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Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13 C‐metabolic flux analysis
Author(s) -
He Lian,
Xiao Yi,
Gebreselassie Nikodimos,
Zhang Fuzhong,
Antoniewicz Maciek R.,
Tang Yinjie J.,
Peng Lifeng
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25124
Subject(s) - biochemistry , metabolic flux analysis , flux (metallurgy) , metabolic engineering , cofactor , escherichia coli , fatty acid , pentose phosphate pathway , phosphoenolpyruvate carboxykinase , metabolic pathway , nad+ kinase , overproduction , metabolism , fatty acid synthesis , flux balance analysis , chemistry , citric acid cycle , biology , glycolysis , enzyme , gene , organic chemistry
We engineered a fatty acid overproducing Escherichia coli strain through overexpressing tesA (“pull”) and fadR (“push”) and knocking out fadE (“block”). This “pull‐push‐block” strategy yielded 0.17 g of fatty acids (C 12 –C 18 ) per gram of glucose (equivalent to 48% of the maximum theoretical yield) in batch cultures during the exponential growth phase under aerobic conditions. Metabolic fluxes were determined for the engineered E. coli and its control strain using tracer ([1,2‐ 13 C]glucose) experiments and 13 C‐metabolic flux analysis. Cofactor (NADPH) and energy (ATP) balances were also investigated for both strains based on estimated fluxes. Compared to the control strain, fatty acid overproduction led to significant metabolic responses in the central metabolism: (1) Acetic acid secretion flux decreased 10‐fold; (2) Pentose phosphate pathway and Entner–Doudoroff pathway fluxes increased 1.5‐ and 2.0‐fold, respectively; (3) Biomass synthesis flux was reduced 1.9‐fold; (4) Anaplerotic phosphoenolpyruvate carboxylation flux decreased 1.7‐fold; (5) Transhydrogenation flux converting NADH to NADPH increased by 1.7‐fold. Real‐time quantitative RT‐PCR analysis revealed the engineered strain increased the transcription levels of pntA (encoding the membrane‐bound transhydrogenase) by 2.1‐fold and udhA (encoding the soluble transhydrogenase) by 1.4‐fold, which is in agreement with the increased transhydrogenation flux. Cofactor and energy balances analyses showed that the fatty acid overproducing E. coli consumed significantly higher cellular maintenance energy than the control strain. We discussed the strategies to future strain development and process improvements for fatty acid production in E. coli . Biotechnol. Bioeng. 2014;111: 575–585. © 2013 Wiley Periodicals, Inc.