A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

Adaptive evolution in the Saccharomyces kudriavzevii Aro4p promoted a reduced production of higher alcohols.

The use of unconventional yeast species in human-driven fermentations has attracted a lot of attention in the last few years. This tool allows the alcoholic beverage industries to solve problems related to climate change or the consumer demand for newer high-quality products. In this sense, one of the most attractive species is Saccharomyces kudriavzevii, which shows interesting fermentative traits such as the increased and diverse aroma compound production in wines. Specifically, it has been observed that different isolates of this species can produce higher amounts of higher alcohols such as phenylethanol compared with Saccharomyces cerevisiae. In this work, we have shed light on this feature relating it to the S. kudriavzevii aromatic amino acid anabolic pathway in which the enzyme Aro4p plays an essential role. Unexpectedly, we observed that the presence of the S. kudriavzevii ARO4 variant reduces phenylethanol production compared with the S. cerevisiae ARO4 allele. Our experiments suggest that this can be explained by increased feedback inhibition, which might be a consequence of the changes detected in the Aro4p amino end such as L26 Q24 that have been under positive selection in the S. kudriavzevii specie.

Functional divergence in the proteins encoded by ARO80 from S. uvarum, S. kudriavzevii and S. cerevisiae explain differences in the aroma production during wine fermentation.

Phenylethanol (PE) and phenylethyl acetate (PEA) are commonly desired compounds in wine because of their rose-like aroma. The yeast S. cerevisiae produces the PE either through de novo biosynthesis by shikimate pathway followed by the Ehrlich pathway or the direct phenylalanine catabolism via Ehrlich pathway, and then converted into PEA. Previous work demonstrated that, compared to S. cerevisiae, other Saccharomyces species, such as S. kudriavzevii and S. uvarum, produce higher concentrations of PE and PEA from the precursor phenylalanine, which indicates differential activities of the biosynthetic-involved enzymes. A previous in-silico analysis suggested that the transcriptional activator Aro80p is one of the best candidates to explain these differences. An improved functional analysis identified significant radical amino acid changes in the S. uvarum and S. kudriavzevii Aro80p that could impact the expression of the catabolic genes ARO9 and ARO10, and hence, the production of PE from phenylalanine. Indeed, wine S. cerevisiae strains carrying the S. uvarum and S. kudriavzevii ARO80 alleles increased the production of both compounds in the presence of phenylalanine by increasing the expression of ARO9 and ARO10. This study provides novel insights of the unidentified Aro80p regulatory region and the potential usage of alternatives ARO80 alleles to enhance the PE and PEA concentration in wine.

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

Molecular profiling of beer wort fermentation diversity across natural Saccharomyces eubayanus isolates.

The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating 'end-to-end' genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2-methylpropanol and 3-methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2 S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.

Distinct Transcriptional Changes in Response to Patulin Underlie Toxin Biosorption Differences in Saccharomyces Cerevisiae.

Patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one) is a mycotoxin produced by a suite of fungi species. Patulin is toxic to humans and is a sporadic contaminant in products that were made from fungi-infected fruits. The baker yeast Saccharomyces cerevisiae (S. cerevisiae) has been shown to decrease patulin levels likely by converting it to the less harmful E-ascladiol, yet this capacity is dependent on the strain utilized. In this study we show that four representative strains of different S. cerevisiae lineages differ in their ability to tolerate and decrease patulin levels in solution, demonstrating that some strains are better suitable for patulin biocontrol. Indeed, we tested the biocontrol capacities of the best patulin-reducer strain (WE) in contaminated apple juice and demonstrated their potential role as an efficient natural biocontrol solution. To investigate the mechanisms behind the differences between strains, we explored transcriptomic changes of the top (WE strain) and worst (WA strain) patulin-biocontroller strains after being exposed to this toxin. Large and significant gene expression differences were found between these two strains, the majority of which represented genes associated with protein biosynthesis, cell wall composition and redox homeostasis. Interestingly, the WE isolate exhibited an overrepresentation of up-regulated genes involved in membrane components, suggesting an active role of the membrane towards patulin detoxification. In contrast, WA upregulated genes were associated with RNA metabolism and ribosome biogenesis, suggesting a patulin impact upon transcription and translation activity. These results suggest that different genotypes of S. cerevisiae encounter different stresses from patulin toxicity and that different rates of detoxification of this toxin might be related with the plasma membrane composition. Altogether, our data demonstrates the different molecular mechanisms in S. cerevisiae strains withstanding patulin exposure and opens new avenues for the selection of new patulin biocontroller strains.

GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation.

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA - Sake, NA - North American, and WA - West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA , GPD1SA , ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE . We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background.

Genetic basis of mycotoxin susceptibility differences between budding yeast isolates.

Micophenolic acid (MPA) is an immunosuppressant mycotoxin which impairs yeast cell growth to variable degrees depending on the genetic background. Such variation could have emerged from several phenomena, including MPA gene resistance mutations and variations in copy number and localisation of resistance genes. To test this, we evaluated MPA susceptibility in four S. cerevisiae isolates and genetically dissected variation through the identification of Quantitative Trait Loci. Via linkage analysis we identified six QTLs, majority of which were located within subtelomeres and co-localised with IMD2, an inosine monophosphate dehydrogenase previously identified underlying MPA drug resistance in yeast cells. From chromosome end disruption and bioinformatics analysis, it was found that the subtelomere localisation of IMD2 within chromosome ends is variable depending on the strain, demonstrating the influence of IMD2 on the natural variation in yeast MPA susceptibility. Furthermore, GxE gene expression analysis of strains exhibiting opposite phenotypes indicated that ribosome biogenesis, RNA transport, and purine biosynthesis were impaired in strains most susceptible to MPA toxicity. Our results demonstrate that natural variation can be exploited to better understand the molecular mechanisms underlying mycotoxin susceptibility in eukaryote cells and demonstrate the role of subtelomeric regions in mediating interactions with the environment.

Identification of Nitrogen Consumption Genetic Variants in Yeast Through QTL Mapping and Bulk Segregant RNA-Seq Analyses.

Saccharomyces cerevisiae is responsible for wine must fermentation. In this process, nitrogen represents a limiting nutrient and its scarcity results in important economic losses for the wine industry. Yeast isolates use different strategies to grow in poor nitrogen environments and their genomic plasticity enables adaptation to multiple habitats through improvements in nitrogen consumption. Here, we used a highly recombinant S. cerevisiae multi-parent population (SGRP-4X) derived from the intercross of four parental strains of different origins to identify new genetic variants responsible for nitrogen consumption differences during wine fermentation. Analysis of 165 fully sequenced F12 segregants allowed us to map 26 QTL in narrow intervals for 14 amino acid sources and ammonium, the majority of which represent genomic regions previously unmapped for these traits. To complement this strategy, we performed Bulk segregant RNA-seq (BSR-seq) analysis in segregants exhibiting extremely high and low ammonium consumption levels. This identified several QTL overlapping differentially expressed genes and refined the gene candidate search. Based on these approaches, we were able to validate ARO1, PDC1, CPS1, ASI2, LYP1, and ALP1 allelic variants underlying nitrogen consumption differences between strains, providing evidence of many genes with small phenotypic effects. Altogether, these variants significantly shape yeast nitrogen consumption with important implications for evolution, ecological, and quantitative genomics.