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Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025., The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity., This work was supported by the Strategic Programme UID/BIA/04050/2020 and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET, both funded by Portuguese funds through the FCT-IP. J.A. acknowledges the FCT and the Doctoral Program in Applied and Environmental Microbiology for the PD/BD/150584/2020 PhD grant and a COST Action CA18113 Short-Term Scientific Mission grant (EuroMicropH). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the re ference NORTE-08-5369-FSE-000060. I.S-S. was supported by the program contract FCTUMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC).

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories., This study was supported by the strategic program UID/BIA/04050/2019, funded by Portuguese funds through the FCT I.P.; the projects PTDC/BIAMIC/5184/2014, funded by national funds through the FCT I.P.; and the European Regional Development Fund (ERDF) through the COMPETE 2020–Programa Operacional Competitividade e Internacionalização (POCI) and EcoAgriFood: Innovative green products and processes to promote AgriFood BioEconomy (operação NORTE-01–0145-FEDER-000009), supported by Norte Portugal Regional Operational Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the reference NORTE-08–5369-FSE-000060, and J.A. acknowledges the FCT for the PD/BD/150584/2020 PhD grant. PS acknowledges FCT for contract CEECINST/0007772018. I.S-S. was supported by the program contract FCT-UMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC).

Organic acids are industrially relevant chemicals with application in polymer, food, agricultural and pharmaceutical sectors. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Since organic acid export over the plasma membrane represents one of the key steps in microbial production of these compounds, organic acid transporters started receiving greater attention in metabolic engineering strategies. Ato1 is the main transporter responsible for uptake of acetic acid into the cytosol in S. cerevisiae, while also being able to mediate organic acid transport in the opposite direction, as it was shown to be involved in the export of lactic acid from S. cerevisiae cells engineered for lactic acid production. Ato1 is a member of the Acetate Uptake Transporter Family (AceTR), with several functionally characterized homologues in yeast, fungi, and bacteria. Recently solved crystal structure of its bacterial homologue, SatP, depicts a hexameric anion channel. In this work, we studied the relationship between structure and function of Ato1 via rational mutagenesis and identified residues critical for Ato1 substrate specificity and transport activity. By utilizing computer-assisted three-dimensional modelling tools, we provide possible explanations of acquired features. Our final goal is to test applicability of these transporters in yeast cell factories that produce organic acids., Supported by strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and TransAcids (PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood (NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement. TC acknowledges Yeastdoc European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 764927.

Considering the global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are needed as current model of natural resource exploitation is unsustainable. Novel strategies to boost bio-based production of organic acids are based on the expression of carboxylate transporters in microbial cell factories. In this work we have focused on the identification and characterization of novel carboxylate transporters in the Cyberlindnera jadinii yeast. The transportome of C. jadinii was analysed by two approaches. First, the C. jadinii homologs of the carboxylate transporters Jen1p (Major Facilitator Superfamily) and Ady2p (AceTr Family) were identified and expressed in Saccharomyces cerevisiae. The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used for the heterologous expression. Genes were identified through sequence alignment and homology prediction. In a parallel bioinformatic approach, the proteome from C. jadinii NRRL-1542 was downloaded from NCBI database and explored using a pipeline developed together with the CBMA bioinformatic team. This tool was designed to retrieve data from a specific database: a) that contains a single representative genome/proteome on the species level; b) where multiple matches within a species directly reflect homologs within the same genome, and c) e-values from BLAST searches that are statistically more reliable. A set of genes were selected using this tool and expressed in the IMX1000 strain, which is deleted in 25 genes related to carboxylic acid transport [1]. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of several radiolabelled CAs. The full characterization of the Ady2 and Jen1 homologs as well as others candidate CAs transporters from C. jadinii is ongoing.

Carboxylic acids (CAs) are a group of organic compounds that play a central role in cellular metabolism of many organisms, using it as unique sources of carbon and energy [1]. In order to replace conventional petroleum-based methods for the obtainment of CAs, alternatives are required for more sustainable way of producing these compounds. The exploitation of yeast biodiversity has received great interest from food, pharmaceuticals and even fuels companies, due to the interesting properties of some microorganisms in producing these compounds in a “greener” trait [2]. Non-Saccharomyces yeasts, also called unconventional yeasts, have recently gained prominence in the biotech industry, and are increasingly being used for the heterologous production of valuable products [3]. New strategies for increasing the production of bio-based organic acids are based on the expression of carboxylate transporters in Saccharomyces cerevisiae strains. In this work, we focus on the identification of new carboxylate transporters present in several yeasts. The strategy involved the search of homologs to known carboxylate transporters characterized in several microorganisms from yeast, fungi and bacteria. The S. cerevisiae IMX1000 Δ25 strain, without carboxylate uptake capacity, was used as a host for the heterologous expression of putative genes encoding CAs transporters [4]. Transport activity was determined by growth phenotypes in different medias containing sole carbon and energy sources, namely mono, di and tricarboxylic acids. The full characterization of the newly identified putative CAs transporters is currently undergoing.

In nature, a wide diversity of yeast species can be found even in wastes from food industries. The exploration of this yeast biodiversity has captured great interest from food, pharmaceutical and even fuel companies due to the interesting properties of such microorganisms [1]. These microorganisms can transform sugars present in raw materials into different valuable compounds as several chemical building-blocks and biofuels, in a process more sustainable than those based on fossil fuels and refineries [2]. Within yeasts, Saccharomyces cerevisiae is considered the model organism, being the most widely used industrially for the production of added-value products [3]. Properties that range from its simple cultivation, short replication period, sporulation efficiency, easy genetic manipulation and rare pathogenicity have turned it in an ideal organism for various biotechnological processes [3]. Nevertheless, other non-Saccharomyces yeasts are being increasingly used for the heterologous production of valuable products [4]. In this work, a group of isolates from the TransBio collection (Project FP7 KBBE–Nº289603) was selected based on their ability to grow in organic acids. Microorganisms were identified by molecular typing (DNA sequencing of the ITS regions) and characterized regarding morpho- and physiological features. The morphological traits and sporulation patterns were evaluated for cell cycle determination. The selected yeasts revealed interesting physiological features regarding growth profiles using carboxylic acids as sole carbon and energy source. The full characterization of these wild yeast strains is underway.

Due to global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are required, as the current model of natural resource exploitation is unsustainable. Novel strategies to boost bio-based production of organic acids are based on the expression of carboxylate transporters in microbial cell factories. In this work we have focused on the identification and characterization of novel carboxylate transporters in the yeast Cyberlindnera jadinii. C. jadinii transportome was analysed by two approaches. First, the C. jadinii homologs of the carboxylate transporters Jen1p (Major Facilitator Superfamily) and Ady2p (AceTr Family) were identified and expressed in Saccharomyces cerevisiae. The S. cerevisiae strain W303 1A jen1Δ ady2Δ was used for the heterologous expression. Genes were identified through sequence alignment and homology prediction. In a parallel bioinformatic approach, the proteome from C. jadinii NRRL 1542 was downloaded from the NCBI database and explored using a pipeline. This tool was designed to retrieve data from a specific database: a) that contains a single representative genome/proteome on the species level; b) where multiple matches within a species directly reflect the presence of homologs within the same genome, and c) e-values from BLAST searches that are statistically more reliable [1]. A set of genes were selected using this tool and expressed in the IMX1000 strain, which is deleted in 25 genes related to carboxylic acid transport [2]. Protein expression and localization was determined by microscopy evaluation of GFP- fused transporter proteins. Transporter activity was evaluated through growth on different carbon sources and measurement of the uptake of several radiolabelled CAs. The full characterization of the Ady2 and Jen1 homologs as well as other candidate CAs transporters from C. jadinii is ongoing.

Background: The wide applicability of organic acids for direct use as commodity chemicals and as polymer building blocks has evidenced their importance in diverse types of industries. In Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates (CA) at the plasma membrane, Jen1p a monocarboxylate proton symporter (Major Facilitator Superfamily) and Ady2p an acetate permease (AceTr Family). Objectives: In Cyberlindnera jadinii, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this study, CA transporter homolog genes from C. jadinii were identified and expressed in S. cerevisiae. Methods: The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used to express C. jadinii ScADY2 homologs. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of labelled CAs, namely D,L-[U14C] lactic acid, [2,3-14C] succinic acid and [1-14C] acetic acid. Results: In C. jadinii, 4 genes homolog to ScADY2 were identified. These are functional carboxylate transporters in S. cerevisiae, localized at the plasma membrane, presenting different specificities for the mono- and di-carboxylates. Further studies are underway to fully characterize these four new plasma membrane transporters, including molecular docking of these transporters to unveil the amino acids that play a major role in the substrate binding of CAs tested., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. UMINHO/BD/25/2016

Concerning the global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are needed as the current model of natural resource exploitation is unsustainable. Cell factories with specific genetic and physiological traits, namely suitable protein transporters, may be key players in the bio-based production of organic acids, as an alternative approach to the production of these chemical building-blocks from petrochemical derivatives. The present work focused on the identification and characterization of novel organic acid transporters from the Cyberlindnera jadinii yeast. C. jadinii homologues of the monocarboxylate proton symporter Jen1p (Major Facilitator Superfamily) and the acetate permease Ady2p (AceTr Family) were identified and expressed in S. cerevisiae. The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used as an expression host. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of CAs, namely [1-14C] acetic acid, D,L-[U-14C] lactic acid and [2,3-14C] succinic acid. Molecular docking of these transporters was performed to unveil the amino acids that play a major role in the substrate binding of CAs tested. In this study, 4 CjADY2 and 6 CjJEN1 homologs were identified and revealed to be functional carboxylate transporters in S. cerevisiae. Further studies are underway to fully characterize these ten new plasma membrane transporters., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060

Novel strategies to boost bio-based production of organic acids are focused in the expression of specific transporter proteins, to improve adequate uptake and export mechanisms. This study focused on the identification and characterization of novel carboxylate (CA) transporters in the yeast Cyberlindnera jadinii. Transporters homologous to Jen1p and Ady2p, the lactate and acetate permeases from Saccharomyces cerevisiae, were identified and expressed in the S. cerevisiae W303-1A jen1Δ ady2Δ strain. Genes were identified by homology prediction and expressed under the control of a GPD constitutive promoter. GFP-fusions were used to determine protein expression and localization. The transformants were characterized physiologically by growth tests in different carbon sources, and evaluation of mediated transport systems for CAs. Molecular docking studies were performed, to unveil the residues involved in substrate binding in these transporters. We have identified 4 CjADY2 and 6 CjJEN1 homologs that are functional carboxylate transporters in S. cerevisiae. All permeases transport CAs, presenting different specificities. The full characterization of these plasma membrane transporters is underway., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060

Objectives: Chronic kidney disease (CKD) is a public health problem worldwide. Currently, the link between oral health status, dialysis modality, and dialysis vintage is still not clear. The aim of this study was to evaluate periodontal disease, dental caries, and Candida colonization among patients under hemodialysis (HD) therapy, peritoneal dialysis (PD) therapy, and PD with previous history of HD (HD/PD). Method and materials:The clinical history, smoking, and oral hygiene habits were recorded. Decayed, missing, or filled teeth (DMFT) index, Visible Plaque Index (VPI), clinical attachment level (CAL), bleeding on probing, saliva flow rate, saliva pH, and oral yeast colonization were assessed. Results: HD/PD patients were generally submitted to longer periods of dialysis therapy than the other groups. The number of decayed and filled teeth did not differ between groups; HD patients presented a higher number of teeth, but poor periodontal status. Among the three groups, HD patients presented higher VPI, CAL, and oral Candida colonization, independently of the time under dialysis therapy. Candida albicans (HD and PD), Candida krusei (HD), and Candida carpophila (PD) were isolated in these patients. Conclusion: HD presented a more adverse impact on oral health than PD, particularly periodontal disease and oral Candida colonization; however, this impact on oral health appears to be reduced or ameliorated when patients change from HD to PD therapy., LSS is supported by SFRH/BD/84837/2012 and ISS is supported by SFRH/BPD/101016/2014 from FCT/QREN-POPH/FSE. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT -Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145F-EDER-007274 and PTDC/MEC-MCI/29777/2017). This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Faculty of Dental Medicine, University of Porto, also supported this investigation.

Organic acids are industrially relevant chemicals obtainable from renewable feedstocks via microbial cell factories. Microbially produced organic acids have a wide variety of applications, including bioplastic synthesis. Thus, they possess the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for microbial production of organic acids, namely due to their tolerance of low pH environments. Such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in low pH conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Our current objective is to deepen the knowledge on organic acid transporters from the AceTR family (1,2,3). We performed functional characterization by studying transporter kinetics, energetics and specificity as well as site-directed mutagenesis to acquire insight into the structural features of transporters. Finally, we aim to improve organic acid production in S. cerevisiae cell factories via expression of engineered AceTR transporters with altered activity and substrate specificity., UID/BIA/04050/2013(POCI-01-0145-FEDER-007569) and TransAcids(PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood(NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement.TCacknowledgesYeastdocEuropean Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curie grant agreement No 764927

Organic acids are industrially relevant building-block chemicals obtainable from renewable feedstocks by utilization of microbial cell factories. With a wide variety of applications, including bioplastics synthesis, microbially produced organic acids have the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments, since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in such conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Here, we are characterizing and engineering plasma membrane transporters with the final aim to improve production of dicarboxylic acids, namely succinic acid, in yeast. This includes transporters that have already been described as efficient dicarboxylate transporters, as well as promising transporters from the AceTr family. First, we perform functional characterization by studying transporter kinetics, energetics and specificity, as well as site-directed mutagenesis, to acquire insight into functional-structural relationship of transporters. This insight further uncovers engineering targets that can lead to improved transporter activity as well as altered substrate specificity. Finally, the performance of these transporters can be assessed via their expression in S. cerevisiae that is engineered for succinic acid production., This work was supported by the strategic programme UID/BIA/ 04050 2019 funded by Portuguese funds through the FCT IP, the project TransAcids (PTDC/ 5184 2014 funded by FCT IP and ERDF by COMPETE 2020 POCI and the project EcoAgriFood (NORTE 01 0145 FEDER 000009 supported by NORTE 2020 under the PORTUGAL 2020 Partnership Agreement

In Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates (CA) at the plasma membrane, Jen1p a monocarboxylate proton symporter (Major Facilitator Superfamily) and Ady2p an acetate permease (AceTr Family). In Cyberlindnera jadinii, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this work, CA transporter homolog genes from C. jadinii were identified and expressed in S. cerevisiae. The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used to express C. jadinii ScJEN1 and ScADY2 homologs. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of labelled CAs, namely D,L-[U-14C] lactic acid, [2,3-14C] succinic acid and [1-14C] acetic acid. In C. jadinii, 6 genes homolog to ScJEN1 (Cjj23088, Cjj21966, Cjj22358, Cjj21989, Cjj21602, Cjj25129) and 4 genes homolog to ScADY2 (Cja24587, Cja20823, Cja20690, Cja20822) were identified. All proteins are being expressed to uncover their subcellular localization and the characterization of transporter specificity is currently underway. In this work, we identified 6 CjJEN1 and 4 CjADY2 homologs that are functional carboxylate transporters in S. cerevisiae. All the CjJEN1 homologs are lactate transporters and CjADY2 homologs present different specificities. Further studies are underway to fully characterize these ten new plasma membrane transporters from C. jadinii., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060

Introduction In Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates (CA) at the plasma membrane, Jen1p a monocarboxylate proton symporter (Major Facilitator Superfamily) and Ady2p an acetate permease (AceTr Family). In Cyberlindnera jadinii, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this work, CA transporter homolog genes from C. jadinii were identified and expressed in S. cerevisiae. Materials and Methods The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used to express C. jadinii ScJEN1 and ScADY2 homologs. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of labelled CAs, namely D,L-[U-14C] lactic acid, [2,3-14C] succinic acid and [1-14C] acetic acid. Results In C. jadinii, 6 genes homolog to ScJEN1 (Cjj23088, Cjj21966, Cjj22358, Cjj21989, Cjj21602, Cjj25129) and 4 genes homolog to ScADY2 (Cja24587, Cja20823, Cja20690, Cja20822) were identified. All proteins were expressed and localized at the plasma membrane. Regarding transporter specificity CJJEN1-6 and CJAD3 encode lactate transporters, CJAD1 and 4, lactate and acetate transporters, and CJAD2 encodes a lactate, acetate and succinate transporter. Conclusions In this work, we identified 6 CjJEN1 and 4 CjADY2 homologs that are functional carboxylate transporters in S. cerevisiae. All the CjJEN1 homologs are lactate transporters and CjADY2 homologs present different specificities. Further studies are underway to fully characterize these ten new plasma membrane transporters from C. jadinii. References Soares-Silva, I., et al. (2007). Mol Membr Biol. 24(5-6), 464-474., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060

Organic acids, such as acetate, are recognized as one of the most prevalent compounds in ecosystems and the transport, biosynthesis, and assimilation of these molecules represent an adaptive advantage for organisms. The majority of AceTr family members so far characterized were associated with the active transport of acetate. AceTr proteins have six predicted transmembrane segments (TMS), sharing the conserved motif NPAPLGL(M/S) located at the beginning of the first TMS. Gpr1 from Yarrowia lipolytica was the first AceTr family member identified in yeasts, being involved in acetic acid sensitivity, cell and colony morphology, yeast-to-hyphae transition and cell lifespan. The Saccharomyces cerevisiae has 3 homologous genes, ADY2, FUN34 and ATO3 was latter characterized as an acetate proton symporter. In this work, we identified the residues of the conserved motif NPAPLGL(M/S) as essential for substrate uptake and binding, but not for membrane targeting. Furthermore, we demonstrated that the Gpr1 from Y. lipolytica is an acetate permease and the S. cerevisiae Fun34 does not transport acetate. Phylogenetic analysis of AceTr family shows that it is dispersed in the tree of life. In eukaryotes, however, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, where it may play an essential role in fungal survival., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060

A broad diversity of yeast species can be found in nature, including in wastes from food industries. The exploration of this biodiversity has captured great interest from food, pharmaceutical and even fuel companies due to the interesting features of these microorganisms [1]. Yeasts can convert sugars present in raw materials into different chemical building-blocks, as well as biofuels, a process more sustainable than those based on fossil fuels and refineries [2]. Among yeasts, Saccharomyces cerevisiae is considered the model organism. Characteristics that range from its simple cultivation, short replication period, sporulation efficiency, easy genetic manipulation and rare pathogenicity have turned it in an ideal organism for various biotechnological processes [3]. In this work, a group of isolates from the TransBio collection (Project FP7 KBBE–Nº289603) were selected, based on their ability to grow in organic acids. The microorganisms, were identified by molecular typing (DNA sequencing of the ITS regions) and characterized morpho- and physiologically. Morphological traits and sporulation patterns were evaluated for cell cycle determination. Phenotypically, evaluated yeasts revealed interesting physiological features regarding growth profiles using carboxylic acids as sole carbon and energy source. The full characterization of these yeast strains is currently ongoing., PTDC/BIAMIC/5184/2014. NORTE-08-5369-FSE-000060

Puromycin aminonucleoside-induced nephrotic syndrome (PAN-NS) is characterized by cardiac remodeling and increased local inflammatory activity. Patients with NS and animal models of NS have vitamin D3 deficiency. The aim of the present study was to evaluate the influence of calcitriol on cardiac remodeling and local inflammatory state in PAN-NS rat model. Male Sprague-Dawley rats were injected with PAN or vehicle on day 0. PAN and control rats were divided into two subgroups for the administration of calcitriol (PAN-D and Ct-D groups) or the vehicle (PAN-V and Ct-V groups) during 21 days. On day 21, the renal function, metabolic balance, calcitriol and FGF-23 plasma levels, prohypertrophy and proinflammatory markers (ET-1, TGF-ß1, TNF-a, and IL-1ß), and calcium signaling molecules (PLB and SERCA-2a) were evaluated. Twenty-one days after injection, PAN-V group presented cardiac hypertrophy and a modulation of proinflammatory markers local expression. Calcitriol treatment of PAN rats prevented cardiac hypertrophy and was associated with marked reduction in the cardiac expression levels of proinflammatory markers. Our results suggest that vitamin D3 deficiency in PAN-NS may contribute to cardiac remodeling and to the increase in local inflammatory activity. Calcitriol treatment prevents both cardiac repercussions and local inflammatory processes in PAN-NS. This work was financed by FEDER (Fundo Europeu de Desenvolvimento Regional) funds, through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT (Fundac¸ão para a Ciência e a Tecnologia)/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). Quelhas-Santos Janete is supported by project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The work was also awarded in 2015 by the Portuguese Society of Nephrology (Roche Grant).

Carboxylic acids are important platform chemicals used traditionally and industrially in food and pharmaceutical companies. In the yeast Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates at the plasma membrane. The JEN1 gene encodes a monocarboxylate proton symporter, with specificity for lactate, pyruvate acetate and propionate, that belongs to the Major Facilitator Superfamily (TC 2.A.1.12.2) [1]. The ADY2 gene is a member of the Acetate Uptake Transporter (AceTr) Family (TC 2.A.96.1.4) and encodes an acetate transporter [2]. In the yeast Candida utilis, different uptake systems for carboxylic acids were functionally characterized [3] however until now the genes encoding these transporters remain unidentified. In this work, carboxylic acid transporter homolog genes from C. utilis were identified and expressed in Saccharomyces cerevisiae. The C. utilis ScJEN1 and ScADY2 homologs were identified through sequence alignment with BlastP and phylogenetic analysis of putative transporters. In Candida utilis, 5 genes homolog to ScJEN1 (Cjj23088, Cjj21966, Cjj22358, Cjj21989, Cjj21602) and 4 genes homolog to ScADY2 (Cja24587, Cja20823, Cja20690, Cja20822) were identified. These genes were expressed under the control of a GPD constitutive promoter, in a S. cerevisiae jen1∆ady2∆ strain, that presents no activity for plasma membrane carboxylate permeases. The functional characterization of these proteins is currently underway., PTDC/BIAMIC/5184/2014. NORTE-01-0145-FEDER-000009. NORTE-08-5369-FSE-000060