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

本课题由两部分组成：一是分析酵母对短脂肪酸残基磷脂的摄取和酰基链重构。在之前的研究中，我们发现含有8- 12碳脂肪酸残基的磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）被酿酒酵母突变体吸收并利用，这些突变体在合成这些磷脂方面存在缺陷。在这个项目中，我们证明了这些可溶性磷脂是。通过追踪稳定同位素标记的PE和PC与癸酸的变化，发现它们的酰基链实际上被正常长度的酰基链所取代。因此，磷脂脂肪酸残基的重塑是真核细胞的共同特性。另一部分是在脂性耶氏菌中发现烷烃诱导基因表达的负转录因子Yas3。在我们之前的研究中，我们报道了两个具有bHLH基序的协同正调控因子Yaslp和Yas2p。我们发现Yas3p改变了它的细胞内定位。当脂质体细胞在有烷烃存在的情况下生长时，在内质网中发现了Yas3p-GFP，而在没有烷烃存在的情况下，在细胞核中发现了Yas3p-GFP，可能通过与Yas2p结合抑制了Yas1p和Yas2p的功能。我们还发现与磷脂酸特异性结合的Yas3p和PA磷酸酶突变增加了烷烃诱导的ALK1基因的表达，表明PA量的变化对Yas3p的功能有重要影响。我们还通过RT-PCR研究了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。我们项目的后半部分导致在Y. lipolytica中发现了一个非常强的诱导启动子和一个适合表达细胞色素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.