17-02: Engineering yeasts for next generation ethanol production

Conversion of cellulose or starch sugars to ethanol via a biological route is preceded by enzymatic conversion of the substrate to soluble sugars that can be assimilated by a fermenting organism.  Consolidation of these events in a single process step via a cellulolytic or amylolytic microorganism(s) is a promising approach to low-cost production of fuels and chemicals.  One strategy for developing a microorganism capable of such consolidated bioprocessing involves engineering Saccharomyces cerevisiae so that it expresses a heterologous enzyme system enabling cellulose or starch utilization. We have developed the fundamental principles behind consolidated bioprocessing as a microbial phenomenon through the successful expression of the three major cellulase activities (β‑glucosidase, β‑endoglucanase and cellobiohydrolase) and two amylolytic activities (glucoamylase and α-amylase) in S. cerevisiae.  It was also shown that different heterologous cellulases yielded varying degrees of cellular stress indicated by induction of the unfolded protein response.  Host cell engineering could also be demonstrated to yield higher secreted cellulase titers. We have subsequently enabled S. cerevisiae to grow on cellobiose, amorphous cellulose and crystalline cellulose through the combinational expression of the three cellulase activities in commercial yeast strains. We could demonstrate efficient conversion of unwashed pretreated hardwoods to ethanol with a 2.5-fold reduction in cellulase loading at pilot scale using proprietary CBP-yeast strains. The conversion of raw starch could also be demonstrated at lab scale by an amylolytic S. cerevisiae recombinant.  These breakthroughs bring the application of CBP at commercial scale ever closer.