Engineering Thermoanaerobacter ethanolicus for co-culturing with Clostridium thermocellum for efficient utilization of cellulosic biomass

Clostridium thermocellum is a candidate microorganism for consolidated bioprocessing since it can rapidly hydrolyze cellulosic material, with the aid of a complexed cellulase system termed the cellulosome, and ferment the hydrolysis products to ethanol and organic acids. Genetic tools have been developed and successfully applied to increase its ethanol yield. However, current C. thermocellum strains do not utilize hemicellulose although they can solubilize it together with cellulose. Hemicellulose consists a significantly fraction of carbohydrate in most cellulosic biomass. It needs to be converted together with cellulose for high yield of product. Thermoanaerobacter ethanolicus, a natural hemicellulose utilizer, has good overlapping growth temperature and pH with C. thermocellum. Wild-type T. ethanolicus strains produce lactic acid and acetic acid along with ethanol. Production of these acids need to be eliminated for high product yield. We will report development of a clean gene deletion and integration system for T. ethanolicus, characterization of its alcohol dehydrogenases and hydrogenases, deletion of acid production pathways, strain evolution for high substrate utilization, and performance of co-culturing with C. thermocellum using high concentration of solid substrate.