(712d) Design and Engineering of a Xylose Sensing-Regulation Gene Circuit in Saccharomyces Cerevisiae: A Potential “Master Switch” for Efficient Xylose Utilization

The goal of efficiently and economically converting cheap biomass into biofuels and industrial chemicals presents both a great opportunity and a challenge in the field of synthetic biology. Xylose is the most abundant sugar except glucose in cellulosic biomass. Currently, xylose can only be utilized after the depletion of glucose. Therefore, in theory, it would be ideal for the xylose catabolic pathway to be turned on when intracellular xylose is abundant. Furthermore, a xylose uptake dependent, dynamically regulated xylose consumption pathway would significantly improve the efficiency of xylose utilization. A robust intracellular xylose sensing-regulation gene circuit is at the heart of the development of such dynamically regulated xylose consumption pathway. We harnessed xylose dependent repressors from bacteria and constructed a panel of xylose sensing-regulation gene circuits in S. cerevisiae. We demonstrated that the output (fluorescence) of these circuits is dependent on the extracellular xylose concentration. In addition, we showed that various properties (induction ratio, basal level expression and dynamic range, etc.) of these gene circuits can be fine-tuned at three different control nodes (expression level of the repressor, insertion position of the operator and operator sequence itself). As a proof of concept, we first applied our xylose sensing-regulation gene circuit as a direct readout of the xylose transportation capacity of xylose transporters. By coupling fluorescence intensity with the xylose transportation capacity, we developed a fluorescence activated cell sorting based enrichment method and engineered sugar transporters with enhanced xylose transportation capacity via directed evolution. Finally, we constructed a dynamically regulated xylose consumption pathway which may significantly improve xylose utilization in S. cerevisiae.