Studies on structure-function relationship of P450-multi-gene family from yeast and regulatory mechanism of their expression

一些酵母物种可以同化正烷烃作为碳和能量的唯一来源。本研究以麦芽糖假丝酵母（Candida maltosa）和解脂耶氏酵母（Yarrowia lipolytica）为研究对象，探讨了正构烷烃对酵母细胞的代谢、基因表达的调控以及细胞膜增殖的机制。我们已经发现，细胞色素P450是正构烷烃代谢的主要氧化酶，在正构烷烃存在的情况下，细胞色素P450所在的细胞质膜的增殖与细胞色素P450的诱导合成一起进行，并且在麦芽糖假丝酵母的基因组中存在两个以上的P450基因。在本研究中，我们分析了麦芽糖假丝酵母的基因表达和膜增殖的调控机制，以及细胞色素P450的基因结构和解脂耶氏酵母的基因表达调控机制，以了解这些细胞在分子水平上对最疏水底物正烷烃的反应。实验结果由以下六章组成.麦芽糖假丝酵母细胞色素P450基因的分离、鉴定及其编码酶的底物特异性研究.麦芽假丝酵母细胞色素P450基因表达调控的顺式元件分析.我们构建的系统用于分离麦芽糖假丝酵母细胞色素P450基因表达所需的反式因子基因.正构烷烃诱导麦芽糖假丝酵母细胞质膜增殖的机制研究.解脂耶氏酵母细胞色素P450基因的分离与鉴定.一些相关的实验。

Some yeast species can assimilate n-alkane as a sole source of carbon and energy. We have been using Candida maltosa (an industrial microbe for dicarboxylic acid production) and Yarrowia lipolytica (a haploid-type yeast) to investigate the metabolism of n-alkane, regulation of gene expression and the mechanism of membrane proliferation in the cells when they are grown on n-alkane. We had already found that cytochromes P450, which are the primary oxigenases for n-alkane metabolism, are inducibly synthesized together with the proliferation of intracellular membrane where cytochromes P450 locate in the presence of n-alkane and that there are more than two genes for P450 in the genome of Candida maltosa. In the present investigation, we have analyzed the mechanisms of regulation of gene expression and of membrane proliferation in Candida maltosa and the gene structures for cytochromes P450 and the regulation of gene expression in Yarrowia lipolytica to understand the response of these cells at the molecular level to the most hydrophobic substrate, n-alkane. The results of our experiments consist of the following 6 chapters.1. Isolation and characterization of several cytochromes P450 genes, and the substrate specificity of the encoded enzymes in Candida maltosa.2. Analysis of the cis-elements involved in the regulation of expression of cytochromes P450 genes in Candida maltosa.3. The system we have constructed to isolate genes for the trans-factors required for cytochrome P450 gene expression in Candida maltosa.4. The mechanisms of intracellular membrane proliferation induced by n-alkane in Candida maltosa.5. Isolation and characterization of several cytochromes P450 genes in Yarrowia lipolytica.6. Some related experiments.