The domestication of plants, animals and microbes has transformed our diet and represents a lever for adapting to future global changes. While the domestication of plants and animals is the subject of much research, that of microorganisms remains poorly understood. IDOK proposes to study the domestication of two yeast species, Kazachstania bulderi and Kazachstania humilis, frequently found in bread-making sourdoughs and belonging to a little-known yeast genus. These two species occur frequently in French sourdoughs and are supposed to have had different histories and selection pressures because the first one is found mainly in bakers with farmer practices, while the second one is encountered mainly in artisanal bakers. We propose to take advantage of past projects where we collected a unique collection of strains of these two species to study their genetic and phenotypic diversity. Through a population genomics approach, we will study their past evolution and search for domestication signatures. With an experimental evolutionary approach, we also want to analyze their short-term adaptation dynamics and better understand their potential, alone or together, to respond to different resources in a bread-making sourdough environment. This project will provide new knowledge on the evolutionary dynamics of yeasts, but also characterize biological resources and develop innovative strategies for the scientific community and the bakery sector. Overall, the results should help to establish recommendations for the development of a more sustainable bakery sector.

Facing climate change, the development of winegrowing practices means taking up major challenges from the vineyard to the winemaking process, while ensuring consistent production quality. In vineyards, grapevine roots are the soil-plant interface and develop close associations with microbial communities, forming the root microbiome that plays an important role in nutrition and confers stress tolerance to plant. In viticulture, nitrogen (N) fertilization influences vigor, plant biomass production and grape composition, but its influence on root microbiota remains unknown. The agroecological transition of viticultural practices may affect soil N availability and in consequence lead to an imbalance of root microbiome, grapevine N nutrition, grape berry quality and by extension some changes in fermentation kinetics and wine flavors. In the INFINITY project, the composition of root microbial communities associated with the main grapevine rootstock in a network of 50 parcels of the Cognac region will be characterized, taking care of soil properties and technical itineraries. Then, 6 parcels will be selected having contrasted soil N availability and microbial communities to functionally analyze root microbial communities, but also the plant response by evaluating indicators of the root physiology, the grape and must quality, and the vinification and aromatic profile of wine distillate. The three objectives, along the N continuum, are to (i) understand on how soil N availability influence the continuum root-grape berry, from the composition and functioning of the root microbiome to the grapevine response, (ii) optimize N availability in must through agroecological practices while guaranteeing high-quality aromatic profile of wine, and (iii) develop recommendations for good winemaking practices, from soil to alcoholic fermentation management, adapted to Cognac vineyard to meet the challenges of climate change.

Complex phenotypes are regulated by multiple interacting quantitative trait loci (QTLs). Dissection of the genetic mechanisms underlying the phenotypic variations remains a major conceptual and experimental challenge, due to the complex genetic architecture with many loci contributing to phenotypic effects, low penetrance, gene-gene and gene-environment interactions. Over the past years, the budding yeast Saccharomyces cerevisiae has become an important model. This success is partially due to its intrinsic biological features, such as its short sexual generation time, high meiotic recombination rate, and small genome size. Precise reverse genetics technologies offer the unique opportunity to experimentally measure the phenotypic effect of genetic variants. Furthermore, intensive efforts have provided the genome sequence of numerous S. cerevisiae isolates and related species allowing powerful comparative genomics and new perspectives for functional and evolutionary studies. So far, nearly all of the yeast QTL mapping studies have used classical F1 meiotic segregants but novel strategies to produce powerful mapping population is needed. This proposal provides an innovative approach to link complex yeast phenotypes with high-resolution mapping of the genetics factors. We will implement an innovative strategy, named return to growth (RTG) that is rapid and does not require classical crosses to generate a large mapping population. This method takes advantage of the natural capacity of S. cerevisiae, to reverse its meiotic progression and yield diploid cells. Most importantly, our preliminary data, based on Next Generation Sequencing (NGS) of a yeast polymorphic strain, demonstrate that the process of RTG gives rise to recombined diploid cells. The repair of the meiotic DSBs upon gene conversion or crossover resolution leads to the maintenance or loss of heterozygosity (LOH) in various ratios and locations. Cells derived from RTG are all genetically different, a resource for quantitative trait analyses and mapping. Our preliminary phenotypic analysis of few multi-factorial traits (colony morphology, sporulation efficiency, growth at high temperature) revealed quantitative variation, providing a proof of concept for using the RTG process in QTL mapping. To optimize and utilize this approach for laboratory and industrial yeast strains, we will collaborate to: (i) isolate, NGS and bioinformatically determine the allelic genotype of 96 RTG diploids of the S288c/SK1 hybrid strain as well as 96 strains derived from spores, (ii) perform high throughput phenotypic analyses of recombined strains for multiple traits, map and validate the QTLs and, (iii) develop a genetic system that allows one to follow the progress of the meiotic cycle and the frequency of LOH in a large variety of strains. We will generate several sets of diploid recombined strains for identifying QTLs of medical and biotechnological relevance. Importantly, we will test if RTG is able to produce recombinant hybrids between different reproductively isolated Saccharomyces species. These experiments will allow us to understand the contribution of RTG to genome structure evolution, where inter-specific introgressions are prevalent in the Saccharomyces species. We envisage that the RTG process is likely to have profound implications in terms of genome evolution, since yeasts have to survive in fluctuating environments where they must also undergo rapid diversification. This scenario offers an attractive alternative to the full meiotic cycle where sequence divergence and chromosomal rearrangements can impair the process, resulting in poor gamete viability and therefore reducing the individual fitness. This ambitious project will bring a new dimension to QTL mapping, strengthen budding yeast as a model organism for quantitative genetics and provide a GMO-free innovation to improve the performance of industrial yeast strains.

Sustaining a growing population while preserving nature is challenging, especially when climates are increasingly unpredictable. Wine is one of the main commodities of the French economy, accounting for 1.3% of total exports in 2012 (5.6 billion euros of wine exports). In the context of global change and a highly competitive international market, maintaining high production levels of fine wine while limiting chemical inputs will require biological innovations. Such breakthroughs are likely to arise from experimenting with the grape must’s microbial community, a neglected actor of winemaking which impacts many wine characteristics. Much has yet to be learned about the main wine yeast, Saccharomyces cerevisiae, which can still fail to carry out alcoholic fermentation to fruition, despite modern microbiological techniques aimed at controlling fermentations. Although there is evidence that S. cerevisiae carries specific adaptations for growing in grape must, its overall level of adaptation to grape must is unknown. This matter can only be resolved with selection experiments, which assess the effect of new genetic variation on adaptation. We propose to study the adaptive potential of S. cerevisiae in natural grape must, including their microbial communities. We will analyze the impact of i) different amount of standing genetic variation and ii) experimental horizontal gene transfers (HGT) on yeast adaptation to different biotic and abiotic environments. These two genetic mechanisms of adaptation (standing genetic variation and HGT) correspond to previously described modes of adaptation to grape must by S. cerevisiae. Specifically, adaptation in S. cerevisiae will be studied in a factorial selection experiment involving five microbial community treatments (T. delbrueckii, H. uvarum, both species, whole microbial community, or sterile grape must) and three recombinant populations of S. cerevisiae (wine strains alone, wine strains and closely related domesticated strains, or strains from all known S. cerevisiae sub-groups). The competitive fitness, fermentation traits, and genomics of adaptation will be studied in the evolved populations. The prevalence of HGT as a mechanism of adaptation to grape musts will be studied among non-Saccharomyces yeasts isolated from natural grape musts, as well as with experimental evolution in S. cerevisiae. Two strategies of experimental HGT will be implemented to test if new HGT can be beneficial for S. cerevisiae in grape must. Our findings will be applicable to oenological research because all experiments will be performed in natural grape must using species and strains isolated from that environment, instead of using single-clones with undefined evolutionary history in standard laboratory media (typical for microbial evolutionary experiments). This innovative approach will allow us to test if previous laboratory studies are transposable to complex natural environments. Our work can impact wine starter technologies by providing new alleles, allele combinations for improving wine technological traits (i.e. fermentation completion). Better knowledge of the ecological niche of wine yeasts, including the effect of biotic interactions on S. cerevisiae growth and evolution, will help stabilize alcoholic fermentations. Moreover, experimental horizontal gene transfer (eHGT) has never been studied as extensively as in our project. Our results will increase dramatically our knowledge of the prevalence and functions of horizontal gene transfers. Sequencing genomes from species isolated from grape must’s microbial communities will fill a major gap in our knowledge of these communities, including the importance of HGT. The project will benefit from our broad consortium of experts because it requires the integration of new challenging approaches (eHGT, experimental evolution in complex environments) and advanced tools (phenomics robotic platform, genomics).

Enzyme reactions have long been analyzed in vitro, using pure enzymes and diluted buffer conditions. Due to the large amount of data generated and collected with thousands of enzymes, enzymology has made tremendous progress on understanding the incredible power of biocatalysts. However, dilute, in vitro conditions are far from the surroundings of natural enzymatic reactions that take place inside cells. The cellular medium is more accurately described as a heterogeneous crowded gel, dense and filled with all sorts of macromolecules and cellular lipidic organelles which may result in some partitioning effects and changes in diffusion. Therefore, enzymatic parameters determined using classical enzymology setups may not perfectly represent the real, in vivo based, rate and equilibrium constants. Although some advances have been made toward the comprehension of viscosity and crowding effects, we are still far to derive rate and equilibrium parameters from in vivo enzymatic reactions. The ENZINVIVO project will combine the most up-to-date and advanced techniques in enzymology, synthetic biology, systems biology and theoretical modelling to setup an advanced system capable of mimicking substrate and enzyme variation in classical enzymology. To exemplify our approach, we will use two isoforms of phytoene synthase (carotenoid biosynthesis) as model enzymes for several reasons. First, carotenoid biosynthesis pathway can be reconstituted in microorganisms such as yeast which will allow the use of genetic engineering tools. Second, phytoene synthase is the pivotal enzyme in the carotenoid pathway, converting the natural soluble precursor geranylgeranyl pyrophosphate (GGPP) into phytoene, the first lipophilic carotenoid molecule. This enzyme is therefore perfectly suited to study a complex enzymatic step for which the cell ultrastructure is of crucial importance. Our project will then consist of a series of increasing complexity experiments, combining simple to complex in vitro experiments, in vivo modulation of substrate and enzyme concentration and analyzing the biological data with mathematical models integrating the critical parameters mentioned in the previous paragraph. In vitro experiments, performed in media of increasing complexity (viscosity, crowding and partial cell structures) will generate pseudo enzymatic constants integrating the complexity factors. Metabolic engineering, by providing advanced techniques to smoothly control gene expression, will allow the construction of a set of yeast strains with: (1) defined (but variables) amounts of GGPP, mimicking the variation of substrate concentration; (2) variable amounts of phytoene synthase, mimicking the variation of enzyme concentration; and (3) absence of presence of the full enzymatic pathway, mimicking the displacement of the thermodynamic equilibrium. In vivo experiments will be performed using the state-of-the-art techniques in systems biology (parallelized chemostats, fast gene regulation, 13C labelling, metabolomics, fluxomics and mathematical/statistical analysis) to determine the in vivo phytoene synthase enzymatic parameters. Although the results of our project will be derived from a single enzymatic reaction, the developed tools and methods will be quite generic and therefore will serve as a first demonstration that synthetic biology and genetic engineering can trigger new developments in enzymology, challenging the in vitro conceptual field and ultimately proposing new concepts for studying the complexity of enzymatic reactions inside the cell. Furthermore our approach will define the optimal concentrations of substrate and enzymes to achieve for maximum efficiency and perfect homeostasis of the synthetic and natural metabolic pathways. These metabolic engineering tools should then be useful for the scientific community to improve the production, by microorganisms, of natural and synthetic molecules.