ATP Regeneration Research Articles

Differential Regulation of ATP- and UTP-Evoked Prostaglandin E2 and IL-6 Production from Human Airway Epithelial Cells.

The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.

Enhancing l-glutamine production in Corynebacterium glutamicum by rational metabolic engineering combined with a two-stage pH control strategy

l-glutamine is a semi-essential amino acid widely used in the food and pharmaceutical industries. The microbial synthesis of l-glutamine is limited by lack of effective strains with high titer and safety. First, ARTP mutagenesis combined with high-throughput screening generated an l-glutamine-producing strain of Corynebacterium glutamicum with titer of 25.7 ± 2.7 g/L. Subsequently, a series of rational metabolic approaches were used to further improve l-glutamine production, which included increasing the carbon flow to l-glutamine (proB and NCgl1221 knockout), enhancing the catalytic efficiency of the key enzyme (glnE knockout and glnA screening and overexpression) and reinforcement of ATP regeneration (ppk overexpression and RBS optimization). Finally, we proposed a two-stage pH control strategy to address the inconsistent effect of pH on cell growth and l-glutamine production. These combined strategies led to a 186.0% increase of l-glutamine titer compared to that of the initial strain, reaching 73.5 ± 3.1 g/L with a yield of 0.368 ± 0.034 g/g glucose.

RESEARCH ON ATHLETE’S AEROBIC AND ANAEROBIC EXERCISE ABILITY WITH BALANCED NUTRITION

ABSTRACT Introduction: The need for a lactic acid cycle eliminates lactic acid produced during exercise. This process requires energy consumption. D-ribose supplementation can increase muscle cell energy, accelerate the synthesis of PRPP in the heart and skeletal muscle, and eliminate the pentose phosphate pathway in the low limit of glucose-6-phosphate dehydrogenase activity; it doubles the speed of ATP recovery, so supplementing ribose can improve exercise capacity and accelerate the elimination of lactic acid to improve recovery ability. Objective: Supplementing D-ribose can increase muscle cell energy and accelerate the regeneration of ATP in the myocardium and skeletal muscle. This experiment intends to explore the effects of anaerobic and aerobic exercise and anaerobic exercise capacity and recovery ability after supplementing D-ribose granules by observing the changes in exercise tests before and after nutritional supplementation and recovery indicators after exercise. Methods: The thesis used a paired design to randomly divide 24 male amateur tennis players into two groups (12 in each group): physical training group (control group), physical training + nutrition D-ribose group (test group), and the D- The effect of ribose on the aerobic and anaerobic exercise capacity of amateur tennis players. Results: The observation indexes of the two groups before the test were not statistically significant (P>0.05); after the test for eight weeks, the aerobic capacity indexes of the test group were higher than those of the control group (P<0.05), and also higher than those before the test (P<0.05)); The recovery of 3minHR and 5minHR of the experimental group after exercise was significantly faster than that of the control group (P<0.05). Conclusions: Nutritional D-ribose supplementation can enhance the aerobic training effect of amateur tennis players, improve aerobic and anaerobic exercise capacity, and accelerate heart rate recovery after exercise. Level of evidence II; Therapeutic studies - investigation of treatment results.

Methionine biosynthesis pathway genes affect curdlan biosynthesis of Agrobacterium sp. CGMCC 11546 via energy regeneration

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Characterization of polyphosphate kinases for the synthesis of GSH with ATP regeneration from AMP

Polyphosphate kinase (PPK) is important for industrial processes involving ATP regeneration. While a variety of methods have been reported for regenerating ATP from ADP, few have explored enzyme catalyzed ATP regeneration from cheaper and stable AMP. In this work, PPKs from different sources were expressed and their catalytic activity were tested at different reaction temperatures, reaction pH and with different polyphosphate (polyPn) types. The ATP regeneration system for glutathione (GSH) synthesis was established using a single PPK capable of phosphorylating AMP to synthesize ATP from AMP and short chain polyPn. GSH yield was obtained using adenosine mono-, di- and triphosphates, which confirmed the flexibility of our constructed ATP regeneration system coupled with GSH synthesis via bifunctional GSH synthase. Finally, optimization of the GSH synthesis yielded conversion value above 80 %. Overall, these results illustrate that PPK is suitable for a broader range of substrates than previously expected, and has great untapped potential for applications involving ATP regeneration.

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis.

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

Comparative 13 C-metabolic flux analysis indicates elevation of ATP regeneration, carbon dioxide, and heat production in industrial Saccharomyces cerevisiae strains.

Various industrial Saccharomyces cerevisiae strains are used for specific processes, such as sake, wine brewing and bread making. Understanding mechanisms underlying the fermentation performance of these strains would be useful for further engineering of the S. cerevisiae metabolism. However, the relationship between the fermentation performance, intra-cellular metabolic states, and other phenotypic characteristics of industrial yeasts is still unclear. In this study, 13 C-metabolic flux analysis of four diploid yeast strains-laboratory, sake, bread, and wine yeasts-was conducted. While the Crabtree effect was observed for all strains, the metabolic flux level of glycolysis was elevated in bread and sake yeast. Furthermore, increased flux levels of the TCA cycle were commonly observed in the three industrial strains. The specific rates of CO2 production, net ATP regeneration, and metabolic heat generation estimated from the metabolic flux distribution were two to three times greater than those of the laboratory strain. The elevation in metabolic heat generation was correlated with the tolerance to low-temperature stress. These results indicate that the metabolic flux distribution of sake and bread yeast strains contributes to faster production of ethanol and CO2 . It is also suggested that the generation of metabolic heat is preferable under the actual industrial fermentation conditions.

Mitochondrial Ca2+ Signaling in Health, Disease and Therapy.

The divalent cation calcium (Ca2+) is considered one of the main second messengers inside cells and acts as the most prominent signal in a plethora of biological processes. Its homeostasis is guaranteed by an intricate and complex system of channels, pumps, and exchangers. In this context, by regulating cellular Ca2+ levels, mitochondria control both the uptake and release of Ca2+. Therefore, at the mitochondrial level, Ca2+ plays a dual role, participating in both vital physiological processes (ATP production and regulation of mitochondrial metabolism) and pathophysiological processes (cell death, cancer progression and metastasis). Hence, it is not surprising that alterations in mitochondrial Ca2+ (mCa2+) pathways or mutations in Ca2+ transporters affect the activities and functions of the entire cell. Indeed, it is widely recognized that dysregulation of mCa2+ signaling leads to various pathological scenarios, including cancer, neurological defects and cardiovascular diseases (CVDs). This review summarizes the current knowledge on the regulation of mCa2+ homeostasis, the related mechanisms and the significance of this regulation in physiology and human diseases. We also highlight strategies aimed at remedying mCa2+ dysregulation as promising therapeutical approaches.

Design and optimizing a new CDP-choline in vitro multienzyme producing process starts from d-ribose.

This work designs an in vitro multienzyme system to produce CDP-choline from d-ribose and develop an optimization procedure for one-pot multienzyme catalytic system. The entire process integrated 10 enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then, some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline from 0.2 to 6g/L (CDP-choline titer 12.2mM). This new one-pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one-pot multienzyme system for industrial production of energy rich compounds.

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in vitro Applications.

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over‐reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post‐translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110‐fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 μM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 μM of NADPH and 86.06 % at 10.62 μM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8O30} Determined by Single‐Cell Proteomics Analysis

AbstractThe 10‐nuclear heteroatom cluster modified {SbW8O30} was successfully synthesized and exhibited inhibitory activity (IC50=0.29 μM). Based on proteomics analysis, Na4Ni2Sb2W2‐SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN‐binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4Ni2Sb2W2‐SbW8, which was consistent with the effect of polyoxometalates (POMs) and FMN‐binding proteins on ATP formation. An electrochemical study showed that Na4Ni2Sb2W2‐SbW8 combined with FMN to form Na4Ni2Sb2W2‐SbW8‐2FMN complex through a one‐electron process of the W atoms. Na4Ni2Sb2W2‐SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 μM of NADPH and 86.06 % at 10.62 μM of 2‐hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration

SummaryHuman milk oligosaccharides (HMOs), the third most abundant solid component of human milk, are reported to be beneficial to infant health. The biosynthesis of lacto-N-biose (LNB), the building block for HMOs, suffers from excessive addition of cofactors and intermediate inhibition. Here, we developed an in vitro multienzyme cascade composed of LNB module, ATP regeneration, and pyruvate oxidase-driven phosphate recycling to produce LNB. The integration between ATP regeneration and Pi alleviation increased the LNB conversion ratio and resulted in a ΔG'° decrease of 540 KJ/mol. Under optimal conditions, the LNB conversion ratio was improved from 0.34 to 0.83 mol/mol GlcNAc and the ATP addition decreased to 50%. Finally, 0.96 mol/mol GlcNAc and 71.6 mg LNB g−1 GlcNAc h−1 of LNB yield was achieved in a 100-mL reaction system. The synergistic strategy not only paves the way for producing LNB but also facilitates other chemicals with multienzyme cascades.

Overview of Inositol and Inositol Phosphates on Chemoprevention of Colitis-Induced Carcinogenesis.

Chronic inflammation is one of the most common and well-recognized risk factors for human cancer, including colon cancer. Inflammatory bowel disease (IBD) is defined as a longstanding idiopathic chronic active inflammatory process in the colon, including ulcerative colitis and Crohn’s disease. Importantly, patients with IBD have a significantly increased risk for the development of colorectal carcinoma. Dietary inositol and its phosphates, as well as phospholipid derivatives, are well known to benefit human health in diverse pathologies including cancer prevention. Inositol phosphates including InsP3, InsP6, and other pyrophosphates, play important roles in cellular metabolic and signal transduction pathways involved in the control of cell proliferation, differentiation, RNA export, DNA repair, energy transduction, ATP regeneration, and numerous others. In the review, we highlight the biologic function and health effects of inositol and its phosphates including the nature and sources of these molecules, potential nutritional deficiencies, their biologic metabolism and function, and finally, their role in the prevention of colitis-induced carcinogenesis.

Professor Daniel I.C. Wang: A Legacy of Education, Innovation, Publication, and Leadership.

Professor Daniel I.C. Wang: A Legacy of Education, Innovation, Publication, and Leadership.

Differential Gene Expression Analysis of Aspergillus terreus Reveals Metabolic Response and Transcription Suppression under Dissolved Oxygen and pH Stress

Aspergillus terreus, an acid tolerant fungus that grows over a wide range of pH from 2 to 8, was employed in this study. Submerged fermentation was conducted under low dissolved oxygen (10% DO) and pH (pH 2). The fermentation kinetics was investigated together with transcriptome to identify key genes associated with metabolic responses to such environmental stresses by high-throughput RNA-seq technology. It was clear that under low dissolved oxygen, glycolysis was uncoupled from oxidative phosphorylation due to limited ATP regeneration, as observed from the downregulation of genes in the mitochondrial respiratory chain complex and slow growth. On the contrary, the transcriptional levels of genes encoding ATPases and cation transporters remained unchanged at –1 ≤ log2 fold change ≤ 1, revealing that A. terreus possessed an excellent defense mechanism to low pH. In addition, genes associated with cell wall synthesis and repair, including chitin synthase and 1,3-beta-glucan synthase genes, and genes in the cluster for cell wall protection and repair were upregulated. This evidence clearly demonstrated that A. terreus was able to survive under the acidic environment. In summary, we comprehensively analyzed and discussed the transcriptome of A. terreus. The findings obtained here provide a theoretical basis for better understaning the molecular mechanism of the fungal response to the environmental factors, providing thereby an insight into the process of designing and optimizing the production of biological products of interest. This approach can be applicable to other metabolic pathways of interest in particular microorganisms.

Metabolic Features of Mouse and Human Retinas: Rods versus Cones, Macula versus Periphery, Retina versus RPE.

SummaryPhotoreceptors, especially cones, which are enriched in the human macula, have high energy demands, making them vulnerable to metabolic stress. Metabolic dysfunction of photoreceptors and their supporting retinal pigment epithelium (RPE) is an important underlying cause of degenerative retinal diseases. However, how cones and the macula support their exorbitant metabolic demand and communicate with RPE is unclear. By profiling metabolite uptake and release and analyzing metabolic genes, we have found cone-rich retinas and human macula share specific metabolic features with upregulated pathways in pyruvate metabolism, mitochondrial TCA cycle, and lipid synthesis. Human neural retina and RPE have distinct but complementary metabolic features. Retinal metabolism centers on NADH production and neurotransmitter biosynthesis. The retina needs aspartate to sustain its aerobic glycolysis and mitochondrial metabolism. RPE metabolism is directed toward NADPH production and biosynthesis of acetyl-rich metabolites, serine, and others. RPE consumes multiple nutrients, including proline, to produce metabolites for the retina.

Electrocatalytic CO2 fixation by regenerating reduced cofactor NADH during Calvin Cycle using glassy carbon electrode

In this study, an enzymatic pathway has been developed to replicate the Calvin Cycle by creating the individual steps of the carbon cycle in a bioreactor. The technology known as “artificial photosynthesis” converts CO2 emissions into a variety of intermediates that serve as precursors to high-value products. CO2, light, water, and electricity were used as feedstock. An electrochemical reactor was also studied for the regeneration of active NADH operating at constant electrode potential. Initially, a batch electrochemical reactor containing 80 mL of 0.2 mM NAD+ in Tris-buffer (pH 7.40) was used to evaluate the electrode material operating at normal temperature and pressure. The results showed that the cathode is highly electrocatalytically efficient and selective to regenerate 97.45±0.8% of NADH from NAD+ at electrode potential of -2.3 V vs. mercury standard electrode (MSE). The NADH regeneration system was then integrated with ATP regeneration system and bioreactor containing Ribulose bisphosphate carboxylase/oxygenase (RuBisCO). NADH was regenerated successfully during the process electrochemically and then was used by the enzymatic reaction to produce triose phosphate and 3-Phosphoglycerate (3GPA).

ATP regulation strategy and its application in the synthesis of microbial metabolites

Cofactor engineering, as a new branch of metabolic engineering, mainly involves ATP/ADP, NADH/NAD⁺, NADPH/NADP⁺ and other cofactors. Cofactor engineering can maximize metabolic flow by directly regulating the concentration and form of the cofactor of key enzymes in cells, and quickly direct carbon flow to target metabolites. ATP, as an important cofactor, is involved in many enzyme-catalyzed reactions in microbial cells, and leads to the restriction of the distribution of metabolic pathways by connecting or linking them into a complex network. Therefore, ATP regulation strategy is expected to be a favorable tool for industrial strain modification, to improve the concentration and production capacity of target metabolites, strengthen microbial tolerance to the environment and promote substrate utilization rate. The present review focuses on the recently used effective ATP regulation strategies and the effects of ATP regulation on cell metabolism in order to provide references for the efficient construction of microbial cell factories.

ATP dynamic regeneration strategy for enhancing co-production of glutathione and S-adenosylmethionine in Escherichia coli.

In general, a sufficient supply of ATP can promote the synthesis of ATP-driven metabolites, but excessive ATP will lead to the inhibition of cell growth. For enhancing the co-production of glutathione(GSH) and S-adenosylmethionine(SAM), a dynamic ATP regeneration strategy was developed. The novel ATP regeneration strategy consisting of ATP-sensing riboswitch ydaO motif, polyphosphate kinase (PPK), and Vitreoscilla hemoglobin (VHb) was successfully applied in Escherichia coli. The intracellular ATP level was always around 0.60mg/g dry cell weight during the fermentation process, resulting in significantly enhanced co-production of GSH and SAM. The GSH titer and SAM titer in the strain CGS-2 increased by 137.40% and 82.18% after fermentation for 24h, compared with the control strain. The ATP regulation strategy is expected to be a favorable tool to improve the efficiency of microbial cell factories. The proposed ATP dynamic regeneration approach may be applicable for cost-effective, high-yield production of ATP-driven metabolites.