RESUMEN
The use of non-conventional yeast species to obtain interesting flavors and aromas has become a new trend in the fermented beverages industry. Among such species, Brettanomyces bruxellensis (B. bruxellensis) has been reported as capable of producing desirable or at least singular aromas in fermented beverages like beer and wine. However, this yeast can also produce an aromatic defect by producing high concentrations of phenolic compounds like, 4-ethylguaiacol and particularly 4-ethylphenol (4-EP). In the present study, we designed a mutant screening method to isolate B. bruxellensis mutants with reduced 4-EP production. More than 1000 mutants were screened with our olfactory screening method, and after further sensory and chemical analysis we were able to select a B. bruxellensis mutant strain with a significant reduction of 4-EP production (more than threefold) and less phenolic perception. Notably, the selected strain also showed higher diversity and concentration of ethyl esters, the most important group of odor active compounds produced by yeasts. Based on these results, we consider that our selected mutant strain is a good candidate to be tested as a non-conventional yeast starter (pure or in co-inoculation) to obtain wines and beers with novel aromatic properties.
Asunto(s)
Brettanomyces/genética , Brettanomyces/aislamiento & purificación , Mutagénesis , Fenoles/metabolismo , Cerveza/análisis , Fermentación , Microbiología de Alimentos , Odorantes/análisis , Gusto , Compuestos Orgánicos Volátiles/análisis , Vino/análisisRESUMEN
The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d-xylose and l-arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d-xylose and l-arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l-arabinose was not consumed under oxygen limitation, d-xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d-xylose and l-arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.
Asunto(s)
Arabinosa/metabolismo , Brettanomyces/metabolismo , Etanol/metabolismo , Xilosa/metabolismo , Aerobiosis , Biomasa , Brettanomyces/genética , Brettanomyces/crecimiento & desarrollo , Medios de Cultivo/metabolismo , FermentaciónRESUMEN
Wine production is an important commercial issue for the liquor industry. The global production was estimated at 275.7 million hectoliters in 2015. The loss of wine production due to Brettanomyces bruxellensis contamination is currently a problem. This yeast causes a "horse sweat" flavor in wine, which is an undesired organoleptic attribute. To date, 6 B. bruxellensis annotated genome sequences are available (LAMAP2480, AWRI1499, AWRI1608, AWRI1613, ST05.12/22, and CBS2499), and whole genome comparisons between strains are limited. In this article, we reassembled and reannotated the genome of B. bruxellensis LAMAP2480, obtaining a 27-Mb assembly with 5.5 kb of N50. In addition, the genome of B. bruxellensis LAMAP2480 was analyzed in the context of spoilage yeast and potential as a biotechnological tool. In addition, we carried out an exploratory transcriptomic analysis of this strain grown in synthetic wine. Several genes related to stress tolerance, micronutrient acquisition, ethanol production, and lignocellulose assimilation were found. In conclusion, the analysis of the genome of B. bruxellensis LAMAP2480 reaffirms the biotechnological potential of this strain. This research represents an interesting platform for the study of the spoilage yeast B. bruxellensis.
Asunto(s)
Brettanomyces/genética , Brettanomyces/metabolismo , Genoma Fúngico , Biotecnología , Mapeo Cromosómico , Microbiología de Alimentos , Genes Bacterianos , Genes Fúngicos , Lignina/metabolismo , Proteómica , Secuenciación Completa del Genoma , Vino/microbiologíaRESUMEN
Volatile phenols are aromatic compounds produced by some yeasts of the genus Brettanomyces as defense against the toxicity of hydroxycinnamic acids (p-coumaric acid, ferulic acid and caffeic acid). The origin of these compounds in winemaking involves the sequential action of two enzymes: coumarate decarboxylase and vinylphenol reductase. The first one converts hydroxycinnamic acids into hydroxystyrenes, which are then reduced to ethyl derivatives by vinylphenol reductase. Volatile phenols derived from p-coumaric acid (4-vinylphenol and 4-ethylphenol) have been described as the major contributors to self-defeating aromas associated with stable, gouache, wet mouse, etc., which generates large economic losses in the wine industry. The gene responsible for the production of 4-vinylphenol from p-coumaric acid has been identified as PAD1, which encodes a phenylacrylic acid decarboxylase. PAD1 has been described for many species, among them Candida albicans, Candida dubliniensis, Debaryomyces hansenii and Pichia anomala. In Brettanomyces bruxellensis LAMAP2480, a 666 bp reading frame (DbPAD) encodes a coumarate decarboxylase. Recent studies have reported the existence of a new reading frame belonging to DbPAD called DbPAD2 of 531 bp, which could encode a protein with similar enzymatic activity to PAD1. The present study confirmed that the transformation of Saccharomyces cerevisiae strain BY4722 with reading frame DbPAD2 under the control of the B. bruxellensis ACT1 promoter, encodes an enzyme with coumarate decarboxylase activity. This work has provided deeper insight into the origin of aroma defects in wine due to contamination by Brettanomyces spp.
Asunto(s)
Brettanomyces/enzimología , Brettanomyces/genética , Carboxiliasas/genética , Microbiología de Alimentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Carboxiliasas/metabolismo , Fenoles/metabolismo , Sistemas de Lectura/genética , Saccharomyces cerevisiae/genética , Compuestos Orgánicos Volátiles/metabolismo , Vino/microbiologíaRESUMEN
Dekkera bruxellensis is one of the main contaminating yeasts in wine due to its ability to metabolize cinnamic acids into volatile phenols. This yeast metabolizes p-coumaric acid into 4-vinylphenol through a coumarate decarboxylase (CD) and then transforms it into to 4-ethylphenol (EF) through a vinylphenol reductase. In this work we investigated the influence of the interaction between the concentration of p-coumaric acid, ferulic acid and ethanol as well as growth temperature on the production of CD activity and the expression of a putative gene that codes for this enzymatic activity. For this, a Box Behnken experimental design was used. The concentration of p-coumaric acid (5-26 ppm) and ferulic acid (3-9 ppm) alone did not show any significant effect on any of the studied response variables. However, the interaction between (ethanol concentration * cinnamic acid concentration) and (ethanol concentration * temperature) had a significant statistical effect on the production of CD activity. Additionally, a higher growth temperature negatively affected the expression of the putative cd gene and the production of CD activity. This is the first work that studies the effect of cinnamic acids on the production of CD activity and the relative expression of its putative gene, using natural concentrations of cinnamic acid found in wine.
Asunto(s)
Brettanomyces/enzimología , Brettanomyces/genética , Carboxiliasas/metabolismo , Dekkera/enzimología , Dekkera/genética , Etanol , Expresión Génica , Reacción en Cadena de la Polimerasa , Temperatura , VinoRESUMEN
AIM: To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern. METHODS AND RESULTS: CD and VR activities were quantified, as well, the growth rate, intracellular protein profile and molecular analysis (RAPD) were determined in 12 isolates of D. bruxellensis. All the isolates studied showed CD activity, but only some showed VR activity. Those isolates with the greatest growth rate did not present a different protein profile from the others. The FASC showed a relationship between RAPD molecular patterns and VR activity. CONCLUSION: CD activity is common to all of the D. bruxellensis isolates. This was not the case with VR activity, which was detected at a low percentage in the analysed micro-organisms. A correlation was observed between VR activity and the RAPD patterns. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that quantifies the CD and VR enzyme activities in D. bruxellensis, demonstrating that these activities are not present in all isolates of this yeast.