Toward conversion of corn stover to ethanol by Saccharomyces cerevisiae displaying a cellulosome

Cellulosomes, synergistic complexes of cellulase enzymes bound to a protein scaffold, are found in many anaerobic species of cellulose degrading microbes. Engineering strains of the ethanologenic yeast Saccharomyces cerevisiae to display such a structure would create microbes well suited for the consolidated bioprocessing of pretreated lignocellulosic biomass to ethanol. Efficient hydrolysis of cellulose to fermentable sugar requires the synergistic activities of exoglucanases and endoglucanases to cleave from the end and middle of the chain, respectively and a β-glucosidase to convert cellobiose to glucose. A series of well characterized glycoside hydrolases were chemically synthesized and fused to the dockerin domain from Ruminococcus flavefaciens endoglucanase Cel44A in an expression vector. Each construct that resulted in the secretion of a highly active heterologous enzyme was subsequently integrated into a separate chromosome of a yeast strain expressing a scaffold from R. flavefaciens. Using the Green Monster method, we enriched for multiple genetic changes through rounds of yeast mating and meiosis, generating a single strain that expresses these enzymes complexed in a cellulosome. Growth and ethanol production capabilities of this strain are being evaluated with phosphoric-acid swollen cellulose, Avicel, and acid-pretreated corn stover as substrates, to see if this strain is capable of direct conversion of cellulose to ethanol. With this methodology firmly established to construct yeast strains that display multifunctional cellulosomes, new strains with different enzyme combinations can be rapidly made and screened for novel synergies.