Nachla Rojas-Torres

Fungal nitrogen metabolism is a central determinant of yeast growth, fermentative performance, and secondary metabolism in fungi associated with fermented environments. For Saccharomyces cerevisiae, the assimilation of nitrogen sources found in grape must, including ammonium, free amino acids, and peptides, has been widely characterized, establishing a consumption hierarchy in which proline is considered a non-preferential nitrogenous source. However, the regulation and hierarchy of nitrogen-source utilization in non-conventional fungi remain poorly characterized, particularly in yeasts adapted to anthropized fermentative niches. In this context, Brettanomyces bruxellensis represents a useful fungal model to study the different nitrogen sources that are used, strain-dependent physiology, and survival under nutrient-limited fermentative conditions. We hypothesized that apparent depletion of amino acid and the expression of selected amino acid permease genes are strain-dependent traits linked to the survival of B. bruxellensis. In this study, we evaluated the strain-dependent physiology and amino acid depletion patterns of two B. bruxellensis strains from wine (LAMAP2480 and LAMAP1359). In both cases, the highest apparent depletion values under the 20 amino acid condition were observed for arginine, glutamine, tryptophan and proline, indicating that these compounds contributed substantially to the organic nitrogen pool under the assayed conditions. Only LAMAP2480 isolate was able to grow in the condition without added amino acids, indicating strain and nitrogen source dependence under limited organic nitrogen availability. Transcriptional analysis of GAP1, GNP1, and TAT1 permeases showed expression patterns dependent on both the strain and the available nitrogen source. This is the first study to combine the evaluation of physiological performance, amino acid consumption, apparent nitrogen depletion derived from amino acids normalized to OD₆₀₀ (OD-AA-N depletion), and the transcriptional responses of selected amino acid permease genes. This knowledge may contribute to understanding the survival of B. bruxellensis in fermentation environments with limited nutritional resources.

Brettanomyces bruxellensis has been described as the main spoilage microorganism in wines due to its ability to produce volatile phenols, which negatively impact the final product’s organoleptic properties. This yeast can grow and survive in environments that are too nutritionally poor and stressful for other microorganisms, and one of the stressful conditions it can endure is the high alcohol content in wine. In this study, cell wall morphology and the expression of some genes related to its composition were characterized under increasing ethanol concentrations to establish a possible ethanol resistance mechanism. B. bruxellensis LAMAP2480 showed greater resistance to β-1,3-glucanase activity when grown in media supplemented with 5% or 10% ethanol compared with the control assay (without ethanol). Transmission electron microscopy showed no significant differences in cell wall thickness during the different adaptation stages. However, the amount of wall polysaccharides and chitin briefly increased at 1% ethanol but returned to baseline at 5% and 10%. The amount of wall-associated protein increased progressively with each increment in ethanol concentration. In addition, overexpression of the ROM2 and KNR4/SMI1 genes was observed at 10% ethanol. These results suggest that the integrity of the cell wall might play an important role in the adaptation of B. bruxellensis to an ethanol-containing medium.