Controlling the transcription levels of argGH redistributed l-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum
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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.
KeywordsCorynebacterium crenatum l-arginine Feedback inhibition argGH l-citrulline l-ornithine
This work was supported by the High Tech Development Program of China (No. 2012AA022102), the National Natural Science Foundation of China (31300028) and the Jiangsu Provincial National Basic Research Program (BK20130137).
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Conflict of interest
The authors declare no financial or commercial conflict of interest.
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