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Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of l-isoleucine biosynthesis

  • Metabolic Engineering and Synthetic Biology
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Previously we have characterized a threonine dehydratase mutant TDF383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHASP176S, D426E, L575W (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AKA279T (encoded by lysC1) and a homoserine dehydrogenase mutant HDG378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AKA279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HDG378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TDF383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HDG378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (NSFC31370131), National Key Basic Research Program of China (973 Program 2012CB725202) and Fundamental Research Funds for the Central Universities (JUDCF11025). We thank Prof. Makoto Nishiyama (University of Tokyo) for providing information on inactivation of intrinsic ribosome binding site for the β subunit of AK, and Ms. Shuying Wang (Jiangnan University) for providing technical advice on HPLC analysis.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Xunyan Dong & **aoyuan Wang

  2. Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Xunyan Dong, Yue Zhao & Jianxun Zhao

  3. Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China

    Xunyan Dong, Yue Zhao, Jianxun Zhao & **aoyuan Wang

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  1. Xunyan Dong
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  2. Yue Zhao
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  4. **aoyuan Wang
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Correspondence to **aoyuan Wang.

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Dong, X., Zhao, Y., Zhao, J. et al. Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of l-isoleucine biosynthesis. J Ind Microbiol Biotechnol 43, 873–885 (2016). https://doi.org/10.1007/s10295-016-1763-5

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