Table_2_Regulation of autophagy and lipid accumulation under phosphate limitation in Rhodotorula toruloides.xls

BackgroundIt is known that autophagy is essential for cell survival under stress conditions. Inorganic phosphate (Pi) is an essential nutrient for cell growth and Pi-limitation can trigger autophagy and lipid accumulation in oleaginous yeasts, yet protein (de)-phosphorylation and related signaling events in response to Pi limitation and the molecular basis linking Pi-limitation to autophagy and lipid accumulation remain elusive.
ResultsHere, we compared the proteome and phosphoproteome of Rhodotorula toruloides CGMCC 2.1389 under Pi-limitation and Pi-repletion. In total, proteome analysis identified 3,556 proteins and the phosphoproteome analysis identified 1,649 phosphoproteins contained 5,659 phosphosites including 4,499 pSer, 978 pThr, and 182 pTyr. We found Pi-starvation-induced autophagy was regulated by autophagy-related proteins, but not the PHO pathway. When ATG9 was knocked down, the engineered strains produced significantly less lipids under Pi-limitation, suggesting that autophagy required Atg9 in R. toruloides and that was conducive to lipid accumulation.
ConclusionOur results provide new insights into autophagy regulation under Pi-limitation and lipid accumulation in oleaginous yeast, which should be valuable to guide further mechanistic study of oleaginicity and genetic engineering for advanced lipid producing cell factory.

背景：已知自噬（autophagy）对于胁迫条件下的细胞存活至关重要。无机磷酸盐（Pi）是细胞生长所必需的营养物质，而磷限制可触发产油酵母的自噬与脂质积累，但响应磷限制的蛋白质（去）磷酸化修饰及相关信号事件，以及磷限制与自噬、脂质积累之间关联的分子机制仍尚未明确。
结果：本研究对红冬孢酵母（Rhodotorula toruloides）CGMCC 2.1389在磷限制与磷充足培养条件下的蛋白质组（proteome）与磷酸化蛋白质组（phosphoproteome）开展了比较分析。蛋白质组分析共鉴定得到3556种蛋白质，磷酸化蛋白质组分析则鉴定出1649种磷酸化蛋白质，包含5659个磷酸化位点，其中磷酸化丝氨酸（pSer）位点4499个、磷酸化苏氨酸（pThr）位点978个、磷酸化酪氨酸（pTyr）位点182个。研究发现，磷饥饿诱导的自噬由自噬相关蛋白调控，而非依赖PHO通路。当敲低ATG9基因后，工程菌株在磷限制条件下的脂质产量显著降低，这表明红冬孢酵母中的自噬过程需要Atg9参与，且该过程有助于脂质积累。
结论：本研究结果为产油酵母在磷限制条件下的自噬调控与脂质积累机制提供了新的研究视角，该成果可为进一步解析产油性的分子机制，以及通过遗传工程构建高产脂质细胞工厂的相关研究提供重要指导。