A physiological portrait of the non-conventional wine yeast Hanseniaspora guilliermondii guided by genomic analyses
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2022-11-15
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Atualmente, os consumidores procuram vinhos com uma complexidade de sabor distinta que pode ser conferida por leveduras não-Saccharomyces. As espécies de levedura Hanseniaspora, incluindo H. guilliermondii, são abundantes em mosto de uvas e desempenham um papel crucial na modulação do perfil sensorial do vinho. Apesar disso, a genética e fisiologia das leveduras Hanseniaspora continuam pouco estudadas.
Neste trabalho é apresentado o genoma de uma estirpe de H. guilliermondii (UTAD222) e as perspetivas que o mesmo fornece. Uma reconstrução metabólica revelou que a levedura H. guilliermondii não possui gluconeogénese nem um ciclo do glioxilato funcionais, assim como não contém enzimas essenciais para catabolismo de glicerol ou galactose, nem para biossíntese de biotina ou tiamina. Uma análise comparativa entre H. guilliermondii, H. uvarum, H. opuntiae e Saccharomyces cerevisiae permitiu identificar 14 genes específicos de H. guilliermondii e 870 proteínas encontradas apenas nos proteomas de Hanseniaspora.
Apesar da capacidade da estirpe H. guilliermondii UTAD222 para produzir elevados níveis de acetato de etilo e acetato de 2-pheniletilo durante a fermentação de sumo de uva natural, uma análise comparativa de aminoácidos não identificou ortólogos das proteínas codificadas pelos genes ATF1 e ATF2, envolvidos na formação de ésteres de acetato em S. cerevisisae. Porém, foram encontradas 4 proteínas contendo motivos conservados da família das álcool acetiltransferases (HgAATs) e foi estabelecida uma correlação positiva entre a expressão de genes HgAAT e a produção de acetato de 2-pheniletilo. Embora mais estudos sejam necessários, estas correlações implicam as proteínas HgAAT na biossíntese de ésteres de acetato, fornecendo dados que permitem clarificar o seu papel bioquímico e a sua função ainda pouco conhecida.
Diferenças na disponibilidade de azoto e açúcar exercem efeitos opostos nos perfis de acumulação de ésteres de acetato entre S. cerevisiae e H. guilliermondii. Enquanto um rácio de carbono-azoto (C/A) leva a uma maior produção em S. cerevisiae, é um menor rácio C/A que resulta numa maior produção de álcoois superiores e ésteres de acetato em H. guilliermondii, indicando uma possível diferença regulatória na produção destes compostos. Variações no conteúdo de amónia também parecem afetar a sua biossíntese em H. guilliermondii, pelo que entender o impacto de fontes de azoto na produção de aromas é crucial para gerir a sua disponibilidade e beneficiar por completo das características metabólicas de H. guilliermondii na caracterização do estilo do vinho.
Posteriormente estudou-se a preferência de utilização de azoto na estirpe H. guilliermondii UTAD222. Foi verificado o efeito de diferentes aminoácidos e amónia, fornecidos como fonte única de azoto ou em conjunto, na produção de biomassa e na cinética de crescimento, seguido do estudo da ordem de assimilação de cada composto numa mistura de fontes de azoto em mosto sintético. Foram encontras preferências semelhantes a S. cerevisiae para a maioria dos compostos quando disponibilizados como única fonte de azoto, mas a ausência de consumo de amónia em H. guilliermondii representa uma diferença chave. Foi ainda determinado que apenas aminoácidos classificados como “boa” fonte de azoto reprimem o consumo de amónia.
O impacto do etanol em H. guilliermondii foi também investigado através de uma análise transcritómica durante a resposta a stress ao etanol (6% v/v). Foi encontrada uma ativação de um número significativo de genes envolvidos em vias energéticas, assim como a repressão de genes envolvidos em respiração. O metabolismo de lípidos foi particularmente afetado, evidenciando-se pela ativação ou repressão, parcial ou total, de várias das suas vias metabólicas, podendo representar uma adaptação ao nível da membrana celular. Estes dados oferecem indicações importantes para clarificar a adaptação de H. guilliermondii ao etanol, permitindo esclarecer possíveis razões para a sua menor tolerância comparando com S. cerevisiae.
Este trabalho mostra pela primeira vez uma visão integrada da resposta adaptativa de uma estirpe de H. guilliermondii ao ambiente desafiante das fermentações. Uma análise transcritómica no início do processo fermentativo revelou que a presença de S. cerevisiae resulta na expressão diferencial de um elevado número de genes em H. guilliermondii, envolvidos no metabolismo de azoto e remodelação de membrana. A expressão de genes de floculação é também induzida, possivelmente de forma a aumentar a capacidade de trocas metabólicas com S. cerevisiae. Foi ainda determinado que S. cerevisiae acelera a resposta ao stress de H. guilliermondii, assim como aparenta estimular uma resposta típica de limitação de azoto, possivelmente devido à competição por nutrientes.
Em conclusão, os dados apresentados nesta tese providenciam conhecimento valioso acerca da fisiologia e genética da levedura não-Saccharomyces, H. guilliermondii UTAD222. Espera-se que estes estudos contribuam para acelerar a investigação em leveduras da espécie Hanseniaspora, permitindo aumentar as suas aplicações na indústria vínica e igualmente noutras bio-indústrias em que possam ser exploradas como fábricas celulares.
Nowadays, there is a broadly recognized consumer demand for wines with differentiated flavour complexity achieved by non-Saccharomyces yeasts activity. Hanseniaspora species, including H. guilliermondii, are known to be abundant in wine grape-musts and to play a critical role in modulating the wine sensory profile. Nonetheless, the genetics and physiology of Hanseniaspora species remains poorly understood. In this work, the first genomic sequence of a H. guilliermondii strain (UTAD222) and the perspectives it unlocks are presented. Metabolic reconstruction revealed that H. guilliermondii is not equipped with a functional gluconeogenesis or glyoxylate cycle, it does not harbour key enzymes for glycerol or galactose catabolism, nor for biotin and thiamine biosynthesis. Comparative analysis involving H. guilliermondii, H. uvarum, H. opuntiae and Saccharomyces cerevisiae allowed the identification of 14 H. guilliermondii-specific genes, and a set of 870 proteins only found within the Hanseniaspora proteomes. Given the recognized ability of H. guilliermondii UTAD222 strain to produce high levels of ethyl acetate and 2-phenylethyl acetate during fermentation of natural grape-juice, we aimed to identify genes putatively involved in aroma formation. Surprisingly, amino acid comparative analysis failed to detect orthologues of ATF1- and ATF2-encoded proteins involved in acetate ester formation in S. cerevisiae. Nevertheless, four proteins harbouring conserved alcohol acetyltransferase family (HgAATs) motifs, were found. A significant positive correlation between expression of HgAAT genes and 2-phenylethyl acetate production was established. Although additional studies are required, these correlations implicate HgAATs in acetate ester biosynthesis, thus providing important clues to elucidate their yet elusive biochemical role and function. Furthermore, differential nitrogen and sugar availability was found to exert opposite effects on acetate ester accumulation profiles between S. cerevisiae and H. guilliermondii. While a higher carbon-to-nitrogen (C/N) ratio leads to higher acetate ester production by S. cerevisiae, it is a lower C/N ratio that stimulates higher alcohol and acetate ester production in H. guilliermondii, highlighting a possible regulatory difference in the production of these compounds. Also, variability in ammonium content appears to impact, to some extent, the biosynthetic pathway of higher alcohol and acetate ester in H. guilliermondii. Therefore, understanding the impact of nitrogen sources on aroma production is crucial to rationally manage nitrogen nutrition and to fully benefit from H. guilliermondii metabolic traits in shaping wine style. We proceeded to study nitrogen utilization preferences of H. guilliermondii UTAD222. The impact of different amino acids and ammonium, provided as sole nitrogen sources or in combination, on biomass and growth kinetics was investigated, followed by examining the assimilation order of each compound in a mixture of nitrogen sources during fermentation of synthetic must. Although we found similar preferences to S. cerevisiae for the majority of nitrogen compounds when provided as the sole nitrogen source, the non-consumption of ammonia by H. guilliermondii represents a crucial difference. Moreover, we discovered that only amino acids classified as “good” nitrogen sources seem to repress ammonium consumption. The impact of ethanol on H. guilliermondii cells was also investigated by transcriptomic analysis during response to short-term ethanol stress (6% v/v). The presence of ethanol caused activation of a significant number of genes involved in energy metabolism, while repressing genes involved in respiration. Moreover, lipid metabolism was particularly affected by ethanol, either through the activation or shutdown of partial and entire pathways, possibly representing membrane adaptation. Altogether, these findings provide important clues to elucidate H. guilliermondii adaptation to ethanol, and to understand its lower ethanol tolerance compared to S. cerevisiae. For the first time, this work also provided an integrated view into the adaptive responses of a H. guilliermondii strain to the challenging environment of fermentations. Transcriptomic analysis uncovered a large set of differentially expressed genes associated with the presence of S. cerevisiae during fermentation, namely, stimulation of nitrogen metabolism and membrane remodelling events. Moreover, flocculation genes were induced in H. guilliermondii, possibly as a way to increase the likelihood of metabolic exchange with S. cerevisiae. S. cerevisiae was found to accelerate H. guilliermondii response to stress and seems to elicit a typical nitrogen limitation response in this yeast, as a result of competition for nutrients between both species. In sum, the data presented in this thesis provides valuable insights on the physiology and genetics of the non-Saccharomyces yeast H. guilliermondii UTAD222. This data is expected to accelerate research focused on Hanseniaspora species and to broaden their application in the wine industry and other bio-industries in which they could be explored as cell factories.
Nowadays, there is a broadly recognized consumer demand for wines with differentiated flavour complexity achieved by non-Saccharomyces yeasts activity. Hanseniaspora species, including H. guilliermondii, are known to be abundant in wine grape-musts and to play a critical role in modulating the wine sensory profile. Nonetheless, the genetics and physiology of Hanseniaspora species remains poorly understood. In this work, the first genomic sequence of a H. guilliermondii strain (UTAD222) and the perspectives it unlocks are presented. Metabolic reconstruction revealed that H. guilliermondii is not equipped with a functional gluconeogenesis or glyoxylate cycle, it does not harbour key enzymes for glycerol or galactose catabolism, nor for biotin and thiamine biosynthesis. Comparative analysis involving H. guilliermondii, H. uvarum, H. opuntiae and Saccharomyces cerevisiae allowed the identification of 14 H. guilliermondii-specific genes, and a set of 870 proteins only found within the Hanseniaspora proteomes. Given the recognized ability of H. guilliermondii UTAD222 strain to produce high levels of ethyl acetate and 2-phenylethyl acetate during fermentation of natural grape-juice, we aimed to identify genes putatively involved in aroma formation. Surprisingly, amino acid comparative analysis failed to detect orthologues of ATF1- and ATF2-encoded proteins involved in acetate ester formation in S. cerevisiae. Nevertheless, four proteins harbouring conserved alcohol acetyltransferase family (HgAATs) motifs, were found. A significant positive correlation between expression of HgAAT genes and 2-phenylethyl acetate production was established. Although additional studies are required, these correlations implicate HgAATs in acetate ester biosynthesis, thus providing important clues to elucidate their yet elusive biochemical role and function. Furthermore, differential nitrogen and sugar availability was found to exert opposite effects on acetate ester accumulation profiles between S. cerevisiae and H. guilliermondii. While a higher carbon-to-nitrogen (C/N) ratio leads to higher acetate ester production by S. cerevisiae, it is a lower C/N ratio that stimulates higher alcohol and acetate ester production in H. guilliermondii, highlighting a possible regulatory difference in the production of these compounds. Also, variability in ammonium content appears to impact, to some extent, the biosynthetic pathway of higher alcohol and acetate ester in H. guilliermondii. Therefore, understanding the impact of nitrogen sources on aroma production is crucial to rationally manage nitrogen nutrition and to fully benefit from H. guilliermondii metabolic traits in shaping wine style. We proceeded to study nitrogen utilization preferences of H. guilliermondii UTAD222. The impact of different amino acids and ammonium, provided as sole nitrogen sources or in combination, on biomass and growth kinetics was investigated, followed by examining the assimilation order of each compound in a mixture of nitrogen sources during fermentation of synthetic must. Although we found similar preferences to S. cerevisiae for the majority of nitrogen compounds when provided as the sole nitrogen source, the non-consumption of ammonia by H. guilliermondii represents a crucial difference. Moreover, we discovered that only amino acids classified as “good” nitrogen sources seem to repress ammonium consumption. The impact of ethanol on H. guilliermondii cells was also investigated by transcriptomic analysis during response to short-term ethanol stress (6% v/v). The presence of ethanol caused activation of a significant number of genes involved in energy metabolism, while repressing genes involved in respiration. Moreover, lipid metabolism was particularly affected by ethanol, either through the activation or shutdown of partial and entire pathways, possibly representing membrane adaptation. Altogether, these findings provide important clues to elucidate H. guilliermondii adaptation to ethanol, and to understand its lower ethanol tolerance compared to S. cerevisiae. For the first time, this work also provided an integrated view into the adaptive responses of a H. guilliermondii strain to the challenging environment of fermentations. Transcriptomic analysis uncovered a large set of differentially expressed genes associated with the presence of S. cerevisiae during fermentation, namely, stimulation of nitrogen metabolism and membrane remodelling events. Moreover, flocculation genes were induced in H. guilliermondii, possibly as a way to increase the likelihood of metabolic exchange with S. cerevisiae. S. cerevisiae was found to accelerate H. guilliermondii response to stress and seems to elicit a typical nitrogen limitation response in this yeast, as a result of competition for nutrients between both species. In sum, the data presented in this thesis provides valuable insights on the physiology and genetics of the non-Saccharomyces yeast H. guilliermondii UTAD222. This data is expected to accelerate research focused on Hanseniaspora species and to broaden their application in the wine industry and other bio-industries in which they could be explored as cell factories.
Descrição
Este trabalho foi expressamente elaborado com vista à obtenção de grau de Doutor em Genética Molecular Comparativa
Palavras-chave
Leveduras vínicas não-Saccharomyces , Hanseniaspora guilliermondii