P101: Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae through simultaneous engineering of multiple proteins

Optimizing metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One significant pathway for biofuel production is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the pathway engineering of a cellobiose utilization pathway by a combinatorial homologous protein search for optimal proteins and the simultaneous directed evolution of all proteins within the pathway. Through the homologous protein search, the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1­) from Neurospora crassa, as previously reported, were discovered as the optimal pathway activity. These proteins were then simultaneously engineered for improved activity, with the final mutant exhibiting a 42% increase over the wild-type pathway. Metabolite analysis of the engineered pathway revealed a 54% increase in cellobiose consumption (1.68 to 2.82 g cellobiose/(L·h)) and a 74% increase in ethanol productivity (0.59 to 1.03 g ethanol/(L·h)). By simultaneously engineering multiple proteins in the pathway, cellobiose utilization by S. cerevisiae was improved, a strategy which can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype.