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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

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

Tese de Doutoramento em Biologia Molecular e Ambiental, As the market demand for sustainable energy increases, production of platform chemicals, such as organic acids, from renewable feedstocks via microbial fermentation has become a more attractive alternative to petroleum-based chemicals. The biosynthesis of organic acids in high and sustainable yields requires the engineering of not only the microorganisms enzymatic and metabolic machinery, but also of plasma membrane transporters. These are able to export organic acids to the extracellular space, improving the host’s tolerance and decreasing downstream processing costs. Thus, to allow a significant impact in future applications envisaging the increase of organic acid production, the study of membrane transporters with specificity for these solutes is of utmost importance. Organic acids are reported to be toxic to cell factories, being associated with trans-membrane potential disruption, cytosol acidification, and anion accumulation. Due to its tolerance to low pH, reducing the need for maintenance of neutral pH and being less vulnerable to contamination, Saccharomyces cerevisiae is taken as one of the most appealing hosts to produce these platform chemicals. In yeasts, the transport of organic acids is played by several membrane proteins, namely the ones belonging to the Jen1 and the Ady2 families. In this study, we functionally characterized Jen1 homologues by heterologous expression in a S. cerevisiae jen1 ady2 strain. We identified an acetate (Dh27), malate (Dh17) and two succinate transporters (Dh18 and Dh24) from the yeast Debaryomyces hansenii. Additionally, we found evidences that saccharate is a substrate of the transporters ScJen1-S271Q and KlJen2, gluconate of CaJen2 and KlJen2, and xylarate and mucate of CaJen2. Organic acid analogues may have therapeutic applications such as 3-Bromopyruvate (3BP) which is reported as an anticancer and antimicrobial drug. We evaluated the role of Jen1 homologues of the human pathogen Cryptococcus neoformans in the transport of 3BP. The Cn04 protein plays an important role in the 3BP uptake and sensitivity of C. neoformans cells. In another approach, several members of the AceTr family were functionally characterized: we show that the YaaH protein from Escherichia coli is a secondary active organic acid/proton symporter, which led to its assignment as SatP, a succinate-acetate transporter; the aceP gene from the archaea Methanosarcina acetivorans was found to be an acetate/proton transporter; and we elucidated the role of Gpr1 from Yarrowia lipolytica as an acetate transport. A phylogenetic analysis of the AceTr family uncovered the presence of homologues in almost all analyzed fungal organisms. Reports about the role of these homologues suggest the importance of these transporters during fungal evolution, associated with sexual reproduction by sporulation. Moreover, we report the essential role of the NPAPLGL(M/S) “signature” motif of this family of transporter, since substitutions in any of the residues of this domain abolished acetate transport. Overall, the present work underlines the wide range of applications of carboxylate transporters in the industrial biotechnology. It also contributed to the deepen the knowledge on microbial carboxylate transporters and provided insights into the evolution, phylogeny and structure of AceTr family members., À medida que a procura de fontes de energia renováveis aumenta, a produção de compostos químicos por fermentação microbiana, tais como os ácidos orgânicos, torna-se uma alternativa atrativa à síntese química a partir de derivados da indústria petrolífera. A biossíntese de ácidos orgânicos, em concentrações e rendimentos economicamente sustentáveis, requer a manipulação de vias metabólicas e da maquinaria enzimática dos microrganismos, assim como a exploração de transportadores de membrana. Estes são essenciais no efluxo dos ácidos orgânicos para o meio de fermentação, permitindo aumentar a tolerância da célula microbiana em relação a estes compostos e, concomitantemente, diminuir os custos de processamento pós-fermentação. De forma a garantir um progresso significativo em futuras aplicações que visem o aumento de produção de ácidos orgânicos, reveste-se de particular importância aprofundar o conhecimento sobre as proteínas transportadoras destes compostos. Os ácidos orgânicos têm um efeito tóxico nas células, associado à dissipação do potencial transmembranar, acidificação do citosol e acumulação de aniões. Devido à tolerância a condições acídicas, e à reduzida necessidade de manutenção e controlo do pH extracelular, a levedura Saccharomyces cerevisiae é considerada um dos microrganismos mais atrativos para a produção de ácidos orgânicos. Em leveduras, o transporte destes ácidos é realizado por diversas proteínas de membrana, nomeadamente as pertencentes às famílias Jen1 e Ady2. Efectuámos uma caracterização funcional dos homólogos da proteína Jen1 através da expressão heteróloga na estirpe S. cerevisiae jen1 ady2. Foram identificados transportadores de acetato (Dh27), de malato (Dh17) e de succinato (DH18 e Dh24) da levedura Debaryomyces hansenii. Estes estudos indicam ainda que o sacarato é substrato dos transportadores ScJen1-S271Q e KlJen2, o gluconato dos transportadores CaJen2 e KlJen2, e o xilarato e o mucato do transportador CaJen2. Alguns análogos de ácidos orgânicos podem apresentar mais valias a nível terapêutico como é o caso do 3-Bromopiruvato (3BP), um composto com propriedades anticancerígenas e antimicrobianas. Desta forma, avaliou-se também o papel de homólogos de Jen1 pertencente ao microrganismo patogénico Cryptococcus neoformans, no transporte de 3BP. Verificou-se que a proteína Cn04 tem um papel relevante no transporte e sensibilidade das células de C. neoformans ao 3BP. Neste trabalho foram caracterizados vários membros da família AceTr. Verificou-se que a proteína YaaH de Escherichia coli é um transportador ativo secundário de acetato e de sucinato por simporte com protões, e por esse motivo foi proposta a sua designação como SatP (succinate-acetate transporter); o gene aceP da arquea Methanosarcina acetivorans foi identificado como sendo específico para o transporte de acetato por cotransporte com protões; elucidámos ainda o papel da proteína Gpr1 de Yarrowia lipolytica, como sendo um transportador de acetato. A análise filogenética da família AceTr revelou a presença de homólogos na maioria das espécies de fungos analisadas. Dados sobre o seu papel fisiológico sugerem a importância destes transportadores ao longo da evolução, associada à reprodução sexuada por esporulação. Os resíduos de aminoácidos do motivo conservado NPAPLGL(M/S) da família AceTr, foram identificados como essenciais para atividade destes transportadores, uma vez que a substituição de qualquer um destes resíduos de aminoácidos eliminou o transporte de acetato. Globalmente, o presente estudo destacou o amplo número de aplicações para os transportadores de ácidos carboxílicos na biotecnologia industrial. Realça-se o aumento do conhecimento sobre transportadores de ácidos orgânicos e a contribuição sobre a evolução, filogenia e a estrutura dos membros da família AceTr., The financial support was given by the Fundação para a Ciência e Tecnologia by means of a grant, SFRH/BD/96166/2013, funded by Fundação para a Ciência e a Tecnologia (FCT) and co-funded by Fundo Social Europeu (FSE) and Programa Operacional Potencial Humano (POPH). Financial support for part of the work was received by FCT I.P. through the strategic funding UID/BIA/04050/2013 and the project PTDC/BIAMIC/5184/2014

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

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

This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jenl, Ady2, Fpsl and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jenl and Ady2 can mediate the uptake of the anionic form of these substrates by a 1-1 -symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fpsl channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions., This work was supported by FCT I.P. through the strategic funding UID/BIA/04050/2013 and the project PTDC/BIA-MIC/5184/2014. D.R and G.T. acknowledge FCT for the SFRH/BD/96166/2013 and SFRH/BD/86221/2012, respectively., info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Molecular Genetics, The rising global energy demands in last century drive to worrying depletion levels of fossil fuels, leading to a growing interest in microbial biofuel synthesis, particularly in model organisms like Saccharomyces cerevisiae. The microbial conversion of sugars to biofuels is a promising technology, thus envisaging for more efficient metabolic pathways has been attempted. Indeed byproducts of biomass pretreatment processes and the biofuels themselves are often toxic at industrially-relevant levels., therefore efforts to improve production yield by engineering efflux systems to overcome toxicity problems and secret inhibitory chemicals from the cell has been revealed as a crucial alternative. In this scope, the aim of the present work was to screen a wide range of promising membrane transporters for the transport of carboxylic acids, particularly for xylonic acid, mucic acid, saccharic acid, gluconic acid and xylaric acid in the yeast S. cerevisiae. During this study a considerable number of membrane proteins, known as carboxylic acid transporters from different yeasts species were screened and functionally characterized in regard to the transport of mono- and dicarboxylic acids with biotechnological application, such as xylonic and gluconic acids, and saccharic, mucic and xylaric acids, respectively. Among the transporters tested, we have found evidences for the transport of four carboxylic acids, mucic, xylaric, gluconic and saccharic acids with the following specificities: gluconic acid (Ki of 13.2 mM and 40.6 mM), xylaric acid (Ki of 27.6 mM), mucic acid (Ki of 32.9 mM) and saccharic acid (Ki of 24.1 mM and 24.7 mM). These results revealed to be very promising for future work aiming at engineering proper microbial strains with increased ability to export biofuel acids to external medium., A crescente procura de combustíveis fósseis a que se assistiu no último século levou a que se atingissem limites de exploração preocupantes, redirecionando a atenção para a síntese microbiana de biocombustíveis, em particular através organismo modelo Saccharomices cerevisiae. A conversão microbiana de açúcares em biocombustíveis temse revelado uma tecnologia promissora, pelo que novas tentativas de melhorar a eficiência das vias metabólicas envolvidas no processo de síntese têm sido testadas. Tanto os produtos resultantes do processamento de biomassa como os próprios biocombustíveis exercem regularmente um efeito tóxico na produção à escala industrial. Nesse sentido, importantes esforços têm sido levados a cabo para desenvolver novos sistemas de efluxo com o intuito de reduzir problemas de toxicidade e conduzir para o exterior da célula diferentes compostos químicos. Neste contexto, o objetivo do presente trabalho é desvendar o potencial de diferentes transportadores de membrana para o transporte de ácidos carboxílicos em S. cerevisiae. Ao longo deste estudo, várias proteínas de membrana, conhecidas como transportadores de ácidos carboxílicos provenientes de diferentes espécies de leveduras foram testadas e caracterizadas funcionalmente no que respeita ao transporte de ácidos mono e dicarboxílicos com interesse biotecnológico, tais como ácidos xilónico e glucónico, e ácidos sacárico, mucico e xilárico, respetivamente. De entre os transportadores testados foram encontradas evidências para o transporte de quatro ácidos carboxílicos, a saber os ácidos mucico, xilárico, glucónico e sacárico com as respetivas especificidades: ácido glucónico (Ki de 13,2 mM e 40,6 mM), ácido xilárico (Ki de 27,6 mM), ácido mucico (Ki de 32,9 mM) e ácido sacárico (de 24,1 mM e 24,7 mM). Estes resultados revelam-se bastante promissores para futuros estudos que envolvam a obtenção de estirpes microbianas com capacidade desenvolvida para excretar biocombustíveis.