Investigation of the Effects of Stress Related Genes on Escherichia coli Fermentation

The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a sustainable future. One potential option is the utilization of metabolically engineered bacteria to produce value-added chemicals during fermentation. Currently, numerous strains of metabolically engineered Escherichia coli have shown great capacity to specialize in the production of high titers of a desired chemical. These metabolic systems, however, are constrained by the biological limits of E. coli itself. During fermentation, E. coli grows to less than one twentieth of the density that aerobically growing cultures can reach. I hypothesized that this decrease in growth during fermentation is due to cellular stress associated with fermentative growth, likely caused by stress related genes. These genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress response, may have an impact on fermentative growth constraints. Through transcriptional analysis, I identified that the genes pspC and relE are highly expressed in fermenting strains of both wild type and metabolically engineered E. coli. Fermentation of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX, yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ, yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN significantly arrested cell growth in both tube and fermentation tests, thus proving these toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli fermentation