2-38: Knocking out ldhA in E. coli JP07 to increase ethanol production during fermentation of pectin-rich materials

            Pectin-rich biomass is an abundant and underused resource that consists of biomass such as whole fruits and vegetables and the wastes leftover from industrial processes, like sugar beet pulp and citrus waste.  These biomasses are categorized as pectin-rich because they contain roughly equal amounts of the polysaccharides cellulose, hemicellulose and pectin based on dry weight.  A strain of Escherichia coli has been engineered from E. coli KO11 (containing pdc and adh from the Zymomonas mobilis ethanol pathway) to tailor it for ethanol production from pectin-rich materials.  These strains contain casAB from Klebseilla oxytoca for cellobiose uptake and metabolism and a suite of genes (pelE, ogl, and out secretion genes) from Erwinia chrysanthemi for the degradation and uptake of pectin.  This engineered strain has been shown to ferment cellobiose and pectin, but like wild type strains of E. coli begins to produce increasing concentrations of lactic acid as pH decreases.  Therefore, ldhA (lactate dehydrogenase) was knocked out of the genome of E. coli JP07 using Red/ET recombination.  The fermentation capabilities of the new strain, E. coli JP07 ΔldhA, was examined through fermentations of 10% w/v model substrate (17% w/v glucose, 17% w/v cellobiose, 33% w/v arabinose, and 33% w/v galacturonic acid).  After removal of ldhA, lactic acid production had significantly decreased.