System biologic approach to lipid traffic and conversion in eukaryotic microorganisms and its application

本课题主要包括两个部分：一是酵母菌对短脂肪酸残基磷脂的摄取和酰基链重塑的研究。在以往的研究中，我们发现，磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）的8-至12-碳脂肪酸残基的整合和利用的S。酿酒酵母突变体在这些磷脂的合成中有缺陷。在这个项目中，我们证明了这些可溶性磷脂。通过用癸酸追踪稳定同位素标记的PE和PC的变化，它们的酰基链实际上被正常长度的酰基链取代。因此磷脂在其脂肪酸残基中的重构是真核细胞的共同特性。另一部分是在解脂耶氏酵母中发现了烷烃诱导基因表达的负转录因子Yas 3。在我们以前的研究中，我们报道了两个具有bHLH基序的协同正调控因子Yaslp和Yas 2 p。我们发现Yas 3 p改变了其细胞内定位。当在烷烃存在下生长的脂解细胞中，在ER中发现Yas 3 p-GFP，但在烷烃不存在下，在细胞核中发现它，在那里它可能通过与Yas 2 p结合来抑制Yas 1 p和Yas 2 p的功能。我们还发现Yas 3 p特异性结合磷脂酸和PA磷酸酶突变增加了烷烃诱导的ALK 1基因的表达，表明PA量的变化在Yas 3 p的功能中是重要的。我们还通过RT-PCR检测了12个ALK基因的表达，发现并非所有的ALK基因都对烷烃有反应。和适于表达细胞色素P450基因的突变菌株。

This project was comprised of two parts : One is the analysis on the uptake and acyl-chain remodeling of phospholipids with short fatty acid residues by yeast Saccharomyces cerevisiae. In previous research, we showed that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with 8-to 12-carbon-fatty acid residues were incorporated and utilized by S. cerevisiae mutants defective in the synthesis of these phospholipids. In this project, we proved that these soluble phospholipids were. transported into the cells and their acyl chains are actually replaced with those of normal length by tracing the change of stable isotope-labeled PE and PC with decanoic acids. Thus the remodeling of phospholipids in their fatty acid residues is a common property of eukaryotic cells.The other part is the finding of a negative transcription factor, Yas3, for alkane-induced gene expression in Yarrowia lipolytica. In our previous research, we reported two cooperative positive regulatory factors, Yaslp and Yas2p, with bHLH motifs. We found that Yas3p changed its intracellular localization. When the lipolytica cells were grown in the presence of alkane, Yas3p-GFP was found in ER, but in the absence of alkane, it was found in nucleus, where it likely represses the function of Yas1p and Yas2p by binding to Yas2p. We also found Yas3p specifically bound to phosphatidic acid and PA phosphatase mutation increased expression of the alkane-inducible ALK1 gene, suggesting the change in the amount of PA is important in the function of Yas3p. We also investigated expression of 12 ALK genes by RT-PCR and found not all ALK genes are responsive to alkane.The latter part of our project resulted in a discovery of a very strong inducible promoter in Y. lipolytica and a mutant strain that is suitable for the expression of cytochrome P450 genes.