Aromatic Amino Acid-Derived Compounds Induce Morphological Changes and Modulate the Cell Growth of Wine Yeast Species

Abstract

Yeasts secrete a large diversity of compounds during alcoholic fermentation, which affect growth rates and developmental processes, like filamentous growth. Several compounds are produced during aromatic amino acid metabolism, including aromatic alcohols, serotonin, melatonin, and tryptamine. We evaluated the effects of these compounds on growth parameters in 16 different wine yeasts, including non-Saccharomyces wine strains, for which the effects of these compounds have not been well-defined. Serotonin, tryptamine, and tryptophol negatively influenced yeast growth, whereas phenylethanol and tyrosol specifically affected non-Saccharomyces strains. The effects of the aromatic alcohols were observed at concentrations commonly found in wines, suggesting a possible role in microbial interaction during wine fermentation. Additionally, we demonstrated that aromatic alcohols and ethanol are able to affect invasive and pseudohyphal growth in a manner dependent on nutrient availability. Some of these compounds showed strain-specific effects. These findings add to the understanding of the fermentation process and illustrate the diversity of metabolic communication that may occur among related species during metabolic processes.

Keywords: aromatic alcohols; invasive growth; non-Saccharomyces; pseudohyphal growth; quorum sensing; serotonin; tryptamine.

FIGURE 1

Effects of aromatic amino acid-derived compounds on the growth of wine yeast species at different nitrogen concentrations. The effects of Mel, Ser, Trpm, TrpOH, PheOH, and TyrOH on the growth of four strains of S. cerevisiae and four of non-Saccharomyces were determined. Yeast were grown for 48 h at 28°C in minimal medium with two different nitrogen concentrations [10 mM or 1 mM (NH₄)₂SO₄] and supplemented with 1000 mg/L of each compound. Non-supplemented cultures were used as controls. Experiments were carried out in triplicate. Growth curves of S. cerevisiae QA23 (A) and S. bacillaris Cz4 (B), with the different compounds added at 10 mM (NH₄)₂SO₄ medium are shown. For each nitrogen condition and compound, maximal growth (C), and generation time (D) was calculated. The fold-change for each growth parameter was determined in relation to its control condition. Statistical analysis was performed using Tukey’s test by comparing the effects of each compound in the different strains; asterisk denotes a p-value < 0.05.

FIGURE 2

Effects of increasing serotonin (Ser) concentrations on yeast growth. Ser was added to minimal medium [1 mM (NH₄)₂SO₄] at increasing concentrations (50, 100, 250, 500, 750, and 1000 mg/L). (A) Growth curves obtained with S. cerevisiae Sc20. (B) Correlation between the generation time and maximal growth fold-changes obtained with different yeast species. (C) Generation time fold-change for each strain at different Ser concentrations. Statistical analysis was performed, using Tukey’s test and comparing the effects of Ser concentrations in each strain; asterisk denotes a p-value < 0.05. The fold-change for each growth parameter was determined in relation to the control (no-supplemented condition, w/o).

FIGURE 3

Effects of increasing tryptophol (TrpOH) concentrations on yeast growth. TrpOH was added to minimal medium [1 mM (NH₄)₂SO₄] at increasing concentrations (50, 100, 250, 500, 750, and 1000 mg/L). (A) Growth curves obtained with H. uvarum Hu4. (B) Correlation between the generation time and maximal growth fold-changes obtained with different yeast species. (C) Generation time fold-change for each strain at different TrpOH concentrations. Statistical analysis was performed using the Tukey’s test and comparing the effects of TrpOH concentrations in each strain; asterisk denotes a p-value < 0.05. The fold-change for each growth parameter was determined in relation to the control (no-supplemented condition, w/o).

FIGURE 4

Effects of increasing tryptamine (Trpm) concentrations on yeast growth. Trpm was added to minimal medium [1 mM (NH₄)₂SO₄] at increasing concentrations (50, 100, 250, 500, 750, and 1000 mg/L). (A) Growth curves obtained with S. cerevisiae Vin7 and M. pulcherrima FLAVIA. (B) Lag phase fold-change for each strain at different Trpm concentrations. (C) Maximal growth fold-change for each strain at different Trpm concentrations. Statistical analysis was performed comparing the effects of Trpm concentrations in each strain, using Tukey’s test statistical method; asterisk denotes a p-value < 0.05. The fold-change for each growth parameter was determined in relation to the control (no-supplemented condition, w/o).

FIGURE 5

Invasive growth phenotypes of different wine yeast species. (A) In a plate washing assay (PWA), equal concentrations of cells were spread on media with different nutrient contents and incubated for 5 days at 28°C. (B) Quantification of invasive growth was performed after washing the plate via densitometry analysis. Cells were spotted in triplicate, and the average values are shown. Statistical analysis was carried out by comparing each strain with respect to rich media (SAD), using Tukey’s test statistical method; asterisk denotes a p-value < 0.05.

FIGURE 6

Pseudohyphal growth phenotypes of different wine yeast species. Cells were spotted on rich medium (SAD) and nutrient limitation media (SALG and SLAD). Colony peripheries were photographed after incubation for 5 days at 28°C. Scale bar is 50 μm. Arrows mark examples of pseudohyphae.

FIGURE 7

Invasive growth of wine yeast species in the presence of aromatic alcohols and ethanol. (A) In a plate washing assay (PWA), equal concentrations of cells were spread on SAD, SALG, and SLAD media in the presence of the aromatic alcohol (TyrOH, PheOH, or TrpOH) at 500 μM or 2% (v/v) EtOH and incubated for 3 days at 28°C. Panel (A) shows the results from the washed plate. The invasive growth obtained with different wine yeast species in SAD (B), SALG (C), and SLAD (D) was obtained via densitometry. Cells were spotted in triplicate, and the average agar invasion values were calculated. Relative invasion values were obtained by dividing the agar invasion in presence of each compound and the one of the controls (no-supplemented condition, w/o). Statistical analysis was performed comparing the effects of the alcohols in each strain relative to the control, p-value < 0.05.

FIGURE 8

Pseudohyphal growth phenotypes of different wine yeast species in response to ethanol and aromatic alcohols. Cells were spotted on SLAD medium. Colony peripheries were photographed after incubation for 3 days at 28°C. Scale bar is 50 μm.