Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

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+/Scura3?::TDH3p-hygro, SbURA3+/Sbura3?::KanMX), which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura homozygous disruptant (Scura3?::TDH3p-hygro/Scura3?::TDH3p-hygro Sbura3?::KanMX/Sbura3?::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. Copyright (C) 2012 John Wiley & Sons, Ltd.