Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Abstract

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His(-), Met(-), Lys(-), Trp(-), Leu(-), Arg(-), and Ura(-) auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

“…This result also suggested that K. marxianus DMKU3-1042 is haploid, because there was no 0.8-kb band corresponding to the wild-type allele in the lys1::ScURA3 strain. If it is diploid, two bands, one containing the insert and other the wild type, should have appeared (18). Further, complementation of the lys1 mutant with ScLYS1 was tested.…”

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

“…Unfortunately, because wine yeast strains are usually prototrophic (and auxotrophic mutants are not easily obtained) the use of these markers has been precluded for industrial strains. This view has been challenged by a recent work by Hashimoto et al (2005) that were able to obtain auxotrophic mutants from industrial wine yeast strains by UV mutagenesis, these mutants would greatly facilitate genetic engineering by allowing the use of recessive selectable markers.…”

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

“…These frequencies are lower than the previously reported ϳ100% for gene disruption in a haploid strain. While the targeted gene disruption efficiency of the CRISPR-Cas system was indeed affected by the increased ploidy number, 15 to 60% is still a fairly impressive percentage that could significantly reduce the laborious screening process of other marker gene disruption strategies, which require either large-scale screening (frequencies ranged from 10 Ϫ4 to 10 Ϫ3 ) or time-consuming procedures (27,33). In addition, CRISPR-Cas directed gene disruption does not leave a scar or undesirable selection marker in the yeast genome.…”

“…However, because of the presumed polyploidy of most industrial yeast strains (25,26), regular gene deletion methods, which have been routinely used with laboratory S. cerevisiae strains to generate auxotrophic markers, are not applicable to polyploid S. cerevisiae strains. The target gene deletion efficiencies of the regular method are not high enough to achieve the deletion of multiple alleles and overcome high frequencies of reversion of the mutated allele through homologous recombination with the wild-type allele from extra copies of the genome (27). Recently, transcriptionalactivator-like effector nuclease (TALENs) has been widely adopted as a genome-editing tool that outperforms the traditional methods (28)(29)(30).…”