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Enhancing l-Isoleucine Production by thrABC Overexpression Combined with alaT Deletion in Corynebacterium glutamicum

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Abstract

l-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for l-isoleucine synthesis. C. glutamicum YILWΔalaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for l-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher l-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of l-lysine, l-alanine, and l-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to l-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly.

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Acknowledgments

This work was supported by the National High Technology Research and Development Program (grant no. 2012AA02A703, 2012AA022102, 2013AA102106), by Tianjin Municipal Science and Technology Commission (grant no. 12ZCZDSY01900, 12ZXCXSY05900), by the Tianjin Municipal Education Commission (grant no. 20120630) and by Program for Changjiang Scholars and Innovative Research Team in University (IRT 1166).

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Authors and Affiliations

  1. National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin, 300457, China

    Jing Wang, Bing Wen, Qingyang Xu, Chenglin Zhang, Ning Chen & Xixian Xie

  2. Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin, 300457, China

    Jing Wang, Bing Wen, Qingyang Xu, Chenglin Zhang, Ning Chen & Xixian Xie

  3. Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin University of Science and Technology, Tianjin, 300457, China

    Jing Wang, Bing Wen, Qingyang Xu, Chenglin Zhang, Ning Chen & Xixian Xie

  4. Tianjin Research Institute of Industrial Microbiology, Tianjin, 300462, China

    Bing Wen

  5. College of Biological and Agricultural Engineering, Jilin University, Changchun, 130022, China

    Jian Wang

  6. Metabolic Engineering Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Area, Tianjin, 300457, People’s Republic of China

    Ning Chen & Xixian Xie

Authors
  1. Jing Wang
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  2. Bing Wen
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  3. Jian Wang
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  4. Qingyang Xu
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  5. Chenglin Zhang
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  6. Ning Chen
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  7. Xixian Xie
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Correspondence to Ning Chen or Xixian Xie.

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Wang, J., Wen, B., Wang, J. et al. Enhancing l-Isoleucine Production by thrABC Overexpression Combined with alaT Deletion in Corynebacterium glutamicum . Appl Biochem Biotechnol 171, 20–30 (2013). https://doi.org/10.1007/s12010-013-0321-0

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  • DOI: https://doi.org/10.1007/s12010-013-0321-0

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