Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

Lactic acid excretion via carrier-mediated facilitated diffusion in Lactobacillus helveticus

Summary During growth on a complex medium containing 2% (w/v) lactose, Lactobacillus helveticus produced about 180 mm lactate. Due to the acidification, the external pH decreased to 3.7. The pH remained constant at a level of 0.5–0.7 units (40 mV), and µ_Lac decreased gradually from –60 to 0 mV. The mechanism of lactate extrusion was studied with resting cells. Upon dilution of lactate-loaded cells in a buffer containing [¹⁴C]-lactate, a typical counterflow was observed, suggesting that a carrier system was employed in lactate excretion. Influx of lactate could not be driven by an artificial membrane potential, indicating that lactate was electroneutrally transported. By examining efflux under various lactate anion and lactic acid concentrations, the undissociated form of the acid was shown to influence the velocity of the transport process. A pH-dependent apparent K _m value of the carrier system was observed in efflux experiments with increasing internal lactate concentrations. It was concluded that the mode of end-product excretion can be defined as a carrier-mediated facilitated diffusion with the undissociated lactic acid or the lactate anion in symport with one proton, respectively, as the object of transport.Abbreviations L _tota total lactate - L _undissb free lactic acid - L _dissc lactate anion - pH_ed external pH - pH_ie internal pH - pH transmembrane H⁺ gradient - µ_Lacf transmembrane gradient of total lactate - µ_HLg transmembrane gradient of the free lactic acid - µ_Lh transmembrane gradient of the lactate anion - V _Effii efflux velocity Offprint requests to: G. Gottschalk     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Lactate transport and glycolytic activity in the freshly isolated rabbit cornea.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 mM iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

Aerobic electron transport in Propionibacterium shermanii effects of cyanide

Cyanide inhibited d- and l-lactate and NADH oxidase activities of membrane particles from Propionibacterium shermanii but only at relatively high concentrations. Inhibition occurred at two different sites in the electron transport pathway. One site, with a half-maximal inhibition concentration (I _0.5) of 2 to 3 mM KCN, is located at the terminal oxidase involved in cytochrome b oxidation; the evidence is consistent with cytochrome d being the major oxidase involved. At high concentrations, cyanide inhibited reduction of cytochrome b by d-lactate (I _0.5 value 20–25 mM cyanide). A proportion of the oxygen-uptake remained uninhibited even by 100 mM cyanide; this proportion was about 80% for succinate, 30% for l-lactate, 15% for d-lactate and 10% for NADH. The oxygen uptake per mol of substrate oxidised increased with increasing cyanide concentration and was accompanied by the formation of hydrogen peroxide as a product of a cyanide-insensitive oxidase system.Abbreviations PMS Phenazine methosulphate     

Methylglyoxal Bypass Identified as Source of Chiral Contamination in l(+) and d(−)-lactate Fermentations by Recombinant Escherichia coli

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both l(+)- and d(−)-lactate. Strain TG113 contains only native genes and produced optically pure d(−)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only l(+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Membrane-bound <Emphasis Type="SmallCaps">l</Emphasis>- and <Emphasis Type="SmallCaps">d</Emphasis>-lactate dehydrogenase activities of a newly isolated <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain

Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate. Ma and Gao contributed equally to this work.     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^– pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono--lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase

Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, dl-lactate or l-lactate. Unlike growing cultures, washed cells excreted significant amounts of pyruvate. The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production. The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA). Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP. Citrate synthase was purified and shown to be a “large” form of the enzyme (M _r 227,000), comprising a single type of subunit (M _r 47,000) as found in several other gram-negative aerobes. The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed. Received: 14 March 1997 / Accepted: 9 July 1997