基于多酶级联协调表达策略高效催化合成(S)-2-羟基丁酸  

Efficient cascade biosynthesis of(S)-2-hydroxybutyric acid

作　　者：田灵芝 周俊平 杨套伟[1] 张显[1] 邵明龙[1] 徐美娟[1] 饶志明[1] Lingzhi Tian;Junping Zhou;Taowei Yang;Xian Zhang;Minglong Shao;Meijuan Xu;Zhiming Rao(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2021年第12期4231-4242,共12页Chinese Journal of Biotechnology

基　　金：国家轻工技术与工程一流学科自主课题(No.LITE2018-06);国家自然科学基金(Nos.32070035,31770058);国家重点研发计划(No.2018YFA0900300);江苏省自然科学基金(No.BK20181205);宁夏回族自治区重点研发计划(No.2019BCH01002)资助。

摘　　要：2-羟基丁酸(2-hydroxybutyric acid,2-HBA)是合成生物可降解材料和各种药物的重要中间体,化学法合成的外消旋2-HBA需要去消旋才能获得光学纯对映异构体,应用于工业。文中通过在大肠杆菌Escherichiacoli BL21(DE3)中共表达苏氨酸脱氨酶(Threoninedeaminase,TD)、L-乳酸脱氢酶(L-lactatedehydrogenase,LDH)和甲酸脱氢酶(Formatedehydrogenase,FDH),构建(S)-2-HBA的合成途径及其辅因子NADH的循环系统,实现了基于三酶级联反应催化底物L-苏氨酸合成(S)-2-HBA。为了解决多酶级联催化反应中中间产物2-酮丁酸的生成速率和消耗率不匹配的问题,文中通过启动子工程策略来调控TD和FDH的表达水平,获得了多酶催化速率平衡的重组大肠杆菌P21285FDH-T7V7827。在5 L发酵罐水平,全细胞催化反应16 h,(S)-2-HBA的最高产量为143g/L,摩尔转化率为97%,为迄今报道的最高产量的1.83倍,使其具有较强的工业化应用潜力。此外,结果表明,在单细胞中构建可调节的多酶协调表达系统对生物催化制备羟基酸类化合物具有重要意义。2-Hydroxybutyric acid(2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase(TD), NAD-dependent L-lactate dehydrogenase(LDH) and formate dehydrogenase(FDH) for production of optically pure(S)-2-HBA from bulk chemical L-threonine(L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285 FDH-T7 V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285 FDH-T7 V7827 was able to efficiently produce(S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application.Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.

关 键 词：级联生物催化 (S)-2-羟基丁酸 多酶协调表达系统 NADH循环 启动子工程 

分 类 号：TQ225.41[化学工程—有机化工] TQ426.97