Eduardo I. Kessi-Pérez

Horizontal gene transfer is the movement of genetic material across species. In Saccharomyces cerevisiae, a DNA segment known as Region B was acquired horizontally from a distant yeast species. This region (∼17 Kb) encodes five genes and is present in the genomes of yeast strains from different phylogenetic clades. Interestingly, the presence of Region B is not restricted to yeast strains isolated from fermentative environments, leaving its contribution to yeast niche-specific adaptation unclear. In this work, the genomic structure of Region B was analyzed in yeast strains from the ScRAP (S. cerevisiae Reference Assembly Panel) collection, identifying ten structural variants that maintain a circular continuity. To assess the role of Region B in yeast adaptation, we performed a high-throughput phenotyping of the ScRAP collection under different growth conditions, identifying that Region B is associated with higher tolerance to oxidative stress. Then, we characterized the transcriptional activity of each gene within Region B using a fluorescent reporter. The results revealed that gene expression depends on the host's genetic background and transcription factors encoded within Region B. To identify the genetic determinants involved in Region B expression within different genetic backgrounds, three expression quantitative trait loci were mapped and validated. Finally, by performing the deletion of Region B in two different strains, we determined a background-dependent contribution of this region to various fermentative phenotypes. Altogether, our results suggest a complex regulatory interaction between the horizontally acquired genes and the host genome that contributes to yeast adaptation under fermentation conditions.

Optogenetic switches are molecular systems enabling light‐controlled gene expression. These systems are based on the reconstitution of chimeric Transcription Factors (TFs) including a DNA‐binding Domain (DBD), a photoreceptor domain, and an Activation Domain (AD). Thus, depending on the light‐mediated homodimerization or heteromerization of the chimeric TF, the optogenetic switches can be classified as single‐component or two‐component systems, respectively. In the budding yeast Saccharomyces cerevisiae , optogenetic switches have shown multiple applications in metabolic engineering and biotechnology. Here, we expand the repertory of optogenetic switches available in yeast, developing a collection of plasmids and yeast strains carrying single‐component or two‐component optogenetic switches based on the BcWCL1 protein, a blue‐light photoreceptor from Botrytis cinerea that contains a LOV (Light Oxygen Voltage) domain. By dissecting the N‐terminal, C‐terminal, and both protein regions simultaneously, we developed 18 plasmids encoding the BcWCL1 versions fused to the Gal4‐DBD and Gal4‐AD. Assembled plasmids were combined and transformed into yeast to generate single‐component and two‐component optogenetic switches, whose blue‐light response was measured as transcriptional activity of the luciferase reporter. In general, we observed that the two‐component configuration increased the luciferase expression in response to blue‐light compared to single‐component switches. Importantly, BcWCL1 versions with deletions in the C‐terminal region showed the highest luciferase expression upon blue‐light stimulation with the lowest background in the dark condition, suggesting that the C‐terminal region modulates the blue‐light response of this protein. Altogether, we generated new single‐component and two‐component optogenetic switches, each of them achieving different levels of light‐activated luciferase expression, enabling their potential applications in yeast biotechnology.

Saccharomyces cerevisiae is a species of industrial significance in the production of alcoholic beverages; it is the main species responsible for the fermentation of grape must. One of the main current problems in the wine industry is high alcohol levels caused by climate change. Pre- and post-fermentation strategies are used to reduce the alcohol content in wines; however, they are inefficient, affect organoleptic properties, face legal restrictions, and/or increase production costs, which has motivated efforts to obtain microbiological solutions. In the present work, we carried out a two-round breeding program to obtain improved yeast strains with lower ethanol yield. The trait under study showed high heritability (0.619), and we were able to lower the ethanol yield by 10.7% in just one generation. We finally obtained a population composed of 132 strains, of which 6 were used to produce wine from natural grape musts on a pilot scale, highlighting improved strains “C2-1B4” and “C7-1B7” as those that showed the best results (alcohol levels between 0.3 and 1.5% ABV less than expected). Further studies are required to understand the connection between initial sugar concentration and ethanol yield, as well as the genetic variants underlying this phenotype.

Saccharomyces cerevisiae is a yeast species of industrial importance due to its role in the production of several alcoholic beverages, including wine. During wine fermentation, S. cerevisiae must be able to cope with several stresses, such as limited nitrogen availability; nitrogen deficiencies in grape must could lead to stuck or sluggish fermentations, generating for the wine industry significant economic losses. A microbiological alternative to this problem is the generation of yeast strains with low nitrogen requirements for fermentation. In this context, it is important to study the natural diversity regarding adaptation to low nitrogen conditions, especially in terms of searching for beneficial alleles with potential industrial applications. In the present work, we validated the impact of specific SNPs present in the RRT5 gene in an industrial genetic background (T73 strain), analyzing both growth kinetics under microculture conditions and fermentative phenotypes in a larger volume. In this way, we were able to validate that these SNPs do indeed have an impact on the growth kinetics and fermentative capabilities of this strain, although the particular effect depends on the type of must used (synthetic or natural) and the nitrogen content (limiting or non-limiting). In particular, one mutation (601A>C) caused slower fermentation kinetics in the nitrogen-limited natural must but did not affect the ethanol produced or the amount of sugars consumed, in addition to producing more glycerol and consuming less phenylalanine, which could have a positive impact on the organoleptic properties of the wine produced and therefore an industrial potential. To our knowledge, this is the first work linking RRT5 to fermentative phenotypes, and the genetic variants validated could have industrial potential for the wine industry.

Nitrogen limitations in the grape must be the main cause of stuck fermentations during the winemaking process. In Saccharomyces cerevisiae , a genetic segment known as region A, which harbors 12 protein-coding genes, was acquired horizontally from a phylogenetically distant yeast species. This region is mainly present in the genome of wine yeast strains, carrying genes that have been associated with nitrogen utilization. Despite the putative importance of region A in yeast fermentation, its contribution to the fermentative process is largely unknown. In this work, we used a wine yeast strain to evaluate the contribution of region A to the fermentation process. To do this, we first sequenced the genome of the wine yeast strain using long-read sequencing and determined that region A is present in a single copy. We then implemented an optogenetic system in this wine yeast strain to precisely regulate the expression of each gene, generating a collection of 12 strains that allow for light-activated gene expression. To evaluate the role of these genes during fermentation, we assayed this collection using microculture and fermentation experiments in synthetic must with varying amounts of nitrogen concentration. Our results show that changes in gene expression for genes within this region can impact growth parameters and fermentation rate. We additionally found that the expression of various genes in region A is necessary to complete the fermentation process and prevent stuck fermentations under low nitrogen conditions. Altogether, our optogenetics-based approach demonstrates the importance of region A in completing fermentation under nitrogen-limited conditions. IMPORTANCE: Stuck fermentations due to limited nitrogen availability in grape must represent one of the main problems in the winemaking industry. Nitrogen limitation in grape must reduces yeast biomass and fermentation rate, resulting in incomplete fermentations with high levels of residual sugar, undesired by-products, and microbiological instability. Here, we used an optogenetic approach to demonstrate that expression of genes within region A is necessary to complete fermentations under low nitrogen availability. Overall, our results suggest that region A is a genetic signature for adaptation to low nitrogen conditions.

The target of rapamycin complex 1 (TORC1) is a highly conserved protein complex whose primary function is to link nutrient availability to cell growth in eukaryotes, particularly nitrogen sources. It was originally identified during the screening of Saccharomyces cerevisiae strains resistant to rapamycin treatment. For its part, S. cerevisiae is well known for being a key model organism in biological research and an essential microorganism for the fermentation of food and beverages. This yeast is widely distributed in nature, with domesticated and wild strains existing. However, little is known about what effects domestication has had on its different phenotypes; for example, how nitrogen sources are sensed for TORC1 activation and what impact domestication has had on TORC1 activation are questions that still have no complete answer. To study the genetic basis of TORC1 activation associated with domestication through approaches such as quantitative trait loci (QTL) mapping or genome-wide association studies (GWAS), and more generally for any study requiring TORC1 activity as a readout for a large number of individuals, it is necessary to have a high-throughput methodology that allows monitoring the activation of this pathway in numerous yeast strains. In this context, the present protocol was designed to assess phenotypical differences in TORC1 activation using a new reporter plasmid, the pTOMAN-G plasmid, specifically designed to monitor TORC1 activation. As a proof of concept, this methodology allowed phenotyping a large population of yeast strains derived from the 1002 Yeast Genomes Project, the most complete catalog of genetic variation in yeasts. This protocol proved to be an efficient alternative to assess TORC1 pathway activation compared to techniques based on immunoblot detection, which, although effective, are considerably more laborious. Briefly, the protocol involves the design and construction of the pTOMAN-G plasmid, which carries a construct containing the firefly luciferase gene (Luc) under the control of the TORC1-regulated RPL26A gene promoter (P_RPL26A ). The protocol then details the process for selecting subgroups of yeasts based on their ability to grow under nutrient-limited conditions, using proline as the sole nitrogen source. These yeasts are then transformed with the TOMAN-G plasmid, using two alternative transformation methods. Finally, those yeasts that emit luminescence are selected, whose phenotype for TORC1 activation is measured by a nitrogen-upshift experiment in microculture. This approach, using the pTOMAN-G plasmid, offers a rapid and consistent method for assessing TORC1 signaling pathway activation in a large number of yeast strains, highlighting its usefulness to study the activation of the TORC1 pathway and the domestication process associated with it. In the future, a redesign of the plasmid could extend its use as a reporter tool to monitor the activation of the TORC1 pathway, or other pathways, in other yeast species.

Domestication can be understood as a symbiotic relationship that benefits both domesticator and domesticated species, involving multiple genetic changes that configure the phenotype of the domesticated species. One of the most important domesticated species is the yeast Saccharomyces cerevisiae, with both domesticated strains used for different fermentations processes for thousands of years and wild strains existing only in environments without human intervention; however, little is known about the phenotypic effects associated with its domestication. In the present work, we studied the effect of domestication on yeast TORC1 activation, a pleiotropic signalling pathway conserved across the eukaryotic domain. To achieve this goal, we improved a previously generated methodology to assess TORC1 activation, which turned out to be as effective as the original one but also presents several practical advantages for its application (such as facilitating confirmation of transformants and putting the Luc reporter gene under the control of the same P_RPL26A promoter for each transformed strain). We then generated a mapping population, the so-called TOMAN-G population, derived from the “1002 Yeast Genomes Project” population, the most comprehensive catalogue of the genetic variation in yeasts. Finally, strains belonging to the TOMAN-G population were phenotyped for TORC1 activation, and then we compared the results obtained between yeast strains with different ecological origins, finding differences in TORC1 activation between wild and domesticated strains, particularly wine strains. These results are indicative of the effect of domestication on TORC1 activation, specifically that the different evolutionary trajectories of wild and domesticated strains have in fact caused differences in the activation of this pathway; furthermore, the phenotypic data obtained in this work could be used to continue underlying the genetic bases of TORC1 activation, a process that is still not fully understood, using techniques such as GWAS to search for specific genetic variants underlying the observed phenotypic variability and phylogenetic tree inferences to gain insight into the evolutionary relationships between these genetic variants.

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.

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.