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
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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。
This work aims to construct a co-expression system of S-imine reductase(S-IRED)and glucose dehydrogenase(GDH)inEscherichia coli Bl21,and to efficiently synthesize chiral secondary amine by regenerating coenzyme NADPH. A co-expression system withdouble promoters in a single plasmid was designed and constructed by seamless cloning. Whole cells of the recombinant strain were adapted asa catalyst to synthesize chiral secondary amine S-2-methylpyrrolidine(S-2MP),and the effects of temperature,pH and organic solvent on thereaction were studied. As results,the recombinant co-expressed plasmid was successfully constructed and intracellular co-expression of S-IREDand 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 pHwere 37℃ and 8 respectively. Methanol of <10% had a positive role in promoting the reaction. In sum,the construction of co-expression systemin E. coli Bl21 allows in-situ regeneration of coenzyme NADPH,which reduces the synthesis cost of chiral amine catalyzed by imine reductaseand provides a basis for further scale preparation of chiral amine.
This work aims to construct a co-expression system of S-imine reductase(S-IRED)and glucose dehydrogenase(GDH)inEscherichia coli Bl21,and to efficiently synthesize chiral secondary amine by regenerating coenzyme NADPH. A co-expression system withdouble promoters in a single plasmid was designed and constructed by seamless cloning. Whole cells of the recombinant strain were adapted asa catalyst to synthesize chiral secondary amine S-2-methylpyrrolidine(S-2MP),and the effects of temperature,pH and organic solvent on thereaction were studied. As results,the recombinant co-expressed plasmid was successfully constructed and intracellular co-expression of S-IREDand 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 pHwere 37℃ and 8 respectively. Methanol of <10% had a positive role in promoting the reaction. In sum,the construction of co-expression systemin E. coli Bl21 allows in-situ regeneration of coenzyme NADPH,which reduces the synthesis cost of chiral amine catalyzed by imine reductaseand provides a basis for further scale preparation of chiral amine.
引文
[1]Mitsukura K, Kuramoto T, Yoshida T, et al. A NADPH-dependent(S)-imine reductase(SIR)from Streptomyces sp. GF3546 forasymmetric synthesis of optically active amines:purification,characterization, gene cloning, and expression[J]. Appl MicrobiolBiotechnol, 2013, 97:8079-8086.
[2]Ward J, Wohlgemuth R. High-Yield biocatalytic amination reactionsin organic synthesis[J]. Current Organic Chemistry, 2010, 14(17):1915.
[3]Guo C, Sun DW, Yang S, et al. Iridium-catalyzed asymmetrichydrogenation of 2-pyridyl cyclic imines:A highly enantioselectiveapproach 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 research areas-a perspective from pharmaceutical manufacturers[J].Green Chemistry, 2007, 9(5):411-420.
[5]Fuchs M, Kozelewski D, Tauber K, et al. Chemoenzymatic asymmetric total synthesis of(S)-Rivastigmine usingω-transaminases[J].Chem Commun, 2010, 46(30):5500-5502.
[6]Ghislieri D, Turner N. Biocatalytic approaches to the synthesis ofenantiomerically pure chiral amines[J]. Topics in Catalysis,2014, 57(5):284-300.
[7]Abrahamson MJ, Wong JW, Bommarius AS. The evolution of anamine dehydrogenase biocatalyst for the asymmetric production ofchiral 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 inimine reductase-catalyzed reactions[J]. World J MicrobiolBiotechnol, 2017, 33(11):199.
[10]Man H, Wells E, Hussain S, et al. Structure, activity and stereoselectivity of NADPH-dependent oxidoreductases catalysing the S-selective reduction of the imine substrate 2-methylpyrroline[J].Chembiochem, 2015, 16:1052-1059.
[11]Mitsukura K, Suzuki M, Tada K, et al. Asymmetric synthesis ofchiral cyclic amine from cyclic imine by bacterial whole-cellcatalyst of enantioselective imine reductase[J]. Org BiomolChem, 2010, 8:4533-4535.
[12]Grogan G, Turner NJ. InspIRED by Nature:NADPH-dependentimine reductases(IREDs)as catalysts for the preparation ofchiral 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 ofthree novel enzymes with imine reductase activity[J]. Journal ofMolecular Catalysis B:Enzymatic, 2014, 110(8):126-132.
[16]Aleku GA, Man H, France SP, et al. Stereoselectivity and structuralcharacterization 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 purificationof 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 highlyefficient for overproduction of protein complexes[J]. ProteinScience, 2004, 13(6):1698-1703.
[20]Leipold F, Hussain S, Ghislieri D, et al. Asymmetric reduction ofcyclic 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 characterization of a novel(R)-imine reductase from Streptomyces sp.GF3587[J]. Biosci Biotechnol Biochem, 2011, 75:1778-1782.
[2]Ward J, Wohlgemuth R. High-Yield biocatalytic amination reactionsin organic synthesis[J]. Current Organic Chemistry, 2010, 14(17):1915.
[3]Guo C, Sun DW, Yang S, et al. Iridium-catalyzed asymmetrichydrogenation of 2-pyridyl cyclic imines:A highly enantioselectiveapproach 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 research areas-a perspective from pharmaceutical manufacturers[J].Green Chemistry, 2007, 9(5):411-420.
[5]Fuchs M, Kozelewski D, Tauber K, et al. Chemoenzymatic asymmetric total synthesis of(S)-Rivastigmine usingω-transaminases[J].Chem Commun, 2010, 46(30):5500-5502.
[6]Ghislieri D, Turner N. Biocatalytic approaches to the synthesis ofenantiomerically pure chiral amines[J]. Topics in Catalysis,2014, 57(5):284-300.
[7]Abrahamson MJ, Wong JW, Bommarius AS. The evolution of anamine dehydrogenase biocatalyst for the asymmetric production ofchiral 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 inimine reductase-catalyzed reactions[J]. World J MicrobiolBiotechnol, 2017, 33(11):199.
[10]Man H, Wells E, Hussain S, et al. Structure, activity and stereoselectivity of NADPH-dependent oxidoreductases catalysing the S-selective reduction of the imine substrate 2-methylpyrroline[J].Chembiochem, 2015, 16:1052-1059.
[11]Mitsukura K, Suzuki M, Tada K, et al. Asymmetric synthesis ofchiral cyclic amine from cyclic imine by bacterial whole-cellcatalyst of enantioselective imine reductase[J]. Org BiomolChem, 2010, 8:4533-4535.
[12]Grogan G, Turner NJ. InspIRED by Nature:NADPH-dependentimine reductases(IREDs)as catalysts for the preparation ofchiral 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 ofthree novel enzymes with imine reductase activity[J]. Journal ofMolecular Catalysis B:Enzymatic, 2014, 110(8):126-132.
[16]Aleku GA, Man H, France SP, et al. Stereoselectivity and structuralcharacterization 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 purificationof 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 highlyefficient for overproduction of protein complexes[J]. ProteinScience, 2004, 13(6):1698-1703.
[20]Leipold F, Hussain S, Ghislieri D, et al. Asymmetric reduction ofcyclic 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 characterization of a novel(R)-imine reductase from Streptomyces sp.GF3587[J]. Biosci Biotechnol Biochem, 2011, 75:1778-1782.
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