(341a) Directed Evolution of a Highly Efficient Arabinose/Xylose Utilization Pathway in Saccharomyces Cerevisiae

Bioconversion of plant derived lignocellulosic materials into biofuels has drawn increasing attention for being an attractive candidate for replacement of fossil fuels. Saccharomyces cerevisiae, also known as baker's yeast, is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which make up to 30% of biomass hydrolysate, cannot be utilized by S. cerevisiae. Heterologous pentose utilization pathways from bacterial and fungal resources have been introduced into S. cerevisiae to enable the assimilation of pentose sugars. However, pentose utilization of recombinant S. cerevisiae strains are inefficient due to the low expression level and activity of heterologous genes, redox imbalance resulted from different preference of cofactor for oxidation and reduction reactions, and suboptimal metabolic flux through different catalytic steps. A lot of research has been done to improve the pentose utilization by S. cerevisiae targeting different aspect of these issues, but it is very challenging to come up with a single strategy which can solve all three problems at the same time. Here we report the directed evolution of a highly efficient pentose utilization pathway in S. cerevisiae. Specifically, we chose the fungal five-step arabinose/xylose utilization pathway as our target pathway. For each catalytic step, 10 to 20 different enzyme homologues from various fungal species with different catalytic efficiency and cofactor preference have been cloned, and the whole pathway was assembled into a random library using our recently developed DNA assembler method. The resultant library exhibited a very high efficiency for correct assembly of the complete multi-gene pathway and good diversity of different homologues within the same catalytic step. Using the library generated, clones with better combination of enzyme homologues can be selected by faster cell growth. This method can also be applied to directly search for the pathway with a better fit in industrial yeast strains, which may have different metabolic flux patterns compared to the laboratory S. cerevisiae strains.