2009年2月
Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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- 巻
- 82
- 号
- 2
- 開始ページ
- 387
- 終了ページ
- 395
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1007/s00253-008-1833-3
- 出版者・発行元
- SPRINGER
Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.
- リンク情報
- ID情報
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- DOI : 10.1007/s00253-008-1833-3
- ISSN : 0175-7598
- eISSN : 1432-0614
- J-Global ID : 200902296320528672
- Web of Science ID : WOS:000263058000022