WO2010003659A1

WO2010003659A1 - Pseudomonas putida styrene monooxygenase variants

Info

Publication number: WO2010003659A1
Application number: PCT/EP2009/004943
Authority: WO
Inventors: Kevin O'connor; Lucas Gursky; Jasmina Nikodinovic
Original assignee: University College Dublin
Current assignee: University College Dublin
Priority date: 2008-07-08
Filing date: 2009-07-08
Publication date: 2010-01-14
Anticipated expiration: 2011-01-08
Also published as: GB0812468D0; GB2461706A

Abstract

The present invention provides isolated polynucleotides derived from Pseudomonas putida, and encoding polypeptides having styrene monooxygenase activity. The polynucleotides of the present invention are derived from the styAB gene from Pseudomonas putida CA-3, using error prone PCR-based in vitro evolution. The polypeptides encoded by these polynucleotides are useful as biocatalysts for the synthesis of oxygenated compounds, as they exhibit improved properties, such as enzymatic activity and enantiospecificity. Methods for obtaining oxygenated compounds are also provided, as are organisms that express the polypeptides and are thus useful for carrying out said biocatalytic syntheses.

Description

Pseudomonas putida styrene monooxygenase variants

Background of the Invention

Oxidoreductase enzymes are potentially attractive biocatalysts as they exhibit a high degree of regio- and stereo-specificity, and they perform reactions under mild conditions, which offers an advantage over many chemical processes. Styrene monooxygenase (SMO) is a two-component NADH- dependent flavoenzyme, made up of a 45kDa oxygenase (StyA) and an 18kDa reductase (StyB). The Pseudomonas putida SMO is encoded by a StyAB gene, which comprises the two coding regions for StyA and StyB, separated by an intervening section of DNA. Expression of the StyAB gene has not yet been fully elucidated, but the gene is most likely transcribed to a dicistronic mRNA molecule, which is in turn translated to the individual StyA and StyB components. No function has yet been identified for the intervening DNA section, which is thought to remain untranslated.

SMO oxygenates the vinyl side chain of styrene to form styrene epoxide, predominantly to S-styrene oxide. For example, the formation of S-styrene oxide from styrene has been shown using E.coli expressing wild-type styAB from P. putida VLB120 with an enantiomeric excess (ee) of 99%. SMO is also known to oxygenate indole and other alkenes to produce a range of epoxides. Indeed, a wide variety of bacteria expressing SMO are able to convert indole to indigo, due to structural similarities between indole and styrene. The rate of styrene oxide formation by a recombinant E. coli expressing xylene monooxygenase has been reported maximally as 90μmoles/min/g cell dry weight. While SMO can offer advantages in terms of stereospecificity, the rate of reactions catalysed by this enzyme needs to be improved to enhance the potential of SMO as a biocatalyst.

Directed evolution is an in vitro random mutagenesis approach, based on error prone PCR techniques, which has led to a number of successes in the engineering of proteins with improved function or stability. Directed evolution requires a discriminatory screen, which is often the limiting factor for strategy design. Few target substrates or products of an enzyme are coloured, fluoresce or have a convenient colorimetric assay designed for their detection. The ability to form indigo from indole provides a convenient colorimetric method for assaying styrene monooxygenase activity and tracking its evolution over generations of random mutagenesis. However, screening for improved SMO activity towards indole may not yield an enzyme with improved activity towards styrene or indene i.e. the evolutionary path of the enzyme is dictated by the screen, and so the selection of mutants based on a screening substrate (such as indole) rather than a target substrate may drive the evolution of the enzyme down a path towards improved activity with indole but not other substrates. Given the structural similarities between indole, styrene, and indene, and the convenience of the indigo formation assay, an improvement in SMO activity by screening for indigo formation was attempted.

Briefly, the styAB gene from Pseudomonas putida CA-3, which encodes SMO₁ was subjected to three rounds of in vitro evolution using error prone PCR, and improvements in styrene monooxygenase activity were monitored using the 'indole to the blue dye, indigo' assay with whole cells expressing StyAB variants.

It is an object of the present invention to circumvent at least some of the described disadvantages associated with known polypeptides having styrene monooxygenase activity.

Summary of the Invention

According to a first aspect of the present invention there is provided an isolated polynucleotide derived from Pseudomonas putida, and encoding a polypeptide having styrene monooxygenase activity, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO 1 , and at least one nucleic acid substitution selected from the group comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751; with the proviso that: a) if there is a single nucleic acid substitution at position 262, the nucleic acid is not a T, b) if there is a single nucleic acid substitution at position 449, the nucleic acid is not an A, and c) if there is a single nucleic acid substitution at position 1751 , the nucleic acid is not an A.

Optionally, the nucleic acid substitution at position 262 is selected from A, C, and G. Optionally, the nucleic acid substitution at either or both of positions 449 and 1751 is independently selected from C, G, and T.

By "a polypeptide having styrene monooxygenase activity" is meant a polypeptide having either or both of a reductase activity and an oxygenase activity. Preferably, the polypeptide is capable of reducing flavin and/or is capable of using the reduced flavin to perform an oxygenation reaction. Styrene monooxygenase activity involves the introduction of a single atom of oxygen into a substrate to produce an oxygenated product. Such polypeptides having styrene monooxygenase activity are useful for the biocatalytic synthesis of organic oxygenated compounds.

A monooxygenase enzyme has a dual activity, wherein one oxygen atom is transferred to a substrate, and one oxygen atom is reduced to, for example, water. Styrene monooxygenase transfers one oxygen atom to, for example, styrene, and reduces the other oxygen atom to water. Although styrene is the primary substrate acted on by styrene monooxygenase, other substrates can also be oxygenated in the practice of the present invention. As examples, when expressed in E. coli cells, the styrene monooxygenase of the present invention oxygenates substrates comprising cyclic or heterocyclic rings, or fused ring systems, containing an oxygenatable moiety, such as a thioether moiety and/or double or triple bond, either within the cyclic or heterocyclic ring, or fused ring system, or attached thereto. Optionally, the ring, or one ring of the fused ring system is a phenyl ring. Further optionally, the substrate is selected from the group comprising, but not limited to, indene, indole, benzothiophene, thioanisole, and naphthalene.

Optionally, the isolated polynucleotide comprises two or more of the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751.

Further optionally, the isolated polynucleotide comprises at least the three nucleic acid substitutions comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751.

Preferably, the nucleic acid at position 262 is substituted for a C, the nucleic acid at position 449 is substituted for a G, and the nucleic acid at position 1751 is substituted for a G.

Preferably, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO 4.

Optionally, the isolated polynucleotide further comprises a nucleic acid modification after position 1241. Further optionally, the nucleic acid modification comprises a polynucleotide insertion after position 1241.

Optionally, the polynucleotide insertion occurs directly after position 1241. The polynucleotide insertion may or may not result in a change in the reading frame of the isolated polynucleotide. Optionally, the polynucleotide insertion results in a change in the reading frame of the isolated polynucleotide. Optionally, the polynucleotide insertion results in a change of one nucleic acid, relative to the original reading frame. Alternatively, the polynucleotide insertion results in a change of two nucleic acids, relative to the original reading frame.

Optionally, the polynucleotide insertion after position 1241 comprises (3x-1) nucleic acids, wherein x is an integer, greater than or equal to 1. Optionally, the polynucleotide insertion after position 1241 is a nucleic acid sequence comprising ATTGC. Optionally, the polynucleotide insertion comprises 8 nucleic acids. Further optionally, the polynucleotide insertion is a nucleic acid sequence comprising either NNNATTGC or ATTGCNNN, wherein each N is independently selected from the group consisting of A, T, G, and C. Optionally, the polynucleotide insertion comprises a nucleic acid sequence comprising ACTATTGC.

Optionally, the isolated polynucleotide further comprises a nucleic acid substitution at position 1674. Preferably, the nucleic acid at position 1674 is substituted with a nucleic acid selected from A, T, and C, optionally a T. Further preferably, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO 7. Optionally, the isolated polynucleotide further comprises a nucleic acid substitution at position 114. Preferably, the nucleic acid at position 114 is substituted for an A.

Further optionally, the isolated polynucleotide still further comprises at least one nucleic acid substitution selected from the group comprising, but not limited to, a nucleic acid substitution at position 27, a nucleic acid substitution at position 147, a nucleic acid substitution at position 192, a nucleic acid substitution at position 259, a nucleic acid substitution at position 465, a nucleic acid substitution at position 535, a nucleic acid substitution at position 702, a nucleic acid substitution at position 733, a nucleic acid substitution at position 907, a nucleic acid substitution at position 1008, a nucleic acid substitution at position 1250, a nucleic acid substitution at position 1261 , a nucleic acid substitution at position 1270, and a nucleic acid substitution at position 1524.

Optionally, the isolated polynucleotide still further comprises the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 27, a nucleic acid substitution at position 1 14, a nucleic acid substitution at position 733, and a nucleic acid substitution at position 1250.

Alternatively, the isolated polynucleotide still further comprises the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 114, a nucleic acid substitution at position 147, a nucleic acid substitution at position 192, a nucleic acid substitution at position 465, a nucleic acid substitution at position 535, a nucleic acid substitution at position 702, a nucleic acid substitution at position 1261 , and a nucleic acid substitution at position 1270.

Further alternatively, the isolated polynucleotide still further comprises the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 259, a nucleic acid substitution at position 907, a nucleic acid substitution at position 1008, and a nucleic acid substitution at position 1524.

Optionally, the isolated polynucleotide still further comprises at least one nucleic acid substitution adjacent or at position 907. Further optionally, the isolated polynucleotide still further comprises at least one nucleic acid substitution at position 907.

Still further optionally, the isolated polynucleotide comprises the nucleic acid sequence selected from the group comprising, but not limited to, SEQ ID NO 10, SEQ ID NO 13, and SEQ ID NO 16.

Optionally, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO 10.

Alternatively, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO 13.

Further alternatively, the isolated polynucleotide comprises the nucleic acid sequence of SEQ ID NO 16. According to a second aspect of the present invention there is provided an isolated polypeptide having styrene monooxygenase activity, wherein the isolated polypeptide comprises at least a first component and a second component.

Preferably, the first component comprises an amino acid sequence selected from the group comprising, but not limited to, SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 11 , SEQ ID NO 14, and SEQ ID NO 17; and the second component comprises an amino acid sequence selected from the group comprising, but not limited to, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 15, and SEQ ID NO 18; with the proviso that if the first component comprises the amino acid sequence of SEQ ID NO 2, then the second component does not comprise the amino acid sequence of SEQ ID NO 3.

Further preferably, the isolated polypeptide comprises at least a first component and a second component, wherein the pair of first and second components comprise the respective amino acid sequences selected from the group comprising, but not limited to, SEQ ID NO 5 and SEQ ID NO 6; SEQ ID NO 8 and SEQ ID NO 9; SEQ ID NO 11 and SEQ ID NO 12; SEQ ID NO 14 and SEQ ID NO 15; and SEQ ID NO 17 and SEQ ID NO 18.

Most preferably, the isolated polypeptide comprises the amino acid sequence encoded by a nucleic acid sequence selected from the group comprising, but not limited to, SEQ ID NO 4, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 13, and SEQ ID NO 16.

According to a third aspect of the present invention there is provided a recombinant expression vector comprising an insert having a nucleic acid sequence selected from the group comprising, but not limited to, SEQ ID NO 4, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 13, and SEQ ID NO 16.

Preferably, the recombinant expression vector can replicate or be maintained within a host cell. Further preferably, the recombinant expression vector is capable of transferring the monooxygenase activity of the polypeptide of the present invention to a host cell.

Preferably, the recombinant expression vector is operably associated with a promoter.

According to a further aspect of the present invention there is provided a host cell, which is stably transformed using the recombinant expression vector of the present invention.

Preferably, the host cell is a competent host cell. Further preferably, the host cell is a competent microorganism. A variety of microorganisms can be modified to contain a styrene monooxygenase enzyme of the present invention. In preferred embodiments, the microorganism is a bacterial species that is transformed with a plasmid vector of the present invention. In alternative embodiments, the microorganism is a bacterial species that is transduced with a viral vector of the present invention. According to a still further aspect of the present invention there is provided a method for the biocatalytic synthesis of oxygenated compounds, the method comprising the steps of providing an oxygen source, contacting at least one oxygenatable compound with a polypeptide, wherein the polypeptide is a polypeptide of the present invention, under reaction conditions sufficient to form the oxygenated compound.

By "oxygenatable compound" is meant a chemical compound, such as an organic chemical compound, containing, for example, a thioether moiety, or a double or triple bond. The oxygenatable compound is, optionally, a cyclic or heterocyclic ring or fused ring system, and the oxygenatable moiety can form part of the ring or ring system, or can be attached thereto. Optionally, the oxygenatable compound is an unsaturated compound.

By "unsaturated compound" is meant a chemical compound, such as an organic chemical compound, containing at least one of either, or both, of a double bond or a triple bond between carbon atoms. The unsaturated compound contains at least one of either or both of a carbon-carbon single pi bond or a carbon-carbon double pi bond. Examples include an alkene, or an alkyne, as well as aromatic rings, aromatic ring systems, and heterocyclic ring systems.

By "oxygenated compound" is meant a chemical compound, such as an organic chemical compound, which has a chemical structure comprising at least one oxygen atom. It is also understood that an oxygenated compound can be a product of an oxidation reaction involving a reductant, wherein the oxidation number of the reductant has been altered. Preferably, the oxidation number of the product is greater than that of the reductant.

Preferably, the at least one oxygenatable compound is contacted with the polypeptide of the present invention in an aqueous medium.

Optionally, the at least one oxygenatable compound is selected from the group compirisng, but not limited to, styrene, indene, indole, benzothiophene, thioanisole, and naphthalene.

Preferably, the at least one oxygenatable compound is contacted with the polypeptide of the present invention until at least a portion of the corresponding oxygenated compound is produced in an isolatable amount. Preferably, the contacting step is carried out at about 3O⁰C for at least 30min, further preferably at least 20 min, still further preferably at least 10min.

Advantageously, the styrene monooxygenase polypeptides of the present invention are stereospecific. Optionally, the styrene monooxygenase polypeptide is enantiospecific. Further optionally, the styrene monooxygenase polypeptide is regiospecific. By "enantiospecific" is meant capable of synthesising a product having high enantiomeric purity, i.e. that a product comprising a single enantiomer is synthesised. However, it is understood that the product does not have to be exclusively enantiopure, but may be partially enantiopure, and that enantioselective capabilities also fall within the scope of this definition.

By "regiospecific" is meant being capable of synthesising a product in which one structural isomer is produced in favour of other isomers are also theoretically possible. However, it is understood that the given structural isomer product does not have to be exclusively synthesised, but other structural isomers may be synthesised, and that regioselective capabilities also fall within the scope of this definition.

By "stereospecific" is meant capable of synthesising products having the same atomic connectivity, wherein a product having a given atomic arrangement in space, or structural configuration, is synthesized in favour of other products of different atomic arrangement in space. However, it is understood that the product having a given atomic arrangement in space does not have to be exclusively synthesised, but products with other structural configuration may be synthesised, and that stereoselective capabilities also fall within the scope of this definition.

Preferably, the synthesised organic oxygenated compounds are homochiral. By homochiral is meant an enantiomeric species comprising only one enantiomeric form (R or S).

Alternatively, the synthesised oxygenated compounds are at least heterochiral. By heterochiral is meant an enantiomeric species wherein one enantiomeric form (R or S) is in excess of the other. Preferably, the heterochiral synthesised oxygenated compounds have an enantiomeric excess of at least 70%. Further preferably, the heterochiral synthesised oxygenated compounds have an enantiomeric excess of at least 90%, still further preferably 95%.

Preferably, the polynucleotide comprises fragments of the nucleic acid sequence. Optionally, the polypeptide comprises fragments of the amino acid sequence.

Preferably, fragments of one or both of the polynucleotide and polypeptide are of a length so as to retain at least part of the monooxygenase activity of the full-length sequence.

It is understood that, polypeptide variants or fragments thereof, comprising amino acid substitutions that preserve the structure and functional properties of the polypeptides described herein fall within the scope of the present invention.

It is also understood that, polynucleotide variants or fragments thereof, which are at least 75%, optionally at least 85%, further optionally at least 90% homologous to the polynucleotides described herein fall within the scope of the present invention. The present invention additionally includes within its scope the use of one or more other microorganisms in combination with one or more of the microorganisms described herein or microorganisms genetically modified to express a polypeptide having styrene monooxygenase activity of the present invention. The styrene monooxygenase is preferably maintained within a host microorganism that is contacted with the oxygenatable compound. However, it is also possible to isolate the polypeptide from the microorganism and use the isolated and purified polypeptide, if desired.

For the purposes of the present specification, the nucleic acid positions outlined herein are made with reference to the nucleic acid positions identified in SEQ ID NO 1. All nucleic acid positions, whether or not a polynucleotide insertion is present, are calculated from the nucleic acid position in the wild-type SMO, i.e., by ignoring the length of any such insertion.

Materials and Methods

Reagents

Indole, indene and styrene oxide were of analytical grade and purchased from Fluka (Buchs, Switzerland). Indene oxide was purchased from Advanced Synthesis Technologies, SA (San Ysidro, CA, USA). Styrene, ether, dimethylsulfoxide (DMSO) and acetonitrile were of GC grade and purchased from Sigma-Aldrich (Dublin, Ireland). Pfu Turbo DNA polymerase and T4 DNA ligase were purchased from Stratagene (La JoIIa, CA, USA). Restriction and DNA-modifying enzymes HinD\\\, Xba\, Nde\, were obtained from Invitrogen (Paisley, UK). Glucose, tryptone, yeast extract, casamino acids and other media components were purchased from Sigma-Aldrich (Dublin, Ireland). Carbenicillin, IPTG, other salts and reagents were purchased from Sigma (Dublin, Ireland). Oligonucleotide primers were obtained from Sigma Genosys (Dublin, Ireland). The QIAprep spin plasmid mini-prep kit, QIAEX Il gel purification kit, and QIAquick PCR purification kit were purchased from QIAGEN (Hilden, Germany).

Strains, plasmids, primers and growth conditions Table 1. Strains, plasmids and primers used in this study.

Strain, plasmid or primer Relevant characteristics Source or reference

Pseudomonas outida CA- Wild-type, styrene degrader O'Connor et a/.,

3 1995 Applied and

(NCIMB41162 of the Environmental

National Collection of Microbiology

Industrial, Marine and Food 61:544-548

Bacteria)

E.coli strains DH5α F^' , recA1 endA1, general cloning host Invitrogen

XLIO-GoId F^' , recA1, endA1, relA1, cloning host Stratagene

BL21(DE3) F^', ompT, high level expression of genes Novagen regulated by T7 promoter

Plasmids pBluescript Il KS expression under lac promoter , amp^R Stratagene (pBSKS) pBSKS-styABRO 1.8 kbp styAB fragment in pBSKS, amp^R This work pBSKS-styABR1 pBSKS-styABRO derivative, positive clone This work from the first round of mutagenesis, amp^R pBSKS-styABR2 pBSKS-styABR1 derivative, positive clone This work from the second round of mutagenesis, amp^R pBSKS-styABR3-5 pBSKS-styABR2 derivative, positive clone This work from the third round of mutagenesis, amp^R pBSKS-styABR3-10 pBSKS-styABR2 derivative, positive clone This work from the third round of mutagenesis, amp^R pBSKS-styABR3-11 pBSKS-styABR2 derivative, positive clone This work

R from the third round of mutagenesis, amp pRSET-B expression under T7 promoter, amp^R Invitrogen pRSET-styABRO pBSKS-styABRO derivative, 1.8 kbp styAB This work fragment in pRSET-B, amp^R pRSET-styABR1 pBSKS-styABR1 derivative, 1.8 kbp styAB This work fragment in pRSET-B, amp^R pRSET-styABR2 pBSKS-styABR2 derivative, 1.8 kbp styAB This work fragment in pRSET-B, amp^R pRSET-styABR3-5 pBSKS-styABR3-5 derivative, 1.8 kbp This work styAB fragment in pRSET-B, amp^R pRSET-styABR3-10 pBSKS-styABR3-10 derivative, 1.8 kbp This work styAB fragment in pRSET-B, amp^R pRSET-styABR3-11 pBSKS-styABR3-11 derivative, 1.8 kbp This work styAB fragment in pRSET-B, amp^R

Primers (5'-3')^a MCS F_seq AAGTAAAACGACGGCCAGTGAGCGCG SEQ ID NO 19 MCS R_seq GGAAACAGCTATGATCATGATTACGCCA SEQ ID NO 20 StyA F_seq CTATGTCCTTCTCGCCAGG SEQ ID NO 21 StyA R_seq CGAGCACCAGCGCTGTG SEQ ID NO 22 StyB F_seq GAACGTATCGGTCAGTGGTG SEQ ID NO 23 StyB R_seq CTCCGCTGGCCTTGACC SEQ ID NO 24 KSStyAB F_HinDIII CGCGAAGCTTAGGAGGAAGCCATGAAA SEQ ID NO 25 AAGCGTATGG KSStyAB R_Xbal CACATCTAGAGATCGGCACAGAAAGGC SEQ ID NO 26

CTC SETStyAB F_Ndel AGGTTGGCATATGAAAAGCGTATCGGTA SEQ ID NO 27

TTGTTG SETStyAB R_HinDIII ATCTTGAAGCTTCAATTCAGCGGCAACG SEQ ID NO 28

GGTTGCC a seq= primers used for sequencing, underlined are restriction nuclease recognition sites

For culture propagation, Luria-Bertani (LB) complex medium or M9 minimal medium was used as described by Sambrook et a/., 1989 (Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory). M9 medium was supplemented with 1 ml/l of US trace element solution (1 M hydrochloric acid and the following salts, per liter: 1.50 g of MnCI₂-4H₂O, 1.05 g of ZnSO₄, 0.30 g of H₃BO₃, 0.25 g of Na₂MoO₄-2H₂O, 0.15 g of CuCI₂-2H₂O, and 0.84 g of Na₂EDTA-2H₂O). When necessary, the medium was solidified by adding 1.5 % (w/v) bacteriological agar (Difco, Detroit, USA). Alternatively, M9^* mineral medium was used as described by Panke et a/., 1998 (Towards a biocatalyst for (S)-styrene oxide production: Characterisation of the styrene degradation pathway of Pseudomonas sp. Strain VLB 120. Applied & Environmental Microbiology 64:2032-2043), which was identical to M9 minimal medium except that it contained three times more phosphate salts to increase the buffer capacity and did not contain calcium chloride. Glucose was added as a carbon source to each mineral medium at a concentration of 2% (w/v) and casamino acids were added at a concentration of 0.5% (w/v). Carbenicillin (50 μg/ml) was routinely used to select for ampicillin resistance (amp^R). Stock cultures of all strains used in this study were maintained at - 8O⁰C in LB broth with glycerol (20%, v/v). Cultures were prepared for use in experiments by inoculation of cells from frozen stock culture into broth or onto agar plates, and incubated at 37⁰C. Cultures used to determine enzyme activities were grown at 30°C. Unless otherwise stated, cultures were induced by the addition of 1 mM (final concentration) IPTG.

DNA techniques and plasmid construction

Unless otherwise specified, standard recombinant DNA techniques were performed as described by Sambrook ef a/., 1989 (Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory). Small-scale isolation of plasmid DNA from E. coli was carried out by using the miniprep procedure (QIAGEN). Plasmids were transformed into E. coli electrocompetent cells using Gene Pulser Il (Bio-Rad, Hercules, USA) according to manufacturer's instructions. For the random mutagenesis experiments, E. coli XL-10 Gold ultracompetent cells were used and transformed according to the manufacturer's instructions (Stratagene, 2005).

The styAB fragment from P. putida CA-3 was amplified from chromosomal DNA template using KSStyAB F_HinDIII and KSStyAB R_Xbal primers (Table 1). PCR was carried out on a DNA Engine Thermal Cycler (Bio-Rad, Hercules, USA) at 1 min, 94°C; followed by 30 cycles of 30 s at 94°C; 30 s at 55°C; 2 mins at 72°C; and a final extension of 10 min at 72°C. The 1.8kbp fragment generated in the PCR was digested with H/πDIII and Xba\ and introduced into the corresponding sites of pBluescript KS to generate pBSKS-styABRO (Table 1). Similarly, all other recombinant vectors were generated using appropriate primers and restriction sites as described in Table 1. To verify plasmid constructs, DNA sequencing was conducted by GATC Biotech (Hamburg, Germany) using appropriate primers as described in Table 1. Sequence data was aligned and compared using Sequencher 4.7 using the default alignment settings (Gene Codes Corp, Mi, USA).

StyAB in vitro evolution

In vitro evolution of styAB was performed using a GeneMorph Il Random Mutagenesis kit (Stratagene, La JoIIa, USA) according to the manufacturer's instructions. Briefly, mutant megaprimer synthesis was carried out by error prone PCR amplification of styAB fragment from the pBSKS- styABO vector for the first round of mutagenesis. For all subsequent rounds, plasmid template for the megaprimer synthesis was chosen from the previous round of mutagenesis from the variants identified as positive by indigo production assay and the sequence verification. The megaprimers were synthesised by 30 cycles of PCR (as above) using IOOOng of purified plasmid template for Round 1 (low mutation frequency), 200ng for Round 2 (medium mutation frequency), and 100ng for Round 3 (high mutation frequency). The megaprimers generated during the PCR reaction, were subsequently used to amplify the rest of the plasmid using the high fidelity polymerase, thus generating the library of recombinant plasmids with introduced mutations within the styAB fragment. The resulting plasmids from each round of mutagenesis were used to transform XL-10 Gold ultracompetent cells, which were then plated on LB agar plates containing carbenicillin (50μg/ml), indole (1mM), and IPTG (1 mM) and grown for 16 h at 37⁰C.

Screening for styAB gene variants Libraries of variants generated by each round of directed evolution were screened for improved indigo production rates. Colonies grown on LB agar plates containing carbenicillin (50μg/ml), indole (1mM), and IPTG (1mM) were visually screened for the most rapid blue color (indigo) production. The single colonies chosen for further screening were grown in 200μl M9 broth supplemented with carbenicillin (50μg/ml), and IPTG (1mM) for 24h at 37⁰C in a 96-well microtitre plate. Each MT plate contained uninoculated wells as a negative control, while the parent strain (from the previous generation) was used as a positive control. Following 24h of growth, each well in the 96-well plate was monitored at 600nm to obtain the cell culture density before the MT plate was centrifuged (3000 x Sf) at 3O⁰C for 15 min, and the supernatant removed by vacuum aspiration. The pelleted cells were resuspended in 100μl of 5OmM potassium phosphate buffer (pH 7) containing 1 mM indole. Cell resuspensions were incubated for 1 to 4 h at 3O⁰C with shaking at 200rpm. The protocol for the spectrophotometric monitoring of indigo formation was adapted from that described by O'Connor et a/., 1998 (Indigo formation by microorganisms expressing styrene monooxygenase activity. Applied & Environmental Microbiology 63:4287-4291), to allow for easy screening in the plastic 96-well microtitre plates. Following incubation with indole 200μl of DMSO was added to each well, and incubated for 5min at room temperature to lyse the cell cultures and terminate the reaction. The assay plate was then centrifuged (3000 x g) in a benchtop centrifuge with swinging buckets adapted for MT plate for 10min and 200μl of the supernatant was transferred to a new 96-well plate. The absorbance reading at 610nm was determined for all wells. The negative control values were averaged and subtracted from the values obtained for each test well. Cultures showing increased indigo formation above the positive control were selected for further screening and confirmation of the increased activity. Confirmation of increased indigo production was attained by repeating the indigo assay in triplicate. Cultures exhibiting verified improved rates of indigo formation were grown in LB broth supplemented with carbenicillin and frozen as stock cultures. Recombinants with the highest rates of indigo formation were cultured for plasmid isolation and then sequenced (GATC Biotech, Hamburg, Germany). The recombinant with the greatest increase in indigo formation rate was chosen as the parental strain for the next round of directed evolution.

Whole cell biotransformations: Determination of substrate depletion and product formation rates by E. coli cells expressing styrene monooxygenase

SMO activity was determined by whole-cell assays as described by Panke ef a/., 1998 (Towards a biocatalyst for (S)-styrene oxide production: Characterisation of the styrene degradation pathway of Pseudomonas sp. Strain VLB120. Applied & Environmental Microbiology 64:2032-2043). E. coli XL- 10 Gold cells expressing styAB on pBluescript were used during the screening phase of the in vitro evolution. To enhance expression of SMO, the styAB genes were sub-cloned into pRSETB to generate a pRSETB-styAB construct and transformed into E. coli cells BL21(DE3) cells as described in Table 1. E. coli cells were grown as starter cultures (3ml) in M9* mineral medium (supplemented with glucose, casamino acids, carbenicillin, and US trace element solution) at 3O⁰C for 16h, and then inoculated into larger cultures (250ml, 0.1% inoculum) and grown in an identical medium at 3O⁰C with shaking at 200rpm. Once the culture had reached an optical density of 0.5 (600 nm; Helios Gamma UV-visible spectrophotometer (Thermo Scientific)), the cells were induced with 1mM lsopropyl β-D-1-thiogalactopyranoside (IPTG) and further incubated under the same conditions for 12-14h. The cells were then harvested by centrifugation at 300Ox g for 20min in a benchtop 5810R centrifuge (Eppendorf), and resuspended to a dry biomass concentration of 2.5g/l in 10OmM potassium phosphate buffer (pH 7.4) containing 1%(w/v) glucose. Aliquots of 2ml were distributed into 15ml Pyrex tubes and incubated horizontally on a rotary shaker at 200rpm. After 5min, the substrate was added to a final concentration of 1.5mM from a 3OmM stock solution in ethanol. The reaction continued for 45min at 3O⁰C (shaking at 200rpm) and was stopped at various time points (one vial per time point) by immediate addition of ice-cold ether (2ml) containing 0.1mM 1-dodecanol as an internal standard and incubating the sample on ice. After addition of saturating amounts of sodium chloride, the water phase was extracted by vigorous shaking for 5min at 30⁰C and the phases were separated by centrifugation at 3000* g for 5min at 4°C. The organic phase was analyzed by gas chromatography. Indole depletion was monitored as previously by Hollmann et a/., 2003 (Stereospecific biocatalytic epoxidation: The first example of direct regeneration of a FAD- dependent monooxygenase for catalysis. JACS 125:8209-8217). Indigo formation was monitored spectrophotometrically as described above.

Analytical procedures Gas chromatography (GC) was used for separation of the styrene, styrene oxide, indene and indene oxide. A Fison 8000 series GC equipped with an Agilent HP-1 capillary column (3Om₁ 0.25mm inner diameter, 0.25μm film thickness; J&W Scientific, USA) was used with splitless injection and hydrogen as the carrier gas. A temperature gradient of 50-180⁰C was implemented with an increment of 10°C/min. Detection was done using a flame ionisation detector. Alternatively, a Supelco Beta-DEX 120 column (30m, 0.25mm inner diameter, 0.25mm film thickness; Supelco, Buchs, Switzerland) was used with splitless injection and an isothermal oven temperature profile at 90⁰C for separation of styrene oxide enantiomers as described by Panke et a/., 1998 (Towards a biocatalyst for (S)-styrene oxide production: Characterisation of the styrene degradation pathway of Pseudomonas sp. Strain VLB120. Applied & Environmental Microbiology 64:2032-2043).

Compounds were identified by comparison of retention times with commercially available standards. HPLC analysis of indole depletion was performed using a Hypersil C-18 column ODS 5μ column (125 x 3 mm) (Hypersil, Runcorn, UK) and a Hewlett Packard HP1100 instrument equipped with an Agilent 1100 Series diode array detector. The samples were isocratically eluted using an acetonitrile and water mix (1 :1) at a flow rate of 0.5ml/min.

Protein expression analysis

The pRSET-derived expression constructs (Table 4) encoded hisδ: :styAB fusion as this would allow to rapidly assess protein expression through western blotting. To test the level of expression of the His tagged protein, E. coli BL21(DE3) cultures harboring pRSET-styAB constructs were grown and treated as described for the whole cell biotransformation experiments (cells harvested, washed in 50 mM phosphate buffer pH 7.0 and resuspended in 50 mM phosphate buffer pH 7 to a concentration of 0.5 g CDW/I). 1 ml of cell suspension was used for the western blot analysis. Cells (0.5 g CDW/I) were centrifuged and resuspended in 1 ml of BugBuster solution containing benzonase (2 U/ml; Novagen) and incubated at 3O⁰C for 15 tmin with shaking (200 rpm). A 15 μl quantity of the whole-cell lysate from each sample containing approximately 0.35 mg ml-1 of protein was separated by SDS- polyacrylamide (10 %, w/v) gel electrophoresis and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Biosciences). The membrane was probed with Penta-His HRP conjugate antibody (Qiagen, Hilden, Germany). The blot was developed using ImmobilonTM Western chemiluminescent HRP substrate according to the manufacturer's protocol (Millipore, Ireland), lmmunoluminescence was detected and evaluated using chemiluminescence compatible FluorChemTM imaging system equipped with AlphaEase FC2 software (Alpha Innotech, San Leandro, USA).

Brief Description of the Drawings

Figure 1 is a diagrammatic representation of substrates and products of SMO biotransformations; Figure 2 is a schematic diagram depicting the Pseudomonas putida styrene monooxygenase variants generated by successive rounds of in vitro directed evolution; Figure 3 is a plot depicting indigo formation by E. coli BL21(DE3) cells expressing StyAB wildtype, and selected polypeptide variants; Figure 4 is a plot depicting styrene depletion (A) and styrene oxide formation (B) over time by E. coli BL21 (DE3) cells expressing wildtype StyAB, and selected polypeptide variants; Figure 5 is a plot depicting indene depletion (A) and indene oxide formation (B) over time by E. coli BL21 (DE3) cells expressing StyAB wildtype, and selected polypeptide variants; and Figure 6 is a Western Blot of cell lysates from E.coli BL21(DE3) cells expressing StyAB wildtype, and selected polypeptide variants. Examples

The following examples are given, with reference to the accompanying drawings, as illustrative of the present invention.

Example 1 : Identification of mutations in SMO variants

Using in vitro evolution by error-prone PCR, mutations in the P. putida CA-3 styAB genes were generated. A mutant library of the 1.8-kb styAB gene was created using GeneMorph Il EZCIone random mutagenesis kit as described herein. E. coli XL-10 Gold cells expressing styrene monooxygenase on pBluescript (pBSKSstyAB) were using during the screening phase of in vitro evolution. The mutant libraries expressed in E. coli were screened for increased indigo formation. A schematic representation of the changes to styAB through directed evolution is provided in Figure 2. Sequence data shows that each round of in vitro evolution resulted in 2-3 amino acid substitutions compared to the P.putida CA-3 wild-type styAB sequence, designated RO, and each of the substitutions was carried forward for further rounds of mutagenesis without reversion.

In Round 1 of the random mutagenesis, two mutations (F88L and K150R - positions within styA itself) occurred in the styA sequence, designated R1. Sequencing of the R1 variant also revealed the N150S substitution within the reductase styB.

During the second round of in vitro mutagenesis, an insertion of 8bp occurred near the C-terminal region of styA (at nucleotide position 1242 - i.e. immediately after nucleotide position 1241). This insertion was a repeat of the ATTGCNNN (N=A₁T₁G₁C) motif that occurs three times in this region in the wild-type gene (RO). This resulted in a change of the reading frame, and subsequently in a protein that is 19 amino acids (aa) longer. In addition to the 19 aa tail, a substitution A415L also occurred within styA. The frameshift did not affect the start codon or reading frame of styB. A substitution E124D was introduced within styB in the second round of mutagenesis.

The first mutation in StyA in the Round 3 variant R3-11 (R87C) occurs at the aa residue just preceding the mutation in variant R1 (F88L), suggesting that this may be a hotspot for mutation. The R3-11 variant contains the substitution V303I in the amino acid just following the GDX₆P, a conserved motif that has been shown to have dual function in FAD/NAD(P)H binding in flavoprotein hydroxylases. The variants R3-5 and R3-10 both contain mutations in styA, each of which occur in the extra N-terminal 'tail' added by the frameshift from Round 2. However, other mutations also occur in these variants, Figure 2, and so the effect of the mutations in the tail cannot be determined. Known monooxygenases and hydroxylases generally do not share a high degree of sequence similarity. However, the conserved glycine residues of the sequence GXGXXG (positions 9 to 14) within the N-terminal region of StyA, did not undergo mutational changes. All amino acid positions in this Example refer to the translated polypeptide.

Example 2: Whole cell biotransformation, and rates of substrate depletion and product formation

The SMO wild type and variant styAB genes were subcloned into the high expression vector pRSETB and transformed into E. coli BL21(DE3) cells (see Example 5). A single round of error- prone PCR generated a sty AB variant with 3.4-fold improvement in the rate of indole depletion and 2.5-fold improvement in the rate of indigo formation, Figure 3 and Table 1 , variant R1. The ability of variant R1 to consume styrene and indene increases 3.2- and 1.6-fold respectively compared to cells expressing the wild-type enzyme.

Table 2. Initial rates (μmol/min/g CDW) of substrate utilization and product formation by E. coli BL21 (DE3) cells expressing wild type and variants of styrene monooxygenase (pRSET- styAB)

RO 53.21 ± 0.05 21.28 ± 0 .07 46.62 ± 0 .12 36.83 ± 0.08 12.83 ± 5.86 ± 0.02

0.07

R1 169.44 ± 74.83 ± 0 .1 1 74.01 ± 0 .08 49.33 ± 0.03 44.11 ± 14.56 ±

0.09 0.09 0.06

R2 52.01 ± 0.03 20.46 ± 0 .02 45.59 ± 0 .04 29.66 ± 0.07 44.55 ± 14.92 ±

0.11 0.09

R3-5 336.10 ± 181.57 ± 370.26 ± 326.56 ± 32.04 ± 12.03 ±

0.13 0.08 0.15 0.13 0.04 0.04

R3-10 420.70 ± 266.41 ± 545.65 ± 433.87 + 21.17 ± 9.68 ± 0.09

0.42 0.20 0.24 0.45 0.07

R3-11 418.11 ± 252.10 ± 205.82 ± 174.93 ± 25.74 ± 12.39 +

0.21 0.18 0.18 0.22 0.08 0.07

A further round of in vitro evolution generated an E. coli variant (variant R2) that was visibly darker on plates and exhibited a 1.2-fold higher rate of indigo formation compared to variant R1 in MT plate assays, Figure 3. However, in the test tube biotransformations used to compare all variants, the rate of the reaction was the same as variant R1 , Table 2. Thus the data arising from experiments with variant R2 suggest that the alteration in styAB did not affect the activity of the SMO towards indole, but negatively affected activity towards styrene and indene. Due to the decrease in the ability of this variant to consume styrene, Figure 4, and indene, Figure 5, as also seen in Table 2, and the unusual 8bp insertion in the DNA sequence, it was decided to bring this variant forward for a third round of in vitro evolution.

A third round of error prone PCR generated variants of styAB (R3) with decreased indigo formation rates in test tube assays despite the selection of variants with apparently darker (blue) colonies on agar plates, Figure 3 and Table 2. This observation indicates a limitation in the agar plate screen and suggests that a thorough liquid-based microtitre plate screen is required to find variants that are improved for indigo formation in liquid culture. The rate of indigo formation with the evolved SMO (variant R1 and R2) is 2.0-fold higher than that previously reported for P. putida CA-3 by O'Connor et a/., 1998 (Indigo formation by microorganisms expressing styrene monooxygenase activity. Applied & Environmental Microbiology 63:4287 -4291) and O'Connor & Hartmans, 1998 (Indigo formation by aromatic hydrocarbon-degrading bacteria. Biotechnology Letters 20:219-223). The rate of indigo formation with the evolved SMO (variant R1 and R2) is also 11.5-fold higher than SMO from

Pseudomonas fluorescens ST expressed in E. coli JM109, as reported by Beltrametti et al., 1997 (Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Applied and Environmental Microbiology 63: 2232-2239). Furthermore whole cells expressing styAB R2 variant consume indole at a rate close to that for styrene consumption by the wild-type enzyme.

The rate of conversion of styrene, Figure 4, and indene, Figure 5, to their respective oxides was dramatically increased in the three R3 variants, indicating that the mutations having a negative effect on indole transformation to indigo, affected styrene and indene transformation positively. E. coli cells expressing either SMO variant R3-10 or R3-11 utilised styrene at a rate 8-fold higher than cells expressing wild-type enzyme (RO). Variant R3-10 exhibited the highest rate of indene utilization, Table 2, which is 11.7-fold higher than the rate exhibited by cells expressing the wild-type enzyme.

The rate of styrene consumption is 1.14-fold higher than indene consumption by wild-type StyAB. However, cells expressing the variant R3-10 have a 1.3-fold higher rate of reaction with indene compared to styrene, and thus the evolution of this enzyme is more towards indene than styrene epoxidation. Conversely, variant R3-11 has evolved towards styrene oxidation with a 2-fold higher rate of styrene consumption over indene, as seen in Table 2. Both variants R3-10 and R3-11 show disimproved activity towards indigo. The data shows that the screening strategy (selection with indigo formation) does not direct the evolution towards styrene or indene logically but represents a random event.

Example 3: Styrene and indene biotransformation progress, stoichiometry and enantiomeric excess In general, a linear relationship between alkene (styrene and indene) consumption and epoxide formation occurs for E.coli cells expressing wild-type and variants of SMO (styAB) over a period of between 5- and 60-minutes, depending upon the variant used in the biotransformation, Figures 4 and 5.

The best variant SMO (R3-10) consumed 1.4mM styrene, which was completely consumed within 15min. The rate of styrene consumption dropped sharply after 5min, when the concentration of styrene fell below 0.11mM. The maximum amount of styrene oxide also appeared after 5min in these biotransformations (Figure 4). In styrene biotransformations, (S)-styrene oxide was the only enantiomer detected by chiral GC analysis. This is in keeping with previous reports for SMO expressed in E.coli, which show an ee of greater than 99%. In all biotransformations with styrene, approximately 65% of the styrene transformed appeared as styrene oxide (66.25% (RO), 69.7% (R1), 65% (R2), 64.5% (R3-10)). Another product, phenylacetaldehyde, was also detected by GC analysis of biotransformations. 2-phenylethanol has previously been reported by Panke et a/., 1998 (Towards a biocatalyst for (S)-styrene oxide production: Characterisation of the styrene degradation pathway of Pseudomonas sp. Strain VLB120. Applied & Environmental Microbiology 64:2032-2043), to be a byproduct of styrene transformation by whole cells expressing SMO.

The transformation of indene by cells expressing wild-type and variants of styAB produced a single product indene oxide. Between 88.2% and 94.5% of the indene transformed appeared as indene oxide in biotransformations catalysed by the E. coli cells expressing wild-type and variants of StyAB. In the best variant (R3-10), the rate of indene transformation is fastest within the first 5min, and slows dramatically after this time point, with indene oxide appearance following the same pattern (Figure 5). (1 S,2R)-\ndene oxide is the predominant enantiomer formed from indene biotransformation with an enantiomeric excess (ee) of 97%. A similar ee was reported by Schmid et a/., 2001 (Integrated biocatalytic synthesis on gram scale: the highly enantioselective preparation of chiral oxiranes with styrene monooxygenase. Advanced synthesis and catalysis 343: 732-737), using E coli JM101 (pSPZ10) expressing SMO from P. putida VLB120. A 35-fold higher rate of indene oxide formation than that reported for SMO from P. putida VLB 120 expressed in E coli cells is disclosed herein.

Despite the limitations of the indole to indigo screen for directing the evolution of SMO towards specific alkene substrates, whole cell biocatalysts, expressing SMO variants that oxygenate styrene and indene at a rate 8-fold and 11.7-fold higher than the same cells expressing wild-type enzyme, have been generated.

Example 4: BENZOTHIOPHENE, THIOANISOLE and NAPHTHALENE biotransformation progress, stoichiometry and enantiomeric excess

Benzothiophene, thioanisole and naphthalene were also transformed by E.coli BL21 (DE3) strains expressing wild-type and variants of SMO. Whole cell biotransformations were carried out as described previously herein. Table 3. Initial rates (μmol/min/g CDW) of substrate utilization and product formation by E. coli BL21 (DE3) cells expressing wildtype and variants of styrene monooxygenase (pRSET- styAB)

Variant Benzothiophene Thioanisole Naphthalene consumption consumption consumption

RO 8.8 ± 0.5 83.3 ± 0.9 0.012 ± 0.003

R1 21.1 ± 0.7 38.2 ± 0.3 0.018 ± 0.002

R2 61.2 ± 0.7 54.4 ± 0.4 0.02 ± 0.004

R3-10 88.6 ± 0.3 0 0.043 ± 0.006

R3-11 89.0 ± 0.8 98.1 ± 0.4 0.044 ± 0.005

The rate of conversion of benzothiophene was increased 10-fold in the two R3 variants, indicating that the mutations that had a negative effect on indole-to-indigo transformation affected styrene and indene transformation positively. Variants R3-10 and R3-11 utilised styrene at a rate 8-fold higher than cells expressing wild-type enzyme (RO). Thioanisole was utilised by cells expressing wild-type SMO (RO) at 85% of the rate exhibited by variant R3-11 , Table 2. Variant R3-10 was not able to transform thioanisole despite being much improved towards styrene and indene.

Naphthalene was utilised at much lower rate in comparison to all other substrates (in an order of 1000-fold). However, the variants are improved compared to wild-type enzyme and thus in vitro mutagenesis has generated variants with the improved activity towards naphthalene (3.6-fold improvement for R3 variant compared to RO).

Example 5: expression level of SMO wild type and variants in E. coli BL21(DE3) cells.

While screening for indigo formation allowed for selection of mutants with higher styrene monooxygenase activity, an aspect of the present invention also provides whole cell biocatalysts with enhanced epoxide-synthesizing capability. To maximize the expression of styrene monooxygenase, the styAB genes were subcloned into the high expression vector pRSETB and transformed into E. coli BL21 (DE3) cells (Table 1 ). The expression level of SMO wild type and variants in E. coli BL21(DE3) cells were compared using SDS-PAGE analysis of the cell lysates and the His tag antibody specific for histidine tagged proteins (Figure 6). Using chemiluminescence detection, a single band was observed in the cell lysates. Using densitometry software, wild type (RO) expression levels were found to be slightly higher (2-7 %) in comparison to the variants (Figure 6).

Discussion

The present invention provides new styrene monooxygenase variants that oxidize styrene and indene at rates 8-fold and 11.7-fold higher than the same cells expressing the wild type enzyme. The in vitro evolution of styAB from P. putida CA-3 has generated mutations in both StyA and StyB. The C-terminal extended 'tail' that first appears in R2 is maintained roughout all of the R3 variants. Without being bound by theory, it is postulated that the tail may help to maintain a closer interaction between the StyA and StyB proteins, or possibly enable a more efficient electron/proton transfer. However, previous reports have shown that StyA and StyB do not interact and thus the promotion of more efficient electron/proton transfer is more likely. Other amino acid changes occur throughout StyAB such as A179T and K245N in variant R3-10, which exhibits a higher activity towards indene compared to styrene but it is difficult to predict the exact effect of these amino acid changes as they appear at apparently random locations. Nevertheless, previous studies have found that a large number of mutations occur outside the active site of enzymes that have been improved through random mutagenesis - indicating that amino acid changes, that affect enzyme activity, are difficult to model and predict.

Round 3 variants R3-10 and R3-11 utilized styrene at a rate 8-fold higher than cells expressing wild type enzyme (RO). Variant R3-10 also exhibited the highest rate of indene utilization (Table 2) which is 11.7-fold higher than that exhibited by cells expressing the wild type enzyme. Variant R3-10 generated herein exhibit a 35-fold higher rate of indene oxide formation than that reported for SMO from P. putida VLB120 expressed in E. coli cells (12.5 μmol/min/g CDW). Thus, the rate of styrene oxide and indene oxide formation by E. coli cells expressing P. putida CA-3 styrene monooxygenase variants of the present invention is the highest reported for any biocatalyst. (S)-styrene oxide was the only enantiomer detected by chiral GC analysis using wild type and variants of styrene monooxygenase. In conclusion, the present invention provides a number of improved variants exhibiting the highest known rate of (S)-styrene epoxide and (1S-2R) indene epoxide production by a biocatalyst.

Claims

1. An isolated polynucleotide derived from Pseudomonas putida, and encoding a polypeptide having styrene monooxygenase activity, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO 1 , and at least one nucleic acid substitution selected from the group comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751 ; with the proviso that: a) if there is a single nucleic acid substitution at position 262, the nucleic acid is not a T, b) if there is a single nucleic acid substitution at position 449, the nucleic acid is not an A, and c) if there is a single nucleic acid substitution at position 1751 , the nucleic acid is not an A.

2. An isolated polynucleotide according to Claim 1 , wherein the nucleic acid substitution at position 262 is selected from A, C, and G.

3. An isolated polynucleotide according to Claim 1 or 2, wherein the nucleic acid substitution at position 449 is selected from C, G, and T.

4. An isolated polynucleotide according to any one of Claims 1-3, wherein the nucleic acid substitution at position 1751 is selected from C, G, and T.

5. An isolated polynucleotide according to any preceding claim, and comprising two or more of the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751.

6. An isolated polynucleotide according to any preceding claim, and comprising at least the three nucleic acid substitutions comprising, but not limited to, a nucleic acid substitution at position 262, a nucleic acid substitution at position 449, and a nucleic acid substitution at position 1751.

7. An isolated polynucleotide according to any preceding claim, wherein the nucleic acid at position 262 is substituted for a C.

8. An isolated polynucleotide according to any preceding claim, wherein the nucleic acid at position 449 is substituted for a G.

9. An isolated polynucleotide according to any preceding claim, wherein the nucleic acid at position 1751 is substituted for a G.

10. An isolated polynucleotide according to any preceding claim, and comprising the nucleic acid sequence of SEQ ID NO 4.

11. An isolated polynucleotide according to any preceding claim, and further comprising a nucleic acid modification after position 1241.

12. An isolated polynucleotide according to Claim 11 , wherein the nucleic acid modification comprises a polynucleotide insertion after, optionally directly after, position 1241.

13. An isolated polynucleotide according to Claim 12, wherein the polynucleotide insertion after, optionally directly after, position 1241 comprises (3x-1) nucleic acids, wherein x is an integer, greater than or equal to 1.

14. An isolated polynucleotide according to Claim 12 or 13, wherein the polynucleotide insertion after, optionally directly after, position 1241 is a nucleic acid sequence comprising ATTGC.

15. An isolated polynucleotide according to any one of Claims 12-14, wherein the polynucleotide insertion is a nucleic acid sequence comprising NNNATTGC, wherein each N is independently selected from the group consisting of A, T, G, and C.

16. An isolated polynucleotide according to any one of Claims 12-14, wherein the polynucleotide insertion is a nucleic acid sequence comprising ATTGCNNN, wherein each N is independently selected from the group consisting of A, T, G, and C.

17. An isolated polynucleotide according to any one of Claims 12-15, wherein the polynucleotide insertion comprises a nucleic acid sequence comprising ACTATTGC.

18. An isolated polynucleotide according to any preceding claim, and further comprising a nucleic acid substitution at position 1674.

19. An isolated polynucleotide according to Claim 18, wherein the nucleic acid at position 1674 is substituted with a nucleic acid selected from A, T, and C.

20. An isolated polynucleotide according to Claim 18 or 19, wherein the nucleic acid at position 1674 is substituted with a T.

21. An isolated polynucleotide according to any one of Claims 18-20, and comprising the nucleic acid sequence of SEQ ID NO 7.

22. An isolated polynucleotide according to any preceding claim, and further comprising a nucleic acid substitution at position 114.

23. An isolated polynucleotide according to Claim 22, wherein the nucleic acid at position 114 is substituted for an A.

24. An isolated polynucleotide according to any preceding claim, and further comprising at least one nucleic acid substitution selected from the group comprising, but not limited to, a nucleic acid substitution at position 27, a nucleic acid substitution at position 147, a nucleic acid substitution at position 192, a nucleic acid substitution at position 259, a nucleic acid substitution at position 465, a nucleic acid substitution at position 535, a nucleic acid substitution at position 702, a nucleic acid substitution at position 733, a nucleic acid substitution at position 907, a nucleic acid substitution at position 1008, a nucleic acid substitution at position 1250, a nucleic acid substitution at position 1261 , a nucleic acid substitution at position 1270, and a nucleic acid substitution at position 1524.

25. An isolated polynucleotide according to Claim 24, and comprising the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 27, a nucleic acid substitution at position 114, a nucleic acid substitution at position 733, and a nucleic acid substitution at position 1250.

26. An isolated polynucleotide according to Claim 24, and comprising the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 114, a nucleic acid substitution at position 147, a nucleic acid substitution at position 192, a nucleic acid substitution at position 465, a nucleic acid substitution at position 535, a nucleic acid substitution at position 702, a nucleic acid substitution at position 1261 , and a nucleic acid substitution at position 1270.

27. An isolated polynucleotide according to Claim 24, and comprising the nucleic acid substitutions selected from the group comprising, but not limited to, a nucleic acid substitution at position 259, a nucleic acid substitution at position 907, a nucleic acid substitution at position 1008, and a nucleic acid substitution at position 1524.

28. An isolated polynucleotide according to any preceding claim, and further comprising at least one nucleic acid substitution adjacent or at position 907.

29. An isolated polynucleotide according to Claim 24 or 25, and comprising the nucleic acid sequence of SEQ ID NO 10.

30. An isolated polynucleotide according to Claim 24 or 26, and comprising the nucleic acid sequence of SEQ ID NO 13.

31. An isolated polynucleotide according to Claim 24 or 27, and comprising the nucleic acid sequence of SEQ ID NO 16.

32. An isolated polypeptide having styrene monooxygenase activity, wherein the isolated polypeptide comprises at least a first component and a second component.

33. An isolated polypeptide according to Claim 32, wherein the first component comprises an amino acid sequence selected from the group comprising, but not limited to, SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 11 , SEQ ID NO 14, and SEQ ID NO 17; and the second component comprises an amino acid sequence selected from the group comprising, but not limited to, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 15, and SEQ ID NO 18; with the proviso that if the first component comprises the amino acid sequence of SEQ ID NO 2, then the second component does not comprise the amino acid sequence of SEQ ID NO 3.

34. An isolated polypeptide according to Claim 32 or 33, wherein the pair of first and second components comprise the respective amino acid sequences selected from the group comprising, but not limited to, SEQ ID NO 5 and SEQ ID NO 6; SEQ ID NO 8 and SEQ ID NO 9; SEQ ID NO 11 and SEQ ID NO 12; SEQ ID NO 14 and SEQ ID NO 15; and SEQ ID NO 17 and SEQ ID NO 18.

35. An isolated polypeptide according to any one of Claims 32-34, wherein the isolated polypeptide comprises an amino acid sequence encoded by a nucleic acid sequence according to any one of Claims 1-31.

36. A recombinant expression vector comprising an insert having a nucleic acid sequence according to any one of Claims 1-31.

37. A host cell, which is stably transformed using a recombinant expression vector according to Claim 36.

38. A method for the biocatalytic synthesis of oxygenated compounds, the method comprising the steps of providing an oxygen source, contacting at least one oxygenatable compound with a polypeptide, under reaction conditions sufficient to form the oxygenated compound, wherein the polypeptide is a polypeptide according to any one of Claims 32-35.

39. A method according to Claim 38, wherein the at least one oxygenatable compound is, optionally, a cyclic or heterocyclic ring or fused ring system, and the oxygenatable moiety forms part of the ring or ring system, or can be attached thereto.

40. A method according to Claim 38 or 39, wherein the at least one oxygenatable compound is an unsaturated compound.

41. A method according to Claim 40, wherein the unsaturated compound is selected from an alkene, an alkyne, aromatic rings, aromatic ring systems, and heterocyclic ring systems.

42. A method according to any one of Claims 38-41 , wherein the at least one oxygenatable compound comprises cyclic or heterocyclic rings, or fused ring systems, containing an oxygenatable moiety, such as a thioether moiety and/or double or triple bond, either within the cyclic or heterocyclic ring, or fused ring system, or attached thereto.

43. A method according to Claim 42, wherein a ring, or one ring of the fused ring system is a phenyl ring.

44. A method according to any one of Claims 38-43, wherein the at least one oxygenatable compound is selected from the group comprising, but not limited to, styrene, indene, indole, benzothiophene, thioanisole, and naphthalene.

45. A method according to any one of Claims 38-44, wherein the at least one oxygenatable compound is contacted with the polypeptide in an aqueous medium.

46. A method according to any one of Claims 38-45, wherein the the contacting step is carried out at about 3O⁰C for at least 30min, further preferably at least 20 min, still further preferably at least 10min.