Uncovering novel plasma membrane carboxylate transporters in the yeast Cyberlindnera jadinii

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)

Engineering the activity and specificity of Saccharomyces cerevisiae Acetate Transporter Ady2/Ato1

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

Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196

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)

Expanding the knowledge on the skillful yeast Cyberlindnera jadinii

Cyberlindnera jadinii is widely used as a source of single-cell protein and is known for its ability to synthesize a great variety of valuable compounds for the food and pharmaceutical industries. Its capacity to produce compounds such as food additives, supplements, and organic acids, among other fine chemicals, has turned it into an attractive microorganism in the biotechnology field. In this review, we performed a robust phylogenetic analysis using the core proteome of C. jadinii and other fungal species, from Asco- to Basidiomycota, to elucidate the evolutionary roots of this species. In addition, we report the evolution of this species nomenclature over-time and the existence of a teleomorph (C. jadinii) and anamorph state (Candida utilis) and summarize the current nomenclature of most common strains. Finally, we highlight relevant traits of its physiology, the solute membrane transporters so far characterized, as well as the molecular tools currently available for its genomic manipulation. The emerging applications of this yeast reinforce its potential in the white biotechnology sector. Nonetheless, it is necessary to expand the knowledge on its metabolism, regulatory networks, and transport mechanisms, as well as to develop more robust genetic manipulation systems and synthetic biology tools to promote the full exploitation of C. jadinii.This work 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 Fundação para a Ciência e Tecnologia (FCT) I.P. and by 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

The Cyberlindnera jadinii carboxylate transporters Ady2 and Jen1 homologs are functional in Saccharomyces cerevisiae

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-00006

Exploring the Cyberlindnera jadinii transportome for the identification of novel carboxylate transporters

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

Characterization of Cyberlindnera jadinii carboxylate permeases by heterologous expression in Saccharomyces cerevisiae

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/25/201

Functional characterization of Cyberlindnera jadinii carboxylate transporters in Saccharomyces cerevisiae

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-00006

Exploring the Cyberlindnera jadinii yeast transportome to uncover novel carboxylate transporters for biotechnological applications

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

Characterization of Cyberlindnera jadinii carboxylate transporters by heterologous expression in S. cerevisiae

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-00006