This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following their weight loss over the time. This dispositive was coupled with robotized enzymatic assays for measuring metabolites of enological interest in natural grape juices. Despite the small volume used, kinetic parameters and fermentation end products measured are similar with those observed in larger scale vats. The vessel used also offers the possibility to assay 32 volatiles compounds using a headspace solid-phase micro-extraction coupled to gas chromatography and mass spectrometry. The vessel shaking applied strongly impacted most of the phenotypes investigated due to oxygen transfer occuring in the first hours of the alcoholic fermentation. The impact of grape must and micro-oxygenation was investigated illustrating some relevant genetic x environmental interactions. By phenotyping a wide panel of commercial wine starters in five grape juices, broad phenotypic correlations between kinetics and metabolic end products were evidentiated. Moreover, a multivariate analysis illustrates that some grape musts are more able than others to discriminate commercial strains since some are less robust to environmental changes.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.