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 MTH1 ^A81D&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.