Please use this identifier to cite or link to this item: http://hdl.handle.net/10348/9785
Title: Tracking the genes and pathways involved in Saccharomyces cerevisiae response and tolerance to the wine preservatives S02 and chitosan: from molecular analysis to OMICS approaches
Authors: Lage, Patricia Pinheiro
Advisor: Ferreira, Ana Alexandra Mendes
Mira, Nuno Pereira
Keywords: S02 and chitosan
Com2 (YER130C)
Issue Date: 20-Dec-2019
Abstract: Sulfur dioxide (S02) is widely used as a preservatives in winemaking to control the activity of undesired spoilage species. The emergence of highly tolerant strains, from different species, have been leading to a persistent increase in the concentrations of S02 used, a practice that has adverse effects for the health of more susceptible consumers, beside exerting pressure for selection of more tolerant strains, and may impact activity of Saccharomyces cerevjsjae, the leading species used to conduct the vinification process. Evidences obtained by others (and reinforced by results described herein) show that S. cerevjsjae commercial strains used for wine production exhibit considerable phenotypic heterogeneity concerning tolerance to S02. The high tolerance is usually attributed to increased basal expression of the SSUJ efflux pump as the result of genomic rearrangements that exchange the endogenous SSUJ promoter for the stronger ECM34promoter (VIII-t-XVI translocation). Nonetheless, it is known that some S02-tolerant strains do not exhibit this trait sustaining the idea that other mechanisms can be involved in S. cerevjsjae adaptation to S02-induced stress. Exploring a combination of transcriptomics and genome-wide phenotypic analysis it is herein demonstrated that the poorly characterized transcription factor Com2 (ORF YER130c) is essential for response and tolerance of the laboratory strain S. cerevjsjae BY 4 7 41 to S02 at the enologically relevant pH of 3.5. Com2 was found to control, directly or indirectly, the expression of more than 80% of the S02-induced genes. Around fifty of the S02-responsive genes up-regulated by Com2 were found to provide protection against this chemical including all the genes that compose the sulfate reduction pathway (SULJ, SUL2, MET3, MET14, MET16, METS and METJO) and genes required for biosynthesis of lysine (LYS20, LYS21, LYS4, LYS2, LYS9 and LYSJ) or arginine (ARG2, ARG3, ARG7, ARG4, ARGS/6, CPAJ and ARGBJ). Not only this was the first biological function attributed to Com2 until so far, but this also opened the range of molecular responses to S02 in S. cerevjsjae, besides up-regulation of SSUJ. Phenotyping of a cohort of S. cerewsjae commercial strains for their tolerance to S02 reinforced the previously observed heterogeneity, the higher tolerance exhibited by strains BM45, AWRI R2, VIN13 and T73 being attributable to these strains harboring a VIIl-t-XVI translocation, homozygous for the first two strains and heterozygous for the exhibited by VL1, VIN13 or T73) and showing increased basal expression of SSU1, we could not detect any alteration of the SSU1 endogenous promoter. In strain QA23, also exhibiting high tolerance to S02, no over-expression of SSU1 was observed leaving open the possibility of Com2 playing a role in determining response and tolerance of this strain to S02. Besides the above-mentioned disadvantages of S02 in the winemaking context, the increasing pressure of consumers for "green" products has been paving the way for the search of alternatives. Chitosan emerges as one of the more interesting alternatives. In this work we have examined the effect of a commercially available form of chitosan (No Brett Inside®) in growth of S. cerevisiae and of the spoilage species Zygossacharomyces bailii, Saccharomycodes Judwigii and Brettanomyces bruxellensis. Under the experimental conditions tested chitosan showed a strong inhibitory potential against S. Judwigii and, less significantly, Z. bailii, at concentrations were no significant effects were detected in S. cerevisiae growth. To better understand the chitosan-yeast cells interaction, a chemogenomic analysis was performed. The results obtained sustain previous evidences indicating that the main target of chitosan is the cellular envelope, namely, the structure of the cell wall and plasma membrane. However, critical players in control of S. cerevisiae response to chitosan were identified in our screening such as all the members of the Rim101 pathway. Around 207 mutants exhibited higher tolerance to chitosan than wild-type cells including mutants devoid encoding of enzymes of the ergosterol biosynthetic pathway or of proteins required for assembly and function of the vacuolar ATPase. Further studies are required to better understand why the deletion of these genes result in increased tolerance to chitosan, however, the overall combination of results described in this chapter might help to understand the differential levels of tolerance exhibited by the spoilage species. In sum, the results gathered in this work provide a broad and integrated view of the molecular mechanisms of tolerance to sulfur dioxide and chitosan, opening an auspicious future in the improvement of the fermentation process and the quality of wines obtained. remaining. Remarkably, for strain UCD522, also highly tolerant to S02 (similar to the one
Sulfur dioxide (S02) is widely used as a preservatives in winemaking to control the activity of undesired spoilage species. The emergence of highly tolerant strains, from different species, have been leading to a persistent increase in the concentrations of S02 used, a practice that has adverse effects for the health of more susceptible consumers, beside exerting pressure for selection of more tolerant strains, and may impact activity of Saccharomyces cerevjsjae, the leading species used to conduct the vinification process. Evidences obtained by others (and reinforced by results described herein) show that S. cerevjsjae commercial strains used for wine production exhibit considerable phenotypic heterogeneity concerning tolerance to S02. The high tolerance is usually attributed to increased basal expression of the SSUJ efflux pump as the result of genomic rearrangements that exchange the endogenous SSUJ promoter for the stronger ECM34promoter (VIII-t-XVI translocation). Nonetheless, it is known that some S02-tolerant strains do not exhibit this trait sustaining the idea that other mechanisms can be involved in S. cerevjsjae adaptation to S02-induced stress. Exploring a combination of transcriptomics and genome-wide phenotypic analysis it is herein demonstrated that the poorly characterized transcription factor Com2 (ORF YER130c) is essential for response and tolerance of the laboratory strain S. cerevjsjae BY 4 7 41 to S02 at the enologically relevant pH of 3.5. Com2 was found to control, directly or indirectly, the expression of more than 80% of the S02-induced genes. Around fifty of the S02-responsive genes up-regulated by Com2 were found to provide protection against this chemical including all the genes that compose the sulfate reduction pathway (SULJ, SUL2, MET3, MET14, MET16, METS and METJO) and genes required for biosynthesis of lysine (LYS20, LYS21, LYS4, LYS2, LYS9 and LYSJ) or arginine (ARG2, ARG3, ARG7, ARG4, ARGS/6, CPAJ and ARGBJ). Not only this was the first biological function attributed to Com2 until so far, but this also opened the range of molecular responses to S02 in S. cerevjsjae, besides up-regulation of SSUJ. Phenotyping of a cohort of S. cerewsjae commercial strains for their tolerance to S02 reinforced the previously observed heterogeneity, the higher tolerance exhibited by strains BM45, AWRI R2, VIN13 and T73 being attributable to these strains harboring a VIIl-t-XVI translocation, homozygous for the first two strains and heterozygous for the exhibited by VL1, VIN13 or T73) and showing increased basal expression of SSU1, we could not detect any alteration of the SSU1 endogenous promoter. In strain QA23, also exhibiting high tolerance to S02, no over-expression of SSU1 was observed leaving open the possibility of Com2 playing a role in determining response and tolerance of this strain to S02. Besides the above-mentioned disadvantages of S02 in the winemaking context, the increasing pressure of consumers for "green" products has been paving the way for the search of alternatives. Chitosan emerges as one of the more interesting alternatives. In this work we have examined the effect of a commercially available form of chitosan (No Brett Inside®) in growth of S. cerevisiae and of the spoilage species Zygossacharomyces bailii, Saccharomycodes Judwigii and Brettanomyces bruxellensis. Under the experimental conditions tested chitosan showed a strong inhibitory potential against S. Judwigii and, less significantly, Z. bailii, at concentrations were no significant effects were detected in S. cerevisiae growth. To better understand the chitosan-yeast cells interaction, a chemogenomic analysis was performed. The results obtained sustain previous evidences indicating that the main target of chitosan is the cellular envelope, namely, the structure of the cell wall and plasma membrane. However, critical players in control of S. cerevisiae response to chitosan were identified in our screening such as all the members of the Rim101 pathway. Around 207 mutants exhibited higher tolerance to chitosan than wild-type cells including mutants devoid encoding of enzymes of the ergosterol biosynthetic pathway or of proteins required for assembly and function of the vacuolar ATPase. Further studies are required to better understand why the deletion of these genes result in increased tolerance to chitosan, however, the overall combination of results described in this chapter might help to understand the differential levels of tolerance exhibited by the spoilage species. In sum, the results gathered in this work provide a broad and integrated view of the molecular mechanisms of tolerance to sulfur dioxide and chitosan, opening an auspicious future in the improvement of the fermentation process and the quality of wines obtained. remaining. Remarkably, for strain UCD522, also highly tolerant to S02 (similar to the one maior tolerancia exibida pelas estirpes BM45, AWRI R2, VIN13 e T73 e atribuida ao facto de estas estirpes possuirem uma translocac;ao homozig6tica, para as primeiras duas estirpes, e heterozig6tica para as restantes. Notavelmente, para a estirpe UCD522, corn elevada tolerancia ao S02 (semelhante a exibida pela VL1, VIN13 ou T73), e corn uma expressao basal de SSUJ elevada, nao foi possivel detectar nenhuma alterac;ao do promotor end6geno do SSU1. Na estirpe QA23, tambem corn elevada tolerancia ao S02, nao foi observada uma super-expressao do SSUJ, deixando em aberto a possibilidade do COM2 desempenhar um papel na determinac;ao da resposta e tolerancia desta estirpe ao S02. Alem das desvantagens do S02 no contexto da vinificac;ao, acima mencionadas, a crescente pressao dos consumidores por produtos "verdes" tern vindo a abrir caminhos para a procura de alternativas. A quitosana surge como uma das alternativas mais interessantes. Neste trabalho, examinamos o efeito de um tipo de quitosana comercialmente disponivel (No Brett inside®) no crescimento de leveduras de S. cerevisjae e em especies de contaminac;ao de Zygossacharomyces bailii, Saccharomycodes Judwigii e Brettanomyces bruxellensis. Nas condic;oes experimentais testadas neste trabalho, a quitosana mostrou um forte potencial inibit6rio contra S. Judwigii e, menos significativa em Z. bailii, para concentrac;oes onde nao foram detectados efeitos significativos no crescimento de S. cerevisiae. Para compreender melhor a interacc;ao entre a quitosana e as celulas de levedura, foi realizada uma analise quimogen6mica. Os resultados obtidos sustentam evidencias previas indicando que o principal alvo da quitosana e o envelope celular, nomeadamente a estrutura da parede celular e a membrana plasmatica. No entanto, elementos importantes no controlo da resposta da S. cerevisiae a quitosana foram identificados no nosso "screening", tais como todos os membros da via Rim101. Cerca de 207 mutantes exibiram maior tolerancia a quitosana do que as celulas da estirpe selvagem, incluindo mutantes sem enzimas de codificac;ao da via biossintetica do ergosterol ou de proteinas necessarias para a montagem e func;ao da ATPase vacuolar. Mais estudos serao necessarios de modo a entender o porque de a exclusao desses genes resultar numa maior tolerancia a quitosana; no entanto a combinac;ao geral dos resultados descritos neste capitulo pode ajudar a perceber os diferentes niveis de tolerancia exibidos pelas especies de contaminac;ao. Em suma, os resultados obtidos neste trabalho fornecem uma visao ampla e integrada dos mecanismos moleculares de tolerancia ao di6xido de enxofre e a quitosana, abrindo um futuro auspicioso na melhoria do processo de fermentac;ao e na qualidade dos vinhos obtidos.
Description: Tese de Doutoramento em Genética Molecular Comparativa apresentada à Universidade de Trás-os-Montes e Alto Douro
URI: http://hdl.handle.net/10348/9785
Document Type: Doctoral Thesis
Appears in Collections:DGB - Teses de Doutoramento
TD - Teses de Doutoramento

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