Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli.
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 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield.
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China.
Xinna
X
Zhu
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China; University of Chinese Academy of Sciences, China.
Zaigao
Z
Tan
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China.
Hongtao
H
Xu
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China; College of Biotechnology, Tianjin University of Science & Technology, China.
Jing
J
Chen
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China.
Jinlei
J
Tang
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China. Electronic address: zhang_xl@tib.cas.cn.
Xueli
X
Zhang
D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D009243:NAD / Q000235:genetics / Q000378:metabolism; D061086:NADP Transhydrogenase, B-Specific / Q000235:genetics / Q000378:metabolism; D010427:Pentose Phosphate Pathway / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D019802:Succinic Acid; D061086:NADP Transhydrogenase, B-Specific; C556052:sthA protein, E coli
Pentose phosphate pathway; Pyruvate dehydrogenase; Reducing equivalent; Succinate; Transhydrogenase