Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae)...Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae) as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCRl and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99% ee) and(R)-δ-decalactone(98% ee) in 85% and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.展开更多
Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA...Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.展开更多
Foxtail millet (Setaria italica L.) is a drought-tolerant millet crop of arid and semi-arid regions. Aldo-keto reductases (AKRs) are significant part of plant defence mechanism, having an ability to confer multiple st...Foxtail millet (Setaria italica L.) is a drought-tolerant millet crop of arid and semi-arid regions. Aldo-keto reductases (AKRs) are significant part of plant defence mechanism, having an ability to confer multiple stress tolerance. In this study, AKR1 gene expression was studied in roots and leaves of foxtail millet subjected to different regimes of PEG- and NaCl-stress for seven days. The quantitative Real-time PCR expression analysis in both root and leaves showed upregulation of AKR1 gene during PEG and salt stress. A close correlation exits between expression of AKR1 gene and the rate of lipid peroxidation along with the retardation of growth. Tissue-specific differences were found in the AKR1 gene expression to the stress intensities studied. The reduction in root and shoot growth under both stress conditions were dependent on stress severity. The level of lipid peroxidation as indicated by MDA formation was significantly increased in roots and leaves along with increased stress levels. Finally, these findings support the early responsive nature of AKR1 gene and seem to be associated at least in part with its ability to contribute in antioxidant defence related pathways which could provide a better protection against oxidative stress under stress conditions.展开更多
We screened 15 Agromyces strains from the Microbacteriaceae family and 16 Gordonia strains from the Gordoniaceae family to investigate their biocatalytic ability to reduce carbonyl compounds. Two Agromyces strains (A....We screened 15 Agromyces strains from the Microbacteriaceae family and 16 Gordonia strains from the Gordoniaceae family to investigate their biocatalytic ability to reduce carbonyl compounds. Two Agromyces strains (A. soli NBRC109063 and A. humatus NBRC109085) and two Gordonia strains (G. hydrophobica NBRC16057 and G. malaquae NBRC108250) grew well in 230 medium. The stereoselective reduction of various carbonyl compounds using these four strains was investigated. We discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide. On the basis of the conversion rate and stereoselectivity of the alcohols produced, G. hydrophobica NBRC16057 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols. Our results also suggest that the reduction of ethyl 2-methylacetoacetate by wet G. hydrophobica NBRC16057 cells in the presence of L-glutamate is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate.展开更多
A novel nicotinamide adenine dinucleotide phosphate（NADPH）-dependent carbonyl reductase from Kluyverornyces marxianus（KmCR） was identified, which can convert various prochiral ketone esters and ketone substrates t...A novel nicotinamide adenine dinucleotide phosphate（NADPH）-dependent carbonyl reductase from Kluyverornyces marxianus（KmCR） was identified, which can convert various prochiral ketone esters and ketone substrates to their corresponding chiral alcohols. KmCR was over-expressed in E. coli BL21（DE3）, purified to homogeneity, and characterized. The purified enzyme exhibits the highest activity at 40℃ and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel eiectrophoresis（SDS-PAGE） analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. Vmax and Km of KmCR are 4.28 μmol.min^-1·mg^-1 and 0.41 mmol/L for ketone ester substrate ethyl 2-oxo-4-phenylbutyrate（OPBE）, 3.09μmol.min^-1·mg^-1 and 1.21 mmol/L for cofactor NADPH, respectively. Cofactor recycle was achieved by co-expression of KmCR and glucose dehydrogenase（GDH） in E. coli. Recombinant E. coli harboring KmCR and GDH showed moderate asymmetric reduction activity towards various α- and β-ketoesters, diaryl ketone substrates. In an aqueous/butyl acetate biphasic system, the whole-cell biocatalyst was used to prepare ethyl （R）-2-hydroxy-4- phenylbutanoate[（R）-HPBE] in an e.e. of 99.5% with a space-time yield of 433.6 g.L-1.d-1 and a yield of 80.3% at 270 g/L OPBE.展开更多
Prostaglandins （PGs） play a critical role in porcine reproduction, of which prostaglandin E2 （PGE2） and prostaglandin F2a （PGF2a） exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enz...Prostaglandins （PGs） play a critical role in porcine reproduction, of which prostaglandin E2 （PGE2） and prostaglandin F2a （PGF2a） exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 （CBR1） catalyzes the conversion of PGE2 to PGF2a. A high ratio of PGE2：PGF2a is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor KB （NF-KB） is involved in the process. Bioinformatic analysis has shown that NF-KB is a possible factor bound to two cis-regulatory elements in CBRI promoter. In this study, we cloned the 2997 bp （-2875/＋122） of the promoter, and constructed six 5＇-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 （-16401＋7） exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 （-2089/＋7）. The activity of P1, P2, and P3 （-1019/＋7） was highly significantly higher than that of others （P〈0.01）, suggesting that two positive regulatory elements were likely present in the regions of -1640/-1019 and -1019/-647. The results also showed that the -1640/ -647 region was indispensable for the promoter. The results of chromatin immunoprecipitation （CHIP） demonstrated that the NF-KB subunit p65 binds to one site around -15451-1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 （P〈0.05） in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBRI expression. These results indicated that NF-KB （p65） could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-KB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.展开更多
The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding ...The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa,and its nucleotide sequence showed 99%similarity to those of other members of the alcohol dehydrogenase superfamily.The rCR gene could express in recombinant strain Escherichia coli JM109,and the expression plasmid could produce(R)-1-pheny-1,2-ethanediol(100%e.e.,80.14%yield)from b-hydroxyacetophenone without any additive to regenerate NAD+from NADH.展开更多
A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified t...A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified through ammonium sulfate,Diethylamino Ethanol(DEAE)sepharose Fast flow(FF),phenyl-sepharose FF and blue sepharose FF chromato graphy from cell-free extract.The molecular mass of the enzyme was about 30 kDa.The optimum pH and temperature for reduction were 4.5℃ and 35℃,respectively.The Cu2+had strong restrictive effect on enzyme activity.In addition,the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity,and showed high asymmetric reduction activity towards a-hydroxyacetophenone and ethyl 4-chloro acetoacetate.For the asymmetric reduction of a-hydroxyacetophenone and ethyl 4-chloro acetoacetate,(S)-1-phenyl-1,2-ethanediol and(R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme,with the 100% and 94.3%e.e.value,respec-tively.Therefore,the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols.The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS,and the car-bonyl reductase showed some identity to the hypothetical protein CaO19.10414 reported.展开更多
As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-...As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate(OPBE)with high R-enantiose-lectivity.This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis.Using this strategy,4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets,generating variant Mu27 with 99%conversion and 98%(R)ee value at substrate loading of up to 500 mmol/L.MD simu-lations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key rea-sons for the excellent R-enantioselectivity of Mu27 towards OPBE.The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.展开更多
基金financially sponsored by the National Key Research and Development Program of China (2016YFA0204300, 2019YFA09005000)the National Natural Science Foundation of China (21536004, 21776085, 21871085)+2 种基金the Natural Science Foundation of Shanghai (18ZR1409900)Key Project of the Shanghai Science and Technology Committee (18DZ1112703)the Fundamental Research Funds for the Central Universities (WF1714026)。
文摘Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae) as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCRl and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99% ee) and(R)-δ-decalactone(98% ee) in 85% and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.
基金supported by grants from China Na-tional Basic Research Program (NO. 2004CB117302)National Natural Science Foundation of China (No.30470171)the Sigrid Jusélius Foundation Finland and the Academy of Finland
文摘Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.
文摘Foxtail millet (Setaria italica L.) is a drought-tolerant millet crop of arid and semi-arid regions. Aldo-keto reductases (AKRs) are significant part of plant defence mechanism, having an ability to confer multiple stress tolerance. In this study, AKR1 gene expression was studied in roots and leaves of foxtail millet subjected to different regimes of PEG- and NaCl-stress for seven days. The quantitative Real-time PCR expression analysis in both root and leaves showed upregulation of AKR1 gene during PEG and salt stress. A close correlation exits between expression of AKR1 gene and the rate of lipid peroxidation along with the retardation of growth. Tissue-specific differences were found in the AKR1 gene expression to the stress intensities studied. The reduction in root and shoot growth under both stress conditions were dependent on stress severity. The level of lipid peroxidation as indicated by MDA formation was significantly increased in roots and leaves along with increased stress levels. Finally, these findings support the early responsive nature of AKR1 gene and seem to be associated at least in part with its ability to contribute in antioxidant defence related pathways which could provide a better protection against oxidative stress under stress conditions.
文摘We screened 15 Agromyces strains from the Microbacteriaceae family and 16 Gordonia strains from the Gordoniaceae family to investigate their biocatalytic ability to reduce carbonyl compounds. Two Agromyces strains (A. soli NBRC109063 and A. humatus NBRC109085) and two Gordonia strains (G. hydrophobica NBRC16057 and G. malaquae NBRC108250) grew well in 230 medium. The stereoselective reduction of various carbonyl compounds using these four strains was investigated. We discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide. On the basis of the conversion rate and stereoselectivity of the alcohols produced, G. hydrophobica NBRC16057 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols. Our results also suggest that the reduction of ethyl 2-methylacetoacetate by wet G. hydrophobica NBRC16057 cells in the presence of L-glutamate is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate.
基金the National Basic Research and Development Program of China，the National Natural Science Foundation of China，the Project of New Century Excellent Talents in University of China，the Natural Science Foundation of Jiangsu Province,China，the Program of Introducing Talents of Discipline to Universities,China，the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘A novel nicotinamide adenine dinucleotide phosphate（NADPH）-dependent carbonyl reductase from Kluyverornyces marxianus（KmCR） was identified, which can convert various prochiral ketone esters and ketone substrates to their corresponding chiral alcohols. KmCR was over-expressed in E. coli BL21（DE3）, purified to homogeneity, and characterized. The purified enzyme exhibits the highest activity at 40℃ and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel eiectrophoresis（SDS-PAGE） analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. Vmax and Km of KmCR are 4.28 μmol.min^-1·mg^-1 and 0.41 mmol/L for ketone ester substrate ethyl 2-oxo-4-phenylbutyrate（OPBE）, 3.09μmol.min^-1·mg^-1 and 1.21 mmol/L for cofactor NADPH, respectively. Cofactor recycle was achieved by co-expression of KmCR and glucose dehydrogenase（GDH） in E. coli. Recombinant E. coli harboring KmCR and GDH showed moderate asymmetric reduction activity towards various α- and β-ketoesters, diaryl ketone substrates. In an aqueous/butyl acetate biphasic system, the whole-cell biocatalyst was used to prepare ethyl （R）-2-hydroxy-4- phenylbutanoate[（R）-HPBE] in an e.e. of 99.5% with a space-time yield of 433.6 g.L-1.d-1 and a yield of 80.3% at 270 g/L OPBE.
基金Project supported by the National Natural Science Foundation of China(No.31201771)the Earmarked Fund for China Agriculture Research System(No.CARS-36)
文摘Prostaglandins （PGs） play a critical role in porcine reproduction, of which prostaglandin E2 （PGE2） and prostaglandin F2a （PGF2a） exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 （CBR1） catalyzes the conversion of PGE2 to PGF2a. A high ratio of PGE2：PGF2a is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor KB （NF-KB） is involved in the process. Bioinformatic analysis has shown that NF-KB is a possible factor bound to two cis-regulatory elements in CBRI promoter. In this study, we cloned the 2997 bp （-2875/＋122） of the promoter, and constructed six 5＇-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 （-16401＋7） exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 （-2089/＋7）. The activity of P1, P2, and P3 （-1019/＋7） was highly significantly higher than that of others （P〈0.01）, suggesting that two positive regulatory elements were likely present in the regions of -1640/-1019 and -1019/-647. The results also showed that the -1640/ -647 region was indispensable for the promoter. The results of chromatin immunoprecipitation （CHIP） demonstrated that the NF-KB subunit p65 binds to one site around -15451-1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 （P〈0.05） in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBRI expression. These results indicated that NF-KB （p65） could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-KB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.
基金This study was supported by the National Basic Research Program of China(No.2003CB716008)the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,IRT0532).
文摘The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa,and its nucleotide sequence showed 99%similarity to those of other members of the alcohol dehydrogenase superfamily.The rCR gene could express in recombinant strain Escherichia coli JM109,and the expression plasmid could produce(R)-1-pheny-1,2-ethanediol(100%e.e.,80.14%yield)from b-hydroxyacetophenone without any additive to regenerate NAD+from NADH.
基金This work was supported by the National Natural Science Foundation of China(Grant No.20376031)the National Key Basic Research and Development Program of China(973 Program)(Grant No.2003CB716008)+1 种基金the Program for New Century Excellent Talents in University,Ministry of Education,China(Grant No.NCET-04-0498)the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.PCSIRT,IRT0532).
文摘A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified through ammonium sulfate,Diethylamino Ethanol(DEAE)sepharose Fast flow(FF),phenyl-sepharose FF and blue sepharose FF chromato graphy from cell-free extract.The molecular mass of the enzyme was about 30 kDa.The optimum pH and temperature for reduction were 4.5℃ and 35℃,respectively.The Cu2+had strong restrictive effect on enzyme activity.In addition,the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity,and showed high asymmetric reduction activity towards a-hydroxyacetophenone and ethyl 4-chloro acetoacetate.For the asymmetric reduction of a-hydroxyacetophenone and ethyl 4-chloro acetoacetate,(S)-1-phenyl-1,2-ethanediol and(R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme,with the 100% and 94.3%e.e.value,respec-tively.Therefore,the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols.The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS,and the car-bonyl reductase showed some identity to the hypothetical protein CaO19.10414 reported.
基金This study is financially supported by the National Key Research and Development Program of China(No.2018YFA0901800 to H.Yu and L.Ye)Zhejiang Provincial Natural Science Foundation of China(No.LY18B060001 to L.Ye and No.LZ20B060002 to H.Yu)Natural Science Foundation of China(No.21776244 to H.Yu).We are grateful to the constructive suggestions from academician Fen-Er Chen(Department of Chemistry,Fudan University,Shanghai,China)and the support of Discovery Studio software from Fuzhou University platform.
文摘As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate(OPBE)with high R-enantiose-lectivity.This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis.Using this strategy,4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets,generating variant Mu27 with 99%conversion and 98%(R)ee value at substrate loading of up to 500 mmol/L.MD simu-lations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key rea-sons for the excellent R-enantioselectivity of Mu27 towards OPBE.The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.
