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

  • Qinqin Zhao
  • Yuchang Luo
  • Wenfang Dou
  • Xian Zhang
  • Xiaomei Zhang
  • Weiwei Zhang
  • Meijuan Xu
  • Yan Geng
  • Zhiming RaoEmail author
  • Zhenghong XuEmail author
Genetics and Molecular Biology of Industrial Organisms

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.

Keywords

Corynebacterium crenatum l-arginine Feedback inhibition argGH l-citrulline l-ornithine 

Notes

Acknowledgments

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).

Compliance with ethical standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Supplementary material

10295_2015_1692_MOESM1_ESM.docx (428 kb)
Supplementary material 1 (DOCX 428 kb)
10295_2015_1692_MOESM2_ESM.docx (195 kb)
Supplementary Fig. S1 Analysis of intracellular (filled circles) and extracellular (filled squares) l-arginine concentration during the fermentation course. (All assays were performed by triplicate cultures, standard deviations of the biological replicates were represented by error bars.) (DOCX 194 kb)
10295_2015_1692_MOESM3_ESM.docx (90 kb)
Supplementary Fig. S2 l-lysine concentration of SYPA5-5, H-7 and R-8, respectively after culturing for 88 h in shake flask batch fermentation. (All assays were performed by triplicate cultures, standard deviations of the biological replicates were represented by error bars.) (DOCX 89 kb)
10295_2015_1692_MOESM4_ESM.docx (146 kb)
Supplementary Fig. S3 Biomass (black bars), l-arginine concentration (gray bars) and glucose concentration (white bars) of H-7-GH fermented in different concentration of yeast extract. (DOCX 428 kb) (DOCX 145 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2015

Authors and Affiliations

  • Qinqin Zhao
    • 1
  • Yuchang Luo
    • 1
  • Wenfang Dou
    • 1
  • Xian Zhang
    • 2
  • Xiaomei Zhang
    • 1
  • Weiwei Zhang
    • 1
  • Meijuan Xu
    • 2
  • Yan Geng
    • 1
  • Zhiming Rao
    • 2
    Email author
  • Zhenghong Xu
    • 1
    Email author
  1. 1.School of Pharmaceutical ScienceJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.Key Laboratory of Industrial Biotechnology of Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiPeople’s Republic of China

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