苯乙酮转化为(S)-a-苯乙醇的工程菌构建

Construction of engineering bacteria for conversion of acetophenone to (S)-α-phenylethanol

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(Rhodococcus erythropolis)和博伊丁假丝酵母(Candida boidinii)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。

Abstract:

[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 (R1F2) and pRSFDuet-FDH-ReADH (F1R2) 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 R1F2 were 6.7 U/mL and 7.6 U/mL, respectively. R1F2 had higher catalytic activity than F1R2. 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 R1F2 is 7.5 and 30 °C. R1F2 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.