Reengineering of the feedback-inhibition enzyme N-acetyl-l-glutamate kinase to enhance l-arginine production in Corynebacterium crenatum

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• 作者：Jingjing Zhang ; Meijuan Xu ; Xiaoxun Ge…
• 关键词：N ; acetyl ; l ; glutamate kinase (NAGK) ; Protein engineering ; Mutagenesis ; l ; Arginine ; Corynebacterium crenatum
• 刊名：Journal of Industrial Microbiology & Biotechnology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：44
• 期：2
• 页码：271-283
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Microbiology; Biochemistry, general; Inorganic Chemistry; Genetic Engineering; Biotechnology; Bioinformatics;
• 出版者：Springer Berlin Heidelberg
• ISSN：1476-5535
• 卷排序：44

文摘

N-acetyl-l-glutamate kinase (NAGK) catalyzes the second step of l-arginine biosynthesis and is inhibited by l-arginine in Corynebacterium crenatum. To ascertain the basis for the arginine sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Typically, the E19Y mutant displayed the greatest deregulation of l-arginine feedback inhibition. An equally important strategy is to improve the catalytic activity and thermostability of CcNAGK. For further strain improvement, we used site-directed mutagenesis to identify mutations that improve CcNAGK. Results identified variants I74V, F91H and K234T display higher specific activity and thermostability. The l-arginine yield and productivity of the recombinant strain C. crenatum SYPA-EH3 (which possesses a combination of all four mutant sites, E19Y/I74V/F91H/K234T) reached 61.2 and 0.638 g/L/h, respectively, after 96 h in 5 L bioreactor fermentation, an increase of approximately 41.8% compared with the initial strain.