Genetic Engineering of Long Chain Dicarboxylic Acid Production in Yeast

酵母生产长链二元酸的基因工程

• 批准号: 06556013
• 负责人: OHTA Akinori
• 金额: $ 4.54万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06556013/
• 关键词: Candida maltosa; Cytochrome P-450; n-Alkane; Dicarboxylic Acid; Gene Disruption; Acyl-CoA Oxidase; Endoplasmic Reticulum; Peroxisome; チトクローム P-450; 遺伝子工学; 酵母; チトクロームP450; アシルCoA オキシダーゼ; Candida tropicalis

The research plan constituted of following three parts. (1) Construction of new dicarboxylic acid-producing yeast by using genetic engineering system which was developed for n-alkane-assimilating yeast Candida maltosa in our laboratory. (2) Improvement of production efficiency and purity of the dicarboxylic acid by overproducing specific cytochrome P-450. (3) Improvement of dicarboxylic acid production by disruption of acyl-CoA oxidase gene which works in degradation of the product.In 1994, we analyzed various genes encoding cytochromes P-450 in C.maltosa.and attained overproduction of specific cytochrome P-450 by GAL gene promoters. We also analyzed the substrate specificity and catalyzed products of the cytochromes. In 1995, disruption of C.maltosa POX2 gene encoding acyl-CoA oxidase was successfully accomplished. Production of dicarboxylic acid from n-tetradecane by the disrupted strain was subsequently confirmed by the Japan Energy group who had fermentation and detection equipments. We constructed a vector that was stably maintained in C.maltosa and isolated cytochrome P-450 reductase gene, of which overexpression will further improve alkane oxidation by C.maltosa.

本研究计划由以下三部分组成。(1)利用本实验室建立的同化正构烷烃酵母麦芽糖假丝酵母基因工程体系构建新型二元酸酵母。(2)通过过量生产特异性细胞色素P-450来提高二羧酸的生产效率和纯度。(3)1994年，我们分析了麦芽糖C.maltosa中编码细胞色素P-450的各种基因，并通过GAL基因启动子获得了特异性细胞色素P-450的过量生产。我们还分析了细胞色素的底物特异性和催化产物。1995年，成功地破坏了编码酰基辅酶A氧化酶的麦芽糖梭菌POX 2基因。由破坏的菌株从正十四烷生产二羧酸随后被具有发酵和检测设备的日本能源集团证实。我们构建了一个载体，在麦芽糖和分离的细胞色素P-450还原酶基因，其过表达将进一步提高烷烃氧化麦芽糖。

项目成果

Moriya Ohkuma: "Evidence that the expression of the gene for NADPH-cytochrome P-450 reductase is alkane-inducible in Candidamaltosa." Bioscience, Biotechnology and Biochemistry. 59. 1328-1330 (1995)

Moriya Ohkuma：“有证据表明 NADPH-细胞色素 P-450 还原酶基因的表达在念珠菌中是烷烃诱导型的。”

Yutaka Masuda: "Cloning and characterization of the POX2 gene in Candida maltosa." Gene. (印刷中). (1995)

Yutaka Masuda：“麦芽糖酵母 POX2 基因的克隆和表征”（出版中）。

Hiroki Sugiyama: "In vivo evidence for non-universal usage of the codon CUG in Candida maltosa." Yeast. 11. 43-52 (1995)

Hiroki Sugiyama：“麦芽糖念珠菌中密码子 CUG 非普遍使用的体内证据。”

Hiroki Sugiyama: "Invitro evidence for non-universal usage of the coden CUG in Candida maltosa" Yeast. 11. 43-52 (1995)

Hiroki Sugiyama：“麦芽糖念珠菌中密码子 CUG 非普遍使用的体外证据”酵母。

Moriya Ohkuma: "Proliferation of intracellular membrane structure upon homologous overproduction of cytochrome P-450 in Candiola maltosa." Biochimica et Biophysica Acta. 1236. 163-169 (1995)

Moriya Ohkuma：“麦芽糖中细胞色素 P-450 同源过量产生，导致细胞内膜结构增殖。”