Optogenetic Manipulation of Saccharomyces cerevisiae Metabolism to Produce Advanced Biofuels Isoprenol and Limonene

Abstract

At a time when growing concerns over energy security and alarming accumulation of greenhouse gases pervade the national discussion, research into sources of renewable energy is more important than ever. This project investigates the viability of using a combination of metabolic engineering and optogenetics to produce advanced biofuels in Saccharomyces cerevisiae, such as isoprenol and limonene. This project aimed to achieve four main goals: 1) Develop a large toolset for metabolic engineering using an optogenetic system2) Design a strain of yeast that produces high titers of mevalonate (≥ 2 g/L)3) Build the pathways to synthesize isoprenol and limonene in yeast4) Synthesize high titers of isoprenol and limonene using the EL222 optogenetic circuit The first goal was achieved by developing a library of seven GAL promoters that operate within the optogenetic system and that yield a range of activities. One promoter that was designed even outperforms the constitutive TEF1 promoter, achieving greater levels of activity in the light while remaining virtually inactive in the dark. The second goal was also met as several strains of yeast that produced titers exceeding mevalonate concentrations of 2 g/L were created, ranging as high as 2.57 g/L. The third goal was similarly achieved as the pathways of isoprenol and limonene were designed and both were successfully assembled into plasmids. Finally, the fourth goal has yet to be completely achieved. Though isoprenol production has been demonstrated, the yields were very low and only reached a maximum of 10.3 mg/L. These low yields were expected as they were observed in a strain of yeast that did not produce much mevalonate. Upon transforming the plasmids into a strain of yeast that encodes the optogenetic circuit and also produces high levels of mevalonate (≥ 2 g/L), we hope to produce large titers of both isoprenol and limonene.