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Applied and Environmental Microbiology, January 2005, p. 312-319, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.312-319.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Shinji Hashimoto,1,{dagger} Mayumi Ogura,1 Kazuo Aritomi,2 Hisashi Hoshida,1 Yoshinori Nishizawa,1 and Rinji Akada1*

Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai,1 Yamaguchi Prefectural Industrial Technology Institute, Asutopia, Ube, Japan2

Received 7 April 2004/ Accepted 29 August 2004

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His–, Met–, Lys–, Trp–, Leu–, Arg–, and Ura– auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.

* Corresponding author. Mailing address: Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan. Phone: 81-836-85-9292. Fax: 81-836-85-9201. E-mail: rinji{at}yamaguchi-u.ac.jp.

{dagger} Present address: Research and Development Department, Sanwa Kagaku Kenkyusho Co. Ltd., Hokusei, Inabe, Mie 511-0406, Japan.

Applied and Environmental Microbiology, January 2005, p. 312-319, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.312-319.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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