Figure 1.
Maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 different yeast strains.
(A) Distribution of relative maximal ethanol production capacity of 68 different yeast strains compared to the wine strain V1116. The semi-static fermentations were performed in 250 mL of YP+33% glucose at 25°C. The V1116 strain produced 18.4% (±0.4%) (v/v) ethanol. (B) Ethanol tolerance of cell proliferation (X-axis) versus maximal ethanol accumulation capacity (Y-axis), expressed as maximal ethanol titer reached, in the 68 yeast strains. The highest ethanol concentration for which there was growth in all dilutions was taken as the maximal ethanol tolerance of cell proliferation. The possible correlation between the two traits was tested with a Spearman test, because of the non-normality of the ethanol accumulation trait. The (one-tailed) Spearman test indicated a weak correlation (90% confidence interval, P-value = 0.0984).
Table 1.
Fermentation results for representative strains from the screen of 68 yeast strains.
Figure 2.
Maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in the superior parent and its segregant.
(A) Identification of a segregant with the same high ethanol accumulation capacity of CBS1585. A segregant, Seg5 (n), derived from CBS1585 (2n) showed better attenuation of the fermentation medium compared to the laboratory strain BY710. The diploid (Seg5/BY710) showed similar final attenuation as the superior strains CBS1585 and Seg5. Strains: (•) Seg5, (○) CBS1585, (▪) Seg5/BY710 and (□) BY710. (B) Maximal ethanol production capacity in 250 mL of YP+33% glucose at 25°C. The strains CBS1585 (2n), Seg5 (n), Seg5/BY710 (2n) showed much higher ethanol accumulation capacity compared to BY710 (n). (C) Growth assays on plates containing YP or YPD plus ethanol (18 and 20% v/v). The strains CBS1585 (2n), Seg5 (n), Seg5/BY710 (2n) showed much higher ethanol tolerance of cell proliferation compared to BY710 (n).
Figure 3.
Maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in meiotic segregants.
(A) Cell proliferation assays on solid media containing YP or YPD plus ethanol (18% and 20% v/v). Stationary phase cells were diluted ten-fold from OD600: 0.5 and 4 µL were spotted on the different media. Seg5 (n) showed much higher ethanol tolerance than BY710 (n) and the segregants derived from the diploid Seg5/BY710 presented different cell proliferation capacity (e.g. Seg11C showed high ethanol tolerance whereas Seg11D was ethanol sensitive). The performance of the segregants in this assay received scores from 0 till 5 according to the growth in the different dilutions. (B) Distribution of maximal ethanol accumulation capacity within 101 meiotic segregants derived from Seg5/BY710. The 101 segregants were preselected based on the assay for ethanol tolerance of cell proliferation (minimum score of 2). The semi-static fermentations were performed in 250 mL of YP+33% glucose at 25°C. (C) Ethanol tolerance of cell proliferation (X-axis) versus maximal ethanol accumulation capacity (Y-axis), expressed as maximal ethanol titer reached, in the 101 segregants. The score for ethanol tolerance of cell proliferation was determined as explained in (A).
Figure 4.
QTL mapping of maximal ethanol accumulation capacity (pool 1) and high ethanol tolerance of cell proliferation (pool 2).
22 selected segregants (pool 1) with high ethanol accumulation capacity and 32 selected segregants (pool 2) with high ethanol tolerance of cell proliferation were pooled for whole genome sequencing analysis, which was performed by two independent companies utilizing the Illumina platform (BGI in green and GATC Biotech in red). An unselected pool composed of 237 segregants (pool 3) was also sequenced twice to assess proper segregation of all chromosomes and possible linkage to inadvertently selected traits. The probability of linkage to the superior or the inferior parent, as determined with the HMM, is indicated on the right.
Table 2.
QTLs identified for maximal ethanol accumulation capacity (pool 1, 22 segregants) by pooled-segregant whole-genome sequencing.
Table 3.
QTLs identified for tolerance of cell proliferation to high ethanol (pool 2, 32 segregants) by pooled-segregant whole-genome sequencing.
Figure 5.
Fine-mapping and bulk RHA of QTL2.
(A) Genes present in QTL2 (pool 1), located on chromosome I, as determined by markers scored in the 22 segregants individually. (B) Bulk RHA (bRHA 1.1) of genes NUP60, ERP1, SWD1, RFA1 and SEN34. Two heterozygous diploids for the five genes were constructed: Seg5/BY710-bRHA1.1Δ (○) and Seg5-bRHA1.1Δ/BY710 (▪). These two diploids were compared with the original strain Seg5/BY710 (•) in semi-static fermentations performed in 250 mL of YP+33% glucose at 25°C. (C) Bulk RHA (bRHA 1.2) of genes YARCdelta3/4/5, YARCTy1-1, YAR009c, YAR010c, tA(UGC), BUD14, ADE1, KIN3, and CDC15. Two heterozygous diploids for the previous genes were constructed: Seg5/BY710-bRHA1.2Δ (○) and Seg5-bRHA1.2Δ/BY710 (▪). These two diploids were compared with the original strain Seg5/BY710 (•) in semi-static fermentations performed in 250 mL of YP+33% glucose at 25°C.
Figure 6.
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.
Figure 7.
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.
Table 4.
Occurrence of the SNPs in the causative genes ADE1 and KIN3 in other yeast strains.