title: Reengineering of the feedback-inhibition enzyme N-acetyl-L-glutamate kinase to enhance L-arginine production in Corynebacterium crenatum.
abstract: 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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China. xumeijuan@jiangnan.edu.cn.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Xiaoxun
authors.initials: X
authors.lastname: Ge
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, People's Republic of China. raozhm@jiangnan.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003352:Corynebacterium / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D007218:Industrial Microbiology; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase
keywords: Corynebacterium crenatum; L-Arginine; Mutagenesis; N-acetyl-L-glutamate kinase (NAGK); Protein engineering