Spectrometry (GC MS). The putative identification of each metabolic product is shown in Table 2.
  • Table 2 Putative identification of metabolic intermediates produced by S. yanoikuyae when growing on 1 ,8-cineole as a sole carbon source.
  • the intermediates were purified from solvent extracts of culture supernatants and partially characterised using NMR and GC MS.
  • P1 was observed in stationary phase culture supernatant analysed using GC, but could not be recovered by solvent extraction.
  • P1 was suspected to be the intermediate Baeyer-Vi Niger oxidation product of the oxo- and hydroxycineole and could rearrange to the lactone (P2) in organic solvents. Therefore in order to structurally elucidate P2, P1 and P2 were purified from the aqueous culture supernatant.
  • the genomic DNA of the S. yanoikuyae isolate was extracted using a standard DNA extraction technique. Illumina® HiSeq sequencing of the isolated DNA and assembly using Velvet delivered a draft genome sequence of approximately 5.9 Mbp containing 263 contigs.
  • the Sphingobium yanoikuyae strain genome was automatically annotated using the PROKKA software (http://www.vicbioinformatics.com) and 9 putative P450 genes were identified in the draft genome.
  • Example 4 Production of 1 ,8-cineole-hydroxylating enzymes in Sphingobium yanoikuyae
  • a (fed-) batch bioprocess was developed.
  • a single S. yanoikuyae colony grown on NA supplemented with ca. 1 mL L "1 1 ,8-cineole was used to inoculate a primary seed culture (10 mL of DM3 in a 50 mL tube) and grown for ca. 3 days at 30°C shaking at 200 rpm.
  • a secondary seed culture 500 mL of DM3 in a 2 L baffled Erlenmeyer flask was inoculated with 9 mL primary seed culture and grown for 24 h at 30°C shaking at 200 rpm.
  • the main culture (1 .6 L of DM3 in a 3 L glass stirred tank bioreactor connected to a Sartorius Biostat B controller (Sartorius, Germany) was inoculated with 101 mL secondary seed to obtain an initial calculated OD 6 oo of 0.25.
  • 0.8 ml_ 1 ,8-cineole was added to the medium.
  • S. yanoikuyae cells were initially cultivated in batch mode. The temperature was maintained at 30 °C and the pH at 7.0 via automatic addition of either 10% (v/v) H 3 P0 4 and 10% (v/v) ammonia solution.
  • Dissolved oxygen control was via a cascade control system (stirrer followed by airflow) with the dissolved oxygen maintained at 30% of saturation.
  • OD 60 o and the level of glucose were measured regularly. When the OD 6 oo had reached about 28 and the level of glucose had decreased to approximately 2 g ⁇ 1 , glucose and cineole feeds were started at rates of 3.5 g h _1 and 0.32 ml h _1 , respectively. (Cineole was fed to the culture to "induce" proteins involved in cineole metabolism.) When an OD 6 oo of ca.
  • Example 5 Isolation and Identification of Cineole-binding P450s from S. yanoikuyae Cell Extracts
  • proteins that could bind 1 ,8-cineole were purified from cell lysates.
  • S. yanoikuyae cells expressing enzymes involved in cineole metabolism were purified from a clarified cell lysate using a multistep purification process. Cells resuspended in buffer were cooled to less than 10°C and lysed by three passages through a high pressure homogeniser at 700 bar. The cell lysate was then clarified by centrifugation (12,000 x g, 30 minutes, 4°C).
  • Ammonium sulphate precipitation was used as the first purification step and the lysate was partitioned into three different fractions (0-20%, 20-40% and 40-60% ammonium sulphate cuts). These fractions were loaded onto an ion-exchange column (IEX) and upon addition of 1 ,8-cineole, a transition from the low-spin (LS) to the high-spin (HS) state (Luthra et al., 201 1 ) was observed. Two distinct peak fractions, PF1 and PF2 were further purified using gel filtration. After the 3-step purification procedure, PF1 was purified to apparent homogeneity on SDS- PAGE.
  • IEX ion-exchange column
  • Example 6 Identification of gene encoding P450 Monooxygenases from S. yanoikuyae
  • P450 B ui P450 B u2 and P450 B u3-
  • P450 B ui shares more than 40% sequence identity with P450s that are member of the CYP101 family
  • pairwise amino acid alignment was performed using the amino acid sequence of P450 B ui and other members of the CYP101 protein family as well as CYP176A1 (P450 cin from C. braakii, the only bacterial P450 known to hydroxylate 1 ,8-cineole).
  • sequence similarities were calculated from the alignment and are summarised Table 4.
  • Table 4 Amino acid sequences similarities between P450 B ui , P450 B u2 and P450BU3 and other members of the CYP101 family and CYP176A1 (P450 cin from C. braakii). The values in the table are expressed as "alignment length (sequence identity) (sequence similarity)".
  • P450 B ui shares the highest amino acid similarity to CYP101 B1 which has been shown to oxidise ⁇ -ionone (Bell and Wong, 2007).
  • the sequence identities of the P450 proteins of the present invention with the P450 from C. braakii (CYP176A1 ) is very low indicating that the P450 proteins of the present invention are is very different to this protein.
  • Example 7 Comparative analysis of the S. yanoikuyae isolate of the Present Invention and S. yanoikuyae DSM 7462
  • Table 5 Growth of S. yanoikuyae strains on a range of terpenes. Cultures were grown in 50 mL screw-capped tubes at 30°C. Growth was scored by visual examination of the cultures: +, growth; -, no growth.
  • the gene encoding the P450 monooxygenases of the present invention were amplified using polymerase chain reaction (PCR) using S. yanoikuyae genomic DNA as the template and the oligonucleotide primers used for the amplification are set out in Table 6. Restriction sites for Ndel and Xhol were introduced into the PCR product by the two primers (sequences for the restriction enzymes sites are underlined) for cloning into the multiple cloning site of the vector pET28a(+).
  • PCR polymerase chain reaction
  • the pET28a(+) vector introduced an in-frame N-terminal 6x histidine tag to P450 B ui , this 6xHis tag allows the purification of the expressed the P450 monooxygenases of the present invention using immobilised metal affinity chromatography (IMAC).
  • IMAC immobilised metal affinity chromatography
  • Table 6 DNA sequences of the oligonucleotide primers used to amplify the P450 monooxygenases of the present invention from DNA. Underlined nucleotides are for introducing Ndel and Xhol restriction enzyme sites into the final PCR product.
  • the PCR cycling conditions were as follows (30 cycles of steps b and c): a. Initial denaturation at 98 °C for 1 min; b. Denaturation at 98 °C for 10 seconds; c. Annealing and extension at 72 °C for 40 seconds; and d. Final extension step at 72 °C for 10 minutes.
  • Table 7 DNA sequences of the oligonucleotide primers used to confirm the correct insertion of the P450 genes of the present invention into pET28a(+).
  • DNA sequencing and base calling was performed by MICROMON, Monash University, Victoria, Australia. Sequencing was used to confirm that the insert was in- frame and had the correct sequence.
  • the seed cultures were incubated for 18 h at 30°C and 180 rpm and then used to inoculate (to an initial OD 6 oo of approximately 0.1 ) 500 mL of TB supplemented with 50 g mL "1 Kan in a 2 L baffled flask. The cultures were incubated under the same conditions until the OD 6 oo reached 0.6-0.8. Protein expression was induced with 1 mM isopropyl ⁇ -D-l - thiogalactopyranoside (IPTG) and incubated for a further 18 h and then harvested by centrifugation at 6000 x g for 20 minutes at 4°C.
  • IPTG isopropyl ⁇ -D-l - thiogalactopyranoside
  • the cell pellets were washed in 50 mM Tris, 150 mM NaCI buffer, pH 7.4 and then stored at -80 °C.
  • the cell pellets were resuspended in the same buffer and then disrupted by two passages through an Avestin cell disruptor. After centrifugation, the clarified cell lysates were each loaded onto a separate column with cobalt-charged resin (TALON® Superflow metal affinity resin; Clontech) equilibrated with 50 mM Tris, 150 mM NaCI and 5 mM imidazole, pH 7.4.
  • cobalt-charged resin TALON® Superflow metal affinity resin
  • the His-tagged P450 B ui , P450 B u2 or P450 B u3 were eluted by a stepwise increase (20, 60, 100 and 200 mM) in imidazole concentration. Fractions with similar purities as judged by SDS-PAGE were pooled and buffer-exchanged into 50 mM Tris, pH 7.4 by repeated cycles of concentration and dilution using Amicon Ultra-15 Centrifugal Filter Units with a molecular weight cut of 10 kDa.
  • P450 B ui , P450 B U2 or P450 B u3's estimated molecular weights inclusive of the 6xHis tag were approximately 48.7 kDa, 47.8 kDa and 48.4 kDa, respectively.
  • the purified proteins were visualised on a Coomassie-stained SDS-PAGE gel (4-12% Bis-Tris, MOPS running buffer) and the size of the main expression product corresponded with the estimated size (see Figure 2).
  • the purity of the P450 BU i , P450 BU 2 or P450 BU 3 preparations were reflected by the RZ values of 1 .6, 1 .0 and 1 .5, respectively.
  • the yield of active protein was estimated in the purified pool by performing carbon monoxide difference spectroscopy in the presence of 1 ,8-cineole (see Figure 3 and Example 7).
  • P450 BU i and P450 BU 2 were both recovered in good yields with more than 30 mg purified protein per litre original culture, while P450 BU 3 was expressed in lower quantities with approximately 1 .5 mg of purified protein per litre of original culture.
  • the concentration of functional P450's were estimated using CO difference spectroscopy (Omura and Sato, 1964). This value is based on the extinction coefficient of another P450 and is used extensively by P450 researchers to estimate P450 concentration.
  • P450 BU i , P450 BU 2 and P450 BU 3 from S. yanoikuyae had properties of a P450 enzyme P450 BU i , P450 BU 2 and P450 BU 3 protein solutions were prepared in 50 mM Tris, pH 7.4. Substrate-induced absorbance shifts were demonstrated by adding 1 ⁇ of undiluted 1 ,8-cineole to 1 mL of protein solution. Each of the P450 proteins were then reduced by the addition of a small amount (a few milligrams) of solid sodium dithionite (Sigma Aldrich, USA) and incubation for 1 minute. Subsequently, carbon monoxide (CO) was bubbled through the cuvette for 30 seconds. Spectra were recorded from 350 to 700 nm. The purified P450's showed the characteristic spectroscopic properties as shown in Figure 3.
  • Table 8 Characteristic absorbance maxima of the four different forms of P450 B ui , P450 B U2 and P450 B u3- All three P450 proteins showed the typical Soret absorbance maximum ( ⁇ 41 7 nm) which shifts to a lower wavelength ( ⁇ 392 nm) upon addition of the substrate, 1 ,8-cineole. The addition of 1 , 8-cineole results in a very large shift indicating that this compound may be the preferred substrate for these P450 proteins.
  • Purified P450 BU 1 , P450 BU 2 and P450 BU 3 were diluted in 50 mM Tris, pH 7.4, or 50 mM Tris, 200 mM KCI, pH 7.4 buffer to approximately 2 ⁇ .
  • 1 ,8-cineole stock solutions in ethanol (EtOH) in the concentration range of 1 - 600 mM were prepared by serial dilution.
  • EtOH ethanol
  • To 1 ml_ of each protein solution, 1 ⁇ _ aliquots of undiluted 1 ,8- cineole stock solutions of varying concentrations were added sequentially. After mixing, an absorbance spectrum between 350 and 450 nm was measured.
  • the dissociation constants (K D ) were determined with 1 ,8-cineole as the substrate and are summarised in Table 9. The dissociation constant was decreased by a factor of approximately 2 when 0.2 M KCI was present.
  • Table 8 The dissociation constant of P450 B ui , P450 B u2 and P450 B u3 for 1 ,8-cineole in the presence or absence of 0.2 M KCI.
  • Table 9 The dissociated constant of P450 B ui and P450 B u 2 proteins for 1 ,8- cineole, 2-adamantanone, ⁇ -ionone, (1 S)-(-)-camphor and (1 R)-(+)-camphor in the present and absence of 0.2 M KCI.
  • E. coli BL21 (DE3) was transformed with the P450 B ui -expressing vector (pET28a(+)) to demonstrate whole-cell biotransformation of 1 ,8-cineole and gas chromatography-mass spectrometry (GC-MS) analysis was used to demonstrate hydroxylation of 1 ,8-cineole.
  • E. coli BL21 (DE3) transformed with an empty pET28a(+) vector was used as the negative control.
  • Transformed E. coli was cultured in a 500 ml_ of TB (30°C and shaking at 180 rpm) until the OD 6 oo reached 0.6 to 0.8. The culture was then induced with 1 mM IPTG and at the same time 250 ⁇ _ of undiluted 1 ,8-cineole were added to the culture. The culture was incubated for a further 45 hours and then the cells were removed by centrifugation at 6000 x g for 20 minutes at 4°C. The culture supernatant was extracted with ethyl acetate (EtOAc) and the organic phase was dried over Na 2 S0 4 and concentrated under reduced pressure.
  • EtOAc ethyl acetate
  • the crude solvent extract was dissolved in 3 ml_ of EtOAc, and then diluted 1000-fold in EtOAc and analysed using GC-MS.
  • GC-mass spectra were obtained with a ThermoQuest MAT95XL GC mass spectrometer using electron impact ionisation in the positive ion mode with ionisation energy of 70 eV.
  • the gas chromatography was performed with a SGE SOLGEL-1 MS column (30 m x 0.25 mm ID, 0.25 pm film thickness), with a temperature program of 50°C for 2 minutes, then heating at 23°C min "1 to 300°C where the temperature was held for 7 minutes with a splitless injection, an injector temperature of 300°C and the transfer line was set to 300°C.
  • High-purity helium was used as carrier gas with a flow rate of 0.8 ml_ min "1 .
  • the hydroxycineole from this study can be distinguished from (1 R)- ⁇ -hydroxy-l ,8-cineole using non-chiral gas chromatography the product of the biotransformation of 1 ,8-cineole by P450 B ui could be either (1 S)-2a-hydroxy-1 ,8- cineole or (1 R)-6a-hydroxy-1 ,8-cineole.
  • Example 10 S. yanoikuyae Ferredoxins and Ferredoxin Reductases
  • Putative ferredoxins and ferredoxin reductases were identified from the genome of S. yanoikuyae on the basis of sequence homology to known ferredoxin and ferredoxin reductases.
  • ferredoxin [Sphingomonas sp. PR0901 1 1 -T3T-6A] - 100 70 WP 019830942.1
  • Variovorax paradoxus B4 chromosome 1 complete 16 94 sequence - CP00391 1 .1
  • ferredoxin [Sphingobium yanoikuyae] - 100 99 WP 03751 1909.1
  • ferredoxin [Sphingobium yanoikuyae] - 100 99 WP 010339184.1
  • Rhizobium etli bv. mimosae str. IE4771 complete 16 89 genome - CP006986.1
  • Rhizobium etli CIAT 652 complete genome - 16 89 CP001074.1
  • ferredoxin [Sphingomonas sp. DC-6] - WP_030090025.1 100 67 ferredoxin [Sphingomonas sp. YL-JM2C] - 100 67 WP 029992007.1
  • ferredoxin [Sphingobium sp. AP49] - WP_007712740.1 100 99 ferredoxin [Sphingobium czechense LL01 ] - 100 91 KMS54512.1
  • Sphingobium quisquiliarum strain DC-2 hypothetical 100 85 protein, Red1 , and potassium transporter Kup genes
  • Sphingomonas sp. XLDN2-5 insertion sequence IS6100, 100 99 complete sequence; and CarR (carR), CarAa (carAa),
  • CarBb (carBa), CarBb (carBb), CarC (carC), and CarAc
  • FdR2 polypeptide - SEQ ID NO: 19 Query Identity
  • MULTISPECIES pyridine nucleotide-disulfide 100 99 oxidoreductase [Sphingobium] - WP_010338887.1
  • FdR4s polynucleotide - SEQ ID NO: 26 Query Identity
  • Example 11 Biotransformation of 1 ,8-cineole with P450 B ui in Combination with Recombinant Ferredoxins from S. yanoikuyae
  • the S. yanoikuyae ferredoxins are as described in Example 10 and their polynucleotide sequences are as follows: FdX1 (SEQ ID NO: 4), FdX2 (SEQ ID NO: 6), FdX3 (SEQ ID NO: 8), FdX4 (SEQ ID NO: 10), FdX5 (SEQ ID NO: 12), FdX6 (SEQ ID NO: 14) and FdX7 (SEQ ID NO: 16).
  • the genes were amplified by PCR from S. yanoikuyae genomic DNA and cloned into pRSFDuet expression vector.
  • the experimental groups are summarised in Table 1 1 .
  • Table 11 Summary of the expression vectors and the genes they express for each experimental group.
  • the transformed E. coli of some of the experimental groups were co-expressing P450 B ui with specific S. yanoikuyae ferredoxins.
  • E. coli was transformed with three empty vectors was used as a negative control and did not hydroxylate 1 ,8-cineole.
  • a 10 mL TB seed culture in a 50 mL Falcon tube containing 30 g mL "1 Kan, 50 g mL "1 Ampicillin sodium salt (Amp) and 50 g mL "1 streptomycin sulphate (Sm) was inoculated with a single transformant and incubated at 180 rpm and 30°C for 18-20 hours.
  • 1 mL of the seed culture was then used to inoculate 50 mL TB containing 30 g mL "1 Kan, 50 g mL "1 Amp and 50 g mL “1 Sm in a 250 mL non- baffled shake flask.
  • the culture was grown for 3 hours and then induced with 1 mM IPTG. 18 hours post induction, the cells were harvested by centrifugation at 6000 x g for 10 min and the supernatant discarded. The pellet was washed with 5 mL of EM medium and centrifuged again. EM medium was discarded and the pellet centrifuged again for 2 mins to remove any residual EM medium.
  • the pellet was then weighed and resuspended in EM medium containing 30 g mL "1 Kan, 50 g mL "1 Amp and 50 g mL “1 Sm (1 g pellet was resuspended in 1 mL EM medium).
  • 500 ⁇ of the suspension were then transferred into a fresh 15 ml_ tube and 2 ⁇ _ of undiluted 1 ,8- cineole were added (final concentration of 4 mM).
  • the reaction was then incubated for 3 hours at 180 rpm and 30°C. After 3 hours, an internal standard was added and the suspension mixed thoroughly. Then 990 ⁇ _ EtOAc were added and the suspension was mixed thoroughly.
  • the suspension was then centrifuged at 2000 xg, 2 min, 4 °C to separate phases, the organic phase was removed and analysed using GC-MS split 10.
  • the GC-MS peak areas were standardised using an internal standard (alpha-Terpineol).
  • Example 12 Biotransformation of 1 ,8-cineole with P450 B ui in Combination with Recombinant Ferredoxin Reductases from S. yanoikuyae
  • the aim of this experiment was to determine whether the efficiency of the whole-cell hydroxylation of 1 ,8-cineole can be improved by recombinantly co- expressing S. yanoikuyae ferredoxin reductases with P450 BU i .
  • the S. yanoikuyae ferredoxin reductases are as described in Example 10 and their polynucleotides sequences are as follows: FdR1 (SEQ ID NO: 18), FdR2 (SEQ ID NO: 20), FdR3 (SEQ ID NO: 22), FdR4 (SEQ ID NO: 24) and FdR4s (SEQ ID NO: 26) were amplified from S. yanoikuyae genomic DNA by PCR and cloned into pETDuet expression vectors. The experimental groups were as summarised in Table 12. [0276] Table 12: Summary of the expression vectors and the genes they express for each experimental group.
  • the transformed E. coli of some of the experimental groups were co-expressing P450 B ui with specific S. yanoikuyae ferredoxin reductases.
  • the method used was the same as that described in Example 1 1 .
  • Table 13 Summary of the expression vectors and the genes they express for each experimental group.
  • the aim of this experiment was to establish the kinetics of the biotransformation of 1 ,8-cineole to hydroxycineole by P450 B ui when recombinantly expressed in combination with any one of FdR2-FdX5, FdR3-FdX2 or FdR4s-FdX6.
  • Table 14 Summary of the expression vectors and the genes they express for each experimental group.
  • the transformed E. coli of each experimental group were co-expressing P450 B ui with a specific combination of S. yanoikuyae ferredoxin and a specific S. yanoikuyae ferredoxin reductase.
  • a 50 ml_ TB seed culture in 250 ml_ non-baffled shake flask containing 30 g ml_ "1 Kan, 50 g ml_ "1 Amp and 50 g ml_ "1 Sm was inoculated with a single colony of transformant and incubated at 180 rpm and 30°C for 18 hours.
  • the seed culture was used to inoculate 500 ml_ TB containing 30 g ml_ "1 Kan, 50 g ml_ “1 Amp and 50 g ml_ "1 Sm in a 2 L baffled shake flask.
  • the culture was grown for 3 hours and then induced with 1 mM IPTG. 18 hours post induction, the cells were harvested by centrifugation at 6000 x g for 20 min and the supernatant removed The pellet was washed with 20 ml_ EM medium and centrifuged again for 10 min. EM medium was removed and the pellet centrifuged again for 2 mins to remove any residual EM medium.
  • the pellet was then weighed and resuspended in EM medium containing 30 g mL "1 Kan, 50 g mL "1 Amp and 50 g mL "1 Sm (1 g pellet was resuspended in 1 mL EM medium).
  • 1 mL aliquots of the suspension were then transferred to fresh 15 mL tubes and 4 ⁇ of 1 ,8-cineole were added (final concentration of 4 mM). Aliquots were extracted at certain times between 0 and 3 hours. To aliquots that had not been extracted after 3 hours, an additional 4 ⁇ 1 ,8-cineole was added. To aliquots that had not been extracted after 6 hours, an additional 12 ⁇ 1 ,8-cineole was added.
  • ferredoxin-expressing pRSFDuet vectors were the same as those in Example 1 1 and the ferredoxin reductase-expressing pETDuet vectors were the same as those in Example 12.
  • the rates in 2-hydroxycineole production by each of the tested enzyme combinations are shown in Figure 7.
  • Example 15 Biotransformation of 1,8-cineole with P450 B ui in combination with FdR3 and FdX2 in a bioreactor #1
  • 500 ⁇ of the primary seed culture were then used to inoculate a secondary seed culture (500 mL TB containing 30 g mL "1 Kan, 50 g mL “1 Amp and 50 g mL “1 Sm in a 2L baffled shake flask) and incubated at 180 rpm and 37°C for 16 hours.
  • the main culture (1 .4 L of DM3) was inoculated with the secondary seed to a OD 6 oo of 0.25 in a 2 L glasses bioreactor controlled by a Biostat B (Sartorius, Germany) control unit.
  • the temperature was maintained at 37 °C and the pH of 7.0 was automatically controlled using 10% (v/v) H 3 PO 4 and 10% (v/v) ammonia solution.
  • Foaming was controlled using 10% (v/v) Sigma Antifoam 204.
  • the bioreactor was operated in cascade mode maintaining a dissolved oxygen of at least 30% by increasing stirrer speed, airflow and oxygen flow as required.
  • a Glucose/Mg feed 800 mL 660 g L "1 Glucose supplemented with 80 mL 1 M MgSO 4 .7H 2 O
  • the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at a flow rate of 0.9 mL h "1 . 14 h post induction, when the OD 6 oo reached ca. 67 the 1 ,8-cineole feed was increased to 1 .8 ml_ h "1 and 16 h post induction further increased to 2.7 ml_ h "1 before the cineole feed was stopped 18 h post induction and then restarted at 1 .8 ml_ h "1 18.5 h post induction.
  • a total of 38.7 ml_ 1 ,8-cineole was fed into the culture. 40 h post induction, the culture was harvested and the cells were removed by centrifugation at 38,400 xg for 30 min at 4°C. The supernatant was then acidified with cone. HCI to precipitate extracellular proteins/proteins released during cell lysis and centrifuged again. The resulting supernatant was neutralised with 4 M NaOH and sterile filtered through a Sartopore 2 300 filter capsule (Sartorius, Germany) and stored at 4°C. Products of 1 ,8-cineole conversion were then extracted using dichloromethane using a continuous extraction process. The combined organic phase was then filtered through a phase separation filter and concentrated under reduced pressure. This procedure yielded 23 g of crude 2-hydroxycineole (confirmed by 1 H-NMR and GC-MS analysis) and small amounts of impurities (e.g. residual 1 ,8-cineole and oxocineole).
  • Example 15 Biotransformation of 1,8-cineole with P450 B ui in combination with FdR3 and FdX2 in a bioreactor #2
  • Example 14 10.4 h post induction the culture was harvested and the cells removed as per Example 14 which resulted in 1 .65 L of culture supernatant. All further downstream processing was done as per Example 15. This process yielded ca. 39 g of crude 2-hydroxycineole.
  • Example 16 Biotransformation of 1,8-cineole with P450 B u2 in combination with FdR3 and FdX2 in a bioreactor #2
  • 500 ⁇ of the primary seed culture were then used to inoculate a secondary seed culture (500 mL TB containing 30 g mL "1 Kan, 50 g mL “1 Amp and 50 g mL “1 Sm in a 2L baffled shake flask) and incubated at 180 rpm and 37°C for 16 hours.
  • the main culture (1 .4 L of DM3) was inoculated with the secondary seed to a OD 6 oo of 0.25 in a 2 L glasses bioreactor controlled by a Biostat B (Sartorius, Germany) control unit.
  • the temperature was maintained at 37 °C and the pH of 7.0 was automatically controlled using 10% (v/v) H 3 P0 4 and 10% (v/v) ammonia solution. Foaming was controlled using 10% (v/v) Sigma Antifoam 204.
  • the bioreactor was operated in cascade mode maintaining a dissolved oxygen of at least 30% by increasing stirrer speed, airflow and oxygen flow as required.
  • the glucose/Mg feed (800 mL 660 g L "1 Glucose supplemented with 80 mL 1 M MgS0 4 .7H 2 0) was started 8 hours post inoculation at a flow rate of 36 mL h "1 which was decreased to 18 mL h "1 after further 4 h when the temperature was decreased to 30°C. After another 15 min, at an OD 6 oo of ca 97, the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at 1 mL h "1 and increased by 1 mL h "1 every hour up to 8 mL h ⁇ 1 .
  • the 1 ,8- cineole feed of 8 mL h "1 was maintained until 10 hours post induction and then turned off, when the accumulation of glucose became apparent and the OD 6 oo had decreased to ca. 68.
  • samples were taken to determine production of hydroxylated 1 ,8-cineole.
  • a total of 52 mL of 1 ,8-cineole was fed to the culture.
  • the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at 1 ml_ h "1 and increased by 1 ml_ h "1 every hour up to 8 ml_ h ⁇ 1 .
  • the 1 ,8-cineole feed of 8 ml_ h "1 was maintained until 10 hours post induction and then turned off, when the accumulation of glucose became apparent and the OD 6 oo had decreased to ca. 108.
  • samples were taken to determine production of hydroxylated 1 ,8-cineole. A total of 52 mL of 1 ,8-cineole was fed to the culture.
  • Example 18 Biotransformation of 1,8-cineole with P450 B u3 in combination with FdR3 and FdX2 in a bioreactor (reduced temperature and later addition of 1,8- cineole)

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Abstract

La présente invention concerne des mono-oxygénases P450, de la ferrédoxine et des ferrédoxines réductases provenant d'une nouvelle souche de Sphingobium yanoikuyae. L'invention concerne également des produits et des procédés de fonctionnalisation de 1,8-cinéol, ainsi que des polynucléotides, des polypeptides, des cellules hôtes et des vecteurs permettant de mettre en œuvre ces procédés. Dans un mode de réalisation, du 1,8-cinéol biohydroxylé ou fonctionnalisé permet d'obtenir un 2-hydroxycinéol, produit à l'aide des mono-oxygénases conjointement à des partenaires de transport d'électrons décrits ici.
PCT/AU2016/050654 2015-07-23 2016-07-22 Mono-oxygénases et procédé de production de cinéol hydroxylé WO2017011879A1 (fr)

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CN114292862A (zh) * 2022-01-12 2022-04-08 华南农业大学 OsCEN2基因在调控水稻种子萌发速率中的应用
CN115786408A (zh) * 2022-10-26 2023-03-14 易安蓝焰煤与煤层气共采技术有限责任公司 一种提高低阶煤生物产气量的方法

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CN112961844A (zh) * 2021-03-02 2021-06-15 江南大学 一种细胞色素p450单加氧酶突变体及其应用
CN112961844B (zh) * 2021-03-02 2022-03-01 江南大学 一种细胞色素p450单加氧酶突变体及其应用
CN114292862A (zh) * 2022-01-12 2022-04-08 华南农业大学 OsCEN2基因在调控水稻种子萌发速率中的应用
CN114292862B (zh) * 2022-01-12 2022-10-04 华南农业大学 OsCEN2基因在调控水稻种子萌发速率中的应用
CN115786408A (zh) * 2022-10-26 2023-03-14 易安蓝焰煤与煤层气共采技术有限责任公司 一种提高低阶煤生物产气量的方法

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