Development of growth selection systems to isolate a-type or α-type of yeast cells spontaneously emerging from MATa/α diploids

Abstract

BackgroundManufacture of MATa and MATα yeast cells is required for crossbreeding, a procedure that permits hybridization and the generation of new heterozygous strains. Crossbreeding also can be performed with a- and α-type of cells, which have the same mating abilities as MATa and MATα haploid cells, respectively.ResultsIn this work, we describe a method to generate a- and α-type of cells via the naturally-occurring chromosomal aberration in parental MATa/α diploids. We successfully designed suitable genetic circuits for expression of the URA3 selection marker gene to permit isolation of a- and α-type of cells, respectively, on solid medium lacking uracil. Furthermore we succeeded in generation of zygotes by mating of both the manufactured a- and α-type of yeast cells.ConclusionsThis process does not require exposure to mutagens such as UV irradiation, thereby avoiding the accumulation of undesirable mutations that would detract from the valuable traits that are under study. All the genetic modifications in the current study were introduced into yeast cells using plasmids, meaning that these traits can be removed without altering the genome sequence. This approach provides a reliable and versatile tool for scientific research and industrial yeast crossbreeding.

An efficient method for isolating mating‐competent cells from bottom‐fermenting yeast using mating pheromone‐supersensitive mutants

“…In this study, we developed a novel efficient method for the isolation of MCCs for yeast crossbreeding, and isolated a/a‐ and α / α ‐type cells from bottom‐fermenting yeast. Although several methods for isolation of MCCs have been previously reported (Alexander et al, ; Fukuda et al, ; Hashimoto, Aritomi, Minohara, et al, ; Mertens et al, ; Nakazawa et al, ; Steensels et al, ), the present method has two advantages. First, it does not depend on DNA recombination techniques.…”

“…Previously, we developed an efficient method for conferring mating ability to the meiotic segregants of bottom‐fermenting yeast by forced expression of the exogenous HO gene controlled by the GAL1 promoter (in preparation). In addition, Nakazawa et al (), Fukuda and Honda (), and Alexander et al () developed methods for isolation of MCCs based on artificial genetic modification. However, these approaches depend on DNA recombination techniques and cannot be put to practical use in terms of public acceptance.…”

Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells

“…In addition, T PGK1 was amplified from genomic DNA derived from strain BY4742 using oligonucleotide pair o5 and o6, and the obtained fragment was digested with Bam HI and Xho I. The two digested DNA fragments, P TDH3 - hygro and T PGK1 , were inserted at the Sac I- Xho I sites of pLY-3U to replace the P STE3 - URA3 - T CYC1 cassette (Fukuda et al 2013a ), yielding a plasmid designated pLY-hygro.…”

“…Using pK6 (Fukuda et al 2013b ) as a template, the expression cassette of the kanMX4 marker (consisting of P TEF1 , ORF and terminator) was amplified with oligonucleotide pair o7 and o8, and inserted in place of the P PGK1 - EGFP - T ADH1 cassette at the Sac II- Xho I sites of pHY-PGA (Fukuda et al 2013a ), yielding a plasmid designated pHY-kan.…”

“…Subsequently, a DNA fragment containing the P PGK1 - a1 - T ADH1 cassette was amplified with oligonucleotide pair o10 and o11 from pHPY-a1 (Fukuda et al 2013b ). Using the In-Fusion kit, the two amplified DNA fragments, P TDH3 - hygro - T PGK1 and P PGK1 - a1 - T ADH1 , were inserted into the Sac I- Sac II sites of pLS-2 K (Fukuda et al 2013a ), yielding a plasmid designated pLhyS-2 K-Pa1.…”