Study on molecular mechanisms of uptake, conversion, and excretion of lipids in yeast.

为了开发用于从烷烃和脂肪酸生产有用化学品的酵母系统，有必要研究酵母如何利用这些自然资源。在这个项目中，我们分析了两种酵母菌，可以利用正构烷烃和脂肪酸作为碳源，解脂耶氏酵母和麦芽糖假丝酵母。解脂耶氏酵母具有最近完成序列分析的单倍体基因组。麦芽糖C.maltosa具有二倍体基因组，并已用于生产长链二羧酸（DCA）。结果表明：1.解脂耶氏酵母对正十六烷的吸收具有能量依赖性。通过增加编码ABC转运蛋白的CmCDR 1基因的表达，提高了麦芽糖梭菌对长链DCA的产量; 3.在烷烃诱导基因的启动子上鉴定了一个烷烃响应元件ARE 1。构建了依赖ARE 1的lacZ报告基因表达系统，并将其整合到解脂耶氏酵母基因组中。通过对重组菌株进行诱变，获得了一株YAS 1基因缺陷突变株。yas 1突变体和Δ yas 1不能在正癸烷上生长，但能在油酸上生长。Yas 1 p定位于细胞核，具有基本螺旋环螺旋（bHLH）基序。CHIP检测证明Yas 1 p-HA与ALK 1启动子区域结合。从这些结果，我们得出结论，Yas 1 p是一个转录因子的正烷烃诱导基因表达。由于Yas 1 p bHLH区与酿酒酵母的Ino 4p bHLH具有同源性，而Ino 4p与另一个具有bHLH基序的蛋白Ino 2 p形成异源二聚体，因此我们通过搜索解脂耶氏酵母基因组序列数据鉴定了一个基因YAS 2，并证明Yas 2 p与Yas 1 p结合，负责烷烃相关基因的表达。

To develop yeast systems for production of useful chemicals from alkanes and fatty acids, it is necessary to study how these natural resources are utilized by yeasts. In this project, we analyzed two yeast species that can utilize n-alkanes and fatty acids as carbon sources, Yarrowia lipolytica and Candida maltosa. Y.lipolytica has a haploid genome of which sequence analysis was completed recently. C.maltosa has a diploid genome and has been used for production of long chain dicarboxylic acids (DCA). We obtained following new results.1.Y.lipolytica takes up n-hexadecane in an energy dependent manner. This uptake is induced by alkane and repressed by glycerol and by mutations in peroxin genes.2.The production of long chain DCA by C.maltosa was improved by the increased expression of CmCDR1 gene that encodes an ABC transporter.3.We identified an alkane-responsive element ARE1 on the promoter of alkane-inducible genes. ARE1 dependent lacZ reporter expression system was constructed and integrated into the Y.lipolytica genome. By mutagenizing the resultant recombinant strain, we obtained one mutant defective in YAS1 gene. The yas1 mutant and Δyas1 did not grow on n-decane but grow on oleic acid. Yas1p is localized in nucleus and has a basic helix loop helix (bHLH) motif. CHIP assay proved that Yas1p-HA bound the ALK1 promoter region. From these results, we concluded that Yas1p is a transcription factor for n-alkane inducible gene expression. Since Yas1p bHLH region has homology to Ino4p bHLH of S.cerevisiae and Ino4p forms heterodimer with Ino2p, another protein with bHLH motif, we identified a gene YAS2 by searching Y.lipolytica genome sequence data and proved that Yas2p binds to Yas1p and is responsible for the alkane-responsible gene expression.