Multiomics Analysis of Mitochondrial Versus Cytosolic Compartmentalization of the Isobutanol Pathway in Saccharomyces Cerevisiae

Isobutanol is a promising alternative biofuel due to its higher energy density and lower hygroscopicity than ethanol. Saccharomyces cerevisiae has been proposed as a suitable host for this process because of its industrial robustness and native capability to produce low levels of isobutanol via valine catabolism; valine biosynthesis and its degradation pathway are separated into two subcellular compartments, the mitochondria and the cytosol, respectively. One engineering strategy to enhance isobutanol production is to localize all the enzymes into a single compartment, however there is debate as to what cellular compartment is optimal for the pathway. In our work, we have equipped yeast with either the cytosolic or mitochondrial localized isobutanol pathway and performed a multiomics analysis to elucidate which compartmentalization strategy favors isobutanol production. We have collected samples for metabolomics, proteomics, and transcriptomics analyses at three different time points during an anaerobic fermentation. Preliminary metabolite data shows a buildup of the first three isobutanol pathway intermediates in the cytosolic localized pathway; these metabolites are upstream of a Fe-S cluster requiring enzyme, Ilv3. Since Fe-S cluster biosynthesis begins with the mitochondrial iron-sulfur cluster (ISC) assembly machinery, we hypothesize that Fe-S cluster loading is limited in the cytosol. We are actively exploring this hypothesis and others to understand why a mitochondria-localized isobutanol pathway has enhanced production in Saccharomyces cerevisiae.