Design and Construction of Synthetic Fungal-Bacterial Consortia for Direct Production of Biofuels and Commodity Chemicals From Cellulosic Feedstocks

In nature microbes usually live in synergistic communities in which individual species with specialized roles cooperate to perform complex tasks.  The powerful features of natural consortia have inspired great interest in engineering synthetic consortia for biotechnology applications.  However, there have been few reports of synthetic consortia being deployed in real-world applications, and stability, robustness, and regulation of population composition remain key barriers.  In this work we bridge ecology theory with engineering principles to develop robust synthetic fungi-bacteria consortia for efficient biosynthesis of valuable products from lignocellulosic feedstocks.  The required biological functions are divided between two specialists: the fungus Trichoderma reesei, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into desired products.  We developed and experimentally validated a comprehensive modeling framework for T. reesei / E. coli (TrEc) consortia, providing insights on key determinants of performance.  We show that ecological dynamics within the TrEc consortium lead to stable coexistence between T. reesei and E. coli, and provide mechanisms for tuning population composition. To illustrate bioprocessing applications, we demonstrate direct conversion of microcrystalline cellulose and AFEX pre-treated corn stover to isobutanol with the TrEc consortium, achieving titers up to 1.86 g/L and 62% of theoretical yield, which represent the highest reported to date for conversion of cellulosic substrates to next-generation biofuels.  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 microbial strains metabolically engineered to produce valuable products, and we are currently validating the consortia platform for production of specialty and commodity chemicals.