Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses.
During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1P247Sand PRO1E415Kgene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses.
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Ariunzaya
A
Tsolmonbaatar
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Keisuke
K
Hashida
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Yukiko
Y
Sugimoto
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Daisuke
D
Watanabe
Laboratory of Yeast & Fermentation, Food Division, Oriental Yeast Co., Ltd., 3-6-10 Azusawa, Itabashi-ku, Tokyo 174-8505, Japan.
Shuhei
S
Furukawa
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Electronic address: hiro@bs.naist.jp.
Hiroshi
H
Takagi
D001939:Bread / Q000382:microbiology; D003296:Cooking; D005285:Fermentation; D005615:Freezing; D006358:Hot Temperature; D009154:Mutation; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D013395:Sucrose; D011392:Proline; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase
Baking associated stress; Bread dough; Fermentation ability; Industrial baker's yeast; Proline; γ-Glutamyl kinase