Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid.

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.

The acetate uptake transporter family motif "NPAPLGL(M/S)" is essential for substrate uptake.

Organic acids are recognized as one of the most prevalent compounds in ecosystems, thus the transport and assimilation of these molecules represent an adaptive advantage for organisms. The AceTr family members are associated with the active transport of organic acids, namely acetate and succinate. The phylogenetic analysis shows this family is dispersed in the tree of life. However, in eukaryotes, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, with an essential role in spore development. Aiming at deepening the knowledge in this family, we studied the acetate permease AceP from Methanosarcina acetivorans, as the first functionally characterized archaeal member of this family. Furthermore, we demonstrate that the yeast Gpr1 from Yarrowia lipolytica is an acetate permease, whereas the Ady2 closest homologue in Saccharomyces cerevisiae, Fun34, has no role in acetate uptake. In this work, we describe the functional role of the AceTr conserved motif NPAPLGL(M/S). We further unveiled the role of the amino acid residues R122 and Q125 of SatP as essential for protein activity.

The microbiome in chronic kidney disease patients undergoing hemodialysis and peritoneal dialysis.

Chronic kidney disease (CKD) is associated with an imbalanced human microbiome due not only to CKD-associated factors such as uremia, increased inflammation and immunosuppression, but also to pharmacological therapies and dietary restrictions. End-stage renal disease patients require renal replacement therapies commonly in the form of hemodialysis (HD) or peritoneal dialysis (PD). HD implies the existence of a vascular access, such as an arteriovenous fistula/graft or a venous catheter, whereas PD implies a long-term peritoneal catheter and the constant inflow of peritoneal dialysate. Also, dietary adaptations are mandatory in both therapies. This revision explores the impact of HD or PD therapies on human microbiome. HD and PD appear to be associated with different changes in the gut microbiome, for example a decrease in Proteobacteria relative abundance in HD patients and increase in PD patients. Both therapies may also have an impact on the human microbiome beyond the gut, leading to increased relative abundance of specific bacteria in the blood microbiome of HD patients and increased relative abundance of other bacteria in the peritoneal microbiome of PD patients. HD and PD catheter biofilms may also play an important role in the changes observed in these microbiomes. A more interdisciplinary approach is needed to further clarify the role of microbial groups other than bacteria in all body habitats to allow the complete understanding of the impact of HD or PD on the microbiome of CKD patients. Moreover, strategies that promote a healthy balance of the human microbiome on these patients should be explored.

Oral Colonization of Staphylococcus Species in a Peritoneal Dialysis Population: A Possible Reservoir for PD-Related Infections?

Peritoneal dialysis-related infections are important morbidity/mortality causes, being staphylococci the most prevalent agents. Since Staphylococcus aureus nasopharynx carriage is a known risk factor for PD infections and the oral cavity is a starting point for systemic diseases development, we aimed at comparing the oral staphylococci colonization between PD patients and controls and studying the association with PD-related infections. Saliva samples were plated in Mannitol salt, and isolates were identified by DnaJ gene sequencing. Staphylococci PD-related infections were recorded throughout the 4-year period following sample collection. Staphylococcus colonization was present in >90% of the samples from both groups (a total of nine species identified). PD patients presented less diversity and less prevalence of multispecies Staphylococcus colonization. Although all patients presenting Staphylococcus epidermidis PD-related infections were also colonized in the oral cavity by the same agent, only 1 out of 7 patients with ESI caused by S. aureus presented S. aureus oral colonization. Staphylococci are highly prevalent in the oral cavity of both groups, although PD patients presented less species diversity. The association between oral Staphylococcus carriage and PD-related infections was present for S. epidermidis but was almost inexistent for S. aureus, so, further studies are still necessary to evaluate the infectious potential of oral Staphylococcus carriage in PD.

Yeast as a tool to express sugar acid transporters with biotechnological interest.

Sugar acids can be used as platform chemicals to generate primary building blocks of industrially relevant products. Microbial production of these organic compounds at high yields requires the engineering of the enzymatic machinery and the presence of plasma membrane transporters able to export them outside the cells. In this study, several yeast carboxylic acid transporters belonging to the Jen family were screened for the transport of biotechnologically relevant sugar acids, namely gluconic, saccharic, mucic, xylaric and xylonic acid, and functionally characterised in Saccharomyces cerevisiae. We show that Jen permeases are capable of transporting most of these sugar acids, although with different specificities. Saccharate is a substrate of the transporters ScJen1-S271Q and KlJen2, gluconate of CaJen2 and KlJen2, and xylarate and mucate of CaJen2. A molecular docking approach of these transporters identified the residues that play a major role in the substrate binding of these sugar acids, namely R188 (ScJen1), R122 (CaJen2) and R127 (KlJen2), all equivalent residues (TMS II). The identification of Jen members as sugar acid transporters can contribute to engineering efficient microbial cell factories with increased sugar acid production, as the ScJen1 is able to promote substrate efflux.

Oral Yeast Colonization and Fungal Infections in Peritoneal Dialysis Patients: A Pilot Study.

Peritonitis and exit-site infections are important complications in peritoneal dialysis (PD) patients that are occasionally caused by opportunistic fungi inhabiting distant body sites. In this study, the oral yeast colonization of PD patients and the antifungal susceptibility profile of the isolated yeasts were accessed and correlated with fungal infection episodes in the following 4 years. Saliva yeast colonization was accessed in 21 PD patients and 27 healthy controls by growth in CHROMagar-Candida® and 18S rRNA/ITS sequencing. PD patients presented a lower oral yeast prevalence when compared to controls, namely, Candida albicans. Other species were also isolated, Candida glabrata and Candida carpophila. The antifungal susceptibility profiles of these isolates revealed resistance to itraconazole, variable susceptibility to caspofungin, and higher MIC values of posaconazole compared to previous reports. The 4-year longitudinal evaluation of these patients revealed Candida parapsilosis and Candida zeylanoides as PD-related exit-site infectious agents, but no correlation was found with oral yeast colonization. This pilot study suggests that oral yeast colonization may represent a limited risk for fungal infection development in PD patients. Oral yeast isolates presented a variable antifungal susceptibility profile, which may suggest resistance to some second-line drugs, highlighting the importance of antifungal susceptibility assessment in the clinical practice.

Renalase regulates peripheral and central dopaminergic activities.

Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

The Debaryomyces hansenii carboxylate transporters Jen1 homologues are functional in Saccharomyces cerevisiae.

We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1Δ ady2Δ strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (Km 0.94 ± 0.17 mM; Vmax 0.43 ± 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (Km 0.27 ± 0.04 mM; Vmax 0.11 ± 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, Km 0.31 ± 0.06 mM; Vmax 0.83 ± 0.04 nmol s(-1) mg(-1)and Km 0.16 ± 0.02 mM; Vmax 0.19 ± 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii.

A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H⁺ symporter Jen1p.

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H⁺ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence ³79NXX[S/T]HX[S/T]QD³87 in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a 'trajectory-like' substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.

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.

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae.

Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO2 in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study, we reported a new group of succinic acid transporters from the AceTr family, which were also described as anion channels. Here, we expressed these transporters in a succinic acid overproducing strain and compared their impact on extracellular succinic acid accumulation with that of the Dct-02 transporter. The results show that the tested transporters of the AceTr family hinder succinic acid accumulation in the extracellular medium at low pH, which is in strong contrast to Dct-02. Data suggests that the AceTr transporters prefer monovalent succinate, whereas Dct-02 prefers divalent succinate anions. In addition, the results provided deeper insights into the characteristics of Dct-02, showing its ability to act as a succinic acid importer (thus being bidirectional) and verifying its capability of exporting malate.

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.

New insights into the acetate uptake transporter (AceTr) family: Unveiling amino acid residues critical for specificity and activity.

Aiming at improving the transport of biotechnologically relevant carboxylic acids in engineered microbial cell factories, the focus of this work was to study plasma membrane transporters belonging to the Acetate Uptake Transporter (AceTr) family. Ato1 and SatP, members of this family from Saccharomyces cerevisiae and Escherichia coli, respectively, are the main acetate transporters in these species. The analysis of conserved amino acid residues within AceTr family members combined with the study of Ato1 3D model based on SatP, was the rationale for selection of site-directed mutagenesis targets. The library of Ato1-GFP mutant alleles was functionally analysed in the S. cerevisiae IMX1000 strain which shows residual growth in all carboxylic acids tested. A gain of function phenotype was found for mutations in the residues F98 and L219 located at the central constrictive site of the pore, enabling cells to grow on lactic and on succinic acid. This phenotype was associated with an increased transport activity for these substrates. A dominant negative acetic acid hypersensitivity was induced in S. cerevisiae cells expressing the E144A mutant, which was associated with an increased acetic acid uptake. By utilizing computer-assisted 3D-modelling tools we highlight structural features that explain the acquired traits found in the analysed Ato1 mutants. Additionally, we achieved the proper expression of the Escherichia coli SatP, a homologue of Ato1, in S. cerevisiae. To our knowledge, this constitutes the first report of a fully functional bacterial plasma membrane transporter protein in yeast cells.

Transport of carboxylic acids in yeasts.

Carboxylic acid transporters form a heterogeneous group of proteins, presenting diverse mechanisms of action and regulation, and belonging to several different families. Multiple physiological and genetic studies in several organisms, from yeast to mammals, have allowed the identification of various genes coding for carboxylate transporters. Detailed understanding of the metabolism and transport of these nutrients has become more important than ever, both from a fundamental and from an applied point of view. Under a biotechnological perspective, the increasing economic value of these compounds has boosted this field of research considerably. Here we review the current knowledge on yeast carboxylate transporters, at the biochemical and molecular level, focusing also on recent biotechnological developments.

Peritoneal Microbiome in End-Stage Renal Disease Patients and the Impact of Peritoneal Dialysis Therapy.

Factors influencing the occurrence of peritoneal dialysis (PD)-related infections are still far from fully understood. Recent studies described the existence of specific microbiomes in body sites previously considered microbiome-free, unravelling new microbial pathways in the human body. In the present study, we analyzed the peritoneum of end-stage kidney disease (ESKD) patients to determine if they harbored a specific microbiome and if it is altered in patients on PD therapy. We conducted a cross-sectional study where the peritoneal microbiomes from ESKD patients with intact peritoneal cavities (ESKD non-PD, n = 11) and ESKD patients undergoing PD therapy (ESKD PD, n = 9) were analyzed with a 16S rRNA approach. Peritoneal tissue of ESKD patients contained characteristically low-abundance microbiomes dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Patients undergoing PD therapy presented lower species richness, with dominance by the Pseudomonadaceae and Prevotelaceae families. This study provides the first characterization of the peritoneal microbiome in ESKD patients, bringing new insight to the human microbiome. Additionally, PD therapy may induce changes in this unique microbiome. The clinical relevance of these observations should be further explored to uncover the role of the peritoneal microbiome as a key element in the onset or aggravation of infection in ESKD patients, especially those undergoing PD.

Carboxylic Acid Transporters in Candida Pathogenesis.

Opportunistic pathogens such as Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence. This review also presents an evolutionary analysis of orthologues of the Saccharomyces cerevisiae Jen1 lactate and Ady2 acetate transporters, including a phylogenetic analysis of 101 putative carboxylate transporters in twelve medically relevant Candida species. These proteins are assigned to distinct clades according to their amino acid sequence homology and represent the major carboxylic acid uptake systems in yeast. While Jen transporters belong to the sialate:H+ symporter (SHS) family, the Ady2 homologue members are assigned to the acetate uptake transporter (AceTr) family. Here, we reclassify the later members as ATO (acetate transporter ortholog). The new nomenclature will facilitate the study of these transporters, as well as the analysis of their relevance for Candida pathogenesis.

Influence of dialysis therapies on oral health: a pilot study.

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.

Phase Angle Predicts Arterial Stiffness and Vascular Calcification in Peritoneal Dialysis Patients.

OBJECTIVES: Fluid overload (FO) is frequently present in peritoneal dialysis (PD) patients and is associated with markers of malnutrition, inflammation, and atherosclerosis/calcification (MIAC) syndrome. We examined the relationships in stable PD patients between phase angle (PhA) and the spectrum of uremic vasculopathy including vascular calcification and arterial stiffness and between PhA and changes in serum fetuin-A levels. METHODS: Sixty-one stable adult PD patients were evaluated in a cross-sectional study (ST1). Phase angle was measured by multifrequency bioimpedance analysis (InbodyS10, Biospace, Korea) at 50 kHz. Augmentation index (AI), a surrogate marker of arterial stiffness, was assessed by digital pulse amplitude tonometry (Endo PAT, Itamar Medical, Caesarea, Israel). Vascular calcification was assessed by simplified calcification score (SCS). Serum fetuin-A levels were measured by ELISA (Thermo scientific; Waltham, MA, USA). Serum albumin was used as a nutritional marker, and serum C-reactive protein (CRP) was used as an inflammatory marker. The same assessments were carried out longitudinally (ST2) in the first 33 patients who completed 1 year of evaluation in ST1. RESULTS: In ST1, patients with PhA < 6° had higher CRP levels, AI, and SCS and lower serum albumin and fetuin-A levels, in comparison with patients with PhA ≥ 6°. In addition, PhA was a predictor of both AI (ß = -0.351, p = 0.023) and SCS ≥ 3 (EXP (B) = 0.243, p = 0.005). In ST2, the increase of PhA over time was associated with decreases in both AI (r = -0.378, p = 0.042) and CRP levels (r = -0.426, p = 0.021), as well as with the increase in serum fetuin-A levels (r = 0.411, p = 0.030). CONCLUSIONS: Phase angle predicts both arterial stiffness and vascular calcification in stable PD patients.

Asymptomatic Effluent Protozoa Colonization in Peritoneal Dialysis Patients.

Currently, chronic kidney disease (CKD) is a global health problem. Considering the impaired immunity of CKD patients, the relevance of infection in peritoneal dialysis (PD), and the increased prevalence of parasites in CKD patients, protozoa colonization was evaluated in PD effluent from CKD patients undergoing PD. Overnight PD effluent was obtained from 49 asymptomatic stable PD patients. Protozoa analysis was performed microscopically by searching cysts and trophozoites in direct wet mount of PD effluent and after staining smears. Protozoa were found in PD effluent of 10.2% of evaluated PD patients, namely Blastocystis hominis, in 2 patients, and Entamoeba sp., Giardia sp., and Endolimax nana in the other 3 patients, respectively. None of these patients presented clinical signs or symptoms of peritonitis at the time of protozoa screening. Our results demonstrate that PD effluent may be susceptible to asymptomatic protozoa colonization. The clinical impact of this finding should be further investigated.

Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris.

In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.