(257c) Directed Evolution of a Cellobiose Utilization Pathway for Biofuel Production

Lignocellulosic biofuel production in S. cerevisae can be made more economical by pentose sugar utilization. However, slow growth occurs if pentose sugars are the sole carbon source. Cofermentation with glucose inhibits effective utilization of the pentose sugars, called glucose repression. One strategy to address glucose repression is to utilize cellobiose rather than glucose. The cellobiose utilization pathway consists of a cellodextrin transporter (CDT-1) coupled with an intracellular β-glucosidase (GH1-1). The process was optimized through the application of a new directed evolution technique. The pathway was constructed with error-prone libraries of the CDT-1 and GH1-1 via the DNA Assembler. Mutants with improved activities were identified by higher growth rate and then subsequently screened for increased ethanol yield. The results of this optimization produced a non-intuitive mutation in GH1-1, improving cellobiose utilization by over 30%. Further rounds of directed evolution identified mutants with reduced cellodextrin accumulation, a major setback in cellobiose utilization. This is one of the first examples of engineering multiple proteins simultaneously on the pathway level in vivo instead of independently engineering single proteins.