Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts.

BACKGROUND

Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains.

RESULTS

It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production.

CONCLUSIONS

Overall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.