Development of intra-strain self-cloning procedure for breeding baker's yeast strains.
Previously reported self-cloning procedures for breeding of industrial yeast strains require DNA from other strains, plasmid DNA, or mutagenesis. Therefore, we aimed to construct a self-cloning baker's yeast strain that exhibits freeze tolerance via an improved self-cloning procedure. We first disrupted the URA3 gene of a prototrophic baker's yeast strain without the use of any marker gene, resulting in a Δura3 homozygous disruptant. Then, the URA3 gene of the parental baker's yeast strain was used as a selection marker to introduce the constitutive TDH3 promoter upstream of the PDE2 gene encoding high-affinity cyclic AMP phosphodiesterase. This self-cloning procedure was performed without using DNA from other Saccharomyces cerevisiae strains, plasmid DNA, or mutagenesis and was therefore designated an intra-strain self-cloning procedure. Using this self-cloning procedure, we succeeded in producing self-cloning baker's yeast strains that harbor the TDH3p-PDE2 gene heterozygously and homozygously, designated TDH3p-PDE2 hetero and TDH3p-PDE2 homo strains, respectively. These self-cloning strains expressed much higher levels of PDE2 mRNA than the parental strain and exhibited higher viability after freeze stress, as well as higher fermentation ability in frozen dough, when compared with the parental strain. The TDH3p-PDE2 homo strain was genetically more stable than the TDH3p-PDE2 hetero strain. These results indicate that both heterozygous and homozygous strains of self-cloning PDE2-overexpressing freeze-tolerant strains of industrial baker's yeast can be prepared using the intra-strain self-cloning procedure, and, from a practical viewpoint, the TDH3p-PDE2 homo strain constructed in this study is preferable to the TDH3p-PDE2 hetero strain for frozen dough baking.
Division of Life and Agricultural Sciences, Faculty of Life and Environmental Sciences, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan. Electronic address: youji@yamanashi.ac.jp.
Youji
Y
Nakagawa
Division of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
Hiroyuki
H
Ogihara
Division of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
Chisato
C
Mochizuki
Division of Life and Agricultural Sciences, Faculty of Life and Environmental Sciences, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Hideki
H
Yamamura
Division of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan.
Yuzuru
Y
Iimura
Division of Life and Agricultural Sciences, Faculty of Life and Environmental Sciences, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Masayuki
M
Hayakawa
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism; D001939:Bread / Q000382:microbiology; D002999:Clone Cells / Q000145:classification* / Q000166:cytology / Q000378:metabolism*; D003296:Cooking; D005285:Fermentation; D005615:Freezing; D005818:Genetic Engineering / Q000379:methods*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000145:classification* / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); C465226:TDH3 protein, S cerevisiae; D015105:3',5'-Cyclic-AMP Phosphodiesterases
Baker's yeast; Freeze tolerance; Frozen dough; PDE2; Saccharomyces cerevisiae; Self-cloning procedure