S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成

doi:10.13560/j.cnki.biotech.bull.1985.2018-0806

摘要/Abstract

摘要： 旨在构建S-亚胺还原酶（S-IRED）和葡萄糖脱氢酶（GDH）在大肠杆菌中的一菌双酶共表达系统,实现辅酶NADPH的再生,高效合成手性仲胺。利用无缝克隆的手段设计构建一种单质粒双启动子共表达系统,以全细胞为催化剂催化手性仲胺S-2-甲基吡咯烷（S-2MP）的合成,并研究温度、pH及有机溶剂对双酶反应的影响。成功构建了S-IRED和GDH的重组共表达质粒,实现了S-IRED与GDH在大肠杆菌中的胞内共表达,以亚胺2-甲基吡咯啉（2MPN）为模式底物,以工程菌全细胞催化手性仲胺S-2MP的合成,在低辅酶添加时催化手性胺的产率和光学纯度均高于95%。该双酶共表达体系的最适温度和pH分别为37℃和pH 8,10%以下的甲醇对双酶反应有正向促进作用。大肠杆菌胞内双酶共表达系统的构建实现了辅酶NADPH的原位再生,降低了亚胺还原酶催化合成手性胺的成本,为手性胺的规模制备奠定了基础。

关键词: 亚胺还原酶, 葡萄糖脱氢酶, 共表达, 手性胺

Abstract: This work aims to construct a co-expression system of S-imine reductase（S-IRED）and glucose dehydrogenase（GDH）in Escherichia coli Bl21,and to efficiently synthesize chiral secondary amine by regenerating coenzyme NADPH. A co-expression system with double promoters in a single plasmid was designed and constructed by seamless cloning. Whole cells of the recombinant strain were adapted as a catalyst to synthesize chiral secondary amine S-2-methylpyrrolidine（S-2MP）,and the effects of temperature,pH and organic solvent on the reaction were studied. As results,the recombinant co-expressed plasmid was successfully constructed and intracellular co-expression of S-IRED and GDH in E. coli was achieved. Using imine 2-methyl pyrroline（2MPN）as the model substrate and catalyzed by whole cells of the strain,S-2MP was synthesized;both the production rate and optical purity of chiral amine were higher than 95%. The optimal temperature and pH were 37℃ and 8 respectively. Methanol of <10% had a positive role in promoting the reaction. In sum,the construction of co-expression system in E. coli Bl21 allows in-situ regeneration of coenzyme NADPH,which reduces the synthesis cost of chiral amine catalyzed by imine reductase and provides a basis for further scale preparation of chiral amine.

Key words: imine reductase, glucose dehydrogenase, co-expression, chiral amine

李骥璇, 余磊, 李京美, 郑桂兰, 王洪钟. S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成[J]. 生物技术通报, 2019, 35(1): 105-111.

LI Ji-xuan, YU Lei, LI Jing-mei, ZHENG Gui-lan, WANG Hong-zhong. Construction of Co-expression System of S-imine Reductase and Glucose Dehydrogenase and Synthesis of Chiral Amine[J]. Biotechnology Bulletin, 2019, 35(1): 105-111.

参考文献

[1] Mitsukura K, Kuramoto T, Yoshida T, et al.A NADPH-dependent(S)-imine reductase(SIR)from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines:purification, characterization, gene cloning, and expression[J]. Appl Microbiol Biotechnol, 2013, 97:8079-8086.
[2] Ward J, Wohlgemuth R.High-Yield biocatalytic amination reactions in organic synthesis[J]. Current Organic Chemistry, 2010, 14(17):1915.
[3] Guo C, Sun DW, Yang S, et al.Iridium-catalyzed asymmetric hydrogenation of 2-pyridyl cyclic imines:A highly enantioselective approach to nicotine derivatives[J]. J Am Chem Soc, 2015, 137(1):90-93.
[4] Constable DJC, Dunn PJ, Hayler JD, et al.Key green chemistry rese-arch areas - a perspective from pharmaceutical manufacturers[J]. Green Chemistry, 2007, 9(5):411-420.
[5] Fuchs M, Kozelewski D, Tauber K, et al.Chemoenzymatic asymme-tric total synthesis of(S)-Rivastigmine using ω-transaminases[J]. Chem Commun, 2010, 46(30):5500-5502.
[6] Ghislieri D, Turner N.Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines[J]. Topics in Catalysis, 2014, 57(5):284-300.
[7] Abrahamson MJ, Wong JW, Bommarius AS.The evolution of an amine dehydrogenase biocatalyst for the asymmetric production of chiral amines[J]. Advanced Synthesis & Catalysis, 2013, 355(9):1780-1786.
[8] Mangassanchez J, France SP, Montgomery SL, et al.Imine reductases(IREDs)[J]. Current Opinion in Chemical Biology, 2016, 37:19-25.
[9] Lenz M, Borlinghaus N, Weinmann L, et al.Recent advances in imine reductase-catalyzed reactions[J]. World J Microbiol Biotechnol, 2017, 33(11):199.
[10] Man H, Wells E, Hussain S, et al.Structure, activity and stereosele-ctivity of NADPH-dependent oxidoreductases catalysing the S-sele-ctive reduction of the imine substrate 2-methylpyrroline[J]. Chembiochem, 2015, 16:1052-1059.
[11] Mitsukura K, Suzuki M, Tada K, et al.Asymmetric synthesis of chiral cyclic amine from cyclic imine by bacterial whole-cell catalyst of enantioselective imine reductase[J]. Org Biomol Chem, 2010, 8:4533-4535.
[12] Grogan G, Turner NJ.InspIRED by Nature:NADPH-dependent imine reductases(IREDs)as catalysts for the preparation of chiral amines[J]. Chemistry, 2016, 22:1900-1907.
[13] 杨兴龙, 穆晓清, 聂尧, 等. 亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸[J]. 微生物学报, 2016, 56(11):1709-1718.
[14] 王玺, 段胜林, 熊舒莉, 等. 自诱导系统在酶促合成2'-脱氧胞苷中的应用[J]. 生物技术通报, 2014, 30(11):225-232.
[15] Ganda M, Müllera H, Wardengab R, et al.Characterization of three novel enzymes with imine reductase activity[J]. Journal of Molecular Catalysis B:Enzymatic, 2014, 110(8):126-132.
[16] Aleku GA, Man H, France SP, et al.Stereoselectivity and structural characterization of an imine reductase(IRED)from Amycolatopsis orientalis[J]. Acs Catalysis, 2016, 6:3380-3889.
[17] Lenz M, Scheller PN, Richter SM, et al.Cultivation and purification of two stereoselective imine reductases from Streptosporangium roseum and Paenibacillus elgii[J]. Protein Expression & Purification, 2017, 133:199-204.
[18] 胡媛周, 文瑜, 魏东芝, 等. gdh和Ldh基因共表达重组大肠杆菌合成D-乳酸研究[J]. 药物生物技术, 2014, 21(1):13-17.
[19] Kim JK, Kim HE, Lee KH, et al.Two-promoter vector is highly efficient for overproduction of protein complexes[J]. Protein Science, 2004, 13(6):1698-1703.
[20] Leipold F, Hussain S, Ghislieri D, et al.Asymmetric reduction of cyclic imines catalyzed by a whole-cell biocatalyst containing an(S)-imine reductase[J]. Chemcatchem, 2013, 5(12):3505-3508.
[21] Mitsukura K, Suzuki M, Shinoda S, et al.Purification and character-ization of a novel(R)-imine reductase from Streptomyces sp. GF3587[J]. Biosci Biotechnol Biochem, 2011, 75:1778-1782.