Rewiring regulation on respiro-fermentative metabolism relieved Crabtree effects in Saccharomyces cerevisiae

The respiro-fermentative metabolism in the yeast Saccharomyces cerevisiae,also called the Crabtree effect,results in lower energy efficiency and biomass yield which can impact yields of chemicals to be produced using this cell factory.Although it can be engineered to become Crabtree negative,the slow growth and glucose consumption rate limit its industrial application.Here the Crabtree effect in yeast can be alleviated by engineering the transcription factor Mth1 involved in glucose signaling and a subunit of the RNA polymerase II mediator complex Med2.It was found that the mutant with the MTH1A81D&MED2*432Y allele could grow in glucose rich medium with a specific growth rate of 0.30 h^(-1),an ethanol yield of 0.10 g g^(-1),and a biomass yield of 0.21 g g^(-1),compared with a specific growth rate of 0.40 h^(-1),an ethanol yield of 0.46 g g^(-1),and a biomass yield of 0.11 g g^(-1) in the wild-type strain CEN.PK 113-5D.Transcriptome analysis revealed significant downregulation of the glycolytic process,as well as the upregulation of the TCA cycle and the electron transfer chain.Significant expression changes of several reporter transcription factors were also identified,which might explain the higher energy efficiencies in the engineered strain.We further demonstrated the potential of the engineered strain with the production of 3-hydroxypropionic acid at a titer of 2.04 g L^(-1),i.e.,5.4-fold higher than that of a reference strain,indicating that the alleviated glucose repression could enhance the supply of mitochondrial acetyl-CoA.These results suggested that the engineered strain could be used as an efficient cell factory for mitochondrial production of acetyl-CoA derived chemicals.

Effect of Glucose Concentrations on the Growth and Metabolism of Brettanomyces bruxellensis under Aerobic Conditions

Acetic acid can be directly produced from glucose in one-step fermentation by using yeasts of the genus Brettanomyces bruxellensis, hence increasing the industrial application to manufacture products with simplified bioprocesses. Thereby, this work evaluates the influence of initial glucose concentration on the growth and acetic acid production by B. bruxellensis. The results obtained confirmed the presence of Crabtree effect on B. bruxellensis under low glucose concentrations. The maximum acetic acid concentration reached was 15.4 g·L-1 starting with 100 g·L-1 leading to a product yield of 0.154 g·g-1 and a specific acetic acid production rate of 0.05 g·g-1·h-1. The results also indicate that after reaching the acetic acid critic threshold of 4 g·L-1 the metabolism can induce the growth second phase even residual glucose was present on the culture media at high starting glucose concentrations. Additionally, it was observed a lineal relationship between cell viability and acetic acid production.

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.