Novel stereoselective carbonyl reductase from Kluyveromyces marxianus for chiral alcohols synthesis

Abstract

A novel nicotinamide adenine dinucleotide phosphate(NADPH)-dependent carbonyl reductase from Kluyveromyces 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 °C and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. V _max and K _m 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), 3.09 μmol·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.

Keywords

Carbonyl reductase / Asymmetric reduction / Chiral alcohol / co-Expression / Kluyveromyces marxianus

References

[1] Woodley J M. Trends Biotechnol., 2008, 26(6): 321.

[2] Patel R N. Adv. Synth. Catal., 2001, 343(6/7): 527.

[3] Ni Y, Xu J H. Biotechnol. Adv., 2012, 30(6): 1279.

[4] Bornscheuer U T, Huisman G W, Kazlauskas R J, Lutz S, Moore J C, Robins K. Nature, 2012, 485(7397): 185.

[5] Luo X J, Yu H L, Xu J H. Enz. Eng., 2012, 1(1): 104.

[6] Zheng G W, Xu J H. Curr. Opin. Biotechnol., 2011, 22(6): 784.

[7] Wang L J, Li C X, Ni Y, Zhang J, Liu X, Xu J H. Bioresour. Technol., 2011, 102(14): 7023.

[8] Kerstin M E, Frank H, Yogan K, Olena P, Reinhard K, Daniel K, Juürgen H, Rolf M, Rita B. J. Biol. Chem., 2009, 284(42): 28590.

[9] Fraaije M W, Wu J, Heutset D P H M, Hellemond E W, Spelberg J H L, Janssen D B. Appl. Microbiol. Biotechnol., 2005, 66(4): 393.

[10] Seffernick J L, Samanta S K, Louie T M, Wackett L P, Subramanian M. J. Biotechnol., 2009, 143(1): 17.

[11] Kataoka M, Kita K, Wada M, Yasohara Y, Hasegawa J, Shimizu S. Appl. Microbiol. Biotechnol., 2003, 62(5/6): 437.

[12] Shen N D, Ni Y, Ma H M, Wang L J, Li C X, Zheng G W, Zhang J, Xu J H. Org. Lett., 2012, 14(8): 1982.

[13] Ni Y, Li C X, Ma H M, Zhang J, Xu J H. Appl. Microbiol. Biotechnol., 2011, 9(4): 1111.

[14] Ni Y, Li C X, Zhang J, Shen N D, Bornscheuer U T, Xu J H. Adv. Synth. Catal., 2011, 353(8): 1213.

[15] Ni Y, Pan J, Ma H M, Li C X, Zhang J, Zheng G W, Xu J H. Tetrahedron Lett., 2012, 53(29): 4715.

[16] Zhang W, Ni Y, Sun Z H, Zheng P, Lin W Q, Zhu P, Ju N F. Process Biochem., 2009, 44(11): 1270.

[17] Li N, Ni Y, Sun Z H. J. Mol. Catal. B: Enzym., 2010, 66(1): 190.

[18] Su Y N. Two-enzyme Coexpressed Recombinant Strain for Asymmetric Synthesis of Ethyl (R)-2-Hydroxy-4-phenylbutyrate, 2013, Wuxi: Jiangnan University.

[19] Koh H Y, Han Y S, Cha J H, Kim W H, Lee J S, Yun H C. Carbonyl Reductase of Kluyveromyces Marxianus and Its Isolation and Purification Method, US 7091016, 2006.

[20] Cha M, Kim E J, Park J, Kim B G. Enzyme Microb. Technol., 2008, 43(7): 480.

[21] Vitale P, Perna F M, Perrone M G, Scilimati A. Tetrahedron: Asymmetry, 2011, 22(23): 1985.

[22] Xu G C, Yu H L, Zhang X Y, Xu J H. ACS Catalysis, 2012, 2(12): 2566.

[23] Lacerda P S, Ribeiro J B, Leite S G F, Coelho R B, Lima E L, Antunes O A C. Biochem. Eng. J., 2006, 28(3): 299.

[24] Ni Y, Zhou J Y, Sun Z H. Process Biochem., 2012, 47(7): 1042.

[25] Bradford M M. Anal. Biochem., 1976, 72(1): 248.

[26] Zhu D M, Yang Y, Buynak J D, Hua L. Org. Biomol. Chem., 2006, 4(14): 2690.

[27] Zelinski T, Peters J, Kula M R. J. Biotechnol., 1994, 33(3): 283.

[28] Wada M, Kawabata H, Kataoka M, Yasohara Y, Kizaki N, Hasegawa J, Shimizu S. J. Mol. Catal. B: Enzym., 1999, 6(3): 333.

[29] Nie Y, Xu Y, Yang M, Mu X Q. Lett. Appl. Microbiol., 2007, 44(5): 555.

[30] Singh A, Bhattacharyya M S, Banerjee U C. Process Biochem., 2009, 44(9): 986.

[31] Kosjek B, Stampfer W, Pogorevc M, Goessler W, Faber K, Kroutil W. Biotechnol. Bioeng., 2004, 86(1): 55.

[32] Soni P, Kansal H, Banerjee U C. Process Biochem., 2007, 42(12): 1632.

[33] Ma Y H, Lü D Q, Zhou S, Lai D Y, Chen Z M. Biotechnol. Lett., 2013, 35(5): 757.

[34] Zou S P, Guo S Y, Kaleem I, Li C. J. Chem. Technol. Biotechnol., 2013, 88(10): 1913.

[35] Wang Z Y, Liu J Z, Xu L S, Zhang H J, Liu Q, Jiao Q C. Chem. Res. Chinese Universities, 2013, 29(1): 95.

[36] Tan Z L, Jia J R, Zhao Y. Chem. J. Chinese Universities, 2009, 30(12): 2404.

[37] Peters J, Minuth T, Kula M R. Enzyme Microb. Technol., 1993, 15(11): 950.

[38] Li S B, Nie H L, Xue Y, Zhang H T, Zhu L M. Chemical Reagents, 2013, 32(2): 97.

[39] Ni Y, Li C X, Wang L J, Zhang J, Xu J H. Org. Biomol. Chem., 2011, 9(15): 5463.

[40] Yun H, Choi H L, Fadnavis N W, Kim B G. Biotechnol. Progr., 2005, 21(2): 366.

[41] Carballeira J D, Quezada M A, Hoyos P, Simeó Y, Hernaiz M J, Alcantara A R, Sinisterra JV. Biotechnol. Adv., 2009, 27(6): 686.

[42] Xu H, Jin Y Q, He B F. Production of R-CHBE Using the Recombinant Carrying Aldehyde Reductase Coupling GDH Gene for Efficient NADPH Regeneration. 5th Chinese National Chemical and Biochemical Engineering Annual Meeting, Xi’an, 2008.

[43] Ye Q, Cao H, Yan M, Cao F, Zhang Y Y, Li X M, Xu L, Chen Y, Xiong J, Ouyang P K, Ying H J. Bioresour. Technol., 2010, 101(17): 6761.