(509d) Post-Translational Control of Central Carbon Metabolism in Yeast (Industry Candidate)

The baker’s yeast Saccharomyces cerevisiae is one of the most widely-used microorganisms for industrial chemical production. However, economically-viable production of commodity chemicals with this organism is made challenging by the yeast’s overwhelming bias to produce ethanol. Because of the Crabtree effect, fermentation is necessary for the yeast’s growth on glucose and other fermentable sugars, making genetic knockouts of the ethanol pathway challenging for growth on many industrially-relevant carbon sources. With dynamic control of the ethanol pathway, production of competing chemicals of interest could be improved without sacrificing biomass accumulation, by splitting the fermentation into a growth stage and a production stage. Our previous efforts to induce the switch from growth to production have focused on transcriptional regulation of the ethanol pathway; while transcriptional circuits can allow for high-fold changes in gene expression, they do not affect proteins already accumulated within cells, creating an inherent lag in circuit response. In this talk, I present a novel genetically-encoded inhibitor of a key enzyme in ethanol production, which allows post-translational control of ethanol production and cell growth. Biochemical and structural characterization of this inhibitor revealed that it inhibits its target with low-nanomolar affinity, with fast association on the order of 10⁴ M^-1s^-1, and slow dissociation on the order of 10^-5 s^-1. The inhibitor shows potent, noncompetitive inhibition of its target. Constitutive expression of the inhibitor in vivo cuts growth by 1/3 in a wild-type laboratory strain, and virtually eliminates growth in a strain engineered with optogenetic transcriptional control of the ethanol pathway. Finally, inducible expression of the inhibitor shows improved production of chemicals of interest compared to purely transcriptional control of the ethanol pathway, demonstrating the advantage of post-translational regulation of metabolic pathways. This new technology enables faster dynamic control of metabolic enzymes, making it possible to more rapidly shut down essential pathways, leaving a greater pool of substrate to be converted to products of interest.