Engineering a Synthetic Regulon in Saccharomyces Cerevisiae for Efficient Xylose Utilization

Saccharomyces cerevisiae is one of the most widely used microbes for generating biofuels using lignocellulosic feedstock. Since this yeast cannot utilize xylose, the second most abundant sugar in lignocellulose, several metabolic engineering approaches have been attempted in the recent past for efficient xylose consumption. However, growth on xylose is still poor and economically unfeasible for biofuel production. We hypothesize that further enhancement in xylose utilization and biofuel production can be made by addressing the issue from a regulatory perspective rather than a metabolic perspective. Therefore, our work aims at developing a regulon (genome-wide regulatory infrastructure) to enhance the growth and biocatalytic fitness of this yeast for enhanced respiratory growth and advanced biofuel (non-ethanolic) production using xylose. To achieve this, we developed a protein sensor-actuator system that regulates the expression of aerobic xylose metabolism based on its concentration. The well-characterized galactose sensor protein, Gal3p, was engineered for increased affinity towards xylose through semi-rational protein engineering. Targets for mutagenesis were identified using in silico docking studies and high-throughput screens were developed based on G418 resistance and GFP fluorescence. In these screens, expression of kanMX and gfp, placed under GAL promoters, were triggered by a regulatory cascade initiated by the binding of Gal3p to xylose. Initial selection was performed in G418 selection plates and the strains that showed resistance were further quantitatively screened for increased fluorescence. The results reveal further scope for increasing xylose sensitivity in Gal3p mutant variants. Future work involves up- and down-regulating genes and pathways required for enhanced aerobic growth on xylose under the control of this synthetic regulatory system. Such a strain will act as a platform to engineer advanced biofuels using lignocellulosic biomass.