Saccharomyces cerevisiae is one of the most widely used microbes for generating value added biochemicals like alcohols, antimicrobials, fatty acids etc. A major shortcoming of this yeast is that it cannot utilize xylose, the second most abundant sugar in lignocellulosic plant biomass. In the past, several metabolic engineering approaches have been attempted to engineer xylose consumption. However, growth on xylose is still poor and economically unfeasible. We hypothesize that further enhancement in xylose utilization can be made by addressing the issue from a regulatory perspective rather than metabolic. 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 using xylose. To achieve this, we are engineering a novel 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 protein engineering approaches (viz semi-rational and random combinatorial). To identify advantageous mutants, we developed high-throughput screens 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 mutants. 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 value added biochemicals using lignocellulosic biomass. Keywords: xylose, advanced biofuels, regulatory system engineering, protein engineering

Synthetic regulon in Saccharomyces cerevisiae for efficient xylose assimilation

251st National Meeting (2016)

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