High Ethanol Titers from Cellulose using Metabolically Engineered Thermophilic, Anaerobic Microbes Running Title: High Titer Cellulosic Ethanol from Thermophiles

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene, and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The ∆ldh ∆pta mutant was evolved for 2,000 hrs, resulting in a stable strain with 40:1 ethanol selectivity and 4.2 fold increase in ethanol yield over the wild type strain. Ethanol production from cellulose was investigated with an engineered co-culture of organic acid deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/L Avicel by this co-culture resulted in 38 g/L ethanol with acetic and lactic acids below detection limits in 146 hours. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.