In order to understand metabolic functions essential for methane assimilation, we investigate dribulose monophosphate pathway and adjacent pathways in gammaproteobacterial Methylomicrobium alcaliphilum 20Z by using combined approaches of RNA-seq, LC-MS, and 13C-labeled techniques. The absolute quantification of metabolome showed that the concentrations of intermediates, such as glucose-6-phosphate and 2-dehydro-3-deoxy-phosphogluconate, involved in Entner-Doudoroff (EDD) pathway were (150.95 +/- 28.75) micromol/L and below the limit of detection of mass spectrometry. In contrast, fructose-1, 6-bisphosphate, glyceraldehyde-3-phosphate/dihydroxyacetone and phosphoenolpyruvate in Embden-Meyerhof-Parnas (EMP) pathway had significantly higher concentrations with (1 142.02 +/- 302.88) micromol/L, (1 866.76 +/- 388.55) micromol/L and (3 067.57 +/- 898.13) micromol/L, respectively. 13C-labeling experiment further indicated that the enrichment of [3-13C1]-pyruvate involved in EMP pathway was 4-6 fold higher than [1,13C1]-pyruvate in EDD pathway in a dynamic course. Moreover, gene expression profile showed that the expression levels of genes in EMP pathway (e.g. fbaA, tpiA, gap and pykA) were 2 479.2, 2 493.9, 2 274.6 and 1 846.0, respectively, but gene expressionlevels in EDD pathway (e.g. pgi, eda and edd) were only 263.8, 341.2 and 225.4, respectively. Overall our current results demonstrated that EMP pathway was the main route for methane assimilation in M. alcaliphilum 20Z. This discovery challenged our understanding of methane assimilation pathway in gammaproteobacterial methanotrophic bacteria, and further provided an important insight for efficient methane biocatalysis in the future.
Glycolysis ; Industrial Microbiology ; Methane ; metabolism ; Methylococcaceae ; metabolism ; Pyruvic Acid ; metabolism

Pyruvate, an important organic acid, is widely used in the industries of pharmaceuticals, chemicals, agrochemicals, food additives and so on. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost and high product quality. In this article, biosynthesis of pyruvate, including direct fermentative production and resting cell method as well as enzymatic method, was discussed. Furthermore, a comparison of these different methods was proposed. Since, a multi-vitamin auxotrophic strain of Torulopsis glabrata is the most competitive strain for industrial production of pyruvate, emphasis was therefore placed on the development of strains screening and fermentation optimization. Finally, some suggestions were put forward to improve the research in this field in the near future.
Biotechnology ; Candida glabrata ; metabolism ; Fermentation ; Oxygen ; pharmacology ; Pyruvic Acid ; metabolism

BACKGROUNDStreptococcus (S.) oligofermentans is a newly identified bacteria with a yet to be defined mechanism of sucrose metabolism that results in acid production. This study aimed to investigate the biochemical mechanisms of S. oligoferm-entans glucose metaolism.

METHODSThe S. oligofermentans LMG21532, Lactobacillus (L.) fermentum 38 and the S. mutans UA140 were used to characterize sucrose metabolism by measuring lactate dehydrogenase (LDH) activity and lactic acid production. Continuous dynamics and high performance capillary electrophoresis were used to determine LDH activity and lactic acid production, respectively, from bacteria collected at 0, 10 and 30 minutes after cultured in 10% sucrose.

RESULTSThese analyses demonstrated that LDH activity of the three bacterial strains examined remained stable but significantly different throughout the sucrose fermentation process. The S. oligofermentans LDH activity ((0.61 ± 0.05) U/mg) was significantly lower than that of L. fermentum ((52.91 ± 8.97) U/mg). In addition, the S. oligofermentans total lactate production ((0.048 ± 0.021) mmol/L) was also significantly lower than that of L. fermentum ((0.958 ± 0.201) mmol/L). Although the S. oligofermentans LDH production was almost double of that produced by S. mutans ((0.32 ± 0.07) U/mg), lactic acid production was approximately one sixth that of S. mutans ((0.296 ± 0.058) mmol/L). Additional tests examining pyruvic acid production (the LDH substrate) demonstrated that lactic acid concentrations correlated with pyruvic acid production. That is, pyruvic acid production by S. oligofermentans was undetectable following sucrose incubation, however, (0.074 ± 0.024) and (0.175 ± 0.098) mmol/L pyruvic acid were produced by S. mutans and L. fermentum, respectively.

CONCLUSIONS. oligofermentans is incapable of fermenting carbohydrates to produce enough pyruvic acid, which results in reduced lactic acid production.

L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; metabolism ; Pyruvic Acid ; metabolism ; Streptococcus ; metabolism ; Sucrose ; metabolism

The capability of utilizing the intermediates of TCA-cycle as the sole carbon source by the multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019 under the conditions of vitamins limitation was demonstrated. Furthermore, the colony numbers grown on medium supplemented with glucose, acetate and one of the intermediates of TCA-cycle was higher than that of medium used glucose and acetate or medium used one of the intermediates of TCA-cycle carbon source. Among the intermediates of TCA-cycle used in this study, oxaloacetate was the best carbon source for the yeast and it was found that its presence stimulated the conversion of acetate to acetyl-CoA. In batch fermentation with glucose medium, the addition of 10 g/L of oxaloacetate improved the dry cell weight from 11.8 g/L to 13.6 g/L, and the productivity of pyruvate from 0.96 g x L(-1) x h(-1) to 1.19 g x L(-1) x h(-1), a 24% increase after 56 h growth. The yield of pyruvate on glucose was also improved as well, from 0.63 g/g to 0.66 g/g.
Candida glabrata ; growth & development ; metabolism ; Citric Acid Cycle ; Culture Media ; Fermentation ; Pyruvic Acid ; metabolism

Temperature plays an important role in pyruvate biosynthesis by Torulopsis glabrata 620. The effects of temperature on the substrate consumption, cell growth, pyruvate biosynthesis and level of energy charge and oxidation-reduction state have been investigated. During the constant temperature fermentation, higher temperature can enhance the rate of glucose consumption, cell growth and pyruvate production. However, it also leads to higher energy charge in the prophase of fermentation, too much consumption of glucose by cell and deficient throughput of pyruvate in the anaphase of fermentation, which brings on decrease of pyruvate yield. Oppositely, maintaining lower temperature during the fermentation can offer constant production capacity of pyruvate in the anaphase of fermentation. But the disadvantages are lower cell growth and higher level of NADH/NAD+ in vivo during the anaphase of fermentation, which leads to lower productivity inevitably. Disaccords above-mentioned indicate that the higher production, higher yield and higher productivity cannot be achieved at one time during a constant temperature fermentation.
Candida glabrata ; growth & development ; metabolism ; Energy Metabolism ; Fermentation ; Kinetics ; Oxidation-Reduction ; Pyruvic Acid ; metabolism ; Temperature

The hemodynamic and metabolic changes during induced hypotension with isoflurane (isoflurane group) or sodium nitroprusside (SNP group) were observed in twelve mongrel dogs. These hypotensive effects were evaluated at 30 and 60 minutes after the mean arterial blood pressure was lowered to 50% from the control. Hemodynamic changes were evaluated by measuring systemic arterial blood pressure, heart rate, central venous pressure, pulmonary capillary wedge pressure, cardiac output, systemic vascular resistance and pulmonary vascular resistance. Metabolic changes were evaluated by measuring serum lactate and pyruvate, arterio-venous oxygen content difference and oxygen extraction rate. We also compared the ventilatory effect of hypotensive anesthesia by blood gas analysis. The results were as follows: 1. Isoflurane inhalation 2-4% or SNP infusion 10-20 micrograms/kg/min was required to reduce the mean arterial pressure to 50% of the control. 2. Heart rate was decreased slightly in the isoflurane group but significantly decreased in the SNP group. 3. There were no significant changes in central venous pressure and pulmonary capillary wedge pressure in either group. 4. Cardiac output was reduced in both groups but was more severe in the isoflurane group. 5. Systemic vascular resistance was decreased by 36% in the isoflurane group and 47% in the SNP group. 6. Acidosis was apparent and did not recover to the control until 30 minutes after recovery in the SNP group. 7. Arterio-venous oxygen difference was increased during hypotension in the isoflurane group probably due to decreased cardiac output. 8. The lactate/pyruvate ratio increased slightly in the SNP group.
Anesthesia ; Animal ; Dogs ; *Hemodynamics ; *Hypotension, Controlled ; *Isoflurane ; Lactates/metabolism ; *Nitroprusside ; Pyruvates/metabolism ; Pyruvic Acid

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t_1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T¹⁰₅₀) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry* ; Molecular Docking Simulation ; Amines/chemistry* ; Pyruvic Acid/metabolism* ; Enzyme Stability

To understand the effects of sugar whose uptake is dependent or independent on the phosphotransferase system (PTS), two-stage culture of Escherichia coli strain NZN111 that was constructed by disruption of IdhA and pflB encoding the fermentative lactate dehydrogenase (LDH) and pyruvate: formate lyase (PFL) of E. coli W1485, was carried out for organic acids production. When NZN111 was aerobically cultured on fructose (PTS dependent) or maltose (PTS independent), it fermented glucose with succinic acid and pyruvic acid as the major products in subsequent anaerobic culture. The experiments were also performed in a 5-L fermentor. The yields of succinic acid by the fructose-and maltose-grown NZN111 were 0.84 and 0.75 mol/mol, whereas the yields of pyruvic acid were 0.65 and 0.83 mol/mol, respectively. The final ratio of succinic acid to pyruvic acid in the anaerobic stage reached 1.73:1 and 1.21:1, respectively. The different behaviors in anaerobic fermentation by the fructose-, maltose- and glucose-grown NZN111 were likely caused by the regulation of catabolite repression in the aerobic culture stage.
Aerobiosis ; Anaerobiosis ; Carbon ; metabolism ; Escherichia coli ; classification ; metabolism ; Fermentation ; Fructose ; metabolism ; Maltose ; metabolism ; Phosphotransferases ; metabolism ; Pyruvic Acid ; metabolism ; Succinic Acid ; metabolism

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. GBMs, like other tumors, rely relatively less on mitochondrial oxidative phosphorylation (OXPHOS) and utilize more aerobic glycolysis, and this metabolic shift becomes augmented under hypoxia. In the present study, we investigated the physiological significance of altered glucose metabolism and hypoxic adaptation in the GBM cell line U251 and two newly established primary GBMs (GBM28 and GBM37). We found that these three GBMs exhibited differential growth rates under hypoxia compared to those under normoxia. Under normoxia, the basal expressions of HIF1α and the glycolysis-associated genes, PDK1, PDK3, and GLUT1, were relatively low in U251 and GBM28, while their basal expressions were high in GBM37. Under hypoxia, the expressions of these genes were enhanced further in all three GBMs. Treatment with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), induced cell death in GBM28 and GBM37 maintained under normoxia, whereas DCA effects disappeared under hypoxia, suggesting that hypoxic adaptation dominated DCA effects in these GBMs. In contrast, the inhibition of HIF1α with chrysin suppressed the expression of PDK1, PDK3, and GLUT1 and markedly promoted cell death of all GBMs under both normoxia and hypoxia. Interestingly, however, GBMs treated with chrysin under hypoxia still sustained higher viability than those under normoxia, and chrysin and DCA co-treatment was unable to eliminate this hypoxia-dependent resistance. Together, these results suggest that hypoxic adaptation is critical for maintaining viability of GBMs, and targeting hypoxic adaptation can be an important treatment option for GBMs.
Anoxia ; Brain Neoplasms ; Cell Death* ; Cell Line ; Dichloroacetic Acid ; Glioblastoma* ; Glucose ; Glycolysis ; Metabolism ; Oxidative Phosphorylation ; Oxidoreductases ; Phosphotransferases ; Pyruvic Acid

PURPOSE: To develop a technique for quantifying the 13C-metabolites by performing frequency-selective hyperpolarized 13C magnetic resonance spectroscopy (MRS) in vitro which combines simple spectrally-selective excitation with spectrally interleaved acquisition. METHODS: Numerical simulations were performed with varying noise level and K(p) values to compare the quantification accuracies of the proposed and the conventional methods. For in vitro experiments, a spectrally-selective excitation scheme was enabled by narrow-band radiofrequency (RF) excitation pulse implemented into a free-induction decay chemical shift imaging (FIDCSI) sequence. Experiments with LDH / NADH enzyme mixture were performed to validate the effectiveness of the proposed acquisition method. Also, a modified two-site exchange model was formulated for metabolism kinetics quantification with the proposed method. RESULTS: From the simulation results, significant increase of the lactate peak signal to noise ratio (PSNR) was observed. Also, the quantified K(p) value from the dynamic curves were more accurate in the case of the proposed acquisition method compared to the conventional non-selective excitation scheme. In vitro experiment results were in good agreement with the simulation results, also displaying increased PSNR for lactate. Fitting results using the modified two-site exchange model also showed expected results in agreement with the simulations. CONCLUSION: A method for accurate quantification of hyperpolarized pyruvate and the downstream product focused on in vitro experiment was described. By using a narrow-band RF excitation pulse with alternating acquisition, different resonances were selectively excited with a different flip angle for increased PSNR while the hyperpolarized magnetization of the substrate can be minimally perturbed with a low flip angle. Baseline signals from neighboring resonances can be effectively suppressed to accurately quantify the metabolism kinetics.
Kinetics ; Lactic Acid ; Magnetic Resonance Imaging ; Magnetic Resonance Spectroscopy ; Metabolism* ; NAD ; Noise ; Pyruvic Acid* ; Signal-To-Noise Ratio ; Spectrum Analysis