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

酵母P450多基因家族结构功能关系及其表达调控机制研究

基本信息

批准号：
08406009
负责人：
TAKAGI Masamichi
金额：
$ 22.46万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

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.

一些酵母菌可以吸收正构烷烃作为碳和能量的唯一来源。我们利用麦芽糖假丝酵母（一种生产二羧酸的工业微生物）和多脂耶氏酵母（一种单倍体型酵母）研究了正构烷烃对细胞的代谢、基因表达的调控以及细胞在正构烷烃上生长时膜增殖的机制。我们已经发现，细胞色素P450是正构烷烃代谢的主要加氧酶，在正构烷烃存在的情况下，细胞色素P450所在的细胞膜增殖过程中被诱导合成，并且在麦氏假丝酵母的基因组中存在两个以上的P450基因。在本研究中，我们分析了麦氏假丝酵母中基因表达和膜增殖的调控机制，以及多脂耶氏菌中细胞色素P450的基因结构和基因表达的调控，以了解这些细胞在分子水平上对最疏水的底物正构烷烃的反应。我们的实验结果包括以下6章。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麦芽糖假丝酵母细胞色素P450基因表达调控的顺式元件分析。我们构建的系统分离了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膜增殖的机制。脂化耶氏菌细胞色素P450基因的分离与鉴定一些相关实验。