Gene mining-based identification of aldo–keto reductases for highly stereoselective reduction of bulky ketones

Abstract

Background

Aldo–keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones.

Results

Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding K_m values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (> 99%e.e.) were obtained through the identified AKRs.

Conclusion

The stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.

Keywords

Aldo–keto reductase / Carbonyl compound / Gene mining / Stereoselectivity / Substrate specificity

References

[1] Athanasiou N, Smallridge AJ, Trewhella MA. Baker’s yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system. J Mol Catal B-Enzym, 2001, 11: 893-896.

[2] de Lacerda PSB, Ribeiro JB, Leite SGF, Ferrara MA, Coelho RB, Bon EPS, da Silva Lima EL, Antunes OAC. Microbial reduction of ethyl 2-oxo-4-phenylbutyrate. Searching for R-enantioselectivity. New access to the enalapril like ACE inhibitors. Tetrahedron Asymmetry, 2006, 17: 1186-1188.

[3] Engelking H, Pfaller R, Wich G, Weuster-Botz D. Reaction engineering studies on β-ketoester reductions with whole cells of recombinant Saccharomyces cerevisiae. Enzyme Microb Technol, 2006, 38: 536-544.

[4] Guo JL, Mu XQ, Xu Y. Integration of newly isolated biocatalyst and resin-based in situ product removal technique for the asymmetric synthesis of (R)-methyl mandelate. Bioprocess Biosyst Eng, 2010, 33: 797-804.

[5] Guo RY, Nie Y, Mu XQ, Xu Y, Xiao R. Genomic mining-based identification of novel stereospecific aldo–keto reductases toolbox from Candida parapsilosis for highly enantioselective reduction of carbonyl compounds. J Mol Catal B-Enzym, 2014, 105: 66-73.

[6] Jez JM, Bennett MJ, Schlegel BP, Lewis M, Penning TM. Comparative anatomy of the aldo–keto reductase superfamily. Biochem J, 1997, 326: 625-636.

[7] Kaluzna IA, Matsuda T, Sewell AK, Stewart JD. Systematic investigation of Saccharomyces cerevisiae enzymes catalyzing carbonyl reductions. J Am Chem Soc, 2004, 126: 12827-12832.

[8] Kasprzak J, Rauter M, Denter S, Becker K, Baronian K, Bode R, Schauer F, Piontek M, Vorbrodt H-M, Kunze G. Synthesis of ethyl (R)-mandelate using recombinant Carboxydothermus hydrogenoformans alcohol dehydrogenase produced by two yeast species. J Mol Catal B-Enzym, 2016, 133: 176-186.

[9] Kizaki N, Yasohara Y, Nagashima N, Hasegawa J. Characterization of novel alcohol dehydrogenase of Devosia riboflavina involved in stereoselective reduction of 3-pyrrolidinone derivatives. J Mol Catal B-Enzym, 2008, 51: 73-80.

[10] Ma YH, Lv DQ, Zhou S, Lai DY, Chen ZM. Characterization of an aldo–keto reductase from Thermotoga maritima with high thermostability and a broad substrate spectrum. Biotechnol Lett, 2013, 35: 757-762.

[11] Nie Y, Xiao R, Xu Y, Montelione GT. Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones. Org Biomol Chem, 2011, 9: 4070-4078.

[12] Nikaido T, Matsuyama A, Ito M, Kobayashi Y, Oonishi H. Stereospecific reduction of 2-oxo-4-phenylbutanoate to (R)-2-hydroxy-4-phenylbutanoate with microbial cells. Biosci Biotechnol Biochem, 1992, 56: 2066-2067.

[13] Ou Z, Chen X, Ying G, Shi H, Sun X. Continuous preparation of (S)-3-hydroxy-3-phenylpropionate by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with Saccharomyces cerevisiae CGMCC No. 2266 in a membrane reactor. Biotechnol Bioprocess Eng, 2011, 16: 320-326.

[14] Schoemaker HE, Mink D, Wubbolts MG. Dispelling the myths—biocatalysis in industrial synthesis. Science, 2003, 299: 1694-1697.

[15] Wang LJ, Li CX, Ni Y, Zhang J, Liu X, Xu JH. Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor. Bioresour Technol, 2011, 102: 7023-7028.

[16] Wang YJ, Liu XQ, Luo X, Liu ZQ, Zheng YG. Cloning, expression and enzymatic characterization of an aldo–keto reductase from Candida albicans XP1463. J Mol Catal B-Enzym, 2015, 122: 44-50.

[17] Yamada-Onodera K, Fukui M, Tani Y. Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum. J Biosci Bioeng, 2007, 103: 174-178.

[18] Zhao M, Gao L, Zhang L, Bai Y, Chen L, Yu M, Cheng F, Sun J, Wang Z, Ying X. Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy. Biotechnol Lett, 2017, 39: 1741-1746.

Funding

National Natural Science Foundation of China(21336009)

Natural Science Foundation of Jiangsu Province(BK20151124)

High-end Foreign Experts Recruitment Program(GDT20183200136)

National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-09)