Salicylate 2-O-β-d-glucoside (SAG) is a derivative of salicylate in plants. Recent reports showed that SAG could be considered as a potential anti-inflammatory substance due to its anti-inflammatory and analgesic effects, and less irritation compared with salicylic acid and aspirin. The biological method uses renewable resources to produce salicylic acid compounds, which is more environmentally friendly than traditional industry methods. In this study, Escherichia coli Tyr002 was used as the starting strain, and a salicylic acid producing strain of E. coli was constructed by introducing the isochorismate pyruvate lyase gene pchB from Pseudomonas aeruginosa. By regulating the expression of the key genes in the downstream aromatic amino acid metabolic pathways, the titer of salicylic acid reached 1.05 g/L in shake flask fermentation. Subsequently, an exogenous salicylic acid glycosyltransferase was introduced into the salicylic acid producing strain to glycosylate the salicylic acid. The newly engineered strain produced 5.7 g/L SAG in shake flask fermentation. In the subsequent batch fed fermentation in a 5 L fermentation tank, the titer of SAG reached 36.5 g/L, which is the highest titer reported to date. This work provides a new route for biosynthesis of salicylate and its derivatives.
Escherichia coli/genetics* ; Glucosides ; Metabolic Engineering ; Salicylic Acid ; Pyruvic Acid

OBJECTIVE: The purpose of this study was to examine the effects on in vitro development of early preimplantation mouse embryos in DMEM medium with glutamine which was controlled by different composition of glucose and pyruvate. METHODS: Four hundred and nineteen mouse 2-cell embryos were cultured in four different media with different composition of glucose and pyruvate for 96 hours. The DMEM-G contained L-glutamine for energy sources was used for control group. Group I embryos were cultured in the medium that mixed one volume of DMEM-GGP contained L-glutamine, D-glucose and sodium pyruvate for energy sources with three volume of DMEM-G, and group II embryos were cultured in the medium that mixed with same volume of DMEM-G and DMEM-GGP, and group III embryos were cultured in DMEM-GGP. RESULTS: At 24 hours, the development into >or=3-cell was significantly higher (p<0.05) in group I (93.3%) than control (84.6%). The development into >or=8-cell was significantly higher in group I (73.1%) than control (44.2%), group II (59.6%) and III (45.8%), and also group II was significantly higher than control and group III. At 48 hours, the development into >or=morula was significantly higher in group I (90.4%) and II (86.5%) than control (73.0%). However, the development into blastocyst, in group III (15.0%) was significantly lower than control, group I and II. At 72 hours, the development into >or=expanded blastocyst was significantly higher in group I (69.2%) than group III (47.7%), and total blastocyst was significantly higher in group I (80.8%) than control (66.3%) and group III (67.3%). At 96 hours, the development into >or=hatching blastocyst was significantly higher in group I (78.8%) than control (61.5%) and group III (57.9%), and also, total blastocyst was significantly higher in group I (85.6%) than control (69.2%) and group III (72.0%). CONCLUSION: The development of early preimplantation mouse embryos cultured in group I medium that mixed one volume of DMEM-GGP with three volume of DMEM-G was better than other groups during the culture period.
Animals ; Blastocyst* ; Embryonic Structures ; Glucose* ; Glutamine* ; Mice* ; Pyruvic Acid* ; Sodium

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

PURPOSE: To investigate the exchange and redistribution of hyperpolarized ¹³C metabolites between different pools by temporally analyzing the relative fraction of dual T₂* components of hyperpolarized ¹³C metabolites. MATERIALS AND METHODS: A dual exponential decay analysis of T₂* is performed for [1-¹³C] pyruvate and [1-¹³C] lactate using nonspatially resolved dynamic ¹³C MR spectroscopy from mice brains with tumors (n = 3) and without (n = 4) tumors. The values of shorter and longer T₂* components are explored when fitted from averaged spectrum and temporal variations of their fractions. RESULTS: The T₂* values were not significantly different between the tumor and control groups, but the fraction of longer T₂* [1-¹³C] lactate components was more than 10% in the tumor group over that of the controls (P < 0.1). The fraction of shorter T₂* components of [1-¹³C] pyruvate showed an increasing tendency while that of the [1-¹³C] lactate was decreasing over time. The slopes of the changing fraction were steeper for the tumor group than the controls, especially for lactate (P < 0.01). In both pyruvate and lactate, the fraction of the shorter T₂* component was always greater than the longer T₂* component over time. CONCLUSIONS: The exchange and redistribution of pyruvate and lactate between different pools was investigated by dual component analysis of the free induction decay signal from hyperpolarized ¹³C experiments. Tumor and control groups showed differences in their fractions rather than the values of longer and shorter T₂* components. Fraction changing dynamics may provide an aspect for extravasation and membrane transport of pyruvate and lactate, and will be useful to determine the appropriate time window for acquisition of hyperpolarized ¹³C images.
Animals ; Brain ; Lactic Acid ; Magnetic Resonance Spectroscopy ; Membranes ; Mice ; Pyruvic Acid

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

Metabolic aberrations in the form of altered flux through key metabolic pathways are the major hallmarks of several life-threatening malignancies including malignant gliomas. These adaptations play an important role in the enhancement of the survival and proliferation of gliomas at the expense of the surrounding normal/healthy tissues. Recent studies in the field of neurooncology have directly targeted the altered metabolic pathways of malignant tumor cells for the development of anti-cancer drugs. Aerobic glycolysis due to elevated production of lactate from pyruvate regardless of oxygen availability is a common metabolic alteration in most malignancies. Aerobic glycolysis offers survival advantages in addition to generating substrates such as fatty acids, amino acids and nucleotides required for the rapid proliferation of cells. This review outlines the role of pyruvate dehydrogenase kinase (PDK) in gliomas as an inhibitor of pyruvate dehydrogenase that catalyzes the oxidative decarboxylation of pyruvate. An in-depth investigation on the key metabolic enzyme PDK may provide a novel therapeutic approach for the treatment of malignant gliomas.
Amino Acids ; Decarboxylation ; Dichloroacetic Acid ; Fatty Acids ; Glioma* ; Glycolysis ; Lactic Acid ; Metabolic Networks and Pathways ; Nucleotides ; Oxidoreductases* ; Oxygen ; Phosphotransferases* ; Pyruvic Acid*

High extracellular glucose concentration was reported to suppress intracellular Ca2+ clearing through altered sarcoplasmic reticulum (SR) function. In the present study, we attempted to elucidate the effects of pyruvate and fatty acid on SR function and reveal the mechanistic link with glucose-induced SR dysfunction. For this purpose, SR Ca2+-uptake rate was measured in digitonin-permeabilized H9c2 cardiomyocytes cultured in various conditions. Exposure of these cells to 5 mM pyruvate for 2 days induced a significant suppression of SR Ca2+-uptake, which was comparable to the effects of high glucose. These effects were accompanied with decreased glucose utilization. However, pyruvate could not further suppress SR Ca2+-uptake in cells cultured in high glucose condition. Enhanced entry of pyruvate into mitochondria by dichloroacetate, an activator of pyruvate dehydrogenase complex, also induced suppression of SR Ca2+-uptake, indicating that mitochondrial uptake of pyruvate is required in the SR dysfunction induced by pyruvate or glucose. On the other hand, augmentation of fatty acid supply by adding 0.2 to 0.8 mM oleic acid resulted in a dose-dependent suppression of SR Ca2+-uptake. However, these effects were attenuated in high glucose-cultured cells, with no significant changes by oleic acid concentrations lower than 0.4 mM. These results demonstrate that (1) increased pyruvate oxidation is the key mechanism in the SR dysfunction observed in high glucose-cultured cardiomyocytes; (2) exogenous fatty acid also suppresses SR Ca2+-uptake, presumably through a mechanism shared by glucose.
Diabetic Cardiomyopathies* ; Dichloroacetic Acid ; Glucose ; Hand ; Mitochondria ; Myocytes, Cardiac* ; Oleic Acid ; Pyruvate Dehydrogenase Complex ; Pyruvic Acid* ; Sarcoplasmic Reticulum*

OBJECTIVE: The objective of this study was to examine the effect on development of mouse preimplantation embryos in culture media with different composition of energy sources in vitro culture. METHODS: Two hundred and seventy one two-cell embryos were cultured in four different culture system for 96 hours. Group I (n=61) was cultured in DMEM-G (DMEM with glutamine) only, groupII (n=64) was cultured in DMEM-GGP (DMEM with glutamine, glucose and pyruvate) only, group III (n=72) was cultured for 48 hours in DMEM-G and then transferred to DMEM-GGP and group IV (n=74) was cultured for 48 hours in DMEM-GGP and then transferred to DMEM-G. Development of embryos in each group was observed every 24 hours. RESULTS: After 24 hours, the rate of development > or = 3-cell was significantly higher in groupII (87.5%) and IV (86.5%) compared with group I (59.0%) and III (62.5%). After 48 hours, the rate of development into > or = morula stage was significantly higher in GroupII (79.7%) and IV (86.5%) compared with group I (34.4%) and III (37.5%). After 72 hours, the rate of development into blastocyst was significantly higher in group IV (74.3%) compared with group I (49.2%) and III (45.8%). After 96 hours, the rate of development into > or = expanded blastocyst was significantly higher in group IV (70.3%) compared with group I (32.8%),II (53.1%), and group III (40.3%). CONCLUSION: Mouse preimplantation embryos development was the most effective in culture system with DMEM-GGP for 48 hours and then transferred to DMEM-G.
Animals ; Blastocyst* ; Culture Media* ; Embryonic Structures ; Glucose ; Glutamine ; Mice* ; Morula ; Pyruvic Acid