Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate.
By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387-395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.
Sahara
Hiroshi
H
Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto 612-8385, Japan. sahara@gekkeikan.co.jp
Kotaka
Atsushi
A
Kondo
Akihiko
A
Ueda
Mitsuyoshi
M
Hata
Yoji
Y
D000123:Acetyltransferases / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D000431:Ethanol / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D007218:Industrial Microbiology / Q000379:methods; D019656:Loss of Heterozygosity / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D000439:Pentanols; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics; D014920:Wine / Q000382:microbiology*
D008562:Membrane Glycoproteins; D000439:Pentanols; C077394:SED1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000431:Ethanol; D000123:Acetyltransferases; C116217:ATF2 protein, S cerevisiae; C020377:isoamyl acetate