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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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Loss of function assessment and complementation assay with the causative gene URA3 in QTL3.(A) URA3 loss-of-function assay. The strain Seg5/BY710 (•) had its URA3 copy deleted, Seg5-ura3Δ/BY710 (○). Both strains were tested in 250 mL of YP+33% glucose at 25°C. (B) URA3 complementation study. The URA3 auxotrophic strain BY4741-ura3Δ (▪) had the URA3 gene inserted in its original position, BY4741-URA3 (□). The performance of both strains were assessed 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-ura3Δ, Seg5-ura3Δ/BY710-ura3Δ.
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pgen-1003548-g007: Loss of function assessment and complementation assay with the causative gene URA3 in QTL3.(A) URA3 loss-of-function assay. The strain Seg5/BY710 (•) had its URA3 copy deleted, Seg5-ura3Δ/BY710 (○). Both strains were tested in 250 mL of YP+33% glucose at 25°C. (B) URA3 complementation study. The URA3 auxotrophic strain BY4741-ura3Δ (▪) had the URA3 gene inserted in its original position, BY4741-URA3 (□). The performance of both strains were assessed 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-ura3Δ, Seg5-ura3Δ/BY710-ura3Δ.

Mentions: We also analysed in more detail QTL3, located on chromosome V. In the same chromosomal region, Swinnen et al. [5] previously identified URA3 as a causative gene in tolerance of cell proliferation to high ethanol levels of VR1, a Brazilian bioethanol production strain, in comparison with BY4741 as inferior parent strain. Since we crossed Seg5 with an ura3 auxotrophic laboratory strain (BY710), we first tested whether deletion of URA3 in Seg5 affected maximal ethanol accumulation in this genetic background. The fermentation profile and maximal ethanol accumulation of the strain Seg5-ura3Δ/BY710-ura3Δ (which is thus homozygous for ura3Δ) compared with the Seg5/BY710-ura3Δ diploid (which is heterozygous for ura3Δ) are shown in Figure 7A. Double deletion of URA3 resulted in a strain with a reduced ethanol fermentation rate, lower maximal ethanol accumulation and higher glucose leftover. We have also tested the effect of introducing URA3 in the ura3 auxotrophic strain BY4741, which accumulates only low amounts of ethanol under VHG conditions (±12% v/v). Introduction of URA3 enhanced the fermentation rate in the later stages of the fermentation and resulted in a clearly higher maximal ethanol titer and lower glucose leftover (Figure 7B). These results show that URA3 positively affects maximal ethanol accumulation capacity. The URA3 gene was located in QTL3, which was not significantly linked with ethanol tolerance of cell proliferation. When we tested the hybrid diploid strains previously used in RHA for maximal ethanol accumulation for determination of ethanol tolerance of cell proliferation, we observed slightly better growth for the strain with the URA3 allele from Seg5 (Figure 7C). This confirms that URA3 has only a minor contribution to this phenotype in this genetic background and suggests that the very weak upward deviation in the SNP variant frequency plot observed in this position for ethanol tolerance of cell proliferation might have been due to the URA3 gene.


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)

Loss of function assessment and complementation assay with the causative gene URA3 in QTL3.(A) URA3 loss-of-function assay. The strain Seg5/BY710 (•) had its URA3 copy deleted, Seg5-ura3Δ/BY710 (○). Both strains were tested in 250 mL of YP+33% glucose at 25°C. (B) URA3 complementation study. The URA3 auxotrophic strain BY4741-ura3Δ (▪) had the URA3 gene inserted in its original position, BY4741-URA3 (□). The performance of both strains were assessed 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-ura3Δ, Seg5-ura3Δ/BY710-ura3Δ.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003548-g007: Loss of function assessment and complementation assay with the causative gene URA3 in QTL3.(A) URA3 loss-of-function assay. The strain Seg5/BY710 (•) had its URA3 copy deleted, Seg5-ura3Δ/BY710 (○). Both strains were tested in 250 mL of YP+33% glucose at 25°C. (B) URA3 complementation study. The URA3 auxotrophic strain BY4741-ura3Δ (▪) had the URA3 gene inserted in its original position, BY4741-URA3 (□). The performance of both strains were assessed 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-ura3Δ, Seg5-ura3Δ/BY710-ura3Δ.
Mentions: We also analysed in more detail QTL3, located on chromosome V. In the same chromosomal region, Swinnen et al. [5] previously identified URA3 as a causative gene in tolerance of cell proliferation to high ethanol levels of VR1, a Brazilian bioethanol production strain, in comparison with BY4741 as inferior parent strain. Since we crossed Seg5 with an ura3 auxotrophic laboratory strain (BY710), we first tested whether deletion of URA3 in Seg5 affected maximal ethanol accumulation in this genetic background. The fermentation profile and maximal ethanol accumulation of the strain Seg5-ura3Δ/BY710-ura3Δ (which is thus homozygous for ura3Δ) compared with the Seg5/BY710-ura3Δ diploid (which is heterozygous for ura3Δ) are shown in Figure 7A. Double deletion of URA3 resulted in a strain with a reduced ethanol fermentation rate, lower maximal ethanol accumulation and higher glucose leftover. We have also tested the effect of introducing URA3 in the ura3 auxotrophic strain BY4741, which accumulates only low amounts of ethanol under VHG conditions (±12% v/v). Introduction of URA3 enhanced the fermentation rate in the later stages of the fermentation and resulted in a clearly higher maximal ethanol titer and lower glucose leftover (Figure 7B). These results show that URA3 positively affects maximal ethanol accumulation capacity. The URA3 gene was located in QTL3, which was not significantly linked with ethanol tolerance of cell proliferation. When we tested the hybrid diploid strains previously used in RHA for maximal ethanol accumulation for determination of ethanol tolerance of cell proliferation, we observed slightly better growth for the strain with the URA3 allele from Seg5 (Figure 7C). This confirms that URA3 has only a minor contribution to this phenotype in this genetic background and suggests that the very weak upward deviation in the SNP variant frequency plot observed in this position for ethanol tolerance of cell proliferation might have been due to the URA3 gene.

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