(210e) In Silico and Experimental Studies of Cofactor Balance in the Engineered Pentose Sugar Utilization Pathways in Saccharomyces Cerevisiae

Biofuels such as ethanol derived from lignocellulosic biomass offer promising alternate renewable energy sources for transportation fuels. The breakdown of lignocellulosic biomass results in a mixture of hexoses and pentoses, but wild type S. cerevisiae only ferments hexoses. Significant effort has been made to engineer yeast to efficiently ferment pentose sugars such as D-xylose and L-arabinose into ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in those pathways is that the cofactors are not balanced because some enzymes such as xylose reductase and L-xyllulose reductase prefer NADPH, whereas others like xylitol dehydrogenase and L-arabinitol prefer NAD⁺. This cofactor imbalance in the engineered pathways contributes to inefficient utilization of pentose sugars since the imbalance of the cofactors leads to systemic perturbation of the metabolic network.

We utilized a genome-scale metabolic network of Saccharomyces cerevisiae to predict the maximal achievable growth rate for cofactor balanced/imbalanced D-xylose and L-arabinose utilization pathways. The cofactor balanced pathways were shown to improve efficient utilization of the pentose sugars in the genome scale metabolic network. Additionally, flux variability analysis identified those reactions that were most affected by the cofactor balancing of the engineered pathways. Maximum variation in fluxes was observed for glycolysis and phospholipid biosynthesis. Furthermore, random sampling of the steady-state flux solution space was done to characterize the allowed metabolic fluxes. A decrease in metabolic network flexibility was observed when the introduced pathways were not cofactor balanced. Cofactor balancing the engineered pathways restored network flexibility and the correlation structure between the reactions to a state was very similar to that of the wild type strain. The D-xylose pathway was introduced in vivo and experiments are in progress for the comparison with in silico predictions. Taken together, the incorporation of balanced cofactors increased pentose sugar utilization efficiency while retaining the flexibility and flux correlation structure of the wild type strain.