Pyruvate Secretion Research Articles

Thiamine-Starved Lactococcus lactis for Producing Food-Grade Pyruvate.

Lactococcus lactis is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, L. lactis can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of L. lactis into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient L. lactis strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and α-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an α-acetolactate decarboxylase deficient L. lactis strain, pyruvate could be converted to α-acetolactate and diacetyl. Summing up, by starving L. lactis for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.

Urea reduces the sustainability of soil Cd immobilization by upregulating the expression of AmSTOP1 and AmMATE genes in edible amaranth roots

After cadmium (Cd) immobilization remediation in contaminated farmland soil, which forms of nitrogen fertilizer should be implemented to keep its sustainability? Urea and nitrate were used to compare for their effects on the remobilization of stabilized Cd in the rhizosphere soil of edible amaranth at nitrogen concentrations of 60, 95, and 130 mg kg−1. The results showed that compared to nitrate nitrogen, the Cd content in shoots increased by 76.2%, 65.6%, and 148% after applying three different concentrations of urea, and the total remobilization amount of Cd also increased by 16.0%, 24.9%, and 14.0% respectively. Urea application promotes root secretion of citric acid, malic acid, pyruvate, and γ-aminobutyric acid, crucial in remobilizing stable Cd. The application of urea promoted the expression of genes involved in sucrose transport, glycolysis, the TCA cycle, amino acid secretion, citric acid efflux, and proton efflux. Arabidopsis heterologous expression and yeast one-hybrid assays identify critical roles of AmMATE42 and AmMATE43 in citric acid and fumaric acid efflux, with AmSTOP1 activating their transcription. Inhibition of SIZ1 expression in urea treatment reduce AmSTOP1 SUMOylation, leading to increased expression of AmMATE42 and AmMATE43 and enhanced organic acids efflux. Using edible amaranth as a model vegetable, we discovered that urea is not beneficial to preserving the sustainability of stabilized Cd during the reuse of remediated farmlands contaminated with Cd.

Staphylococcus aureus adapts to the immunometabolite itaconic acid by inducing acid and oxidative stress responses including S-bacillithiolations and S-itaconations

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus.In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.

The Commensal Anaerobe Veillonella dispar Reprograms Its Lactate Metabolism and Short-Chain Fatty Acid Production during the Stationary Phase.

Veillonella spp. are obligate, anaerobic, Gram-negative bacteria found in the human oral cavity and gut. Recent studies have indicated that gut Veillonella promote human homeostasis by producing beneficial metabolites, specifically short-chain fatty acids (SCFAs), by lactate fermentation. The gut lumen is a dynamic environment with fluctuating nutrient levels, so the microbes present shifting growth rates and significant variations of gene expression. The current knowledge of lactate metabolism by Veillonella has focused on log phase growth. However, the gut microbes are mainly in the stationary phase. In this study, we investigated the transcriptomes and major metabolites of Veillonella dispar ATCC 17748T during growth from log to stationary phases with lactate as the main carbon source. Our results revealed that V. dispar reprogrammed its lactate metabolism during the stationary phase. Lactate catabolic activity and propionate production were significantly decreased during the early stationary phase but were partially restored during the stationary phase. The propionate/acetate production ratio was lowered from 1.5 during the log phase to 0.9 during the stationary phase. Pyruvate secretion was also greatly decreased during the stationary phase. Furthermore, we have demonstrated that the gene expression of V. dispar is reprogrammed during growth, as evidenced by the distinct transcriptomes present during the log, early stationary, and stationary phases. In particular, propionate metabolism (the propanediol pathway) was downregulated during the early stationary phase, which explains the decrease in propionate production during the stationary phase. The fluctuations in lactate fermentation during the stationary phase and the associated gene regulation expand our understanding of the metabolism of commensal anaerobes in changing environments. IMPORTANCE Short-chain fatty acids produced by gut commensal bacteria play an important role in human physiology. Gut Veillonella and the metabolites acetate and propionate, produced by lactate fermentation, are associated with human health. Most gut bacteria in humans are in the stationary phase. Lactate metabolism by Veillonella spp. during the stationary phase is poorly understood and was therefore the focus of the study. To this end, we used a commensal anaerobic bacterium and explored its short-chain fatty acid production and gene regulation in order to provide a better understanding of lactate metabolism dynamics during nutrient limitation.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer

BackgroundGlutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.MethodsA panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.ResultsThe anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.ConclusionThis study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Pyruvate secretion by oral streptococci modulates hydrogen peroxide dependent antagonism.

Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.

Biocharacteristics and draft genome sequence of Halomonas hydrothermalis C22, a pyruvate-producing halophilic bacterium isolated from a commercial Spirulina culture in Vietnam.

Halophilic bacteria are receiving increasing attention for industrial chemical production processes due to their unique properties. Herein, an alkaliphilic and halophilic bacterium was isolated from a commercial Spirulina culture at Nghe An province in Vietnam and found to secrete pyruvate. Pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals, and is employed for production of crop protection agents, polymers, cosmetics, and food additives. Phenotypic and chemotaxonomic characterization, and the 16S rRNA gene sequence homology with Halomonas hydrothermalis strain DSM 15,725 (99.2%) predicted that the strain belongs to the Halomonas genus, thus we named this strain as H. hydrothermalis strain C22. We investigated the biocharacteristics and capacity of strain C22 and determined the draft genome sequence comprising 3,934,166bp with a G + C content of 60.2% encoding 3,668 proteins, 58 tRNAs, 9 rRNAs, and 1 tmRNA. Maximal pyruvate secretion reached 51.1g/l after 84h of cultivation. The results will facilitate future studies on the genetic and metabolic diversity of halophilic bacteria and expand our understanding of important bioprocesses in this microorganism.

Metabolic Phenotyping of Adipose-Derived Stem Cells Reveals a Unique Signature and Intrinsic Differences between Fat Pads.

White adipose tissues are functionally heterogeneous and differently manage the excess of energy supply. While the expansion of subcutaneous adipose tissues (SAT) is protective in obesity, that of visceral adipose tissues (VAT) correlates with the emergence of metabolic diseases. Maintained in fat pads throughout life, adipose stem cells (ASC) are mesenchymal-like stem cells with adipogenesis and multipotent differentiation potential. ASC from distinct fat pads have long been reported to present distinct proliferation and differentiation potentials that are maintained in culture, yet the origins of these intrinsic differences are still unknown. Metabolism is central to stem cell fate decision in line with environmental changes. In this study, we performed high-resolution nuclear magnetic resonance (NMR) metabolomic analyses of ASC culture supernatants in order to characterize their metabolic phenotype in culture. We identified and quantified 29 ASC exometabolites and evaluated their consumption or secretion over 72 h of cell culture. Both ASC used glycolysis and mitochondrial metabolism, as evidenced by the high secretions of lactate and citrate, respectively, but V-ASC mostly used glycolysis. By varying the composition of the cell culture medium, we showed that glutaminolysis, rather than glycolysis, supported the secretion of pyruvate, alanine, and citrate, evidencing a peculiar metabolism in ASC cells. The comparison of the two types of ASC in glutamine-free culture conditions also revealed the role of glutaminolysis in the limitation of pyruvate routing towards the lactate synthesis, in S-ASC but not in V-ASC. Altogether, our results suggest a difference between depots in the capacity of ASC mitochondria to assimilate pyruvate, with probable consequences on their differentiation potential in pathways requiring an increased mitochondrial activity. These results highlight a pivotal role of metabolic mechanisms in the discrimination between ASC and provide new perspectives in the understanding of their functional differences.

Pyruvate secreted from patient-derived cancer-associated fibroblasts supports survival of primary lymphoma cells.

Cancer‐associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor‐cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse‐Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.

Potential pyruvate and poly (3- hydroxybutyrate) production of halophiles bacteria isolated from Giao Thuy, Nam Dinh’s mangrove forest

Pyruvic acid (pyruvate) the most important α-oxocarboxylic acid, plays a central role in energy metabo-lism in living organisms. It is used mainly as a starting material in the biosynthesis of pharmaceuticals as well as employed in the production of crop protection agents, polymers, cosmetics and food additives. Four Gram-negative, moderately halophilic, pyruvate and poly (3-hydroxybutyrate)- producing strains were isolated from soil samples collected from mangrove forest at Giao Thuy, Nam Dinh province. Preliminary screening results showed that among fours strain, the ND7 strain was able to produce the highest bioplastic-poly (3-hydroxybutyrate) (PHB) and extracellular secretion of pyruvate, respectively, reaching 50.23% dry weight and 6.87 g/L after 60 hours of cultivation. When cultured in high alkaliphilic medium (pH 9.4), the pyruvate secretion increased significantly (21.02 g/L after 60 hours of cultivation). Based on the morphological, bio-chemical characteristics and nucleotide sequence analysis of partial 16S rRNA gene, the ND7 strain was iden-tified as Halomonas maura. The results allow us to conclude that ND7 strain is a potential target for the ex-ploitation of pyruvate in a larger scale.

Pharmacologic Reprogramming Designed to Induce a Warburg Effect in Porcine Fetal Fibroblasts Alters Gene Expression and Quantities of Metabolites from Conditioned Media Without Increased Cell Proliferation.

The Warburg effect is a metabolic phenomenon characterized by increased glycolytic activity, decreased mitochondrial oxidative phosphorylation, and the production of lactate. This metabolic phenotype is characterized in rapidly proliferative cell types such as cancerous cells and embryonic stem cells. We hypothesized that a Warburg-like metabolism could be achieved in other cell types by treatment with pharmacological agents, which might, in turn, facilitate nuclear reprogramming. The aim of this study was to treat fibroblasts with CPI-613 and PS48 to induce a Warburg-like metabolic state. We demonstrate that treatment with both drugs altered the expression of 69 genes and changed the level of 21 metabolites in conditioned culture media, but did not induce higher proliferation compared to the control treatment. These results support a role for the reverse Warburg effect, whereby cancer cells induce cancer-associated fibroblast cells in the surrounding stroma to exhibit the metabolically characterized Warburg effect. Cancer-associated fibroblasts then produce and secrete metabolites such as pyruvate to supply the cancerous cells, thereby supporting tumor growth and metastasis. While anticipating an increase in the production of lactate and increased cellular proliferation, both hallmarks of the Warburg effect, we instead observed increased secretion of pyruvate without changes in proliferation.

Efficient production and secretion of oxaloacetate from Halomonas sp. KM-1 under aerobic conditions

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize glucose for the intracellular storage of the bioplastic poly-(R)-3-hydroxybutyric acid (PHB) and extracellular secretion of pyruvate under aerobic conditions. In this study, we investigated the effects of sodium chloride concentration on PHB accumulation and pyruvate secretion in the KM-1 strain and, unexpectedly, observed that oxaloacetate, an important intermediate chemical in the TCA cycle, glycogenesis, and aspartic acid biosynthesis, was secreted. We then further analyzed oxaloacetate productivity after changing the sodium chloride additive concentration, additive time-shift, and culture temperature. In 42-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 39.0 g/L oxaloacetate at a rate of 0.93 g/(L h). The halophilic bacteria Halomonas has already gained attention for industrial chemical-production processes owing to its unique properties, such as contamination-free culture conditions and a tolerance for high substrate concentrations. Moreover, no commercial scale oxaloacetate production was previously reported to result from bacterial fermentation. Oxaloacetate is an important intermediate chemical in biosynthesis and is used as a health food based on its role in energy synthesis. Thus, these data provided important insights into the production of oxaloacetate and other derivative chemicals using this strain.

Nigella sativa, a traditional Tunisian herbal medicine, attenuates bleomycin-induced pulmonary fibrosis in a rat model

The present study investigated the effects of Nigella sativa oil (NSO) on bleomycin (BLM)-induced lung fibrosis in rats. The rat model of pulmonary fibrosis (PF) was established by intratracheal instillation of BLM, and the effect of 1ml/kg oral NSO treatment once daily observed. The effect of NSO was studied over a period of 50daysusing 1H RMN analysis on the urine and broncho alveolar lavage fluid (Balf) of the rats. Histopathological (inflammation and fibrosis) and immunohistochemical (TGF-β1 density) changes were evaluated.Results found that the BLM group showed a significant increase in inflammatory index (II), fibrosis score (FS) and TGF-β1 distribution in the lung inflammatory infiltrate, accompanied by a decreased urinary secretion of Krebs cycle intermediates, including acetate, pyruvate, carnitine, trimethylamine-N-oxide and succinate. However, at the same time point, NSO treated rats had a reduced II and FS, and had an increased urinary secretion of histidine, fumarate, allantoin and malate.In conclusion, NSO treatment attenuated the effects of BLM-induced PF, by supporting lung, liver and kidney activity in resisting PF. These findings provide an insight into the preventive and therapeutic potential of NSO in the treatment of PF.

Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate.

We have developed a method for rapid quenching of samples taken from chemostat cultures of Escherichia coli that gives reproducible and reliable measurements of extracellular and intracellular metabolites by 1H NMR and have applied it to study the major central metabolites during the transition from anaerobic to aerobic growth. Almost all metabolites showed a gradual change after perturbation with air, consistent with immediate inhibition of pyruvate formate-lyase, dilution of overflow metabolites and induction of aerobic enzymes. Surprisingly, although pyruvate showed almost no change in intracellular concentration, the extracellular concentration transiently increased. The absence of intracellular accumulation of pyruvate suggested that one or more glycolytic enzymes might relocate to the cell membrane. To test this hypothesis, chromosomal pyruvate kinase (pykF) was modified to express either PykF-green fluorescent protein or PykF-FLAG fusion proteins. Measurements showed that PykF-FLAG relocates to the cell membrane within 5 min of aeration and then slowly returns to the cytoplasm, suggesting that on aeration, PykF associates with the membrane to facilitate secretion of pyruvate to maintain constant intracellular levels.

Efficient production and secretion of pyruvate from Halomonas sp. KM-1 under aerobic conditions.

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain.

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts.

Physiology and global gene expression of a Corynebacterium glutamicum ΔF1FO-ATP synthase mutant devoid of oxidative phosphorylation

A mutant of Corynebacterium glutamicum ATCC 13032 with a deletion of the atpBEFHAGDC genes encoding F1FO-ATP synthase was characterized. Whereas no growth was observed with acetate as sole carbon source, the ΔF1FO mutant reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium. Initially, the mutant strain showed a strongly increased glucose uptake rate accompanied by a high oxygen consumption rate and pyruvate secretion into the medium. When oxygen became limiting, the glucose consumption rate was reduced below that of the wild type and pyruvate was consumed again. The ΔF1FO mutant had increased levels of b- and d-type cytochromes and a significantly increased proton motive force. Transcription of genes involved in central carbon metabolism was essentially unchanged, whereas genes for cytochrome bd oxidase, pyruvate:quinone oxidoreductase, oxidative stress response, and others showed increased mRNA levels. On the other hand, genes for amino acid biosynthesis and ribosomal proteins as well as many genes involved in transport displayed decreased mRNA levels. Several of the transcriptional changes were reflected at the protein level, but there were also discrepancies between the mRNA and protein levels suggesting some kind of posttranscriptional regulation. The results prove for the first time that F1FO-ATP synthase and oxidative phosphorylation are in general not essential for growth of C. glutamicum.

Changes in sertoli cell function in vitro induced by nitrobenzene

Nitrobenzene (NB) has been identified as a testicular toxicant in vivo, but its site of action remains unknown. In the present study, the effect of NB on the Sertoli cell was assessed in vitro using Sertoli cell and Sertoli-germ cell cocultures. The parameters measured were the exfoliation of germ cells; the secretion of lactate, pyruvate, and inhibin; and gross cellular morphology. The effect of meta-dinitrobenzene (mDNB), a related compound which is a known Sertoli cell toxicant, was assessed for comparison. Gross morphological changes including vacuolation of Sertoli cells were observed following treatment of cultures with 10 −3 m NB. Exposure of cocultures to NB also resulted in dose-dependent exfoliation of predominantly viable germ cells. NB (> 5 × 10 −4 m) and mDNB at the single dose level used (10 −4 m) stimulated the secretion of lactate and pyruvate significantly by Sertoli cells, an effect that was more marked in the absence of germ cells. Comparable changes were observed in follicle stimulating hormone (FSH)-stimulated cultures. Inhibin secretion by Sertoli cells was also altered by exposure to NB but in a biphasic manner, with low (10 −8 to 10 −6 m) and high (10 −4 to 10 −3 m) doses enhancing inhibin secretion while intermediate (10 −5 m) doses had no effect. These effects were evident in both culture systems but inhibin secretion by Sertoli-germ cell cocultures was always greater than that by Sertoli cell cultures. However, these effects of NB on inhibin secretion were not evident in FSH-stimulated cultures. In contrast to the effects of NB, mDNB had no effect on basal secretion of inhibin but blocked the stimulatory effect of FSH. It is concluded that NB, like mDNB, is probably a Sertoli cell toxicant in view of its similar disruptive effects on various parameters of Sertoli cell function. However, NB is far less toxic than mDNB at equivalent concentrations in vitro. The present study is the first to evaluate the potential of inhibin secretion by Sertoli cells in culture as an additional marker of toxicant action, and concludes that it merits further study in this context.