Construction of terminal oxidation systems for hydrocabon chain A use of a variety of yeast cytochrome P450 genes

多种酵母细胞色素P450基因的烃链末端氧化系统的构建

• 批准号: 10556015
• 负责人: OHTA Akinori
• 金额: $ 8.38万
• 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 (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10556015/
• 关键词: endoplasmic reticulum; yeast; Candida maltosa; cytochrome P450Alk; Yarrowia lipolytica; dicarboxylic acid; cytochrome P450 reductase; heterologous expression

The aim of this research was to construct microbial systems which produce oxidized hydrocabons, such as long chain dicarboxylic acids. For this purpose, we cloned various genes which are encoding cytochromes P450 with different specificities, but of CYP52 family members. Those were 8 ALK genes from the yeast Candida maltosa and also 8 YlALK genes from Yarrowia lipolytica. The product of ALK1 gene that is prominently induced and expressed in C. maltosa oxidized very end of n-alkanes, whereas ALK5 gene product also oxidized omega position of fatty acids. ALK7 and ALK8 gene products only oxidize fatty acids. These gene were functionally expressed in heterologous yeast Saccharomyces cerevisiae. The eight cytochromes P450ALK from Y. lipolytica form a different CYP52 sub family. Among the products of YlALK genes, Ylalklp seemed principally to oxidize n-decane and Ylalk2p had some activity to n-hexadecane.These genes encoding different kind of cytochromes P450, properties of which have been elucidated by our current research, thus can be introduced into a new host and be utilized to construct a new production system of oxidized alkanes.

本研究的目的是构建生产氧化碳氢化合物，如长链二元酸的微生物系统。为此目的，我们克隆了不同的基因编码细胞色素P450具有不同的特异性，但CYP52家族成员。这些是来自酵母麦芽糖假丝酵母的8个ALK基因和来自解脂耶氏酵母的8个YlALK基因。ALK 1基因在C.麦芽糖氧化正构烷烃的最末端，而ALK 5基因产物也氧化脂肪酸的ω位。ALK 7和ALK 8基因产物仅氧化脂肪酸。这些基因在异源酿酒酵母中得到功能性表达。从Y. Lipolytica形成不同的CYP 52亚家族。在YlALK基因的产物中，Ylalklp主要氧化正癸烷，Ylalk2p具有一定的氧化正十六烷的活性，这些基因编码不同的细胞色素P450，我们的研究已经阐明了这些细胞色素的性质，因此可以将这些基因导入到新的宿主中，并利用它们构建一个新的氧化烷烃生产系统。

项目成果

Iida, T., Sumita, T., et al.: "The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast Yarrowia lipolytica: cloning and characterization of genes encoding for new CYP52 family members"Yeast. (in press).

Iida, T.、Sumita, T. 等人：“正烷烃同化酵母解脂耶氏酵母的细胞色素 P450ALK 多基因家族：编码新 CYP52 家族成员的基因的克隆和表征”酵母。

Iida, T., Ohta, A., et al.: "Cloning and Characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica"Yeast. 14. 1387-1397 (1998)

Iida, T., Ohta, A. 等人：“解脂耶氏酵母对正癸烷同化至关重要的正烷烃诱导型细胞色素 P450 基因的克隆和表征”酵母。

Zimmer, T., Iida, T., et al.: "Relation between evolutionary distance and enzymatic properties among the members of the CYP52A subfamily of Candida maltosa"Biochemical and Biophysical Research Communications. 251. 244-247 (1998)

Zimmer, T.、Iida, T. 等人：“麦芽糖假丝酵母 CYP52A 亚家族成员之间的进化距离和酶特性之间的关系”生物化学和生物物理研究通讯。

Iida, T., Sumita, T., et al.: "The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast Yarrowia lipolytica : cloning and characterization of genes encoding for new CYP52 family members"Yeast. (in press).

Iida, T., Sumita, T., et al.：“正烷烃同化酵母解脂耶氏酵母的细胞色素 P450ALK 多基因家族：编码新 CYP52 家族成员的基因的克隆和表征”酵母。

Ohkuma, M., Zimmer, T., et al.: "Isozyme function of n-alkane-inducible cytochrome P450 in Candida maltosa revealed by sequential gene disruption"Journal of Biological Chemistry. 273. 3948-3953 (1998)

Ohkuma, M.、Zimmer, T. 等人：“通过连续基因破坏揭示麦芽糖念珠菌中正烷烃诱导型细胞色素 P450 的同工酶功能”生物化学杂志。