2,3-BUTANEDIOL PRODUCTION FROM CELLOBIOSE BY ENGINEERED SACCHAROMYCES CEREVISIAE BY HONG NAN THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Human Nutrition with a concentration in Food Science

Production of renewable chemicals from cellulosic biomass is a critical step towards energy sustainability and reduced greenhouse gas emissions. Microbial cells have been engineered for producing fuels and chemicals from cellulosic sugars. Among these chemicals, 2,3-butanediol (2,3-BDO) is a compound of interest due to its diverse applications. While microbial production of 2,3-BDO with high yields and productivities has been reported, there are concerns with the use of potential pathogenic bacteria and inefficient utilization of cellulosic sugars. To address these problems, we engineered Saccharomyces cerevisiae to produce 2,3-BDO, especially from cellobiose which is a prevalent sugar in cellulosic hydrolyzates. Specifically, we overexpressed alsS and alsD from Bacillus subtilis to convert pyruvate to 2,3-BDO via α-acetolactate and acetoin in engineered S. cerevisiae capable of fermenting cellobiose directly. Under oxygen-limited conditions, the resulting strain was able to produce 2,3-BDO. Still, the majority of carbon flux in the strain went to ethanol, resulting in significant amounts of ethanol production. To enhance pyruvate flux to 2,3-BDO through elimination of the pyruvate decarboxylation (PDC) reaction, we employed a deletion mutant of both PDC1 and PDC5 for producing 2,3-BDO from cellobiose. The subsequent strain was able to produce only 2,3-BDO without ethanol production from cellobiose under oxygen-limited conditions. These results suggest the possibility of producing 2,3-BDO safely and sustainably from cellulosic hydrolyzates. iii To my parents iv ACKNOWLEDGEMENTS