Mizoguchi Hiroko, Yamauchi Takahiro, Watanabe Mutsumi, Yamanaka Hiroshi, Nishimura Akira, Hanamoto Hideo
Research & Development Department, Hakutsuru Sake Brewing Co. Ltd., 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe 658-0041, Japan.
J Biosci Bioeng. 2002;93(2):221-7. doi: 10.1263/jbb.93.221.
Series of gain-of-function missense mutations in PDR1 and PDR3 that encode transcription factors are known to increase the expression of ABC transporters that extrude various kinds of drugs out of a cell, leading to pleiotropic drug resistance (PDR). Previously, the PDR1-H176 (L309S) allele from a clotrimazole (CTZ)-resistant sake yeast mutant has been cloned and found to be sufficient to confer PDR and improved fermentative activity on the arginase-deficient (car1/car1) sake yeast HL163. In this study, another PDR1 and three PDR3 a!leles were cloned from other CTZ-resistant mutants. These alleles contain different missense mutations, PDR1-2 (M308I), PDR3-C21 (L950S), PDR3-C231 (G948D), and PDR3-11 (G957D). The gene recombinants containing each of the mutations found in CTZ-resistant mutants were constructed using the wild-type sake yeast K1001. The gene recombination was designed to generate only a nucleotide replacement without any other DNA changes such as insertion of external DNA fragments. The resultant recombinants that were heterozygous for each of the mutations showed various degrees of drug resistance that were comparable to those of the CTZ-resistant mutants. The recombinants, except one introduced with PDR1-H176, also fermented sake mash faster and produced higher amounts of alcohol than K1001 did. These results demonstrated that PDR1-2, PDR3-C21, PDR3-C231, and PDR3-11 are sufficient to confer PDR and improved fermentative activity on K1001, and suggested that previous results in which fermentative activity of HL163 was significantly improved by PDR1-H176 must have depended on its genetic background, probably arginase deficiency.
已知编码转录因子的PDR1和PDR3中的一系列功能获得性错义突变会增加ABC转运蛋白的表达，这些转运蛋白将各种药物排出细胞，导致多药耐药性（PDR）。此前，已从抗克霉唑（CTZ）的清酒酵母突变体中克隆出PDR1-H176（L309S）等位基因，并发现其足以赋予精氨酸酶缺陷型（car1/car1）清酒酵母HL163多药耐药性并提高发酵活性。在本研究中，从其他抗CTZ突变体中克隆了另一个PDR1和三个PDR3等位基因。这些等位基因包含不同的错义突变，即PDR1-2（M308I）、PDR3-C21（L950S）、PDR3-C231（G948D）和PDR3-11（G957D）。使用野生型清酒酵母K1001构建了含有在抗CTZ突变体中发现的每种突变的基因重组体。基因重组设计为仅产生核苷酸替换，而无任何其他DNA变化，如插入外部DNA片段。所得的每个突变杂合的重组体表现出不同程度的耐药性，与抗CTZ突变体相当。除了导入PDR1-H176的重组体之外，其他重组体发酵清酒醪的速度也比K1001更快，并且产生的酒精量更高。这些结果表明，PDR1-2、PDR3-C21、PDR3-C231和PDR3-11足以赋予K1001多药耐药性并提高发酵活性，并表明先前PDR1-H176显著提高HL163发酵活性的结果一定依赖于其遗传背景，可能是精氨酸酶缺陷。
