The engineering of a multivalent cellulosome on the cell surface of Saccharomyces cerevisiae

Consolidated bioprocessing (CBP) has the potential to increase the efficiency of cellulosic biofuel production.  In this approach, enzyme production, saccharification, and fermentation all occur in a single reactor, which reduces capital and operating costs associated with the process.  Toward efficient CBP, we aim to develop a cellulolytic Saccharomyces cerevisiae strain capable of direct biomass utilization and ethanol production at industrially relevant rates.  Some cellulolytic bacteria and fungi display on their cell surface large biomass-active protein complexes, termed cellulosomes.  The structural component of cellulosome is the scaffoldin protein, which consists of multiple cohesin domains separated by linker sequences.  Cellulases and other enzymes are loaded onto the scaffoldin via dockerin-cohesin interactions, which are specific to each species and to different types within a species.  With the goal of constructing multivalent cellulosomes on the surface of S. cerevisiae as a platform for displaying combinations of enzymes, we are testing dockerin-cohesin pairs from two cellulosomal bacteria, Clostridium thermocellum and Ruminococcus flavefaciens. I will discuss design considerations and challenges related to the engineered cellulosomes.  The generated scaffoldin platforms will be loaded with cellulases and auxiliary activity enzymes sourced from various origins and expressed in yeast. These enzymes will be shuffled using multiple rounds of yeast mating and meiotic gene assortment followed by selection to produce a superior S. cerevisiae strain capable of growth on biomass.  In addition, we expect that our experimental approach will promote better understanding of synergistic interactions in the enzymatic digestion of biomass by complex mixtures of cellulases and other enzymes.