title: Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus.
abstract: The bottom-fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)-type and two S. bayanus (Sb)-type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p-hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G-418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p-hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura⁻ homozygous disruptant (Scura3Δ::TDH3p-hygro/Scura3Δ::TDH3p-hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom-fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1-overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3∆ lager strain will be a useful host for constructing strains of relevance to brewing.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan.
authors.forename: Nobutada
authors.initials: N
authors.lastname: Murakami
authors.forename: Sae
authors.initials: S
authors.lastname: Miyoshi
authors.forename: Ryo
authors.initials: R
authors.lastname: Yokoyama
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
authors.forename: Tomoo
authors.initials: T
authors.lastname: Ogata
mesh_terms: D015825:Chromosomes, Fungal / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D011123:Polyploidy; D012440:Saccharomyces / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D005656:Fungal Proteins