(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 μmol/min/mg, ten times of that of the wild-type. Its kcat/Km increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.

The synthesis of optically pure secondary epoxy alcohols from racemic allylic alcohols using a single whole-cell biocatalyst of recombinant Escherichia coli coexpressing three oxidoreductases is described. The cascade involves the concurrent action of a styrene monooxygenase that catalyzes the formation of the chiral epoxy group, and two alcohol dehydrogenases that fulfil the epimerisation of the hydroxy group. Two sets of alcohol dehydrogenases were each applied to couple with styrene monooxygenase in order to realize the epimerisation in a stereo-complementary manner. Excellent enantio- and diastereo-selectivities were achieved for most of the 12 substrates.

•CYP108N7 was characterized as the first reported CYP108N subfamily member.•CYP108N7 accepted surrogate redox partners.•CYP108N7 was expressed together with redox partners and GDH in E.coli.•CYP108N7 catalyzed epoxidation, hydroxylation, demethylation and dehalogenation.•CYP108N7 catalyzed asymmetric sulfoxidation with excellent stereoselectivity.Cytochrome P450 enzymes are versatile biocatalysts with great potential in biotechnology. A new bacterial P450 was identified from the genome of Rhodococcus wratislaviensis NBRC 100605 and annotated as CYP108N7. The enzyme accepted the ferredoxin and ferredoxin reductase from spinach as surrogate redox partners for improved electron transfer efficiency. It was heterologous expressed in Escherichia coli together with the redox partners and a glucose dehydrogenase which supplied the reduced cofactor NADPH. The resulting whole-cell biocatalyst catalyzed a variety of reactions including sulfoxidation, epoxidation, hydroxylation, demethylation and dehalogenation. Remarkable stereoselectivity was observed in asymmetric sulfoxidation reaction, which could deliver chiral sulfoxides with >99% ee from thioanisole and derivatives.

•Ketoreductases READH and ChKRED20 were applied in asymmetric bioreduction.•Saturated N-heterocyclic ketones with a five- to seven-membered ring were reduced.•Stereo-complementary reduction was achieved with excellent selectivity.•Isopropanol was used as both the ultimate reducing agent and co-solvent.The asymmetric bioreduction of several saturated N-heterocyclic ketones is demonstrated in a stereo-complementary fashion using the ketoreductases READH and ChKRED20 for the production of (S)- and (R)-alcohols, respectively. The reaction accepts substrates with a five-, six- or seven-membered ring, and exhibits excellent stereoselectivity when using 2-propanol as both the ultimate reducing agent and cosolvent, achieve >99% ee in the majority of cases for both enantiomers.

Efficient asymmetric bio-epoxidation of electron-deficient α,β-unsaturated ketones was realized via a tandem reduction-epoxidation-dehydrogenation cascade, which proceeds in a switchable manner to afford either chiral epoxy ketones or allylic epoxy alcohols with up to >99% yield and >99%ee.

Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of “blockbuster” drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.

Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed a one-pot two-step enzymatic cascade using prochiral α,β-unsaturated ketones as the substrates. An S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de > 99%) were achieved for the majority of the substrates, and product yields reached up to >99%.

A putative enoate reductase, Achr-OYE4, was mined from the genome of Achromobacter sp. JA81, expressed in Escherichia coli, and was characterized. Sequence analysis and spectral properties indicated that Achr-OYE4 is a typical flavin mononucleotide-dependent protein; it preferred NADH over NADPH as a cofactor. The heterologously expressed protein displayed good activity and excellent stereoselectivity toward some activated alkenes in the presence of NADH, NADPH, or their recycling systems. The glucose dehydrogenase-based recycling system yielded the best results in most cases, with a product yield of up to 99 % and enantiopurity of >99 % ee. Achr-OYE4 is an important addition to the asymmetric reduction reservoir as an “old yellow enzyme” from Achromobacter.

The strain Achromobacter sp. JA81, which produced enoate reductase, was applied in the asymmetric reduction of activated alkenes. The strain could catalyze the bioreduction of alkenes to form enantiopure (R)-β-aryl-β-cyano-propanoic acids, a precursor of (R)-γ-amino butyric acids, including the pharmaceutically active enantiomer of the chiral drug (R)-baclofen with excellent enantioselectivity. It could catalyze as well the stereoselective bioreduction of other activated alkenes such as cyclic imides, β-nitro acrylates, and nitro-alkenes with up to >99 % ee and >99 % conversion. The draft genome sequencing of JA81 revealed six putative old yellow enzyme homologies, and the transcription of one of them, Achr-OYE3, was detected using reverse transcription polymerase chain reaction. The recombinant Escherichia coli expressing Achr-OYE3 displayed enoate reductase activity toward (Z)-3-cyano-3-phenyl-propenoic acid (2a).

Enantiomerically enriched glycidol derivatives with contiguous stereogenic centers were obtained in a highly diastereo- and enantio-selective epoxidation catalyzed with the styrene monooxygenase StyAB2.

Recombinant Escherichia coli expressing a styrene monooxygenase, StyAB2, from Pseudomonas sp. LQ26 was applied to synthesize a range of chiral epoxides from conjugated styrene derivatives with excellent (>99%) enantioselectivity in most cases. The substrate preference was studied with a special focus on the steric effect of α- and β-substituents.(S)-Styrene oxideC8H8OEe >99%[α]D25=+32.1 (c 1.02, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(2S,3S)-2-Methyl-3-phenyloxiraneC9H10OEe >99%[α]D25=-41.8 (c 1.00, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (2S,3S)(S)-2-Methyl-2-phenyloxiraneC9H10OEe >99%[α]D25=+16 (c 0.20, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(S)-2,2-Dimethyl-3-phenyloxiraneC10H12OEe >99%[α]D25=-23.6 (c 0.85, benzene)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(2S,3S)-2-Phenyl-3-vinyloxiraneC10H10OEe 96%[α]D25=-42.3 (c 0.35, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (2S,3S)(S)-2-Ethyl-2-phenyloxiraneC10H12OEe >99%[α]D25=+23.1 (c 0.34, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(2S,3S)-2-Isopropyl-3-phenyloxiraneC11H14OEe >99%[α]D25=-62.4 (c 0.25, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (2S,3S)(S)-2-Phenyl-2-propyloxiraneC11H14OEe >99%[α]D25=+32.3 (c 0.26, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(2S,3S)-3-Phenyloxiran-2-yl methanolC9H10O2Ee >99%[α]D25=-48 (c 0.3, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (2S,3S)(1S,2S)-1-Phenylcyclohexene oxideC12H14OEe 65%[α]D25=-30.9 (c 1.01, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (1S,2S)(S)-2-(Naphthalen-2-yl)oxiraneC12H10OEe >99%[α]D25=+17 (c 0.72, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)(S)-4-(Oxiran-2-yl)-2, 3-dihydrobenzofuranC10H10O2Ee >99%[α]D25=+40.8 (c 1.0, CHCl3)Source of chirality: enzymatic catalysisAbsolute configuration: (S)

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.

To elucidate the effects of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s (P450) in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold, respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperones GroEL/ES, the expression level increased up to 14-fold for P450 27C1, and 3- to 5-fold for P450s 2B1, 2S1, and 2W1. Simultaneous application of these two techniques resulted in synergetic effects.

•A novel “thermophilic-like” Old Yellow Enzyme was mined from genome data.•The half-life of thermal inactivation of Chr-OYE3 was 233.5 h at 35 °C.•The midpoint for thermal inactivation of Chr-OYE3 was 70 °C.•Chr-OYE3 showed high resistance toward various organic solvents.•Chr-OYE3 catalyzed the reduction of variety of activated alkenes.A putative Old Yellow Enzyme (OYE) homologue, Chr-OYE3, was identified from the genome of Chryseobacterium sp. CA49 as a new member of the “thermophilic-like” OYE subfamily. Chr-OYE3 is most closely related to YqjM from Bacillus subtilis with 51% identity. The heterologously expressed enzyme adopted two oligomeric states in solutions, dimers and tetramers. It can reduce a spectrum of activated alkenes, and displayed increased thermal resistance with half-lives of thermal inactivation (t1/2) of 233.5 and 21.8 h at 35 °C and 55 °C, respectively. The enzyme also exhibited high resistance toward various organic solvents, especially water-miscible solvents with higher polarity, such as ethylene glycol and DMSO, which would facilitate its application in biocatalysis where such reaction conditions are often encountered.Download full-size image

Enantiopure epoxides are remarkably versatile intermediates for the synthesis of numerous biologically active targets, to which considerable efforts have been devoted either chemically or biologically during the past few decades. This review will emphasize the application of biocatalysis as an efficient alternative that complements conventional chemical reactions, with a special focus on the epoxidation reactions catalyzed with monooxygenases and chloroperoxidases and the hydrolytic kinetic resolution catalyzed with epoxide hydrolases. Their scopes and limitations will be elaborately discussed as compared with their chemical counterparts. These biocatalytic approaches have not only provided environmentally friendly alternatives, but also displayed advantages for certain types of enantiopure epoxides, and could serve as potential tools for synthetic chemists.Graphical abstract.Download full-size imageHighlights► The review summarizes several biocatalytic approaches that lead to chiral epoxides with high enantio-purity. ► The scopes and limitations of these biocatalytic approaches are discussed as compared with classic chemo-catalysis. ► Biocatalytic approaches display advantages over chemo-catalysis for some types of enantiopure epoxides. ► Biocatalytic approaches can serve as an alternative to classic methods for synthetic chemists to acquire chiral epoxides.

•Ketoreductase ChKRED15 catalyzed the bioreduction of five duloxetine intermediates.•(S)-Alcohols were achieved with >99% ee for all five intermediates.•The S12G mutant was constructed to recover the G-rich motif.•The S12G mutant displayed improved activity and stability.•Up to 50 g substrate/l could be completely reduced within 24 h.The blockbuster antidepressant drug duloxetine contains one stereo-center derived from chiral alcohol intermediates. The stereoselective bioreductive production of five of such intermediates could be achieved using the recombinant ketoreductase ChKRED15, yielding the enantiopure (S)-alcohols with >99% ee. Sequence alignment indicated that ChKRED15 lacks the conserved G-rich motif, which was then amended by a single mutation of S12G. The resulting S12G mutant displayed significantly improved catalytic activity and protein stability. When coupled with a cofactor recycling system, the S12G mutant was able to catalyze the complete conversion of ethyl 3-oxo-3-(thiophen-2-yl)propanoate within 6 h and N-methyl-3-oxo-3-(thiophen-2-yl)propanamide within 24 h at substrate concentrations of 10 and 50 g/l, respectively, without the compromise of enantioselectivity.Download full-size image

•Bioreduction of NBPO afforded the chiral intermediate of ibrutinib, (S)-NBHP.•The reaction was catalyzed with ChKRED03 with excellent stereoselectivity.•Using 3 g/L crude enzyme, 200 g/L substrate was completely reduced in 3 h.Ibrutinib is an anticancer drug targeting B-cell malignancies. The key chiral intermediate for ibrutinib synthesis is the alcohol (S)-N-Boc-3-hydroxypiperidine ((S)-NBHP), which can be produced via ketoreductase (KRED)-catalyzed bioreduction. After screening a small inventory of 27 KREDs mined from the genome of Chryseobacterium sp. CA49, ChKRED03 was selected as the best performer, leading to the complete conversion of 100 g substrate/L within 10 h to yield (S)-NBHP with high enantiomeric excess ( > 99% ee). The enzyme was NADPH dependent, and the highest enzymatic activity was observed at 30 °C in potassium phosphate buffer (pH 7.0). At a substrate/catalyst ratio of 66.7 (w/w), ChKRED03 catalyzed the complete conversion of 200 g/L substrate within 3 h to yield (S)-NBHP with >99% ee, demonstrating great potential for industrial application.Download full-size image

•A novel short chain dehydrogenase ChKRED20 was characterized.•The half-life of thermal inactivation of ChKRED20 was 163 h at 40 °C.•ChKRED20 was resistant to a variety of additives and organic solvents.•Excellent stereoselectivity was achieved for a variety of acetophenone derivatives.ChKRED20 is a short-chain dehydrogenase/reductase (SDR) cloned from Chryseobacterium sp. CA49 for the anti-Prelog bioreduction of 3,5-bis(trifluoromethyl)acetophenone to produce the chiral alcohol intermediate for aprepitant. Purified ChKRED20 showed broad pH adaptability and stability with 91% of the maximal activity retained at pH 10.0. The temperature dependence of activity reached the maxima at 50 °C. Its half-lives of thermal inactivation were 163 and 9.8 h at 40 °C and 50 °C, respectively. The enzyme was resistant to a variety of metal ions, additives, and organic solvents. The enzymatic activity could be enhanced by the addition of particular metal ions or detergents to up to 168%. ChKRED20 also displayed good activity and excellent anti-Prelog stereoselectivity toward a spectrum of acetophenone derivatives, providing chiral alcohols with >99% ee for the majority of the substrates.Download full-size image

•Two new “classic” Old Yellow Enzymes were mined from genome data.•Chr-OYE1 catalyzed the reduction of 18 of the tested substrates.•Chr-OYE1 catalyzed the reduction of an ynone via a two-step sequential process.•Chr-OYE2 catalyzed 3 of the substrates with lower activity than Chr-OYE1.•Mutant Chr-OYE2 showed significantly enhanced activity and stereoselectivity.Two putative Old Yellow Enzyme (OYE) homologues, Chr-OYE1 and Chr-OYE2, were identified from the genome of Chryseobacterium sp. CA49 as new members of the “classical” subfamily. Chr-OYE1 and Chr-OYE2 were most closely related to the SYE4 from Shewanella oneidensis and NerA from Agrobacterium radiobacter with 41% and 45% identity, respectively. Both enzymes were expressed in Escherichia coli in soluble form, but their catalytic abilities as ene-reductases were quite different. Among the 19 substrate tested, Chr-OYE1 could catalyze the reduction of 18 of them including an ynone with excellent stereoselectivity for several prochiral ones, and its specific activity was roughly 1100-fold high than Chr-OYE2, which only catalyzed 3 of the substrates. After restoring the conserved tyrosine, Chr-OYE2 remained the same substrate spectrum, but showed significantly enhanced activity and stereoselectivity.Download full-size image

Feruloyl esterases (FAEs) are a group of important industrial enzymes, which could hydrolyze the ester bonds between hydroxycinnamic acids and arabinose or galactose in hemicellulose present in plant cell walls. To establish a practical high-throughput screening system of feruoyl esterases, two new substrates with modified chromo- or fluoro-phore, 2-chloro-4-nitrophenyl ferulate (CNPF; 1b) and umbelliferyl 5-O-feruolyl-α-L-arabinofuranoside (UFA; 2a), were designed and synthesized. Both substrates provided significantly improved signal-to-noise ratio compared with previously known structural analogues. The chromogenic substrate CNPF could be readily adapted to the high-throughput screening of feruloyl esterase A from Aspergillus niger, one of the most studied FAEs, with the coefficient of variance of as low as 10.3% as determined for a single sequence library.Graphical abstractDownload full-size imageHighlights► Two new mimic substrates with modified chlorophore or fluorophore were synthesized. ► Each new substrate provides significantly improved signal-to-noise ratio. ► A high-throughput screening system was developed for feruloyl esterases.

A novel styrene monooxygenase (SMO) was isolated from Pseudomonas sp. LQ26, a styrene degrader from activated sludge. Sequence alignment demonstrated that it was the most distant member of all SMOs originating from the genus of Pseudomonas. The substrate spectrum of this enzyme extended beyond typical SMO substrates to 1-allylbenzene analogues, previously reported as non-substrates for the SMO from Pseudomonas fluorescens ST. The results demonstrate for the first time the asymmetric epoxidation of both conjugated and unconjugated alkenes catalyzed by SMO and suggest that a much broader substrate spectrum is expected for SMOs.Graphical abstractDownload full-size imageResearch highlights▶ Styrene monooxygenase StyAB2 is isolated from Pseudomonas sp. LQ26. ▶ Both conjugated and unconjugated alkenes serve as substrates. ▶ Enantiomeric excesses of up to >99% are achieved for conjugated alkenes. ▶ Enantiomeric excesses of up to 86% are achieved for unconjugated alkenes.

•A strain of Chryseobacterium sp. CA49 was isolated to catalyze anti-Prelog reduction.•Enantiopure (R)-3,5-bis(trifluoromethyl)-1-phenylethanol with >99.9%ee was obtained.•Draft genome sequencing revealed 27 putative short chain dehydrogenases/reductases.•The key enzyme ChKRED20 was identified with excellent activity and stereoselectivity.•Substrate of 150 g/l was completely converted to enantiopure product within 24 h.A strain of Chryseobacterium sp. CA49 was isolated to perform efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone (1a) to enantiopure (R)-3,5-bis(trifluoromethyl)-1-phenylethanol ((R)-1b), a key intermediate for the chiral drug Aprepitant. The draft genome sequencing of the strain revealed 27 putative short chain dehydrogenases/reductases of COG1028. Their activity and stereoselectivity were assayed after expression in Escherichia coli as recombinant proteins, and the key enzyme ChKRED20 was identified with excellent activity and stereoselectivity. The lyophilized powder of the crude recombinant enzyme was applied to generate (R)-1b with >99% conversion and >99.9% enantiomeric excess at a substrate concentration of 150 g/l within 24 h by using 2-propanol as the co-substrate. The results indicate great potential for industrial-scale application of ChKRED20.Download full-size image

Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-β-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene.Highlights► Rational design of styrene monooxygenase was performed via molecular docking. ► Three mutants with enhanced activity were identified for styrene monooxygenase. ► One mutant displayed reversed enantioselectivity toward 1-phenylcyclohexene. ► Mutants retained excellent enantioselectivity toward styrene.

Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (kcat/Km) and 2-fold increases in kcat were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for “indigo assay” to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme.

Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed a one-pot two-step enzymatic cascade using prochiral α,β-unsaturated ketones as the substrates. An S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de > 99%) were achieved for the majority of the substrates, and product yields reached up to >99%.

Enantiomerically enriched glycidol derivatives with contiguous stereogenic centers were obtained in a highly diastereo- and enantio-selective epoxidation catalyzed with the styrene monooxygenase StyAB2.

The synthesis of optically pure secondary epoxy alcohols from racemic allylic alcohols using a single whole-cell biocatalyst of recombinant Escherichia coli coexpressing three oxidoreductases is described. The cascade involves the concurrent action of a styrene monooxygenase that catalyzes the formation of the chiral epoxy group, and two alcohol dehydrogenases that fulfil the epimerisation of the hydroxy group. Two sets of alcohol dehydrogenases were each applied to couple with styrene monooxygenase in order to realize the epimerisation in a stereo-complementary manner. Excellent enantio- and diastereo-selectivities were achieved for most of the 12 substrates.

Efficient asymmetric bio-epoxidation of electron-deficient α,β-unsaturated ketones was realized via a tandem reduction-epoxidation-dehydrogenation cascade, which proceeds in a switchable manner to afford either chiral epoxy ketones or allylic epoxy alcohols with up to >99% yield and >99%ee.