Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli |
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| Institution: | 1. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China;2. University of Chinese Academy of Sciences, China;3. College of Biotechnology, Tianjin University of Science & Technology, China;1. The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China;2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China;3. School of Medicine and Pharmaceuticals, Jiangnan University, Wuxi, Jiangsu 214122, PR China;1. Department of Bioengineering, Rice University, Houston, TX, USA;2. Technology Holding, Salt Lake City, UT, USA;3. Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA;1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;3. Hangzhou Bioking Biochemical Engineering Co. Ltd, Hangzhou 311106, China;4. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA;1. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Department of Biochemistry, Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, Nigeria;1. School of Life Science, Linyi University, Linyi 276000, China;2. State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China;1. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China;2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China;3. Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, PR China |
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| Abstract: | Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31 mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33 mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5 mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. |
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| Keywords: | Succinate Reducing equivalent Pyruvate dehydrogenase Pentose phosphate pathway Transhydrogenase |
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