Optogenetic control to manipulate a blend of branched-chain higher alcohols produced by Saccharomyces cerevisiae

Abstract

This thesis investigates the use of metabolic engineering and optogenetics to produce three branched chain higher alcohols (BCHAs), which are isobutanol, isopentanol, and 2-methyl-1-butanol, using the yeast Saccharomyces cerevisiae. Key genes in the BCHA pathway (LEU4, ILV1, and ILV6) were put individually under the control of a light inducible or light repressible promoter in different yeast strains, so that they are expressed only in the light or dark. Therefore, it became possible to control overproduction of certain key genes using only light or dark. The specific aims reached in this thesis are creating novel yeast strains, determining the BCHA production ability of yeast strains containing only one key gene overexpressed, and achieving control over biofuel blends of isopentanol and isobutanol. This is a critical first step in the larger aim of controlling multiple key genes in a single yeast strain, because the BCHA pathways rely on many of the same upstream resources for production. Therefore, when one pathway is overexpressed, another will be repressed; when one alcohol is maximized, another is minimized. This larger aim will allow for more fine control over the ratio of BCHAs in a blend. The ability to achieve a blend of BCHAs solely by changing the light given to yeast during fermentation has the potential of leading to biofuels with blends of desirable properties, since each alcohol has different chemical and physical properties. This research is especially important today, because BCHAs represent a potential renewable energy source with properties superior to ethanol and are cleaner burning than traditional fossil fuel sources.