Development of an NAD(H)-Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids
Development of an NAD(H)-Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids
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DOI：
10.1002/adsc.202101441
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发表时间：
2022-03-18
影响因子：
5.4
通讯作者：
Zheng, Yu-Guo
中科院分区：
文献类型：
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作者：
Cheng, Feng;Zhang, Jia-Min;Zheng, Yu-Guo
手性氨基酸广泛应用于医药、食品、化妆品和农业等行业。以l-phosphinothricin （l-PPT，一种手性非天然氨基酸）为代表，是一种广谱除草剂。一种高效、低成本的NAD(H)驱动生物催化系统用于手性氨基酸（以l-PPT为重点）的不对称合成。开发这种生物催化体系的关键是获得对l-PPT前酮PPO具有高催化性能的nadh依赖性生物催化剂。本研究通过基因挖掘和蛋白工程技术，从堆肥赖氨酸芽胞杆菌（Lysinibacillus composti, LcGluDH）中获得了具有预期活性的谷氨酸脱氢酶。硅分析表明，底物结合袋的体积从330.5埃(3)大幅增加到409.6埃(3)。通过在375位引入带正电的氨基酸或芳香氨基酸，提高了LcGluDH的稳定性（T-m值从47.3℃提高到65.3℃）。通过将工程的LcGluDH与NAD（+）依赖性的FDH偶联构建所需的生物催化体系。通过该生物催化体系，批量生产l-PPT的时空产率高达207.3 g。l - 1。day(-1))具有严格的立体选择性（l-PPT>的ee为99%）。此外，开发的NAD(H)驱动生物催化体系还合成了8种具有高ee值的手性氨基酸。
Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l-phosphinothricin (l-PPT, a chiral non-natural amino acid) is a broad-spectrum herbicide. An NAD(H)-driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l-PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH-dependent biocatalyst with high catalytic performance toward l-PPT pro-ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate-binding pocket was substantially enlarged from 330.5 angstrom(3) to 409.6 angstrom(3). The stability of LcGluDH was increased (T-m value increased from 47.3 degrees C to 65.3 degrees C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD(+)-dependent FDH. Through this biocatalytic system, the batch production of l-PPT exhibited high space-time yield (207.3 g . L-1 . day(-1)) with strict stereoselectivity (ee of l-PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)-driven biocatalytic system with high ee values.