Ethanologenic bacterial biocatalysts were generated: one series was engineered specifically for grass fermentations and a second series was designed for pectin-rich biomass fermentations. Ethanologenic Escherichia coli KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) for transport and metabolism of cellobiose, a dimer of glucose, and was designated LY40A. This strain was further engineered for production of xylanase, useful for degradation of grass biomass. In order to optimize pectin degradation, a pectate lyase gene (pelB) from Paenibacillus amylolyticus, the Sec-dependent pathway out genes from E. chrysanthemi, and an oligogalacturonide lyase from E. chrysanthemi were added.
Using energy cane and sugar cane as substrates, monomeric sugars were liberated and varied amongst the nine cultivars examined. Cellobiase and xylanase activities were present and ethanol was produced. With sugar beet pulp as feedstock, xylose, arabinose, glucose, and galacturonic acid were released via enzymatic digestion. Engineered biocatalysts expressed plant cell wall degrading activity, transport and metabolism of cellobiose, and reduced the commercial enzyme needed during fermentations, although not completely replacing them.