4-06: Genetic modification of E. coli KO11 for improved ethanol yield from gluconate

The conventional biochemical platform for cellulosic ethanol involves five distinct steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as the reactive intermediate for the subsequent fermentation.  Our research efforts aim to develop a novel biochemical route for fuels and chemical production which replaces the three most expensive steps in the conventional platform (pretreatment, cellulase production, enzymatic hydrolysis) with a single biological step.  Cellulolytic microorganism(s) that can secrete all the enzymes needed to hydrolyze cellulose and hemicellulose in spite of the presence of lignin can be modified to convert most of the carbohydrate contained in the cellulosic biomass to sugar aldonates.  By over-expressing cellobiose dehydrogenase (CDH) and knocking down copies of B-glucosidase, we can enhance cellobionate production.  In a second step, sugar aldonates are utilized as the carbon source to produce ethanol and other products.  Experiments in our lab have demonstrated that the ethanologen Escherichia coli KO11 can metabolize gluconate via the Entner-Doudoroff pathway and produce ethanol at high yield and a rate even faster than that of glucose.  However, a significant portion of the carbon flow is directed to lactic and acetic acid production.  By knocking out the gene for the competing lactic acid pathway (ldh), the carbon flow can be further directed to ethanol production.  In this study, we report the effects of knocking out the ldh gene to improve ethanol yield.