Browsing by Author "Mardones, Wladimir"

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    Fungal Diversity Analysis of Grape Musts from Central Valley-Chile and Characterization of Potential New Starter Cultures
    (2020) Mandakovic, Dinka; Pulgar, Rodrigo; Maldonado, Jonathan; Mardones, Wladimir; Gonzalez, Mauricio; Cubillos, Francisco A.; Cambiazo, Veronica
    Autochthonous microorganisms are an important source of the distinctive metabolites that influence the chemical profile of wine. However, little is known about the diversity of fungal communities associated with grape musts, even though they are the source of local yeast strains with potential capacities to become starters during fermentation. By using internal transcribed spacer (ITS) amplicon sequencing, we identified the taxonomic structure of the yeast community in unfermented and fermented musts of a typicalVitis viniferaL. var. Sauvignon blanc from the Central Valley of Chile throughout two consecutive seasons of production. Unsurprisingly,Saccharomycesrepresented the most abundant fungal genus in unfermented and fermented musts, mainly due to the contribution ofS. uvarum(42.7%) andS. cerevisiae(80%). Unfermented musts were highly variable between seasons and showed higher values of fungal diversity than fermented musts. Since microbial physiological characterization is primarily achieved in culture, we isolated nine species belonging to six genera of fungi from the unfermented must samples. All isolates were characterized for their potential capacities to be used as new starters in wine. Remarkably, onlyMetschnikowia pulcherrimacould co-exist with a commercialSaccharomyces cerevisiaestrain under fermentative conditions, representing a feasible candidate strain for wine production.
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    Molecular profiling of beer wort fermentation diversity across natural Saccharomyces eubayanus isolates
    (2020) Mardones, Wladimir; Villarroel, Carlos A.; Krogerus, Kristoffer; Tapia, Sebastian M.; Urbina, Kamila; Oporto, Christian I.; O'Donnell, Samuel; Minebois, Romain; Nespolo, Roberto; Fischer, Gilles; Querol, Amparo; Gibson, Brian; Cubillos, Francisco A.
    The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating 'end-to-end' genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2-methylpropanol and 3-methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.
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    Rapid selection response to ethanol in Saccharomyces eubayanus emulates the domestication process under brewing conditions
    (2022) Mardones, Wladimir; Villarroel, Carlos A.; Abarca, Valentina; Urbina, Kamila; Pena, Tomas A.; Molinet, Jennifer; Nespolo, Roberto F.; Cubillos, Francisco A.
    Although the typical genomic and phenotypic changes that characterize the evolution of organisms under the human domestication syndrome represent textbook examples of rapid evolution, the molecular processes that underpin such changes are still poorly understood. Domesticated yeasts for brewing, where short generation times and large phenotypic and genomic plasticity were attained in a few generations under selection, are prime examples. To experimentally emulate the lager yeast domestication process, we created a genetically complex (panmictic) artificial population of multiple Saccharomyces eubayanus genotypes, one of the parents of lager yeast. Then, we imposed a constant selection regime under a high ethanol concentration in 10 replicated populations during 260 generations (6 months) and compared them with propagated controls exposed solely to glucose. Propagated populations exhibited a selection differential of 60% in growth rate in ethanol, mostly explained by the proliferation of a single lineage (CL248.1) that competitively displaced all other clones. Interestingly, the outcome does not require the entire time-course of adaptation, as four lineages monopolized the culture at generation 120. Sequencing demonstrated that de novo genetic variants were produced in all propagated lines, including SNPs, aneuploidies, INDELs and translocations. In addition, the different propagated populations showed correlated responses resembling the domestication syndrome: genomic rearrangements, faster fermentation rates, lower production of phenolic off-flavours and lower volatile compound complexity. Expression profiling in beer wort revealed altered expression levels of genes related to methionine metabolism, flocculation, stress tolerance and diauxic shift, likely contributing to higher ethanol and fermentation stress tolerance in the evolved populations. Our study shows that experimental evolution can rebuild the brewing domestication process in 'fast motion' in wild yeast, and also provides a powerful tool for studying the genetics of the adaptation process in complex populations.