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Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol levels of cell proliferation in yeast.

Pais TM, Foulquié-Moreno MR, Hubmann G, Duitama J, Swinnen S, Goovaerts A, Yang Y, Dumortier F, Thevelein JM - PLoS Genet. (2013)

Bottom Line: From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced.Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different.Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Flanders, Belgium.

ABSTRACT
The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance.

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Single gene RHA and loss of function assessment for the causative genes ADE1 and KIN3 in QTL2.(A) RHA of genes ADE1 and KIN3. The diploid strain Seg5/BY710 (•) had ADE1 or KIN3 deleted in one of the alleles separately. The resulting strains Seg5/BY710-ade1Δ (○), Seg5-ade1Δ/BY710 (▴), Seg5/BY710-kin3Δ (Δ) and Seg5-kin3Δ/BY710 (▪) were compared with the original diploid Seg5/BY710 (•) in semi-static small-scale fermentations in YP+33% glucose at 25°C. The deletion of the alleles present in Seg5 resulted in diploids with lower ethanol accumulation capacity in comparison to the original strain and the deletion of the alleles from BY710. (B) ADE1 and KIN3 loss-of-function assays. The genes ADE1 and KIN3 were deleted in the haploid strains Seg5 (•) and BY4742 (Δ) separately. The strains Seg5-ade1Δ (○), Seg5-kin3Δ (▴), BY4742-ade1Δ (▪) and BY4742-kin3Δ (□) were evaluated by semi-static fermentations in 250 mL of YP+33% glucose at 25°C. (C) Determination of ethanol tolerance of cell proliferation with the hybrid diploid strains Seg5/BY710-ade1Δ, Seg5-ade1Δ/BY710, Seg5/BY710-kin3Δ and Seg5-kin3Δ/BY710.
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pgen-1003548-g006: Single gene RHA and loss of function assessment for the causative genes ADE1 and KIN3 in QTL2.(A) RHA of genes ADE1 and KIN3. The diploid strain Seg5/BY710 (•) had ADE1 or KIN3 deleted in one of the alleles separately. The resulting strains Seg5/BY710-ade1Δ (○), Seg5-ade1Δ/BY710 (▴), Seg5/BY710-kin3Δ (Δ) and Seg5-kin3Δ/BY710 (▪) were compared with the original diploid Seg5/BY710 (•) in semi-static small-scale fermentations in YP+33% glucose at 25°C. The deletion of the alleles present in Seg5 resulted in diploids with lower ethanol accumulation capacity in comparison to the original strain and the deletion of the alleles from BY710. (B) ADE1 and KIN3 loss-of-function assays. The genes ADE1 and KIN3 were deleted in the haploid strains Seg5 (•) and BY4742 (Δ) separately. The strains Seg5-ade1Δ (○), Seg5-kin3Δ (▴), BY4742-ade1Δ (▪) and BY4742-kin3Δ (□) were evaluated by semi-static fermentations in 250 mL of YP+33% glucose at 25°C. (C) Determination of ethanol tolerance of cell proliferation with the hybrid diploid strains Seg5/BY710-ade1Δ, Seg5-ade1Δ/BY710, Seg5/BY710-kin3Δ and Seg5-kin3Δ/BY710.

Mentions: YARCdelta3/4/5, YARCTy1-1, YAR009c and YAR010c are transposable elements, while tA(UGC)A encodes one of the sixteen tRNAs for the amino acid alanine. BUD14 is involved in bud-site selection [26], ADE1 is involved in de novo purine biosynthesis [27], KIN3 encodes a non-essential serine/threonine protein kinase involved in a.o. DNA damage repair [28] and CDC15 encodes a protein kinase involved in control of the cell division cycle [29]. In order to identify the genes(s) involved in ethanol accumulation capacity, we investigated the most likely candidate genes individually with the classical one-gene RHA [10]. Involvement of the transposable elements appeared unlikely and was not evaluated by RHA. The other genes, BUD14, ADE1, KIN3 and CDC15, have polymorphisms (SNPs and/or indels) within their ORFs and/or promoter regions. RHA with the genes ADE1 and KIN3 showed that deletion of the Seg5 alleles resulted in strains with clearly lower ethanol accumulation capacity and higher glucose leftover compared to the strain with deletion of the respective BY allele, indicating that ADE1 and KIN3 are causative genes for high ethanol accumulation capacity in Seg5 (Figure 6A). For both genes, the hybrid parent strain Seg5/BY710 behaved in a similar way as the strain with the deleted BY710 allele. For CDC15 and BUD14 there was no difference in the performance of the two reciprocally deleted diploid strains (not shown). Deletion of ADE1 and KIN3 in the Seg5 and BY backgrounds caused a more pronounced effect in the Seg5 sake genetic background (Figure 6B).


Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol levels of cell proliferation in yeast.

Pais TM, Foulquié-Moreno MR, Hubmann G, Duitama J, Swinnen S, Goovaerts A, Yang Y, Dumortier F, Thevelein JM - PLoS Genet. (2013)

Single gene RHA and loss of function assessment for the causative genes ADE1 and KIN3 in QTL2.(A) RHA of genes ADE1 and KIN3. The diploid strain Seg5/BY710 (•) had ADE1 or KIN3 deleted in one of the alleles separately. The resulting strains Seg5/BY710-ade1Δ (○), Seg5-ade1Δ/BY710 (▴), Seg5/BY710-kin3Δ (Δ) and Seg5-kin3Δ/BY710 (▪) were compared with the original diploid Seg5/BY710 (•) in semi-static small-scale fermentations in YP+33% glucose at 25°C. The deletion of the alleles present in Seg5 resulted in diploids with lower ethanol accumulation capacity in comparison to the original strain and the deletion of the alleles from BY710. (B) ADE1 and KIN3 loss-of-function assays. The genes ADE1 and KIN3 were deleted in the haploid strains Seg5 (•) and BY4742 (Δ) separately. The strains Seg5-ade1Δ (○), Seg5-kin3Δ (▴), BY4742-ade1Δ (▪) and BY4742-kin3Δ (□) were evaluated by semi-static fermentations in 250 mL of YP+33% glucose at 25°C. (C) Determination of ethanol tolerance of cell proliferation with the hybrid diploid strains Seg5/BY710-ade1Δ, Seg5-ade1Δ/BY710, Seg5/BY710-kin3Δ and Seg5-kin3Δ/BY710.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675000&req=5

pgen-1003548-g006: Single gene RHA and loss of function assessment for the causative genes ADE1 and KIN3 in QTL2.(A) RHA of genes ADE1 and KIN3. The diploid strain Seg5/BY710 (•) had ADE1 or KIN3 deleted in one of the alleles separately. The resulting strains Seg5/BY710-ade1Δ (○), Seg5-ade1Δ/BY710 (▴), Seg5/BY710-kin3Δ (Δ) and Seg5-kin3Δ/BY710 (▪) were compared with the original diploid Seg5/BY710 (•) in semi-static small-scale fermentations in YP+33% glucose at 25°C. The deletion of the alleles present in Seg5 resulted in diploids with lower ethanol accumulation capacity in comparison to the original strain and the deletion of the alleles from BY710. (B) ADE1 and KIN3 loss-of-function assays. The genes ADE1 and KIN3 were deleted in the haploid strains Seg5 (•) and BY4742 (Δ) separately. The strains Seg5-ade1Δ (○), Seg5-kin3Δ (▴), BY4742-ade1Δ (▪) and BY4742-kin3Δ (□) were evaluated by semi-static fermentations in 250 mL of YP+33% glucose at 25°C. (C) Determination of ethanol tolerance of cell proliferation with the hybrid diploid strains Seg5/BY710-ade1Δ, Seg5-ade1Δ/BY710, Seg5/BY710-kin3Δ and Seg5-kin3Δ/BY710.
Mentions: YARCdelta3/4/5, YARCTy1-1, YAR009c and YAR010c are transposable elements, while tA(UGC)A encodes one of the sixteen tRNAs for the amino acid alanine. BUD14 is involved in bud-site selection [26], ADE1 is involved in de novo purine biosynthesis [27], KIN3 encodes a non-essential serine/threonine protein kinase involved in a.o. DNA damage repair [28] and CDC15 encodes a protein kinase involved in control of the cell division cycle [29]. In order to identify the genes(s) involved in ethanol accumulation capacity, we investigated the most likely candidate genes individually with the classical one-gene RHA [10]. Involvement of the transposable elements appeared unlikely and was not evaluated by RHA. The other genes, BUD14, ADE1, KIN3 and CDC15, have polymorphisms (SNPs and/or indels) within their ORFs and/or promoter regions. RHA with the genes ADE1 and KIN3 showed that deletion of the Seg5 alleles resulted in strains with clearly lower ethanol accumulation capacity and higher glucose leftover compared to the strain with deletion of the respective BY allele, indicating that ADE1 and KIN3 are causative genes for high ethanol accumulation capacity in Seg5 (Figure 6A). For both genes, the hybrid parent strain Seg5/BY710 behaved in a similar way as the strain with the deleted BY710 allele. For CDC15 and BUD14 there was no difference in the performance of the two reciprocally deleted diploid strains (not shown). Deletion of ADE1 and KIN3 in the Seg5 and BY backgrounds caused a more pronounced effect in the Seg5 sake genetic background (Figure 6B).

Bottom Line: From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced.Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different.Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Flanders, Belgium.

ABSTRACT
The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance.

Show MeSH
Related in: MedlinePlus