Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis
| Autor: | Mayumi Ogura, Shinji Hashimoto, Kazuo Aritomi, Yoshinori Nishizawa, Hisashi Hoshida, Rinji Akada |
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| Rok vydání: | 2005 |
| Předmět: |
Ultraviolet Rays
Auxotrophy Saccharomyces cerevisiae Mutant Mutagenesis (molecular biology technique) Wine Haploidy Applied Microbiology and Biotechnology Industrial Microbiology Japan Gene Selectable marker Genetics Ecology biology Physiology and Biotechnology biology.organism_classification Diploidy Yeast Culture Media Transformation (genetics) Genetic Techniques Mutagenesis Ethyl Methanesulfonate Mutation Amino Acids Essential Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 71:312-319 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.71.1.312-319.2005 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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