Genes restoring redox balance in fermentation-deficient E. coli NZN111

The objectives of this study were to improve understanding of the biochemical mechanisms underlying the growth defects resulting from deletion of pflB and ldhA in E. coli (strain NZN111) and identify genes for which overexpression would relieve this growth defect. Our approach involved the application of a mixed library selection method [Lynch et al., 2007. SCALEs: multiscale analysis of library enrichment. Nature Methods 4, 87–93.] to identify genes for which increased copy number improved growth of E. coli NZN111 under microaerobic conditions. This method employs libraries that cover the genome at a higher resolution relative to the conventional library methods. Our results indicate that NZN111 is growth impaired primarily due to unusually high intracellular NADH/NAD+ ratios as opposed to limitations in intracellular acetyl-coA pools or pyruvate accumulation. We report the effect of several genetic and biochemical methods for decreasing the intracellular redox ratio on NZN111 growth and succinate production, including strategies that result in a 5-fold increase in growth in M9 media under microaerobic conditions and up to a 20% increase in succinate production. This work provides new insights into the growth defects resulting from ldhA and pflB growth defects in E. coli as well as a list of genes that can be used to improve growth and production of succinate and other metabolic products where redox imbalances are a key limitation.