INRA, Institut Micalis, INRA-AgroParisTech, UMR1319, domaine de Vilvert, Jouy-en-Josas, France.
Metab Eng. 2011 Sep;13(5):482-91. doi: 10.1016/j.ymben.2011.05.002. Epub 2011 May 23.
The oleaginous yeast Yarrowia lipolytica can accumulate up to 38% of its dry weight (DW) as lipids. Factors involved in lipid accumulation, particularly triglycerides, are not well identified. Using different mutations in the glycerol-3-phosphate (G3P) shuttle pathway (Δgut2 affecting the anabolic dehydrogenase or overexpressing GPD1 affecting the catabolic dehydrogenase), we were able to modulate G3P concentration. We show that in a Po1d genetic background, GPD1 overexpression, GUT2 inactivation or both mutations together result in 1.5, 2.9, and 5.6-fold respective increases in the level of G3P leading to an increase of triacylglyceride (TAG) accumulation. Moreover, our results indicate that each strain with an increased concentration of G3P, also presented a decreased concentration of glycerol. Analysis of the different genes involved in glycerol metabolism indicated that Y. lipolytica does not possess a gene for glycerol-3-phosphatase. These findings suggest that Y. lipolytica has a modified and unique metabolism of glycerol that is dedicated to G3P synthesis (and also to TAG synthesis) which may contribute to its oleaginous character. Furthermore, coupling the G3P shuttle disorders to a deficient β-oxidation pathway (by inactiving POX1-6 or MFE1 genes) increased TAG and free fatty acids content. Finally, we obtained strains that accumulated up to 65-75% of their DW as lipid. Transcriptional analysis in these strains, revealed that the high levels of lipids resulted from over-expression of genes involved in TAG synthesis (SCT1, encoding a sn-1 acyltransferase; and DGA1, encoding an acylCoA diacylglycerol acyltransferase) and the repression of genes involved in the degradation of TAG (TGL3 and TGL4, encoding triacylglycerol lipases). These findings indicate that TAG synthesis is limited by the availability of G3P and fatty acids, and that the expression of genes involved in TAG homeostasis is regulated by the G3P shuttle and the β-oxidation pathway. Finally, the synergistic contribution of acyltransferase gene expression to G3P synthesis is required for high levels of TAG synthesis and lipid accumulation in Y. lipolytica.
产油酵母解脂耶氏酵母可以将其干重的 38%积累为油脂。参与脂质积累的因素，特别是甘油三酯，尚未得到很好的鉴定。使用甘油-3-磷酸 (G3P) 穿梭途径中的不同突变（影响合成代谢脱氢酶的 Δgut2 或影响分解代谢脱氢酶的 GPD1 过表达），我们能够调节 G3P 浓度。我们表明，在 Po1d 遗传背景下，GPD1 过表达、GUT2 失活或两者突变共同导致 G3P 水平分别增加 1.5、2.9 和 5.6 倍，导致三酰基甘油 (TAG) 积累增加。此外，我们的结果表明，每种 G3P 浓度增加的菌株，甘油浓度也降低。对甘油代谢中涉及的不同基因的分析表明，解脂耶氏酵母没有甘油-3-磷酸酶基因。这些发现表明，解脂耶氏酵母具有独特的甘油代谢途径，专门用于 G3P 合成（也用于 TAG 合成），这可能有助于其产油特性。此外，将 G3P 穿梭障碍与β-氧化途径缺陷（通过失活 POX1-6 或 MFE1 基因）相结合，增加了 TAG 和游离脂肪酸的含量。最后，我们获得了积累高达 65-75%干重为脂质的菌株。在这些菌株中的转录分析表明，高水平的脂质是由于参与 TAG 合成的基因（编码 sn-1 酰基转移酶的 SCT1 和编码酰基辅酶 A 二酰甘油酰基转移酶的 DGA1）的过表达和参与 TAG 降解的基因（编码三酰基甘油脂肪酶的 TGL3 和 TGL4）的抑制所致。这些发现表明，TAG 合成受到 G3P 和脂肪酸可用性的限制，并且参与 TAG 动态平衡的基因的表达受到 G3P 穿梭和β-氧化途径的调节。最后，酰基转移酶基因表达对 G3P 合成的协同贡献是解脂耶氏酵母中高水平的 TAG 合成和脂质积累所必需的。
