多酶组合催化制备L-高苯丙氨酸

A multi-enzyme cascade reaction for the production of L-homophenylalanine

【目的】L-高苯丙氨酸(L-HPA)是许多医药化学品的重要中间体,化学合成法生产L-HPA反应复杂、环境污染严重,本研究旨在开发高效环保的L-HPA酶法合成路线。【方法】采用模块化组装的方法,构建了一条以甘氨酸和苯乙醛为底物高产L-HPA的新途径。【结果】首先,根据文献挖掘设计了一条由苏氨酸醛缩酶(TA)、苏氨酸脱氨酶(TD)、苯丙氨酸脱氢酶(PheDH)和甲酸脱氢酶(FDH)组成的多酶组合催化途径,用于L-HPA的合成。其次,根据氨基基团的引入和重构,将L-HPA多酶组合催化途径分为基础单元和扩增单元,基础单元包括TA和TD,扩增单元包括PheDH和FDH。然后,利用不同表达水平的质粒,对基础单元和扩增单元进行蛋白表达的组合调节,获得最优工程菌BL21-C-M1-R-M2,使L-HPA产量达到208.6 mg/L。最后,我们对全细胞转化体系进行优化,使L-HPA产量进一步提高到1226.6 mg/L,苯乙醛摩尔转化率为34.2%。【结论】该工艺路线绿色高效,为未来大规模生产L-HPA奠定基础。

[Objective]L-homophenylalanine(L-HPA)is a key intermediate for many pharmaceuticals and pesticides.As chemical synthesis of L-HPA was complicated and environmentally unfriendly,this study aimed to develop an efficient and environmentally friendly route for the biosynthesis of L-HPA.[Methods]A multi-enzyme cascade reaction for L-HPA production from L-glycine and phenylacetaldehyde was constructed by modular assembly method.[Results]Firstly,the multi-enzyme cascade reaction,which consisted of threonine aldolases(TA),threonine deaminase(TD),phenylalanine dehydrogenase(PheDH),and formate dehydrogenase(FDH),was constructed by literature mining.Then,according to the introduction and deletion of chiral-OH/-NH2 groups,the whole cascade reaction was divided into two parts,in which the basic parts included TA and TD and the extender parts included PheDH and FDH.Next,the two modules were assembled and optimized with different plasmids,enhancing the L-HPA production up to 208.6 mg/L in the optimal engineered strain BL21-C-M1-R-M2.Finally,the whole cell catalytic system was optimized to improve the L-HPA production up to 1226.6 mg/L and the molar conversion of phenylacetaldehyde reached 34.2%.[Conclusion]This environmental-friendly and efficient process represented a promising strategy for large-scale L-HPA production in the future.