Alleviating glucose repression and enhancing respiratory capacity to increase itaconic acid production

The Crabtree effect products ethanol and acetic acid can be used for itaconic acid(IA)production in Saccharomyces cerevisiae.However,both the IA synthesis and oxidative phosphorylation pathways were hampered by glucose repression when glucose was used as the substrate.This study aimed to improve IA titer by increasing gene expressions related to glucose derepression without impairing yeast growth on glucose.Engineering the acetyl-CoA synthesis pathway increased the titer of IA to 257 mg/L in a urea-based medium.Instead of entire pathway overexpression,we found that some signaling pathways regulating glucose repression were effective targets to improve IA production and respiratory capacity.As a consequence of the reduced inhibition,IA titer was further increased by knocking out a negative regulator of the mitochondrial retrograde signaling MKS1.SNF1/MIG1 signaling was disturbed by deleting the hexokinase HXK2 or an endoplasmic reticulum membrane protein GSF2.The shaking results showed that XYY286(BY4741,HO::cadA,Y::Dz.ada,208a::Mt.acs,Δhxk2,pRS415-cadA,pRS423-aac2)accumulated 535 mg/L IA in 168 h in the YSCGLU medium.qRT-PCR results verified that deletion of MKS1 or HXK2 upregulated the gene expressions of the IA synthesis and respiratory pathways during the growth on glucose.

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.De-grading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene(nerolidol)pro-duction 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 investi-gated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway,at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δbackgrounds.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.Seven-teen 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 syner-gistic effect of Ash1 and Hxk2-mediated repression.Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δbackground,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.