Biocontrol of Brettanomyces/Dekkera bruxellensis in alcoholic fermentations using saccharomycin-overproducing Saccharomyces cerevisiae strains
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Microbial contamination of alcoholic fermentation processes (e.g. winemaking and fuel-ethanol production) is a serious problem for the industry since it may render the product unacceptable and/or reduce its productivity, leading to large economic losses. Brettanomyces/Dekkera bruxellensis is one of the most dangerous microbial contaminant of ethanol industrial fermentations. In the case of wine, this yeast species can produce phenolic compounds that confer off-flavours to the final product. In fuel-ethanol fermentations, D. bruxellensis is a persistent contaminant that affects ethanol yields and productivities. We recently found that Saccharomyces cerevisiae secretes a biocide, which we named saccharomycin, composed of antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Saccharomycin is active against several wine-related yeast species, namely D. bruxellensis. However, the levels of saccharomycin naturally secreted by S. cerevisiae during alcoholic fermentation are not sufficient to ensure the complete death of D. bruxellensis. Therefore, the aim of the present work was to construct genetically modified S. cerevisiae strains to overproduce these GAPDH-derived AMPs. The expression levels of the nucleotides sequences encoding the AMPs were evaluated in the modified S. cerevisiae strains by RT-qPCR, confirming the success of the recombinant approach. Furthermore, we confirmed by immunological tests that the modified S. cerevisiae strains secreted higher amounts of the AMPs by comparison with the non-modified strain, inducing total death of D. bruxellensis during alcoholic fermentations.
KeywordsAntimicrobial peptides, glyceraldehyde 3-phosphate dehydrogenase Microbial contamination Genetically-modified yeasts Wine Preservatives Fuel-ethanol Bioethanol
The authors thank Fundação para a Ciência e Tecnologia for financial support of this work through the project PTDC/BII-BIO/31761/2017 and also through the Research Units “Linking Landscape, Environment, Agriculture and Food” (LEAF) and “Unidade de Ciências Biomoleculares Aplicadas” (UCIBIO). P. Branco was the recipient of a PhD fellowship (SFRH/ BD/ 89673/ 2012) funded by FCT, Portugal.
The present work was financed by FEDER funds through POFC-COMPETE in the scope of project FCOMP-01-0124-FEDER-014055 and by national funds through Fundação para a Ciência e Tecnologia (FCT) in the scope of project PTDC/BII-BIO/31761/2017 and also through the research units LEAF (UID/AGR/04129/2013) and UCIBIO (UID/Multi/04378/2013). P. Branco received a PhD fellowship (SFRH/ BD/ 89673/ 2012) funded by FCT, Portugal.
Compliance with ethical standards
Conflict of interest
The authors declare they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Albergaria H, Branco P, Francisco D, Coutinho R, Monteiro M, Malfeito-Ferreira M, Arneborg N, Almeida MG, Caldeira J (2013) Dominance of Saccharomyces cerevisiae in wine fermentations: secretion of antimicrobial peptides and microbial interactions. In: Gallego JB, Cardinalli G, Casella S, Cocolin L, Neviani E (eds) Proceedings of the 2nd international conference on microbial diversity: microbial interactions in complex ecosystems. Società Italiana di Microbiologia Agraria-Alimentare e Ambientale, Firenze, pp 98–101Google Scholar
- Barros Pita W, Leite FCB, Souza Liberal AT, Simoes DA, Morais MA (2011) The ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes. Antonie Van Leeuwenhoek 100:99–107. https://doi.org/10.1007/s10482-011-9568-z CrossRefGoogle Scholar
- Branco P, Francisco D, Chambon C, Hébraud M, Arneborg N, Almeida MG, Caldeira J, Albergaria H (2014) Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions. Appl Microbiol Biotechnol 98:843–853. https://doi.org/10.1007/s00253-013-5411-y CrossRefGoogle Scholar
- Branco P, Francisco D, Monteiro M, Almeida MG, Caldeira J, Arneborg N, Prista C, Albergaria H (2017a) Antimicrobial properties and death-inducing mechanisms of saccharomycin, a biocide secreted by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 101:159–171. https://doi.org/10.1007/s00253-016-7755-6 CrossRefGoogle Scholar
- Branco P, Kemsawasd V, Santos L, Diniz M, Caldeira J, Almeida MG, Arneborg N, Albergaria H (2017b) Saccharomyces cerevisiae accumulates GAPDH-derived peptides on its cell surface that induce death of non-Saccharomyces yeasts by cell-to-cell contact. FEMS Microbiol Ecol 93:fix055. https://doi.org/10.1093/femsec/fix055 CrossRefGoogle Scholar
- Delgado ML, O'Connor JE, Azorín I, Renau-piqueras J, Gil ML, Gozalbo D (2001) The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the genes Saccharomyces cerevisiae TDH1, TDH2 and TDH3 are also cell wall proteins. Microbiol 147:411–417. https://doi.org/10.1099/00221287-147-2-411 CrossRefGoogle Scholar
- Geitz RD, Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269Google Scholar
- Ribéreau-Gayon P, Dubourdieu D, Donèche B, Lonvaud A (2006) The use of sulfur dioxide in must and wine treatment. In: Ribéreau-Gayon P, Dubourdieu D, Donèche B, Lonvaud A (eds) Handbook of Enology: The Microbiology of Wine and Vinifications. John Wiley and Sons Ltd., Chichester, pp 193–221CrossRefGoogle Scholar
- Romano P, Suzzi G (1993) Sulfur dioxide and wine microorganisms. In: Fleet GH (ed) Wine microbiology and biotechnology. Harwood Academic Publishers, Chur, pp 373–393Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2th edn). Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Souza Liberal AT, Basilio ACM, Resende AD, Brasileiro BTV, da Silva EA, de Morais JOF, Simoes DA, de Morais MA (2007) Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel-ethanol fermentation. J Appl Microbiol 102:538–547. https://doi.org/10.1111/j.1365-2672.2006.03082.x CrossRefGoogle Scholar