Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum

• Published in: Journal of industrial microbiology & biotechnology 2016-01, Vol.43 (1), p.55-66
• Main Authors: Zhao, Qinqin, Luo, Yuchang, Dou, Wenfang, Zhang, Xian, Zhang, Xiaomei, Zhang, Weiwei, Xu, Meijuan, Geng, Yan, Rao, Zhiming, Xu, Zhenghong
• Format: Article
• Language: English
• Subjects: Amino acids; Analysis; arginine; Arginine - biosynthesis; Arginine - metabolism; Argininosuccinate Lyase - metabolism; Argininosuccinate Synthase - metabolism; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; batch fermentation; biochemical pathways; Biochemistry; Bioinformatics; Biomedical and Life Sciences; Bioreactors; Biosynthesis; Biosynthetic Pathways - genetics; Biotechnology; Chromosomes; citrulline; Citrulline - metabolism; Corynebacterium; Corynebacterium - genetics; Corynebacterium - metabolism; Enzymes; Feedback, Physiological; Fermentation; Gene Expression Regulation, Bacterial; Genes; Genes, Bacterial - genetics; Genetic Engineering; Genetics and Molecular Biology of Industrial Organisms; Glucose; Inorganic Chemistry; Kinases; Life Sciences; Metabolic Flux Analysis; Metabolism; Metabolites; Microbiology; Mutagenesis; Mutation; Ornithine - metabolism; Pharmaceutical industry; Phosphotransferases (Carboxyl Group Acceptor) - metabolism; Plasmids; point mutation; Productivity; Studies; Transcription factors; Transcription, Genetic
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-015-1692-8

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.