TECHNICAL FIELD
This invention relates to biocatalytic reactions, and more particularly to the use of enantiomer selective hydrolases to obtain optically active epoxides and vicinal diols.
BACKGROUND
Optically active epoxides and vicinal diols are versatile fine chemical intermediates for use in the production of Pharmaceuticals, agrochemicals, ferro-electric liquid crystals and flavours and fragrances. Epoxides are highly reactive electrophiles because of the strain inherent in the three-membered ring and the electronegativity of the oxygen. Epoxides react readily with various O-, N-, S-, and C-nucleophiles, acids, bases, reducing and oxidizing agents, allowing the production to bifunctional molecules. Vicinal diols, employed as their highly reactive cyclic sulfites and sulfates, act like epoxide-like synthons with a broad range of nucleophiles. The possibility of double nucleophilic displacement reactions with amidines and azide, allow access to dihydroimidazole derivatives, aziridines, diamines and diazides. Since enantiopure epoxides and vicinal diols can stereospecifically be interconverted, they can be regarded as synthetic equivalents.
Styrene oxide derivatives are among the most useful terminal epoxides from a synthetic standpoint. For example, Styrene oxide and phenylethanediol derivatives with substitutents in the meta- and/orp#r3-chl6foSbO grid (RJ?3-chl6r6PEU b"y VafioUS1 yeast strains transformed with vectors expressing YESH polypeptides from selected wild type yeast strains. The A panel in each figure is a line graph showing the change in concentrations of the epoxide enantiomers with time and the B panel in each figure is a line graph showing the enantiomeric excess of the (S)-epoxide at different conversions.
Figs. 29 and 30 (Samples 143-144) are line graphs showing the hydrolysis of (±)-2-chloroSEO to produce optically active (S)-2-chloroSEO and (R)-2-chIoroPED by two yeast strains transformed with vectors expressing YESH polypeptides from selected wild type yeast strains. The A panel in each figure is a line graph showing the change in concentrations of the epoxide enantiomers with time and the B panel in each figure is a line graph showing the enantiomeric excess of the epoxide at different conversions.
Figs. 31 - 36 (Samples 145-150) are line graphs showing the hydrolysis of (±)-4-chloroSEO to produce optically active (S)-4-chloroSEO and (R)-4-chloroPED by various t yeast strains transformed with vectors expressing YESH polypeptides from selected wild type yeast strains. The A panel in each figure is a line graph showing the change in concentrations of the epoxide enantiomers with time and the B panel in each figure is a line graph showing the enantiomeric excess of the (S)-epoxide at different conversions.
Figs. 37 - 44 (Samples 151-158) are line graphs showing the hydrolysis of (±)-2, 3-, or-4-nitroSEO to produce optically active (S) or (R)-2, 3-, or 4-nitroSEO (and associated nitroPED) by a wild-type yeast strain (Fig. 37) and various t yeast host strains (Figs, 38-44) transformed with vectors expressing YESH polypeptides from selected wild type yeast strains. The A panel in each figure is a line graph showing the change in concentrations of the epoxide enantiomers with time and the B panel in each figure is a line graph showing the enantiomeric excess of the excess epoxide enantiomer at different conversions.
Fig. 45 is a depiction of the amino acid sequence (SEQ ID NO:1) of a YESH polypeptide encoded by cDNA derived from a Rhodosporidium toruloides strain
18

(assigned accession no. NCYC 3181).
Fig. 46 is a depiction of the amino acid sequence (SEQ ID NO:2) of a YESH polypeptide encoded by cDNA derived from a Rhodosporidium toruloides strain (assigned identification no. UOFS Y-0471).
Fig. 4 /TS a de^lcUoH oTTfre aTOno acfiJ sequence (SbQ llTNUl.iJ'Sr a YLSH polypeptide encoded by cDNA derived from a Rhodotorula araucariae strain (assigned accession no. NCYC 3183).
Fig. 48 is a depiction of the amino acid sequence (SEQ ID NO:4) of a YESH polypeptide encoded by cDNA derived from a Rhodosporidium paludigenum strain (assigned accession no. NCYC 3179).
Fig. 49 is a depiction of the amino acid sequence (SEQ ID NO:5) of a YESH polypeptide encoded by a cDNA derived from a Rhodotorula imtcilaginosa strain (assigned accession no. NCYC 3190).
Fig. 50 is a depiction of the nucleotide sequence (SEQ ID NO:6) of a YESH polypeptide encoded by cDNA derived from a Rhodosporidium toruloides strain (assigned accession no. NCYC 3181).
Fig. 51 is a depiction of the nucleotide sequence (SEQ ID NO:7) of a YESH poiypeptide-encoding cDNA derived from a Rhodosporidium toruloides strain (assigned identification no. UOFS Y-0471
Fig. 52 is a depiction of the nucleotide sequence (SEQ ID NO:8) of a YESH poiypeptide-encoding cDNA derived from a Rhodotorula araucariae strain (assigned accession no. NCYC 3183).
Fig. 53 is a depiction of the nucleotide sequence (SEQ ID NO:9 of a YESH poiypeptide-encoding cDNA derived from a Rhodosporidium paludigenum strain (assigned accession no. NCYC 3179).
Fig. 54 is a depiction of the nucleic acid sequence (SEQ 1DNO:10) of a YESH poiypeptide-encoding cDNA derived from a Rhodotorula mucilagwosa strain (assigned accession no. NCYC 3190).
Fig. 55 is a table showing the homology at the amino acid level of the YESH polypeptides with SEQ ID NOs: 1-5.
19

Fig. 56 is a table showing the homology at the nucleotide level of YESH-encoding cDNA molecules with SEQ ID NOs: 6-10.
Fig. 57 is a depiction of the amino acid sequences of eight enantioselective epoxide hydrolases aligned for maximum homology. Also shown are consensus amino Acids': ThJe""sTquence?1al?eled:'#ir#4'6; #25,~#6'92 and #23 correspond to SEQ" ID NOs:" f-5' and the sequences labeled CarO54 (SEQ ID NO. 27), Jen46-2 (SEQ ID NO. 28), and #777 (SEQ ID NO. 29) correspond to enantioselective hydrolases catalyzing the hydrolysis of non-SOE epoxides. The consensus catalytic triad is composed of a nucleophile, an acid and a base, the positions of which are indicated by N, A and B, respectively. "HGXP" represents the region of the oxy-anion hole of the enzymes. "sxNxss" represents the genetic motif found in ct/p-hydrolase fold enzymes, (| = homology; f = identity of 75-100%; | = identity of 50-75%;. = gap).
DETAILED DESCRIPTION
Various aspects of the invention are described below.
Nucleic Acid Molecules
The YESH nucleic acid molecules of the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Segments of these molecules are.also considered within the scope of the invention, and can be produced by, for example, the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases. A ribonucleic acid (RNA) molecule can be produced by in vitro transcription. Preferably, the nucleic acid molecules encode polypeptides that, regardless of length, are soluble under normal physiological conditions.
The nucleic acid molecules of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide (for example, one of the polypeptides with SEQ ID NOS: 1 - 5). In addition, these nucleic acid molecules are not limited to coding sequences, e.g., they can include some or all of the non-coding sequences that lie upstream or downstream from a coding sequence.
20

The nucleic acid molecules of the invention can be synthesized (for example, by phosphoramidite-based synthesis) or obtained from a biological cell, such as the cell of a eukaryote (e.g., a mammal such as human or a mouse or a yeast such as any of the genera, species, and strains of yeast disclosed herein) or a prokaryote (e.g., a bacterium such as Escherichia coli). The nucleic aci 3 siiart" E>e~tho se "o"t" a yeast such a"s any of the ^* genera, species, and strains of yeast disclosed herein. Combinations or modifications of the nucleotides within these types of nucleic acids are also encompassed.
In addition, the isolated nucleic acid molecules of the invention encompass segments that are not found as such in the natural state. Thus, the invention encompasses recombinant nucleic acid molecules (for example, isolated nucleic acid molecules encoding the polypeptides of SEQ ID NOs: 1-5) incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location). Recombinant nucleic acid molecules and uses therefor are discussed further below.
Techniques associated with detection or regulation of genes are well known to skilled artisans. Such techniques can be used, for example, to test for expression of a YESH gene in a test cell (e.g., a yeast cell) of interest.
A YESH family gene or protein can be identified based on its similarity to the relevant YESH gene or protein, respectively. For example, the identification can be based on sequence identity. The invention features isolated nucleic acid molecules which are, or are at least 50% (e.g., at least: 55%; 60%; 65%; 75%; 85%; 95%; 98%; or 99%) identical to: (a) a nucleic acid molecule that encodes the polypeptide of SEQ ID NOs: 1-5; (b) the nucleotide sequence of SEQ ID NOs: 6- 10; (c) a nucleic acid molecule which includes a segment of at least 15 (e.g., at least: 20; 25; 30; 35; 40; 50; 60; 80; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 600; 700; 800; 900; 1,000; 1,100; 1,150; 1,160; 1,170; 1,175; 1,178; 1,180; 1,181; 1,200; 1,220; 1,225; 1,226; 1,228; 1,230; 1,231; or 1,232) nucleotides of SEQ ID NOs: 6-10; (d) a nucleic acid molecule encoding any of the polypeptides or fragments thereof disclosed below; and (e) the complement of any of the above nucleic acid molecules. The complements of the above molecules can be full-length complements or segment complements containing a segment of at least 15 (e.g., at least: 20; 25; 30; 35; 40; 50; 60; 80; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500;
21

600; 700; 800; 900; 1,000; 1,100; 1,200; 1,220; 1,225; 1,228; 1,230; 1,231; or 1,232) consecutive nucleotides complementary to any of the above nucleic acid molecules. Identity can be over the full-length of SEQ ID NOs: 6-10 or over one or more contiguous or non-contiguous segments.
The deferminatiorir5f percent identify between1 two^equenceTis accomplished using the mathematical algorithm of Karlin and Altschul, Proc. Natl Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990)7. Mot Biol 215, 403-410. BLAST nucleotide searches are performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to HIN-1-encoding nucleic acids. BLAST protein searches are performed with the BLASTP program, score — 50, wordlength = 3, to obtain amino acid sequences homologous to the HIN-1 poiypeptide. To obtain gap alignments for comparative purposes, Gap BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gap BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used.
Hybridization can also be used as a measure of homology between two nucleic acid sequences. A YESH-encoding nucleic acid sequence, or a portion thereof, can be used as a hybridization probe according to standard hybridization techniques. The hybridization of a YESH probe to DNA or RNA from a test source (e.g., a mammalian cell) is an indication of the presence of YESH DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in 1 X SSC, 0.1% SDS at 50°C. Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1% SDS at 65°C.
The invention also encompassesr(a) vectors (see below) that contain any of the foregoing YESH coding sequences (including coding sequence segments) and/or their complements (that is, "antisense" sequences); (b) expression vectors that contain any of
22

the foregoing YESH coding sequences (including coding sequence segments) operably linked to one or more transcriptional and/or translational regulatory elements (TRE; examples of which are given below) necessary to direct expression of the coding sequences; (c) expression vectors encoding, in addition to a YESH polypeptide (or a TragnrenTth~ere~of)7 alfequerice unrelated to'YESH,"suchxas a reporter*a^rriarkerToTa^ighal peptide fused to YESH; and (d) genetically engineered host cells (see below) that contain any of the foregoing expression vectors and thereby express the nucleic acid molecules of the invention.
Recombinant nucleic acid molecules can contain a sequence encoding a YESH polypeptide or a YESH polypeptide having an heterologous signal sequence. The full length YESH polypeptide, or a fragment thereof, can be fused to such heterologous signal sequences or to additional polypeptides, as described below. Similarly, the nucleic acid molecules of the invention can encode a YESH that includes an exogenous polypeptide that facilitates secretion.
The TRE referred to above and further described below include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphogIycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast a-mating factors. Other useful TRE are listed in the examples below.
Similarly, the nucleic acid can form part of a hybrid gene encoding additional polypeptide sequences, for example, a sequence that functions as a marker or reporter. Examples of marker and reporter genes include p-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydrofotate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding p-galactosidase), xanthine guanine phosphoribosyltransferase (XGPRT), and green, yellow, or blue
23

fluorescent protein. As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter. Generally, the hybrid polypeptide will include a first portion and a second portion; the first portion being a Y-ESH'poiypepttae (5f any ofYESH'ffagments described -"Below) and the second portion being, for example, the reporter described above or an Ig heavy chain constant region or part of an Ig heavy chain constant region, e.g., the CH2 and CH3 domains of IgG2a heavy chain. Other hybrids could include an antigenic tag or a poIy-His tag to facilitate purification.
The expression systems that can be used for purposes of the invention include, but are not limited to, microorganisms such as yeasts (e.g, any of the genera, species or strains listed herein) or bacteria (for example, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention; yeast (for example, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, Arxula and Candida, and other genera, species, and strains listed herein) transformed with recombinant yeast expression vectors containing the nucleic acid molecule of the invention; insect cell systems infected with recombinant virus expression vectors (for example, baculovirus) containing the nucleic acid molecule of the invention; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid) containing a YESH nucleotide sequence; or mammalian cell systems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter and the vaccinia virus 7.5K promoter). Also useful as host cells are primary or secondary cells obtained directly from a mammal and transfected with a plasmid vector or infected with a viral vector.
The invention includes wild-type and recombinant cells including, but not limited to, yeast cells (e.g., any of those disclosed herein) containing any of the above YESH
24

genes, nucleic acid molecules, and genetic constructs. Other cells that can be used as host cells are listed herein. The cells are preferably isolated cells. As used herein, the term "isolated" as applied to a microorganism (e.g., a yeast cell) refers to a microorganism which either has no naturally-occurring counterpart (e.g., a recombinant microorganism stfch as a recombinant yeast) or has been extracted and/or purified from an environment in which it naturally occurs. Thus, an "isolated microorganism" does not include one residing in an environment in which it naturally occurs, for example, in the air, outer space, the ground, oceans, lakes, rivers, and streams and the like, ground at the bottom of oceans, lakes, rivers, and streams and the like, snow, ice on top of the ground or in/on oceans lakes, rivers, and streams and the like, man-made structures (e.g., buildings), or in natural hosts (e.g., plant, animal ormicrobial hosts) of the microorganism, unless the microorganism (or a progenitor of the microorganism) was previously extracted and/or purified from an environment in which it naturally occurs and subsequently returned to such an environment or any other environment in which it can survive. An example of an isolated microorganism is one in a substantially pure culture of the microorganism.
Moreover the invention provides a substantially pure culture of a microorganism (e.g., a microbial cell such as a yeast cell). As used herein, a "substantially pure culture" of a microorganism is a culture of that microorganism in which less than about 40% (i.e., less than about: 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%; 0.001%; 0.0001%; or even less) of the total number of viable microbial (e.g., bacterial, fungal (including yeast), mycoplasmal, or protozoan) cells in the culture are viable microbial cells other than the microorganism. The term "about" in this context means that the relevant percentage can be 15% percent of the specified percentage above or below the specified percentage. Thus, for example, about 20% can be 17% to 23%. Such a culture of microorganisms includes the microorganisms and a growth, storage, or transport medium. Media can be liquid, semi-solid (e.g., gelatinous media), or frozen. The culture includes the cells growing in the liquid or in/on the semi-solid medium or being stored or transported in a storage or transport medium, including a frozen storage or transport medium. The cultures are in a culture vessel or storage vessel or substrate (e.g., a culture dish, flask, or tube or a storage vial or tube).
25

The microbial cells of the invention can be stored, for example, as frozen cell suspensions, e.g.. in buffer containing a cryoprotectant such as glycerol or sucrose, as lyophilized cells. Alternatively, they can be stored, for example, as dried cell preparations obtained, e.g., by fluidised bed drying or spray drying, or any other suitable *drying'metTiodr^>irni1arly'th"eenzymelpreparations can bTirozen, lyoprTNtsedTor^ immobilized and stored under appropriate conditions to retain activity.
Polypeptides and Polvpeptide Fragments
The YESH polypeptides of the invention include all the YESH and fragments of YESH disclosed herein. They can be, for example, the polypeptides with SEQ ID N0s:l-5 and functional fragments of these polypeptides. The polypeptides embraced by the invention also include fusion proteins that contain either full-length or a functional fragment of it fused to unrelated amino acid sequence. The unrelated sequences can be additional functional domains or signal peptides.
The invention features isolated polypeptides which are, or are at least 50% (e.g., at least: 55%; 60%; 65%; 75%; 85%; 95%; 98%; or 99%) identical to the polypeptides with SEQ ID NOs: 1-5. The identity can be over the full-length of the latter polypeptides or over one or more contiguous or non-contiguous segments.
Fragments of YESH polypeptide are segments of the full-length YESH polypeptide that are shorter than full-length YESH. Fragments of YESH can contain 5-410 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 250, 300, 350, 380, 390, 391, 392, 393, 400, 405, 406,407, 408, 409, or 410) amino acids of SEQ ID NOs: 1 -5. Fragments of YESH can be functional fragments orantigenic fragments.
The polypeptides can be any of those described above but with not more 50 (e.g., not more than 50, 45, 40, 35, 30, 25, 20, 17, 14, 12, 10, nine, eight, seven, six, five, four, three, two, or one) conservative substitution(s). Such substitutions can be made by, for example, site-directed mutagenesis or random mutagenesis of appropriate YESH coding sequences
"Functional fragments" of a YESH polypeptide (and, optionally, any of the above-described YESH polypeptide variants) have at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more) of the ability of the full-length,
26

wild-type YESH polypeptide to enantioselectively hydrolyse a SEO of interest. One of skill in the art wili be able to predict YESH functional fragments using his or her own knowledge and information provided herein, e.g., the amino acid alignments in Fig. 57 showing highly conserved domains and residues required for epoxide hydrolase activity.
TragmentiTof intefesf can be maWeither1 b'y're'cSrh'bihant; syntnetifc, or prbr^oTytrc" digestive methods and tested for their ability to enantioselectively hydrolyse a SEO.
Antigenic fragments of the polypeptides of the invention are fragments that can bind to an antibody. Methods of testing whether a fragment of interest can bind to an antibody are known in the art.
The polypeptides can be purified from natural sources (e.g., wild-type or recombinant yeast cells such as any of those described herein). Smaller peptides (e.g., those less than about 100 amino acids in length) can also be conveniently synthesized by standard chemical means. In addition, both polypeptides and peptides can be produced by standard in vitro recombinant DNA techniques and in vivo transgenesis, using nucleotide sequences encoding the appropriate polypeptides or peptides. Methods well-known to those skilled in the art can be used to construct expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals. See. for example, the techniques described in Sambrook et al., Molecular Cloning: A Laboratoiy Manual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y., 1989], and Ausubel et al., Current Protocols in Molecular Biology [Green Publishing Associates and Wiley Interscience, N.Y., 1989].
Polypeptides and fragments of the invention also include those described above, but modified by the addition, at the amino- and/or carboxyi-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide. This can be useful in those situations in which the peptide termini tend to be degraded by proteases. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyi terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA lechnology by methods familiar to artisans of average skill.
Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyi terminal residues, or the
27

amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the peptides can be covalentiy or non-covalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
Also of interest "are pe]stidomimetic^om~pounds~"tKat are"*de signed based upon the amino acid sequences of the functional peptide fragments. Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide. The peptide motif provides the peptidomimetic compound with the ability to enantioselectively hydrolyse a SEO of interest in a manner qualitatively identical to that of the YESH functional fragment from which the peptidomimetic was derived. Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased cell permeability and prolonged biological half-life.
The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of pro tease-resistant peptidomimetics.
The invention also provides compositions and preparations containing one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, or more) of the above-described polypeptides, polypeptide variants, and polypeptide fragments. The composition or preparation can be, for example a crude cell (e.g., yeast cell) extract or culture supernatant, a crude enzyme preparation, a highly purified enzyme preparation. The compositions and preparations can also contain one or more of a variety of carriers or stabilizers known in the art. Carriers and stabilizers are known in the ait and include, for example: buffers, such as phosphate, citrate, and other non-organic acids; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose,
28

mannose, or dextrans; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol, or sorbitol; sait-forming counterions such as sodium; and/or nonionic surfactants such as Tween and Pluronics,.
1vr5tnotts~or'F(IbaucingcupticaiiV"A'ctive K.poxibeTan"a 'upiically Active ViffitlaTttiCTff
The invention provides methods for obtaining enantiopure, or substantially enantiopure, optically active SEO and optically active PED. Enantiopure optically active SEO or PED preparations are preparations containing one enantiomer of the SEO or PED and none of the other enantiomer of the SEO or PED. "Substantially enantiopure" optically active SEO or PED preparations are preparations containing at least 55% (e.g., at least: 60%; 70%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%; 99.8%; or 99.9%), relative to the total amount of both SEO or PED enantiomers, of the particular enantiomer of the SEO or the PED.
The method involves exposing a SEO sample containing a mixture of both enantiomers of the SEO to a YESH polypeptide (e.g., an isolated YESH polypeptide or one in a microbial cell), which selectively catalyzes the conversion of one of the enantiomers of the SEO to a corresponding PED. In this way the desired PED is produced, the selective SEO enantiomer substrate for the YESH is selectively depleted, and the relative proportion (of the total amount of the SEO) of the other SEO enantiomer is increased. YESH poiypeptides useful for the invention (i.e., those with SEO enantioselective activity) will catalyze the conversion of one enantiomer of a SEO to its corresponding PED with less than 80% (e.g., less than: 70%, 60%, 50%, 40%, 30%; 20%; 10%; 5%; 2.5%; 1%; 0.5%; 0.01%) of the efficiency that its catalyzes the conversion of the other enantiomer of the SEO to its corresponding PED. The starting enantiomeric mixtures can be racemic with respect to the two SEO enantiomers or they can contain various proportions of the two SEO enantiomers ((e.g., 95:5, 90:10, 80:20, 70:30, 60:40 or 50:50) In addition, optimal concentrations of the SEO and conditions of incubation will vary from one YESH polypeptide to another and from one SEO to another. Given the teachings of the working examples contained herein, one skilled in the art will know how to select working conditions for the production of a desired enantiomer of a desired PED and/or SEO.
29

The method can be implemented by, for example, incubating (cuituring) the S enantiomeric mixtures with a wild-type yeast cell or a recombinant cell (yeast or any other host species listed herein) containing a nucleic acid sequence (e.g.. a gene or a recombinant nucleic acid sequence) encoding a YESH polypeptide, a crude extract fr< such ctllS,la"^e'rhi-l^tifiHedllpreparaiion 6f"a" Vhsi-i jibl^ffeptifie1,1 or amsoiail£a"'Y'fc,5hi polypeptide, all of which exhibit epoxide hydrolase activity with chiral preference.
The strain of the yeast cell may be selected from the following genera: Arxula, Brettanomyces, Bullera, Bulleromyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Exophiala, Geotrichum, Hormonema, Issatchenkia, Kluyveromyces, Lipomyces, Mastigomyces, Myxozyma, Pichia, Rhodosporidium, Rhodotorula, Sporidiobolus, Sporobolomyces, Trichosporon, Wingea, or Yarrowia.
Yeast strains innately capable of producing a polypeptide that converts or hydrolyses mixtures of SEO to optically active (i.e. enantiopure or substantially enantiopure) SEO and/or PED include the following exemplary genera and species:

Genus Species
Bullera B. dendrophila
Candida C. magnoliae
C. rugosa
Cryptococcus C. albidus <
C. cwvatus
C. hungaricus
C. laurentii
C. podzolicus
Debaryomyces D. hansenii
Pichia P. fmlandica
P. guillermondii
P. haplophila
Rhodosporidium R. paludigenum
R. toruloides
Rhodotorula R. aurantiaca
30

R. araucariae
R. aurantiaca
R. glutinis
R. minuta

R. species (e.g.,UOFS Y-2043)
Sporidiobolus 5". salmonicolor
Sporobolomyces S. holsaticus
S. roseus
S. tsugae
Trichosporon T.cutaneum var. cutaneum
T. montevideense
T. ovoides
T. species (e.g.UOFS Y-0533)
Yarrowia Y. lipolytica
The yeast strain can be, for example, one selected from Tables 2 or 3 (see below).
Cultivation in bioreactors (fermenters) of yeast strains expressing a YESH polypeptide, or fragment thereof, (with the purpose of preparing yeasts stocks or for the enantioselective preparative methods of the invention) can be carried out under conditions that provide useful biomass and/or enzyme titer yields. Cultivation can be by batch, fed-batch or continuous culture methods. Useful cultivation conditions are dependent on the yeast strain used. General procedures for establishing useful growth conditions of yeasts, fungi and bacteria in bioreactors are known to those skilled in the art. The mixture of epoxides can be added directly to the culture. The concentration of the SEO enantiomeric mixture in the reaction matrix can be at least equal to the soluble concentration of the SEO enantiomeric mixture in water. The preferred epoxide level in the reaction matrix is greater than the solubility limit in the aqueous reaction medium thereby resulting in a two phase reaction system. The starting amount of epoxide added to the reaction mixture is not critical, provided that the concentration is at least equal to the solubility of the specific epoxide in the aqueous reaction medium. The epoxide can
31

be metered out continuously or in batch mode to the reaction mixture. The relative proportions of (R)- and (S)-epoxide in the mixture of enantiomers of the epoxide shown by the general formula (I) is not critical but it is advantageous for commercial purpose to employ a racemic form of the epoxide shown by the general formula (I). The epoxide ~can be added m""a r'acemicloTm or as a mixiure OT enahtlomers in "differentTatio?.
The amount of the yeast cells, crude yeast cell extract, or partially purifie^er isolated polypeptide having SEO enantioselective activity added to the reaction depends on the kinetic parameters of the specific reaction and the amount of epoxide that is to be hydrolysed. In the case of product inhibition, it can be advantageous to remove the formed vicinal diol from the reaction mixture or to maintain the concentration of the vicinal diol at levels that allow reasonable reaction rates. Techniques used to enhance enzyme and biomass yields include the identification of useful (or optimal) carbon sources, nitrogen sources, cultivation time, dilution rates (in the case of continuous culture) and feed rates, carbon starvation, addition of trace elements and growth factors to the culture medium, and addition of inducers for example substrates or substrate analogs of the epoxide hydrolases during cultivation. In the case of recombinant hosts, the conditions under which the promoters function workably for transcription of the gene encoding the polypeptide with epoxide hydrolase activity are taken into account. At the end of fermentation (culture), biomass and culture medium can be separated by methods known to one skilled in the art, such as filtration or centrifugation.
The processes are generally performed under mild conditions. For example, the reactions can be carried out at a pH from 5 to 10, preferably from 6.5 to 9, and most preferably from 7 to 8.5. The temperature for hydrolysis can be from 0 to 70 °C, preferably from 0 to 50 °C, most preferably from 4 to 40 °C. It is also known that lowering of the temperature of the reaction can enhance enantioselectivity of an enzyme.
The reaction mixture can contain mixtures of water with at least one water-miscible solvents (e.g., water-miscible organic solvents). Preferably, water-miscible solvents are added to the reaction mixture such that epoxide hydrolase activity remains measurable. Water-miscible solvents are preferably organic solvents and can be, for example, acetone, methanol, ethanol, propanol, isopropanol, acetonitrile, dimethylsulfoxide, A^jV-dimethylformamide, JV-methylpyrolidine5 and the like,
32

The reaction mixture can also, or alternatively, contain mixtures of water with at least one water-immiscible organic solvent. Examples of water-immiscible solvents that can be used include, for example, toluene, l,l;2-trich!orotrifluoroethane, methyl tert-butyl ether, methyl isobutyl ketone, dibutyl-o-phtalate, aliphatic alcohols containing 6 to 9 carbon atoms (for example hexanol, octanol), aliphatic hydrocarbons containing 6 to 16 earben-atoms-ffeF-ejframpI e-eyel©hexaflerw-he»ener«^oetanerM-deeaner«-dodeean er/? tetradecane and n -hexadecane or mixtures of the aforementioned hydrocarbons), and the like. Thus, the reaction mixture can include water with at least one water-immiscible organic solvent selected from the group consisting of toluene, 1,1,2-trichlorotrifluoroethane, methyl ter/-butyl ether, methyl isobutyl ketone, dibutyl-o-phtalate, aliphatic alcohols containing 6 to 9 carbon atoms, and aliphatic hydrocarbons containing 6 to 16 carbon.
The reaction mixture can also contain surfactants (for example, Tween 80), cyclodextrins or any agent that can increase the solubility, selectively or otherwise, of the epoxide enantiomers in the aqueous reaction phase.
The reaction mixture can also contain a buffer. Buffers are known in the art and include, for example, phosphate buffers, Tris buffer, and HEPES buffers.
The production of the YESH polypeptides, including functional fragments, can be, for example, as recited above in the section o^ Polypeptides and Polypeptide Fragments. Thus they can made by production in a natural host cell, production in a recombinant host cell, or synthetic production. Recombinant production can be carried out in host cells of microbial origin. Preferred yeast host cells are selected from, but are not limited to, the genera Saccharomyces, Kluyveromyces, Hansenuia, Pichia, Yarrowia and Candida. Preferred bacterial host cells include Escherichia coli, Agrobacterium species, Bacillus species and Streptomyces species. Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, and Fusarium. The production of the polypeptide can be, e.g., intra- or extra- cellular production and can be by, e.g., secretion into the culture medium.
In these fermentation reactions of the invention, the polypeptides (including functional fragments) can be immobilized on a solid support or free in solution. Procedures for immobilization of the yeast or preparation thereof include, but are not
33

limited to, adsorption; covalent attachment; cross-linked enzyme aggregates; cross-linked enzyme crystals; entrapment in hydrogels; and entrapment into reverse micelles.
The progress of the reaction can be monitored by standard procedures known to one skilled in the art, which include, for example, gas chromatography or high-pressure liquid chromatography on columns containing chiral stationary phases. The vicinal diol formed can be removed from the reaction mixture at one or more stages of the reaction
The reaction can be terminated when one enantiomer of the epoxide and/or vicinal diol is found to be in excess compared to the other enantiomer of the epoxide and/or vicinal diol. Preferably, the reaction is terminated when one enantiomer of an epoxide of general formula (I) and/or vicinal diol of general formula (II) is found to be in an enantiomeric excess of at least 90%. In a more preferred embodiment of the invention, the reaction is terminated when one enantiomer of an epoxide of general formula (I) and/or vicinal of general formula (II) is found to be in an enantiomeric excess of at least 95%. The reaction can be terminated by the separation (for example centrifugation, membrane filtration and the like) of the yeast, or a preparation thereof, from the reaction mixture or by inactivation (for example by heat treatment or addition of salts and/or organic solvents) of the yeast or polypeptide, or preparation thereof. Thus, the reaction can be stop for by, for example, the separation of the catalytic agent from the reactants and products in the mixture, or by ablation or inhibition of the catalytic activity, by techniques known to one skilled in the art.
The optically active epoxides and/or vicinal diols produced by the reaction can be recovered from the reaction mixture, directly or after removal of the yeast, or preparation thereof. Preferably, the process can include continuously recovering the optically active epoxide and/or vicinal diol produced by the reaction directly from the reaction mixture. Methods of removal of the optically active epoxide and/or vicinal diol produced by the reaction include, for example, extraction with an organic solvent (such as hexane, toluene, diethyl ether, petroleum ether, dichloromethane, chloroform, ethyl acetate and the like), vacuum concentration, crystallisation, distillation, membrane separation, column chromatography and the like.
Thus, the present invention provides an efficient process with economical advantages compared to other chemical and biological methods for the production, in
34

high enantiomeric purity, of optically active epoxides of the general formula (I) and vicinal diols of the general formula (II) in the presence of a yeast strain having enantioselective epoxide hydrolase activity or a polypeptide having such activity.
Yeast Epoxide Hydrolase Antibodies
The invention features antibodies that bind to yeast epoxide hydrolase polypeptides or fragments (e.g., antigenic or functional fragments) of such polypeptides. The polypeptides are preferably yeast epoxide polypeptides with enantioselective activity, and in particular those with styrene-type epoxide enantioseiective activity (i.e.,YESH), e.g., those with SEQ ID NOs: 1, 2, 3, 4, or 5. The antibodies preferably bind specifically to yeast epoxide hydrolase polypeptides, i.e., not to epoxide hydrolase polypeptides of species other than yeast species. More preferably, they can bind specifically to yeast epoxide polypeptides with enantioselective activity, and in particular to YESH polypeptides, e.g., those with SEQ ID NOs: 1, 2, 3, 4, or 5. They can moreover bind specifically to one or more of polypeptides with SEQ ID NOs: 1, 2, 3, 4, or 5.
Antibodies can be polyclonal or monoclonal antibodies; methods for producing both types of antibody are known in the art. The antibodies can be of any class (e.g., IgM, IgG, IgA, IgD, or IgE).1 They are preferably IgG antibodies. Moreover, polyclonal antibodies and monoclonal antibodies can be generated in, or generated from B cells from, animals any number of vertebrate (e.g., mammalian) species, e.g., humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, goats, camels, sheep, pigs, bovine animals (e.g., cows, bulls, or oxen), dogs, cats, rabbits, gerbils, hamsters, guinea pigs, rats, mice, birds (such as chickens or turkeys), or fish.
Recombinant antibodies specific for YESH polypeptides, such as chimeric monoclonal antibodies composed of portions derived from different species and humanized monoclonal antibodies comprising both human and non-human portions, are also encompassed by the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in Robinson et al., International Patent Publication PCT/US86/02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application
35

173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240, 1041-43; Liu et al. (1987) J. Immunol. 139, 3521-26; Sun et al. (1987) PNAS 84, 214-18; Nishimura et ai. (1987) Cane. Res. 47, 999-1005; Wood et al. (1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80, 1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al. (1986) BioTechniques 4, 214; Winter, U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534; andBeidleretal.(1988)J. Immunol. 141,4053-60.
Also useful for the invention are antibody fragments and derivatives that contain at least the functional portion of the antigen-binding domain of an antibody that binds to a YESH polypeptide. Antibody fragments that contain the binding domain of the molecule can be generated by known techniques. Such fragments include, but are not limited to: F(ab')2 fragments that can be produced by pepsin digestion of antibody molecules; Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments; and Fab fragments that can be generated by treating antibody molecules with papain and a reducing agent. See, e.g., National Institutes of Health, I Current Protocols In Immunology, Coligan et al., ed. 2.8, 2.10 (Wiley Interscience, 1991). Antibody fragments also include Fv fragments, i.e., antibody products in which there are few or no constant region amino acid residues. A single chain Fv fragment (scFv) is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. Such fragments can be produced, for example, as described in U.S. Patent No. 4,642,334, which is incorporated herein by reference in its entirety. The antibody can be a "humanized" version of a monoclonal antibody originally generated in a different species.
The above-described antibodies can be used for a variety of purposes including, but not limited to, YESH polypeptide purification, detection, and quantitative measurement.
The following examples serve to illustrate, not limit, the invention.
36

EXAMPLES
Example I. Materials and Methods Preparation of frozen yeast cells for screening
Yeasts were grown at 30 °C in 1 L shake-flask cultures containing 200 ml yeast extract/malt extract (YM) medium (3 % yeast extract, 2 % malt extract, 1 % peptone w/v) supplemented with 1 % glucose (w/v). At late stationary phase (48 - 72 h) the cells were harvested by cenlnfugation (10 000 g, 10 min, 4 °C), washed with phosphate buffer (50 mM, pH7.5); pelleted by centrifugation, and frozen in phosphate buffer containing glycerol (20%) at -20 °C as 20% (w/v) cell suspensions. The cells were stored for several months without significant loss of activity.
Yeast isolate (strain) screening
Epoxide (10 u.1 of a 1M stock solution in EtOH) was added to a final concentration of 20 mM to 500 }il cell suspension (20% w/v) inphosphate buffer (50 mM, pH 7.5). The reaction mixtures were incubated at 30 °C for 5 hours, extracted with EtOAc (300 ul). and centrifuged. Vicinal diol formation was evaluated by thin layer chromatography (TLC) (silica gel Merck 60 F254)- Compounds were visualized by spraying with vanillin/cone. H2SO4 (5g/l). Reaction mixtures that showed substantial vicinal diol formation were evaluated for asymmetric hydrolysis of the epoxide by chiral GLC analysis. Some reactions were repeated over longer or shorter times in order to analyse the reactions at suitable conversions.
General procedure for the hydrolysis of (substituted) stvrene epoxides
Frozen yeast cells were thawed, washed with phosphate buffer (50 mM, pH 7.5) and resuspended in buffer. Cell suspensions (10 ml, 20 % or 50 % w/v) were placed in 20 ml glass bottles with screw caps fitted with septa. The substrate (100 or 250 uj of a
37

2M (v/v) stock solution in ethanol) was added to final concentrations of 20 mM or 50 mM. The mixtures were agitated on a shaking water bath at 30°C. The course of the bioconversions of epoxides was followed by withdrawing samples (500 ul) at appropriate time intervals. Samples were extracted with 300 u.1 EtOAc. After centrifugation (3000 x g, 2 min), the organic layer was dried over anhydrous MgSCU, and the products analyzed by chiral GLC.
Determination of the absolute configuration of residual stvrene epoxide and phenyl ethanediol
Absolute configurations were deduced from reported elution orders of the epoxide and diol enantiomers on cyclodextrin columns and verified by co-injection of the commercially available (R)-3-chIorostyrene oxide (Aldrich).
Determination of concentrations and enantiomeric excesses
In the various working examples below, quantitative determinations of the compounds and determination of enantiomeric excesses were carried out by GC. Gas
chromatography (GLC) was performed on a Hewlett-Packard 6890 gas chromatograph
'i
equipped with a FID detector and using H2 as carrier gas. Chiral analysis of styrene epoxides and phenylethanediol was done on fused capillary cyclodextrin columns (30 m length, 25 mm ID and 25 pm film thickness) from Supelco (head pressure of 10-14 psi) at isotherms given below:

Compound Chiral column Columntemperature Retention time (min)
Styrene oxide: P-Dex 225 90°C (S)-: 8.00; (R)-: 8.23
2-Chlorostyrene oxide ct~Dex 110 90°C (S)-2.69; (R)-: 23.12
3-ChIorostyrene oxide (3-Dex 225 100°C (S)-: 7.20; (R)-: 7.40
4-Chlorostyrene oxide 3-Dex 225 100° C (S)- 9.35 (R)-: 9.50
2-Mitrostyrene oxide P-Dex 225 130°C (S)-:12.12; (R)-: 12.36
3-Nitrostyrene oxide P-Dex 225 150°C (S)-: 11.98; (R)-: 12.25
4-Nitrostyrene oxide P-Dex 225 150°C (S)-:18.16; (R)-: 18.99
38

Phenylethanediol (3-Dex 120 150 °C (S)-: 11 .76; (R>: 12.25
Synthesis of chloro-substituted styrene epoxides
(a) Synthesis of 2-chIorostyrene epoxide
A solution of 2-chlorostyrene (10.820 g, 78.066 mmol) in chloroform (200 cm3, 0.4 M) was treated with 1 Og dried magnesium sulphate and cooled to 0 °C. meta-Chloroperoxybenzoic acid (23.094 g, 93.680 mmol, 1.2 eq.) was then added, with effervescence, and the resultant yellow suspension left to stir for lh at 0 °C. The mixture was decanted into 250 cm saturated aqueous sodium bicarbonate solution and extracted with chloroform (3 x 100 cm3). Drying and concentration afforded a yellow oil, which was purified by column chromatography [1:10 (v/v) ethyl acetate:hexane aseluent] to afford a pale yellow oil, 2-chlorostyrene epoxide (8.693 g, 72 %). <5k (200 MHz, CDC13) 7.34-7.41 (1H, m, H-3), 7.27-7.30 (3H, m, H-4, H-5 and H-6), 4.23 (1H, dd, ArCtf-, J4.0 and 2.4), 3.21 (1H, dd, CHC//aHb, J6.0 and 4.2) and 2.69 (1H, dd, CHCHatfb, -Z5.8 and 2.4).
(b) Synthesis of 3-chlorostyrene epoxide
A solution of 3-chlorostyrene (52.72 g, 0.380 mol) in dichloromethane (250 cm3, 1.5 M) was treated with 50g dried magnesium sulphate and cooled to 0 °C. meta-Chloroperoxybenzoic acid (140.66 g, 0.571 moi, 1.5 eq.) was then added, with effervescence, and the resultant yellow suspension left to stir for 3h at 0 °C. The mixture was decanted into 250 cm3 saturated aqueous sodium carbonate solution and extracted with dichloromethane (3 x 100 cm ). Drying and concentration afforded a yellow oil, which was purified by column chromatography [1:10 (v/v) ethyl acetate:hexane as eluent] to afford a pale yellow oil, 3-chlorostyrene epoxide (38.24 g, 65 %). SH (200 MHz, CDC13) 7.15-7.36 (4H, m, aryl H), 3.86 (IH, dd, ArCtf, J4.0 and 2.4), 3.16 [1H, dd, -C/faHb, J5.6 and 4.0] and 2.78 [IH, dd, -CHa//b, J5.4 and 2.4].
(c) Synthesis of 4-chlorostyrene epoxide
39

A solution of 4-chlorostyrene (10.876 g, 78.470 mmol) in chloroform (200 cm3, 0.4 M) was treated with lOg dried magnesium sulphate and cooled to 0 °C. meta-Chloroperoxybenzoic acid (22.229 g, 94.225 mmol, 1.2 eq.) was then added, with effervescence, and the resultant yellow suspension left to stir for lh at 0 °C. The mixture was decanted into 250 cm3 saturated aqueous sodium bicarbonate solution and extracted with chloroform (3 x 100 cm3). Drying and concentration afforded a yellow oil, which was purified by column chromatography [1:10 (v/v) ethyl acetate:hexane as eluent] to afford a pale yellow oil, 4-chlorostyrene epoxide (7.632 g, 63%). <5H (200 MHz, CDCI3) 7.31-7.37 (2H, m, H-3 and H-5), 7.19-7.26 (2H, m, H-2 and H-6), 3.85 (1H, dd, ArCtf-, 74.2 and 2.6), 3.16 (1H, dd, CHC//aHb, J5A and 4.2) and 2.77 (1H, dd, CHCHa//b,./5.4 and 2.4).
Synthesis of nitro-substituted styrene epoxides
2-nitrostyrene epoxide and 4-nitrostyrene epoxide was synthesized from 2-bromo-2' nitroacetophenone and 2-bromo-4' nitroacetophenone as previously reported (Pedragosa-Moreau et al., 1996). 3-nitro styrene epoxide was synthesized from 3-nitroacetophenone as described below.
(a) Synthesis of 3-Nitroacetophenone
A mixture of acetophenone (30.018 g, 0.250 mol) in concentrated sulphuric acid (75 cm3, 6.9 M) was cooled to 0 °C in an ice-salt bath. An ice cold mixture of 65 % nitric acid (20 cm3, 0.206 mol, 0.83 eq.) in concentrated sulfuric acid (30 cm3, 6.9 M) was then added dropwise with vigorous stirring over 45 minutes while maintaining the internal temperature below 0 °C, affording a dense orange-yellow gum. This was poured onto 300 cm3 of crushed ice in,750 cm3 water, stirred for 30 minutes and the resultant yellow powder, 3-nitroacetophenone (19.293 g, 57%) was isolated by filtration. <5H (200 MHz, CDCb) 8.74 (1H, d, H-2,72.0), 8.35 (1H, dd, H-6,78.2 and 2.4), 8.29 (1H, dd, H-4, J 7.8 and 2.2), 7.69 (1H, dd, H-5, .78.2 and 7.8) and 2.69 (3H, s, C//3CO).
(b) Synthesis of 3-nitrostvrene epoxide
40

3-Nitroacetophenone (5.048 g, 30.567 mmol) in anhydrous tetrahydrofuran (230 cm3, 0.1 M) was cooled to 0°C. Aluminium chloride (0.155 g, 3 g.mol"1) was then added to the solution, followed by dropwise addition of bromine (2.0 cm3, 39.4 mmol, 1.3 eq.) in anhydrous tetrahydrofuran (38 cm3, 1 M) over lh to afford a yellow solution. The mixture was warmed over lh to room temperature, then concentrated to a viscous orange oil. This was crystallised from ethyl acetate:hexane to afford a beige soiid, the bromoacetophenone, which was approximately 80 % monobromide to 20 % starting material. Monobromide: <5H (200 MHz, CDC13) 8.49 (1H, d, H-2, J2.0), 8.10 (2H, dd, H-4 and H-6, J7.6 and 2.2), 7.69 (1H, d, H-5, J 8.2) and 4.42 (3H, s, C//3CO). The material was dissolved in methanol (46 cm , 0.4 M) and cooled to 0 °C. Sodium borohydride (0.905 g, 23.924 mmol, 1.3 eq.) was then added, with much effervescence, and left for 20 minutes whereupon TLC analysis reveaied complete reduction had occurred. A chilled solution of 2M aqueous sodium hydroxide (65 cm3, 0:13 mol, 7 eq.) was then added, and the orange mixture left to stir for 2h at 0 °C. Extraction with dichloromethane (3 x 50 cm3), drying, and concentration afforded an orange oil. Column chromatography using 1:10 (v/v) ethyl acetate:hexane as eluent afforded a yellow oil, S-nitrostyrene epoxide (1.377 g, 44% over two steps). $H (200 MHz, CDC13) 8.15-8.21 (2H, m, H-2 and H-6), 7.53-7.68 (2H, m, H-4 and H-5), 3.99 (1H, dd, ArC//-,74.0 and 2.4), 3.23 (1H, dd, CHC//aHb, J 5.4 and 4.0) and 2.82 (1H, dd, CHCHatfb, J5.4 and 2.6).
Yeast strains
Yeast strains with the "Jen" designation and numerical screen numbers were obtained from the Yeast Culture Collection of the University of the Free State. Yeast strains with "AB" or "Car" or "Alf' or "Poh" designations were isolated from soil from specialised ecological niches. "AB" and "Alf strains were isolated from Cape Mountain fynbos, an ecological environment unique to South Africa, "Car" strains were isolated from soil under pine trees, and "Poh" strains from soil contaminated by high concentrations of cyanide. It seemed likely that microorganisms existing in these contaminated soils would have alternative respiratory mechanisms.
41

All new isolates that produced optically active epoxides or vicinal diols during hydrolysis of SEO were identified by known biochemical characterisation methods, and most of the isolates were also subjected to molecular identification by sequence analyses of the D1/D2 region of the large subunit rDNA. These new isolates were subsequently deposited at the Yeast Culture Collection of the University of the Orange Free State (UOFS) and assigned UOFS numbers. Some of the isolates were deposited at the National Collection of Yeast Cultures (NCYC) under the Budapest Treaty.
Cultivation of Rhodosooridium toruloides (NCYC 3181) in a 10 L volume in a 15 L fed-batch bioreactor
Cultivation was performed at 25 °C in 15 L Braun Biostat C bioreactors (working volume 10 L). An overpressure of 500 mbar was applied to the reactor. Dissolved oxygen was continuously monitored and maintained at 30% saturation by adjustment of the stirrer speed. Airflow rate was controlled at 5.5 L.min'1 by use of a mass flow meter. pH was automatically maintained at 5.5 ± 0.05 by the addition of 25 % (w/v) NH4OH. Excessive foam formation was avoided by the addition of antifoam (Pluriol P 2000).
A Fernbach shake flask inoculum (10 %, v/v) of Rhodosporidium toruloides (NCYC 3181) was transferred into a 15 L Braun Biostat C bioreactor containing 6 L medium with the following composition (per litre): citric acid, 2.5 g; yeast extract, 7 g; (NH4)2SO4, 58 g; KH2PO4, 11.3 g; MgSO4.7H2O, 8.2 g; CaCl2.2H2O, 0.88 g; NaCl, 0.1 g; H3PO4, 13.4; vitamin solution, 1.7 ml; trace element solution, 1 ml; antifoam (Plurioi P-2000), 0.5 ml; and glucose, 20 g. Glucose (60 % m/m) was fed to maintain a residua! glucose concentration of 5 g/1 after the batch phase. Glucose feed was stopped when the glucose uptake rate decreased. Cultivation was continued for 12 hours after the residual glucose concentration was zero. The biomass was harvested by centrifugation and frozen at -20 °C in phosphate buffer pH 7.5 containing 20 % glycerol until use.
42

Example II. Sequencing, cloning and overexpression of wild type yeast enoxide hydrolases in Yarrowia lipolvtica as production host under the control of different
promoters
1. Vectors, Strains and Primers (Table 1)
The following features are common to all the E. coliJY. lipolytica auto-cloning integrative vectors used:
• LIP2 terminator
• Zeta regions
• Kanamycin resistance for E. coli selection
• mono-copy auto cloning vectors (pINA 1311, pINA 1313, pINA 3313) with a
fully functional selection marker gene carry the fully functional ura3dl allele
from the URA3 selection marker gene
• multi-copy auto cloning vectors (pINA 1291, pINA 1293, pTNA 3293) with a
defective selection marker gene (copy number amplification) carry the
defective ura3d4 allele from the URA3 selection marker gene.
Table 1. Vectors, strains and primers

,t • , , ,, A * Description,, l-rlJ',. Cloning sites ¦:
Rectors',, ^ Promoter Selection marker* 5 Targeting sequence A Upstream/down f \stream j&.i ;',, A ' Reference/ Origin f
pINA1291 (pYLHmA) hp4d ura3d4 none Pml\ (blunt)/ BamU\,Kpn\AvrW Nicaud et al (2002)
pYL3313 (1313) (pYLTsA) TEF ura3dl none Xmn\ (in pro)/ BamUl, Kpnl, AvrU This study
Host Strain5 i , *,&, - ,-;„,. ,;J ,,Sl ,,,£ !;, ^Description '.a » ' * ,- " % ' Reference/. ; CJrigin
Yarrowia lipolytica Polh MATA, ura3-302, uxpr2-322, axpl-2 (deleted for both extracellular proteases and growth on sucrose) CLIB882
Primers * ", I ;•'• Sequence^ ;' ** V - w ^ * %'-™-* •• zf ;¦ ''': Specifications "';.,_ fJ *
YL-fwd 5'-GGA GTT CTT CGC CCA C-3' (SEQIDNO:11) amplification of expression
YL-rev 5'-GAT CCC CAC CGG AAT TG-3' (SEQ1DNO:12) casette between Notl sites
43

pINA-1 5'- CAT ACA ACC ACA CAC ATC CA-3' (SEQIDNO:13) PYLHmA fwd primer
pINA-2 5'-TAA ATA GCT TAG ATA CCA CAG-3' (SEQIDN0:14) pYLTsA/pYLHmA rev primer
pTNA-3 5'-CTC TCT CTC CTT GTC AAC T-31 (SE0IDN0:I51- pYLTsA fwd primer
2. Transform ants (multi-copy and single-copy)

jpjransformants^ml (Bill
TEF promoter Vector: pYL3313 (1313) = (pYLTsA) I
YL23TsA Rhodotorula mucilaginosa NCYC 3190
YL25TsA Rhodotorula araucariae NCYC 3183
YL46TsA Rhodosporidium toruloides UOFS Y-0471
YL692TsA Rhodosporidium paludigenum NCYC 3179
YLlTsA Rhodosporidium toruloides NCYC 3181
YLCar54 TsA Cryptococcus curvatus NCYC 3158
hp4d promoter Vector: pINA1291=( pYLHmA) "
YL25HmA Rhodotorula araucariae NCYC 3183
YL46HmA Rhodosporidium toruloides UOFS Y-0471
YL692HmA Rhodosporidium paludigenum NCYC 3179
3. Vector preparation
pINA1291 (Fig. 1) was obtained from Dr Madzak of Labo de Genetique, INRA, CNRS. This vector was renamed pYLHmA (Yarrowia Lipolytica expression vector, with Hp4d promoter, multi-copy integration selection, Absent secretion signal; e.g. for construction of recombinant strain YL-25HmA).
plNA3313 (pKOV93) (Fig. 2) was prepared by the inventors. This vector was renamed pYLTsA {Yarrowia Lipolytica expression vector, with TEF promoter, single-copy integration selection, Absent secretion signal ; e.g. for construction of recombinant strain YL-25TsA).
44

To prepare the vectors for ligation with an epoxide hydrolase coding sequence (or other insert to be expressed in Y. lipolytica), DNA was digested with BamWl and Avrll.
4. Insert preparation
Total RNA was isolated "from selected yeast strain cells and messenger RNA (mRNA) was purified from it. The mRNA was used as a template to synthesise complementary DNA (cDNA) using reverse transcriptase. The cDNA was then used as a template for Polymerase Chain Reaction (PCR) using appropriate primers. PCR primers were selected by repeated experimentation using multiple test primers for each yeast strain, the sequences of which were based on previously described epoxide hydrolase sequences from a variety of species. The nucleotide sequences of the forward and reverse primers used to generate cDNA coding sequences from mRNA from seven different yeast strains with appropriate restriction enzyme recognition sites at their termini are shown below. Restriction enzyme recognition sequences are underlined and the relevant restriction enzymes are shown in parentheses.

Strain 5' primer 3' primer
R. tondoides GTGGATCCATGGCGACACACA GACCTAGGCTACTTCTCC
NCYC3181 (#1) R. toruloides UOFS Y-0471 (#46) C. curvatus NCYC 3158 (Car 054) (BamHY) (SEQIDNO:16) CACA (Blnl) (SEQ ID NO: 17)
R. araucariae NCYC 3183 (#25) GATTAATGATCAATGAGCGAG CA (5c/I)(SEQIDNO:18) GACCTAGGTCACGACGAC AG (Blnl) (SEQ ID NO: 19)
R. paludigenum NCYC 3179 (#692) GTGGATCCATGGCTGCCCA (BamHl) (SEQ ID NO:20) GAGCTAGCTCAGGCCTGG {Nhe\) (SEQJDNO:21)
R. mucilaginosa NCYC GTATATCTATGCCCGCCCGCT GACCTAGGCTACGATT
3190 (#23) (Bglll) (SEQ ID NO: 22) TTTGCT (Blnl) (SEQIDNO:23)
Y. lipoiytica GCAGATCTATGTCATCACTCG GACCTAGGCTACAACTTC
NCYC 3229 (Jen 46-2) (Bglll) (SEQ ID NO:24) GACG (Blnl)
45

(Bg/II) (SEQIDNO:24) GACG (Blnl) (SEQ]DNO:25)
Each PCR reaction contained 200 uM dNTPs, 250 nM of each primer, 2 mM of MgCh, cDNA and 2.5 U of Taq polymerase in a 50 \i\ reaction volume. The PCR profile used was: 95°C for 5 minutes, followed by 30 cycles of: 95°C - 1 min, 50°C - 1 min, 72°C - 2 min, then a final extension of 72°C for 10 minutes. The PCR products were purified and digested with the restriction enzymes whose recognition sites are engineered at the end of the primers. The cDNA fragment was cloned into a vector and sequenced for confirmation.
Coding seqences to be inserted in either pYLHmA or pYLTsA were prepared with BamUl and Avrll at their termini. The above PCR primers were designed with these restriction sites, unless the sites were also present in the gene to be inserted. If this occurred, appropriate compatible restriction enzymes were selected. PCR template DNA was either the insert cloned into a different vector, or cDNA synthesized from the original host organism. PCR reactions consisted of 200 uM dNTP's, 250 nM each primer, IX Taq polymerase buffer, and 2.5 units Taq polymerase per 100 ul reaction. The amplification programme used was: 95°C for 5 minutes, 30 cycles of 95°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes, followed by a single duration at 72°C for 10 minutes.
PCR products were purified and digested with the relevant restriction enzymes. The digested DNA was subsequently repurifled and was ready for ligation into the prepared vector.
5. Preparation of pYLHmA or pYLTsA constructs
Vector and insert were ligated at pmol end ratios of 3:1 - 10:1 (insert:vector), using commercial T4 DNA Ligase. legations were electroporated into any laboratory strain of Escherichia coli, using the Bio-Rad GenePulser, or equivalent electroporator. Transformants were selected on LM media (10 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl), supplemented with kanamycin (50 ug/ml). Transformants were selected based on restriction enzyme digests of purified plasmid DNA.
46

6. Yarrowia Hpolytica transformation
6.1.1. Preparation of DNA - method 1
Digestion of the pINA-series of plasmids with Not\ resulted in the release of a bacterial DNA-free expression cassette, containing the ura3d4 (pYLHmA) or the ura3dl (pYLTsA) marker gene and the promoter-gene-terminator.
Scaled-up quantities of each plasmid were isolated. Noil was used to restrict the plasmid DNA, and the digested DNA was run on an agarose gel. Not\ digests resulted in generation of the bacterial fragment of the plasmid as a band at 2210 bp, and the expression cassette as a band of 2760 bp + size of insert (pYLHmA) or 2596 bp + size of insert (pYLTsA). The expression cassette fragments were excised from the gel and purified from the agarose. The purified fragment was used for transformation of Y. Hpolytica Polh.
6.1.2. Preparation of DNA - method 2
Primers YL-Fwd and YL-Rev (Table 1) were used to amplify the expression cassette. PCR reactions consisted of 200 uM dNTP's, 250 pmol each primer, IX Taq po!ymerase buffer and 2.5 units Taq polymerase per 100 uJ reaction. The amplification programme used was 95°C for 5 minutes, 30 cycles of 95°C for I minute, 50°C for 1 minute, and 72°C for 3 V2 minutes, followed by a single duration at 72°C for 10 minutes. The PCR product was purified from the PCR reaction mix and used for transformation of Y. Hpolytica Polh.
6.1.3. Preparation of carrier DNA
DNA from salmon testes was made up as a 10 mg/mf stock in TE (]0mM Tris-HC1, pH 8.0, 1 mM EDTA) and sonicated to result in fragments that ranged from approximately 15 kb to 100 bp, with most fragments in a range of 6 to 10 kb. The DNA was denatured by boiling. Aliquots were stored at -20°C.
6.1.4. Transformation of Yarrowia Hpolytica with pYLHmA or pYLTsA
47

An adaptation of the method of Xuan et al (1988) was used for the transformation of Y. lipolytica Polh. The yeast was inoculated into 50 ml YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose). The culture was incubated at 30°C, 220 rpm until cell densities of 8 x 107 - 2 x 108 cells/ml were reached. The entire culture was harvested, the pellet resuspended in 10 ml TE and reharvested. 1 ml TE + 0.1 M LiOAc was used to resuspend the pellet and the culture was incubated at 28°C in a ProBlot Jr (Labnet) hybridisation oven, set at 4 rpm (or similar incubator) for 1 hour. Transformation mixes were set up with 0.5-2 u.g of transforming DNA + 5 u.gof carrier DNA with 100 u.1 of treated cells.
Each mix was set up in a 1.5 ml microfuge tube, and incubated in a 28°C heating block for 30 minutes. 7 volumes of PEG reagent (40% PEG 4,000, 0.1 M LiOAc, 10 mM Tris, I mM EDTA, pH 7.5, filter-sterilised) were added to each, mixed carefully and incubated at 28°C for a further 1 hour. The tubes were transferred to a 37°C heating block for 15 minutes, and then the cells were pelleted by centrifugation for 1 minute at 13,000 rpm. The cell pellets were carefully resuspended in 100 pj dFbO. The transformations were plated on Y. lipolytica selective plates (17 g/1 Difco yeast nitrogen base without amino acids and without (NH^SO^ 20 g/L glucose, 4 g/L NH4CI, 2 g/L casamino acids, 300 mg/L leucine) and incubated at 28°C.
Colonies which appeared on the selective plates after 3-7 days were transferred onto fresh plates and regrown.
6.1.5. Confirmation of integration of pYLHmA or pYLTsA
Colonies that grew on the newly-streaked selective plates were inoculated into 5 ml of YPD medium and grown at 30°C, 200 rpm for 24-48 hours. A small-scale genomic DNA isolation was performed.
PCR was performed using this genomic DNA as template, with either pINA-1 and pINA-2 as primers (transformants with pYLHmA), or plNA-3 and plNA-2 (pYLTsA) (Table 1). Each PCR reaction contained 200 uM dNTPs, 250 nM of each primer, 2 mM of MgCh, genomic DNA and 2.5 U/50uJ of Taq polymerase. The PCR profile was as described above in this example. These primer sets gave products the size of the inserted genes.
48

Example III. Selection of wild type yeasts that are able to produce optically active styrene epoxide and phenyl ethanediol from unsubstituted (±)-styrene epoxide.
Yeasts were cultivated, harvested and frozen as described above. Racemic unsubstituted SEO was added and the screening was performed as described above. Strains with the highest activities as judged by TLC from diol formation were subjected (as Samples 1-23) to chiral GC analysis as described above (Table 2). Enantiomeric excesses (ee) were determined from the formula: ee = [[S]-[R]}/{[S]+[R]}. [S] is the concentration at the appropriate time point of the reaction of (S)-SEO enantiomer and [R] is the concentration at the appropriate time point of the reaction of the (R)-SEO enantiomer. A positive ee value indicates the relevant yeast (or yeast-derived epoxide hydrolase) is enantioselective for the R-enantiomer of the SEO and negative ee value indicates the relevant yeast (or yeast-derived epoxide hydrolase) is enantioselective for the S enantiomer of the SEO.
Table 2. Conversion or racemic unsubstituted styrene oxide and enantiomeric excess of the residual (S)-styrene oxide in the reaction after exposure to selected wild type yeast strains. Reaction conditions: 20 mM (R/S) styrene oxide. 20% cells (wet w/v), 25°C.

Sample Internal
No. strain no. Species Culture collection no. Time(min) (S)-Styrcne oxide (R)-Styrenc oxide Conv(%) ce(%)
1 #751 Candida magnohae UOFSY-1040 120 8.26 6.05 28.46 15.40
2 #678 Candida magnoliae UOFS Y-1297 180 9.86 8.04 10.50 10.15
3 Car-54 Cryptococcus curvatus NCYC3158 120 7.04 1.65 56.54 62.07
4 AB-29 Cryptococcus podzolicus UOFS Y-1897 120 9.32 6.12 22.81 20.75
5 AB-37 Cryptococcus podzolicus UOFS Y-1896 180 8.26 3.68 40.32 38.41
6 AB-39 Cryptococcus podzolicus UOFSY-1912 120 9.16 5.35 27.47 26.26
7 AB-50 Cryptococcus podzolicus UOFS Y-1883 180 8.67 4.01 36.60 36.73
8 AB-52 Cryptococcus podzoltcus UOFS Y-1895 120 9.47 5.72 24.05 24.64
9 AB-56 Cryptococcus podzolicus UOFS Y-1913 120 9.38 5.02 27.98 30.26
10 #692 Rhodosporidium paludigenum NCYC3179 120 9.11 4.85 30.22 30.53
11 POH-20 Rhodosporidium toruloides NCYC3216 120 9.00 3.72 63.61 41.46
12 POH-33 Rhodosporidium toruloides NCYC3218 120 8.85 1.85 53.49 65.42
13 POH-38 Rhodosporidium toruloides NCYC3219 120 9.61 4.71 71.60 34.18
14 AB1 Rhodosporidium toruloides NCYC 3181 120 6.56 0.98 62.26 73.95
15 AB2 RhodosDoridium toruloides UOFSY-0518 120 7.08 1.22 58.48 70.57
49

16 Car-52 Rhodospondtum torulotdes UOFS Y-2230 120 7.31 1.59 55.47 64.25
17 Car-200 Rhodosporidium tomloides UOFS Y-2256 180 7.85 3.45 43.51 38.95
18 EP-230 Rhodotorula auranttaca NCYC3185 180 8.55 5.91 27.66 18.24
19 #50 Rhodotoruta glulinis NCYC 3186 120 8.79 6.53 23.42 14.78
20 #680 Rhodotorula glulinis UOFS Y-0459 30 8.74 3.21 40.26 46.32
21 #681 Rhodotorula ghttinis UOFS Y-0653 60 9.10 2.85 40.25 52.23
22 #713 Rhodotorula minuta ' NCYC 3187 120 8.84 4.10 35.26 36.62
23 #232 Trichosporon cutaneum var. cutaneum NCYC 3202 240 8.51 3.42 40.32 42.65
All the yeast strains referred to in this and the following examples are kept and maintained at the University of the Orange Free State (UOFS), Department of Microbial, Biochemical and Food Biotechnology, Faculty of Natural and Agricultural Sciences, P.O. Box 339, Bloemfontein 9300, South Africa (Tet +27 51 401 2396, Fax + 27 51 444 3219) and are readily identified by the yeast species and culture collection number as indicated. Representative examples of strains belonging to the different species have been deposited under the Budapest Treaty at National Collection of Yeast Cultures (NCYC), Institute of Food Research Norwich Research Park Colney, Norwich NR4 7UA, U.K. ( Tel: +44-(0)1603-255274 Fax: +44-(0)1603-4584i4 Email: ncyc@bbsrc.ac.uk) and are readily identified by the yeast species and culture collection accession number as indicated. The samples deposited with the NCYC are taken from the same deposit maintained by the South African Council for Scientific and Industrial Research (CSIR) since prior to the filing date of this application. The deposits will be maintained without restriction in the NCYC depository for a period of 30 years, or 5 years after the most recent request, or for the effective life of the patent, whichever is longer, and will be replaced if the deposit becomes non-viable duringthat period. Samples of the yeast strains not deposited at NCYC will be made available upon request on the same basis and conditions of the Budapest Treaty.
Various wild-type yeast strains selected from Table 2 were used (as Samples 24-30) to produce optically active unsubstituted SEO and optically active unsubstituted ' phenylethanediol from racemic unsubstituted SEO. For each example, a line graph is supplied that shows the change in concentrations of the epoxide and diol enantiomers with time (left y-axis) and the enantiomeric excess (ee) of the epoxide and conversion to diol at different times where the degree of conversion means the molar amount of
50

epoxide converted to diol as a percentage (%) of the starting total molar amount of both epoxide enantiomers (right y-axis) (Figs. 3 - 9). The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be seen from these graphs where the yield (%) = 100% - conversion (%).
Example IV. Production of optically active (S)-stvrene epoxide and (R)-phenylethanediol using host yeast cells transformed with the epoxide hvdrolase genes from selected wild type yeast strains
Figs. 10-12 show the hydrolysis of (±) racemic unsubstituted SEO by recombinant yeast strains (tested in this example as Samples 31-33) expressing, under control of different promoters, exogenous epoxide hydrolases from selected wild-type yeast strains to produce (S)-unsubstituted SEO and (R)-unsubstituted PED.
Example V. Production of optically active (S)-unsubstituted-styrene epoxide and
(R)-unsubstituted phenvlethanediol using whole or Ivsed veast host cells transformed with the epoxide hvdrolase genes from a Rhodotorula araucariae strain
Whole cells and lysed cell suspensions of recombinant expression hosts transformed with the epoxide hydrolase gene from Rhodotorula araucariae (NCYC 3183) strains under control of two different promoters were tested (as Samples 34-37) for their ability to produce optically active (S)-SEO and (R)-PED from (±) unsubstituted SEO (Figs. 13-16). Lysed cell suspensions were prepared by treatment of the cells with Y-PER®, a yeast protein extraction reagent from Sigma-AIdrich (St. Louis, MO, U.S.A) used according to the instructions of the supplier. No significant differences in activity or enantioselectivity between and whole and lysed yeast cells were observed.
Example VI. Hydrolysis of (±)-unsubstituted styrene oxide by a veast cultivated in a 15 L fermenter to produce optically active (R)-unsubstituted phenvlethanediol and
(S)-unsubstituted sryrene oxide
Fig. 17 shows the improvement in yields that can be achieved by process optimisation. The same recombinant strain tested in Example V was cultivated (as Sample 38 in this example) in a 10 L reaction volume in a 15 L fermenter and used to
51

hydrolyse (±)-unsubstituted SEO in a stirred tank reactor at high substrate concentration (1 M SEO in total reaction matrix) at low temperature with inclusion of 2% m/v tri-butyl phosphate (TBP) additive to improve biocatalyst stability.
Example VII. Phenyl-substituted stvrene epoxide reactions
Reactions using phenyl-substituted SEO that were used as substrates to illustrate the ability of different yeast strains with enantioselective epoxide hydrolases to hydrolyse (±)-2-, 3- or 4-susbtituted styrene epoxides represented by the general formula (I) are schematically depicted in Fig 18.
Example VIII. Use of wild-type yeast strains to produce optically active 3-chlorostvrene epoxide and 3-chlorophenyl ethanediol from (±)-3-ehlorostvrene
epoxide
Enantiomeric excesses (ee; calculated as described in Example III) of the remaining epoxide enantiomer obtained after incubation of the different yeast strains (tested in this Example as Samples 39-132) with 3-chlorostyrene epoxide are shown in Table 3.
Reaction conditions: 20 mM 3-chloroSEO5 20 % cells (wet w/v), 25 °C. Reaction time = 300 minutes. Positive ee values indicate that the [S]-epoxide was in excess (i.e., that the relevant yeast strain was enantioselective for the (R) enantiomer), while negative ee values indicate opposite enantioselectivity.
Table 3. Enantiomeric excesses (ee) of the remaining (S)-epoxide enantiomer obtained after incubation of the different yeast strains with 3-chlorostyrene epoxide (negative ee values denote opposite enantioselectivity)

Internal
Sample strain Culture ee (%)
no. no. collection no. (s) .
39 Jen-25 Bullera dendrophila NCYC3152 21.0
40 Jen-26 BulJera dendrophila NCYC 3208 6.1
41 708 Candida rugosa NCYC3155 7.0
52

42 Jen-17 Cryptococcus albidus UOFS Y-0223 5.9
43 Jen-2 Ctyptococcus albidus NCYC3156 -3.6
44 Jen-15 Ciyptococcus hungaricus NCYC3159 11.2
45 AB-24 Cryptococcus laurentii NCYC3161 14.3
46 AB-26 Cryptococcus laurentii UOFS Y-1885 13.5
47 AB-32 Ciyptococcus laurentii UOFS Y-l 887 12.6
48 AB-25 Ciyptococcus laurentii UOFS Y-l 884 10:7
49 AB-33 Ciyptococcus laurentii UOFS Y-l 888 10.2
50 Jen-12 Cryptococcus laurentii UOFSY-0135 9.3
51 AB-27 Ciyptococcus laurentii UOFS Y-l 886 5.6
52 AB-50 Ciyptococcus podzolicus UOFS Y-l 883 20.1
53 AB-28 Ciyptococcus podzolicus UOFS Y-l 889 11.7
54 AB-30 Ctyptococcus podzolicus UOFS Y-1904 11.4
55 AB-39 Ctyptococcus podzolicus UOFSY-1912 8.9
56 AB-29 Cryptococcus podzolicus UOFS Y-l 897 6.4
57 AB-58 Ciyptococcus podzolicus NCYC3164 6.1
58 AB-34 Ciyptococcus podzolicus UOFS Y-l 890 4.1
59 113 Debaiyomyces h'ansenii UOFS Y-0058 4.5
60 520 Pichia finlandica NCYC3173 -41.3
61 674 Pichia guillermondii UOFSY-1030 5.4
62 675 Pichia guillermondii UOFS Y-l033 3.7
63 706 Pichia guillermondii UOFS Y-0057 2.6
64 112 Pichia guillermondii UOFS Y-0053 2.1
65 707 Pichia guillermondii NCYC 3174 1.8
66 28 Pichia haplophila UOFS Y-2136 -3.8
67 673 Pichia haplophila NCYC 3177 -19.8
68 692 Rhodosporidium paludigenum NCYC 3179 10.1
69 Car-052 Rhodosporidium toruloides UOFS Y-2230 83.1
70 Car-020 Rhodosporidium toruloides UOFS Y-2226 74.9
71 Car-059 Rhodosporidium toruloides UOFSY-2231 72.6
72 AB 1 Rhodosporidium toruloides NCYC 3181 72.0
73 Car-126 Rhodosporidium toruloides UOFS Y-2251 70.9
74 Car-118 Rhodosporidium toruloides NCYC 3182 65.2
75 Car-120 Rhodosporidium toruloides UOFS Y-2249 62.6
76 Car-108 Rhodosporidium toruloides UOFS Y-2247 61.8
77 Car-204 Rhodosporidium toruloides UOFS Y-2257 60.8
78 Car-078 Rhodosporidium toruloides UOFS Y-2240 59.1
53

79 Car-134 Rhodosporidium tondoides UOFS Y-2253 58.4
80 Car-077 Rhodosporidium tondoides UOFS Y-2239 56.9
81 Car-038 Rhodosporidium tondoides UOFS Y-2228 55.6
82 Car-006 Rhodosporidium tondoides UOFS Y-2223 48.5
83 Car-076 Rhodosporidium toruloides UOFS Y-2238 47.0
84 Car-092 Rhodosporidium toruloides UOFS Y-2241 46.6
85 Car-067 Rhodosporidium tondoides UOFS Y-2236 45.1
86 Car-093 Rhodosporidium toruloides UOFS Y-2242 44.7
87 Car-003 Rhodosporidium tondoides UOFS Y-2222 42.4
88 Car-142 Rhodosporidium tondoides UOFS Y-2255 40.9
89 Car-210 Rhodosporidium toruloides UOFS Y-2261 38.3
90 Car-121 Rhodosporidium tondoides UOFSY-2250 36.6
91 AB2 Rhodosporidium tondoides UOFS Y-0518 33.9
92 Car-070 Rhodosporidium tondoides UOFS Y-2237 33.8
93 Car-094 Rhodosporidium toruloides UOFS Y-2243 32.2
94 Car-103 Rhodosporidium toruloides UOFS Y-2246 30.8
95 Car-205 A Rhodosporidium toruloides UOFS Y-2258 21.8
96 Car-131 Rhodosporidium toruloides UOFS Y-2252 21.4
97 Car-100 Rhodosporidium tondoides UOFS Y-2245 20.8
98 Car-209 Rhodosporidium tondoides UOFS Y-2260 15.6
99 46 Rhodosporidium tondoides UOFS Y-0471 5.2
100 EP-230 Rhodotorula aurantiaca NCYC3185 62.9
101 50 Rhodotonda glutinis NCYC3186 89.6
102 Car-62 Rhodotorula glutinis UOFS Y-2234 64.5
103 Car-75 Rhodotorula glutinis UOFS Y-2265 49.7
104 713 Rhodotorula glutinis UOFS Y-0489 43.6
105 Car-61 Rhodotonda glutinis UOFS Y-2233 43.3
106 Car-60 Rhodotorula glutinis UOFS Y-2232 42.6
107 Car-66 Rhodotorula glutinis UOFS Y-2235 41.7
108 Car-22 Rhodotonda glutinis UOFS Y-2227 24.8
10© 680 Rhodotonda glutinis UOFS Y-0459 19.6
110 AB6 Rhodotorula glutinis UOFS Y-0513 12.0
111 714 Rhodotorula minuta NCYC3187 32.8
112 712 Rhodotorula minuta UOFS Y-0835 8.2
113 165 Rhodotorula sp. UOFS Y-2043 3.0
114 Jen-31 Sporidiobolus salmonicolor NCYC3196 10.2
115 Jen-32 Sporidiobolus salmonicolor NCYC3195 6.8
54

116 Jen-30 Sporobolomyces holsaticus NCYC3I98 15
117 Jen-29 Sporobolomyces rosens NCYC3197 15
118 Jen-28 Sporobolomyces tsugae NCYC3199 -6.
119 20 Trichosporon cutaneum var. ctttaneum NCYC 3201 -3.
120 21 Trichosporon cutaneum var. cutaneum UOFS Y-0063 -13
121 '9 Trichosporon ovoides NCYC 3207 -34
122 231 Trichosporon sp. UOFS Y-0533 2.;
123 Car-205B Trichosporon montevideense NCYC 3225 -11
124 Jen-39 Yarrowia lipolytica UOFS Y-1698 -4.
125 Jen-38 Yarrowia lipolytica UOFS Y-0809 -4.
126 Jen-33 . Yarrowia lipolytica UOFS Y-0164 -6.
127 Jen-41 Yarrowia lipolytica UOFS Y-1571 -6.
128 Jen 46 Yarrowia lipolytica NCYC 3229 -8.
129 Jen-43 Yarrowia lipolytica UOFS Y-1699 -8.
130 Jen-37 Yarrowia lipolytica UOFS Y-0097 -9.
131 Jen-46 Yarrowia lipolytica UOFS Y-1701 -10
132 Jen-48 Yarrowia lipolytica UOFS Y-l 700 -25
All the yeast strains referred to in this and the following examples are kept and maintained at the University of the Orange Free State (UOFS), Department of Microbial, Biochemical and Food Biotechnology, Faculty of Natural and Agricultural Sciences, P.O. Box 339, Bloemfontein 9300, South Africa (Tel +27 51 401 2396, Fax + 27 51 444 3219) and are readily identified by the yeast species and culture collection number as indicated. Representative examples of strains belonging to the different species have been deposited under the Budapest Treaty at National Collection of Yeast Cultures (NCYC), Institute of Food Research Norwich Research Park Colney, Norwich NR4 7UA, U.K. ( Tel: +44-(0)1603-255274 Fax: +44-(0)1603-458414 Email: ncyc@bbsrc.ac.uk) and are readily identified by the yeast species and culture collection accession number as indicated. The samples deposited with the NCYC are taken from the same deposit maintained by the South African Council for Scientific and Industrial Research (CSIR) since prior to the filing date of this application. The deposits will be maintained without restriction in the NCYC depository for a period of 30 years, or 5 years after the most recent request, or for the effective life of the patent, whichever is longer, and will be replaced if the deposit becomes non-viable during that period. Samples of the yeast strains not deposited at
55

NCYC will be made available upon request on the same basis and conditions of the Budapest Treaty.
Various wild-type yeast strains selected from Table 3 were used (as Samples 133-137 in this example) to produce to produce optically active epoxides and vicinal diols from 3-chloroSEO. For each Sample, two graphs are provided (Figs. 19- 23). The first graph (panel A) shows the change in concentrations of the epoxide enantiomers with time, while the second graph (panel B) shows the enantiomeric excess of the epoxide at different conversions. The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be obtained from these graphs.
Example IX. Production of optically active (S)-3-chlorostyrene epoxide and (R)-3-chlorophenylethanediol using yeast host cells transformed with the epoxide hydrolase genes from selected wild type yeast strains
Figs. 24-28 show the hydrolysis of (±) 3-chloroSEO by recombinant yeast strains (tested in this example as Samples 138-142) expressing, under control of different promoters, exogenous epoxide hydrolases from selected wild-type yeast strains to produce (S)-3-chloroSEO and (R)-3-chloroPED. The first graph (panel A) shows the change in concentrations of the epoxide enantiomers with time, while the second graph (panel B) shows the enantiomeric excess of the residual epoxide at different conversions. The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be obtained from these graphs.
Example X. Production of optically active (S)-2-chlorostyrene epoxide and (R)-2-chlorophenvlethanediol using yeast host cells transformed with the epoxide hydrolase genes from selected wild type yeast strains
Figs. 29-30 show the hydrolysis of (±) 2-chloroSEO by recombinant yeast strains (tested in this example as Samples 143 and 144) expressing, under control of Hp4d promoter, exogenous epoxide hydrolases from selected wild-type yeast strains to produce (S)-2-chloroSEO and (R)-2-chloroPED. The first graph (panel A) shows the change in concentrations of the epoxide enantiomers with time, while the second graph
56

(panel B) shows the enantiomeric excess of the residual epoxide at different conversions. The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be obtained from these graphs.
Example XI. Production of optically active (S)-4-chlorostyrene epoxide and (R)-4-chlorophenylethanedioI using yeast host cells transformed with the epoxide hydrolase genes from selected wild type yeast strains
Figs. 31 -36 show the hydrolysis of (±)-4-chloroSEO by recombinant yeast strains (tested in this example as Samples 145-150) expressing, under control of different promoters, exogenous epoxide hydrolases from selected wild-type yeast strains to produce (S)-4-chloroSEO and (R)-4-chloroPED. The first graph (panel A) shows the change in concentrations of the epoxide enantiomers with time, while the second graph (panel B) shows the enantiomeric excess of the residual epoxide at different conversions. The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be obtained from these graphs.
Example XII. Production of optically active (S or R)-2,-, 3-, or 4-nitrostvrene epoxide and (R or S)-2-, 3-, or 4-nitrophenylethanediol using selected wild-type yeast strains or yeast host cells transformed with the enoxide hydrolase genes from
selected wild type yeast strains
Figs. 37-44 show the hydrolysis of (±)-4-nitroSEO by wild-type yeast strains or recombinant yeast strains (tested in this example as Samples 151-158) expressing, under control of different promoters, exogenous epoxide hydrolases from selected wild-type yeast strains to produce (S or R)-2-, 3-, or -4-nitroSEO and (R or S)-2-, 3-, or -4-nitroPED. The first graph (panel A) shows the change in concentrations of the epoxide enantiomers with time, while the second graph (panel B) shows the enantiomeric excess of the residual epoxide at different conversions. The yield of the optically active epoxide that can be obtained at a particular enantiomeric purity can be obtained from these graphs.
57

References
Harada, H., Hirokawa, Y., Suzuki, K., Hiyama, Y., Oue, M. et al.,(2003). Novel and potent human and rat p3-adrenergic receptor agonists containing substituted 3-indolylalkylaminesMSioorganic and Medicinal Chemistry Letters 13: 1301-1305.
Manoj, K.M., Archelas, A., Baratti, J., Furstoss, R. (2001). Microbiological transformations. Part 45: A green chemistry preparative scale synthesis of enantiopure building blocks of Eliprodil. Tetrahedron 57: 695- 701
Monterde, M.I., Lombard, M., Archelas, A., Cronin, A., Arand, M.3 Furstoss, R. (2004). Enzymatic transformations. Part 58: Enantioconvergent biohydrolysis of styrene oxide derivatives catalysed by the Solarium tuberosum epoxide hydrolase. Tetrahedron: Asymmetry 15: 2801-2805.
Nicaud J-M, Madzak C, Van den Broek P, Gysler C, Duboc P, Niederberger P. Gaillardin C. (2002) Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Research 2, 371-279.
Pedragosa-Moreau, S., Archelas, A. and Furstoss, R. (1996) Microbiological Transformations 32. Use of epoxide hydrolase mediated biohydrolysis as a way to enantiopure epoxides and vicinal diols - Application to substituted styrene oxide derivatives. Tetrahedron 52: 4593-4606.
Xuan J-W, Fournier P, Gaillardin C. (1988) Cloning of the LYS5 gene encoding saccharopine dehydrpgenase from the yeast Yarrowia lipolytica by target integration. Current Genetics 14, 15-21.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
58

SEQ. ID. NO. 1
Rhodospohdium toruloides NCYC 3181 (#1)
Amino acid sequence
ORIGIN
1 MATHTFASPP TRFTVDIPQS ELDELDFRLD KTRWPATEIV PEDGADDPTA FGLGAGPTLP
61 LMKELAKGWR EFDWKKAQDH LNTFEHYTVE IEDLSIHFLH HRSTRPNAVP LILCHGWPGH
121 FGEFLNVIPL LTEPSDPSAQ AFHVVAPSMP GYAWSSPPPS SKWSMPDTAR VFDKLMTGLG
181 YEKYMAQGGD WGSIAARCLG SLHKDHCKAV HLNFLPVFPP VPMWLINPHT LLAWAPRFLV
241 PEKQAARMKR GLAYLEKGSA YYVMQQLTPR TPAYGLTDSP VGLLAWIGEK FEPTIQEASK
301 QAQPTLTRDE LYFTCSLYWF TRSIGTSFLP YSLNPHFTTF LTDSSTTCPT LPCPSTRARS
361 TARQNGTLSV PATSSDQGRA GGRTLCCARE ARRVCRPSQG GVRRHVGEV
SEQ. ID. NO. 2
Rhodosooridium toruloides UOFS Y-0471 (#46)
Amino acid sequence
ORIGIN
1 MKMATHTFAS PPTRFTVDIP QSELDELHSR LDKTRWPATE IVPEDGTDDP TAFGLGAGPT
61 LPLMKELAKG WREFDWKKAQ DHLNTFEHYM VEIEDLSIHF LHHRSTRPNA VPLILCHGWP
121 GHFGEFLNVI PLLTEPSDPS AQAFHVVAPS MPGYAWSLPP PSSKWNMPDT ARVFDKLMTG
181 LGYEKYMAQG GDWGSIAARC LGSLHKDHCK AVHLNFLPVF PPVPMWLINP HTLLAWAPRF
241 LVPEKQAARM KRGLAYLEKG SAYYVMQQLT PRTPAYGLTD SPVGLLAWIG EKFEPTIQEA
301 SKQAQPTLTR DELYFTCSLY WFTRSIGTSF LPYSLNPHFT TFLTDSKYHL PNFALSLYPG
361 EIYCPAERDA KRTGNLKWIK DAPEGGHFAA LEKPDVFVEH LREAFGVMWE K
59

SEQ. ID. NO. 3
Rhodotorula araucariae NCYC 3183 (#25)
Amino acid'sequence
ORIGIN
MSEHSFEAPP QPFTVDFAPH IEDLHRRLDN ARWPTQEIVP VDVSETEHHN AFGLGMGPQL
61 NLMKELANGW RAFDQSALQD HLNSFNNWKV EIEGLSIHFL HHRSTRAGAL PLILCHGWPG
121 GYHEFLHVVQ LLTEPEGADA QAFHLVVPSM PGYAFSSPPP TAKWGMEDTA RVFDKLMTGL
181 GYNKYVAQGG DWGSITARCL GALHKDHCIA VHLNFCPVPP PFPFDQFNPR TLLNWMPRFV
241 ISDQQRAKLE RGLAYLEKGS AYYVMQQLTP RTPAYALNDS PIGLLAWIGE KMIPGINEAS
301 AQPNPTLNRD ALYTTLSLYW FTNSIGTSFL PYSLNPHFST FLTSPRYRLP NFALSSFPGE
361 LFSPTPRDAA RTGVMRWYKE ADDGGHFAAL EKPDVFSQHV REAVKAMLSS
SEQ. ID. NO.4
Rhodosporidium oaludiaenum NCYC 3179 f #692)
Amino acid sequence
ORIGIN
1 MAAHSFTAPP APYNIDFAPQ VNDLHRRLDA ARWPEHDVVP DDVDHGEHGA FGLGAGPSLA
61 LMKELAQEWR GQDQKQLQDH LNSYKNYRVE IEGLNIHFLH YPSSRADAFP LILCHGWPGG
121 YHEFLHVLER LTEPEDQGSR AFHVVVPSMP GYAFSSPPKT AKWGMEDTAR VFDKLMTGLG
181 YAKYAAQGGD WGSITARCLG SLHKENCVAV HLNFCPVPPP FPLNMFNPRT LLDWMPRFVL
241 PDQRRAKIER GVAYIERGSA YYAMQNLTPR TPAYGLNDSP IGLLAWIGEK MIPGIDKAVK
\ 301 HPNATLNREA LFTTLSIYWF TGSIGSSFLP YALNPHFSTF LVSPRHQLPN FALSNFPDEL
361 FTPEERDARR TGNLRWYKDA EDGGHFAALE KPEVFAEHVR EAMGVLLSNQ A
60

SEQ. ID. NO. 5
Rhodotorula mucilactinosa NCYC 3190 (#23)
Amino acid sequence
ORIGIN
1 MPARSLTLRP FSPSFTAPEL DGLARSLESS RLPAETYASR QAKYGIKHAW MKNALQRWKD
61 GFDWKKHEQD INEVDHYMVQ VQSDGIQHDL HVIYHESKDP NAIPLLLLHG WPGSAFEFIE
121 AIKILRKSTS PAFHLIAPME PGYGWSTPPP LDRGFNMNDC TALMNDLMVG LGYGDGYAAQ
181 GGDIGSGLAR LLAVNYDACK CININYMPAV APPEDAPERH QIKPHEEDAL RRADEFQKTG
241 RGYANMHATR PGTVGIVVGS SPVALLAWIA EKYLAWTDED PPLDTILAIC TIWWIRDSYP
301 SSIWAYADFL ETGISALHND PKYKLDKKPF GFSSFKEEIS ATPEAWAGRN GNLQFYRYHD
361 KGGHFAALEQ PEAFAQDMQD CFGKIKPLSQ EQKS
61

SEQUENCE ID NO 6
Rhodosporidium toruloides NCYC 3181 (#11
Nucleotide sequence
ORIGIN
1 ATGGCGACAC ACACATTCGC TTCGCCTCCC ACGCGCTTCA CCGTCGACAT CCCACAGTCA
61 GAACTCGACG AACTCGACTT CCGACTCGAC AAGACCCGCT GGCCGGCGAC AGAGATCGTT
121 CCAGAGGATG GGGCGGACGA CCCGACGGCG TTTGGGCTCG GAGCAGGGCC GACGCTGCCG
181 CTCATGAAGG AACTGGCGAA GGGTTGGCGC GAGTTCGACT GGAAGAAGGC GCAGGACCAC
241 CTCAACACCT TTGAGCACTA CACGGTCGAA ATCGAGGACC TCTCCATCCA CTTCCTCCAC
301 CACCGCTCGA CTCGCCCGAA TGCTGTTCCG CTCATCCTCT GCCACGGCTG GCCAGGCCAC
361 TTCGGCGAGT TCCTGAACGT CATACCGCTC TTGACGGAGC CGTCGGACCC GTCCGCCCAG
421 GCGTTCCACG TCGTCGCGCC TTCGATGCCC GGTTATGCTT GGTCTTCGCC TCCTCCGTCC
481 TCCAAGTGGA GCATGCCTGA TACCGCGAGG GTCTTCGACA AGCTCATGAC CGGGCTTGGC
541 TACGAGAAGT ACATGGCGCA GGGCGGAGAC TGGGGCAGCA TCGCTGCTCG CTGCCTTGGA
601 TCGCTTCACA AAGACCACTG CAAAGCCGTC CACCTCAACT TCCTCCCCGT CTTCCCACCC
661 GTCCCGATGT GGCTTATCAA CCCGCACACG CTCCTTGCCT GGGCACCGCG CTTCCTCGTG
721 CCGGAGAAGC AGGCTGCGCG TATGAAGCGC GGGTTGGCGT ACCTTGAGAA GGGCTCCGCC
781 TACTACGTCA TGCAGCAGTT GACGCCTCGC ACGCCTGCGT ACGGCCTGAC CGACAGTCCC
841 GTCGGCTTGC TGGCCTGGAT CGGCGAGAAG TTCGAGCCGA CCATTCAGGA GGCGAGCAAG
-901- .CAAGCCCAGC-GGACCCTGAC TCGCGACGAG CTCTACTTCA CCTGCTCGCT CTACTGGTTC
961 ACCCGCTCAA TCGGCACCTC CTTCCTTCCC TACTCGCTCA ACCCGCACTT CACCACCTTC
1021 CTGACCGACA GCAGGTACCA CCTGCCCAAC TTTGCCCTGT CCCTCTACCC GGGCGAGATC
1081 TACTGCCCGG CAGAACGGGA CGCCAAGCGT ACCGGCAACC TCAAGTGGAT CAAGGACGCG
1141 CCCGATGGAG GACACTTTGC TGCGCTCGAG AAGCCCGATG TGTTTGTCGA GCATCTCAGG
1201 GAGGCGTTTG GCGTCATGTG GGAGAAGTAG
62

SEQUENCE ID NO 7
Rhodosporidium toruloides UOFS Y-0471 (#46)
Nucleotide sequence
ORIGIN
1 ATGGCGACAC ACACATTCGC TTCGCCTCCC ACCCGCTTCA CCGTCGACAT CCCACAGTCG
61 GAACTCGACG AACTTCACTC GCGACTCGAC AAGACCCGCT GGCCGGCGAC AGAGATCGTT
121 CCAGAGGATG GGACGGACGA TCCGACGGCG TTTGGGCTCG GAGCAGGGCC GACGCTGCCG
181 CTCATGAAGG AATTGGCGAA GGGTTGGCGC GAGTTCGACT GGAAAAAGGC GCAGGACCAC
241 CTCAACACCT TCGAGCACTA CATGGTCGAA ATTGAGGACC TCTCGATCCA CTTCCTCCAC
301 CATCGCTCGA CTCGCCCGAA CGCTGTTCCC CTCATCCTCT GCCACGGCTG GCCAGGCCAC
361 TTTGGCGAGT TCCTGAACGT TATCCCGCTC TTGACGGAGC CGTCGGACCC CTCCGCTCAG
421 GCGTTCCACG TCGTCGCCCC TTCGATGCCT GGCTATGCTT GGTCTTTGCC TCCTCCGTCC
4 81 TCCAAGTGGA ACATGCCTGA CACCGCGAGG GTCTTCGACA AGCTCATGAC CGGGCTTGGC
541 TACGAGAAGT ACATGGCGCA GGGCGGAGAC TGGGGAAGCA TCGCCGCTCG CTGCCTTGGA
601 TCGCTGCACA AGGACCATTG CAAAGCCGTC CACCTCAACT TCCTCCCCGT CTTCCCACCC
661 GTCCCGATGT GGCTTATCAA CCCGCACACG CTCCTTGCCT GGGCACCGCG CTTCCTCGTG
721 CCGGAGAAGC AGGCTGCGCG TATGAAGCGC GGGTTGGCGT ACCTTGAGAA GGGCTCCGCC
781 TACTACGTCA TGCAGCAGTT GACGCCTCGC ACGCCTGCGT ACGGCCTGAC CGACAGTCCC
841 GTCGGCTTGC TGGCCTGGAT CGGCGAGAAG TTCGAGCCGA CCATTCAGGA GGCGAGCAAG
901 CAAGCCCAGC CGACCCTGAC TCGCGACGAG CTCTACTTCA CCTGCTCGCT CTACTGGTTC
961 ACCCGCTCAA TCGGCACCTC CTTCCTTCCC TACTCGCTCA ACCCGCACTT CACCACCTTC
1021 CTGACCGACA GCAAGTACCA CCTGCCCAAC TTTGCCCTCT CGCTTTACCC AGGCGAGATC
1081 TACTGCCCCG CGGAGCGGGA CGCCAAGCGC ACCGGCAACC TCAAGTGGAT CAAGGACGCG
1141 CCTGAGGGAG GACACTTTGC TGCGCTCGAA AAGCCGGATG TGTTTGTCGA GCACCTCAGG
1201 GAGGCGTTTG GCGTCATGTG GGAGAAGTAG
63

SEQUENCE ID NO 8
Rhodotorula araucariae NCYC 3183 (#25)
Nucleotide sequence
ORIGIN
1 ATGAGCGAGC ACAGCTTCGA GGCCCCGCCA CAGCCGTTTA CGGTCGACTT TGCTCCCCAC
61 ATCGAGGATC TCCACCGCCG TCTCGACAAT GCGCGCTGGC CGACGCAAGA GATTGTCCCC
121 GTCGACGTGT CCGAGACGGA GCATCACAAC GCGTTCGGAC TCGGGATGGG CCCGCAGCTC
181 AACCTTATGA AGGAGCTCGC CAACGGCTGG CGCGCGTTCG ACCAGTCGGC GCTCCAGGAC
241 CACCTCAACA GCTTCAACAA CTGGAAGGTC GAGATCGAGG GATTGTCGAT CCACTTCCTC
301 CACCATCGCT CGACGCGCGC CGGCGCTCTC CCGCTCATCC TGTGCCATGG CTGGCCCGGC
361 GGGTACCACG AGTTCCTCCA CGTCGTCCAG CTCCTCACCG AACCAGAGGG GGCGGATGCG
421 CAGGCGTTTC ACCTCGTCGT CCCCTCGATG CCCGGGTACG CCTTCTCGTC TCCGCCGCCG
4 81 ACGGCCAAGT GGGGCATGGA AGACACTGCA AGGGTTTTTG ACAAGCTCAT GACCGGTTTG
541 GGGTACAACA AGTATGTCGC GCAGGGCGGT GACTGGGGGT CCATCACGGC GCGATGCCTC
601 GGCGCGCTGC ACAAGGACCA CTGCATTGCT GTCCACCTCA ACTTCTGCCC CGTCCCGCCG
661 CCGTTCCCAT TCGACCAGTT CAACCCGCGC ACGCTGCTCA ACTGGATGCC GCGCTTCGTG
721 ATCTCGGACC AGCAGCGTGC GAAGCTCGAG CGTGGGCTGG CGTACCTCGA GAAGGGGTCT
781 GCTTACTATG TCATGCAGCA GCTCACACCG CGTACCCCGG CCTACGCTCT CAATGACAGC
841 CCGATTGGCC TGCTCGCCTG GATTGGCGAA AAGATGATCC CAGGCATCAA CGAGGCGAGC
901 GCGCAGCCGA ACCCGACGCT CAATCGCGAT GCGTTGTACA CCACGCTCTC GCTGTACTGG
961 TTCACCAACT CCATCGGCAC CTCTTTCCTC CCCTACTCGC TTAACCCGCA CTTCAGCACG
1021 TTCCTCACCT CGCCCCGCTA TCGCCTGCCG AACTTTGCGC TGTCTTCCTT CCCGGGCGAG
1081 CTGTTCTCGC CGACGCCGCG CGATGCTGCG AGGACGGGCG TGATGCGCTG GTACAAGGAG
1141 GCGGACGATG GCGGGCACTT TGCGGCGCTC GAGAAGCCCG ATGTGTTCAG CCAGCATGTC
1201 AGGGAGGCAG TCAAGGCCAT GCTGTCGTCG TGA
64

SEQUENCE ID NO 9
Rhodosporidium paiudiaenum NCYC 3179 f #692)
Nucleotide sequence
ORIGIN
ATGGCTGCCC ATTCCTTTAC TGCACCTCCT GCACCCTACA ACATCGACTT TGCGCCCCAG
61 GTAAATGACC TGCACCGCCG TCTCGATGCT GCCCGCTGGC CGGAACACGA CGTGGTGCCC
121 GACGATGTGG ATCACGGAGA GCACGGCGCA TTCGGACTCG GCGCTGGTCC CAGCCTCGCC
181 CTCATGAAGG AGCTCGCGCA GGAATGGAGG GGCCAGGACC AGAAGCAGCT GCAGGACCAC
241 CTCAACTCCT ACAAGAACTA TCGCGTCGAG'ATCGAGGGTC TCAACATCCA CTTCCTGCAC
Stfl TACCCGTCGT CTCGCGCCGA TGCGTTCCCG CTCATCCTGT GCCACGGCTG GCCTGGCGGC
361 TACCACGAGT TCCTGCACGT CCTAGAGCGC CTCACGGAGC CCGAGGATCA GGGGTCGCGG
421 GCCTTCCATG TCGTCGTGCC TTCCATGCCG GGTTACGCCT TCTCCTCGCC GCCCAAGACG
481 GCAAAATGGG GCATGGAGGA CACGGCTCGC GTGTTCGACA AGCTCATGAC GGGGCTAGGT
541 TACGCCAAGT ATGCGGCCCA AGGCGGTGAC TGGGGGTCTA TCACGGCGCG CTGCCTAGGT
601 TCGCTGCACA AGGAGAACTG CGTCGCTGTC CACCTCAACT TCTGCCCGGT TCCCCCGCCG
661 TTCCCGCTCA ACATGTTCAA CCCGCGCACA CTTCTGGACT GGATGCCTCG CTTTGTCCTG
721 CCTGATCAAC GGCGGGCCAA GATTGAGCGC GGCGTGGCCT ATATCGAGCG CGGCTCTGCC
781 TACTACGCCA TGCAAAACTT GACGCCGCGC ACGCCTGCGT ACGGCTTGAA CGATAGTCCG
841 ATTGGTTTGC TCGCGTGGAT TGGCGAGAAG ATGATTCCGG GCATTGACAA GGCTGTCAAG
901 CATCCGAACG CAACCCTCAA TCGCGAAGCT CTTTTCACGA CACTCTCGAT CTACTGGTTC
961 ACGGGCTCGA TTGGCTCCTC CTTCCTGCCA TACGCTCTCA ACCCGCACTT CTCTACCTTC
1021 CTCGTCTCGC CGCGGCACCA ACTGCCGAAC TTTGCTCTGT CCAACTTTCC CGACGAGCTG
1081 TTCACGCCCG AAGAACGCGA TGCTCGCCGA ACCGGAAACT TGCGGTGGTA CAAGGATGCA
1141 GAGGATGGAG GGCACTTCGC GGCGCTGGAG AAGCCCGAGG TCTTCGCCGA GCACGTAAGG
1201 GAGGCGATGG GGGTCTTGCT GTCGAACCAG GCCTGA
65

SEQUENCE ID NO 10
Rhodotorula mucilaginosa NCYC 3190 (#23)
Nucleotide sequence
ORIGIN
1 ATGCCCGCCC GCTCGCTCAC GCTGCGCCCG TTCTCGCCGT CGTTCACGGC TCCGGAACTG
61 GACGGTCTCG CTCGCTCGCT CGAGTCGTCG CGCTTGCCCG CCGAGACGTA CGCTTCCCGC
121 CAGGCCAAAT ACGGCATCAA GCATGCTTGG ATGAAGAATG CCCTCCAACG GTGGAAGGAC
181 GGGTTCGATT GGAAGAAGCA CGAGCAGGAC ATCAACGAGG TCGACCACTA TATGGTGCAG
241 GTCCAGTCCG ATGGCATTCA ACACGACCTC CATGTGATCT ATCACGAATC GAAAGACCCG
301 AATGCGATCC CGCTCTTGCT GCTGCATGGG TGGCCCGGTT CCGCGTTCGA GTTTATCGAG
361 ¦ GCGATCAAGA TCCTTCGCAA GAGTACCTCG CCCGCGTTCC ACCTGATCGC GCCCATGGAG
421 CCCGGCTACG GGTGGAGTAC TCCGCCGCCA CTCGACCGCG GTTTCAACAT GAACGATTGC
481 ACAGCGCTCA TGAACGACTT GATGGTTGGA CTCGGGTACG GAGACGGTTA CGCTGCTCAG
541 GGTGGCGACA TCGGTTCGGG ACTCGCAAGA CTCCTCGCCG TCAACTATGA CGCATGCAAA
601 TGCATCAACA TCAACTACAT GCCTGCCGTT GCACCACCAG AGGACGCTCC GGAGCGGCAC
661 CAGATCAAAC CGCACGAGGA GGATGCGCTC CGACGTGCGG ACGAGTTTCA GAAGACGGGC
721 AGGGGGTATG CCAACATGCA TGCAACGAGA CCCGGTACGG TCGGCATCGT CGTCGGTAGT
781 TCGCCGGTCG CACTGCTCGC TTGGATCGCT GAAAAGTACC TCGCTTGGAC CGATGAGGAT
841 CCGCCCCTCG ACACGATCCT CGCAATCTGC ACCATCTGGT GGATCCGCGA CTCGTACCCT
901 TCTTCAATCT GGGCCTACGC CGACTTTCTC GAGACGGGCA TCTCGGCCCT GCACAACGAC
961 CCGAAGTACA AACTTGACAA GAAACCGTTC GGGTTCTCGA GCTTCAAGGA GGAGATCAGC
1021 GCGACTCCCG AGGCGTGGGC GGGCAGGAAC GGCAACTTGC AGTTCTATCG GTACCACGAC
1081 AAGGGAGGTC AGTTTGCGGC GCTCGAGCAG CCGGAAGCGT TCGCGCAAGA CATGCAGGAT
1141 TGCTTCGGCA AAATCTGGCC TCTCTCTCAG GAGCAAAAAT CGTAG
66

WE CLAIM:
A process for obtaining an optically active epoxide or an optically active vicinal diol, which process includes the steps of:
providing an enantiomeric mixture of a styrene epoxide;
creating a reaction mixture by adding to the enantiomeric mixture a polypeptide, or a functional fragment thereof, having enantioselective styrene epoxide hydrolase activity, the polypeptide being a polypeptide encoded by a gene of a yeast cell;
incubating the reaction mixture; and
recovering from the reaction mixture: (a) an enantiopure, or a substantially enantiopure, phenylethanediol; (b) an enantiopure, or a substantially enantiopure, styrene epoxide; or (c) an enantiopure, or a substantially enantiopure, phenylethanediol and an enantiopure, or a substantially enantiopure, styrene epoxide.
2. A process for obtaining an optically active epoxide or an optically active
vicinal diol, which process includes the steps of:
providing an enantiomeric mixture of a styrene epoxide;
creating a reaction mixture by adding to the enantiomeric mixture a cell comprising a nucleic acid encoding, and capable of expressing, a polypeptide having enantioselective styrene epoxide hydrolase activity;
incubating the reaction mixture; and
recovering from the reaction mixture: (a) an enantiopure, or a substantially enantiopure, phenylethanediol; (b) an enantiopure, or a substantially enantiopure, styrene epoxide; or (c) an enantiopure, or a substantially enantiopure, phenylethanediol and an enantiopure, or a substantially enantiopure, styrene epoxide.
3. The process of claims 1 or 2, wherein the cell is a yeast cell.
4. The process of any of claims 1 to 3, wherein the polypeptide is encoded by
an endogenous gene of the cell.
5. The process of claim 2 or 3, wherein the cell is a recombinant cell and the
67

polypeptide is encoded by a nucleic acid sequence with which the cell is transformed.
6. The process of claim 5, wherein the nucleic acid sequence is a
heterologous nucleic acid sequence.
7. The process of claim 5, wherein the nucleic acid sequence is a
homologous nucleic acid sequence.
8. The process of any of claims I to 7, wherein the polypeptide is a full-
length yeast epoxide hydrolase.
9. The process of any of claims 1 to 7, wherein the polypeptide is a
functional fragment of yeast epoxide hydrolase.
10. The process of any of claims 1 to 9, wherein the process is carried out at a
pH from 5 to 10.
11. The process of any of claims 1 to 10, wherein the process is carried out at
a temperature of 0°C to 70°C.
12. The process of any of claims 1 to 11, wherein the concentration of the
styrene epoxide in the reaction matrix is at least equal to the soluble concentration of the
styrene epoxide in water.
13. The process of any of claims 1 to 12, wherein the styrene epoxide of the
enantiomeric mixture and the obtained optically active epoxide is a compound of the
general formula (1) and the vicinal diol produced by the process is a compound of the
general formula (II),
68


wherein,
Xi, X2, X3, X4 and X5 are, independently of each other, selected from: H, halogens, hydroxyl groups, mercapto groups, carboxylates, nitro groups, cyano groups, substituted or unsubstituted amino groups, amide groups, alkoxy groups, alkenyloxy groups, aryloxy groups, aryl alkyloxy groups, alkylthio groups, alkoxycarbonyl groups, substituted or unsubstituted carbamoyl groups, acyl groups, substituted and unsubstituted alkyl groups; substituted and unsubstituted alkenyl groups; and substituted and unsubstituted aryl groups, wherein the number of substituents is one or more than one and wherein the substituents are the same or different; or
Xj and X2, or X2 and X3, or X3 and X4; or X4 and X5 together and independent are a substituted or unsubstituted aryl group selected from the group consisting of: phenyl; biphenyl; naphtyl; anthracenyl groups; and the like; or
Xi and X2) or X2 and X3, or X3 and X4, or X4 and X5 together and independent are a cycloalkyl group with 4 to 8 carbon atoms, wherein the cycloalkyl group is selected from the group consisting of: cyclobutyl-; cyclopentyl-; cyclohexyl-; cycloheptyl-; and cyclooctyl- groups, wherein the cycloalkyl group is unsubstituted or variably substituted at any position of the ring; or
X| and X2. or X2 and X3, or X3 and X4, or X4 and X5 together and independent are a cycloalkenyl group with 4 to 8 carbon atoms, wherein the cycloalkenyl group is selected from the group consisting of: cydobutenyl-; cyclopentenyl-; cyciohexenyl-; cycloheptenyl-; and cyclooctenyl- groups, wherein the cycloalkenyl group is unsubstituted or is variably be substituted at one or more positions in the ring; or
Xi and X2, or X2 and X3, or X3 and X4, or X4 and X5 together and independent are
69

a heterocyclic group consisting of a 5- to 7-membered heterocyclic group containing a nitrogen atom, an oxygen atom; or a sulfur atom, wherein the heterocyclic group is selected from the group consisting of: furyl-; dihydrofuranyl-; tetrahydrofuranyl-; dioxolanyl-; oxazolyl-; dihydrooxazolyl-; oxazolidinyl-; isoxazolyl-; dihydroisoxazolyl-; isoxazolidinyl-; oxathiolanyl-; thienyl-; tetrahydrothienyl-; dithiolanyl-; thiazolyl-; dihydrothiazolyl-; thiazolidinyl-; isothiazolyl-; dihydroisothiazolyl-; isothiazolidinyl-; pyrrolyl-; dihydropyrrolyl-; pyrrolidinyl-; pyrazolyl-; dihydropyrazoiyl-; pyrazolidinyl-; imidazolyl-; dihydroimidazolyl-; imidazolidinyl-; triazolyl-; dihydrotriazolyl-; triazolidinyl-; tetrazolyl-; dihydrotetrazolyl-; tetrazolidinyl-; pyridyl-; dihydropyridyl-; piperidinyl-; morpholinyl-; dioxanyl-; oxathianyl-; trioxanyl-; thiomorpholinyl-; pyridazinyl-; dihydropyridazinyl-; tetrahydropyridazinyl-; hexahydropyridazinyl-; pyrimidinyl-; dihydropyrimadinyl-; tetrahydropyrimadinyl-; hexahydropyrimadinyl-; pyrazinyl-; piperazinyl-; pyranyl-; dihydropyranyl-; tetrahydropyranyl-; thiopyranyl-; dihydrothiopyranyl-; tetrahydrothiopyranyl-; dithianyl-; purinyl-; pyrimidinyl-; pyrrolizinyl-; pyrrolizidinyl; indolyl-; dihydroindolyl-; isoindolyl-; indolizinyl-; indolizidinyl-; quinolyl-; dihydroquinolyl-; tetrahydroquinolyl-; isoquinolyl-; dihydroquinolyl-; tetrahydroquinolyl-; quinolizinyl-; quinolizidinyl-; phenanthroliny!-; chromenyl-; chromanyl-; isochromenyl-; isochromanyl-; benzofuranyl-; and carbazolyl-groups; and the like.
14. The process of any of claims 1 to 13, wherein the aryl group is a
substituted or an unsubstituted phenyl group.
15. The process of any of claims 1 tol4, wherein the cycloalkyl group is a
cycloalkyl group with 5 to 7 carbon atoms
16. The process of any of claims 1 to 15, wherein the cycloalkenyl group is a
cycloalkenyl group with 5 to 7 carbon atoms.
17. The process of any of claims 1 to 16, wherein the heterocyclic group has 5
or 6 carbon atoms.
70

18. The process of any of claims 1 to 17, wherein the enantiomeric mixture is
a racemic mixture or a mixture of any ratio concentrations of the enantiomers.
19. The process of any of claims 1 to 18, which process includes adding to the
reaction mixture water and at least one water-immiscible solvent.
20. The process of any of claims 1 to 19, which process includes adding to the
reaction mixture water and at least one water-miscible organic solvent.
21. The process of any of claims 1 to 20, which process includes stopping the
reaction when one enantiomer of the epoxide and/or vicinal diol is in excess compared to
the other enantiomer of the epoxide and/or vicinal diol.
22. The process of any of claims 1 to 21, which process includes recovering
continuously during the reaction the optically active epoxide and/or the optically active
vicinal diol produced by the reaction directly from the reaction mixture.
23. The process of any of claims 1 to 22, wherein the yeast cell is of a yeast
genus selected from the group consisting of Arxula, Brettanomyces, Bullera,
Bulleromyces, Candida, Cryptococcus, Debaryomyces, Dekkera, Exophiala, Geotrichum,
Hormonema, Jssatchenkia, Kluyveromyces, Lipomyces, Mastigomyces, Myxozyma,
Pichia, Rhodosporidium., Rhodotorula, Sporidiobolus, Sporobolomyces, Trichosporon,
Wingea, and Yarrowia
24. The process of any of claims 1 to 22. wherein the yeast cell of a yeast
species selected from the group consisting of Arxula adeninivorans, Arxula terrestris,
Brettanomyces bruxellensis, Brettanomyces naardenensis ^Brettanomyces anomalus,
Brettanomyces species (e.g.NCYC 3151J, Bullera dendrophila, Bulleromyces albus,
Candida albicans, Candida fabianii, Candida glabrata, Candida haemulonii, Candida
intermedia, Candida magnoliaeCandida parapsilosis, Candida rugosa, Candida tenuis,
71

Candida tropicalis, Candida famata, Candida kruisei, Candida sp. (new) rel to C. sorbophila, Cryptococcus albidus, Cryptococcus amylolentus, Cryptococcus bhutanensis, Cryptococcus cwvatus, Cryptococcus gastricus, Cryptococcus humicola, Cryptococcus hungaricus, Cryptococcus laurentii, Cryptococcus luteolus, Cryptococcus macerans, Ciyptococcus podzolicus, Cryptococcus terreusy Cryptococcus macerans, Debaryomyces hansenii, Dekkera anomala, Exophiala dermatitidis, Geotrichum species (e.g. UOFS Y-0111), Hormonema species (e.g. NCYC 3171), Issatchenkia occidentalism Kluyveromyces marxianus, Lipomyces species (e.g.UOFS Y-2159), Lipomyces tetrasporus, Mastigomycesphilipporii, Myxozyma melibiosi, Pichia anomala,, Pichia finlandica, Pichia guillermondii, Pichia haplophila, Rhodosporidium lusitaniae, Rhodosporidium paludigenum, Rhodosporidium sphaerocarpum, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula araucariae, Rhodotorula glutinis, Rhodofonsla minuta, Rhodotorula minuta var. minuta, Rhodotorula mucilaginosa, Rhodotorula philyla, Rhodotorula rubra, Rhodotorula species (e.g. UOFS Y-2042), Rhodotorula species (e.g. UOFS Y-0448), Rhodotorula species (e.g. NCYC 3193), Rhodotorula species (e.g. UOFS Y-0139), Rhodotorula secies (e.g. UOFS Y-0560), Rhodotorula aurantiaca, Rhodotorula species (e.g. NCYC 3224), Rhodotorula sp. "mucilaginosa", Sporidiobolus salmonicolor, Sporobolomyces holsaticus, Sporobolomyces roseus, Sporobolomyces tsugae, Trichosporon beigelii, Trichosporon cutaneum var. cutaneum., Trichosporon delbrueckii, Trichosporon jirovecii, Trichosporon mucoides, Trichosporon ovoides, Trichosporon pullulans, Trichosporon species (e.g. UOFS Y-0861), Trichosporon species (e.g. UOFS Y-1615), Trichosporon species (e.g. UOFS Y-0451), Trichosporon species (e.g. NCYC 3212), Trichosporon species (e.g. UOFS Y-0449^, Trichosporon species (e.g. NCYC 3211), Trichosporon species (e.g. UOFS Y-2113), Trichosporon species (e.g. NCYC 3210), Trichosporon moniliiforme, Trichosporon montevideense, Wingea robertsiae, and Yarrowia lipolytica.
25. A method for producing a polypeptide, which process includes the steps of:
providing a cell comprising a nucleic acid encoding and capable of expressing a polypeptide that has enantioselective styrene epoxide hydrolase activity;
72

culturing the cell; and recovering the polypeptide from the culture.
26. The method of claim 25, wherein the cell is a yeast cell.
27. The method of claim 25 or 26, wherein the polypeptide is a full-length
yeast epoxide hydrolase.
28. The method of claim 25 or 26, wherein the polypeptide is a functional
fragment of a yeast epoxide hydrolase.
29. The method of any of claims 25 to 28, wherein the polypeptide is encoded
by an endogenous gene of the cell.
30. The method of any of claims 25 to 28, wherein the cell is a recombinant
ceil and the polypeptide is encoded by a nucleic acid sequence with which the cell is
transformed.
31. The method of claim 30, wherein the nucleic acid sequence is a
heterologous nucleic acid sequence.
32. The method of claim 30, wherein the nucleic acid sequence is a
homologous nucleic acid sequence.
33. A crude or pure enzyme preparation which includes an isolated
polypeptide having enantioselective styrene epoxide hydrolase activity.
34.^ A substantially pure culture of cells, a substantial number of which comprise a nucleic acid encoding, and are capable of expressing, a polypeptide having enantioselective styrene epoxide hydrolase activity.
73

35. An isolated cell, the cell comprising a nucleic acid encoding a polypeptide
having enantioselective styrene epoxide hydrolase activity, the cell being capable of
expressing the polypeptide,
36. An isolated DNA comprising:

(a) a nucleic acid sequence that encodes a polypeptide that has enantioselective
styrene epoxide hydrolase activity and that hybridizes under highly stringent conditions
to the complement of a sequence selected from the group consisting of SEQ ID NOs: 6,
7, 8,9, and 10; or
(b) the complement of the nucleic acid sequence.

37. The DNA of claim 36, wherein the nucleic acid sequence encodes a
polypeptide comprising an amino acid sequence selected from the group consisting of
SEQ ID NOs: 1,2, 3,4, and 5.
38. The DNA of claim 36 or 37, wherein the nucleic acid sequence is selected
from the group consisting of SEQ ID NOs: 6,7, 8, 9 and 10.
39. An isolated DNA comprising:

(a) a nucleic acid sequence that is at least 55% identical to a sequence selected
from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10; or
(b) the complement of the nucleic acid sequence,
wherein the nucleic acid sequence encodes a polypeptide that has enantioselective styrene epoxide hydrolase activity.
40. An isolated DNA comprising:
(a) a nucleic acid sequence that encodes a polypeptide consisting of an amino acid
sequence that is at least 55% identical to a sequence selected from the group consisting of
SEQ ID NOs: I, 2, 3, 4 and 5; or
(b) the complement of the nucleic acid sequence,
wherein the polypeptide has enantioselective styrene epoxide hydrolase activity.
74

41. An isolated polypeptide encoded by the DNA of any of claims 32 to 36.
42. An isolated polypeptide comprising an amino acid sequence that is at least
55% identical to SEQ IE) NOs: 1, 2, 3, 4, or 5, the polypeptide having enantioselective
styrene epoxide hydrolase activity.
43. The polypeptide of claim 41 or 42, comprising:
(a) an amino acid sequence selected from the group consisting of SEQ ID NOs; 1, 2, 3, 4, and 5, or a functional fragment of the sequence; or
(b)'the sequence of (a), but with no more than;five conservative substitutions, wherein the polypeptide has enantioseiective styrene epoxide hydrolase activity.
44. An isolated antibody that binds to the polypeptide of any of claims 41 to 43.
45. The antibody of claim 44, wherein the antibody is a polyclona! antibody.
46. The antibody of claim 44,' wherein the antibody is a monoclonal antibody.

75
The invention provides yeast strains, and polypeptides encoded by genes of such yeast strains, that have enantiospeciflc styrene epoxide hydrolase activity. The invention also features nucleic acid molecules encoding such polypeptides, vectors containing such nucleic acid molecules, and cells containing such vectors. Also embraced by the invention are methods for obtaining optically active styrene vicinal diols and optically active styrene epoxides.