Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Cell Biology, whole-genome transformation, whole-genome sequence analysis, allele exchange, industrial yeast, acetic acid tolerance, SNF4, SACCHAROMYCES-CEREVISIAE RESPONSE, XYLOSE FERMENTATION, WEAK ACIDS, DNA, RESISTANCE, GENE, CELLS, OVEREXPRESSION, ADAPTATION, EXPRESSION, AMP-Activated Protein Kinases, Acetic Acid, Alleles, Mutation, Polymorphism, Single Nucleotide, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Streptothricins, Transcription Factors, 06 Biological Sciences, 11 Medical and Health Sciences, Developmental Biology, 31 Biological sciences, 32 Biomedical and clinical sciences, 42 Health sciences

Abstract:

Whole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes-most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805G→T], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.