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WO2004090124A2 - Sequences encoding s-hydroxynitril lyases and their use in methods for the production of cyanohydrins - Google Patents

Sequences encoding s-hydroxynitril lyases and their use in methods for the production of cyanohydrins Download PDF

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WO2004090124A2
WO2004090124A2 PCT/EP2004/003844 EP2004003844W WO2004090124A2 WO 2004090124 A2 WO2004090124 A2 WO 2004090124A2 EP 2004003844 W EP2004003844 W EP 2004003844W WO 2004090124 A2 WO2004090124 A2 WO 2004090124A2
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sequence
nucleic acid
sequences
seq
recombinant
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PCT/EP2004/003844
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WO2004090124A3 (en
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Thomas Friedrich
Maria Kesseler
Bernhard Hauer
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Basf Aktiengesellschaft
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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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C12P13/004—Cyanohydrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to new aminoacid sequences isolated from Adenia racemosa encoding S-hydroxynitrile lyases and their use in methods for the production of nitriles and/or carboxylic acids, preferably optically active nitriles and/or carboxylic acids.
  • the invention further relates to nucleic acid encoding said S-hydroxynitrile lyases, recombinant expression constructs comprising said nucleic acid sequences, recombinant vectors comprising the nucleic acid sequences or the expression constructs, to recombinant organisms, preferably microorganisms or yeasts, comprising the nucleic acid sequences, expression constructs or vectors.
  • the invention additionally relates to a method for the production of cyanohydrins and/or carboxylic acids, preferably opti- cally active cyanohydrins and/or carboxylic acids from racemic aldehydes and cyanide.
  • Optically active cyanohydrins and their secondary products serve as building blocks for producing biologically active substances which are used in the pharmaceutical or agricultural industries.
  • cyanohydrins can be prepared by addition of a cyanide group to the carbonyl carbon of an aldehyde or of an unsymmetrical ketone, resulting in mixtures of enantiomers of optically active cyanohydrins.
  • opti- cally active ⁇ -hydroxycarbonic acids can be easily obtained from said optically active cyanohydrines (e.g., by saponification using HCl).
  • ⁇ -arylpropionic acids like e.g., (S)-lbuprofen and (S)-Naproxen (used as nonsteroidal antiinflammatory drugs) and (R)- ⁇ - aryloxypropionic acid esters (used as herbicides, R. A. Sheldon, Chirotechnology, Mar- eel Dekker, 1993, 130-131).
  • Optically active mandeloacid are derivatives thereof are important intermediates for the synthesis of active compounds. Pyrethroide-type insec- tizides are often esters of (S)-m-phenoxybenzaldehyde cyanohydrines.
  • HNL Hydroxynitrile lyases
  • hydroxynitrile lyases can be used for the synthesis of enantriomerically pure cyanohydrins ( Kruse CG Chiral cyanohydrins, their manufacture and utility as chiral building blocks. In: Chirality in Industry Collins AN, Sheldrake GN, and Crosby J, Eds. . John Wiley and Sons Ltd., New York, 1992, 279-299; Effenberger F (1994) Angew Chem Int Ed Engl 257:1555-1564; Griengl H et al. (1997) Chem Commun 20:1933-1940).
  • R-hydroxynitrile lyase derived from almond (Prunus amigdalus; EP-A1 1 223 220; EP 0 276 375, EP 0 326 063, EP 0 547 655) or R-hydroxynitrile lyase derived from flax (Linum usitatissimum), and methods for synthesizing S-cyanohydrins by using S-hydroxynitrile lyase
  • HbHnl hydroxynitrile lyase from Hevea brasiliensis
  • EC 4.1.2.39 hydroxynitrile lyase from Hevea brasiliensis
  • the enzyme has been purified and characterized (Selmar D (1989) Physiol Plant 75: 97-101; Wajant H & Forster S (1996) Plant Sci 115:25-31).
  • the crystal structure of HbHnl has been determined whereas the proposed mechanism of enzyme catalysis is still under discussion (Wagner UG et al. (1996) Structure 4:811-822; Hasslacher M et al.. (1997) Proteins 27, 438- 449).
  • HbHnl has been cloned and expressed in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae (Hasslacher M et al. (1997) Prot. Expres. Pur. 11:61-71).
  • the enzyme is a versatile tool for the synthesis of optically active cyanohydrins as it accepts aliphatic, aromatic, and heterocyclic aldehydes (Griengl H et al. (1997) Chem Commun 20:1933-1940; Klempier N et al. (1993) Tetrahedron Lett. 34:4769-4772; Klempier N et al. (1995) Tetrahedron Asymmetry 6:845-848; Schmidt M et al.
  • EP 0 632 30, WO 97/03204, EP 0 951561, and EP 0 927 766 are describing processes for the production of optically active compounds utilizing (S)-hydroxynitrile lyase from Hevea brasiliensis.
  • EP-A1 969 095 and US 6,319,697 are disclosing (S)-Hydroxynitrile lyases with an improved substrate acceptance derived from the Hevea brasiliensis and Manihot escu- lenta (S)-hydroxynitrile lyases, wherein one or more bulky amino acid residues within the hydrophobic channel leading to the active center have been replaced with less bulky amino acid residues.
  • EP 0 539 767 describes a process based on (S)-hydroxynitrile lyases for the. preparation of (S)-cyanohydrins, using specific cyanide group donors instead of hydrocyanic acid ⁇ .
  • EP-A1 1 016 712 and US 6,387,659 are disclosing a process for producing S-hydroxynitrile lyase using a recombinant yeast cell into which S-hydroxynitrile lyase coding gene derived from cassava (iVlanihot esculenta) (EC 4.1.2.37) is introduced.
  • EP1026256 is disclosing a method for the production of optically active cyanohydrins by adding recombinant microorganism cells which have been transformed by introducing an hydroxynitrile lyase enzyme gene to a reaction system comprising one or more aldehydes or ketones and hydrogen cyanide or a substance capable of producing cyanide ions in the reaction system.
  • a first embodiment of the invention relates to an isolated polypeptide having (S)-hydroxynitrile lyase activity comprising a polypeptide sequence selected from the group comprising
  • sequences comprising at least 10, preferably et least 20, more preferably et least 30, most preferably et least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5.
  • the polypeptide of the invention preferably has a molecular weight lower than 50 kDa, preferably 30 kDa, more preferably 20 kDa, even more preferably 15 kDa, most preferably an apparent weight of the native polypeptide of 14,5 kDa. This low molecular weight makes the protein especially suitable for recombinant expression systems.
  • Said protein may be modified and comprise for example additional aminoacids like e.g., a methionine start codon and/or a signalpeptide fusion at the N-terminus.
  • the poypeptide is described by a sequence selected from the group comprising SEQ ID NO: 3 and 5.
  • the isolated polypeptide of the invention may also be a fusionprotein or a heterologous protein having (S)-hydroxynitrile lyase activity further comprising at least one sequence encoding for a secretory signal peptide, suit- able for causing secretion of said fusionprotein upon expression in at least one eu- karyotic cell.
  • nucleic acid molecules encoding a polypeptide of the invention.
  • nucleic acid molecules comprise a sequences selected from the group consisting of
  • the isolated nucleic acid molecule of the invention is selected from the group consisting of
  • Another embodiment of the invention related to a recombinant expression construct comprising at least one nucleic acid of the invention and to recombinant expression vectors comprising at least one of said recombinant expression constructs and/or nucleic acids of the invention.
  • Another embodiment of the invention related to recombinant organisms comprising at least one recombinant expression vector, recombinant expression construct and/or nucleic acid of the invention.
  • said organism is selected from the group consisting of bacteria, fungi, algae, or plant organism.
  • said organisms is a bacterium of the genera Escherichia, Rhodococcus, Nocardia, Streptomyces or Mycobacterium, or a yeast selected from the group of yeast genera consisting of Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloekera, Trigonopsis, Trichosporon, Rhodotorula, Sporobolomyces, and Bullera, Saccharo-, Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, and Hanseniaspora.
  • the organism is a yeast selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica.
  • sequences comprising at least 10, preferably et least 20, more preferably at least 30, most preferably at least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5.
  • Another embodiment of the invention relates to a method for the preparation of the (S)- enantiomers of optically active cyanohydrins by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a (S)-hydroxynitrile lyase comprising a polypeptide sequence selected from the group comprising
  • the aldehyde or ketone may be an aliphatic, aromatic or heteroaromatic aldehyde or an unsymmetrical ketone is reacted.
  • the cyanide group donor employed may be hydrocyanic acid or a cyanohydrin of the formula (R1)(R2)C(OH)(CN), in which R1 and R2 are alkyl groups.
  • the method of the invention may comprise adding viable, dormant, immobilized, permeabilized or disrupted cells of a recombinant organism expressing a (S)- hydroxynitrile lyase of the invention.
  • the aldehyde and/or ketone may, for example, be reacted with said recombinant (S)-hydroxynitrile lyase in a reaction system selected from the group consisting of organic systems, aqueous or micro-aqueous systems, 2- phase systems, and emulsion systems.
  • a organic solvent may be employed selected from the group consisting of poorly water-soluble, slightly water-miscible or water- immiscible aliphatic or aromatic hydrocarbons, alcohols, ethers and esters.
  • Said organic solvent may be preferably selected from diethyl ether, di-n-propyl ether, di- isopropyl ether, di-n-butyl ether, di-isobutyl ether, methyl-t-butyl ether, n-propyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, sec-butyl acetate, iso-butyl acetates, iso-amyl acetates, methylethylketone, diethylketone, methylisobutylketone, and a mixture of these solvents with each other or with an apolar diluent or solvent selected from aromatic hydrocarbons, aliphatic hydrocarbons and chlorinated aromatic or aliphatic hydrocarbons.
  • Another embodiment of the invention related to the use of at least one material selected from the group consisting of an isolated polypeptide sequence, an isolated nucleic acid sequence, a recombinant expression vector, a recombinant expression construct, a recombinant organism of the invewntion for the production of cyanohydrins, preferably of optically active cyanohydrines
  • Fig.1 Lane 1. Molecular weight standard (from above: 106kDa, 77kDa, 50,8kDa, 30,6kDa, 28,1 kDa, 20,3kDa); Lane 2: Mono-Q purification of a homogenate. Lane 3: Homogenate of a new preparation; Lane 4: Fractions comprising target protein from Q-sepharose column; Lane 5: Fractions comprising target protein from TSK-Phenyl column; Lane 6 to 9: Fractions comprising target protein from Waters-Q column.
  • Fig. 2 Isoelectric point (pi) determination of the (S)-hydroxynitrile lyase from Adenia racemosa.
  • the pi of the (S)-hydroxynitrile lyase from Adenia racemosa is between pH 3,5 and 4,2.
  • Fig. 3 Alternative method for molecular weight determination for (S)-hydroxynitrile lyasefrom Adenia racemosa: The apparent molecular weight is between 14 and
  • Fig. 4 Sequence encoding (S)-hydroxynitrile lyase from Adenia racemosa as derived by amino acid (peptide) sequencing. Ambiguous amino acid residues at the N- and C-terminus are indicated by the amino acid with the highest probability given in the sequence and other possible variation given below.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers or hybrids thereof in either single-or double-stranded, sense or antisense form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • nucleic acid refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides.
  • a nucleic acid can be a "probe” which is a relatively short nucleic acid, usually less than 100 nucleotides in length.
  • a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length.
  • a "target region” of a nucleic acid is a portion of a nucleic acid that is identified to be of interest.
  • a “coding region” of a nucleic acid is the portion of the nucleic acid which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein.
  • the term “gene” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the polypeptide in some manner.
  • a gene includes untranslated regulatory regions of DNA (e.
  • polypeptide refers to a polymer or oligomer of consecutive amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide refers to amino acids joined to each other by peptide bonds or modified peptide bonds and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide can have many types of modifications.
  • Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin or of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, ⁇ -carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
  • Modifications may also include N- or C-terminal fusions to short peptides ("tags", like e.g., 6xHIS-tag) or larger domains (e.g., maltose-binding protein, GST-protein, thioredoxin).
  • tags like e.g., 6xHIS-tag
  • larger domains e.g., maltose-binding protein, GST-protein, thioredoxin
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e. g., homoserine, norieucine, methionine sulfoxide, methionine methyl sulfonium).
  • amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, may be referred to by their commonly accepted single-letter codes.
  • A alanine
  • B asparagine or aspartic acid
  • C cysteine
  • D aspartic acid
  • E glutamate
  • F phenylalanine
  • G glycine
  • H histidine
  • I isoleucine
  • K lysine
  • L leucine
  • M methionine
  • N asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • W tryptophan
  • Y tyrosine
  • Z glutamine or glutamic acid
  • isolated means that a material has been removed from its original environment.
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be isolated in that such a vector or composition is not part of its original environment.
  • nucleic acid sequence or a organism, expression cassette or vector comprising said nucleic acid sequence
  • recombinant refers to all those constructs originating by recombinant methods in which either
  • nucleic acid sequence a) said nucleic acid sequence, or b) a genetic control sequence linked operably to said nucleic acid sequence a), for example a promoter, or c) (a) and (b)
  • Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp, in length.
  • a naturally occurring expression cassette for example the naturally occurring combination of a promoter with the corresponding gene - becomes a recombinant expression cassette when it is modified by non-natural, synthetic "artificial" methods such as, for example, mutagenization.
  • non-natural, synthetic "artificial" methods such as, for example, mutagenization.
  • mutagenization such methods have been described (US 5,565,350; WO 00/15815; also see above).
  • the term "recombinant" with respect to nucleic acids as used herein means that the nucleic acid is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment.
  • Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques, i. e., produced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein.
  • Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modified, changed, mutated or otherwise manipulated by man.
  • a "recombinant polypeptide” is a non-naturally occurring polypeptide that differs in sequence from a naturally occurring polypeptide by at least one amino acid residue.
  • Preferred methods for producing said recombinant polypeptide and/or nucleic acid may comprise directed or non-directed mutagenesis, DNA shuffling or other methods of recursive recombination, an especially preferred method to obtain such recombinant molecules may involve gene shuffling. Shuffling methods are known in the art and, for example, described in WO 01/12817 and in the references cited therein, employed.
  • shuffling is used herein to indicate recombination between nonidentical sequences, in some embodiments shuffling may include crossover via homologous recombination or via non-homologous recombination, such as via cre/lox and/or flp/frt systems.
  • Shuffling can be carried out by employing a variety of different formats, including, for example, in vitro and in vivo shuffling formats, in silico shuffling formats, shuffling formats that utilize either double-stranded or single-stranded templates, primer-based shuffling formats, nucleic acid fragmentation-based shuffling formats, oligonucleotide mediated shuffling formats, all of which are based on recombination events between non identical sequences and are described in more detail or reference herein below, as well as other similar recombination-based formats.
  • Synthetic polypeptides or proteins are those prepared by chemical synthesis (e. g., solid-phase peptide synthesis). Chemical peptide synthesis is well known in the art (see, e. g., Merrifield (1963), Am. Chem. Soc. 85: 2149-2154; Geysen et al. (1984) Proc Natl Acad Sci USA 81:3998) and synthesis kits and automated peptide synthesizer are commercially available (e.g., Cambridge Research Biochemicals, Cleveland, United Kingdom; Model 431 A synthesizer from Applied Biosystems, Inc., Foster City, CA). Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
  • identity as used herein with respect to two nucleic acid sequences is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389 et seq.), setting the following parameters:
  • Gap weight 50 Length weight: 3
  • sequence which has at least 60% homology with sequence SEQ ID NO: 2 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set, has at least 60% identity.
  • the scoring matrix blosum62 was used.
  • identity as used herein with respect to two polypeptides is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters: Gap weight: 8 Length weight: 2
  • sequence which has at least 60% homology with sequence SEQ ID NO: 1 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 60% identity.
  • homology has the same meaning as “identity” in the context of nucleotide sequences. However, with respect to amino acid sequences, “homology” includes the percentage of identical and conservative amino acid substitutions. Percentages of homology can be calculated according to the algorithms of Smith and Waterman (1981) Adv Appl Math 2:482 and Needleman & Wunsch (1970) J Mol Biol 48:443-453 using the scoring matrix blosum62.
  • two sequences are “substantially identical” when they have at least 99.5% nucleotide identity, when compared and aligned for maximum correspondence, as measured using the known sequence comparison algorithms described above.
  • synonymous codons in a coding region may be treated as identical to account for the degeneracy of the genetic code.
  • the region for determination of substantial identity must span at least about 20 residues, and most commonly the sequences are substantially identical over at least about 25-200 residues.
  • two sequences are “substantially identical” when they have at least 99.5% identity, when compared and aligned for maximum correspondence, as measured using the known sequence comparison algorithms described above.
  • conservative amino acid substitutions may be treated as identical if the polypeptide substantially retains its biological function.
  • Hybridization refers to the process by which a nucleic acid strand joins with a complementary strand through hydrogen bonding at complementary bases. Hybridization assays can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions are defined by concentrations of salt or formamide in the pre- hybridization and hybridization solutions, and/or by the hybridization temperature, and are well known in the art. Stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • the hybridization conditions for DNA: RNA hybrids preferably comprise 0.1 x SSC and temperatures between about 30°C and 55°C, preferably between about 45°C and 55°C.
  • melting temperatures calculated by way of example for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for the DNA hybridization are described in relevant textbooks of genetics such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated by formulae known to the skilled worker, for example depending on the length of the nucleic acids, the nature of the hybrids or the G + C content. The skilled worker can find further information on hybridization in the following textbooks: Ausubel et al.
  • stringent hybridization conditions include 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and 200 ng/ml sheared and denatured salmon sperm DNA, and equivalents thereof. Variations on the above ranges and conditions are well known in the art.
  • variant refers to polynucleotides or polypeptides of the invention modified at one or more nucleotides or amino acid residues (respectively) and wherein the encoded polypeptide or polypeptide retains (S)-hydroxynitrile lyase activity.
  • Variants can be produced by any number of means including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site-saturated mutagenesis or any combination thereof.
  • means including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site-saturated mutagenesis or any combination thereof.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-bases and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res 19:5081 ; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al.
  • nucleic acid variations are "silent variations", which are one species of conservatively modified variations. Every nucleic acid sequence recited herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • racemic or “racemate” means compositions of compounds comrising at least one center of asymmetry (e.g., a chiral C-atom) consisting of a 50:50 mixture of the two enantiomers or of any other mixture with enrichment of one of the two enantiomers in the mixture.
  • center of asymmetry e.g., a chiral C-atom
  • optically active means compositions of compounds comrising at least one center of asymmetry (e.g., a chiral C-atom) showing an enantiomeric enrichment.
  • (S)-hydroxynitrile lyase as used herein includes all polypeptides which exhibit at least (S)-hydroxynitrile lyase activity.
  • (S)-hydroxynitrile lyase activity means in general the ability to hydrolyze at least one (S)-hydroxynitrile into the corresponding aldehyde or ketone and cyanide and/or the ability to catalyze the reverse reaction.
  • a compound of the following general formula (I) wherein the carbon atom bound to the cyanide moiety has S-configuration, can be converted to (or produced from) the aldehyde or ketone of the general formula (II):
  • (S)-hydroxynitrile lyases may include but shall not be limited to are enzyme of the EC- classes EC 4.1.2.11 , EC 4.1.2.37, and EC 4.1.2.39.
  • (S)-Hydroxynitriles may for example comprise 2-hydroxyisobutyronitrile, mandelonitrile, (S)-4-hydroxymandelonitrile (hydroxymandelonitrile)
  • the isolated polypeptide encoding the (S)-hydroxynitrile lyase of the invention can be of natural, synthetic, or recombinant origin.
  • a (S)-hydroxynitrile lyase of the invention is further characterized as being described by a polypeptide sequence selected from the group consisting of:
  • sequences comprising at least 10, preferably et least 20, more preferably et least 30, most preferably et least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1, 3 or 5.
  • a (S)-hydroxynitrile lyase of the invention can be mutated or modified by numerous methods known to the person skilled in the art. Mutagenesis methods may be random or directed and may include, for example, those described in W0 98/42727; site- directed mutagenesis (Ling et al. (1997) Anal Biochem. 254(2): 157-78; Dale et al. (1996) Methods Mol Biol 57:369-74; Smith (1985) Ann Rev Genet 19:423-462; Botstein & Shortle (1985) Science 229:1193-1201; Carter (1986) Biochem J 237 :1-7; Kunkel
  • Modifications of a (S)-hydroxynitrile lyase sequence of the invention may be achieved, for example, by mutagenesis on the sequence encoding an enzyme of natural origin (Skandalis et al. (1997) Chemistry & Biology 4:8889-898; Crameri et al. (1998) Nature 391 :288-291).
  • the mutagenesis may include mutagenic chemicals (Singer and Fraenkel-Conrat (1969) Prog Nucl Acid Res Mol Biol 9:1-29), mutagenesis by error- prone PCR (Leung et al. (1989) Technique 1 :11-15), by combinative PCR (Crameri et al.
  • a (S)-hydroxynitrile lyase to be utilized within the invention may be obtained by screening banks of DNA, in particular cDNA or genomic DNA of various sources, in particular of banks of DNA obtained by recombinations and random changes of (S)-hydroxynitrile lyases, by directed molecular evolution or screening of DNA libraries of ground or other biotopes.
  • Additional suitable methods include point mismatch repair (Kramer et al. (1984) Cell 38: 879-887), mutagenesis using repair deficient host strains (Carter et al. (1985)Nucl Acids Res 13: 4431-4443 (1985); Carter (1987) Methods in Enzymol. 154:382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells et al. (1986) Phil Trans R Soc Lond A317: 415-423), mutagenesis by total gene synthesis (Nambiar et al.
  • Kits for mutagenesis are commercially available.
  • kits are available from, e. g., Stratagene , Bio-Rad, Roche, Clontech Laboratories, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, and Promega Corp.
  • Amino acid changes can be made to a (S)-hydroxynitrile lyase of the invention to achieve new or modified properties.
  • This may be realized by a procedure known in the art as "directed evolution". It combines methods of materially changing the enzyme while selecting in an iterative way the variations which present improved properties (Arnold & Volkov (1999) Current Opin Chem Biol 3:54-59; Kuchner and Arnold (1997) Tibtech 15:523-530). For example, methods like the methods described in WO 01/12817 and the methods cited therein may be employed.
  • Preferred methods for obtaining modified (S)-hydroxynitrile lyase may comprise directed or non-directed mutagenesis, DNA shuffling or other methods of recursive recombination.
  • the shuffling of a "family" of nucleic acids is used to create the library of recombinant polynucleotides.
  • a family of nucleic acids is shuffled, nucleic acids that encode homologous polypeptides from different strains, species, or gene families or portions thereof, are used as the different forms of the nucleic acids.
  • the invention may involve creating recombinant libraries of polynucleotides encoding (S)-hydroxynitrile lyases that are then screened to identify those library members that encode a (S)-hydroxynitrile lyase that exhibits a desired property, e. g., enhanced enzymatic activity, stereospecificity, regiospecificity and enantiospecificity, reduced susceptibility to inhibitors, processing stability (e. g., solvent stability, pH stability, thermal stability, etc.), and the like.
  • the recombinant libraries can be created using any of various methods including but not limited to shuffling protocols as described for example in WO 01/12817 and the references cited therein.
  • a (S)-hydroxynitrile lyase of the invention exhibits an activity with regard to an hydroxyarylacetonitrile, more preferably a substituted or unsubstituted mandelonitrile.
  • the mandelonitrile aryl-group may carry one or more substituents.
  • Preferred substituents may be alkyl (preferably methyl), alkoxy (preferably methoxy), hologen, or nitro.
  • the substituted mandelonitrile may be selected from - but not limited to - the group consisting of o-fluoromandelonitrile, p-fluoromandelonitrile, m- fluoromandelonitrile, o-chloromandelonitrile, p-chloromandelonitrile, m- chloromandelonitrile, o-bromomandelonitrile, p-bromomandelonitrile, m- bromomandelonitrile, o-nitromandelonitrile, p-nitromandelonitrile, m-nitromandelonitrile, o-methyimandelonitrile, p-methylmandelonitrile, m-methylmandelonitrile, o- . methoxymandelonitrile, p-methoxymandelonitrile or m-methoxymandelonitrile. More preferred is o-ch
  • a (S)-hydroxynitrile lyase of the invention exhibits an activity with regard to a optically active hydroxyarylacetonitrile, preferably selected from the group consisting S- mandelonitrile, S-p-chloromandelonitrile, S-m-chloromandelonitrile, S-o- chloromandelonitri le, S-o-bromomandelonitrile, S-p-bromomandelonitrile, S-m- bromomandelonitri le, S-o-methylmandelonitrile, S-p-methylmandelonitrile, S-m- methylmandelonitri le. More preferred is S-o-chloromandelic acid.
  • nucleic sequence encoding a (S)-hydroxynitriie lyase of this invention.
  • nucleic acid sequence is encoding an enzyme with (S)-hydroxynitrile lyase activity, which comprises at least one sequence selected from the group of sequences comprising
  • nucleic acid sequence which is at least 60%, preferably 80%, more preferably 90%, most preferably 95% identical to the nucleic acid sequence of SEQ ID NO: 2 or 4, and
  • nucleic acid sequence comprising at least one fragment of at least 20 consecutive bases, preferably 50 consecutive bases, more preferably 100 consecutive bases of at least one of the sequences described by SEQ ID NO: 2 or 4, and
  • nucleic acid sequence which under stringent conditions hybridize with a sequences described by SEQ ID NO: 2 or 4 or sequences derived therefrom by degeneration of the genetic code
  • nucleic acid sequence described by a sequence derived in consequence of the degeneration of the genetic code from a polypeptidesequence encoded by a sequence selected from the group of sequences a, b, c, and d.
  • a nucleic acid sequence encoding said (S)-hydroxynitrile lyase may be incorporated into an expression construct.
  • expression construct in general means any nucleic acid construct, wherein a nucleic acid molecule whose expression (transcription and, if appropriate, translation) generates a (S)-hydroxynitrile lyase is preferably operably linked to at least one genetic control element.
  • genetic control sequences is to be understood in the broad sense and refers to all those sequences which have an effect on the materialization, propagation or function of the expression cassette or a recombinant microorganism according to the invention.
  • genetic control elements are, for example, sequences to which the inducers or repressors bind and thus regulate the expression of the nucleic acid. Genetic control sequences may enhance, regulate, guarantee, or modify the transcription and/or translation in prokaryotic or eukaryotic organisms.
  • nucleic acid construct may, however, also have a simpler structure, that is to say no additional regulatory signals have been inserted upstream of a nucleic acid sequence of the invention, and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequence is mutated in such a way that the regulation no longer takes place, and gene expression is increased.
  • the nucleic acid construct may additionally advantageously comprise one or more enhancer sequences, which make increased expression of the nucleic acid sequence possible, functionally linked to the promoter.
  • nucleic acids according to the invention may be present in one or more copies in the construct.
  • the construct may also comprise further markers such as antibiotic resistances or auxotrophy-complementing genes where appropriate for selection of the construct.
  • nucleic acid sequence encoding a (S)-hydroxynitrile lyase of the invention is operably linked to at least one promoter sequence which ensures its expression in an organism (e.g., a microorganism or a plant).
  • Operable linkage is to be understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example a (S)-hydroxynitrile lyase) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed recombinantly.
  • a direct linkage in the chemical sense is not always necessarily required.
  • Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules.
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 500 base pairs, especially preferably less than 200 base pairs, very especially preferably less than 100 base pairs.
  • a recombinant expression cassette can be constructed by inserting a transcription promoter at upstream site and a transcription-terminator at downstream site of the(S)- hydroxynitrile lyase encoding sequence of the invention to allow the (S)-hydroxynitrile lyase to express in the recombinant host cell (e.g., a yeast cells), and the constructed recombinant expression cassette is then introduced into a recombinant expression vector.
  • the recombinant host cell e.g., a yeast cells
  • transcription promoter and terminator are already present in an expression vector into which the enzyme gene is to be introduced
  • the transcription promoter and terminator may be used and only the enzyme gene may be introduced therebetween, i.e., there is no need to construct an isolated expression cassette, but the recombinant expression cassette is comprised in said recombinant expression vector.
  • multiple expression cassettes can be present in one expression vector.
  • Operable linkage, and an expression cassette can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual.
  • sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences.
  • the insertion of sequences may also lead to the expression of fusion proteins.
  • the expression cassettes according to the invention encompass a promoter, function in the respective host organism S'-upstream of the nucleic acid sequence in question-to be expressed recombinantly, and 3'-downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly.
  • a promoter function in the respective host organism S'-upstream of the nucleic acid sequence in question-to be expressed recombinantly
  • 3'-downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly.
  • different genetic control sequences are preferred.
  • Advantageous regulatory sequences for carrying out the invention in microorganisms are, for example, present in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl q ' T7, T5, T3, gal, trc, rhaP (rhaP B Ao), ara, SP6, ⁇ -P R or the ⁇ -P promoter, which are advantageously used in Gram-negative bacteria.
  • Further advantageous regulatory sequences are, for example, the Gram-positive promoters amy and SP02, or fungal or yeast promoters.
  • promoters of pyruvate decarboxylase and of methanol oxidase from, for example, Hansenula or Pichia (like e.g., the AOX promoter). It is also possible to use artificial promoters for the regulation. Where the yeast Pichia is used as a host to be transformed, promoter in such an expression cassette as described above may be one which promotes enzyme expression within a methanol-utilizing strain in the yeast Pichia in the presence of methanol carbon source. Terminator in such an expression cassette may be one which allows efficient transcrip- tion-termination to obtain maximum gene expression. Particularly, AOX1 promoter and AOX1 terminator are preferred.
  • Plant-specific promoters suitable for expression of the (S)-hydroxynitrile lyase in plant organisms may include constitutive promoters (e.g., CaMV 35S promoter (Franck et al. (1980) Cell 21:285-294), OCS promoter, ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), promoter of the Arabidopsis thaliana (S)-hydroxynitrile lyase-1 gene (GenBank Ace.
  • each of the promoters described above may have DNA consisting of the nucleotide sequence of a native promoter, or DNA consisting of the native promoter sequence having deletion, substitution and/or addition of one or more bases but still retaining the promoter activity. Deletion, substitution or addition of base(s) may be generated by using any conventional techniques such as site-directed mutagenesis.
  • An expression construct or vector of the invention may also comprise further functional elements.
  • the term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention. The following may be mentioned by way of example, but not by limitation:
  • Selection markers are useful to select and separate successfully transformed or homologous recombined cells and to prevent loss of an extrachromosomal DNA- construct over the time. Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., ampicillin, tetracycline, kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or glyphosate).
  • a metabolic inhibitor e.g., 2-deoxyglucose-6-phosphate, WO 98/45456
  • antibiotics e.g., ampicillin, tetracycline, kanamycin, G 418, bleomycin or hygromycin
  • herbicides e.g., phosphinothricin or glyphosate
  • Selection markers suitable for prokarytic organisms may include, but shall not be limited to: Amp (ampicillin resistance; ⁇ Lactamase), Cab (Carbenicillin resistance), Cam (Chloramphenicol resistance), Kan (kanamycin resistance), Rif (rifampicin resistance), Tet (tetracycline resisteace), Zeo (Zeocin resistance), or Spec (Spectinomycin resistance).
  • the selective pressure is kept by certain levels of the antibiotic in the medium (like, e.g., Ampicillin 100 mg/l, Carbenicillin 100 mg/l; Chloramphenicol 35 mg/l, Kanamycin 30 mg/l, rifampicin 200 mg/l, tetracycline 12,5 mg/l, Spectinomycin 50 mg/l).
  • the antibiotic like, e.g., Ampicillin 100 mg/l, Carbenicillin 100 mg/l; Chloramphenicol 35 mg/l, Kanamycin 30 mg/l, rifampicin 200 mg/l, tetracycline 12,5 mg/l, Spectinomycin 50 mg/l.
  • selection markers are those which confer resistance to herbicides.
  • Phosphinothricin acetyltransferases PAT; de Block et al. (1987) EMBO J 6: 2513-2518
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • S Bromoxynil ® degrading
  • bxn Bromoxynil ® degrading
  • Kanamycin- or. G418- resistance genes NPTIl; NPTI
  • marker gene suitable in yeast include Tn903 kan, Com, Hyg, CUP1 and DHFR though they are not limited thereto.
  • expression cassettes or vectors may contain a marker gene by which the host organism (e.g., a yeast clone) can be selected depending on their auxotrophy and/or drug-resistance when a recombinant organism is prepared.
  • Said kind of selection markers is suitable for complementation of a genetic defect in the host organism, like e.g. a deficiency in amino acid synthesis. Complementation allows the host cell to grow on a medium deficient in said amino acid.
  • a marker gene should be se- lected based on the genomic-type of the host (e.g., the Saccharomyces strain) to be used for gene introduction.
  • Suitable are for example deficiencies in the synthesis of tryptophan (e.g., trpC), leucine (e.g., leuB), or histidine (e.g., hisB).
  • trpC tryptophan
  • leucine e.g., leuB
  • histidine e.g., hisB
  • Corresponding microorganism strains are commercially available (e.g., from Clontech Inc.) and can be complemented by selectable markers like e.g., HIS3, TRP1 , LEU2, URA3, LYS2, respectively.
  • Transcription terminator sequences prevent unintended transcription (e.g., read-through) and enhance plasmid and/or mRNA stability and/or amount.
  • Said transcription-terminator may be present downstream of the enzyme gene to allow efficient transcription-termination to obtain maximum gene expression. Examples of such transcription-terminator include, e.g., for expression in yeast ADH1 , TDH1 , TFF and TRP5.
  • SD Shine-Dalgamo sequences
  • Said sequence may be localized 4 to 14 nucleotides upstream of the ATG start- codon, wherein the optimum is 8 nucleotides.
  • the corresponding region should be preferably A/T-rich.
  • Start codon The start codon is the point of initiation for translation. In E.coli and higher eukaryotic organisms ATG is the most often used start codon. In E.coli GTG may be used alternatively.
  • Tags and fusion proteins N- or C-terminal fusions of the recombinant protein with shorter peptides ("tags”) or other proteins hurryfusion proteins" may be used to allow an improved expression, solubility, detection, or purification.
  • the fusion part comprises a protease (e.g., thrombine or factor X) cleavage site, which allows removal of the fusion part after expression and purification.
  • Fusion protein may also, preferably, comprise signalpeptides allowing transport of the (S)- hydroxynitrile lyase of the invention to certain compartments of a host cell or secretion therefrom.
  • said signal peptide is a secretory signal peptide derived from a the (S)-hydroxynitrile lyase known in the art, more preferably from a plant specie (e.g., a Rosacea specie). Sequences are described for example for the HNL5 gene from Prunus amygdalus, and the glucoseoxidase- gene from Aspergillus niger.
  • MCS Multiple cloning sites
  • Stop codons / translation terminators Among the three possible stop codons TAA is preferred (since TAG and TGA may under certain circumstances allow read-through translation). Multiple stop codons can be used to guarantee translation termination.
  • Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation efficacy, the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenborn E &
  • GFP green fluorescent protein
  • chloramphenicol transferase luciferase
  • luciferase Ow et al. (1986) Science 234:856-859
  • ⁇ -galactosidase ⁇ -galactosidase
  • Origins of replication which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E.coli.
  • Examples which may be mentioned are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof can be effected advantageously using vectors which comprise the expression cassettes.
  • the expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site.
  • the plasmid formed is first introduced into E.coli. Correctly transformed E.coli are selected, grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning step.
  • Examples of vectors may be plasmids, cosmids, phages, viruses or else agrobacteria.
  • the expression cassette is introduced by means of plasmid vectors.
  • plasmids in E.coli are pUC18, PUC19, pBIueScript series, pKK223-3, pJOE2702, pBAD, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-lll 113 -B1 , Dgt11 or pBdCI, in Streptomyces are plJ101 , pIJ364, pl 702 or plJ361 , in Bacillus are pUB110, pC194 or pBD214, in Corynebacterium are pSA77 or pAJ667, in fungi are pALS1 , plL2 or pBB116, in plants are pLGV23, pGHIac + , pBIN19, pAK2004
  • yeast episome expression vector (autonomously replicating plasmid) is used as expression vector.
  • Yeast episome plasmid vector contains 2 mu plasmid sequence, which is native to yeast.
  • the vector can be replicated within a host yeast cell by utilizing the replication origin of the 2 mu plasmid sequence.
  • Yeast episome expression vector to be used in the present inven- tion may not be limited to particular vectors as long as it comprises at least ORI sequence of yeast 2 mu plasmid sequence and can be autonomously replicated in a host yeast cell.
  • yeast episome expression vector examples include in yeasts are 2 ⁇ M, pAG-1, YEp6, YEp13 or pEMBLYe23, YEp51 , pYES2, YEp351 and YEp352 but are not limited thereto.
  • yeast episome expression vector may be a shuttle vector which can replicate in a E.coli cell for subcloning in the recombinant E. coli.
  • yeast episome expression vector to be used in the present invention include: a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YEp352, S-hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (des- ignated as YEp352-GC); a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of GAL 10 promoter in yeast expression vector YEp51 (designated as YEp51-C); a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YE351 , S- hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (designated as YEp351-GC); and a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of
  • yeast integrating expression vector (which can be integrated into chromosomal DNA) is used as expression vector.
  • yeast integrating plasmid vector has a DNA sequence (normally a selective marker gene sequence) homologous to that of yeast chromosome, it cannot be replicated as a plasmid in a yeast cell.
  • Such yeast integrating plasmid vector can remain in yeast cells only when homologous replacement occurs between the sequence on the vector homologous to yeast chromosome and the yeast chromosome gene thereby integrating the plasmid vector into the chromosome.
  • the integrated gene is known to be stably retained within the yeast cell even not under growth conditions where expression of selected marker gene is essential.
  • yeast integrating expression vector to be used in the present invention is not limited to particular ones as long as it allows inte- gration of S-hydroxynitrile lyase coding gene derived from cassava carried by the vector into yeast chromosome.
  • yeast integrating vectors such as pRS303 and pRS304, or modified vectors constructed by excising yeast 2 mu plasmid-derived sequence from vectors derived from yeast 2 mu such as YEp51, pYES2, YEp351 and YEp352 and then cyclizing the vectors may be preferably used.
  • Vectors for integration into the chromosome of a methanol-utilizing strain in the yeast Pichia are not limited to but include pPIC3.5K, pPIC9K and pA0815.
  • Yeast integrating expression vectors described above may be preferably shuttle vectors which can replicate in E. coli cells for subcloning in the recombinant E. coli cells. More preferably, such yeast expression vectors contain selec- five marker genes such as ampicillin-resistant genes. Alternatively, such expression vectors contain marker genes by which yeast clones can be selected depending on auxotrophy and drug resistance when recombinant yeast is prepared.
  • marker gene for introduction into the yeast Saccharomyces examples include HIS3, TRP1 , LEU2, URA3, LYS2, Tn903 kan, Cm, Hyg, CUP1 and DHFR though they are not limited thereto.
  • a marker gene should be selected depending on the genomic-type of the host Saccharomyces strain into which the gene is to be introduced.
  • Examples of marker gene for introduction into the yeast Pichia include HIS4 and kan though they are not limited thereto.
  • a marker gene should be selected depending on the genomic-type of the host Pichia strain into which the gene is to be introduced.
  • Vectors for expression in higher eukaryotic (e.g., mammalian) cells containing viral sequences on the basis of SV40, papilloma-virus, adenovirus or polyomavirus (Rodriquez RL & Denhardt DT, ed. ; Vectors : A survey of molecular cloning vectors and their uses, Butterworths (1988), Lenstra et al. (1990) Arch Virol 110:1-24).
  • Said plasmids represent a small selection of the possible plasmids. Further plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (eds. Pouwels PH et al.
  • the invention furthermore relates to recombinant organisms or tissues, organs, parts, cells or propagation material thereof which comprise a (S)-hydroxynitrile lyase of the invention, a nucleic acid sequence encoding said (S)-hydroxynitrile lyase, a recombinant expression cassette comprising said nucleic acid sequence, or a recombinant vector encompassing said expression cassette.
  • Such a recombinant organism is generated, for example, by means of transformation or transfection with the corresponding proteins or nucleic acids.
  • the generation of a transformed organism requires introducing the DNA in question (for example the expression vector), RNA or protein into the host cell in question.
  • a multiplicity of methods are available for this procedure, which is termed transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185:527-537).
  • transformation or transduction or transfection
  • the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can be permeabilized chemically, for example using polyethylene glycol, so that DNA can enter the cell by diffusion.
  • the DNA can also be introduced by calcium phosphate mediation, or fusion with DNA-containing units such as minicells, lysosomes or liposomes.
  • Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an electrical pulse.
  • Preferred general methods include but shall not be limited to calcium phosphate mediated transformation, DEAE- dextrane mediated transformation, cationic lipid mediated transformation, electroporation, transduction, and infection. These methods are well known to the person skilled in the art (Davis et al.(1986) Basic Methods In Molecular Biology; Sambrook J et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press; Ausubel FM et al. (1994) Current protocols in molecular biology, John Wiley and Sons; Glover DM et al. (1995) DNA Cloning Vol.1, IRL Press ISBN 019-963476-9).
  • transformation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the Agrobacterium-mediated transformation is best suited to dicotyledonous plant cells. The methods are described and well known in the art (Horsch RB et al. (1985) Science 225: 1229f.; EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287).
  • the expression cassette is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector.
  • Binary vectors are preferably used (Holsters et al. (1978) Mol Gen Genet 163:181-187).
  • Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).
  • Transformed cells can be selected from untransformed cells when a selectable marker is part of the DNA introduced. Examples of genes which can act as markers are all those which are capable of conferring resistance to antibiotics or herbicides are given above.
  • the invention also relates to recombinant organisms transformed with at least one of the nucleic acid sequences according to the invention, expression cassette according to the invention or vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation material derived from such organisms.
  • organism is to be understood in the broad sense and refers to prokaryotic and eukaryotic organisms, preferably bacteria, yeasts (like e.g., Saccharomyces, Kluyveromyces or Pichia), fungi (like e.g., Aspergillus or Penicilium), non-human animal organisms and plant organisms. Preferred plant organisms are indicated above.
  • microorganism includes bacteria, yeast, fungi, algae and other uni-cellular organism.
  • bacteria includes gram-positive and gram-negative bacteria. Preferred are all Enterobacteriaceae genera and species, and all Actinomycetales orders and species. Especially preferred are the Enterobacteriaceae speicies Escherichia, Serratia, Proteus, Enterobacter, Klebsiella, Salmonella, Shigella, Edwardsielle, Citrobacter, Morganella, Providencia, and Yersinia.
  • Agrobacterium Pseudomonas, Burkholderia, Nocardia, Acetobacter, Gluconobacter, Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Staphylococcus, Aerobacter, Alcaligenes, Rhodococcus, and Streptomyces.
  • Escherichia species are most preferred, especially Escherichia coli.
  • yeast may include but shall not be limited to yeasts of the families Cryptococcaceae, Sporobolomycetaceae including the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloekera, Trigonopsis, Trichosporon, Rhodotorula, Sporobolomyces, and Bullera, and yeast of the families Endomycetaceae and Saccharomycetaceae, icluding but not limited to the genera Saccharo-, Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora.
  • Saccharomyces cerevisiae Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica.
  • the yeast Saccharomyces is used as a host though the host to be used in the present invention is not limited thereto as long as it can stably retain such an expression cassette after introduction of the cas- sette.
  • Example of the yeast Saccharomyces include Saccharomyces cerevisiae KK4, Y334, Inv-Scl and W303 strains. Further, both haploid and diploid strains of these host yeast may be used.
  • yeast is not limited to particular ones as long as it can retain such an expression cassette after introduction of the cassette.
  • Examples of methanol-utilizing strains in the yeast Pichia include Pichia pastoris KM71 and GS115 strains. Both haploid and diploid of these host yeast may be used.
  • plant or "plant organism” as used herein means any organism capable of photosynthesis.
  • the organism is a differentiated multicellular organization.
  • the term includes all genera and species of higher and lower plants of the Plant Kingdom.
  • monocotyledoneous and dicotyledoneous plants more particularly of the plants of culture intended for animal or human feed or food purpose or for industrial utilization, like corn, wheat, barley, canola, soybean, rice, sugarcane, sugar beet, potato, beet, tobacco, cotton, etc.
  • the recombinant organisms can be generated with the above-described methods for the transformation or transfection of organisms.
  • a microorganisms of this invention can be grown and propagated in a medium, which allows growth of said microorganism.
  • Said medium can be of synthetic or natural origin.
  • Various media are available depending on the microorganism and known to the person skilled in the art.
  • the media comprise a carbon source, a nitrogen source, inorganic salts and optionally small amounts of vitamins and/or trace elements.
  • Preferred carbon sources are, for example, polyoles like, e.g., glycerol, sugars like e.g., mono-, di- or polysaccharides (e.g., glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose), complex sugar sources (e.g., molasses), sugar phosphates (e.g., fructose-1 ,6- bisphosphate), sugar alcohols (e.g., mannit), alcohols (e.g., methanol or ethanol), carbonic acids (e.g., citric acid, lactic acid or acetic acid), oils and fats (e.g., soybean oil or rapeseed oil), amino acids or amino acid mixtures (e.g., Casamino acids; Difco) or distinct amino acids (e.g
  • Preferred nitrogen sources are organic or inorganic nitrogen compounds or materials, comprising said compounds.
  • ammonia salts e.g., NH 4 CI or (NH 4 ) 2 S0 4
  • nitrates e.g., NH 4 CI or (NH 4 ) 2 S0 4
  • complex nitrogen sources e.g., brewers yeast autolysate, soybean flour, wheat gluten, yeast extract, peptone, meat extract, caseine hydrolysate, yeast or potato protein, which may also often also function as carbon sources.
  • inorganic salts examples include calcium, magnesium, sodium, cobalt, molybdenum, manganese, potassium, zinc, copper and iron salts.
  • corresponding anions chlorine, sulfate, sulfide, and phosphate ions are especially preferred.
  • An important issue for enhancing productivity is the control of the Fe 2+ or Fe 3+ -ion concentration of the medium.
  • the medium may comprise additional growth factors like e.g., vitamins or growth promoters like biotin, 2-keto-L-gulonic acid, ascorbic acid, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids (e.g., alanine, cysteine, proline, asparagine, glutamine, serine, phenylalanine, omithine or valine), carbonic acids (e.g., citric acid, formic acid, lactic acid) or substances like dithiothreitole.
  • vitamins or growth promoters like biotin, 2-keto-L-gulonic acid, ascorbic acid, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine
  • amino acids e.g., alanine, cysteine, proline, asparagine, glutamine, serine, phenylalanine, omithine or valine
  • the balance of the individual nutricients depends on the fermentation mode and will be adopted to the individual requirements.
  • the media components may be provided at the beginning of the fermentation, after they have been sterilized before if required, or may be continuously or discontinuously added according to the requirements of the culture during the fermentation process.
  • the fermentation and growth conditions are selected in a way to guarantee optimal yield of the product (e.g., optimal yield of (S)-hydroxynitrile lyase activity).
  • Preferred fermentation conditions are between 15°C to 40°C, preferably 25°C to 37°C.
  • the pH is preferably kept in a range of pH 3 to 9, preferably pH 5 to 8.
  • the fermentation time may take from a few hours to several days, preferably from 8 hours to 21 days, more preferably from 4 hours to 14 days. Processes for optimization of media and fermentations conditions is well known in the art (Applied Microbiol Physiology, "A Practical Approach (Eds. PM Rhodes, PF Stanbury, IRL-Press, 1997, S.53-73, ISBN 0 19 963577 3).
  • the S-hydroxynitrile lyase of the invention can be produced by culturing the obtained recombinant yeast in a appropriate yeast medium.
  • Said medium may be conveniently supplemented with nitrogen sources such as yeast nitrogen base w/o amino acids (Difco Laboratories), essential amino acids and casamino acid, carbon sources such as glucose, galactose, raffinose and other saccharides, or alcohol such as glycerol and ethanol.
  • the medium may be appropriately adjusted to pH 4-7.
  • composition of medium may be preferably altered depending on the selective marker gene on the vector to be used in order to prevent deletion of the enzyme gene present in the recombinant yeast cells.
  • medium which does not substantially contain uracil is selected for recombinant yeast transformed with yeast episome expression vector YEp352-GC where the selection marker gene is URA3.
  • medium which does not substantially contain L-leucine is selected for recombinant yeast transformed with yeast episome expression vector YEp351-GC where the selective marker gene is LEU2.
  • an inducer substrate may be added to the medium depending on the pro- moter to be used where enzyme production is required to be induced.
  • an appropriate carbon source should be selected for each case.
  • GAP promoter which is one of the promoters preferable for the present invention
  • any carbon source which can be utilized by the host yeast cells may be used since the promoter will constitutively express.
  • AOX1 promoter which is one of promoters suitable for the present invention
  • glucose may not be added since it may repress/inhibit the expression though glycerol and raffinose do not affect the enzyme gene expression.
  • methanol may be preferably added to the culture for efficient expression of the enzyme gene and thus for production of a large amount of the enzyme since it will promote the gene expression.
  • medium may be suitably selected for growth of the host yeast to be used.
  • nutritional source nor need to add any antibiotics since the enzyme gene may be stably retained in the recombinant yeast cells.
  • medium may be adjusted to pH4-7.
  • the yeast cells are cultured at 25-35°for several hours to three days, for example, until the growth reaches at its stationary phase.
  • another embodiment of the invention relates to a method for the preparation of a cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a hydroxynitrile lyase of the invention, preferably comprising a polypeptide sequence selected from the group comprising
  • sequences comprising at least 10, preferably et least 20, more preferably at least 30, most preferably at least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1, 3 or 5.
  • Another embodiment of the invention further relates to a method for preparing a optically active cyanhydrin (2-hydroxynitrile), which comprises converting the corresponding aldehyde or ketone and a cyanide donor in the presence of (S)- hydroxynitrile lyase of the invention, or a growing, dormant or disrupted abovementioned recombinant organism which contains said either a (S)-hydroxynitrile lyase of the invention, a nucleic acid sequence encoding said (S)-hydroxynitrile lyase of the invention, a recombinant expression cassette or expression vector according to the invention.
  • Starting materials employed in the process according to the invention are an aldehyde or a ketone, a cyanide group donor, a recombinant hydroxynitrile lyase of the invention, and - preferably - at least one diluent or solvent.
  • the diluent or solvent may be water or an organic diluent or solvent, which may be immiscible or slightly miscible with water.
  • a variety of carbonyl compounds can be used as substrates for the addition reaction of the invention, for example optionally substituted (hetero)aromatic aldehydes such as Aldehydes are in this case understood as meaning aliphatic, aromatic or heteroaromatic aldehydes.
  • Aliphatic aldehydes are in this case understood as meaning saturated or unsaturated aliphatic, straight-chain, branched or cyclic aldehydes.
  • Preferred aliphatic aldehydes are straight-chain aldehydes in particular having 2 to 18 C atoms, preferably from 2 to 12, which are saturated or mono- or polyunsaturated.
  • the aldehyde can in this case have both C-C double bonds and C-C triple bonds.
  • the aldehyde can be unsubstituted or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid es- ter, nitro or azido groups.
  • groups which are inert under the reaction conditions for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid es- ter, nitro or azido groups.
  • aromatic or heteroaromatic aldehydes are ben- ⁇ aldehyde or variously substituted benzaldehydes such as, for example, fluorobenzal- dehyde, hydroxybenzaldehyde, phenoxybenzaldehyde (especially 3- phenoxybenzaldehyde), methoxybenzaldehyde, furfural, methylfurfural, nicotinalde- hyde and piperonal, further aromatic aldehydes like e.g., anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-l-carbaldehyde, phthalalde- hydes, pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridine aldehydes
  • saturated or unsaturated aliphatic aldehydes such as crotonaldehyde, methylthiopropionaidehyde, pivaldehyde, (C1- C6)alkoxy-acetaldehyde and isomeric butyraldehydes, and optionally substituted aral- kyl aldehydes such as (subst.) phenylacetaldehyde and phenoxyacetaldehyde.
  • Suitable substituents for the above carbonyl compounds are (C1-C4)alkyl, hydroxy, (C1- C4)alkoxy, phenoxy, halogen and hydroxy(C1-C4)alkyl.
  • Ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is identically or unidentically substituted.
  • Aliphatic ketones are understood as meaning saturated or unsaturated, straight-chain, branched or cyclic ketones.
  • the ketones can be saturated or mono- or polyunsaturated. They can be unsubstituted, or substituted by groups which are inert under reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halo- gen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups.
  • aromatic or heteroaromatic ketones are acetophenone, benzophenone, indolylacetone etc. Aldehydes and unsymmetrical ketones are preferably reacted.
  • Aldehydes and ketones which are suitable for the process according to the invention are known or can be prepared in the customary manner.
  • aldehydes or ketones as a reactive substrate may be represented by the following formula (II).
  • R1 and R2 may independently of one another be selected from the group consisting of (i) a hydrogen atom, (ii) a substituted or unsubstituted, linear or branched, saturated C1-18 alkyl group, or (iii) a substituted or unsubstituted aromatic group having 5-22 cyclic members; where R1 and R2 do not represent hydrogen atoms at the same time. If is advantageous for one of the R 1 or R 2 substituents in the formulae I and II to be aryl, such as phenyl.
  • Suitable substituents for said R 1 or R 2 substituents are, for example, one or more of halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, imino, hydroxy, alkyl, alkenyl, alkenyloxy, alkynyl, alkoxy (preferably C1-8 alkoxy), carboxyl, C3-20 cycloalkyl, aromatic or other saturated or unsaturated nonaromatic rings or ring systems having carbon number of up to 22 which may be substituted with a heteroa- tom, N, O or S (here, when the substituent is a cyclic substituent, the substituent itself may be substituted with one or more halogen, hydroxy group, linear or branched C1-8 alkyl group, or linear or branched C2-8 alkenyl group).
  • halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, imino, hydroxy, alkyl, alkenyl, alken
  • alkyl radicals such as C Ce-alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, thiophenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino.
  • R1 and/or R2 represent substituted aromatic groups
  • the substituent may be one or more of amirio group, imino group, hydroxy group, C1-8 alkoxy groups allyloxy group, halogen, cal- boxyl group, linear or branched, saturated or unsaturated C1-22 alkyl group (here, an aromatic group may be substituted with at least 2 substituents).
  • the method for preparing cyanohydrine comprises conversion of a, aldehyde or ketone of the general formula III into the cyanohydrine of the general formula IV
  • R3 and R5 may independently be selected from the group consisting of hydrogen, substituted or unsubstituted, branched or unbranched C ⁇ -C ⁇ 0 -alkyl or C 1 -C 10 - alkoxy, substituted or unsubstituted aryl or heteroaryl, hydroxyl, halogen such as fluorine, chlorine or bromine, C ⁇ do-alkylamino and amino.
  • Suitable substituents for said R 3, R 4 or R 5 radicals are, for example, one or more substituents such as halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, or other aromatic or other saturated or unsaturated nonaromatic rings or ring systems.
  • substituents such as halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, or other aromatic or other saturated or unsaturated nonaromatic rings or ring systems.
  • alkyl radicals such as C C 6 -alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, thiophenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino
  • a possible cyanide group donor is hydrocyanic acid, a cyanide salt, or a cyanohydrin of the general formula (I).
  • R1 and R2 independently of one another are hydrogen or a hydrocarbon group which is unsubstituted or substituted by groups which are inert under the reaction conditions, or R1 and R2 together are an alkylene group having 4 or 5 C atoms, where R1 and R2 are not simultaneously hydrogen.
  • the hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups.
  • R1 and R2 are preferably alkyl groups having 1 to 6 C atoms, the cyanide group donor is very preferably acetone cyanohydrin.
  • the cyanide group donor can be prepared according to known processes.
  • Cyanohydrins in particular acetone cyanohydrin, are also commercially available.
  • Preferred cyanide salts are preferably sodium cyanide or potassium cyanide.
  • hydrocyanic acid, a cyanide salt or acetone cyanohydrin is employed as the cyanide group donor.
  • the hydrogen cyanide may be supplied either in the form of liquid or gas.
  • An aqueous solution of hydrogen cyanide, i.e., hydrocyanic acid (or prussic acid) may also be equally employed. Any substance that is capable of producing cyanide ion (CN) upon addition to a reaction system may be used.
  • Such substances include salt of hydrogen cyanide such as sodium cyanide and po- tassium cyanide, or the above mentioned cyanhydrin compounds such as acetone cyanohydrin.
  • the hydrocyanic acid can also be released from one of its salts such as, for example, NaCN or KCN only shortly before the reaction and added to the reaction mixture in undiluted form or in dissolved form.
  • the concentration of the aldehyde or ketone contained in the reaction solvent is preferably within the range of 0.01 mM to 5 M.
  • Hydrogen cyanide or the substance capable of producing cyanide ion in the reaction system is used for 1-20 mol per mol of aldehyde or ketone.
  • Per mole of aldehyde or keto group employed at least 1 mol, prefera- bly 1 to 5 mol, particularly preferably 1 to 2 mol, of cyanide group donor are added.
  • recombinant cells which comprise the nucleic acids, nucleic acid constructs or vectors according to the invention for the method according to the invention.
  • Dormant, immobilized, permeabilized or disrupted cells can also be used.
  • Disrupted cells mean, for example, cells which have been made permeable by a treatment with, for example, solvents, or cells which have been disintegrated by an enzyme treatment, by a mechanical treatment (e.g. French press or ultrasound) or by any other method.
  • the crude exracts obtained in this way are suitable and advantageous for the method according to the invention.
  • Purified or partially purified enzymes can also be used for the process. Immobilized microorganisms or enzymes are likewise suitable and can advantageously be used in the reaction.
  • the recombinant organism e.g., the recombinant yeast expressing the (S)- hydroxynitrile lyase of the invention
  • the recombinant organism may be used as a catalyst of the reaction in the form of wet cells directly obtained from the medium, or in the form of dry cells which are subjected to drying or dehydration before use to remove the excessive amount of moisture to enhance dispersibility in the reaction system.
  • the drying or dehydrating process of the cells is not specifically limited as long as the enzymatic activity of the cells is retained. Examples of such process include hot air drying, vacuum drying, lyophilization, spray drying, or drying with. an organic solvent such as acetone.
  • the dry cells are powdered, or granulated by mixing with a binder.
  • the dry cells may be mixed with insoluble carriers, or may be immobilized according to a known method.
  • the cells may also be encapsulated into alginate.
  • the reaction may be carried out by employing a batch or semi-batch system.
  • a continuous system is advantageous where the cells used are filled in a filling tank that allows a liquid to flow therethrough.
  • the recombinant cells may be disrupted.
  • the cytosolic fraction can be used without further purification, by means of which the expenditure of work is mini- mized.
  • the hydroxynitrile lyase can be employed in purified or unpurified form, as such or immobilized. The preparation and purification of the hydroxynitrile lyase can be carried out, for example, by precipitation with ammonium sulfate and subsequent gel filtration (as described e.g., in Example X, or in Selmar D et al. (1989) Physiologia Planta- rum 75:97-101).
  • the S-hydroxynitrile lyase of the invention may be collected from the culture by using conventional enzyme collection methods including: cell lysis using cell- wall digesting enzyme (zymolyase); ultrasonication; disruption using glass beads; extraction with surfactants; self-digestion; and freezing-thawing method.
  • undis- solved materials may be removed by, for example, filtration or centrifugation to give crude enzyme solution containing S-hydroxynitrile lyase.
  • S-hydroxynitrile lyase may be further purified from the crude enzyme solution by using any conventional protein purification method alone or in combination, including: ammonium sulfate fractionation; organic solvent precipitation; adsorption with ion exchanger; ion exchange chromatography; hydrophobic chromatography; gel filtration chromatography; affinity chromatography; and electrophoresis.
  • the enzyme can also be employed in the method of the invention in an adsorbed or precipated form (e.g., on cellulose, viz. Avicel-cellulose TM).
  • An IU (International Unit) in this case expresses the formation of one micromole of product per minute and per gram of enzyme crude isolation.
  • the amount of the respective hydroxynitrile lyase needed is best determined in an activity test, for example according to Selmar et al. (1987) Anal Biochem 166:208-211.
  • the enzymatic activity of the cells may be calculated as follows. The cells suspended in water or buffer are disrupted, and then subjected to centrifugation to obtain the supernatant. Using the supernatant and DL-mandelonitrile as a substrate, the change of absorption at a wavelength of 249.6 nm upon benzaldehyde production resulting from degradation of the substrate with the enzyme is measured.
  • the reaction may be performed in an organic systems, aquateous systems, 2-phase systems (Griengl H.et al. (1998) Tetrahedron. 54(48): 14477-14486), and emulsion systems.
  • the reaction solvent mainly consists of an organic solvent that has poor water solubility or are water- immiscible or only slightly water-miscible (EP-A1 276 375; Tetr.
  • lipophylic solvents e.g. hydrocarbons
  • hydrocarbons are recommended as organic solvents for many enzymatic conversions.
  • organic solvent is not specifically limited as long as it does not influence the enzymatic reaction for synthesizing optically active cyanohydrins, and may suitably be selected depending upon physical properties of the substrates (i.e., aldehydes or ketones) used for the synthesis reaction and upon the physical properties of the cyano- hydrins to be produced.
  • Organic diluents which can be used are water-immiscible aliphatic or aromatic hydrocarbons which are optionally halogenated, alcohols, ethers or esters.
  • the organic solvent used in the biphasic solvent system of the present invention is preferably selected from the group consisting of di(C1-C6)alkyl ethers, (C1- C5)carboxylic (C1-C5)alkyl eaters, di(C1-C5)alkyl ketones, (C4-C8)aliphatic alcohols, and mixtures of these solvents with each other or with apolar diluents.
  • organic solvent examples include linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic hydrocarbon solvent which may optionally be halogenated, such as pentane, hexane, cyclohexane, trichloroethene, chlorobenzene, toluene, xylene, methylene chloride and the like; linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic alcohol solvent which may optionally be halogenated, such as isopropyl alcohol, n-butanol, isobutanol, t-butanol, hexanol, cyclohexanol, n- amyl alcohol and the like; linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic ether solvent which may optionally be halogenated, such as di- ethylether, di-n-propylether, di-iso-p
  • ethyl acetate, diisopropyl ether, methyl tert-butyl ether and dibutyl ether are used.
  • methylethyl ketone, diethylketone, and methylisobutylketone can be used.
  • These solvents may be used alone, or two or more of them may be used in combination. pH of the reaction solvent does not need to be adjusted if the above-described organic solvent is not saturated with an aqueous buffer.
  • the pH of the aqueous buffer is adjusted to be within the range of 3-7, preferably within the range of 3-6 (e.g., using a citrate buffer, a phosphate buffer or an acetate buffer).
  • a hydroxynitrile lyase mediated transcyanation using e.g., acetone cyanohydrin in a biphasic reaction mixture, consisting of an aqueous buffer solution and a water- immiscible organic solvent, i.e. diethylether
  • a hydroxynitrile lyase mediated transcyanation using e.g., acetone cyanohydrin in a biphasic reaction mixture, consisting of an aqueous buffer solution and a water- immiscible organic solvent, i.e. diethylether
  • the method of the present invention in a biphasic solvent system wherein the volume ratio organic phase : aqueous phase varies between approx. 3:1 and approx. 1 :3.
  • the concentration of the starting carbonyl compound in the organic solvent is preferably more than 5% wtJvol.
  • the hydroxynitrile lyase of the invention can be present either in immobilized form in the organic diluent, but the reaction can also be carried out in the above mentioned two-phase systema, using a nonimmobilized hydroxynitrile lyase, the organic diluent employed being a water-immiscible diluent.
  • the enzyme of the invention may also be solubilized in a lyotropic liquid crystal, using certain tensides for the liquid crystal formation.
  • Organic solvent, aqueous buffer and tenside form a ternary system (EP-A1 446 826).
  • the reaction mixture is shaken or stirred at temperatures from approximately 0°C up to the deactivation temperature of the hydroxynitrile lyase, preferably from 20 to 30°C.
  • the cyanide group is transferred from the cyanide group donor to the carbonyl carbon atom of the aldehyde or ketone employed and the (S)-enantiomer of the optically active cyanohydrin corresponding to the aldehyde or ketone employed is mainly formed.
  • the progress of the reaction can in this case be monitored, inter alia, by gas chromatography.
  • the reaction solution and the cells are separated from each other to obtain a solution containing the reaction product.
  • customary tech- niques which first break the emulsion, such as, for example, filtration, centrifugation or coalescence, are employed.
  • the phases formed are then separated, if necessary with addition of demulsifiers, and the product-containing phase is worked up.
  • Components other than the optically active cyanohydrin are removed from the solution, thereby obtaining the optically active cyanohydrin of interest.
  • the product is separated according to a routine method such as distillation, column chromatography, crystallization, extraction or the like.
  • the cyanohydrin formed can be extracted from the reaction mixture with the aid of an organic solvent which is not miscible with water, for example aliphatic or aromatic optionally halogenated hydrocarbons, e.g. pentane, hexane, benzene, toluene, methylene chloride, chloroform, chlorobenzenes, ethers such as, for example, diethyl ether, diisopropyl ether or esters, for example ethyl acetate or mixtures of such solvents.
  • a dehydrating agent for dehydration, a stabilizer or the like may be added.
  • the cyanohydrins thus obtained can optionally be stabilized e.g., by addition of an acid before further processing.
  • the optical purity of the aldehyde cyanohydrins formed was determined as menthyl carbonate by means of gas chromatography on a capillary column as described by J. W. Westley et al. (1968) J Org Chem 33:3978-3980.
  • the optical purity of the ketone cyanohydrins was determined by gas chromatography using a chiral separating phase as described by V. Schurig et al., (1990) Ang. Chemie 102:969-986.
  • the method of the invention preferably results in enantiomeric purities of at least 70%ee, preferably of min. 90%ee, particularly preferably of min. 98%ee, very particularly preferably min. 99 %ee.
  • SEQ ID NO: 1 Aminoacid sequence encoding (S)-hydroxynitrile lyase from Adenia racemosa .
  • SEQ ID NO: 2 Backtranslated nucleicacid sequence optimized for expression in Pichia pastoris. encoding (S)-hydroxynitrile lyase from Adenia racemosa.
  • SEQ ID NO: 3 Aminoacid sequence modified for expression in Pichia pastoris encoding (S)-hydroxynitrile lyase from Adenia racemosa
  • SEQ ID NO: 4 Backtranslated nucleicacid sequence optimized for expression in enteric bacteria (like e.g., E.coli) encoding (S)-hydroxynitrile lyase from Adenia racemosa.
  • SEQ ID NO: 5 Aminoacid sequence modified for expression in enteric bacteria (like e.g., E.coli) encoding (S)-hydroxynitrile lyase from Adenia racemosa
  • x ⁇ l of a sample comprising the enzyme to be tested are diluted with 600 ⁇ l disttllated, sterilized water and suspended in 200 ⁇ l 1 mol/l sodium citrate (pH 5,0), 200 ⁇ l 250 mmol/I R/S-mandelonitrile (in 0,1 M sodium citrate, comprising 0,2 % Triton X- 100 reduced).
  • Substrate Mandelonitril (Aldrich 11,602-5) 4 mM in 0,1 M sodium citrate buffer, prepared immediately before use. Samples: Dilution in assay buffer, pH has to be controlled afterwards Controls:: Positive control blank (reagents only) Assay formate: in 1 ,5ml Eppendorff tube
  • Microtiterplate assay Assay buffer: Sodium citrate 0,1M pH 5,0
  • Substrate Mandelonitrile (Aldrich 11 ,602-5) 8mM in assay buffer; immediately prepared before use. Samples: Dilution in assay buffer (pH has to be controlled afterwards) Controls: Positive control: enzyme from almonds
  • Negative control sample without substrate Blank (reagents only)
  • Microtiterplate UV Star Fa.Greiner 655801
  • Analytic Measurement for 10min, Kinetik Spectramax at 280nm;
  • Example 2 Isolation and purification of a new S-hydroxynitrile lyase from Adenia racemosa
  • 1200 g tuber from Adenia racemosa is cut into small pieces and is together with dry ice grinded into a dry powder.
  • the resulting powder is dissolved in 2 I 20mM MES, pH 6,5, 0OmM L-Lysin, and 20mM ascorbic acid, stirred vigorously for 5 minutes, and extracted for 16 hours under continuous, slow stirring (small amounts of HCN >120ppm can be detected).
  • the resulting mixture is filtered using a hair sieve.
  • the filtered solution is further centrifugated and adjusted to pH7.0. Using water, the conductivity is adjusted to ⁇ 5mS/cm (3,5 I).
  • Q-Sepharose A Q-Sepharose fast flow column (Pharmacia, 5cm diameter, 200 ml volume) is equilibrated in 20mM Tris/HCI, pH 7.0 and loaded at 15ml/min with the filtered solution. After washing the column a linear gradient is run using the same buffer and 1 M NaCl over 120 min. (10ml/min) until 100%. Samples were taken every minute and samples comprising activity (44-47) were pooled (9,6 mU/mg, 94 mg protein in total).
  • TSK-Phenyl A TSK-Pheny column (8mm diameter, 7,5cm length, 3,8ml volume) was equilibrated using 20mM MES, pH 6.0, saturated with ammonium sulfate to 40%. The pooled Q-Sepharose samples were adjusted to 40% saturation using ammonium sulfate. The resulting precipitation is removed by centrifugation. The supernatant was applied in 20 ml portions to the TSK-phenyl column and eluated using a linear gradient against 20mM MESt. Samples comprising activity were pooled (specific activity 15U/mg). The pooled samples comprising activity was saturated to 90% with ammonium sulfate. The resulting precipitation is separated by centrifugation, redissolved in 20mM Tris/HCI, pH 7.0 and dialyzed against the same buffer.
  • Molecular sieve chromatography For determination of the molecular weight of the active protein a chromatography using Superose TM 12 was performed. As standards catalase (232kDa), aldolase (158kDa), serum albumin (67kDa), chymotrypsinogen (25kDa), and ovalbumine (43kDa) were used. Also under this conditions, for the native, active protein a molecular weight below 20kDa was observed. Using a high- resolution Tricin-gel the apparent molecular weight was determined with 14,5kDa.
  • Trypsine cleavage 0,5mg purified protein were adjusted using 1M Tris/HCI, pH 8,5 and 10% SDS to pH 8.5 and 0,1%SDS (ca 50mM final concentration of Tris) and treated with 0.025mg Trypsin (Typ XIII, Sigma) for 16 h at 35°C. The resulting peptide fragments were separated using a RP C18 column (Fa. Phenomenex). A V8-cleavage was performed in the same manner. The serquence of the resulting peptide fragments was determined and the complete protein sequence assembled using overlapping peptide fragment sequences (SEQ ID NO: 1 ; Fig. 4).
  • C-terminale proteine sequence 690 ⁇ l sample (comprising 2 mg protein) were mixed with 310 ⁇ l sodium citrate buffer (50 mM; pH6.0). 40 ⁇ g carboxypeptidase-Y (2 vials of 20 ⁇ g each, Roche) were dissolved in 50 ⁇ l H 2 0. The entire carboxypeptidase-Y solution was then added at room temperature to the protein sample. After 25s, 50s, 80s, 120s, 200s, 300s, 500s, 800s, 1200, and 2100s, respectively, 100 ⁇ l aliquots of the resulting solution were transferred into a new Eppendorf-tube. The reaction was stopped immediately by adding 10 ⁇ l 1% acetic acid and incubated for min at 80°C.
  • the PCR assembly reaction was carried out using the- High Fidelity Master Mix (25 ⁇ l, Roche), 20 pmol of each primer MKe310, 311 , 312, 313, 314, 316, 100 pmol of Mke317 and 318 each, 9 ⁇ l H 2 0 and the following temperature program: 95°C for 30 sec; 55°C 2 min (Hot start); 55°C 2 min, ;30 cycles with 95°C for 303 sec, 55°C for 30 sec, and 72°C for 30 sec; 72°C for 5 min.; storage at 4°C until further usage. In further 2 samples the 55°C steps were substituted for corresponding 55°C and 60°C steps, respectively.
  • arHNLI from plasmid pBKSarHNLI was cloned via BamHI / Notl into pPIK3.5 and transformed into E. coli XLIBIue (Stratagene). Clones were analyzed for plasmid DNA by sequence analysis. The corresponding plasmid, pPIK3.5arHNL1 was transformed into Pichia strains GS115 and KM71 as described in the Pichia expression kit, Cat. No. 1710-01 , Invitrogen (Manual Version F, 160624, 25-0043). Clones were verified via PCR and sequencing of the PCR product.
  • arHNLI secretion of expressed protein
  • PCR products from primers MKe328-318 and Mke329-318 were 350 bp in size and purified by agarose gel electrophoresis (E-Gel, Invitrogen) and column chromatography (GFX-Kit, Pharmacia). They were subsequently digested with SnaBI/ Notl and Notl, respectively, and cloned into a correspondingly digested pPIK9 (Stratagene) giving pPIK9arHNL1-1 and pPIKarHNL1-2. Clones obtained by transformation into E.coli XLIBIue (Stratagene) using solid LB medium comprising 100 ⁇ g/ml ampicilline were analyzed for plasmid DNA by sequence analysis.
  • arHNLI gene should was generated in a way that should also allow (medium) expression in E.coli.
  • a DNA sequence with optimized codon usage for E.coli could be created by modification of arHNLI .
  • the arHNLI gene was subcloned by PCR amplification and digestion with Ndel and Hindlll into a correspondingly digested pDHE vector (DE1SS4S1 S-A1).
  • the High Fidelity Master Mix (Roche) and the following primers were used:
  • the PCR was carried out using the following temperature program: 95°C for 2 minutes; 30 cycles with 95°C for 45 sec, 54°C for 45 sec, and 72°C for 60 sec; 72°C for 10 min.; storage at 4°C until further usage.
  • the PCR products were purified by agarose gel electrophoresis (E-Gel, Invitrogen) and column chromatography (GFX-Kit, Pharmacia). Isolated PCR products were subsequently digested with Ndel and Hindlll, and cloned into a correspondingly digested pDHE vector (DE19848129-A1). Clones obtained by transformation into E. coli XL1 Blue (Stratagene) using solid LB medium comprising 100 ⁇ g/ml ampicilline were analysed for plasmid DNA.
  • E.coli TG10 is a derivative of E.coli TG1 comprising a deficiency in the rhamnose-isomerase rhaA; pAgro (pBB541 ; Tashifumi Tomo-yasu et al. (2001) Mol Microbiol 40(2):397-413) and pHSG575 (Takeshita S et al. (1987) Gene 61:63-74) are plasmids for chaperon GroELS coexpression). Plasmids were transformed into Pichia strains GS115 and KM71 , respectively, as described in the Pichia expression kit, Cat. No. 1710-01 , Invitrogen (Manual Version F, 160624, 25-0043). Clones were verified via PCR and sequencing of the PCR product.

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Abstract

The present invention relates to new aminoacid sequences isolated from Adenia racemosa encoding S-hydroxynitrile lyases and their use in methods for the production of nitriles and/or carboxylic acids, preferably optically active nitriles and/or carboxylic acids. The invention further relates to nucleic acid encoding said S-hydroxynitrile lyases, recombinant expression constructs comprising said nucleic acid sequences, recombinant vectors comprising the nucleic acid sequences or the expression constructs, to recombinant organisms, preferably microorganisms or yeasts, comprising the nucleic acid sequences, expression constructs or vectors. The invention additionally relates to a method for the production of cyanohydrins and/or carboxylic acids, preferably optically active cyanohydrins and/or carboxylic acids from racemic aldehydes and cyanide.

Description

Sequences encoding S-hydroxynitril lyases and their use in methods for the production of cyanohydrins
FIELD OF THE INVENTION
The present invention relates to new aminoacid sequences isolated from Adenia racemosa encoding S-hydroxynitrile lyases and their use in methods for the production of nitriles and/or carboxylic acids, preferably optically active nitriles and/or carboxylic acids. The invention further relates to nucleic acid encoding said S-hydroxynitrile lyases, recombinant expression constructs comprising said nucleic acid sequences, recombinant vectors comprising the nucleic acid sequences or the expression constructs, to recombinant organisms, preferably microorganisms or yeasts, comprising the nucleic acid sequences, expression constructs or vectors. The invention additionally relates to a method for the production of cyanohydrins and/or carboxylic acids, preferably opti- cally active cyanohydrins and/or carboxylic acids from racemic aldehydes and cyanide.
BACKGROUND OF THE INVENTION
Optically active cyanohydrins and their secondary products, for example optically active α-hydroxycarboxylic acids, α -hydroxy ketones and β-aminoalcohols serve as building blocks for producing biologically active substances which are used in the pharmaceutical or agricultural industries. In general, cyanohydrins can be prepared by addition of a cyanide group to the carbonyl carbon of an aldehyde or of an unsymmetrical ketone, resulting in mixtures of enantiomers of optically active cyanohydrins. Furthermore, opti- cally active α-hydroxycarbonic acids can be easily obtained from said optically active cyanohydrines (e.g., by saponification using HCl). Of special importance are compounds including but not limited to α-arylpropionic acids like e.g., (S)-lbuprofen and (S)-Naproxen (used as nonsteroidal antiinflammatory drugs) and (R)- α- aryloxypropionic acid esters (used as herbicides, R. A. Sheldon, Chirotechnology, Mar- eel Dekker, 1993, 130-131). Optically active mandeloacid are derivatives thereof are important intermediates for the synthesis of active compounds. Pyrethroide-type insec- tizides are often esters of (S)-m-phenoxybenzaldehyde cyanohydrines.
Several processes for the production of (S)-cyanohydrins are known in the art (Grδger H (2001) Adv Synth Cata! 343:547-558). In Makromol. Chem. 186, (1985), 1755-62, for example, a process for obtaining (S)-cyanohydrins by reaction of aldehydes with hydrocyanic acid in the presence of benzyloxycarbonyl-(R)-phenylalanine-(R)-histidine methyl ester as a catalyst is described. The optical purity of the (S)-cyanohydrins obtained, however, is unsatisfactory. Hydroxynitrile lyases (HNL) are enzymes catalyzing the stereoselective addition of cyanide to aldehydes or ketones and - depending from the equilibrium of the reaction - also the reverse reaction (reviews: Effenberger F (1994) Angew Chem 106:1609-1619, Effenberger F (1996) Enantiomer 1 :359-363; Effenberger F et al. (2000) Curr Opin Bio- technol 11(6):532-539; Johnson DV & Griengl H (1997) Chimica oggi Sept/Oct, 9-13; Griengl H et al. (1997) Chem. Commun 20:1933-1940; Griengl H et al. (1997) Chem Comm (20): 1933-1940; Schmidt M & Griengl H (1999) Oxynitrilases: From cyanogene- sis to asymmetric synthesis BIOCATALYSIS - FROM DISCOVERY TO APPLICATION. 200 PG. 193-226; Johnson DV et al. (2000) Curr Opin Chem Biol 4(1): 103-9; Griengl H et al. (2000) Ophthalmic Genetics. 18(6):252-6).
Methods utilizing hydroxynitrile lyases for the production of optically active cyanohydrines (or compounds derived therefrom like e.g., α-hydroxycarbonic acids) are described and result in general in optically active products with high optical purity (Effen- berger F (2000) Hydroxynitrile lyases in Stereoselective Synthesis (in Stereoselective Biocatalysis edited by Patel RN; Marcel Dekker Inc. New York-Basle, p.321-342; EP 1148042, EP 1160235, EP 1160329, DE 19529116, EP 1016712, US 6319697, EP 969095; WO 97/03204, EP 1026256; EP 927766).
The biochemistry of hydroxynitrile lyases in general has been reviewed in the literature (Hickel A et al. (1996) Physiol. Plant 98:891-898; Wajant H & Effenberger F (1996) Biol Chem 377: 611-617). In their natural environment, hydroxynitrile lyases from plants catalyze the cleavage of cyanohydrins from cyanogenic glycosides into the corresponding aldehydes of ketones and HCN (Conn EE Cyanogenic glycosides. In: Biochemistry of Plants: A Comprehensive Treatise Vol. 7 Secondary Plant Products. Stumpf PK and Conn EE Eds. Academic Press/New York, 1981, 479-500). In the reverse reaction, hydroxynitrile lyases can be used for the synthesis of enantriomerically pure cyanohydrins ( Kruse CG Chiral cyanohydrins, their manufacture and utility as chiral building blocks. In: Chirality in Industry Collins AN, Sheldrake GN, and Crosby J, Eds. . John Wiley and Sons Ltd., New York, 1992, 279-299; Effenberger F (1994) Angew Chem Int Ed Engl 257:1555-1564; Griengl H et al. (1997) Chem Commun 20:1933-1940).
Methods for synthesizing R-cyanohydrins by using R-hydroxynitrile lyase (EC 4.1.2.10) derived from almond (Prunus amigdalus; EP-A1 1 223 220; EP 0 276 375, EP 0 326 063, EP 0 547 655) or R-hydroxynitrile lyase derived from flax (Linum usitatissimum), and methods for synthesizing S-cyanohydrins by using S-hydroxynitrile lyase
(EC4.1.2.11) deri ved from sorghum (Sorghum bicolor), S-hydroxynitriie lyase (EC4.1.2.37) deri ved from cassava (Manihot esculenta), or S-hydroxynitrile lyase (EC4.1.2.39) deri ved from tropical rubber tree (Hevea brasiliensis) are known. The enzyme from Hevea (rubber tree) and Manihot spp. (cassava) accepts aliphatic and aromatic hydroxynitriles, unlike the enzyme from Sorghum bicolor which does not act on aliphatic hydroxynitriles.
An enzymatic process for the preparation of optically active (R)- or (S)-cyanohydrins by reaction of aliphatic, aromatic or heteroaromatic aldehydes or ketones with hydrocyanic acid in the presence of (R)-hydroxynitrile lyase (EC 4.1.2.10) from Prunus amygdalis or oxy(S)-hydroxynitrile lyase (EC 4.1.2.11) from Sorghum bicolor is described in EP-A1 0 326 063 and EP-A1 0 350 908. However, no aliphatic (S)-cyanohydrins can be prepared with hydrocyanic acid from the (S)-oxy(S)-hydroxynitrile lyase from Sorghum (Angew. Chemie 102 (1990), No. 4, pp. 423-425; Effenberger F et al. (1990) Tetrahedron Letters 31 (9): 1249-1252; Swiss-Prot P52708, Wajant H et al. (1994) Plant Mol Biol 26:735-746).
Most characterized is the hydroxynitrile lyase from Hevea brasiliensis (HbHnl; EC 4.1.2.39), whose natural substrate is acetone cyanohydrin. The enzyme has been purified and characterized (Selmar D (1989) Physiol Plant 75: 97-101; Wajant H & Forster S (1996) Plant Sci 115:25-31). The crystal structure of HbHnl has been determined whereas the proposed mechanism of enzyme catalysis is still under discussion (Wagner UG et al. (1996) Structure 4:811-822; Hasslacher M et al.. (1997) Proteins 27, 438- 449). HbHnl has been cloned and expressed in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae (Hasslacher M et al. (1997) Prot. Expres. Pur. 11:61-71). The enzyme is a versatile tool for the synthesis of optically active cyanohydrins as it accepts aliphatic, aromatic, and heterocyclic aldehydes (Griengl H et al. (1997) Chem Commun 20:1933-1940; Klempier N et al. (1993) Tetrahedron Lett. 34:4769-4772; Klempier N et al. (1995) Tetrahedron Asymmetry 6:845-848; Schmidt M et al. (1996) Tetrahedron 52:7833-7840). EP 0 632 30, WO 97/03204, EP 0 951561, and EP 0 927 766 are describing processes for the production of optically active compounds utilizing (S)-hydroxynitrile lyase from Hevea brasiliensis.
EP-A1 969 095 and US 6,319,697 are disclosing (S)-Hydroxynitrile lyases with an improved substrate acceptance derived from the Hevea brasiliensis and Manihot escu- lenta (S)-hydroxynitrile lyases, wherein one or more bulky amino acid residues within the hydrophobic channel leading to the active center have been replaced with less bulky amino acid residues.
EP 0 539 767 describes a process based on (S)-hydroxynitrile lyases for the. preparation of (S)-cyanohydrins, using specific cyanide group donors instead of hydrocyanic acid~. Disclosed are methods for synthesizing optically active cyanohydrins using recombinant cells transformed with a (S)-hydroxynitrile lyase (EC 4.1.2.39) gene from tropical rubber tree (Hevea brasiliensis; US 6,337,196, EP-A1 0 951 561 , WO 98/30711) or S- hydroxynitrile lyase (EC 4.1.2.37) gene from cassava (Manihot esculenta) (Angew, Chem. Int. Ed. Engl. 35, 437-439, 1996; Biotechnol. Bioeng. 53, 332-338, 1997; Arch. Biochem, Biophys. 311, 496-502, 1994). EP-A1 1 016 712 and US 6,387,659 are disclosing a process for producing S-hydroxynitrile lyase using a recombinant yeast cell into which S-hydroxynitrile lyase coding gene derived from cassava (iVlanihot esculenta) (EC 4.1.2.37) is introduced. EP1026256 is disclosing a method for the production of optically active cyanohydrins by adding recombinant microorganism cells which have been transformed by introducing an hydroxynitrile lyase enzyme gene to a reaction system comprising one or more aldehydes or ketones and hydrogen cyanide or a substance capable of producing cyanide ions in the reaction system.
Despite the hydroxynitrile lyases known in the arte, there is still a high demand for hydroxynitrile lyases with broader or modified substrate specificity, increased stability (e.g., with regard to low pH or application in organic solvents or 2-phase systems), improved activity and improved applicability with regard to recombinant expressions systems. It is therefore an objective of the present invention, to provide new polypeptide sequences having (S)-hydroxynitrile lyase activity, which fulfill one or more of the above described demands.
SUMMARY OF THE INVENTION
Accordingly, a first embodiment of the invention relates to an isolated polypeptide having (S)-hydroxynitrile lyase activity comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60%, preferably at least 80%, more preferably et least 90%, most preferably et least 95% to the sequence as described by SEQ ID NO: 1 , 3 or 5, and
c) sequences comprising at least 10, preferably et least 20, more preferably et least 30, most preferably et least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5.
The polypeptide of the invention preferably has a molecular weight lower than 50 kDa, preferably 30 kDa, more preferably 20 kDa, even more preferably 15 kDa, most preferably an apparent weight of the native polypeptide of 14,5 kDa. This low molecular weight makes the protein especially suitable for recombinant expression systems.
Said protein may be modified and comprise for example additional aminoacids like e.g., a methionine start codon and/or a signalpeptide fusion at the N-terminus. In an preferred embodiment the poypeptide is described by a sequence selected from the group comprising SEQ ID NO: 3 and 5. The isolated polypeptide of the invention may also be a fusionprotein or a heterologous protein having (S)-hydroxynitrile lyase activity further comprising at least one sequence encoding for a secretory signal peptide, suit- able for causing secretion of said fusionprotein upon expression in at least one eu- karyotic cell.
Another embodiment of the invention relates to isolated nucleic acid molecules encoding a polypeptide of the invention. Preferably said nucleic acid molecules comprise a sequences selected from the group consisting of
a) sequences encoding a polypeptide as claimed in any of claim 1 to 3, and
b) sequences which under stringent conditions hybridize with a sequence encoding a polypeptide as claimed in any of claim 1 to 3.
Preferably, the isolated nucleic acid molecule of the invention is selected from the group consisting of
a) sequences described by SEQ ID NO: 2 or 4 and sequences derived therefrom by degeneration of the genetic code, and
b) sequences which under stringend conditions hybridize with a sequences described by SEQ ID NO: 2 or 4 or sequences derived therefrom by degeneration of the ge- netic code.
Another embodiment of the invention related to a recombinant expression construct comprising at least one nucleic acid of the invention and to recombinant expression vectors comprising at least one of said recombinant expression constructs and/or nucleic acids of the invention.
Another embodiment of the invention related to recombinant organisms comprising at least one recombinant expression vector, recombinant expression construct and/or nucleic acid of the invention. Preferably, said organism is selected from the group consisting of bacteria, fungi, algae, or plant organism. More preferably said organisms is a bacterium of the genera Escherichia, Rhodococcus, Nocardia, Streptomyces or Mycobacterium, or a yeast selected from the group of yeast genera consisting of Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloekera, Trigonopsis, Trichosporon, Rhodotorula, Sporobolomyces, and Bullera, Saccharo-, Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, and Hanseniaspora. Most preferably, the organism is a yeast selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica.
Another embodiment of the invention relates to a method for the preparation of a cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a hydroxynitrile lyase comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60%, preferably at least 80%, more preferably et least 90%, most preferably et least 95% to the sequence as described by SEQ ID NO: 1, 3 or 5, and
c) sequences comprising at least 10, preferably et least 20, more preferably at least 30, most preferably at least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5.
Another embodiment of the invention relates to a method for the preparation of the (S)- enantiomers of optically active cyanohydrins by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a (S)-hydroxynitrile lyase comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60%, preferably at least 80%, more preferably et least 90%, most preferably et least 95% to the sequence as described by SEQ ID NO: 1 , 3 or 5, and
c) sequences comprising at least 10, preferably et least 20, more preferably at least 30, most preferably at least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5. The aldehyde or ketone may be an aliphatic, aromatic or heteroaromatic aldehyde or an unsymmetrical ketone is reacted. The cyanide group donor employed may be hydrocyanic acid or a cyanohydrin of the formula (R1)(R2)C(OH)(CN), in which R1 and R2 are alkyl groups.
The method of the invention may comprise adding viable, dormant, immobilized, permeabilized or disrupted cells of a recombinant organism expressing a (S)- hydroxynitrile lyase of the invention. The aldehyde and/or ketone may, for example, be reacted with said recombinant (S)-hydroxynitrile lyase in a reaction system selected from the group consisting of organic systems, aqueous or micro-aqueous systems, 2- phase systems, and emulsion systems. A organic solvent may be employed selected from the group consisting of poorly water-soluble, slightly water-miscible or water- immiscible aliphatic or aromatic hydrocarbons, alcohols, ethers and esters. Said organic solvent may be preferably selected from diethyl ether, di-n-propyl ether, di- isopropyl ether, di-n-butyl ether, di-isobutyl ether, methyl-t-butyl ether, n-propyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, sec-butyl acetate, iso-butyl acetates, iso-amyl acetates, methylethylketone, diethylketone, methylisobutylketone, and a mixture of these solvents with each other or with an apolar diluent or solvent selected from aromatic hydrocarbons, aliphatic hydrocarbons and chlorinated aromatic or aliphatic hydrocarbons.
Another embodiment of the invention related to the use of at least one material selected from the group consisting of an isolated polypeptide sequence, an isolated nucleic acid sequence, a recombinant expression vector, a recombinant expression construct, a recombinant organism of the invewntion for the production of cyanohydrins, preferably of optically active cyanohydrines
BRIEF DESCRIPTION OF THE DRAWINGS
Fig.1: Lane 1. Molecular weight standard (from above: 106kDa, 77kDa, 50,8kDa, 30,6kDa, 28,1 kDa, 20,3kDa); Lane 2: Mono-Q purification of a homogenate. Lane 3: Homogenate of a new preparation; Lane 4: Fractions comprising target protein from Q-sepharose column; Lane 5: Fractions comprising target protein from TSK-Phenyl column; Lane 6 to 9: Fractions comprising target protein from Waters-Q column.
Fig. 2: Isoelectric point (pi) determination of the (S)-hydroxynitrile lyase from Adenia racemosa. Lane 1 , 2, 7, 8 ff, lEF-marker proteins; Lane 3 and 7, soybean tryp- sin-inhibitor IEP 4,55; Lane 4, 5, 6: Fractions comprising target protein from Wa- ters-Q column. The pi of the (S)-hydroxynitrile lyase from Adenia racemosa is between pH 3,5 and 4,2.
Fig. 3: Alternative method for molecular weight determination for (S)-hydroxynitrile lyasefrom Adenia racemosa: The apparent molecular weight is between 14 and
15 KDa.
Fig. 4: Sequence encoding (S)-hydroxynitrile lyase from Adenia racemosa as derived by amino acid (peptide) sequencing. Ambiguous amino acid residues at the N- and C-terminus are indicated by the amino acid with the highest probability given in the sequence and other possible variation given below.
DETAILED DESCRIPTION OF THE INVENTION
GENERAL DEFINITIONS
The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers or hybrids thereof in either single-or double-stranded, sense or antisense form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide". The phrase "a nucleic acid sequence" as used herein refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A "coding region" of a nucleic acid is the portion of the nucleic acid which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein. The term "gene" refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the polypeptide in some manner. A gene includes untranslated regulatory regions of DNA (e. g., promoters, enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).
The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin or of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins-Structure and Molecular Properties 2nd Ed., Creighton TE, Freeman WH and Company, New York (1993); Posttranslational Covalent Modification of Proteins, Johnson BC, Ed., Academic Press, New York, pp. 1-12 (1983)). Modifications may also include N- or C-terminal fusions to short peptides ("tags", like e.g., 6xHIS-tag) or larger domains (e.g., maltose-binding protein, GST-protein, thioredoxin).
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e. g., homoserine, norieucine, methionine sulfoxide, methionine methyl sulfonium).
As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The abbreviations used herein are conventional one letter codes for the amino acids: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine ; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid (see L. Stryer, Biochemistry, 1988, W. H. Freeman and Company, New York.
The term "isolated" as used herein means that a material has been removed from its original environment. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be isolated in that such a vector or composition is not part of its original environment.
The term "natural" or of "natural origin" means in this context that an organism, polypeptide a nucleic acid sequence available in at least one organism which is not changed, mutated, or otherwise manipulated by man. The term "recombinant" with respect to, for example, a nucleic acid sequence (or a organism, expression cassette or vector comprising said nucleic acid sequence) refers to all those constructs originating by recombinant methods in which either
a) said nucleic acid sequence, or b) a genetic control sequence linked operably to said nucleic acid sequence a), for example a promoter, or c) (a) and (b)
is not located in its natural genetic environment or has been modified by recombinant methods, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp, in length. A naturally occurring expression cassette -for example the naturally occurring combination of a promoter with the corresponding gene - becomes a recombinant expression cassette when it is modified by non-natural, synthetic "artificial" methods such as, for example, mutagenization. Such methods have been described (US 5,565,350; WO 00/15815; also see above). Preferably, the term "recombinant" with respect to nucleic acids as used herein means that the nucleic acid is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment.
"Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques, i. e., produced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein.
Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modified, changed, mutated or otherwise manipulated by man. A "recombinant polypeptide" is a non-naturally occurring polypeptide that differs in sequence from a naturally occurring polypeptide by at least one amino acid residue. Preferred methods for producing said recombinant polypeptide and/or nucleic acid may comprise directed or non-directed mutagenesis, DNA shuffling or other methods of recursive recombination, an especially preferred method to obtain such recombinant molecules may involve gene shuffling. Shuffling methods are known in the art and, for example, described in WO 01/12817 and in the references cited therein, employed. The term "shuffling" is used herein to indicate recombination between nonidentical sequences, in some embodiments shuffling may include crossover via homologous recombination or via non-homologous recombination, such as via cre/lox and/or flp/frt systems. Shuffling can be carried out by employing a variety of different formats, including, for example, in vitro and in vivo shuffling formats, in silico shuffling formats, shuffling formats that utilize either double-stranded or single-stranded templates, primer-based shuffling formats, nucleic acid fragmentation-based shuffling formats, oligonucleotide mediated shuffling formats, all of which are based on recombination events between non identical sequences and are described in more detail or reference herein below, as well as other similar recombination-based formats.
"Synthetic" polypeptides or proteins are those prepared by chemical synthesis (e. g., solid-phase peptide synthesis). Chemical peptide synthesis is well known in the art (see, e. g., Merrifield (1963), Am. Chem. Soc. 85: 2149-2154; Geysen et al. (1984) Proc Natl Acad Sci USA 81:3998) and synthesis kits and automated peptide synthesizer are commercially available (e.g., Cambridge Research Biochemicals, Cleveland, United Kingdom; Model 431 A synthesizer from Applied Biosystems, Inc., Foster City, CA). Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
The term "identity" as used herein with respect to two nucleic acid sequences is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389 et seq.), setting the following parameters:
Gap weight: 50 Length weight: 3
Average match: 10 Average mismatch: 0
For example a sequence which has at least 60% homology with sequence SEQ ID NO: 2 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set, has at least 60% identity. Where appropriate the scoring matrix blosum62 was used.
The term "identity" as used herein with respect to two polypeptides is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters: Gap weight: 8 Length weight: 2
Average match: 2,912 Average mismatch:-2,003
For example a sequence which has at least 60% homology with sequence SEQ ID NO: 1 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 60% identity.
As used herein, "homology" has the same meaning as "identity" in the context of nucleotide sequences. However, with respect to amino acid sequences, "homology" includes the percentage of identical and conservative amino acid substitutions. Percentages of homology can be calculated according to the algorithms of Smith and Waterman (1981) Adv Appl Math 2:482 and Needleman & Wunsch (1970) J Mol Biol 48:443-453 using the scoring matrix blosum62.
As used herein in the context of two or more nucleic acid sequences, two sequences are "substantially identical" when they have at least 99.5% nucleotide identity, when compared and aligned for maximum correspondence, as measured using the known sequence comparison algorithms described above. In addition, for purposes of determining whether sequences are substantially identical, synonymous codons in a coding region may be treated as identical to account for the degeneracy of the genetic code. Typically, the region for determination of substantial identity must span at least about 20 residues, and most commonly the sequences are substantially identical over at least about 25-200 residues.
As used herein in the context of two or more amino acid sequences, two sequences are "substantially identical" when they have at least 99.5% identity, when compared and aligned for maximum correspondence, as measured using the known sequence comparison algorithms described above. In addition, for purposes of determining whether sequences are substantially identical, conservative amino acid substitutions may be treated as identical if the polypeptide substantially retains its biological function.
"Hybridization" refers to the process by which a nucleic acid strand joins with a complementary strand through hydrogen bonding at complementary bases. Hybridization assays can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions are defined by concentrations of salt or formamide in the pre- hybridization and hybridization solutions, and/or by the hybridization temperature, and are well known in the art. Stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature. The term "standard conditions" as used herein means, for example depending on the nucleic acid, temperatures between 42 and 58°C in an aqueous buffer solution with a concentration between 0.1 and 5 x SSC (1 X SSC = 0.15 M NaCl, 15 M sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, such as, for example, 42°C in 5xSSC, 50% formamide. The hybridization conditions for DNA: DNA hybrids preferably comprise 0.1 x SSC and temperatures between about 20°C and 45°C, preferably between about 30°C and 45°C. The hybridization conditions for DNA: RNA hybrids preferably comprise 0.1 x SSC and temperatures between about 30°C and 55°C, preferably between about 45°C and 55°C. These temperatures stated for the hybridization are melting temperatures calculated by way of example for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for the DNA hybridization are described in relevant textbooks of genetics such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated by formulae known to the skilled worker, for example depending on the length of the nucleic acids, the nature of the hybrids or the G + C content. The skilled worker can find further information on hybridization in the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford. In particular, as used herein, "stringent hybridization conditions" include 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and 200 ng/ml sheared and denatured salmon sperm DNA, and equivalents thereof. Variations on the above ranges and conditions are well known in the art.
The term "variant" as used herein refers to polynucleotides or polypeptides of the invention modified at one or more nucleotides or amino acid residues (respectively) and wherein the encoded polypeptide or polypeptide retains (S)-hydroxynitrile lyase activity. Variants can be produced by any number of means including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site-saturated mutagenesis or any combination thereof.
"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-bases and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res 19:5081 ; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al. (1994) Mol Cell Probes 8:91-98). Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one species of conservatively modified variations. Every nucleic acid sequence recited herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which, along with GUG in some organisms, is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The term "racemic" or "racemate" means compositions of compounds comrising at least one center of asymmetry (e.g., a chiral C-atom) consisting of a 50:50 mixture of the two enantiomers or of any other mixture with enrichment of one of the two enantiomers in the mixture.
The term "optically active" means compositions of compounds comrising at least one center of asymmetry (e.g., a chiral C-atom) showing an enantiomeric enrichment. The term "(S)-hydroxynitrile lyase" as used herein includes all polypeptides which exhibit at least (S)-hydroxynitrile lyase activity.
The term "(S)-hydroxynitrile lyase activity" means in general the ability to hydrolyze at least one (S)-hydroxynitrile into the corresponding aldehyde or ketone and cyanide and/or the ability to catalyze the reverse reaction. Preferably a compound of the following general formula (I), wherein the carbon atom bound to the cyanide moiety has S-configuration, can be converted to (or produced from) the aldehyde or ketone of the general formula (II):
Figure imgf000017_0001
(S)-hydroxynitrile lyases may include but shall not be limited to are enzyme of the EC- classes EC 4.1.2.11 , EC 4.1.2.37, and EC 4.1.2.39.
(S)-Hydroxynitriles may for example comprise 2-hydroxyisobutyronitrile, mandelonitrile, (S)-4-hydroxymandelonitrile (hydroxymandelonitrile)
The isolated polypeptide encoding the (S)-hydroxynitrile lyase of the invention can be of natural, synthetic, or recombinant origin.
In a preferred embodiment, a (S)-hydroxynitrile lyase of the invention is further characterized as being described by a polypeptide sequence selected from the group consisting of:
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60%, preferably at least 80%, more preferably et least 90%, most preferably et least 95% to the sequence as described by SEQ ID NO: 1, 3 or 5, and
c) sequences comprising at least 10, preferably et least 20, more preferably et least 30, most preferably et least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1, 3 or 5.
A (S)-hydroxynitrile lyase of the invention can be mutated or modified by numerous methods known to the person skilled in the art. Mutagenesis methods may be random or directed and may include, for example, those described in W0 98/42727; site- directed mutagenesis (Ling et al. (1997) Anal Biochem. 254(2): 157-78; Dale et al. (1996) Methods Mol Biol 57:369-74; Smith (1985) Ann Rev Genet 19:423-462; Botstein & Shortle (1985) Science 229:1193-1201; Carter (1986) Biochem J 237 :1-7; Kunkel
(1987) "The efficiency of oligonucleotide directed mutagenesis" Nucleic Acids & Molecular Biology, Eckstein F and Lilley DMJ eds, Springer Verlag, Berlin) ; mutagenesis using uracil containing templates (Kunkel (1985) Proc Natl Acad Sci USA 82:488-492; Kunkel TA et al. (1987) Methods in Enzymol 154,367-382; Bass SV et al.
(1988) Science 242:240-245); oligonucleotide-directed mutagenesis (for review see, Smith (1985) Ann Rev Genet 19:423-462; Botstein & Shortle (1985) Science 229:1193- 1201; Carter (1986) Biochem J 237:1-7, Zoller & Smith (1982) Nucleic Acids Res 10:6487-6500, Zoller & Smith (1983) Methods in Enzymol 100,468-500, Zoller & Smith (1987) Methods in Enzymol. 154,329-350); phosphothioate-modified DNA mutagenesis (Taylor et al. (1985) Nucl Acids Res 13: 8749-8764; Taylor et al. (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein (1986) Nucl Acids Res 14:9679-9698; Sayers et al. (1988) Nucl Acids Res 16:791-802; Sayers et al. (1988) Nucl Acids Res 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz (1987) Methods in Enzymol 154:350-367; Kramer et al. (1988) Nucl Acids Res. 16:7207; Fritz et al. (1988) Nucl Acids Res 16:6987-6999. Modifications of a (S)-hydroxynitrile lyase sequence of the invention may be achieved, for example, by mutagenesis on the sequence encoding an enzyme of natural origin (Skandalis et al. (1997) Chemistry & Biology 4:8889-898; Crameri et al. (1998) Nature 391 :288-291). The mutagenesis may include mutagenic chemicals (Singer and Fraenkel-Conrat (1969) Prog Nucl Acid Res Mol Biol 9:1-29), mutagenesis by error- prone PCR (Leung et al. (1989) Technique 1 :11-15), by combinative PCR (Crameri et al. (1998) above mentioned; Shao et al.(1998) Nucleic Acids Res 26:681-683), or by directed mutagenesis (Directed Mutagenesis: A Practical Approach (1991) McPherson MJ, ed. IRL PRESS), etc. In addition, a (S)-hydroxynitrile lyase to be utilized within the invention may be obtained by screening banks of DNA, in particular cDNA or genomic DNA of various sources, in particular of banks of DNA obtained by recombinations and random changes of (S)-hydroxynitrile lyases, by directed molecular evolution or screening of DNA libraries of ground or other biotopes.
Additional suitable methods include point mismatch repair (Kramer et al. (1984) Cell 38: 879-887), mutagenesis using repair deficient host strains (Carter et al. (1985)Nucl Acids Res 13: 4431-4443 (1985); Carter (1987) Methods in Enzymol. 154:382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells et al. (1986) Phil Trans R Soc Lond A317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) Science 223:1299-1301; Sakamar & Khorana (1988) Nucl Acids Res 14:6361-6372; Wells et al. (1985) Gene 34: 315-323 (1985); Grundstrom et al. (1985) Nucl Acids Res. 13:3305-3316), double-strand break repair (Mandecki (1986) Proc Natl Acad Sci. USA 83:7177-7181). Additional details on many of the above methods can be found in Methods in Enzymology, Vol. 154, which also describes useful controls for troubleshooting problems with various mutagenesis methods. Furthermore, mutations can be introduced by applying other random mutagenesis methods (e. g., passage through mutagenic bacterial strains, and the like). Kits for mutagenesis are commercially available. For example, kits are available from, e. g., Stratagene , Bio-Rad, Roche, Clontech Laboratories, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, and Promega Corp.
Amino acid changes can be made to a (S)-hydroxynitrile lyase of the invention to achieve new or modified properties. This may be realized by a procedure known in the art as "directed evolution". It combines methods of materially changing the enzyme while selecting in an iterative way the variations which present improved properties (Arnold & Volkov (1999) Current Opin Chem Biol 3:54-59; Kuchner and Arnold (1997) Tibtech 15:523-530). For example, methods like the methods described in WO 01/12817 and the methods cited therein may be employed. Preferred methods for obtaining modified (S)-hydroxynitrile lyase may comprise directed or non-directed mutagenesis, DNA shuffling or other methods of recursive recombination. In a preferred embodiment, the shuffling of a "family" of nucleic acids (e.g., nucleic acid sequences encoding for (S)-hydroxynitrile lyases from different species) is used to create the library of recombinant polynucleotides. When a family of nucleic acids is shuffled, nucleic acids that encode homologous polypeptides from different strains, species, or gene families or portions thereof, are used as the different forms of the nucleic acids.
The invention may involve creating recombinant libraries of polynucleotides encoding (S)-hydroxynitrile lyases that are then screened to identify those library members that encode a (S)-hydroxynitrile lyase that exhibits a desired property, e. g., enhanced enzymatic activity, stereospecificity, regiospecificity and enantiospecificity, reduced susceptibility to inhibitors, processing stability (e. g., solvent stability, pH stability, thermal stability, etc.), and the like. The recombinant libraries can be created using any of various methods including but not limited to shuffling protocols as described for example in WO 01/12817 and the references cited therein.
Preferably a (S)-hydroxynitrile lyase of the invention exhibits an activity with regard to an hydroxyarylacetonitrile, more preferably a substituted or unsubstituted mandelonitrile. The mandelonitrile aryl-group may carry one or more substituents. Preferred substituents may be alkyl (preferably methyl), alkoxy (preferably methoxy), hologen, or nitro. The substituted mandelonitrile may be selected from - but not limited to - the group consisting of o-fluoromandelonitrile, p-fluoromandelonitrile, m- fluoromandelonitrile, o-chloromandelonitrile, p-chloromandelonitrile, m- chloromandelonitrile, o-bromomandelonitrile, p-bromomandelonitrile, m- bromomandelonitrile, o-nitromandelonitrile, p-nitromandelonitrile, m-nitromandelonitrile, o-methyimandelonitrile, p-methylmandelonitrile, m-methylmandelonitrile, o- . methoxymandelonitrile, p-methoxymandelonitrile or m-methoxymandelonitrile. More preferred is o-chloromandelonitrile.
Most preferably, a (S)-hydroxynitrile lyase of the invention exhibits an activity with regard to a optically active hydroxyarylacetonitrile, preferably selected from the group consisting S- mandelonitrile, S-p-chloromandelonitrile, S-m-chloromandelonitrile, S-o- chloromandelonitri le, S-o-bromomandelonitrile, S-p-bromomandelonitrile, S-m- bromomandelonitri le, S-o-methylmandelonitrile, S-p-methylmandelonitrile, S-m- methylmandelonitri le. More preferred is S-o-chloromandelic acid.
Yet another embodiment of the invention comprises a nucleic sequence encoding a (S)-hydroxynitriie lyase of this invention. Preferably said nucleic acid sequence is encoding an enzyme with (S)-hydroxynitrile lyase activity, which comprises at least one sequence selected from the group of sequences comprising
Preferably a nucleic acid sequence of the invention is further characterized as being described by a sequence selected from the group consisting of:
a) a nucleic acid sequence as described by SEQ ID NO: 2 or 4, and
b) a nucleic acid sequence which is at least 60%, preferably 80%, more preferably 90%, most preferably 95% identical to the nucleic acid sequence of SEQ ID NO: 2 or 4, and
c) a nucleic acid sequence comprising at least one fragment of at least 20 consecutive bases, preferably 50 consecutive bases, more preferably 100 consecutive bases of at least one of the sequences described by SEQ ID NO: 2 or 4, and
d) a nucleic acid sequence which under stringent conditions hybridize with a sequences described by SEQ ID NO: 2 or 4 or sequences derived therefrom by degeneration of the genetic code, and
e) a nucleic acid sequence described by a sequence derived in consequence of the degeneration of the genetic code from a polypeptidesequence encoded by a sequence selected from the group of sequences a, b, c, and d.
For recombinant expression of a (S)-hydroxynitrile lyase according to the invention, a nucleic acid sequence encoding said (S)-hydroxynitrile lyase may be incorporated into an expression construct. The term "expression construct" in general means any nucleic acid construct, wherein a nucleic acid molecule whose expression (transcription and, if appropriate, translation) generates a (S)-hydroxynitrile lyase is preferably operably linked to at least one genetic control element. The term "genetic control sequences" is to be understood in the broad sense and refers to all those sequences which have an effect on the materialization, propagation or function of the expression cassette or a recombinant microorganism according to the invention. These genetic control elements are, for example, sequences to which the inducers or repressors bind and thus regulate the expression of the nucleic acid. Genetic control sequences may enhance, regulate, guarantee, or modify the transcription and/or translation in prokaryotic or eukaryotic organisms.
Genetic control sequences are, for example, described in "Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)", "Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press,Boca Raton, Florida, eds.:Glick and Thompson, Chapter 7, 89-108", and in the references cited therein.
In addition to these novel regulatory sequences, it is also possible for the natural regulation of these sequences to be present upstream (in front) of the actual structural genes and, where appropriate, to have been genetically modified so that the natural regulation is switched off and the expression of the genes has been increased. The nucleic acid construct may, however, also have a simpler structure, that is to say no additional regulatory signals have been inserted upstream of a nucleic acid sequence of the invention, and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequence is mutated in such a way that the regulation no longer takes place, and gene expression is increased. The nucleic acid construct may additionally advantageously comprise one or more enhancer sequences, which make increased expression of the nucleic acid sequence possible, functionally linked to the promoter. It is also possible to insert advantageous additional sequences at the 3' end of the DNA sequences, such as other regulatory elements or terminators. The nucleic acids according to the invention may be present in one or more copies in the construct. The construct may also comprise further markers such as antibiotic resistances or auxotrophy-complementing genes where appropriate for selection of the construct.
In a preferred embodiment, the nucleic acid sequence encoding a (S)-hydroxynitrile lyase of the invention is operably linked to at least one promoter sequence which ensures its expression in an organism (e.g., a microorganism or a plant).
Operable linkage is to be understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example a (S)-hydroxynitrile lyase) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed recombinantly. A direct linkage in the chemical sense is not always necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 500 base pairs, especially preferably less than 200 base pairs, very especially preferably less than 100 base pairs.
A recombinant expression cassette can be constructed by inserting a transcription promoter at upstream site and a transcription-terminator at downstream site of the(S)- hydroxynitrile lyase encoding sequence of the invention to allow the (S)-hydroxynitrile lyase to express in the recombinant host cell (e.g., a yeast cells), and the constructed recombinant expression cassette is then introduced into a recombinant expression vector. Alternatively, where transcription promoter and terminator are already present in an expression vector into which the enzyme gene is to be introduced, the transcription promoter and terminator may be used and only the enzyme gene may be introduced therebetween, i.e., there is no need to construct an isolated expression cassette, but the recombinant expression cassette is comprised in said recombinant expression vector. In either cases, multiple expression cassettes can be present in one expression vector.
Operable linkage, and an expression cassette, can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual. However, further sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins.
The expression cassettes according to the invention encompass a promoter, function in the respective host organism S'-upstream of the nucleic acid sequence in question-to be expressed recombinantly, and 3'-downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly. Depending on the host organism to be transformed with an expression cassette or vector of the invention, different genetic control sequences are preferred.
Advantageous regulatory sequences for carrying out the invention in microorganisms are, for example, present in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq' T7, T5, T3, gal, trc, rhaP (rhaPBAo), ara, SP6, λ-PR or the λ-P promoter, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, the Gram-positive promoters amy and SP02, or fungal or yeast promoters. Examples of yeast promoter for efficient expression of the introduced gene in a yeast cell include native promoters such as PGK, GAP, TPI, GAL1, GAL10, ADH, ADH2, PH05, CUP1 and MF alpha 1, recombinant promoters such as PGK/ al- pha 2 operator, GAP/GAL, PGK/GAL, GAP/ADH2, CYC/GRE and PGK/ARE, and mutated promoters such as Leu2-d. Further useful are promoters such as ADC1 , MFα, AC, P-60, CYC1 , GAPDH, TEF, and rp28. Particularly, GAP promoter is preferred. Also advantageous in this connection are the promoters of pyruvate decarboxylase and of methanol oxidase from, for example, Hansenula or Pichia (like e.g., the AOX promoter). It is also possible to use artificial promoters for the regulation. Where the yeast Pichia is used as a host to be transformed, promoter in such an expression cassette as described above may be one which promotes enzyme expression within a methanol-utilizing strain in the yeast Pichia in the presence of methanol carbon source. Terminator in such an expression cassette may be one which allows efficient transcrip- tion-termination to obtain maximum gene expression. Particularly, AOX1 promoter and AOX1 terminator are preferred.
Plant-specific promoters suitable for expression of the (S)-hydroxynitrile lyase in plant organisms may include constitutive promoters (e.g., CaMV 35S promoter (Franck et al. (1980) Cell 21:285-294), OCS promoter, ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), promoter of the Arabidopsis thaliana (S)-hydroxynitrile lyase-1 gene (GenBank Ace. No.: U38846, nucleotides 3862 to 5325 or else 5342), tissue-specific promoters (e.g., phaseolin promoter; US 5,504,200) or chemically inducible promoters (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108).
In principle, all natural promoters with their regulatory sequences like those mentioned above may be used for the method according to the invention. In addition, synthetic promoters may also be used advantageously. Each of the promoters described above may have DNA consisting of the nucleotide sequence of a native promoter, or DNA consisting of the native promoter sequence having deletion, substitution and/or addition of one or more bases but still retaining the promoter activity. Deletion, substitution or addition of base(s) may be generated by using any conventional techniques such as site-directed mutagenesis.
An expression construct or vector of the invention may also comprise further functional elements. The term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention. The following may be mentioned by way of example, but not by limitation:
a). Selection markers
Selection markers are useful to select and separate successfully transformed or homologous recombined cells and to prevent loss of an extrachromosomal DNA- construct over the time. Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., ampicillin, tetracycline, kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or glyphosate).
Selection markers suitable for prokarytic organisms may include, but shall not be limited to: Amp (ampicillin resistance; βLactamase), Cab (Carbenicillin resistance), Cam (Chloramphenicol resistance), Kan (kanamycin resistance), Rif (rifampicin resistance), Tet (tetracycline resisteace), Zeo (Zeocin resistance), or Spec (Spectinomycin resistance). The selective pressure is kept by certain levels of the antibiotic in the medium (like, e.g., Ampicillin 100 mg/l, Carbenicillin 100 mg/l; Chloramphenicol 35 mg/l, Kanamycin 30 mg/l, rifampicin 200 mg/l, tetracycline 12,5 mg/l, Spectinomycin 50 mg/l).
For use in plants, especially preferred selection markers are those which confer resistance to herbicides. Examples are Phosphinothricin acetyltransferases (PAT; de Block et al. (1987) EMBO J 6: 2513-2518), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), sulfonylurea- and imidazolinone-inactivating acetolactate synthases, Bromoxynil® degrading (S)-hydroxynitrile lyases (bxn). In other eukaryotic organisms, Kanamycin- or. G418- resistance genes (NPTIl; NPTI) can be used. Examples of marker gene suitable in yeast include Tn903 kan, Com, Hyg, CUP1 and DHFR though they are not limited thereto.
Also, expression cassettes or vectors may contain a marker gene by which the host organism (e.g., a yeast clone) can be selected depending on their auxotrophy and/or drug-resistance when a recombinant organism is prepared. Said kind of selection markers is suitable for complementation of a genetic defect in the host organism, like e.g. a deficiency in amino acid synthesis. Complementation allows the host cell to grow on a medium deficient in said amino acid. Preferably, a marker gene should be se- lected based on the genomic-type of the host (e.g., the Saccharomyces strain) to be used for gene introduction. Suitable are for example deficiencies in the synthesis of tryptophan (e.g., trpC), leucine (e.g., leuB), or histidine (e.g., hisB). Corresponding microorganism strains are commercially available (e.g., from Clontech Inc.) and can be complemented by selectable markers like e.g., HIS3, TRP1 , LEU2, URA3, LYS2, respectively.
b) Transcription terminator sequences: Transcription terminator sequences prevent unintended transcription (e.g., read-through) and enhance plasmid and/or mRNA stability and/or amount. Said transcription-terminator may be present downstream of the enzyme gene to allow efficient transcription-termination to obtain maximum gene expression. Examples of such transcription-terminator include, e.g., for expression in yeast ADH1 , TDH1 , TFF and TRP5.
c) Shine-Dalgamo sequences (SD) are useful for initiation of translation. A suitable consensus sequence for expression for E.coli is for example: 5'-TAAGGAGG-3\
Said sequence may be localized 4 to 14 nucleotides upstream of the ATG start- codon, wherein the optimum is 8 nucleotides. For preventing secondary RNA structures, which may reduce translation efficacy, the corresponding region should be preferably A/T-rich.
d) Start codon: The start codon is the point of initiation for translation. In E.coli and higher eukaryotic organisms ATG is the most often used start codon. In E.coli GTG may be used alternatively.
e) Tags and fusion proteins: N- or C-terminal fusions of the recombinant protein with shorter peptides ("tags") or other proteins („fusion proteins") may be used to allow an improved expression, solubility, detection, or purification. Preferably, the fusion part comprises a protease (e.g., thrombine or factor X) cleavage site, which allows removal of the fusion part after expression and purification. Fusion protein may also, preferably, comprise signalpeptides allowing transport of the (S)- hydroxynitrile lyase of the invention to certain compartments of a host cell or secretion therefrom. In an preferred embodiment, said signal peptide is a secretory signal peptide derived from a the (S)-hydroxynitrile lyase known in the art, more preferably from a plant specie (e.g., a Rosacea specie). Sequences are described for example for the HNL5 gene from Prunus amygdalus, and the glucoseoxidase- gene from Aspergillus niger.
f) Multiple cloning sites (MCS) allow and facilitate insertion of one or more nucleic acid sequences.
g) Stop codons / translation terminators: Among the three possible stop codons TAA is preferred (since TAG and TGA may under certain circumstances allow read-through translation). Multiple stop codons can be used to guarantee translation termination.
h) Reporter genes: Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation efficacy, the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenborn E &
Groskreutz D (1999) Mol Biotechnol 13(1):29-44) such as the green fluorescent protein (GFP), chloramphenicol transferase, luciferase (Ow et al. (1986) Science 234:856-859), or β-galactosidase.
i) Origins of replication, which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E.coli. Examples which may be mentioned are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
j) Elements which are necessary for Agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.
k) Expression cassettes mediating expression of chaperone proteins (like e.g., GroELS, dnaKJ, grpE, or clpB), known to increase levels of correctly folded proteins especially in recombinant expression systems (US 5,635,391).
I) Elements which facilitate plasmid segregation and distribution during host amplification (like e.g., cer-sites; Wilms B et al. (2001) Biotechnol Bioeng 73(2):95-103).
The introduction of an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof can be effected advantageously using vectors which comprise the expression cassettes. The expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site. The plasmid formed is first introduced into E.coli. Correctly transformed E.coli are selected, grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning step. Examples of vectors may be plasmids, cosmids, phages, viruses or else agrobacteria. In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors.
Examples of suitable plasmids in E.coli are pUC18, PUC19, pBIueScript series, pKK223-3, pJOE2702, pBAD, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-lll113-B1 , Dgt11 or pBdCI, in Streptomyces are plJ101 , pIJ364, pl 702 or plJ361 , in Bacillus are pUB110, pC194 or pBD214, in Corynebacterium are pSA77 or pAJ667, in fungi are pALS1 , plL2 or pBB116, in plants are pLGV23, pGHIac+, pBIN19, pAK2004 or pDH51.
According to one embodiment of the present invention, yeast episome expression vector (autonomously replicating plasmid) is used as expression vector. Yeast episome plasmid vector contains 2 mu plasmid sequence, which is native to yeast. The vector can be replicated within a host yeast cell by utilizing the replication origin of the 2 mu plasmid sequence. Yeast episome expression vector to be used in the present inven- tion may not be limited to particular vectors as long as it comprises at least ORI sequence of yeast 2 mu plasmid sequence and can be autonomously replicated in a host yeast cell. Examples of such vector include in yeasts are 2μM, pAG-1, YEp6, YEp13 or pEMBLYe23, YEp51 , pYES2, YEp351 and YEp352 but are not limited thereto. Preferably, the above-described yeast episome expression vector may be a shuttle vector which can replicate in a E.coli cell for subcloning in the recombinant E. coli. Particular examples of the above-described yeast episome expression vector to be used in the present invention include: a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YEp352, S-hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (des- ignated as YEp352-GC); a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of GAL 10 promoter in yeast expression vector YEp51 (designated as YEp51-C); a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YE351 , S- hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (designated as YEp351-GC); and a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of GAL 1 promoter in yeast expression vector pYES2 (designated as pYES2-C).
According to another embodiment of the present invention yeast integrating expression vector (which can be integrated into chromosomal DNA) is used as expression vector. Although yeast integrating plasmid vector has a DNA sequence (normally a selective marker gene sequence) homologous to that of yeast chromosome, it cannot be replicated as a plasmid in a yeast cell. Such yeast integrating plasmid vector can remain in yeast cells only when homologous replacement occurs between the sequence on the vector homologous to yeast chromosome and the yeast chromosome gene thereby integrating the plasmid vector into the chromosome. The integrated gene is known to be stably retained within the yeast cell even not under growth conditions where expression of selected marker gene is essential. Yeast integrating expression vector to be used in the present invention is not limited to particular ones as long as it allows inte- gration of S-hydroxynitrile lyase coding gene derived from cassava carried by the vector into yeast chromosome. For example, when incorporated into chromosome of Saccharomyces, yeast integrating vectors such as pRS303 and pRS304, or modified vectors constructed by excising yeast 2 mu plasmid-derived sequence from vectors derived from yeast 2 mu such as YEp51, pYES2, YEp351 and YEp352 and then cyclizing the vectors may be preferably used. Vectors for integration into the chromosome of a methanol-utilizing strain in the yeast Pichia are not limited to but include pPIC3.5K, pPIC9K and pA0815. Yeast integrating expression vectors described above may be preferably shuttle vectors which can replicate in E. coli cells for subcloning in the recombinant E. coli cells. More preferably, such yeast expression vectors contain selec- five marker genes such as ampicillin-resistant genes. Alternatively, such expression vectors contain marker genes by which yeast clones can be selected depending on auxotrophy and drug resistance when recombinant yeast is prepared. Examples of marker gene for introduction into the yeast Saccharomyces include HIS3, TRP1 , LEU2, URA3, LYS2, Tn903 kan, Cm, Hyg, CUP1 and DHFR though they are not limited thereto. A marker gene should be selected depending on the genomic-type of the host Saccharomyces strain into which the gene is to be introduced. Examples of marker gene for introduction into the yeast Pichia include HIS4 and kan though they are not limited thereto. A marker gene should be selected depending on the genomic-type of the host Pichia strain into which the gene is to be introduced.
Vectors for expression in higher eukaryotic (e.g., mammalian) cells containing viral sequences on the basis of SV40, papilloma-virus, adenovirus or polyomavirus (Rodriquez RL & Denhardt DT, ed. ; Vectors : A survey of molecular cloning vectors and their uses, Butterworths (1988), Lenstra et al. (1990) Arch Virol 110:1-24). Said plasmids represent a small selection of the possible plasmids. Further plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). All recombinant molecules comprising the nucleic acid sequence under the control of regulating sequences enabling expression of a (S)-hydroxynitrile lyase of the invention are considered to be part of the present invention.
The invention furthermore relates to recombinant organisms or tissues, organs, parts, cells or propagation material thereof which comprise a (S)-hydroxynitrile lyase of the invention, a nucleic acid sequence encoding said (S)-hydroxynitrile lyase, a recombinant expression cassette comprising said nucleic acid sequence, or a recombinant vector encompassing said expression cassette.
Such a recombinant organism is generated, for example, by means of transformation or transfection with the corresponding proteins or nucleic acids. The generation of a transformed organism (or a transformed cell or tissue) requires introducing the DNA in question (for example the expression vector), RNA or protein into the host cell in question. A multiplicity of methods are available for this procedure, which is termed transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185:527-537). For example, the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. Also, the cell can be permeabilized chemically, for example using polyethylene glycol, so that DNA can enter the cell by diffusion. The DNA can also be introduced by calcium phosphate mediation, or fusion with DNA-containing units such as minicells, lysosomes or liposomes. Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an electrical pulse. Preferred general methods include but shall not be limited to calcium phosphate mediated transformation, DEAE- dextrane mediated transformation, cationic lipid mediated transformation, electroporation, transduction, and infection. These methods are well known to the person skilled in the art (Davis et al.(1986) Basic Methods In Molecular Biology; Sambrook J et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press; Ausubel FM et al. (1994) Current protocols in molecular biology, John Wiley and Sons; Glover DM et al. (1995) DNA Cloning Vol.1, IRL Press ISBN 019-963476-9).
In plants, in addition to these "direct" transformation techniques, transformation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The Agrobacterium-mediated transformation is best suited to dicotyledonous plant cells. The methods are described and well known in the art (Horsch RB et al. (1985) Science 225: 1229f.; EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287). When Agrobacteria are used, the expression cassette is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. Binary vectors are preferably used (Holsters et al. (1978) Mol Gen Genet 163:181-187). Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA). Transformed cells can be selected from untransformed cells when a selectable marker is part of the DNA introduced. Examples of genes which can act as markers are all those which are capable of conferring resistance to antibiotics or herbicides are given above. Concerning plants, the skilled worker is familiar with such methods of regenerating intact plants from plant cells and plant parts. Methods to do so are described, for example, by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-
The invention also relates to recombinant organisms transformed with at least one of the nucleic acid sequences according to the invention, expression cassette according to the invention or vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation material derived from such organisms. The term organism is to be understood in the broad sense and refers to prokaryotic and eukaryotic organisms, preferably bacteria, yeasts (like e.g., Saccharomyces, Kluyveromyces or Pichia), fungi (like e.g., Aspergillus or Penicilium), non-human animal organisms and plant organisms. Preferred plant organisms are indicated above.
The term "microorganism" includes bacteria, yeast, fungi, algae and other uni-cellular organism.
The term "bacteria" includes gram-positive and gram-negative bacteria. Preferred are all Enterobacteriaceae genera and species, and all Actinomycetales orders and species. Especially preferred are the Enterobacteriaceae speicies Escherichia, Serratia, Proteus, Enterobacter, Klebsiella, Salmonella, Shigella, Edwardsielle, Citrobacter, Morganella, Providencia, and Yersinia. Further preferred are all species of Agrobacterium, Pseudomonas, Burkholderia, Nocardia, Acetobacter, Gluconobacter, Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Staphylococcus, Aerobacter, Alcaligenes, Rhodococcus, and Streptomyces. For expression of recombinant (S)-hydroxynitrile lyases Escherichia species are most preferred, especially Escherichia coli.
The term "yeast" may include but shall not be limited to yeasts of the families Cryptococcaceae, Sporobolomycetaceae including the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloekera, Trigonopsis, Trichosporon, Rhodotorula, Sporobolomyces, and Bullera, and yeast of the families Endomycetaceae and Saccharomycetaceae, icluding but not limited to the genera Saccharo-, Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora. Especially preferred are Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica.
According to one embodiment of the present invention, the yeast Saccharomyces is used as a host though the host to be used in the present invention is not limited thereto as long as it can stably retain such an expression cassette after introduction of the cas- sette. Example of the yeast Saccharomyces include Saccharomyces cerevisiae KK4, Y334, Inv-Scl and W303 strains. Further, both haploid and diploid strains of these host yeast may be used.
According to another embodiment of the present invention, although a methanol- utilizing strain in the yeast Pichia is used as a host, yeast is not limited to particular ones as long as it can retain such an expression cassette after introduction of the cassette. Examples of methanol-utilizing strains in the yeast Pichia include Pichia pastoris KM71 and GS115 strains. Both haploid and diploid of these host yeast may be used.
The term "plant" or "plant organism" as used herein means any organism capable of photosynthesis. Preferably, the organism is a differentiated multicellular organization. The term includes all genera and species of higher and lower plants of the Plant Kingdom. Furthermore included are the mature plants, seed, shoots and seedlings, and parts, propagation material and cultures derived therefrom, for example cell cultures. Especially preferred are monocotyledoneous and dicotyledoneous plants, more particularly of the plants of culture intended for animal or human feed or food purpose or for industrial utilization, like corn, wheat, barley, canola, soybean, rice, sugarcane, sugar beet, potato, beet, tobacco, cotton, etc.
The recombinant organisms can be generated with the above-described methods for the transformation or transfection of organisms.
A microorganisms of this invention can be grown and propagated in a medium, which allows growth of said microorganism. Said medium can be of synthetic or natural origin. Various media are available depending on the microorganism and known to the person skilled in the art. For growth of microorganism the media comprise a carbon source, a nitrogen source, inorganic salts and optionally small amounts of vitamins and/or trace elements. Preferred carbon sources are, for example, polyoles like, e.g., glycerol, sugars like e.g., mono-, di- or polysaccharides (e.g., glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose), complex sugar sources (e.g., molasses), sugar phosphates (e.g., fructose-1 ,6- bisphosphate), sugar alcohols (e.g., mannit), alcohols (e.g., methanol or ethanol), carbonic acids (e.g., citric acid, lactic acid or acetic acid), oils and fats (e.g., soybean oil or rapeseed oil), amino acids or amino acid mixtures (e.g., Casamino acids; Difco) or distinct amino acids (e.g., glycine, asparagine) or amino-sugars, wherein the later can also be utilized as nitrogen sources. More preferred are glucose and polyoles, especially glycerol.
Preferred nitrogen sources are organic or inorganic nitrogen compounds or materials, comprising said compounds. Examples are ammonia salts (e.g., NH4CI or (NH4)2S04), nitrates, urea, and complex nitrogen sources like e.g., brewers yeast autolysate, soybean flour, wheat gluten, yeast extract, peptone, meat extract, caseine hydrolysate, yeast or potato protein, which may also often also function as carbon sources.
Examples for inorganic salts include calcium, magnesium, sodium, cobalt, molybdenum, manganese, potassium, zinc, copper and iron salts. As corresponding anions chlorine, sulfate, sulfide, and phosphate ions are especially preferred. An important issue for enhancing productivity is the control of the Fe2+ or Fe3+-ion concentration of the medium.
Optionally, the medium may comprise additional growth factors like e.g., vitamins or growth promoters like biotin, 2-keto-L-gulonic acid, ascorbic acid, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids (e.g., alanine, cysteine, proline, asparagine, glutamine, serine, phenylalanine, omithine or valine), carbonic acids (e.g., citric acid, formic acid, lactic acid) or substances like dithiothreitole.
The balance of the individual nutricients depends on the fermentation mode and will be adopted to the individual requirements. The media components may be provided at the beginning of the fermentation, after they have been sterilized before if required, or may be continuously or discontinuously added according to the requirements of the culture during the fermentation process.
The fermentation and growth conditions are selected in a way to guarantee optimal yield of the product (e.g., optimal yield of (S)-hydroxynitrile lyase activity). Preferred fermentation conditions are between 15°C to 40°C, preferably 25°C to 37°C. The pH is preferably kept in a range of pH 3 to 9, preferably pH 5 to 8. In general the fermentation time may take from a few hours to several days, preferably from 8 hours to 21 days, more preferably from 4 hours to 14 days. Processes for optimization of media and fermentations conditions is well known in the art (Applied Microbiol Physiology, "A Practical Approach (Eds. PM Rhodes, PF Stanbury, IRL-Press, 1997, S.53-73, ISBN 0 19 963577 3).
For expression in E.coli the methods as described in WO 01/48178 may be employed.
In an preferred embodiment, the S-hydroxynitrile lyase of the invention can be produced by culturing the obtained recombinant yeast in a appropriate yeast medium. Said medium may be conveniently supplemented with nitrogen sources such as yeast nitrogen base w/o amino acids (Difco Laboratories), essential amino acids and casamino acid, carbon sources such as glucose, galactose, raffinose and other saccharides, or alcohol such as glycerol and ethanol. The medium may be appropriately adjusted to pH 4-7. According to the present invention, for culturing yeast transformed with yeast epi- some expression vector, composition of medium may be preferably altered depending on the selective marker gene on the vector to be used in order to prevent deletion of the enzyme gene present in the recombinant yeast cells. For example, medium which does not substantially contain uracil is selected for recombinant yeast transformed with yeast episome expression vector YEp352-GC where the selection marker gene is URA3. Alternatively, medium which does not substantially contain L-leucine is selected for recombinant yeast transformed with yeast episome expression vector YEp351-GC where the selective marker gene is LEU2.
Preferably, an inducer substrate may be added to the medium depending on the pro- moter to be used where enzyme production is required to be induced. For example, where promoter expression is promoted or inhibited by a particular carbon source, an appropriate carbon source should be selected for each case. Where the expression of the enzyme gene is controlled by GAP promoter, which is one of the promoters preferable for the present invention, any carbon source which can be utilized by the host yeast cells may be used since the promoter will constitutively express. On the other hand, where expression of the enzyme gene is controlled by AOX1 promoter, which is one of promoters suitable for the present invention, preferably glucose may not be added since it may repress/inhibit the expression though glycerol and raffinose do not affect the enzyme gene expression. Moreover, methanol may be preferably added to the culture for efficient expression of the enzyme gene and thus for production of a large amount of the enzyme since it will promote the gene expression. For culturing yeast transformed with yeast integrating expression vector, medium may be suitably selected for growth of the host yeast to be used. There is no limitation to nutritional source nor need to add any antibiotics since the enzyme gene may be stably retained in the recombinant yeast cells. Preferably, medium may be adjusted to pH4-7. The yeast cells are cultured at 25-35°for several hours to three days, for example, until the growth reaches at its stationary phase.
It has now unexpectedly been found that the use of recombinant hydroxynitrile lyase from Adenia racemosa makes possible the reaction of a large number of carbonyl compounds, such as, for example, aliphatic, alicyclic, unsaturated, aromatically substituted aliphatic, aromatic, and also heteroaromatic aldehydes and/or ketones to give the corresponding cyanohydrins.
Accordinglay, another embodiment of the invention relates to a method for the preparation of a cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a hydroxynitrile lyase of the invention, preferably comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60%, preferably at least 80%, more preferably et least 90%, most preferably et least 95% to the sequence as described by SEQ ID NO: 1 , 3 or 5, and
c) sequences comprising at least 10, preferably et least 20, more preferably at least 30, most preferably at least 50 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1, 3 or 5.
Another embodiment of the invention further relates to a method for preparing a optically active cyanhydrin (2-hydroxynitrile), which comprises converting the corresponding aldehyde or ketone and a cyanide donor in the presence of (S)- hydroxynitrile lyase of the invention, or a growing, dormant or disrupted abovementioned recombinant organism which contains said either a (S)-hydroxynitrile lyase of the invention, a nucleic acid sequence encoding said (S)-hydroxynitrile lyase of the invention, a recombinant expression cassette or expression vector according to the invention.
Starting materials employed in the process according to the invention are an aldehyde or a ketone, a cyanide group donor, a recombinant hydroxynitrile lyase of the invention, and - preferably - at least one diluent or solvent. The diluent or solvent may be water or an organic diluent or solvent, which may be immiscible or slightly miscible with water.
A variety of carbonyl compounds can be used as substrates for the addition reaction of the invention, for example optionally substituted (hetero)aromatic aldehydes such as Aldehydes are in this case understood as meaning aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes are in this case understood as meaning saturated or unsaturated aliphatic, straight-chain, branched or cyclic aldehydes. Preferred aliphatic aldehydes are straight-chain aldehydes in particular having 2 to 18 C atoms, preferably from 2 to 12, which are saturated or mono- or polyunsaturated. The aldehyde can in this case have both C-C double bonds and C-C triple bonds. The aldehyde can be unsubstituted or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid es- ter, nitro or azido groups. Examples of aromatic or heteroaromatic aldehydes are ben- ∑aldehyde or variously substituted benzaldehydes such as, for example, fluorobenzal- dehyde, hydroxybenzaldehyde, phenoxybenzaldehyde (especially 3- phenoxybenzaldehyde), methoxybenzaldehyde, furfural, methylfurfural, nicotinalde- hyde and piperonal, further aromatic aldehydes like e.g., anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-l-carbaldehyde, phthalalde- hydes, pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridine aldehydes etc. saturated or unsaturated aliphatic aldehydes such as crotonaldehyde, methylthiopropionaidehyde, pivaldehyde, (C1- C6)alkoxy-acetaldehyde and isomeric butyraldehydes, and optionally substituted aral- kyl aldehydes such as (subst.) phenylacetaldehyde and phenoxyacetaldehyde. Suitable substituents for the above carbonyl compounds are (C1-C4)alkyl, hydroxy, (C1- C4)alkoxy, phenoxy, halogen and hydroxy(C1-C4)alkyl.
Ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is identically or unidentically substituted. Aliphatic ketones are understood as meaning saturated or unsaturated, straight-chain, branched or cyclic ketones. The ketones can be saturated or mono- or polyunsaturated. They can be unsubstituted, or substituted by groups which are inert under reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halo- gen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups. Examples of aromatic or heteroaromatic ketones are acetophenone, benzophenone, indolylacetone etc. Aldehydes and unsymmetrical ketones are preferably reacted.
Aldehydes and ketones which are suitable for the process according to the invention are known or can be prepared in the customary manner.
In an preferred embodiment of the invention, aldehydes or ketones as a reactive substrate may be represented by the following formula (II).
Figure imgf000036_0001
Wherein, R1 and R2 may independently of one another be selected from the group consisting of (i) a hydrogen atom, (ii) a substituted or unsubstituted, linear or branched, saturated C1-18 alkyl group, or (iii) a substituted or unsubstituted aromatic group having 5-22 cyclic members; where R1 and R2 do not represent hydrogen atoms at the same time. If is advantageous for one of the R1 or R2 substituents in the formulae I and II to be aryl, such as phenyl.
Suitable substituents for said R1 or R2 substituents are, for example, one or more of halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, imino, hydroxy, alkyl, alkenyl, alkenyloxy, alkynyl, alkoxy (preferably C1-8 alkoxy), carboxyl, C3-20 cycloalkyl, aromatic or other saturated or unsaturated nonaromatic rings or ring systems having carbon number of up to 22 which may be substituted with a heteroa- tom, N, O or S (here, when the substituent is a cyclic substituent, the substituent itself may be substituted with one or more halogen, hydroxy group, linear or branched C1-8 alkyl group, or linear or branched C2-8 alkenyl group). Preferred are alkyl radicals such as C Ce-alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, thiophenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino. Where R1 and/or R2 represent substituted aromatic groups, the substituent may be one or more of amirio group, imino group, hydroxy group, C1-8 alkoxy groups allyloxy group, halogen, cal- boxyl group, linear or branched, saturated or unsaturated C1-22 alkyl group (here, an aromatic group may be substituted with at least 2 substituents).
In a preferred embodiment the method for preparing cyanohydrine, preferably an optically active cyanohydrine, comprises conversion of a, aldehyde or ketone of the general formula III into the cyanohydrine of the general formula IV
Figure imgf000036_0002
wherein the substituents and variables in the formulae III and IV have the following meanings: n = 0 or 1 m = 0, 1 , 2 or 3, where for m > 2 there is one or no double bond present between two adjacent carbon atoms, A, B, D and E independently of one another are CH, N or CR5 G = O, S, NR4, CH or CR5, when n = 0, or CH, N or CR5, when n = 1 ,
wherein it is possible for two adjacent variables A, B, D, E or G together to form another substituted or unsubstituted aromatic, saturated or partially saturated ring with 5 to 8 atoms in the ring which may contain one or more heteroatoms such as O, N or S, and not more than three of the variables A, B, D, E or G being a heteroatom, and wherein R4 is selected from the group consisting of hydrogen, substituted or unsubstituted, branched or unbranched Cι-C10-alkyl, and
wherein R3 and R5 may independently be selected from the group consisting of hydrogen, substituted or unsubstituted, branched or unbranched Cι-Cι0-alkyl or C1-C10- alkoxy, substituted or unsubstituted aryl or heteroaryl, hydroxyl, halogen such as fluorine, chlorine or bromine, C^do-alkylamino and amino.
Suitable substituents for said R3, R4 or R5 radicals are, for example, one or more substituents such as halogen such as fluorine, chlorine or bromine, mercapto, nitro, amino, hydroxyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, or other aromatic or other saturated or unsaturated nonaromatic rings or ring systems. Preference is given to alkyl radicals such as C C6-alkyl such as methyl, ethyl, propyl or butyl, aryl such as phenyl, thiophenyl, halogen such as chlorine, fluorine or bromine, hydroxyl or amino.
A possible cyanide group donor is hydrocyanic acid, a cyanide salt, or a cyanohydrin of the general formula (I). In the formula I, R1 and R2 independently of one another are hydrogen or a hydrocarbon group which is unsubstituted or substituted by groups which are inert under the reaction conditions, or R1 and R2 together are an alkylene group having 4 or 5 C atoms, where R1 and R2 are not simultaneously hydrogen. The hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups. R1 and R2 are preferably alkyl groups having 1 to 6 C atoms, the cyanide group donor is very preferably acetone cyanohydrin. The cyanide group donor can be prepared according to known processes. Cyanohydrins, in particular acetone cyanohydrin, are also commercially available. Preferred cyanide salts are preferably sodium cyanide or potassium cyanide. Most preferably, hydrocyanic acid, a cyanide salt or acetone cyanohydrin is employed as the cyanide group donor. The hydrogen cyanide may be supplied either in the form of liquid or gas. An aqueous solution of hydrogen cyanide, i.e., hydrocyanic acid (or prussic acid), may also be equally employed. Any substance that is capable of producing cyanide ion (CN) upon addition to a reaction system may be used. Examples of such substances include salt of hydrogen cyanide such as sodium cyanide and po- tassium cyanide, or the above mentioned cyanhydrin compounds such as acetone cyanohydrin. The hydrocyanic acid can also be released from one of its salts such as, for example, NaCN or KCN only shortly before the reaction and added to the reaction mixture in undiluted form or in dissolved form.
The concentration of the aldehyde or ketone contained in the reaction solvent is preferably within the range of 0.01 mM to 5 M. Hydrogen cyanide or the substance capable of producing cyanide ion in the reaction system is used for 1-20 mol per mol of aldehyde or ketone. Per mole of aldehyde or keto group employed, at least 1 mol, prefera- bly 1 to 5 mol, particularly preferably 1 to 2 mol, of cyanide group donor are added.
It is possible to use growing recombinant cells which comprise the nucleic acids, nucleic acid constructs or vectors according to the invention for the method according to the invention. Dormant, immobilized, permeabilized or disrupted cells can also be used. Disrupted cells mean, for example, cells which have been made permeable by a treatment with, for example, solvents, or cells which have been disintegrated by an enzyme treatment, by a mechanical treatment (e.g. French press or ultrasound) or by any other method. The crude exracts obtained in this way are suitable and advantageous for the method according to the invention. Purified or partially purified enzymes can also be used for the process. Immobilized microorganisms or enzymes are likewise suitable and can advantageously be used in the reaction.
The recombinant organism (e.g., the recombinant yeast expressing the (S)- hydroxynitrile lyase of the invention) may be used as a catalyst of the reaction in the form of wet cells directly obtained from the medium, or in the form of dry cells which are subjected to drying or dehydration before use to remove the excessive amount of moisture to enhance dispersibility in the reaction system. The drying or dehydrating process of the cells is not specifically limited as long as the enzymatic activity of the cells is retained. Examples of such process include hot air drying, vacuum drying, lyophilization, spray drying, or drying with. an organic solvent such as acetone. The dry cells are powdered, or granulated by mixing with a binder. Alternatively, the dry cells may be mixed with insoluble carriers, or may be immobilized according to a known method. For example the cells may also be encapsulated into alginate. Where the cells are used in a reaction solvent as a suspension, the reaction may be carried out by employing a batch or semi-batch system. On the other hand, a continuous system is advantageous where the cells used are filled in a filling tank that allows a liquid to flow therethrough.
Alternatively, the recombinant cells may be disrupted. The cytosolic fraction can be used without further purification, by means of which the expenditure of work is mini- mized. The hydroxynitrile lyase can be employed in purified or unpurified form, as such or immobilized. The preparation and purification of the hydroxynitrile lyase can be carried out, for example, by precipitation with ammonium sulfate and subsequent gel filtration (as described e.g., in Example X, or in Selmar D et al. (1989) Physiologia Planta- rum 75:97-101). The S-hydroxynitrile lyase of the invention may be collected from the culture by using conventional enzyme collection methods including: cell lysis using cell- wall digesting enzyme (zymolyase); ultrasonication; disruption using glass beads; extraction with surfactants; self-digestion; and freezing-thawing method. Next, undis- solved materials may be removed by, for example, filtration or centrifugation to give crude enzyme solution containing S-hydroxynitrile lyase. S-hydroxynitrile lyase may be further purified from the crude enzyme solution by using any conventional protein purification method alone or in combination, including: ammonium sulfate fractionation; organic solvent precipitation; adsorption with ion exchanger; ion exchange chromatography; hydrophobic chromatography; gel filtration chromatography; affinity chromatography; and electrophoresis. The enzyme can also be employed in the method of the invention in an adsorbed or precipated form (e.g., on cellulose, viz. Avicel-cellulose TM).
Approximately 50 to 300 g of diluent and 200 to 20,000 IU of hydroxynitrile lyase activity, preferably approximately 500 to 5000 IU, are added per g of aldehyde or ketone. An IU (International Unit) in this case expresses the formation of one micromole of product per minute and per gram of enzyme crude isolation. The amount of the respective hydroxynitrile lyase needed is best determined in an activity test, for example according to Selmar et al. (1987) Anal Biochem 166:208-211. The enzymatic activity of the cells may be calculated as follows. The cells suspended in water or buffer are disrupted, and then subjected to centrifugation to obtain the supernatant. Using the supernatant and DL-mandelonitrile as a substrate, the change of absorption at a wavelength of 249.6 nm upon benzaldehyde production resulting from degradation of the substrate with the enzyme is measured.
The reaction may be performed in an organic systems, aquateous systems, 2-phase systems (Griengl H.et al. (1998) Tetrahedron. 54(48): 14477-14486), and emulsion systems. When a large amount of water is present in the reaction system, the optically active cyanohydrins resulting from the enzyme reactions are likely to be racemizated, or use of aldehydes or ketones having low solubility to water as the raw material will decrease the production efficiency. Accordingly, it is preferable that the reaction solvent mainly consists of an organic solvent that has poor water solubility or are water- immiscible or only slightly water-miscible (EP-A1 276 375; Tetr. Lett., 1990, 31 , 1249- 1252). The use of water-miscible organic solvents to improve the solubility of the substrate and of the product has been reported in the literature (DE-A 1 ,300, 111; DE-A 1 ,593,260; J.Am.Chem.Soc, 1966, 88, 4299; Angew. Chem., 1965, 77, 1139) and by Brussee et al (EP-A-322973; Tetr.Lett., 1988, 29, 4485; Tetrahedron 1990, 46, 979). In reviews by Klibanov (ChemTech., 1986, 16, 354-359; Acc.Chem.Res., 1990, 23, 114; Biotechnology and Bioengeneering, 1977, 19, 1351) lipophylic solvents, e.g. hydrocarbons, are recommended as organic solvents for many enzymatic conversions.
Such organic solvent is not specifically limited as long as it does not influence the enzymatic reaction for synthesizing optically active cyanohydrins, and may suitably be selected depending upon physical properties of the substrates (i.e., aldehydes or ketones) used for the synthesis reaction and upon the physical properties of the cyano- hydrins to be produced. Organic diluents which can be used are water-immiscible aliphatic or aromatic hydrocarbons which are optionally halogenated, alcohols, ethers or esters. The organic solvent used in the biphasic solvent system of the present invention is preferably selected from the group consisting of di(C1-C6)alkyl ethers, (C1- C5)carboxylic (C1-C5)alkyl eaters, di(C1-C5)alkyl ketones, (C4-C8)aliphatic alcohols, and mixtures of these solvents with each other or with apolar diluents. Specifically, examples of such organic solvent include linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic hydrocarbon solvent which may optionally be halogenated, such as pentane, hexane, cyclohexane, trichloroethene, chlorobenzene, toluene, xylene, methylene chloride and the like; linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic alcohol solvent which may optionally be halogenated, such as isopropyl alcohol, n-butanol, isobutanol, t-butanol, hexanol, cyclohexanol, n- amyl alcohol and the like; linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic ether solvent which may optionally be halogenated, such as di- ethylether, di-n-propylether, di-iso-pylether, di-n-butyl ether, di-isobutyl ether, methyl-t- buthylether and the like; and linear or branched or cyclic, saturated or unsaturated, aliphatic or aromatic ester solvent which may optionally be halogenated, such as methyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, isomeric butyl acetates, isomeric amyl acetates, methyl propionate and the like. Preferably, ethyl acetate, diisopropyl ether, methyl tert-butyl ether and dibutyl ether are used. Furthermore, methylethyl ketone, diethylketone, and methylisobutylketone can be used. Most preferred are: n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec. -butyl acetate and amyl acetate. These solvents may be used alone, or two or more of them may be used in combination. pH of the reaction solvent does not need to be adjusted if the above-described organic solvent is not saturated with an aqueous buffer. If the organic solvent is saturated with an aqueous buffer, the pH of the aqueous buffer is adjusted to be within the range of 3-7, preferably within the range of 3-6 (e.g., using a citrate buffer, a phosphate buffer or an acetate buffer). Furthermore a hydroxynitrile lyase mediated transcyanation (using e.g., acetone cyanohydrin in a biphasic reaction mixture, consisting of an aqueous buffer solution and a water- immiscible organic solvent, i.e. diethylether) may be employed (Ognyanov et al. (1991) J Amer Chem Soc 113:6992-6996)
In an preferred embodiment, the method of the present invention in a biphasic solvent system, wherein the volume ratio organic phase : aqueous phase varies between approx. 3:1 and approx. 1 :3. For a technical realization of the process of the present invention it may be desirable to avoid the use of large amounts of organic solvents. Therefore the concentration of the starting carbonyl compound in the organic solvent, as defined above, is preferably more than 5% wtJvol.
The hydroxynitrile lyase of the invention can be present either in immobilized form in the organic diluent, but the reaction can also be carried out in the above mentioned two-phase systema, using a nonimmobilized hydroxynitrile lyase, the organic diluent employed being a water-immiscible diluent. The enzyme of the invention may also be solubilized in a lyotropic liquid crystal, using certain tensides for the liquid crystal formation. Organic solvent, aqueous buffer and tenside form a ternary system (EP-A1 446 826).
The reaction mixture is shaken or stirred at temperatures from approximately 0°C up to the deactivation temperature of the hydroxynitrile lyase, preferably from 20 to 30°C. In the course of this, the cyanide group is transferred from the cyanide group donor to the carbonyl carbon atom of the aldehyde or ketone employed and the (S)-enantiomer of the optically active cyanohydrin corresponding to the aldehyde or ketone employed is mainly formed. The progress of the reaction can in this case be monitored, inter alia, by gas chromatography.
Once the reaction is completed, the reaction solution and the cells are separated from each other to obtain a solution containing the reaction product. For the working-up of the reaction mixture and for the isolation of the cyanohydrin formed, customary tech- niques which first break the emulsion, such as, for example, filtration, centrifugation or coalescence, are employed. The phases formed are then separated, if necessary with addition of demulsifiers, and the product-containing phase is worked up. Components other than the optically active cyanohydrin are removed from the solution, thereby obtaining the optically active cyanohydrin of interest. The product is separated according to a routine method such as distillation, column chromatography, crystallization, extraction or the like. For example, the cyanohydrin formed can be extracted from the reaction mixture with the aid of an organic solvent which is not miscible with water, for example aliphatic or aromatic optionally halogenated hydrocarbons, e.g. pentane, hexane, benzene, toluene, methylene chloride, chloroform, chlorobenzenes, ethers such as, for example, diethyl ether, diisopropyl ether or esters, for example ethyl acetate or mixtures of such solvents. Upon separation, a dehydrating agent for dehydration, a stabilizer or the like may be added. The cyanohydrins thus obtained can optionally be stabilized e.g., by addition of an acid before further processing.
The optical purity of the aldehyde cyanohydrins formed was determined as menthyl carbonate by means of gas chromatography on a capillary column as described by J. W. Westley et al. (1968) J Org Chem 33:3978-3980. The optical purity of the ketone cyanohydrins was determined by gas chromatography using a chiral separating phase as described by V. Schurig et al., (1990) Ang. Chemie 102:969-986. The method of the invention preferably results in enantiomeric purities of at least 70%ee, preferably of min. 90%ee, particularly preferably of min. 98%ee, very particularly preferably min. 99 %ee.
Additional suitable methods which may be employed in the method of the invention are disclosed in US2002052523, EP-A1 1 203 820, US 6,225,095, EP EP-A1 0 927 766, US 5,346,816, EP-A1 0 539 767, EP-A1 0 547 655, US 5,350,871.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.
Sequences
SEQ ID NO: 1 Aminoacid sequence encoding (S)-hydroxynitrile lyase from Adenia racemosa .
SEQ ID NO: 2 Backtranslated nucleicacid sequence optimized for expression in Pichia pastoris. encoding (S)-hydroxynitrile lyase from Adenia racemosa.
SEQ ID NO: 3 Aminoacid sequence modified for expression in Pichia pastoris encoding (S)-hydroxynitrile lyase from Adenia racemosa
SEQ ID NO: 4 Backtranslated nucleicacid sequence optimized for expression in enteric bacteria (like e.g., E.coli) encoding (S)-hydroxynitrile lyase from Adenia racemosa.
SEQ ID NO: 5 Aminoacid sequence modified for expression in enteric bacteria (like e.g., E.coli) encoding (S)-hydroxynitrile lyase from Adenia racemosa
Examples
Example 1 :
1.1 Activity Assay: Determination of enantiomeric excess (ee) for production of R/S- mandelonitrile for assessment of enantiomeric specificity of hydroxynitrile lyases
As positive controls the (R)-mandelonitrile lyase from almonds and the (S)- hydroxynitrile lyase from manioc were used.
Test: x μl of a sample comprising the enzyme to be tested are diluted with 600 μl disttllated, sterilized water and suspended in 200 μl 1 mol/l sodium citrate (pH 5,0), 200 μl 250 mmol/I R/S-mandelonitrile (in 0,1 M sodium citrate, comprising 0,2 % Triton X- 100 reduced).
For chirale HPLC the sample are diluted: 200 μl of the test sample are diluted 1 :5 using 100 mmol/I HCl. The resulting pH should be lower than pH 3,0 within 0 to 1 hour. Centrifugation of the sample. Isolation of the supernatant and analysis by HPLC (10 μl of about 10mmol/l solutions) Controls: Enzyme from Prunus for R-mandelonitrile, enzyme from Manioc for S-mandelonitrile, and zero control. Benzaldehyde Determination-(HPLC): Assay buffer: Sodium citrate buffer 1 M pH 5,0
Substrate: Mandelonitril (Aldrich 11,602-5) 4 mM in 0,1 M sodium citrate buffer, prepared immediately before use. Samples: Dilution in assay buffer, pH has to be controlled afterwards Controls:: Positive control blank (reagents only) Assay formate: in 1 ,5ml Eppendorff tube
Up to 500μl sample (for blank SOOμl IVIES buffer) were diluted with H20 to 600μl. 200 μl 1 M sodium citrate buffer and 10Oμl 40mM mandelonitrile solution were added. The assay was incubated in a thermoblock at 25°C unter shaking for 1 ,5h. The reaction is stopped using 200μl 2M HCl (pH-control !), centrifugated, and the supernatant transferred into HPLC-sample tubes.
Microtiterplate assay: Assay buffer: Sodium citrate 0,1M pH 5,0
Substrate: Mandelonitrile (Aldrich 11 ,602-5) 8mM in assay buffer; immediately prepared before use. Samples: Dilution in assay buffer (pH has to be controlled afterwards) Controls: Positive control: enzyme from almonds
Negative control: sample without substrate Blank (reagents only) Microtiterplate: UV Star Fa.Greiner 655801 Analytic: Measurement for 10min, Kinetik Spectramax at 280nm;
(Values=Vmax=mOD/min); U/ml=(Vmax/1000)*2,643*Verdϋnnung der Probe Assay formate: 100μl diluted sample are added to 100μl substrate solution
Example 2: Isolation and purification of a new S-hydroxynitrile lyase from Adenia racemosa
1200 g tuber from Adenia racemosa is cut into small pieces and is together with dry ice grinded into a dry powder. The resulting powder is dissolved in 2 I 20mM MES, pH 6,5, 0OmM L-Lysin, and 20mM ascorbic acid, stirred vigorously for 5 minutes, and extracted for 16 hours under continuous, slow stirring (small amounts of HCN >120ppm can be detected). The resulting mixture is filtered using a hair sieve. The filtered solution is further centrifugated and adjusted to pH7.0. Using water, the conductivity is adjusted to <5mS/cm (3,5 I). Q-Sepharose: A Q-Sepharose fast flow column (Pharmacia, 5cm diameter, 200 ml volume) is equilibrated in 20mM Tris/HCI, pH 7.0 and loaded at 15ml/min with the filtered solution. After washing the column a linear gradient is run using the same buffer and 1 M NaCl over 120 min. (10ml/min) until 100%. Samples were taken every minute and samples comprising activity (44-47) were pooled (9,6 mU/mg, 94 mg protein in total).
TSK-Phenyl: A TSK-Pheny column (8mm diameter, 7,5cm length, 3,8ml volume) was equilibrated using 20mM MES, pH 6.0, saturated with ammonium sulfate to 40%. The pooled Q-Sepharose samples were adjusted to 40% saturation using ammonium sulfate. The resulting precipitation is removed by centrifugation. The supernatant was applied in 20 ml portions to the TSK-phenyl column and eluated using a linear gradient against 20mM MESt. Samples comprising activity were pooled (specific activity 15U/mg). The pooled samples comprising activity was saturated to 90% with ammonium sulfate. The resulting precipitation is separated by centrifugation, redissolved in 20mM Tris/HCI, pH 7.0 and dialyzed against the same buffer.
Waters Q-HR8: The column (8 ml) was equilibrated in 20mM Tris/HCL, pH 7.0 and loaded with 7,5ml of the dialysate. At aflow rate of 1ml/min the column was eluated using a linear gradient towards 20mM MES, pH 7.0, 750mM NaCl. At this stage, activity and protein mediated absorption eluate together in a single signal. On the SDS- PAGE gel (Fig. 1) and in a isoelectric focusing (IEF; Fig. 2) only a single protein having a apparant molecular weight smaller than 20kDa and a single IEF spot can be detected.
Molecular sieve chromatography: For determination of the molecular weight of the active protein a chromatography using Superose TM 12 was performed. As standards catalase (232kDa), aldolase (158kDa), serum albumin (67kDa), chymotrypsinogen (25kDa), and ovalbumine (43kDa) were used. Also under this conditions, for the native, active protein a molecular weight below 20kDa was observed. Using a high- resolution Tricin-gel the apparent molecular weight was determined with 14,5kDa.
Trypsine cleavage: 0,5mg purified protein were adjusted using 1M Tris/HCI, pH 8,5 and 10% SDS to pH 8.5 and 0,1%SDS (ca 50mM final concentration of Tris) and treated with 0.025mg Trypsin (Typ XIII, Sigma) for 16 h at 35°C. The resulting peptide fragments were separated using a RP C18 column (Fa. Phenomenex). A V8-cleavage was performed in the same manner. The serquence of the resulting peptide fragments was determined and the complete protein sequence assembled using overlapping peptide fragment sequences (SEQ ID NO: 1 ; Fig. 4). For determination of the C-terminale proteine sequence 690μl sample (comprising 2 mg protein) were mixed with 310 μl sodium citrate buffer (50 mM; pH6.0). 40 μg carboxypeptidase-Y (2 vials of 20μg each, Roche) were dissolved in 50 μl H20. The entire carboxypeptidase-Y solution was then added at room temperature to the protein sample. After 25s, 50s, 80s, 120s, 200s, 300s, 500s, 800s, 1200, and 2100s, respectively, 100μl aliquots of the resulting solution were transferred into a new Eppendorf-tube. The reaction was stopped immediately by adding 10 μl 1% acetic acid and incubated for min at 80°C. To each of the resulting mixtures 100 μl acetonitrile were added to precipitate residual proteins. The samples were centrifugated and the supernatant saved for further use. 10 μl of each sample were dervatized and 10% thereof analyzed by HPLC. The remaining sample was concentrated by evaporation until dryness, dissolved in 40 μl borate buffer (Derivatisation kit; Waters) and treated with 20 μl dye -reagent (Waters). 50 μl of the resulting sample were used for HPLC analysis. The anaylsis resulted in a high propability for the C-terminal aminoacids F, T, R, G, P; for which the exact sequence, however is not known. Since the last tryptic peptide ends with an arginine, there is a high propability, that only one aminoacid is missing.
Example 3: Construction of a nucleic acid sequence (arHNLI) encoding (S)- hydroxynitrile lyase from Adenia racemosa (ArHNL)
Using the backtranslate function of the program vnti (vs 7, 2001 , InforMax Inc.) and the most probable codon usage of yeast, the DNA sequence was generated and embedded into restriction sites for cloning and the Kozak nucleotides (Kozak, M. (1987) Nu- cleic Acids Res. 15, 8125-8148; Kozak, M. (1990) Proc. Natl. Acad. Sci. USA 87, 8301- 8305; Cavener DR & Stuart CR (1991) Nucleic Acids Res. 19, 3185-3192) as well as the stop codon TAA for translation initiation and termination, respectively, in Pichia. Furthermore, some adaptations e.g. to avoid long T-stretches were performed manually yielding SEQ ID NO: 2. This sequence was synthesized by assembly of oligonu- cleotides as follows.
Figure imgf000046_0001
Figure imgf000047_0001
The PCR assembly reaction was carried out using the- High Fidelity Master Mix (25 μl, Roche), 20 pmol of each primer MKe310, 311 , 312, 313, 314, 316, 100 pmol of Mke317 and 318 each, 9 μl H20 and the following temperature program: 95°C for 30 sec; 55°C 2 min (Hot start); 55°C 2 min, ;30 cycles with 95°C for 303 sec, 55°C for 30 sec, and 72°C for 30 sec; 72°C for 5 min.; storage at 4°C until further usage. In further 2 samples the 55°C steps were substituted for corresponding 55°C and 60°C steps, respectively. All PCR reactions gave a main product of 350 bp in size, which were purified by agarose gel electrophoresis (E-Gel, Invitrogen), column chromatography (GFX- Kit, Pharmacia) and ethanol precipitation. Isolated PCR products were subsequently digested with Bam HI and Notl, and cloned into a correspondingly digested pBluescrip- tll KS+ (Stratagene) giving pBKSarHNL Clones obtained by transformation into E. coli XLIBIue (Stratagene) using solid LB medium comprising 100 μg/ml ampicilline were analyzed for plasmid DNA by sequence analysis. One of 96 clones showed the desired insert sequence depicted in SEQ ID NO: 2. Example 4: Cloning of arHNLI into the yeast expression vectors pPIK3.5 and pPIK9.
For expression of arHNLI in the yeast Pichia pastoris, arHNLI from plasmid pBKSarHNLI was cloned via BamHI / Notl into pPIK3.5 and transformed into E. coli XLIBIue (Stratagene). Clones were analyzed for plasmid DNA by sequence analysis. The corresponding plasmid, pPIK3.5arHNL1 was transformed into Pichia strains GS115 and KM71 as described in the Pichia expression kit, Cat. No. 1710-01 , Invitrogen (Manual Version F, 160624, 25-0043). Clones were verified via PCR and sequencing of the PCR product.
For cloning into pPIK9 (secretion of expressed protein) arHNLI was subcloned from pBKSarHNLI via PCR using the following primers and Pfu polymerase standard amplification conditions (Invitrogen):
Figure imgf000048_0001
The PCR products from primers MKe328-318 and Mke329-318 were 350 bp in size and purified by agarose gel electrophoresis (E-Gel, Invitrogen) and column chromatography (GFX-Kit, Pharmacia). They were subsequently digested with SnaBI/ Notl and Notl, respectively, and cloned into a correspondingly digested pPIK9 (Stratagene) giving pPIK9arHNL1-1 and pPIKarHNL1-2. Clones obtained by transformation into E.coli XLIBIue (Stratagene) using solid LB medium comprising 100 μg/ml ampicilline were analyzed for plasmid DNA by sequence analysis.
Example 5: Cloning and expression of a gene encoding ArHNL (arHNL2) in E.coli.
The sequence of arHNLI gene should was generated in a way that should also allow (medium) expression in E.coli. For further optimization a DNA sequence with optimized codon usage for E.coli could be created by modification of arHNLI . Here, the arHNLI gene was subcloned by PCR amplification and digestion with Ndel and Hindlll into a correspondingly digested pDHE vector (DE1SS4S1 S-A1). For the subcloning the High Fidelity Master Mix (Roche) and the following primers were used:
Figure imgf000049_0001
The PCR was carried out using the following temperature program: 95°C for 2 minutes; 30 cycles with 95°C for 45 sec, 54°C for 45 sec, and 72°C for 60 sec; 72°C for 10 min.; storage at 4°C until further usage. The PCR products were purified by agarose gel electrophoresis (E-Gel, Invitrogen) and column chromatography (GFX-Kit, Pharmacia). Isolated PCR products were subsequently digested with Ndel and Hindlll, and cloned into a correspondingly digested pDHE vector (DE19848129-A1). Clones obtained by transformation into E. coli XL1 Blue (Stratagene) using solid LB medium comprising 100 μg/ml ampicilline were analysed for plasmid DNA.
The corresponding plasmids were transformed into E.coli TG10 comprising pAgro pHSG575 for expression (E.coli TG10 is a derivative of E.coli TG1 comprising a deficiency in the rhamnose-isomerase rhaA; pAgro (pBB541 ; Tashifumi Tomo-yasu et al. (2001) Mol Microbiol 40(2):397-413) and pHSG575 (Takeshita S et al. (1987) Gene 61:63-74) are plasmids for chaperon GroELS coexpression). Plasmids were transformed into Pichia strains GS115 and KM71 , respectively, as described in the Pichia expression kit, Cat. No. 1710-01 , Invitrogen (Manual Version F, 160624, 25-0043). Clones were verified via PCR and sequencing of the PCR product.
Selection was on LB solid medium comprising 100 μg/ml ampicilline, spectinomycine (50 μg/ml) and chloramphenicole (10 μg/ml). Obtained clones were transferred to 4 ml liquid LB-Amp-Spec-Cm medium and grown over night at 37°C. Part of this culture was used to inoculate 20 ml liquid LB-Amp-Spec-Cm medium comprising 0,5 g/l L- rhamnose and 0,1 mM IPTG for induction of expression. The cultures were grown over night, harvested and analyzed for their catalytic activity for mandelonitrile as decribed above.

Claims

We claim:
1. Isolated polypeptide having (S)-hydroxynitrile lyase activity comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60% to the sequence as described by SEQ ID NO: 1 , 3 or 5, and
c) sequences comprising at least 10 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1 , 3 or 5.
2. Isolated polypeptide as claimed in Claim 1 , wherein said isolated polypeptide is a fusionprotein or a heterologous protein having (S)-hydroxynitrile lyase activity further comprising at least one sequence encoding for a secretory signal peptide, suitable for causing secretion of said fusionprotein upon expression in at least one host cell.
3. Isolated nucleic acid molecule comprising a sequences selected from the group consisting of
a) sequences encoding a polypeptide as claimed in claim 1 or 2, and
b) sequences which under stringent conditions hybridize with a sequence encoding a polypeptide as claimed in claim 1 or2.
4. Isolated nucleic acid molecule as claimed in Claim 3, wherein said molecule is selected from the group consisting of
a) a nucleic acid sequence as described by SEQ ID NO: 2 or 4, and
b) a nucleic acid sequence which is at least 60% identical to the nucleic acid sequence of SEQ ID NO: 2 or 4, and
c) a nucleic acid sequence comprising at least one fragment of at least 20 consecutive bases of the sequence of SEQ ID NO: 2 or 4, and
4 p. Fig + Seq d) a nucleic acid sequence which under stringent conditions hybridize with a sequences described by SEQ ID NO: 2 or 4 or sequences derived therefrom by degeneration of the genetic code, and
e) a nucleic acid sequence described by a sequence derived in consequence of the degeneration of the genetic code from a polypeptidesequence encoded by a sequence selected from the group of sequences a, b, c, and d.
5. Recombinant expression construct comprising at least one nucleic acid as claimed in claim 3 or 4.
6. Recombinant expression vector comprising at least one recombinant expression construct as claimed in claim 5 and/or at least one nucleic acid as claimed in claim
3 or 4
7. Recombinant organism comprising at least one recombinant expression vector as claimed in claim 6, at least one recombinant expression construct as claimed in claim 5 and/or at least one nucleic acid as claimed in claim 3 or 4.
8. Recombinant organism as claimed in claim 7, wherein said organism is selected from the group consisting of bacteria, fungi, algae, or plant organism.
9. Recombinant organism as claimed in claim 7 or 8, wherein the microorganism is a bacterium of the genera Escherichia, Rhodococcus, Nocardia, Streptomyces or Mycobacterium.
10. Recombinant organism as claimed in claim 7 or 8, wherein the microorganism is a yeast is a yeast selected from the group of yeast genera consisting of Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloekera, Trigonopsis, Trichosporon, Rhodotorula, Sporobolomyces, and Bullera, Saccharo-,
Debaro-, Lipomyces, Hansenula, Endomycopsis, Pichia, and Hanseniaspora.
11. Recombinant organism as claimed in any of claim 8 to 10, wherein the microorganism is a yeast selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica.
12. A method for the preparation of a cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a hydroxynitrile lyase com- prising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60% to the sequence as described by
SEQ ID NO: 1, 3 or 5, and
c) sequences comprising at least 10 consecutive aminoacid residues of the sequence as described by SEQ ID NO: 1, 3 or 5.
13. A method for the preparation of the (S)-enantiomers of optically active cyanohydrins by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a (S)-hydroxynitrile lyase comprising a polypeptide sequence selected from the group comprising
a) the sequence as described by SEQ ID NO: 1 , 3 or 5, and
b) sequences having an identity of at least 60% to the sequence as described by SEQ ID NO: 1, 3, or 5, and
c) sequences comprising at least 10 consecutive aminoacid residues of the sequence as described by SEQ ID NO:1, 3, or 5.
14. The method as claimed in claim 12 or 13, wherein an aliphatic, aromatic or het- eroaromatic aldehyde or an unsymmetrical ketone is reacted.
15. The method as claimed in any of claim 12 to 14, wherein the cyanide group donor employed is hydrocyanic acid or a cyanohydrin of the formula (R1)(R2)C(OH)(CN), in which R1 and R2 are alkyl groups.
16. The method as claimed in any of claim 12 to 15, wherein said method comprises adding viable, dormant, immobilized, permeabilized or disrupted cells of a recombinant organism expressing said (S)-hydroxynitrile lyase.
17. The method as claimed in any of claim 12 to 16, wherein said aldehyde and/or ketone is reacted with said recombinant (S)-hydroxynitrile lyase in a reaction system selected from the group consisting of organic systems, aqueous systems, micro- aqueous systems, 2-phase systems, and emulsion systems.
18. The method as claimed in any of claim 12 to 17, wherein the a organic solvent is employed selected from the group consisting of poorly water-soluble, slightly water-miscible or water-immiscible aliphatic or aromatic hydrocarbons, alcohols, ethers and esters.
19. The method as claimed in any of claim 12 to 18, wherein a organic solvent is employed selected from diethyl ether, di-n-propyl ether, di-isopropyl ether, di-n-butyl ether, di-isobutyl ether, methyl-t-butyl ether, n-propyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, sec-butyl acetate, iso-butyl acetates, iso-amyl ace- tates, methylethylketone, diethylketone, methylisobutylketone, and a mixture of these solvents with each other or with an apolar diluent selected from aromatic hydrocarbons, aliphatic hydrocarbons and chlorinated aromatic or aliphatic hydrocarbons.
20. Use of at least one material selected from the group consisting of an isolated polypeptide sequence as claimed in any of claim 1 or 2, a recombinant expression vector as claimed in claim 6, a recombinant expression construct as claimed in claim 5, a nucleic acid as claimed in claim 3 or 4, a recombinant organism as claimed in any of claim 7 to 11 for the production of cyanohydrins.
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WO2008067807A3 (en) * 2006-12-08 2008-09-12 Evocatal Gmbh (r)-hydroxynitrile lyase from brassicaceae

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008067807A3 (en) * 2006-12-08 2008-09-12 Evocatal Gmbh (r)-hydroxynitrile lyase from brassicaceae
US8940516B2 (en) 2006-12-08 2015-01-27 Evocatal Gmbh Method for the synthesis of chiral cyanohydrins via a hydroxynitrile lyase from brassicaceae

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