title: Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum.
abstract: Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Yuchang
authors.initials: Y
authors.lastname: Luo
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Wenfang
authors.initials: W
authors.lastname: Dou
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Geng
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China. raozhm@jiangnan.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, People's Republic of China. zhenghxu@jiangnan.edu.cn.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
mesh_terms: D001120:Arginine / Q000096:biosynthesis / Q000378:metabolism*; D001123:Argininosuccinate Lyase / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D002956:Citrulline / Q000378:metabolism; D003352:Corynebacterium / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D064688:Metabolic Flux Analysis; D009952:Ornithine / Q000378:metabolism; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D002956:Citrulline; D001120:Arginine; D009952:Ornithine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase; D001123:Argininosuccinate Lyase; D001124:Argininosuccinate Synthase
keywords: Corynebacterium crenatum; Feedback inhibition; L-arginine; L-citrulline; L-ornithine; argGH