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In silico profiling of cell growth and succinate production in Escherichia coli NZN111.

BACKGROUND: Succinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB , respectively. However, this double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and consequently disables proper growth under anaerobic conditions.

RESULTS: In this study, genome-scale metabolic core model of E. coli was reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strain E. coli NZN111. Non-optimized artificial centering hit-and-run (ACHR) method and metabolite flux-sum analysis were utilized to evaluate metabolic characteristics of strains. Thus, metabolic characteristics of the strains wild-type E. coli , ldhA mutant, pflB mutant, and NZN111 under anaerobic conditions were successfully unraveled.

CONCLUSIONS: We found a viewpoint contrary to the widespread realization that an NADH/NAD+ in NZN111 mainly resulted from the inactivation of PFL rather than the inactivation of LDH. In addition, the two alternative anaerobic fermentation pathways, lactate and ethanol production pathways, were blocked owing to the disruption of ldhA and pflB , resulting in insufficient NAD+ regeneration to oxidize or metabolize glucose for cell growth. Furthermore, we speculated reaction NADH16, the conversion of ubiquinone-8 (q8) to ubiquinol-8 (q8h2), as a potential amplification target for anaerobically improving cell growth and succinate production in NZN111.

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