SYSTEMS METABOLIC ENGINEERING OF ISOBUTANOL PRODUCTION IN SACCHAROMYCES CEREVISIAE

Abstract

Branched-chain alcohols, including isobutanol, are promising advanced biofuels, as they have a higher energy content, increased compatibility with existing infrastructure, and allow for higher blends with gasoline compared to ethanol. Saccharomyces cerevisiae is arguably the best host organism to produce isobutanol, given its high tolerance to alcohols, robust growth under low oxygen conditions, genetic tractability, and immunity to phage contamination. Although the isobutanol biosynthetic pathway has been characterized, our understanding of how cellular and genetic networks interact with this pathway is non-existing. In this thesis, I describe my work to increase isobutanol tolerance and production in the yeast Saccharomyces cerevisiae. My first project involved a genomic screen to find mutants displaying higher tolerance to isobutanol, which can be toxic in high concentrations. By analyzing RNA-seq data, we found that the most tolerant strain senses a nitrogen starvation signal when grown in isobutanol, which activates the expression of pathways involved in amino acid synthesis, contributing to improved growth. In a follow up study, we subjected tolerant strains to adaptive laboratory evolution under increasing isobutanol concentrations. We sequenced these strains and found the mutations responsible for the increased tolerance. In another study, I introduced a genetically encoded biosensor of isobutanol production to the >5000 strains that form the yeast gene deletion library. After inserting the biosensor into each member of the library, I screened them via flow cytometry. We found that the deletion of genes involved in the modification of histones can significantly boost isobutanol production. My follow-up analysis of proteomics data showed that these mutants display an enrichment of mitochondrial proteins. In addition, we performed untargeted metabolomics, showing an enriched pool of mitochondrial metabolites and precursors that explain the increased isobutanol production. Finally, in another study we explored how mitochondrial morphology can impact isobutanol production when the metabolic pathway is targeted to that organelle. We did fermentations and fluorescence microscopy and found that the deletion of genes involved in either fusion or fission has a significant effect on production. Our results suggest that these effects might depend on the product of interest as a different result was observed in geraniol-producing strains.