Pyruvate Production Research Articles

The emerging role of apoptosis-associated speck-like protein containing a CARD (ASC) in dendritic cell metabolism and function.

Abstract Chlamydia trachomatis (C. trachomatis) is a major infectious bacterial agent that causes sexually transmitted infections (STIs) worldwide. The regulatory functions of dendritic cells (DCs) play a major role in protective immunity against Chlamydia infections. The mechanism(s) underlying this immunomodulation are not fully understood. The inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) regulates the direction of immunity against such bacterial infections. In this study, we assessed the effect of ASC on the metabolism and function of Chlamydia infected DCs. Our results revealed that mitochondria of infected ASC−/− DCs had a gradual disruption of the cristae compared to the normal narrow pleomorphic cristae of uninfected wild type (WT) DCs and ASC−/− and infected WT DCs. In addition to the damaged mitochondria, we observed ASC−/− DCs had significantly reduced pyruvate production; the output of the metabolism of glucose known as glycolysis, following Chlamydia infection. Finally, the DCs from Chlamydia infected ASC−/− mice also displayed defective activation and antigen presentation. Our results suggest that ASC may play a role in cellular respiration of Chlamydia infected DCs, starting from glycolysis to the electron transport chain (ETC) which occurs within the mitochondria, thereby effecting the actions and functions of these DCs.

Loss of ATP13A2 impairs glycolytic function in Kufor-Rakeb syndrome patient-derived cell models

BackgroundKufor-Rakeb syndrome (KRS) is an autosomal recessive, juvenile-onset Parkinson's disease (PD) caused by loss-of-function mutations in ATP13A2 (PARK9). Impaired energy metabolism is considered a pathogenic mechanism in PD and mitochondrial dysfunction resulting from Zn2+ dyshomeostasis has been found in KRS patient-derived cells. In addition to mitochondrial energy production, glycolysis plays an important role in cellular energy metabolism and glucose hypometabolism has been reported in PD. However, glycolytic status in KRS remains undetermined despite its potential importance. MethodsWe assessed glycolytic function in ATP13A2-deficient KRS patient-derived human olfactory neurosphere cells and fibroblasts and determined the effect of pyruvate supplementation on improving cellular energy production. ResultsWe found impaired extracellular acidification, reduction in pyruvate production and a decrease in the NAD+/NADH ratio, indicative of glycolytic dysfunction. In addition, gene expression analysis revealed an altered expression profile for several glycolytic enzymes. Glycolytic dysfunction was aggravated when the intracellular Zn2+ concentration was increased, while ATP13A2 overexpression and pyruvate supplementation blocked the observed Zn2+-mediated toxicity. Moreover, supplementation with pyruvate significantly increased basal mitochondrial ATP production and abolished Zn2+-induced cell death. ConclusionsThese findings indicate that glycolytic dysfunction contributes to pathogenesis and pyruvate supplementation improves overall cellular bioenergetics in our KRS patient-derived cell model, highlighting a therapeutic potential.

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.

Transport of pyruvate into mitochondria is involved in methylmercury toxicity.

We have previously demonstrated that the overexpression of enzymes involved in the production of pyruvate, enolase 2 (Eno2) and D-lactate dehydrogenase (Dld3) renders yeast highly sensitive to methylmercury and that the promotion of intracellular pyruvate synthesis may be involved in intensifying the toxicity of methylmercury. In the present study, we showed that the addition of pyruvate to culture media in non-toxic concentrations significantly enhanced the sensitivity of yeast and human neuroblastoma cells to methylmercury. The results also suggested that methylmercury promoted the transport of pyruvate into mitochondria and that the increased pyruvate concentrations in mitochondria were involved in intensifying the toxicity of methylmercury without pyruvate being converted to acetyl-CoA. Furthermore, in human neuroblastoma cells, methylmercury treatment alone decreased the mitochondrial membrane potential, and the addition of pyruvate led to a further significant decrease. In addition, treatment with N-acetylcysteine (an antioxidant) significantly alleviated the toxicity of methylmercury and significantly inhibited the intensification of methylmercury toxicity by pyruvate. Based on these data, we hypothesize that methylmercury exerts its toxicity by raising the level of pyruvate in mitochondria and that mitochondrial dysfunction and increased levels of reactive oxygen species are involved in the action of pyruvate.

Transporter engineering and enzyme evolution for pyruvate production fromd/l-alanine with a whole-cell biocatalyst expressingl-amino acid deaminase from Proteus mirabilis

Pyruvate, which has been widely used in the food, pharmaceutical, and agrochemical industries, can be produced by “one-step pyruvate production” method fromd/l-alanine with a whole-cellE. colibiocatalyst expressingl-amino acid deaminase (pm1) fromProteus mirabilis.

Conserved Metabolic Changes in Nondiabetic and Type 2 Diabetic Bariatric Surgery Patients: Global Metabolomic Pilot Study.

The goal of this study was to provide insight into the mechanism by which bariatric surgical procedures led to weight loss and improvement or resolution of diabetes. Global biochemical profiling was used to evaluate changes occurring in nondiabetic and type 2 diabetic (T2D) patients experiencing either less extreme sleeve gastrectomy or a full gastric bypass. We were able to identify changes in metabolism that were affected by standard preoperation liquid weight loss diet as well as by bariatric surgery itself. Preoperation weight-loss diet was associated with a strong lipid metabolism signature largely related to the consumption of adipose reserves for energy production. Glucose usage shift away from glycolytic pyruvate production toward pentose phosphate pathway, via glucose-6-phosphate, appeared to be shared across all patients regardless of T2D status or bariatric surgery procedure. Our results suggested that bariatric surgery might promote antioxidant defense and insulin sensitivity through both increased heme synthesis and HO activity or expression. Changes in histidine and its metabolites following surgery might be an indication of altered gut microbiome ecology or liver function. This initial study provided broad understanding of how metabolism changed globally in morbidly obese nondiabetic and T2D patients following weight-loss surgery.

13C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation–reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [13C]lactate labeling from metabolism of [1,2-13C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements.

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease.

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae.

We studied the transcriptomic response of Streptococcus pneumoniae to the fluoroquinolone moxifloxacin at a concentration that inhibits DNA gyrase. Treatment of the wild-type strain R6, at a concentration of 10× the MIC, triggered a response involving 132 genes after 30 min of treatment. Genes from several metabolic pathways involved in the production of pyruvate were upregulated. These included 3 glycolytic enzymes, which ultimately convert fructose 6-phosphate to pyruvate, and 2 enzymes that funnel phosphate sugars into the glycolytic pathway. In addition, acetyl coenzyme A (acetyl-CoA) carboxylase was downregulated, likely leading to an increase in acetyl-CoA. When coupled with an upregulation in formate acetyltransferase, an increase in acetyl-CoA would raise the production of pyruvate. Since pyruvate is converted by pyruvate oxidase (SpxB) into hydrogen peroxide (H2O2), an increase in pyruvate would augment intracellular H2O2. Here, we confirm a 21-fold increase in the production of H2O2 and a 55-fold increase in the amount of hydroxyl radical in cultures treated during 4 h with moxifloxacin. This increase in hydroxyl radical through the Fenton reaction would damage DNA, lipids, and proteins. These reactive oxygen species contributed to the lethality of the drug, a conclusion supported by the observed protective effects of an SpxB deletion. These results support the model whereby fluoroquinolones cause redox alterations. The transcriptional response of S. pneumoniae to moxifloxacin is compared with the response to levofloxacin, an inhibitor of topoisomerase IV. Levofloxacin triggers the transcriptional activation of iron transport genes and also enhances the Fenton reaction.

O-linked N-acetylglucosaminylation of Sp1 interferes with Sp1 activation of glycolytic genes

Glycolysis, the primary pathway metabolizing glucose for energy production, is connected to the hexosamine biosynthetic pathway (HBP) which produces UDP-N-acetylglucosamine (UDP-GlcNAc), a GlcNAc donor for O-linked GlcNAc modification (O-GlcNAc), as well as for traditional elongated glycosylation. Thus, glycolysis and O-GlcNAc are intimately associated. The present study reports the transcriptional activation of glycolytic genes by the transcription factor Sp1 and the O-GlcNAc-mediated suppression of Sp1-dependent activation of glycolytic genes. O-GlcNAc-deficient mutant Sp1 stimulated the transcription of nine glycolytic genes and cellular production of pyruvate, the final product of glycolysis, to a greater extent than wild-type Sp1. Consistently, this mutant Sp1 increased the protein levels of the two key glycolytic enzymes, phosphofructokinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), to a greater extent than wild-type Sp1. Finally, the mutant Sp1 occupied GC-rich elements on PFK and GAPDH promoters more efficiently than wild-type Sp1. These results suggest that O-GlcNAcylation of Sp1 suppresses Sp1-mediated activation of glycolytic gene transcription.

Novel NAD-independent d-lactate dehydrogenases from Acetobacter aceti and Acidocella species MX-AZ02 as potential candidates for in vitro biocatalytic pyruvate production

Pyruvate is a significant platform chemical widely used in the agrochemical and pharmaceutical industries. We discovered FAD-containing lactate dehydrogenases (LDHs) from Acetobacter aceti (Aa-LDH) and Acidocella species MX-AZ02 (As-LDH), expressed them in Escherichia coli, optimized their FAD reconstitution, and characterized the recombinants as NAD-independent D-LDHs that are capable of the in vitro biocatalytic production of pyruvate from lactate. Instead of NAD, both Aa-LDH and As-LDH utilized various organic dyes as the electron acceptor. In addition, Aa-LDH and As-LDH exhibited substrate specificity for d-lactate only. Activity was optimized at pH 7.0 and 65°C. The kinetic parameters of Aa-LDH and As-LDH were examined and both enzymes exhibited higher catalytic efficiency (kcat/Km) for 2,6-dichlorophenolindophenol (DCIP), one of the electron acceptors, than d-lactate due to higher binding affinities. When using 10mM d-lactate as the substrate with stepwise DCIP and D-LDH feeding, Aa-LDH and As-LDH produced 5.48 and 4.09mM pyruvate, respectively, and the conversion was proportional to the DCIP concentration.

Malate dehydrogenase-2 inhibitor LW6 promotes metabolic adaptations and reduces proliferation and apoptosis in activated human T-cells.

Activated T cells rely on aerobic glycolysis and glutaminolysis in order to proliferate and differentiate into effector cells. Therefore, intervention in these metabolic pathways inhibits proliferation. The aim of the present study was to evaluate the effects of Krebs' cycle inhibition at the level of malate dehydrogenase-2 (MDH2) in human activated T cells using the MDH2 inhibitor LW6. Activated T cells from healthy volunteers were cultured in the presence or absence of LW6 and cytotoxicity, cell proliferation and the expression levels of hypoxia-inducible factor (HIF)-1α, c-Myc, p53, cleaved caspase-3 and certain enzymes involved in glucose metabolism and glutaminolysis were evaluated. The results revealed that LW6 was not toxic and decreased apoptosis and the levels of the pro-apoptotic tumor suppressor p53. In addition, LW6 inhibited T-cell proliferation and decreased the levels of c-Myc, HIF-1α, glucose transporter-1, hexokinase-II, lactate dehydrogenase-A and phosphorylated pyruvate dehydrogenase. By contrast, LW6 increased the levels of pyruvate dehydrogenase. These alterations may lead to decreased production of pyruvate, which preferentially enters into the Krebs' cycle. Furthermore, LW6 decreased the levels of glutaminase-2, while increasing those of glutaminase-1, which may preserve glutaminolysis, and possibly pyruvate-malate cycling, potentially protecting the cells from energy collapse. In summary, the inhibition of MDH2 in activated T cells abrogates proliferation without adversely affecting cell survival. Adaptations of cellular glucose and glutamine metabolism may prevent energy collapse.

Speeding up the product release: a second‐sphere contribution from Tyr191 to the reactivity of l‐lactate oxidase revealed in crystallographic and kinetic studies of site‐directed variants

Among α-hydroxy acid-oxidizing flavoenzymes l-lactate oxidase (LOX) is unique in featuring a second-sphere tyrosine (Tyr191 in Aerococcus viridans LOX; avLOX) at the binding site for the substrate's carboxylate group. Y191F, Y191L and Y191A variants of avLOX were constructed to affect a hydrogen-bond network connecting Tyr191 to the carboxylate of the bound ligand via the conserved Tyr40 and to examine consequent effects on enzymatic reactivity. Kinetic studies at 20 °C and pH 6.5 revealed that release of pyruvate product was decreased 4.7-fold (Y191F), 19-fold (Y191L) and 28-fold (Y191A) compared with wild-type enzyme (~ 141 s(-1)) and thus became mainly rate limiting for l-lactate oxidation by the variants at a steady-state under air-saturated conditions. In the Y191L and the Y191A variants, but not in the Y191F variant, l-lactate binding was also affected strongly by the site-directed substitution. Reduction of the flavin cofactor by l-lactate and its reoxidation by molecular oxygen were, however, comparatively weakly affected by the replacements of Tyr191. Unlike the related lactate monooxygenase, which prevents the fast dissociation of pyruvate to promote its oxidative decarboxylation by H2 O2 into acetate, CO2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H2O2. The 1.9 Å crystal structure of the Y191F variant bound with FMN and pyruvate revealed a strictly locally disruptive effect of the site-directed substitution. Product off-rates appear to be dictated by partitioning of residues including Tyr191 from an active-site lid loop into bulk solvent and modulation of the hydrogen bond strength that links Tyr40 with the pyruvate's carboxylate group. Overall, this study emphasizes the possibly high importance of contributions from second-sphere substrate-binding residues to the fine-tuning of reactivity in α-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of α-keto acid product release.

Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain.

BackgroundA Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol.ResultsIn this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h−1, respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH181D allele restored its growth on glucose at a maximum specific rate of 0.053 h−1 in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h−1.ConclusionsIn this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0305-6) contains supplementary material, which is available to authorized users.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2015-1158

Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats.

Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a light-induced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant up-regulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism.

Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate.

Metabolic engineering in methanotrophic bacteria

Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems.

Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin

Lignin represents an untapped feedstock for the production of fuels and chemicals, but its intrinsic heterogeneity makes lignin valorization a significant challenge. In nature, many aerobic organisms degrade lignin-derived aromatic molecules through conserved central intermediates including catechol and protocatechuate. Harnessing this microbial approach offers potential for lignin upgrading in modern biorefineries, but significant technical development is needed to achieve this end. Catechol and protocatechuate are subjected to aromatic ring cleavage by dioxygenase enzymes that, depending on the position, ortho or meta relative to adjacent hydroxyl groups, result in different products that are metabolized through parallel pathways for entry into the TCA cycle. These degradation pathways differ in the combination of succinate, acetyl-CoA, and pyruvate produced, the reducing equivalents regenerated, and the amount of carbon emitted as CO2—factors that will ultimately impact the yield of the targeted product. As shown here, the ring-cleavage pathways can be interchanged with one another, and such substitutions have a predictable and substantial impact on product yield. We demonstrate that replacement of the catechol ortho degradation pathway endogenous to Pseudomonas putida KT2440 with an exogenous meta-cleavage pathway from P. putida mt-2 increases yields of pyruvate produced from aromatic molecules in engineered strains. Even more dramatically, replacing the endogenous protocatechuate ortho pathway with a meta-cleavage pathway from Sphingobium sp. SYK-6 results in a nearly five-fold increase in pyruvate production. We further demonstrate the aerobic conversion of pyruvate to l-lactate with a yield of 41.1±2.6% (wt/wt). Overall, this study illustrates how aromatic degradation pathways can be tuned to optimize the yield of a desired product in biological lignin upgrading.