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Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production

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Abstract

N-Acetyl-l-glutamate kinase (EC 2.7.2.8) is first committed in the specific l-arginine pathway of Corynebacterium sp. A limited increase of l-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that l-arginine feedback inhibition plays an influence on the l-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I R0.5 efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I R0.5 of l-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust l-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The l-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by l-arginine. Thus l-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the l-arginine metabolic pathway by deregulating the key enzyme of the pathway.

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Abbreviations

NAGK:

N-Acetyl-l-glutamate kinase

Ccre_NAGK:

N-Acetyl-l-glutamate kinase of Corynebacterium crenatum

WT:

Wild-type

NAG:

N-Acetyl-l-glutamate

I R0.5 :

Inhibition constant (the l-arginine concentration yields 50% inhibition)

EMS:

Ethylmethane sulfonate

DCW:

Dry cell weight

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgments

This work was supported by the High-tech Research and Development Programs of China (2007AA02Z207), the National Basic Research Program of China (2007CB707804), the National Natural Science Foundation of China (30970056), the Program for New Century Excellent Talents in University (NCET-07-0380, NCET-10-0459), the Fundamental Research Funds for the Central Universities (JUSRP31001), the Program of Introducing Talents of Discipline to Universities (111-2-06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, 214122, People’s Republic of China

    Meijuan Xu, Zhiming Rao, Juan Yang, Jian Jin & Zhenghong Xu

  2. Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Wenfang Dou, Jian Jin & Zhenghong Xu

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  1. Meijuan Xu
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  2. Zhiming Rao
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  4. Juan Yang
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Correspondence to Zhiming Rao or Zhenghong Xu.

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Xu, M., Rao, Z., Dou, W. et al. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production. Amino Acids 43, 255–266 (2012). https://doi.org/10.1007/s00726-011-1069-x

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