Komeda T, Yurimoto H, Kato N, Sakai Y, Kondo K
Central Laboratories for Key Technology, Kirin Brewery Co, Ltd, 1-13-5 Fukuura, Kanazawa-ku, 236-0004 Yokohama-shi, Kanagawa, Japan.
Mol Genet Genomics. 2003 Nov;270(3):273-80. doi: 10.1007/s00438-003-0917-0. Epub 2003 Oct 8.
The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5' non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.
博伊丁假丝酵母的FDH1基因编码一种NAD⁺依赖性甲酸脱氢酶，该酶催化甲醇异化途径中的最后一步反应。FDH1的表达受甲醇强烈诱导，AOD1（醇氧化酶）和DAS1（二羟基丙酮合酶）基因的启动子也是如此。当细胞在以葡萄糖作为碳源的培养基上生长时，FDH1的表达可被甲酸诱导，而在有葡萄糖存在的情况下，AOD1和DAS1的表达则被完全抑制。通过缺失分析，我们在FDH1基因的5'非编码区鉴定出两个顺式作用调控元件，分别称为UAS-FM和UAS-M。这两个元件对于甲醇完全诱导FDH1启动子都是必需的，而只有UAS-FM元件是甲酸完全诱导所必需的。无论诱导是通过甲醇还是甲酸实现，UAS-FM元件都以一种依赖于拷贝数但不依赖于其方向的方式增强FDH1启动子的诱导水平，并且还将ACT1启动子从组成型元件转变为可诱导元件。我们的结果不仅为异源基因表达提供了一个强大的启动子，还在分子水平上深入了解了FDH1表达的调控机制。
