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

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

The yeast protein Gdt1p transports Mn ions and thereby regulates manganese homeostasis in the Golgi.

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis. Using Mn-induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn influx, in addition to its previously reported regulation of Ca influx. The estimated KM values of Gdt1p of 15.6 ± 2.6 μM for Ca and 83.2 ± 9.8 μM for Mn indicated that Gdt1p has a higher affinity for Ca than for Mn In yeast cells, we found that Gdt1p is involved in the resistance to high Mn concentration and controls total Mn stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation

Deciphering Biochemical and Biophysical Properties of UPF0016 Membrane Proteins – A Link to Human Congenital Disorders of Glycosylation - Poster

Congenital Disorders of Glycosylation (CDG) comprise a group of rare inborn human diseases caused by defects in protein glycosylation. Recently, a subtype of CDG has been associated with mutations within the human protein TMEM165. This protein belongs to a family of poorly characterized membrane proteins (UPF0016) which is highly conserved through evolution and widely distributed among kingdoms. Recent results indicate that the UPF0016 proteins play a role in calcium, manganese and pH homeostasis. We have shown the S. cerevisiae UPF0016 member, Gdt1p, to be localized at the Golgi membrane, and to act as calcium and manganese transporter. Bioinformatic analysis of the UPF0016 family predicts three structural states formed through evolution: i) single-domain proteins with 3 transmembrane spans (TMD) which form homodimers, ii) single-domain proteins with 3 TMD (encoded by two adjacent genes on the chromosome) which form heterodimers, or iii) two-domains proteins with 6 TMD. To gain better insight into the conformational topologies of the three evolution states we selected seven genes from different UPF0016 subfamilies: TMEM165 (human), Gdt1 (S. cerevisiae), and five prokaryotic members, Ter1a and Ter2b (Trichodesmium erythraeum), which are predicted to form heterodimers, Dma (Desulfovibrio magneticus), which is predicted to form homodimers, and two (hyper)thermophilic genes from the archea Thermococcus gammatolerans and Pyrococcus furiosus. The codon-optimized synthetic genes were expressed in E. coli and L. lactis. By in vivo transport assays using the fluorescent dye Fura-2 we studied the calcium and manganese transport activity. Furthermore, purification of the proteins is being optimized for reconstitution into liposomes and in vitro transport assays. Altogether, a better understanding of the enzymatic activity, physiological role and structural aspects of the UPF0016 members will enable a better comprehension of the causal link between the development of CDG and the presence of mutations in the human gene TMEM165

The human UPF0016 ortholog TMEM165 shows transport activity towards Ca2+ and Mn2+ ions - Poster

Congenital Disorders of Glycosylation (CDG) comprise a group of rare inborn human diseases caused by defects in protein glycosylation. Recently, a subtype of CDG has been associated with mutations within the human protein TMEM165 (1). This protein belongs to a family of poorly characterized membrane proteins (UPF0016) which is highly conserved through evolution and widely distributed among kingdoms (2). Recent results indicate that the UPF0016 proteins play a role in calcium, manganese and pH homeostasis. We have shown the S. cerevisiae UPF0016 member, Gdt1p, to be localized at the Golgi membrane, and to act as calcium and manganese transporter (3, 4). However, no direct transport of calcium and/or manganese by the human ortholog TMEM165 has been reported so far. The codon-optimized synthetic gene was expressed in L. lactis and by in vivo transport assays using the fluorescent dye Fura-2 we studied the calcium and manganese transport activity of TMEM165. Additionally, we performed site-directed mutagenesis in order to introduce the specific mutations detected in TMEM165-CDG patients and we tested whether this mutations directly affects the transport activity. Altogether, a better understanding of the enzymatic activities, physiological role and structural aspects of the UPF0016 members will enable a better comprehension of the causal link between the development of CDG and the presence of mutations in the human gene TMEM16

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO fixation. Changes in free Ca in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca fluxes in the chloroplasts have been unknown. By employing a Ca reporter-based approach, we identified two chloroplast-localized Ca transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca signal in the chloroplast stroma. BICAT2 mediated Ca uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca ] signal. Conversely, this Ca transient was increased in knockout mutants of BICAT1, which transports Ca into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca transporters