苯乙酮转化为(S)-a-苯乙醇的工程菌构建
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摘要
【背景】(S)-a-苯乙醇是一种重要的药物合成中间体,利用工程菌将苯乙酮转化为(S)-a-苯乙醇的方法具有立体选择性强、转化条件温和等优势,该研究对未来绿色工业化生产有重要意义。【目的】构建能将苯乙酮转化为(S)-a-苯乙醇的工程菌并对其转化条件进行研究。【方法】分别从红平红球菌(Rhodococcuserythropolis)和博伊丁假丝酵母(Candidaboidinii)中克隆得到羰基还原酶基因ReADH以及甲酸脱氢酶基因FDH,构建pRSFDuet-ReADH-FDH(R1F2)和pRSFDuet-FDH-ReADH (F1R2)两个共表达载体,分别在大肠杆菌中表达。测定R1F2和F1R2中ReADH和FDH酶活性及其催化反应的最适反应条件。对利用全细胞转化苯乙酮为(S)-a-苯乙醇反应条件进行研究。【结果】构建了R1F2和F1R2两个共表达载体,其中R1F2中ReADH和FDH的酶活性分别为6.7 U/mL和7.6 U/mL,对苯乙酮有更强的催化还原能力。R1F2中ReADH和FDH的最适pH分别为6.0和8.5,最适温度分别为40°C和35°C。R1F2全细胞转化苯乙酮反应的最适pH和温度分别为7.5和30°C,对高底物有较强耐受性,对400 mmol/L苯乙酮转化率大于98%,产物(S)-α-苯乙醇的光学纯度大于99%。【结论】研究获得的工程菌及其全细胞转化条件为工业应用奠定了基础。
[Background](S)-α-phenylethanol is a kind of important pharmaceutical intermediate. Conversion of acetophenone into(S)-α-phenylethanol by engineering bacteria has the advantages of strong stereoselectivity and mild transformation conditions. The study of this synthetic method is of great significance for the future green industrial production. [Objective] Construction of engineering bacteria capable of transforming acetophenone into(S)-α-phenylethanol, and the study on its transformation conditions. [Methods] ReADH gene encoding carbonyl reductase and FDH gene encoding formate dehydrogenase were cloned from Rhodococcus erythropolis and Candida boidinii, respectively. The constructed pRSFDuet-ReADH-FDH(R1 F2) and pRSFDuet-FDH-ReADH(F1 R2) were transformed into Escherichia coli, respectively. The enzymatic assay of ReADH and FDH were analyzed. The optimized transformation conditions to produce(S)-α-phenylethanol were tested. [Results] The enzyme activity of ReADH and FDH in R1 F2 were 6.7 U/mL and 7.6 U/mL, respectively. R1 F2 had higher catalytic activity than F1 R2. The optimum pH of ReADH and FDH were 6.0 and 8.5, respectively. The optimum temperature of the ReADH and FDH were 40 °C and 35 °C, respectively. The optimum pH and temperature for the reaction of acetophenone catalyzed by R1 F2 is 7.5 and 30 °C. R1 F2 co-expression strain could catalyze the reduction of acetophenone with high substrate concentration, the conversion of 400 mmol/L acetophenone was over 98%, and the e.e. of(S)-α-phenylethanol value was over 99%. [Conclusion] Conversion of acetophenone into(S)-α-phenylethanol by engineered strain has a good prospect for industrial application.
[Background](S)-α-phenylethanol is a kind of important pharmaceutical intermediate. Conversion of acetophenone into(S)-α-phenylethanol by engineering bacteria has the advantages of strong stereoselectivity and mild transformation conditions. The study of this synthetic method is of great significance for the future green industrial production. [Objective] Construction of engineering bacteria capable of transforming acetophenone into(S)-α-phenylethanol, and the study on its transformation conditions. [Methods] ReADH gene encoding carbonyl reductase and FDH gene encoding formate dehydrogenase were cloned from Rhodococcus erythropolis and Candida boidinii, respectively. The constructed pRSFDuet-ReADH-FDH(R1 F2) and pRSFDuet-FDH-ReADH(F1 R2) were transformed into Escherichia coli, respectively. The enzymatic assay of ReADH and FDH were analyzed. The optimized transformation conditions to produce(S)-α-phenylethanol were tested. [Results] The enzyme activity of ReADH and FDH in R1 F2 were 6.7 U/mL and 7.6 U/mL, respectively. R1 F2 had higher catalytic activity than F1 R2. The optimum pH of ReADH and FDH were 6.0 and 8.5, respectively. The optimum temperature of the ReADH and FDH were 40 °C and 35 °C, respectively. The optimum pH and temperature for the reaction of acetophenone catalyzed by R1 F2 is 7.5 and 30 °C. R1 F2 co-expression strain could catalyze the reduction of acetophenone with high substrate concentration, the conversion of 400 mmol/L acetophenone was over 98%, and the e.e. of(S)-α-phenylethanol value was over 99%. [Conclusion] Conversion of acetophenone into(S)-α-phenylethanol by engineered strain has a good prospect for industrial application.
引文
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[15]Li C,Qi NC,Peng G,et al.Screening and identification of S-phenylethanol-producing strain[J].Journal of Microbiology,2013,33(2):43-47(in Chinese)李成,戚南昌,彭果,等.S-苯乙醇高产菌株的筛选及鉴定[J].微生物学杂志,2013,33(2):43-47
[16]Sun Y,Zhang RZ,Xu Y.Co-expression of formate dehydrogenase form Candida boidinii and(R)-specific carbonyl reductase form Candida parapsilosis CCTCCM203011 in Escherichia coli[J].Acta Microbiologica Sinica,2008,48(12):1629-1633(in Chinese)孙莹,张荣珍,徐岩.(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达[J].微生物学报,2008,48(12):1629-1633
[17]Jiang JW,Zhang RZ,Zhou XT,et al.Efficient biosynthesis of(S)-1-phenyl-1,2-ethanediol catalyzed by(S)-carbonyl reductaseⅡand glucose dehydrogenase[J].Acta Microbiologica Sinica,2016,56(10):1647-1655(in Chinese)姜佳伟,张荣珍,周晓天,等.(S)-羰基还原酶Ⅱ与葡萄糖脱氢酶共催化高效合成(S)-苯乙二醇[J].微生物学报,2016,56(10):1647-1655
[18]Weckbecker A,Hummel W.Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase,NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase[J].Biotechnology Letters,2004,26(22):1739-1744
[19]Yun HD,Yang YH,Cho BK,et al.Simultaneous synthesis of enantiomerically pure(R)-1-phenylethanol and(R)-α-methylbenzylamine from racemicα-methylbenzylamine usingω-transaminase/alcohol dehydrogenase/glucose dehydrogenase coupling reaction[J].Biotechnology Letters,2003,25(10):809-814
[20]Hummel W,Abokitse K,Drauz K,et al.Towards a large-scale asymmetric reduction process with isolated enzymes:expression of an(S)-alcohol dehydrogenase in E.coli and studies on the synthetic potential of this biocatalyst[J].Advanced Synthesis&Catalysis,2003,345(1/2):153-159
[2]Ni Y,Xu JH.Biocatalytic ketone reduction:a green and efficient access to enantiopure alcohols[J].Biotechnology Advances,2012,30(6):1279-1288
[3]Pollard DJ,Woodley JM.Biocatalysis for pharmaceutical intermediates:the future is now[J].Trends in Biotechnology,2007,25(2):66-73
[4]Forrest GL,Gonzalez B.Carbonyl reductase[J].Chemico-Biological Interactions,2000,129(1/2):21-40
[5]Ema T,Yagasaki H,Okita N,et al.Asymmetric reduction of ketones using recombinant E.coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity[J].Tetrahedron,2006,62(26):6143-6149
[6]Yasohara Y,Kizaki N,Hasegawa J,et al.Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae,involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate[J].Bioscience,Biotechnology,and Biochemistry,2000,64(7):1430-1436
[7]Schoemaker HE,Mink D,Wubbolts MG.Dispelling the myths--biocatalysis in industrial synthesis[J].Science,2003,299(5613):1694-1697
[8]Schmid A,Dordick JS,Hauer B,et al.Industrial biocatalysis today and tomorrow[J].Nature,2001,409(6817):258-268
[9]Betancor L,Berne C,Luckarift HR,et al.Coimmobilization of a redox enzyme and a cofactor regeneration system[J].Chemical Communications,2006(34):3640-3642
[10]Liu WF,Wang P.Cofactor regeneration for sustainable enzymatic biosynthesis[J].Biotechnology Advances,2007,25(4):369-384
[11]Yu HL,Huang L,Ni Y,et al.Advances in synthesis of chiral alcohols by carbonyl bioreduction[J].Chinese Journal of Bioprocess Engineering,2013,11(3):71-82(in Chinese)郁惠蕾,黄磊,倪燕,等.羰基生物还原法合成手性醇的研究进展[J].生物加工过程,2013,11(3):71-82
[12]Bommarius AS,Schwarm M,Drauz K.Biocatalysis to amino acid-based chiral pharmaceuticals-examples and perspectives[J].Journal of Molecular Catalysis B:Enzymatic,1998,5(1/4):1-11
[13]Bommarius AS,Drauz K,Hummel W,et al.Some new developments in reductive amtnation with cofactor regeneration[J].Biocatalysis,2009,10(1/4):37-47
[14]Yuwen WG,Zhang L,Wang ZX,et al.Co-expression of glucose dehydrogenase and carbonyl reductase in Escherichia coli and its application in asymmetric synthesis of d-ψ-ephedrine[J].Industrial Microbiology,2010,40(3):19-24(in Chinese)宇文伟刚,张梁,王正祥,等.羰基还原酶基因与葡萄糖脱氢酶基因在大肠杆菌中的共表达及其在不对称还原产麻黄碱中的初步应用[J].工业微生物,2010,40(3):19-24
[15]Li C,Qi NC,Peng G,et al.Screening and identification of S-phenylethanol-producing strain[J].Journal of Microbiology,2013,33(2):43-47(in Chinese)李成,戚南昌,彭果,等.S-苯乙醇高产菌株的筛选及鉴定[J].微生物学杂志,2013,33(2):43-47
[16]Sun Y,Zhang RZ,Xu Y.Co-expression of formate dehydrogenase form Candida boidinii and(R)-specific carbonyl reductase form Candida parapsilosis CCTCCM203011 in Escherichia coli[J].Acta Microbiologica Sinica,2008,48(12):1629-1633(in Chinese)孙莹,张荣珍,徐岩.(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达[J].微生物学报,2008,48(12):1629-1633
[17]Jiang JW,Zhang RZ,Zhou XT,et al.Efficient biosynthesis of(S)-1-phenyl-1,2-ethanediol catalyzed by(S)-carbonyl reductaseⅡand glucose dehydrogenase[J].Acta Microbiologica Sinica,2016,56(10):1647-1655(in Chinese)姜佳伟,张荣珍,周晓天,等.(S)-羰基还原酶Ⅱ与葡萄糖脱氢酶共催化高效合成(S)-苯乙二醇[J].微生物学报,2016,56(10):1647-1655
[18]Weckbecker A,Hummel W.Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase,NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase[J].Biotechnology Letters,2004,26(22):1739-1744
[19]Yun HD,Yang YH,Cho BK,et al.Simultaneous synthesis of enantiomerically pure(R)-1-phenylethanol and(R)-α-methylbenzylamine from racemicα-methylbenzylamine usingω-transaminase/alcohol dehydrogenase/glucose dehydrogenase coupling reaction[J].Biotechnology Letters,2003,25(10):809-814
[20]Hummel W,Abokitse K,Drauz K,et al.Towards a large-scale asymmetric reduction process with isolated enzymes:expression of an(S)-alcohol dehydrogenase in E.coli and studies on the synthetic potential of this biocatalyst[J].Advanced Synthesis&Catalysis,2003,345(1/2):153-159