Profiling proteomic responses to hexokinase-II depletion in terpene-producing Saccharomyces cerevisiae

Hexokinase II (Hxk2) is a master protein in glucose-mediated transcriptional repression signaling pathway. Degrading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene (nerolidol) production at gram-per-liter levels in Saccharomyces cerevisiae. Global transcriptomics/proteomics profiles in Hxk2-deficient background are important to understanding genetic and molecular mechanisms for improved nerolidol production and guiding further strain optimization. Here, proteomic responses to Hxk2 depletion are investigated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway, at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δ backgrounds. Carbon metabolic pathways and amino acid metabolic pathways show diversified responses to Hxk2 depletion and growth on ethanol, including upregulation of alternative carbon catabolism and respiration as well as downregulation of amino acid synthesis. De-repression of GAL genes may contribute to improved nerolidol production in Hxk2-depleted strains. Seventeen transcription factors associated with upregulated genes are enriched. Validating Ash1-mediated repression on the RIM4 promoter shows the variation on the regulatory effects of different Ash1-binding sites and the synergistic effect of Ash1 and Hxk2-mediated repression. Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δ background, but much weaker than those in HXK2-wildtype background. In summary, inactivating HXK2 may relieve glucose repression on respiration and GAL promoters for improved bioproduction under aerobic conditions in S. cerevisiae. The proteomics profiles provide a better genetics overview for a better metabolic engineering design in Hxk2-deficient backgrounds.