PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae

Abstract

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

Fig 1. Activation of PKA is needed for xylose fermentation.

A. A brief overview of the PKA signaling pathway. B-C. Average (n = 6 biological replicates) growth (OD600, optical density) and (B) xylose concentration in the medium over time of parental Y184, ira2Δ, bcy1Δ, and ira2Δbcy1Δ strains grown anaerobically on rich medium containing xylose as a carbon source. Asterisks denote significant differences in profiles (p < 0.05, ANOVA); n.s. indicates ‘not significant’ (p > 0.05). D. Growth and fermentation capabilities of strains shown in B-C.

Fig 2. Few transcriptomic patterns correlate with anaerobic xylose growth.

A. Experimental overview. Strains were grown anaerobically in rich glucose medium to early/mid-log phase, then switched to anaerobic rich xylose medium for three hours. B. Expression of 65 genes whose log₂(fold change) upon glucose to xylose shift is different (FDR < 0.05) in at least one of the three non-growing strains (Y184, bcy1Δ, ira2Δbcy1Δ) compared to ira2Δ. Genes (rows) were organized by hierarchical clustering across biological triplicates measured for each strain (columns). Genes discussed in the text are annotated on the figure. C. Hierarchical clustering of 292 genes whose log₂(fold change) upon glucose to xylose shift is different (FDR < 0.05) between the non-fermenting Y184 strain and the two robustly xylose fermenting strains (ira2Δ, bcy1Δ). The blue-yellow heatmap on the left represents the log₂(fold change) in expression upon glucose to xylose shift across biological triplicates (columns). The purple-green heatmap on the right represents the abundance of each transcript (rows) in each strain grown on glucose (G) or xylose (X), relative to the average (n = 3) abundance of that transcript measured in the Y184 YPD sample. Clusters I and II are described in the text. D. Expression of 15 genes from C) that have annotations linked to glycolysis, gluconeogenesis, TCA cycle, and carbohydrate storage.

Fig 3. Genes uniquely expressed in the bcy1Δ strain implicate an integrated response to xylose metabolism and growth coupling.

A. Expression of 654 genes whose log₂(fold change) upon glucose to xylose shift is different (FDR < 0.05) between the ira2Δ and bcy1Δ strains and whose expression change is in the opposite direction across the two strains (see Methods for details). Significant functional enrichments are annotated next to the two main clusters (p < 10⁻⁴, hypergeometric test). Bar graph inset represents the log₂(fold change) of the two phosphatidic acid biosynthesis enzymes in this group, see text for details. B. Regulatory relationships between transcription factors whose targets or known binding sites were enriched in (A). Documented PKA-dependent phosphorylation is indicated by a P. See text for details.

Fig 4. bcy1Δ strains show altered phospholipids after anaerobic xylose shift.

A-B. Abundance of lipids (rows) with a significant difference in log₂(fold change) upon anaerobic glucose-to-xylose shift in (A) Y184 compared to PKA pathway mutants (ira2Δ, bcy1Δ, ira2Δbcy1Δ) analyzed as a group in the statistical model or (B) ira2Δ cells compared to ira2Δbcy1Δ cells. Lipids of interest are annotated. C. Partial phospholipid biosynthesis pathway with transcriptomic and lipidomic data represented. Yellow-blue boxes next to each enzyme name represent the average log₂(fold change) in transcript abundance upon glucose-to-xylose shift for each strain, as outlined in the key. Significant differences compared to the ira2Δ strain (FDR < 0.05) are represented in sharp, bolded boxes, whereas insignificant differences are translucent. Colorized pathway arrows (yellow: induced, blue: repressed) represent the predominant transcript patterns for that enzymatic step when comparing the bcy1Δ and ira2Δ strains. Lipids whose fold-change in abundance is different in specific strains are according to the key. Lipid abbreviations: FFA–free fatty acids; PA–phosphatidic acid; DG–diacylglycerol; TG–triacylglycerol; PI–phosphatidylinositol; PS–phosphatidylserine; PE–phosphatidylethanolamine; PMME–monomethyl-phosphatidylethanolamine; PDME–dimethyl-phosphatidylethanolamine; PC–phosphatidylcholine; CL–cardiolipin. D. Average (n = 4) change in OD₆₀₀ of ira2Δ and bcy1Δ grown anaerobically in rich xylose medium either in the absence (solid lines) or presence (dashed lines, IC) of inositol (75 μM) and choline (10 mM) (* indicates p = 2.4 x 10⁻⁶, ANOVA).

Fig 5. Directed evolution recoupled growth and metabolism on xylose.

A. Average (n = 3) change in OD₆₀₀ of ira2Δ, bcy1Δ, and EWY55 strains grown anaerobically in rich xylose medium (*, p < 10⁻⁴, ANOVA; n.s., not significant). B. Change in OD₆₀₀ (left panel) and xylose concentration (right panel) over 48 hours of EWY55 and EWY55 opi1Δ strains grown anaerobically on rich xylose medium. (*, p < 0.05, ANOVA). C. Expression of 233 genes whose transcript abundance during growth on xylose was significantly different in EWY55 and/or ira2Δ strains compared to the bcy1Δ strain (FDR < 0.05), visualized by hierarchical clustering. Data represent the log₂ transcript abundance in each strain grown anaerobically in xylose compared to bcy1Δ strain. Cluster A (9 genes) and B (13 genes) are annotated, see text for details. D. Bar plot of the average and standard deviation log₂(fold change) (n = 3) in lipid abundance of key lipids with reproducible differences 1.5-fold or greater in EWY55 compared to ira2Δ or bcy1Δ strains. Asterisks denote significant differences by ANOVA.