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Abstract
The dependence on foreign oil in the United States has increased the demand for alternative fuels. Ethanol production from food sources in the United States could create heavy competition for the ultimate utilization of crops. However, ethanol production from pectin-rich lignocellulosic biomass provides a noncompetitive alternative fuel source. Therefore, adaptation of potential ethanologens to such substrates is very important. Ethanologen Escherichia coli strain LY40A containing the cellobiose phosphotransferase system from Klebsiella oxytoca and the alcohol dehydrogenase and pyruvate decarboxylase from Zymomonas mobilis was previously engineered to express a pectate lyase from Erwinia chrysanthemi (casAB; pelE), yielding strain JP07. To obtain effective secretion of PelE, the Sec-independent pathway out genes from E. chrysanthemi on the cosmid pCPP2006 were provided to strain JP07 to construct strain JP07C. E. coli strains LY40A, JP07, and JP07C possessed significant cellobiase activity in cell lysates, while only strain JP07C demonstrated extracellular pectate lyase activity. Fermentation with sugar beet pulp at very low fungal enzyme loads during saccharification revealed significantly higher ethanol production for LY40A and JP07C compared to the parent strain, E. coli KO11. While JP07C ethanol yields were not considerably higher than LY40A, investigation of oligogalacturonide polymerization showed an increased breakdown of biomass to small chain (degree of polymerization 6) oligogalacturonides. The activity of pectate lyases along with other additional enzymes in ethanol fermentations could lead to more efficient breakdown of noncompetitive substrates to produce fuel ethanol.