期刊
MOLECULAR AND CELLULAR BIOLOGY
卷 42, 期 4, 页码 -出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/mcb.00560-21
关键词
whole-genome transformation; whole-genome sequence analysis; allele exchange; industrial yeast; acetic acid tolerance; SNF4
资金
- SBO grant SUPERIORYEAST from IWT-Flanders
- SBO grant SPICY from IWT-Flanders
- KU Leuven Program Financing project NATAR
- Open Bio-Incubator of the Erasmus High School in Brussels (Jette)
- [DBOF/12/020]
Whole-genome transformation can generate strains with superior traits by introducing DNA from other strains. This study successfully obtained stable transformants with acetic acid tolerance by transforming a haploid S. cerevisiae strain with DNA from a tolerant strain, and identified the main causative SNP as c.[805G -> T] mutation in the SNF4 gene.
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. 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 Snf4(E269)*, was the main causative SNP. Allele exchange of SNF4(E269)* or snf4 Delta 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.
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