Pathway optimization and engineering for biofuel production

Optimizing metabolic pathways is paramount for effective and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route for efficient biomass utilization. Here we describe the simultaneous optimization of the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1) through directed evolution of the pathway. The improved pathway was assessed based on specific growth rate on cellobiose, with the final mutant exhibiting a 42% increase over the wild-type pathway. Metabolite analysis of the engineered pathway presented 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. This strategy can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. This improved cellobiose utilization in vivo will not only decrease the in vitro enzyme load in biomass pretreatment, it will also reduce the diauxic shift in pentose sugar utilization, thus significantly reducing the high economics of biofuel processes. More than engineering microbes to more efficiently utilize the biomass sugars, constructing and designing pathway for biofuel production is also very significant. We explored the development of a biodiesel production pathway using a heterologously expressed fatty acid synthase coupled with a wax ester synthase. In this reaction, the esterification of a fatty acyl-CoA and fatty alcohol catalyzed by the wax ester synthase produces free fatty acid ethyl esters, otherwise known as biodiesel. Only initial experiments have been completed in this project, including initial enzyme characterization and plasmid construction