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

All 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 they are integrated to produce global changes in a cell. Past work from our lab, focused on engineering the budding yeast Saccharomyces cerevisiae for fermentation of the non-native pentose sugar xylose, discovered that hyperactivation of the RAS/Protein Kinase A (PKA) pathway was needed for rapid anaerobic xylose fermentation. Interestingly, the mechanism of PKA hyperactivation has a dramatic impact on growth and metabolism on xylose; deletion of the RAS inhibitor IRA2 permits rapid growth and fermentation, while deletion of the PKA regulatory subunit BCY1 allows for fermentation without growth on xylose. To understand how a single deletion in the PKA pathway can decouple growth and metabolism, we performed transcriptomic analysis of these strains, predicting that altered PKA activity would impact global gene expression and identify pathways important for growth and metabolism coordination. Notably, we found enriched differential expression of lipid metabolism genes, targets of the phospholipid biosynthetic gene transcription factor Ino4, and genes containing the Aft1/2 consensus motif. These results suggested that dysfunctional lipid homeostasis may be responsible for decoupling growth and metabolism in the bcy1∆ strain. In parallel work, we also directly evolved the bcy1∆ strain to grow anaerobically on xylose and found point mutations in TPK1, OPI1, RIM8, and TOA1 permitted growth. Interestingly, Opi1 is the inhibitor of Ino4, further supporting the role of lipid homeostasis in growth and metabolism coordination. This work shows that a single genetic change can have dramatic impacts on multiple aspects of cellular physiology.所有生物均演化出精密的生理通路，用以调控生长、增殖、代谢与应激响应。这些通路必须得到恰当协调，才能在不断变化的环境中触发适配性应答。尽管诸多模式系统中已对单一通路开展了充分研究，但关于这些通路如何协同整合以引发细胞内全局性变化的机制，仍有诸多待阐释之处。本实验室过往的研究聚焦于改造出芽酵母（budding yeast，即酿酒酵母（Saccharomyces cerevisiae）），使其能够发酵非天然戊糖木糖（xylose），此项研究发现RAS/蛋白激酶A（Protein Kinase A, PKA）通路的过度激活是实现厌氧快速木糖发酵的必要条件。值得注意的是，PKA通路过度激活的具体机制对木糖环境下的生长与代谢具有显著影响：敲除RAS抑制剂IRA2可使菌株实现快速生长与发酵，而敲除PKA调控亚基BCY1则可使菌株在木糖上仅发酵却不生长。为探究PKA通路的单基因敲除如何实现生长与代谢的解偶联，我们对上述菌株开展了转录组学分析（transcriptomic analysis），并推测PKA活性改变会影响全局基因表达，进而识别出参与生长与代谢协调的关键通路。尤为关键的是，我们发现脂质代谢基因、磷脂生物合成基因转录因子Ino4的靶基因，以及携带Aft1/2共有基序的基因均呈现出显著的差异表达富集。上述结果表明，脂质稳态失衡可能是导致bcy1Δ菌株中生长与代谢解偶联的原因。在平行研究中，我们还对bcy1Δ菌株进行了直接定向进化，使其能够在木糖上厌氧生长，并发现TPK1、OPI1、RIM8与TOA1基因中的点突变可赋予其生长能力。有趣的是，Opi1正是Ino4的抑制剂，这进一步证实了脂质稳态在生长与代谢协调过程中的作用。本研究表明，单基因遗传改变可对细胞生理的多个方面产生显著影响。