Combinatorial Gene Overexpression and Recessive Mutant Gene Introduction in Sake Yeast

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  • ANO Akihiko
    Iwata Chemical Co., Ltd.
  • SUEHIRO Daisuke
    Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine
  • CHA-AIM Kamonchai
    Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine
  • ARITOMI Kazuo
    Yamaguchi Prefectural Industrial Technology Institute
  • PHONIMDAENG Prasart
    Department of Microbiology, Faculty of Science, Khon Kaen University
  • NONTASO Ngarmnit
    Department of Microbiology, Faculty of Science, Khon Kaen University
  • HOSHIDA Hisashi
    Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine
  • MIZUNUMA Masaki
    Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University
  • MIYAKAWA Tokichi
    Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University
  • AKADA Rinji
    Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine

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Industrial yeast strains are generally diploid and are often defective in sporulation. Such strains are hence thought to be less tractable for manipulation by genetic engineering. To facilitate more reliable genetic manipulation of the diploid yeast Japanese sake, we constructed variants of this strain that were homozygous for a URA3 deletion, homozygous for either MATa or MATα, and homozygous for either the his3 or the lys4 mutation. A ura3-null genotype enabled gene targeting to be undertaken more easily. The TDH3 promoter was inserted upstream of six yeast genes that have been implicated in flavor control to drive their constitutive overexpression. The homozygous MAT alleles, combined with the non-complementary auxotrophic mutations in the targeted transformants, allowed for tetraploid selection through mating. This resulted in the combinatorial construction of tetraploid strains that overexpress two different genes simultaneously. In addition, a recessive mutant gene, sah1-1, that is known to overproduce S-adenosylmethionine, was introduced into the diploid sake strain by the replacement of one wild-type allele and subsequent disruption of the other. The resulting sah1-1/sah1Δ::URA3 strain produced higher amounts of S-adenosylmethionine than the wild type. The novel sake yeast diploid strains we generated in this study can thus undergo simple PCR-mediated gene manipulation and mating in a manner analogous to established laboratory strains. Moreover, none of these sake strains had extraneous sequences, and they are thus suitable for use in commercial applications.

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