title: Combinatorial gene overexpression and recessive mutant gene introduction in sake yeast.
abstract: 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 MATalpha, 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/sah1Delta::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.
authors.affiliation: Iwata Chemical Co., Ltd., Nakaizumi, Iwata, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Ano
authors.forename: Daisuke
authors.initials: D
authors.lastname: Suehiro
authors.forename: Kamonchai
authors.initials: K
authors.lastname: Cha-Aim
authors.forename: Kazuo
authors.initials: K
authors.lastname: Aritomi
authors.forename: Prasart
authors.initials: P
authors.lastname: Phonimdaeng
authors.forename: Ngarmnit
authors.initials: N
authors.lastname: Nontaso
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Masaki
authors.initials: M
authors.lastname: Mizunuma
authors.forename: Tokichi
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D004171:Diploidy; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005808:Genes, Recessive / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005819:Genetic Markers; D012436:S-Adenosylmethionine