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

Abstract

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.

Keywords: 3-Hydroxypropionic acid; Crabtree effect; Respiro-fermentation; Saccharomyces cerevisiae.

Fig. 1

Growth, glucose and ethanol profiles of the wild-type strain (A) and the mutants with MTH1^A81D (B), MED2*^432Y (C) and MTH1^A81D&MED2*^432Y (D), respectively. The cultivations were performed in minimal medium with 2% glucose in bioreactors in duplicate and error bars represent ±standard errors.

Fig. 2

Transcriptional analysis of the mutant strains. (A) Principal component analysis (PCA) plot of the transcription data in triplicates. (B) Venn diagram of significantly regulated genes of mutant strains in comparison with the WT strain (p-adj < 0.01). (C) Gene set enrichment analysis of the 245 commonly regulated genes. Fold enrichment indicated the magnitude of enrichment against the genome background of the strain S288C analyzed via DAVID. (D) Venn diagram of highly scored (p-value<0.01) reporter GO terms through analyzing significantly regulated genes of the mutants compared with the WT strain (p-adj < 0.05). (E) The common high scored reporter GO terms in distinct-directional up and down class presented by their significance in the mutants. (F) The high scored reporter transcription factors (TFs) in distinct-directional up and down class presented by their significance. (G) Expression levels of reporter TFs in the mutant strains compared with the wildtype strain.

Fig. 3

Transcriptional representation of genes involved in carbohydrate metabolism. All data was made in comparisons with the WT strain.

Fig. 4

Effects of MTH1 and MED2 mutations on cell respiration. (A) Expression fold changes of the genes involved in mitochondrial electron transport chains. All data were made in comparisons with WT strain. (B) Oxygen consumption rates (OCRs) of the wild-type strain (WT) and mutant strains under two different glucose levels using a Seahorse XF96 analyzer. The measurements were performed in replicate and error bars represent ±standard errors.

Fig. 5

The growth, glucose and ethanol profiles of the wild-type strain (A) and the mutants ZS_mth1(B), ZS_med2 (C) and ZS_mm (D) with (red line) and without (black line) mitochondrial pyruvate transporter inhibitor UK-5099. The cultivations were performed in minimal medium with 2% glucose in shake flasks in triplicate and error bars represent ±standard errors.

Fig. 6

The growth, glucose, ethanol and 3-HP profiles of the strains without (A) and with (B) the mutations of MTH1^A81D&MED2*^432Y. The cultivations were performed in minimal medium with 2% glucose in shake flasks in triplicate and error bars represent ±standard errors.