Different missense mutations in PDR1 and PDR3 genes from clotrimazole-resistant sake yeast are responsible for pleiotropic drug resistance and improved fermentative activity.
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.
Mizoguchi
Hiroko
H
Research & Development Department, Hakutsuru Sake Brewing Co. Ltd., 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe 658-0041, Japan.
Yamauchi
Takahiro
T
Watanabe
Mutsumi
M
Yamanaka
Hiroshi
H
Nishimura
Akira
A
Hanamoto
Hideo
H