Patent Application
20150307833
The disclosure relates to a method for identifying genes that are important determinators of thermotolerance in yeast. The disclosure relates further to genes, identified with the method, especially specific alleles of PRP42 and/or SMD2 and the use of such alleles to increase thermotolerance. It relates further to recombinant strains transformed with such alleles.
Many genetic traits are quantitative and show complex inheritance. Because these traits are so prevalent in nature, understanding the underlying factors is important for various biological fields and applications including agriculture, industrial biotechnology and human disease (Paterson 1998). The study of quantitative traits has its origin in agricultural practice, in which commercially-important features of plants and animals are improved by breeding (Lynch and Walsh 1998). Over the last two decades, much attention has been paid to identifying quantitative trait loci and/or genes contributing to genetic diversity in human disease susceptibility and severity (Risch 2000), since most human diseases seem to be caused by multiple genes instead of single genes (Botstein and Risch 2003). Baker's yeast Saccharomyces cerevisiae has become an important subject for studies in quantitative genetics during the last ten years for several reasons. First, it has a fully sequenced (Goffeau et al., 1996) and well-annotated genome, which enables easy establishment of high-density genetic markers. Second, the ease of making experimental crosses and the relatively high frequency of recombination events in meiosis facilitate identification of QTLs and subsequent fine-mapping (Mancera et al., 2008). Third, the efficiency of genetic engineering strongly simplifies the identification and detailed interaction analysis of causative genes. Fourth, S. cerevisiae displays many quantitative traits that are also important in other cell types, including industrial microorganisms and cells of higher, multicellular organisms. Such properties include thermotolerance (Steinmetz et al., 2002) and oxidative stress tolerance (Diezmann and Dietrich 2011), two factors required for making S. cerevisiae, an opportunistic pathogen, the capacity to produce small molecules such as acetic acid (Marullo et al., 2007), ethanol tolerance (Hu et al., 2007; Swinnen et al., 2012) and many other important features for industrial microorganisms. Other quantitative traits that have been studied in yeast include transcriptional regulation (Brem et al., 2002), sporulation efficiency (Ben-Ari et al., 2006), telomere length (Gatbonton et al., 2006), cell morphology traits (Nogami et al., 2007), mitochondrial genome instability (Dimitrov et al., 2009), global gene expression (Ehrenreich et al., 2009), evolution of biochemical pathways (Bullard et al., 2010) and resistance to chemicals (Ehrenreich et al., 2010).
Despite the recent surge in quantitative trait studies with S. cerevisiae, several issues still remain to be solved. A major challenge is the efficient mapping of minor quantitative trait loci (QTLs) and identification of their causative genes. Minor QTLs have a subtle influence on the phenotype, which is easily masked by epistasis (Carlborg and Haley 2004), gene-environment interactions (Smith and Kruglyak 2008), low association to the phenotype because of limited sample size and complex interactions with QTLs that act in a redundant way. Mainly two methods have been reported to address this problem. Sinha et al., 2008, used a targeted backcross strategy to eliminate the superior allele that plays a major role in the phenotype. A subsequent mapping study enabled the authors to reveal a novel allele that had an epistatic interaction with the previously identified major gene. A disadvantage of this approach is the reduction of genetic diversity in the backcrossing, which easily leads to loss of minor QTLs. In addition, this approach effectively reveals minor QTLs that independently produce additional effects, but may miss QTLs with an epistatic interaction with the major QTL that was eliminated. An important disadvantage of elimination of major QTLs in one parent is the reduction in phenotypic difference between the two parent strains, which makes phenotypic screening much more cumbersome. In a second approach, Swinnen et al., 2012, made use of more stringent phenotyping, tolerance to 17% versus 16% ethanol, which effectively revealed several additional minor QTLs. The disadvantage of this approach is that higher stringency of phenotyping requires much higher numbers of segregants to be phenotyped in order to obtain enough segregants displaying the superior phenotype.
In this work we have established a novel approach for identifying minor QTLs that is based on the observation that superior haploid segregants of heterozygous natural or industrial diploid strains with a superior phenotype often contain recessive mutations that to some extent compromise rather than promote the phenotype of interest. As a result genetic mapping with such segregants often reveals QTLs which are linked to the inferior parent rather than to the superior parent. This allows the construction of two new parent strains, which are both downgraded for the trait of interest by replacement of a superior allele with an inferior allele from the other parent. As a result, the new downgraded parent strains remain sufficiently different in the trait of interest for efficient QTL mapping and also retain all their genetic diversity, in particular all remaining minor QTLs and causative genes. We show the effectiveness of this approach by first mapping QTLs involved in high thermotolerance of a selected yeast strain compared to a control strain, identifying causative genes linked to the superior and inferior parent, constructing two downgraded parent strains and repeating the genetic mapping with the downgraded parent strains. This revealed several new minor QTLs of which we have validated two by identifying the causative gene. Interestingly, the two novel causative genes identified in this study are both involved in pre-mRNA splicing, which suggests an important role for RNA processing in conferring high thermotolerance.
A first aspect of the disclosure is the use of a small nuclear ribonucleoprotein particle protein to obtain thermotolerance in yeast. Preferably, the use is the use of a Prp42 protein and/or a Smd2 protein to obtain thermotolerance in yeast. PRP42 is known to the person skilled in the art and encodes an essential protein for U1 small nuclear ribonucleoprotein (snRNP) biogenesis, which has a high similarity to Prp39 (McLean and Rymond 1998). Similarly, SMD2 is known to the person skilled in the art, and the gene is also encoding a small ribonucleoprotein particle protein. Preferably, the Prp42 protein as well as the Smd2 protein are encoded by a specific allele (indicated as “superior allele”), even more preferably by the Prp42 protein is encoded by the allele as present in strain BY4742 and the Smd2 protein is encoded by the allele as present in strain 21A. More preferably, the Prp42 protein comprises, even more preferably consists of SEQ ID NO:2 and the Smp2 protein comprises, even more preferably consists of SEQ ID NO:4. In one preferred embodiment, the use is the replacement of an endogeneous allele by the superior allele. A preferred embodiment is the replacement by the superior allele of PRP42. Another preferred embodiment is the replacement with the superior allele of SMD2. Still another preferred embodiment is the replacement with both the superior alleles of both PRP42 and SMD2. On another preferred embodiment, the use is overexpression of the gene, preferably of the superior allele, encoding the Prp42 protein and/or the overexpression of the gene, preferably the superior allele, encoding the Smd2 protein. Overexpression, as used herein, means that the expression of the gene in the modified strain is higher than in the parental strain, when grown under the same conditions. As a non-limiting example, overexpression can be obtained by increasing the copy number of the gene, or by replacing the endogeneous promoter by a stronger promoter. Preferably, the yeast is a Saccharomyces sp., even more preferably a Saccharomyces cerevisiae. Thermotolerance, as used herein, means that the yeast can grow at high temperatures, preferably at a temperature of more than 40° C., even more preferably more than 40.5° C., most preferably at 40.7° C. or higher.
Another aspect of the disclosure is a recombinant yeast, preferably a Saccharomyces sp., even more preferably a Saccharomyces cerevisiae, comprising a recombinant gene encoding a Prp42 protein, preferably a protein comprising, even more preferably consisting of SEQ ID NO:2. Preferably, the gene comprises SEQ ID NO:1 and/or comprising a recombinant gene encoding a Smd42 protein, preferably a protein comprising, even more preferably consisting of SEQ ID NO:4. Preferably, the gene comprises SEQ ID NO:3.
Still another aspect of the disclosure is a method to obtain a thermotolerant yeast, comprising the crossing of two parental strains, wherein at least one parental strain encodes a protein comprising SEQ ID NO:2 and/or a protein comprising SEQ ID NO:4.
Another aspect of the disclosure is a method to obtain a thermotolerant yeast, comprising a transformation with a gene encoding a protein consisting of SEQ ID NO:2 and/or SEQ ID NO:4. Preferably, the gene comprises SEQ ID NO:1 and/or SEQ ID NO:3. In one preferred embodiment, the yeast is transformed with a gene encoding a protein consisting of SEQ ID NO:2, preferably a gene comprising SEQ ID NO:1. In another preferred embodiment, the yeast is transformed with a gene encoding a protein consisting of SEQ ID NO:4, preferably a gene comprising SEQ ID NO:3. In still another preferred embodiment, the yeast is transformed with both the genes encoding the protein consisting of SEQ ID NO:2 and SEQ ID NO:4, preferably both the gene comprising SEQ ID NO:1 and the gene comprising SEQ ID NO:3.
Still another aspect of the disclosure is a method for screening thermotolerance in yeast, comprising (1) identifying at least one gene responsible for thermotolerance (2) downgrading the gene in a yeast strain (3) crossing two downgraded strains (4) screening for thermotolerance genes in the offspring of the cross. “Downgrading,” as used herein, means that an allele conferring thermotolerance is replaced by an allele conferring lower thermotolerance, as can be determined by growing the two strains comprising the two different alleles at a critical temperature. Preferably, at least two genes responsible for thermotolerance are identified, and each gene is downgraded in one of the parental strains used in the cross.
Yeast Strains, Growth Conditions and Sporulation
Yeast cells were grown in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone, and 2% (w/v) glucose. 1.5% (w/v) Bacto agar was used to make solid nutrient plates. Transformants were grown on YPD agar plates containing 200 μg/ml geneticin. Mating, sporulation and isolation of haploid segregants were done using standard protocols (Sherman and Hicks 1991).
Phenotyping
Strains were inoculated in liquid YPD and grown in a shaking incubator at 30° C. overnight. The next day the cells were transferred to fresh liquid YPD at an OD600 of 1 and grown for 2 to 4 h to enter exponential phase. The cell cultures were then diluted to an OD600 of 0.5 μl and 5 μl of a fourfold dilution range was spotted on YPD agar plates, which were incubated at different temperatures. Growth was scored after two days incubation for all conditions. All spot tests were repeated at least once, starting with freshly inoculated cultures.
Pooled-Segregant Whole-Genome Sequence Analysis and Determination of SNP Variant Frequency
For each genetic mapping experiment, 58 thermotolerant segregants were grown separately in 50 ml liquid YPD cultures at 30° C. for three days. Cell dry weight was measured for each culture and the cultures were pooled based on the same dry weight. Genomic samples of the pooled culture, together with that of 21A were isolated with standard methods (Johnston 1994). At least 5 μg of each DNA sample was provided to GATC Biotech AG or BGI for sequencing. Paired-end short reads of 100 bp were generated. Sequence alignment was performed using SeqMan NGen. SNP calling, filtering, and frequency prediction was performed using previously described methods (Swinnen et al., 2012).
SNP Scoring in Individual Segregants
SNPs were scored in individual segregants by PCR. At a given chromosomal location, two SNPs spacing between 500 bp and 1,500 bp were chosen for the design of specific primers. For a given SNP, two primers either in the forward or reverse direction, were designed with one mismatch at their 3′ ends. First, a gradient PCR was applied using genomic samples of 21A and BY4742 as templates, with each template tested with two primer combinations (primer pair based on the sequence of BY4742 and primer pair based on the sequence of 21A). The annealing temperature at which the best distinguishing power was obtained with the two parents was used for scoring of the SNPs in the individual segregants.
Reciprocal Hemizygosity Analysis
All the ORFs of non-essential genes in the center of the QTL were deleted separately in both 21A and BY4742. PCR-mediated gene disruption was used (Winzeler et al., 1999). Plasmid pFA6a was used as a template to amplify a linear DNA fragment containing the kanMX4 cassette (Wach et al., 1994), with 50 bp homologous sequences for the target regions at both ends. Transformants growing on YPD geneticin plates were verified by PCR with several combinations of internal and external primers. The verified haploid deletion strains were subsequently crossed with the matching wild-type haploid to generate the hybrid diploids. For RHA with essential genes and fragments containing multiple genes, transformation was performed directly in the hybrid diploid. External SNPs primer pairs together with internal primers within the kanMX4 cassette were used in different combinations to determine in which parent the allele or the fragment had been deleted. For each heterozygous deletion hybrid, at least two isogenic strains were made and evaluated for thermotolerance.
Allele Replacement
The replacement of MKT1-21A with MKT1-BY4742 in 21A was performed by a two-step transformation. For the first transformation, a linear DNA fragment with the AMD1 gene from Zygosaccharomyces rouxii flanked by 50 bp sequences that are homologous to the two sides of the MKT1 ORF was amplified from plasmid pFA6a-AMD1-MX6 (Shepherd and Piper 2010) by PCR, and transformed into 21A. Transformants were grown on YCB (Yeast Carbon Base 1.17%, phosphate buffer 3%, Bacto agar 2%) plates containing 10 mM acetamide. Single colonies were checked for the correct replacement with the use of external and internal primers. For the second transformation, colonies were transformed with a linear DNA fragment containing the MKT1-BY4742 ORF, together with ˜100 bp downstream and upstream. Transformants were grown on YNB galactose (0.17 Yeast Nitrogen Base w/o amino acids and ammonium sulfate, 1.5% Difco agar, 0.01% galactose, pH 6.5) containing 100 mM fluoroacetamide. Colonies were first checked for the presence of MKT1 by PCR, and then confirmed by DNA sequencing. The replacement of PRP42-BY4742 with PRP42-21A in BY4742 was performed in a two-step transformation. For the first transformation, a URA3 gene was inserted ˜50 bp downstream of the PRP42 ORF in BY4742. Colonies growing on -URA plates were confirmed to have a correct insertion by PCR. For the second transformation, a linear DNA fragment containing the ORF of PRP42-21A together with ˜400 bp downstream and upstream was transformed into the previous colonies, and the transformants were grown on 5-FOA plates. Colonies were first checked for the right DNA polymorphism by SNP primer pairs, and then confirmed by DNA sequencing.
We have screened over three hundred natural and industrial isolates of S. cerevisiae for their ability to grow at high temperature, i.e., 40-41° C., on solid YPD plates. Not a single yeast strain was able to grow with a reasonable rate at 42° C. The strain MUCL28177 showed very good growth at 41° C. and was chosen for further analysis. After sporulation, we selected a haploid segregant MUCL28177-21A, further referred to as 21A, which grew almost as well as the parent strain at 41° C. Strain 21A was crossed with the laboratory strain BY4742 which is unable to grow at 41° C. The hybrid 21A/BY4742 diploid strain grew at least as well as the 21A strain at 41° C., indicating that the high thermotolerance of 21A is a dominant characteristic. Over 900 segregants of the 21A/BY4742 diploid strain were phenotyped for thermotolerance. This resulted in 58 segregants with similar growth at high temperature as 21A. The growth of the original strain MUCL28177, the parent strains 21A and BY4742, the hybrid diploid strain 21A/BY4742 and ten representative segregants with varying thermotolerance, is shown in
The 58 thermotolerant segregants were pooled based on dry weight and genomic DNA isolated from the pool. Genomic DNA samples from the pooled segregants and from parent strain 21A were sequenced with an average coverage of 75 and 73, respectively, by IlluminaHiSeq 2000 technology (GATC Biotech, Konstanz). The sequence reads obtained were aligned with the sequence of the reference S288c genome, which is essentially the same as that of the inferior parent strain BY4742. A set of quality-filtered SNPs to be used as genetic markers, was acquired essentially as described before (Swinnen et al., 2012). The SNP variant frequency was plotted against the chromosomal position for each chromosome and smoothened lines through the data points were calculated as described previously (Swinnen et al., 2012). The results are shown in
We have calculated 2-sided P-values for all SNPs (Benjamini and Yekutieli 2005) and selected for further analysis seven putative QTLs with a P-value approaching or lower than the cut-off for significance of 0.05 (
We first fine-mapped QTL1 by scoring eight selected SNPs in individual thermotolerant segregants, which reduced the size of the locus to about 60,000 bp (
The hybrid strain with the MKT1-21A allele showed better growth than the strain with the MKT1-BY4742 allele. We further confirmed the relevance of MKT1 by demonstrating that MKT1 deletion reduced thermotolerance in the 21A strain background (
In a previous QTL mapping study of thermotolerance with a clinical isolate of S. cerevisiae and the lab strain S288c, the MKT1 allele of the clinical isolate was also identified as a causative gene (Steinmetz et al., 2002). In that study, END3 and RHO2, which are located closely to MKT1 in the same QTL, were also reported to have an allele-specific contribution to thermotolerance. However, in the current experimental setup, the alleles from our two genetic backgrounds did not produce a difference in thermotolerance (
QTL3 is linked to the genome of the inferior parent strain, indicating that BY4742 contains a superior genetic element for thermotolerance in this region. This may be consistent with the observation that the hybrid 21A/BY4742 strain is growing slightly better than 21A at 41° C. (
To accelerate identification of the causative genes in this region, we first performed “bulk RHA.” Instead of comparing alleles for each single gene, we first made a reciprocal deletion in the hybrid strain of a fragment with multiple genes. We divided the 40,000 bp region of QTL3 into two fragments, with the first containing 11 genes and the second 14 genes (
We then applied RHA for the six individual genes of FRAGMENT1 that had at least one non-synonymous mutation (
After identifying MKT1-21A and PRP42-BY4742 as causative alleles for high thermotolerance, we confirmed by Sanger sequencing the identity of all SNPs in these genes. MKT1-21A contains two SNPs within the ORF that cause two protein polymorphisms in Mkt1: D30G and K453R. PRP42-BY4742 contains eleven SNPs within the ORF that cause three protein polymorphisms in Prp42: H296Y, F467S, and E526Q.
We next constructed two downgraded parent strains each with their own superior allele replaced by the inferior allele of the other parent: 21ADG: 21A mkt1A::MKT1-BY4742 and BY4742DG: BY4742 prp42Δ::PRP42-21A. Growth at 41° C. of 21ADG was reduced compared to 21A, confirming the importance of MKT1-21A for high thermotolerance in 21A (
From the over 2200 segregants derived from the diploid 21ADG/BY4742DG, composed of the downgraded parent strains, we selected 58 thermotolerant segregants that grew at 40.7° C. at least as well as the 21ADG superior parent strain. The 58 segregants were separately grown in liquid cultures and pooled based on dry weight for genomic DNA isolation. We performed the same genomic DNA isolation from a pool of 58 unselected segregants. The genomic DNA samples from the selected and unselected pools were sequenced (BGI, Hong Kong) with an average coverage of 37 and 36, respectively, and the sequence reads were aligned to the S288c reference sequence. We have used the same set of SNPs as generated in the previous sequencing of the 21A parent strain compared to S288c, for the mapping of QTLs linked to thermotolerance. The SNP variant frequency was plotted against the chromosomal position for the whole genome (
We have focused on QTL8 and QTL9 because they showed the strongest linkage with high thermotolerance (Table 2). In a previous QTL mapping study of thermotolerance (Sinha et al., 2008), the authors identified the NCS2 allele of a clinical isolate as a superior allele compared to the inferior allele from the S288c control strain. Since NCS2 is located in the central region of QTL8 and since the NCS2-21A allele contains the same mutation (A212T) as identified in the previous study, we have tested whether NCS2-21A is also a causative allele in our genetic background. For that purpose, we performed RHA for NCS2 using a hybrid diploid strain constructed from the two downgraded parent strains. We found that the NCS2-21A allele supported higher thermotolerance compared to the NCS2-BY4742 allele, indicating that also in our genetic background the NCS2 allele from the superior strain acted as a causative gene. Deletion of the inferior NCS2-BY4742 allele in the hybrid diploid strain also caused a conspicuous drop in thermotolerance (
We performed Sanger sequencing of both SMD2-21A and SMD2-BY4742, which confirmed the Illumina whole-genome sequencing data that nine point mutations were present in the promoter and terminator region of SMD2-21A versus SMD2-BY4742, and that no mutations were present in the ORF. This indicates that the superior character of the SMD2-21A allele is likely due a difference in expression level. Interestingly, the QTL9 region did not show any indication for linkage to the genome of the original superior parent strain 21A, with only 37 out of 58 thermotolerant segregants from 21A/BY4742 having the SMD2-21A allele. We have also applied RHA for SMD2 in the original 21A/BY4742 hybrid and tested thermotolerance at two temperatures (40.7° C. and 41° C.). However, we could not detect any growth difference at both temperatures for the reciprocal hemizygous deletion strains (
| Number | Date | Country | Kind |
|---|---|---|---|
| 12196785.5 | Dec 2012 | EP | regional |
This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2013/076344, filed Dec. 12, 2013, designating the United States of America and published in English as International Patent Publication WO 2014/090930 A1 on Jun. 19, 2014, which claims the benefit under Article 8 of the Patent Cooperation Treaty and under 35 U.S.C. §119(e) to European Serial No. 12196785.5, filed Dec. 12, 2012.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/076344 | 12/12/2013 | WO | 00 |
