Transporter engineering as a tool for metabolic engineering

The purpose of metabolic engineering is to understand, design, and optimize metabolism. The objective is chemicals synthesis by microbes. To fulfill this purpose and achieve this objective, tools that control metabolism are essential. Molecular transport is a vital metabolic step yet tools to control it are underdeveloped. Therefore, this work aims to establish transporter engineering, a tool that can rewire transport. Efficient xylose utilization is a key component to economical consumption of lignocellulosic biomass, the most abundant source of sugars on the planet. Transport is a limiting step in the metabolism of xylose by the industrial yeast Saccharomyces cerevisiae. In yeast, transport proteins enabling xylose uptake also permit transit of a broad spectrum of other sugars. Furthermore, glucose is preferred as a substrate to the exclusion of xylose. Therefore, the goal of transporter engineering in this context is twofold: improve xylose uptake while reducing glucose uptake. Four strategies were used to accomplish this goal. First, we performed an iterative bioprospecting approach to explore the extant biodiversity of sugar transporters. However, the transporters tested lack efficient and exclusive xylose transport, motivating development of additional engineering strategies. Second, a directed evolution strategy increased xylose transport efficiency, demonstrating the power directed evolution has to improve transport phenotypes. Third, a targeted engineering strategy was used to analyze key residues responsible for the improved xylose transport phenotype, representing the first targeted engineering strategy to improve xylose growth and reduce glucose growth. Finally, rational engineering was explored. With all of the information collected using the previous strategies, design rules could be developed and implemented. A triple mutant of C. intermedia GXS1 was engineered that does not confer growth on glucose, but xylose growth is retained. By implementing this design rule in S. stipitis RGT2 and S. cerevisiae HXT7, additional xylose exclusive variants can be engineered. This demonstrates that a fundamental design component has been identified and can be used to rewire transport. Thus, this work builds the foundation for molecular transporter engineering.