S31: Cellolosic isobutanol production via a synthetic fungus-bacterium consortium

Present approaches to microbial cellulosic biofuel production are largely based on the “superbug” paradigm of aiming to incorporate all required functionalities (optimized biofuel production pathways, cellulase production, etc.) into a single organism. Inspired by the ubiquity of synergistic microbial communities in nature, we explore an alternative approach for biofuel production: the design, construction, and characterization of a synthetic microbial consortium consisting of multiple species which cooperate to directly convert lignocellulosic biomass to isobutanol, a promising next-generation biofuel.  Three microbial specialists are utilized: the fungus Trichoderma reesei RUTC30, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into component hexose and pentose sugars, and engineered Escherichia coli hexose and pentose specialists which ferment hexose and pentose sugars to isobutanol, respectively.  We develop and experimentally validate a comprehensive modeling framework for cellulolytic fungi-bacteria consortia, allowing us to elucidate key ecological interactions.  Using stability and sensitivity analyses, we identify possible population equilibria and key parameters controlling the partition of carbon flow between T. reesei and E. coli.  We demonstrate direct conversion of microcrystalline cellulose and AFEX pre-treated biomass to isobutanol with the T. reesei / E. coli consortium, with titers up to 1.7 g/L and 41% of theoretical yield.  While we offer isobutanol production as a proof-of-concept application, the modularity of our system will allow it to be readily adapted to the large portfolio of existing E. coli strains metabolically engineered to produce biofuels or commodity chemicals.