Genetic and molecular basis of the aroma production in S. kudriavzevii, S. uvarum and S. cerevisiae

En la presente tesis nos hemos centrado en el estudio del papel de las especies S. uvarum y S. kudriavzevii en la síntesis de aromas y como principal aplicación en la elaboración de vinos. En estudios anteriores, estas dos especies, estrechamente relacionadas con S.cerevisiae, mostraron diferencias notables durante la producción de alcoholes superiores y ésteres cuando se comparaban con S. cerevisiae (Gamero et al., 2013; Pérez-Torrado et al., 2015). Los alcoholes superiores y ésteres formados por las levaduras, son componentes claves en el sabor y el aroma de los productos fermentados. Tal y como hemos mencionado previamente, S. kudriavzevii y S. uvarum presentan diferencias muy significativas en la formación de estos compuestos aromáticos al comparar con S. cerevisiae. Por lo tanto, el principal objetivo de la presenten tesis fue profundizar en la comprensión de los aspectos moleculares básicos responsables de las estas diferencias. En la primera parte de la tesis se estudia cómo las tres especies del género Saccharomyces difieren en la producción de aromas a partir de los aminoácidos precursores de dichos compuestos. Este objetivo se llevó a cabo mediante el crecimiento de cepas de S. cerevisiae, S. uvarum y S. kudriavzevii usando como única fuente de nitrógeno, los correspondientes aminoácidos de forma individual, y siguiendo la producción de alcoholes superiores y ésteres de acetato. El análisis global de los resultados obtenidos en este capítulo indicó que S. kudriavzevii produce una mayor cantidad de alcoholes superiores, mientras que S. uvarum se caracteriza por ser la especie que produce mayor cantidad de ésteres de acetato. En concreto, S. uvarum es la especie que produce las mayores cantidades de acetato de 2-feniletilo y S. kudriavzevii del precursor de dicho acetato, el 2-feniletanol. Los resultados obtenidos en este capítulo nos indican diferencias importantes en el metabolismo de los aminoácidos y la producción de alcoholes superiores, así como de ésteres de acetato entre las tres especies del género Saccharomyces, estrechamente relacionadas. A continuación decidimos explorar las divergencias aminoacídicas en 23 enzimas ortólogos que participan en la producción de alcoholes superiores y ésteres de acetato en las especies S.kudriavzevii o S. uvarum, comparadas con las secuencias de S. cerevisiae. Para alcanzar este objetivo se realizó un estudio in silico de un alineamiento múltiple de secuencias y un posterior análisis de las distancias de Grantham, basadas en las diferencias de las propiedades físico-químicas de los reemplazamientos aminoácidos (Grantham, 1974; Li et al., 1984). El análisis reveló que tres secuencias mostraron valores de Grantham significativamente más elevados: la 2-cetoácido descarboxilasa codificada por el gen ARO10, y dos alcohol acetil transferasas, codificadas por los genes ATF1 y ATF2. Los estudios llevados a cabo en la última parte de la tesis se diseñaron para evaluar el efecto de forma individual de los alelos ARO10, ATF1 y ATF2 de las especies S. kudriavzevii y S. uvarum, en la producción de alcoholes superiores y ésteres de acetato respectivamente. En estos capítulos también nos centramos en evaluar las propiedades cinéticas y de especificidad por sustrato de las respectivas enzimas. La expresión individual de los alelos ARO10, ATF1 y ATF2 procedentes de S. kudriavzevii y S. uvarum en una cepa de S. cerevisiae donde se habían disrumpido dichos genes, mostraron diferencias en la producción de alcoholes superiores y ésteres de acetato. Todos los datos descritos y analizados en este trabajo indican que las variaciones de aminoácidos observadas entre las descarboxilasas codificadas por los genes ortólogos ARO10 y entre las AATases codificadas por los genes ortólogos ATF1 y ATF2 podrían ser la razón de las diferencias enzimáticas observadas, y por tanto también podrían ser la razón de la mejora en el aroma de los vinos al fermentar con las especies S. kudriavzevii y S. uvarum respecto a S. cerevisiae.The species of the genus Saccharomyces, S. kudriavzevii and S. uvarum revealed significant differences during the formation of fermentative aroma as compared to S. cerevisiae. The main goal of this PhD thesis was to obtain a deeper fundamental understanding of the molecular aspects behind these differences. First part of the thesis describes observed differences among Saccharomyces kudriavzevii and Saccharomyces uvarum during the production of aroma-active higher alcohols and acetate esters using their amino acidic precursors comparing to Saccharomyces cerevisiae. Subsequent in silico comparative analysis of the enzymes involved in the branched-chain amino acids catabolism in S. cerevesiae, S. kudriavzevii and S. uvarum released ARO10, ATF1 and ATF2 as the candidates for further experiments. The heterologous expression of the individual ARO10, ATF1 and ATF2 alleles in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. All together the data described and discussed in this thesis indicate that the amino acid variations observed between the decarboxylases encoded by the orthologues ARO10 genes and between the AATases encoded by the orthologues ATF1 and ATF2 genes could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The main objects of the research presented in this dissertation were S. kudriavzevii and S.uvarum. In previous studies these two species, closely related to S. cerevisiae, showed remarkable differences during the production of higher alcohols and esters when comparing the three species (Gamero et al., 2013; Pérez-Torrado et al., 2015). Higher alcohols and esters formed by yeast belong to key components of overall flavour and aroma in the fermented products. As already mentioned, S. kudriavzevii and S. uvarum revealed significant differences during the formation of these important flavour-active compounds as compared to S. cerevisiae. The main goal of this PhD thesis was to obtain a deeper fundamental understanding of the molecular aspects behind these differences. First part of the thesis investigates how the three Saccharomyces species differ during the production of higher alcohols and acetate esters that derived directly from their amino acidic precursors. This objective was fulfilled by carrying out cultivations of S.cerevisiae, S. uvarum and S. kudriavzevii strains with individual amino acids as the sole nitrogen source, followed by analysis and comparison of the produced higher alcohols and acetate esters. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S.uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. Altogether, the data discussed in Chapter 1 indicated differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among these closely related Saccharomyces species. Subsequently, in silico comparative analysis of the enzymes involved in the branched-chain amino acids catabolism in S. cerevesiae, S. kudriavzevii and S. uvarum was performed in order to explore the amino acid divergences among the orthologous sequences. In silico screening was based on multiple sequence alignments and the Grantham scoring. The Grantham scoring provides a quantitative assessment of the biochemical distances between amino acids, on the basis of their composition, polarity and molecular volume, and according to increasing dissimilarity classifies the amino acid substitutions as conservative or radical (Grantham, 1974; Li et al., 1984). The analysis released three sequences that were evaluated with significantly higher Grantham scores: 2-keto acid decarboxylase encoded by ARO10, and two alcohol acetyltransferases encoded by ATF1 and ATF2. The last part of the thesis then investigates the impact of the selected genes on the production of higher alcohols and acetate esters, and the catalytic properties of the corresponding enzymes. The heterologous expression of the individual ARO10, ATF1 and ATF2 alleles from S. kudriavzevii and S. uvarum in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. All together the data described and discussed in this thesis indicate that the amino acid variations observed between the decarboxylases encoded by the orthologues ARO10 genes and between the AATases encoded by the orthologues ATF1 and ATF2 genes could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds

Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and Their Impact on Aroma-Active Compounds Production

Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher Km for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the aroma-active acetate ester formation may be due, to some extent, to the distinct properties of Atf enzymes.This work has been supported by the European Commission FP7: Marie Curie Initial Network CORNUCOPIA no. 264717 and by CICYT grant (ref. AGL2015-67504-C3-1-R) from Ministerio de Economía y Competitividad.Peer reviewedPeer Reviewe

The use of mixed populations of Saccharomyces cerevisiae and S. kudriavzevii to reduce ethanol content in wine: limited aeration, inoculum proportions, and sequential inoculation

Saccharomyces cerevisiae is the most widespread microorganism responsible for wine alcoholic fermentation. Nevertheless, the wine industry is currently facing new challenges, some of them associate with climate change, which have a negative effect on ethanol content and wine quality. Numerous and varied strategies have been carried out to overcome these concerns. From a biotechnological point of view, the use of alternative non-Saccharomyces yeasts, yielding lower ethanol concentrations and sometimes giving rise to new and interesting aroma, is one of the trendiest approaches. However, S. cerevisiae usually outcompetes other Saccharomyces species due to its better adaptation to the fermentative environment. For this reason, we studied for the first time the use of a Saccharomyces kudriavzevii strain, CR85, for co-inoculations at increasing proportions and sequential inoculations, as well as the effect of aeration, to improve its fermentation performance in order to obtain wines with an ethanol yield reduction. An enhanced competitive performance of S. kudriavzevii CR85 was observed when it represented 90% of the cells present in the inoculum. Furthermore, airflow supply of 20 VVH to the fermentation synergistically improved CR85 endurance and, interestingly, a significant ethanol concentration reduction was achieved

Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.The help of Dr. P. Ergang with the real-time PCR experiments is gratefully acknowledged. We thank O. Zimmermannova for critical reading of the paper. This work was supported by the following grants: Grant Agency of the Czech Republic P503/ 10/0307, institutional concept RVO:6798582, Grant Agency of the Charles University 299611/2011/B-Bio/PrF, an Lifelong Learning Programme ERASMUS practical placement grant and by Fundo Europeu de Desenvolvimento Regional – Programa Operacional de Fatores de Competitividade – COMPETE and by national funds from Fundação para a Ciência e Tecnologia through the project PEstC/BIA/UI4050/ 2011.info:eu-repo/semantics/publishedVersio

Saccharomyces kudriavzevii and Saccharomyces uvarum differ from Saccharomyces cerevisiae during the production of aroma-active higher alcohols and acetate esters using their amino acidic precursors

Higher alcohols and acetate esters are important flavour and aroma components in the food industry. In alcoholic beverages these compounds are produced by yeast during fermentation. Although Saccharomyces cerevisiae is one of the most extensively used species, other species of the Saccharomyces genus have become common in fermentation processes. This study analyses and compares the production of higher alcohols and acetate esters from their amino acidic precursors in three Saccharomyces species: Saccharomyces kudriavzevii, Saccharomyces uvarum and S. cerevisiae. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S. uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. The present data indicate differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among the closely related Saccharomyces species. This knowledge will prove useful for developing new enhanced processes in fragrance, flavour, and food industries.This work has been supported by the European Commission FP7: Marie Curie Initial Training Network CORNUCOPIA no. 264717.Peer Reviewe

From the Uncharacterized Protein Family 0016 to the GDT1 family: Molecular insights into a newly-characterized family of cation secondary transporters

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn2+, with an extra reported function in Ca2+ and/or H+ transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca2+ signaling in yeast, and Mn2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles

Yeast as a Tool for Deeper Understanding of Human Manganese-Related Diseases.

The biological importance of manganese lies in its function as a key cofactor for numerous metalloenzymes and as non-enzymatic antioxidant. Due to these two essential roles, it appears evident that disturbed manganese homeostasis may trigger the development of pathologies in humans. In this context, yeast has been extensively used over the last decades to gain insight into how cells regulate intra-organellar manganese concentrations and how human pathologies may be related to disturbed cellular manganese homeostasis. This review first summarizes how manganese homeostasis is controlled in yeast cells and how this knowledge can be extrapolated to human cells. Several manganese-related pathologies whose molecular mechanisms have been studied in yeast are then presented in the light of the function of this cation as a non-enzymatic antioxidant or as a key cofactor of metalloenzymes. In this line, we first describe the Transmembrane protein 165-Congenital Disorder of Glycosylation (TMEM165-CDG) and Friedreich ataxia pathologies. Then, due to the established connection between manganese cations and neurodegeneration, the Kufor-Rakeb syndrome and prion-related diseases are finally presented

The human Golgi protein TMEM165 transports calcium and manganese in yeast and bacterial cells

Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi transmembrane protein 165 (TMEM165), belonging to uncharacterized protein family 0016 (UPF0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca2+- and Mn2+-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis. Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively auto-regulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca2+ and Mn2+ transporters abrogates Ca2+- and Mn2+-induced growth defects, excessive Mn2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca2+ and Mn2+ influxes. We also used the yeast and bacterial systems to evaluate theimpact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca2+ and Mn2+, which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photo-synthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H + ions are transported in exchange for Ca 2+ and Mn 2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+ , Mn2+ or H + gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H + ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H + transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts