Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains.

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the "1002 Yeast Genomes Project" population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

Genetically modified organisms: adapting regulatory frameworks for evolving genome editing technologies.

Genetic modification of living organisms has been a prosperous activity for research and development of agricultural, industrial and biomedical applications. Three decades have passed since the first genetically modified products, obtained by transgenesis, become available to the market. The regulatory frameworks across the world have not been able to keep up to date with new technologies, monitoring and safety concerns. New genome editing techniques are opening new avenues to genetic modification development and uses, putting pressure on these frameworks. Here we discuss the implications of definitions of living/genetically modified organisms, the evolving genome editing tools to obtain them and how the regulatory frameworks around the world have taken these technologies into account, with a focus on agricultural crops. Finally, we expand this review beyond commercial crops to address living modified organism uses in food industry, biomedical applications and climate change-oriented solutions.

Yeast as a biological platform for vitamin D production: A promising alternative to help reduce vitamin D deficiency in humans.

Vitamin D is an important human hormone, known primarily to be involved in the intestinal absorption of calcium and phosphate, but it is also involved in various nonskeletal processes (molecular, cellular, immune, and neuronal). One of the main health problems nowadays is the vitamin D deficiency of the human population due to lack of sun exposure, with estimates of one billion people worldwide with vitamin D deficiency, and the consequent need for clinical intervention (i.e., prescription of pharmacological vitamin D supplements). An alternative to reduce vitamin D deficiency is to produce good dietary sources of it, a scenario in which the yeast Saccharomyces cerevisiae seems to be a promising alternative. This review focuses on the potential use of yeast as a biological platform to produce vitamin D, summarizing both the biological aspects of vitamin D (synthesis, ecology and evolution, metabolism, and bioequivalence) and the work done to produce it in yeast (both for vitamin D2 and for vitamin D3 ), highlighting existing challenges and potential solutions.

A multi-modal algorithm based on an NSGA-II scheme for phylogenetic tree inference.

The analysis of evolutionary data allows uncovering information about the organisms and how they have adapted and evolved. This information could provide us with new insights about the specialisation of organisms (or part of them), how they adapt, how similar they are with other species, among others. Unfortunately, this evolutionary history can only be estimated, and for that, several computational methods exist. Among the methods, optimisation methods are one of the main approaches to deal with this problem, with multiobjective optimisation producing promising results. In this paper, we deal with multiobjective phylogenetic inference, using a multi-modal metaheuristic approach that exploits the decision space in the multiobjective formulation of the problem. In particular, we incorporate a new metric based on a topological tree distance. We compare the method with state of the art algorithms in terms of performance. Additionally, we perform a thorough analysis of a study case on a yeast Saccharomyces cerevisiae dataset. Results show that our proposal is able to improve the diversity of solutions while improving or keeping the quality of solutions in terms of hypervolume.

Genetically modified organisms: adapting regulatory frameworks for evolving genome editing technologies

Genetic modification of living organisms has been a prosperous activity for research and development of agricultural, industrial and biomedical applications. Three decades have passed since the first genetically modified products, obtained by transgenesis, become available to the market. The regulatory frameworks across the world have not been able to keep up to date with new technologies, monitoring and safety concerns. New genome editing techniques are opening new avenues to genetic modification development and uses, putting pressure on these frameworks. Here we discuss the implications of definitions of living/genetically modified organisms, the evolving genome editing tools to obtain them and how the regulatory frameworks around the world have taken these technologies into account, with a focus on agricultural crops. Finally, we expand this review beyond commercial crops to address living modified organism uses in food industry, biomedical applications and climate change-oriented solutions.

Generation of a Non-Transgenic Genetically Improved Yeast Strain for Wine Production from Nitrogen-Deficient Musts.

The yeast Saccharomyces cerevisiae is the main species responsible for the process that involves the transformation of grape must into wine, with the initial nitrogen in the grape must being vital for it. One of the main problems in the wine industry is the deficiency of nitrogen sources in the grape must, leading to stuck or sluggish fermentations, and generating economic losses. In this scenario, an alternative is the isolation or generation of yeast strains with low nitrogen requirements for fermentation. In the present study, we carry out a genetic improvement program using as a base population a group of 70 strains isolated from winemaking environments mainly in Chile and Argentina (F0), making from it a first and second filial generation (F1 and F2, respectively) based in different families and hybrids. It was found that the trait under study has a high heritability, obtaining in the F2 population strains that consume a minor proportion of the nitrogen sources present in the must. Among these improved strains, strain "686" specially showed a marked drop in the nitrogen consumption, without losing fermentative performance, in synthetic grape must at laboratory level. When using this improved strain to produce wine from a natural grape must (supplemented and non-supplemented with ammonium) at pilot scale under wine cellar conditions, a similar fermentative capacity was obtained between this strain and a widely used commercial strain (EC1118). However, when fermented in a non-supplemented must, improved strain "686" showed the presence of a marked floral aroma absent for EC1118 strain, this difference being probably a direct consequence of its different pattern in amino acid consumption. The combination of the capacity of improved strain "686" to ferment without nitrogen addition and produce floral aromas may be of commercial interest for the wine industry.

Differential Gene Expression and Allele Frequency Changes Favour Adaptation of a Heterogeneous Yeast Population to Nitrogen-Limited Fermentations.

Alcoholic fermentation is fundamentally an adaptation process, in which the yeast Saccharomyces cerevisiae outperforms its competitors and takes over the fermentation process itself. Although wine yeast strains appear to be adapted to the stressful conditions of alcoholic fermentation, nitrogen limitations in grape must cause stuck or slow fermentations, generating significant economic losses for the wine industry. One way to discover the genetic bases that promote yeast adaptation to nitrogen-deficient environments are selection experiments, where a yeast population undergoes selection under conditions of nitrogen restriction for a number of generations, to then identify by sequencing the molecular characteristics that promote this adaptation. In this work, we carried out selection experiments in bioreactors imitating wine fermentation under nitrogen-limited fermentation conditions (SM60), using the heterogeneous SGRP-4X yeast population, to then sequence the transcriptome and the genome of the population at different time points of the selection process. The transcriptomic results showed an overexpression of genes from the NA strain (North American/YPS128), a wild, non-domesticated isolate. In addition, genome sequencing and allele frequency results allowed several QTLs to be mapped for adaptation to nitrogen-limited fermentation. Finally, we validated the ECM38 allele of NA strain as responsible for higher growth efficiency under nitrogen-limited conditions. Taken together, our results revealed a complex pattern of molecular signatures favouring adaptation of the yeast population to nitrogen-limited fermentations, including differential gene expression, allele frequency changes and loss of the mitochondrial genome. Finally, the results suggest that wild alleles from a non-domesticated isolate (NA) may have a relevant role in the adaptation to the assayed fermentation conditions, with the consequent potential of these alleles for the genetic improvement of wine yeast strains.

Transcriptional Activity and Protein Levels of Horizontally Acquired Genes in Yeast Reveal Hallmarks of Adaptation to Fermentative Environments.

In the past decade, the sequencing of large cohorts of Saccharomyces cerevisiae strains has revealed a landscape of genomic regions acquired by Horizontal Gene Transfer (HGT). The genes acquired by HGT play important roles in yeast adaptation to the fermentation process, improving nitrogen and carbon source utilization. However, the functional characterization of these genes at the molecular level has been poorly attended. In this work, we carried out a systematic analysis of the promoter activity and protein level of 30 genes contained in three horizontally acquired regions commonly known as regions A, B, and C. In three strains (one for each region), we used the luciferase reporter gene and the mCherry fluorescent protein to quantify the transcriptional and translational activity of these genes, respectively. We assayed the strains generated in four different culture conditions; all showed low levels of transcriptional and translational activity across these environments. However, we observed an increase in protein levels under low nitrogen culture conditions, suggesting a possible role of the horizontally acquired genes in the adaptation to nitrogen-limited environments. Furthermore, since the strains carrying the luciferase reporter gene are null mutants for the horizontally acquired genes, we assayed growth parameters (latency time, growth rate, and efficiency) and the fermentation kinetics in this set of deletion strains. The results showed that single deletion of 20 horizontally acquired genes modified the growth parameters, whereas the deletion of five of them altered the maximal CO2 production rate (Vmax). Interestingly, we observed a correlation between growth parameters and Vmax for an ORF within region A, encoding an ortholog to a thiamine (vitamin B1) transporter whose deletion decreased the growth rate, growth efficiency, and CO2 production. Altogether, our results provided molecular and phenotypic evidence highlighting the importance of horizontally acquired genes in yeast adaptation to fermentative environments.

Disentangling the genetic bases of Saccharomyces cerevisiae nitrogen consumption and adaptation to low nitrogen environments in wine fermentation.

The budding yeast Saccharomyces cerevisiae has been considered for more than 20 years as a premier model organism for biological sciences, also being the main microorganism used in wide industrial applications, like alcoholic fermentation in the winemaking process. Grape juice is a challenging environment for S. cerevisiae, with nitrogen deficiencies impairing fermentation rate and yeast biomass production, causing stuck or sluggish fermentations, thus generating sizeable economic losses for wine industry. In the present review, we summarize some recent efforts in the search of causative genes that account for yeast adaptation to low nitrogen environments, specially focused in wine fermentation conditions. We start presenting a brief perspective of yeast nitrogen utilization under wine fermentative conditions, highlighting yeast preference for some nitrogen sources above others. Then, we give an outlook of S. cerevisiae genetic diversity studies, paying special attention to efforts in genome sequencing for population structure determination and presenting QTL mapping as a powerful tool for phenotype-genotype correlations. Finally, we do a recapitulation of S. cerevisiae natural diversity related to low nitrogen adaptation, specially showing how different studies have left in evidence the central role of the TORC1 signalling pathway in nitrogen utilization and positioned wild S. cerevisiae strains as a reservoir of beneficial alleles with potential industrial applications (e.g. improvement of industrial yeasts for wine production). More studies focused in disentangling the genetic bases of S. cerevisiae adaptation in wine fermentation will be key to determine the domestication effects over low nitrogen adaptation, as well as to definitely proof that wild S. cerevisiae strains have potential genetic determinants for better adaptation to low nitrogen conditions.

Disentangling the genetic bases of Saccharomyces cerevisiae nitrogen consumption and adaptation to low nitrogen environments in wine fermentation

The budding yeast Saccharomyces cerevisiae has been considered for more than 20 years as a premier model organ- ism for biological sciences, also being the main microorganism used in wide industrial applications, like alcoholic fermentation in the winemaking process. Grape juice is a challenging environment for S. cerevisiae , with nitrogen deficiencies impairing fermentation rate and yeast biomass production, causing stuck or sluggish fermentations, thus generating sizeable economic losses for wine industry. In the present review, we summarize some recent efforts in the search of causative genes that account for yeast adaptation to low nitrogen environments, specially focused in wine fermentation conditions. We start presenting a brief perspective of yeast nitrogen utilization under wine fermentative conditions, highlighting yeast preference for some nitrogen sources above others. Then, we give an outlook of S. cerevisiae genetic diversity studies, paying special attention to efforts in genome sequencing for population structure determination and presenting QTL mapping as a powerful tool for phenotype-genotype correlations. Finally, we do a recapitulation of S. cerevisiae natural diversity related to low nitrogen adaptation, specially showing how different studies have left in evidence the central role of the TORC1 signalling pathway in nitrogen utilization and positioned wild S. cerevisiae strains as a reservoir of beneficial alleles with potential industrial applications (e.g. improvement of industrial yeasts for wine production). More studies focused in disentangling the genetic bases of S. cerevisiae adaptation in wine fermentation will be key to determine the domestication effects over low nitrogen adaptation, as well as to definitely proof that wild S. cerevisiae strains have potential genetic determinants for better adaptation to low nitrogen conditions.

Comparison of Phylogenetic Tree Topologies for Nitrogen Associated Genes Partially Reconstruct the Evolutionary History of Saccharomyces cerevisiae.

Massive sequencing projects executed in Saccharomyces cerevisiae have revealed in detail its population structure. The recent "1002 yeast genomes project" has become the most complete catalogue of yeast genetic diversity and a powerful resource to analyse the evolutionary history of genes affecting specific phenotypes. In this work, we selected 22 nitrogen associated genes and analysed the sequence information from the 1011 strains of the "1002 yeast genomes project". We constructed a total evidence (TE) phylogenetic tree using concatenated information, which showed a 27% topology similarity with the reference (REF) tree of the "1002 yeast genomes project". We also generated individual phylogenetic trees for each gene and compared their topologies, identifying genes with similar topologies (suggesting a shared evolutionary history). Furthermore, we pruned the constructed phylogenetic trees to compare the REF tree topology versus the TE tree and the individual genes trees, considering each phylogenetic cluster/subcluster within the population, observing genes with cluster/subcluster topologies of high similarity to the REF tree. Finally, we used the pruned versions of the phylogenetic trees to compare four strains considered as representatives of S. cerevisiae clean lineages, observing for 15 genes that its cluster topologies match 100% the REF tree, supporting that these strains represent main lineages of yeast population. Altogether, our results showed the potential of tree topologies comparison for exploring the evolutionary history of a specific group of genes.

KAE1 Allelic Variants Affect TORC1 Activation and Fermentation Kinetics in Saccharomyces cerevisiae.

The eukaryotic domain-conserved TORC1 signalling pathway connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis. In Saccharomyces cerevisiae, TORC1 is activated mainly by the nitrogen sources. Recently, this pathway has gotten renewed attention but now in the context of the alcoholic fermentation, due to its key role in nitrogen metabolism regulation. Although the distal and proximal effectors downstream TORC1 are well characterised in yeast, the mechanism by which TORC1 is activated by nitrogen sources is not fully understood. In this work, we took advantage of a previously developed microculture-based methodology, which indirectly evaluates TORC1 activation in a nitrogen upshift experiment, to identify genetic variants affecting the activation of this pathway. We used this method to phenotype a recombinant population derived from two strains (SA and WE) with different geographic origins, which show opposite phenotypes for TORC1 activation by glutamine. Using this phenotypic information, we performed a QTL mapping that allowed us to identify several QTLs for TORC1 activation. Using a reciprocal hemizygous analysis, we validated GUS1, KAE1, PIB2, and UTH1 as genes responsible for the natural variation in the TORC1 activation. We observed that reciprocal hemizygous strains for KAE1 (ATPase required for t6A tRNA modification) gene showed the greatest phenotypic differences for TORC1 activation, with the hemizygous strain carrying the SA allele (KAE1 SA ) showing the higher TORC1 activation. In addition, we evaluated the fermentative capacities of the hemizygous strains under low nitrogen conditions, observing an antagonistic effect for KAE1 SA allele, where the hemizygous strain containing this allele presented the lower fermentation rate. Altogether, these results highlight the importance of the tRNA processing in TORC1 activation and connects this pathway with the yeasts fermentation kinetics under nitrogen-limited conditions.

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains.

Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S. cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.

RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.