Combinatorial Gene Overexpression and Recessive Mutant Gene Introduction in Sake Yeast

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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 <I>URA3</I> deletion, homozygous for either <I>MAT</I><B>a</B> or <I>MAT</I>α, and homozygous for either the <I>his3</I> or the <I>lys4</I> mutation. A <I>ura3</I>-null genotype enabled gene targeting to be undertaken more easily. The <I>TDH3</I> promoter was inserted upstream of six yeast genes that have been implicated in flavor control to drive their constitutive overexpression. The homozygous <I>MAT</I> 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, <I>sah1-1</I>, that is known to overproduce <I>S</I>-adenosylmethionine, was introduced into the diploid sake strain by the replacement of one wild-type allele and subsequent disruption of the other. The resulting <I>sah1-1/sah1</I>Δ<I>::URA3</I> strain produced higher amounts of <I>S</I>-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.


  • Bioscience, Biotechnology, and Biochemistry

    Bioscience, Biotechnology, and Biochemistry 73(3), 633-640, 2009-03-23

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

References:  37

Cited by:  2


  • NII Article ID (NAID)
  • Text Lang
  • Article Type
    Journal Article
  • ISSN
  • NDL Article ID
  • NDL Source Classification
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL Call No.
  • Data Source
    CJP  CJPref  NDL  J-STAGE 
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