Sakai Y, Rogi T, Takeuchi R, Kato N, Tani Y
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Appl Microbiol Biotechnol. 1995 Mar;42(6):860-4. doi: 10.1007/BF00191182.
The methylotrophic yeast, Candida boidinii, was investigated as a new efficient host for heterologous gene expression. The Saccharomyces cerevisiae adenylate kinase gene (ADK1) was used as the first example for heterologous enzyme production in C. boidinii. C. boidinii cells were transformed with plasmids harboring the S. cerevisiae ADK1 gene under the alcohol oxidase (C. boidinii AOD1) promoter. The chromosome-integrant strains produced adenylate kinase protein corresponding to 22%-28% of the total soluble proteins in an enzymatically active form. When the three-copy integrative transformant was grown for 60 h on methanol-glycerol medium in a 1.5-l jar fermentor, adenylate kinase was produced intracellularly with a yield of up to 2 milligrams culture medium. As the expression of the S. cerevisiae ADK1 in C. boidinii was under similar regulation to that of the C. boidinii AOD1, the previously cloned 1.7-kb AOD1 promoter fragment was proved to harbor sufficient cis elements for AOD1 regulation and found to be an efficient promoter for heterologous gene expression.
对甲基营养型酵母博伊丁假丝酵母作为异源基因表达的新型高效宿主进行了研究。酿酒酵母腺苷酸激酶基因（ADK1）被用作在博伊丁假丝酵母中生产异源酶的首个实例。用携带在乙醇氧化酶（博伊丁假丝酵母AOD1）启动子控制下的酿酒酵母ADK1基因的质粒转化博伊丁假丝酵母细胞。染色体整合菌株产生了腺苷酸激酶蛋白，其以酶活性形式存在，占总可溶性蛋白的22% - 28%。当三拷贝整合转化体在1.5升罐式发酵罐中的甲醇 - 甘油培养基上培养60小时时，腺苷酸激酶在细胞内产生，产量高达每升培养基2毫克。由于酿酒酵母ADK1在博伊丁假丝酵母中的表达与博伊丁假丝酵母AOD1的表达受到相似的调控，先前克隆的1.7 kb AOD1启动子片段被证明含有足够的顺式元件用于AOD1调控，并且是异源基因表达的有效启动子。
