Regulating the metabolic flux of pyruvate dehydrogenase bypass to enhance lipid production in Saccharomyces cerevisiae

Abstract

To achieve high efficiency in microbial cell factories, it is crucial to redesign central carbon fluxes to ensure an adequate supply of precursors for producing high-value compounds. In this study, we employed a multi-omics approach to rearrange the central carbon flux of the pyruvate dehydrogenase (PDH) bypass, thereby enhancing the supply of intermediate precursors, specifically acetyl-CoA. This enhancement aimed to improve the biosynthesis of acetyl-CoA-derived compounds, such as terpenoids and fatty acid-derived molecules, in Saccharomyces cerevisiae. Through transcriptomic and lipidomic analyses, we identified ALD4 as a key regulatory gene influencing lipid metabolism. Genetic validation demonstrated that overexpression of the mitochondrial acetaldehyde dehydrogenase (ALDH) gene ALD4 resulted in a 20.1% increase in lipid production. This study provides theoretical support for optimising the performance of S. cerevisiae as a "cell factory" for the production of commercial compounds.

Fig. 1. Construction of key functional gene analysis platform.

Based on population data, biological information involving multiple genes and traits can be obtained with high throughput. The phenome, genome, transcriptome, and metabolome combination strategy were used to further target key functional genes, and the metabolic pathways in which the genes reside and specific biological function changes were understood in detail. Finally, the function of key genes was verified using gene knockout or overexpression, which confirmed that the candidate genes that were mined truly regulated the target phenotype. (Some icons created in BioRender).

Fig. 2. Lipidome and transcriptome analysis of MRDMs (Resd-1, Resd-2, and Resd-4).

A PCA score plot of the MRDM/control group, PCA primary component analysis, MRDM mitochondrial respiration-deficient mutants. B Univariate analysis, volcano plot of the MRDM/control group data, and DEM screening (FC > 2.0, p < 0.05); DEMs differentially expressed metabolites. Based on the KEGG pathway annotations, the top 20 pathways with the lowest significant Q values were obtained (C: ResD-1, D: ResD-2, E: ResD-4). F Relative contents of fatty acyls (F–J all included six biological replicates). G Relative glycerophospholipid content. H Relative glycerolipid content. I Relative contents of sphingolipids. J Relative contents of prenol lipids. K Heatmap of DEGs related to lipid metabolism; DEGs differentially expressed genes. Error bars represent standard error (SE).

Fig. 3. Growth and fermentation of the ald4-oe and Δald4 strains.

A Growth curves (n = 3 biological replicates) in YPD medium with 2% glucose. Measurements were taken every 3 h for 36 h. B Residual sugar content (n = 3 biological replicates, **p = 0.001, **p = 0.005, ***p < 0.001) in ethanol fermentation medium containing 20% glucose. Measurements were taken at 72 and 96 h. C Ethanol yield (n = 3 biological replicates, *p = 0.017, **p = 0.001, **p = 0.005, ***p < 0.001). D CO₂ weight loss (n = 3 biological replicates, ^n.sp = 0.088, ^n.sp = 0.083, *p = 0.011, **p = 0.004, **p = 0.006, ***p < 0.001). E Growth of the Δald4 strain in ethanol liquid medium (n = 3 biological replicates, *p = 0.044, *p = 0.025, *p = 0.024). F Ethanol stress tolerance of strains (the ethanol volume fractions were 4%, 6%, and 8%). G Schematic diagram of ALD4 overexpression or knockout affecting acetaldehyde flow. Error bars represent standard error (SE).

Fig. 4. Measurement of key enzyme activities and PDH bypass gene expression after 72 h of fermentation in ethanol medium.

A Mitochondrial ALDH enzyme activity (n = 3 biological replicates, ***p < 0.001, **p = 0.002), ALDH acetaldehyde dehydrogenase. B Cytoplasmic ALDH enzyme activity (n = 3 biological replicates, ^n.sp = 0.180, ^n.sp = 0.917, ^n.sp = 0.209). C ADH enzyme activity (n = 3 biological replicates, ***p < 0.001, **p = 0.002), ADH alcohol dehydrogenase. D PDC enzyme activity (n = 3 biological replicates, **p = 0.002), PDC pyruvate decarboxylase. E Acetyl-CoA content (n = 3 biological replicates, ***p < 0.001, **p = 0.009). F qPCR of ALDH-encoding genes. G qPCR of ADH-encoding genes. H qPCR of PDC-encoding genes. I qPCR of ACS-encoding genes; ACS acetyl-CoA synthetase. J qPCR of other genes. Error bars represent standard error (SE).

Fig. 5. Metabolomic analysis of ald4-oe and Δald4 strains after 72 h of fermentation in ethanol medium.

A DEM statistical Venn diagram, DEM screening (FC > 1.0, VIP > 1.0, p < 0.05), DEMs differentially expressed metabolites. G1: ald4-oe vs control, G2: Δald4 vs control, and G3: ald4-oe vs Δald4. B PCA score plot of the ald4-oe/Δald4/control group. C DELM classification statistics, DELMs differentially expressed lipid metabolites, FA fatty acyls, GP glycerophospholipids, PK polyketides, PR prenol lipids, SP sphingolipids, ST sterol lipids. D The relative content of different lipid subclasses in the ald4-oe group vs the control group. E The relative content of different lipid subclasses in the Δald4 vs control groups. F Annotation of all identified metabolites using the KEGG database. The top 20 annotations with the most annotations of the KO pathway-level entries were selected, and a summary bar chart was constructed. G Heatmap of DELMs (FC > 2.0, VIP > 1.0, p < 0.05, n = 6 biological replicates). H Correlation analysis between DELMs in the ald4-oe group. I Correlation analysis between DELMs in the Δald4 group. Error bars represent standard error (SE).

Fig. 6. Schematic representation of acetyl-CoA synthesis through the  PDH bypass and its impact on lipid metabolites.

A The influence of ALD4 overexpression on the PDH bypasses key enzyme activity and key genes. ALD4 overexpression increased mitochondrial ALDH enzyme activity, inhibited ADH enzyme activity, and promoted acetaldehyde conversion to acetic acid, and the generated acetic acid was transferred to the cytoplasm and became a precursor molecule for acetyl-CoA synthesis. The orange circles, green circles, blue circles, yellow ovals, blue arrows, and bold arrows indicate gene upregulation, gene downregulation, nonsignificant gene changes, enzymes encoded by genes, PDH bypass, and increased metabolic flux, respectively. B In the metabolome of ald4-oe strains, significant changes in the levels of different lipid compounds were detected. The black, green, orange, and blue arrows indicate fatty acid synthesis, synthesis of major secondary metabolites, synthesis of hormone molecules responsible for signalling, and biofilm synthesis, respectively. (Some icons created in BioRender).