Improving advanced biofuels production in Saccharomyces cerevisiae via protein engineering and synthetic biology approaches

Cellodextrin transporter engineering;Reversed beta-oxidation cycles;Synthetic biology;Yeast;Cellulosic biofuels

Lian, Jiazhang ; Zhao ; Huimin

关键词: Cellodextrin transporter engineering;    Reversed beta-oxidation cycles;    Synthetic biology;    Yeast;    Cellulosic biofuels;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46944/Jiazhang_Lian.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Saccharomyces cerevisiae has been widely established as a platform microorganism for industrial production of fuels and chemicals from lignocellulosic biomass. However, we are still encountering several challenges to achieve cost-effective production of cellulosic biofuels, such as the sequential utilization of sugar mixtures caused by glucose repression and the low efficiency of synthesizing fatty acid derived advanced biofuels.In this thesis, we aim to improve the production of advanced biofuels in S. cerevisiae using protein engineering and synthetic biology approaches. To overcome glucose repression, a cellobiose utilization pathway consisting of a cellodextrin transporter and a β-glucosidase was introduced into S. cerevisiae to allow co-fermentation of mixed sugars. However, the utilization of cellobiose was still much lower than that of glucose, and the uptake of cellobiose was considered the rate-limiting step for cellobiose fermentation. Therefore, directed evolution of the cellodextrin transporter (CDT2) was carried out to improve the uptake activity and thus cellobiose fermentation. After three rounds of directed evolution, both the specific activity and transporter expression level of CDT2 were increased, leading to 2.15 fold improvement of the cellobiose uptake activity. Using high cell density fermentation under anaerobic conditions, the best mutant conferred 2.67 fold and 4.96 fold increase in the cellobiose consumption rate and ethanol productivity, respectively. Besides bioethanol, we are also interested in advanced biofuels that have similar properties to current transportation fuels. Since most of the advanced biofuels are derived from fatty acids, efficient production is limited by the low efficiency and high energy input of the fatty acid biosynthesis pathways. Reversal of β-oxidation cycles has been engineered to produce a series of fatty acid derived fuels and chemicals in Escherichia coli. Thus, another goal of this thesis is to construct a new fatty acid biosynthesis platform based on the reversal of β-oxidation cycles for advanced biofuel production in S. cerevisiae. Using synthetic biology approaches, reversed β-oxidation pathways were constructed and characterized to be able to produce butanol, indicating the functional reversal of β-oxidation cycles. Future work will focus on expanding this platform to produce other fatty acid derived advanced biofuels such as biodiesel (fatty acid ethyl ester, FAEE).