Fructose-6-phosphate Research Articles

Thermodynamics and Kinetics of Glycolytic Reactions. Part I: Kinetic Modeling Based on Irreversible Thermodynamics and Validation by Calorimetry.

In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

Metabolic mechanism of the mud crab (Scylla paramamosain) adapting to salinity sudden drop based on GC-MS technology

Salinity impacts the respiratory metabolism, growth, and survival of marine crustaceans. Although the S. paramamosain is a euryhaline species, the sudden drop in salinity often causes individual death. The study aimed to reveal the adaptive mechanism of S. paramamosain in response to a sudden drop from 23‰ to 3‰ in salinity based on GC–MS data. We identified 1264 metabolites, and 437 were differentially expressed. Of them, 71 were up-regulated (FC > 1), including taurine, L-homoserine, aspartic acid, fructose 6-phosphate, glucose 6-phosphate, pyruvic acid, and lactic acid, and 74 were down-regulated (FC < 1), including glutamic acid, valine, glycine, fructose, tagatose, and ribose. KEGG enrichment analysis of differential metabolites identified 57 metabolic pathways, of which 29 were statistically significant (P < 0.05), including glycine, serine, and threonine metabolism, the pentose phosphate pathway, and ABC transporters. These metabolic pathways were mostly the amino acid metabolism pathway, carbohydrate metabolism pathway, metabolism of cofactors and vitamins, nucleotide metabolism, energy, metabolism, membrane, transport and translation. The results of this study show that free amino acids play an important role in adaptation to a sudden decrease in salinity and that energy metabolism involving carbohydrates and organic acids provides the energy supply during adaptation. The study provides important information about the osmoregulation of S. paramamosain and even other crustaceans.

Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked β-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages

Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C.d iff Infection.

SummaryClostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

Discovery of PF-06835919: A Potent Inhibitor of Ketohexokinase (KHK) for the Treatment of Metabolic Disorders Driven by the Overconsumption of Fructose.

Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.

Protein biomarkers and risk scores in pulmonary arterial hypertension associated with ventricular septal defect: integration of multi-omics and validation.

The molecular mechanisms underlying pulmonary arterial hypertension (PAH) in congenital ventricular septal defects (VSD) are unclear. We aimed to reveal molecular pathways and potential biomarkers by multi-omics analysis in VSD-PAH. Plasma from 160 children, including 120 VSD patients with/without PAH and 40 healthy children was studied by integrated proteomics, metabolomics, and bioinformatics analyses. Proteomics identified 107 differential proteins (DPs) between patients with/without PAH including significantly increased adiponectin (ADIPO), dopamine β-hydroxylase (DBH), alanyl membrane aminopeptidase (ANPEP), transferrin receptor 1, and glycoprotein Ib platelet α-subunit and decreased guanine nucleotide-binding protein Gs in VSD-PAH. Metabolomics discovered 191 differential metabolites between patients with/without PAH, including elevation of serotonin, taurine, creatine, sarcosine, and 2-oxobutanoate, and decrease of vanillylmandelic acid, 3,4-dihydroxymandelate, 15-keto-prostaglandin F2α, fructose 6-phosphate, l-glutamine, dehydroascorbate, hydroxypyruvate, threonine, l-cystine, and 1-aminocyclopropane-1-carboxylate. The DPs were validated in a new cohort of patients (n = 80). Integrated analyses identified key pathways, including cAMP, ECM receptor interaction, AMPK, hypoxia-inducible factor 1, PI3K-Akt signaling pathways, and amino acid metabolisms. Increased plasma protein levels of DBH, ADIPO, and ANPEP were found to be independently associated with the occurrence of PAH, with a new total risk score from these three proteins developed for clinical diagnosis. In this integrated multi-omics analysis in VSD-PAH patients, we have, for the first time, found that VSD-PAH patients present important differential proteins, metabolites, and key pathways. We have developed a total risk score (based on the plasma concentration of DBH, ANPEP, and ADIPO) as a predictor of development of PAH in CHD-VSD patients. Therefore, these proteins may be used as biomarkers, and the new total risk score has significant clinical implications in the diagnosis of PAH.

Environmental and Intestinal Phylum Firmicutes Bacteria Metabolize the Plant Sugar Sulfoquinovose via a 6-Deoxy-6-sulfofructose Transaldolase Pathway.

SummaryBacterial degradation of the sugar sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) produced by plants, algae, and cyanobacteria, is an important component of the biogeochemical carbon and sulfur cycles. Here, we reveal a third biochemical pathway for primary SQ degradation in an aerobic Bacillus aryabhattai strain. An isomerase converts SQ to 6-deoxy-6-sulfofructose (SF). A novel transaldolase enzyme cleaves the SF to 3-sulfolactaldehyde (SLA), while the non-sulfonated C3-(glycerone)-moiety is transferred to an acceptor molecule, glyceraldehyde phosphate (GAP), yielding fructose-6-phosphate (F6P). Intestinal anaerobic bacteria such as Enterococcus gilvus, Clostridium symbiosum, and Eubacterium rectale strains also express transaldolase pathway gene clusters during fermentative growth with SQ. The now three known biochemical strategies for SQ catabolism reflect adaptations to the aerobic or anaerobic lifestyle of the different bacteria. The occurrence of these pathways in intestinal (family) Enterobacteriaceae and (phylum) Firmicutes strains further highlights a potential importance of metabolism of green-diet SQ by gut microbial communities to, ultimately, hydrogen sulfide.

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study.

Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes.

SummaryInsulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.

Abstract 524: Phosphofructokinase Coordinates Anabolic Pathways in the Heart

Energy provision and biosynthesis are thought to be controlled by amphibolic enzymes such as phosphofructokinase (PFK). Nevertheless, it remains unclear how PFK influences collateral biosynthetic pathways in the heart. Here, we investigated the control exerted by PFK on anabolic pathways in the heart using in vivo deep network tracing. Wild-type (WT) mice and mice overexpressing kinase- or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgenes, termed respectively Glyco Lo or Glyco Hi mice, were fed a liquid diet containing 13 C 6 -glucose for 18 h. From freeze-clamped hearts, metabolite abundance and 13 C enrichment were then assessed by ion chromatography-mass spectrometry. Compared with Glyco Hi and WT hearts, Glyco Lo hearts had remarkably higher levels and 13 C enrichment of glucose 6-phosphate, fructose 6-phosphate (F6P) (p&lt;0.01 each, n =4/gp), and purine and pyrimidine biosynthetic pathway intermediates. In addition to elevated activity of nucleotide biosynthetic pathways, we also found in Glyco Lo hearts higher 13 C-hexose incorporation into N-acetyl-D-glucosamine 6-phosphate (p&lt;0.05); however, the levels of UDP-GlcNAc and its enrichment with 13 C were not different between the groups, which suggests that PFK activity influences the hexosamine biosynthetic pathway primarily by modulating F6P incorporation. Whereas Glyco Lo hearts showed lower 13 C fractional enrichment in triacylglycerols and hexosyl ceramide species, Glyco Hi hearts showed higher 13 C fractional enrichment in ceramide species. Interestingly, Glyco Lo hearts had 2-fold lower levels of alpha-ketoglutarate with concomitant increases in glutamate abundance. Energy charge values ranged from 0.69-0.81 with no differences observed between groups.These results suggest that PFK regulates lipid biosynthesis and collateral biosynthetic pathways of glucose metabolism in the heart. Understanding how these pathways contribute to cardiac remodeling could be important for developing metabolic therapies to improve cardiac health.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

SummaryButenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

Chronic heat stress affects the photosynthetic apparatus of Solanum lycopersicum L. cv Micro-Tom

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated crops in the world. Tomato is a plant model and the relationship between yield and biotic/abiotic stress has attracted increasing scientific interest. Tomato cultivation under sub-optimal conditions usually negatively impacts growth and development; in particular, heat stress affects several cellular and metabolic processes, such as respiration and photosynthesis. In this work, we studied the effects of chronic heat stress on various cytological and biochemical aspects using the Micro-Tom cultivar as a model. Photosynthetic efficiency decreased during heat stress while levels of post-photosynthetic sugars (sucrose, fructose, glucose and glucose 6-phosphate) oscillated during stress. Similarly, photosynthetic pigments (lutein, chlorophyll a, chlorophyll b and β-carotene) showed an oscillating downward trend with partial recovery during the stress-free phase. The energetic capacity of leaves (e.g. ATP and ADP) was altered, as well as the reactive oxygen species (ROS) profile; the latter increased during stress. Important effects were also found on the accumulation of Rubisco isoforms, which decreased in number. Heat stress also resulted in a decreased accumulation of lipids (oleic and linoleic acid). Photosynthetically alterations were accompanied by cytological changes in leaf structure, particularly in the number of lipid bodies and starch granules. Prolonged heat stress progressively compromised the photosynthetic efficiency of tomato leaves. The present study reports multi-approach information on metabolic and photosynthetic injuries and responses of tomato plants to chronic heat stress, highlighting the plant's ability to adapt to stress.

Metabolomics study of polysaccharide extracts from Polygonatum sibiricum in mice based on 1 H NMR technology.

Polygonatum sibiricum Liliaceae perennial herb, as a commonly used medicine and food homologous plant, has been widely used in clinical practice of Chinese medicine since ancient times, with a history of 2000 years. As the main active ingredient, P. sibiricum polysaccharides have important pharmacological effects in blood sugar reduction and antitumor, antioxidant and liver protection. Mouse models of P. sibiricum polysaccharides were used in combination with 1 H NMR to investigate the metabolic regulation mechanism in mouse tissue and blood. The metabolite maps of the control group and the drug group in the liver had significant changes. The main differential metabolites were glucose 6-phosphate, inositol, lactose, glutamylglycine, galactose, rhamnose, cis-aconitic acid and histidine, indicating that there was definite correlation between the metabolic detection based on 1 H NMR and the metabolic characteristics of P. sibiricum. The common differential metabolites obtained by overall metabolism analysis were 3-hydroxybutyric acid, d-ribose, adenosine phosphate, inositol, fructose 6-phosphate, histidine, aspartic acid and cis-aconitic acid. This work forms the basis for identification of metabolic states combined with metabolic pathways, which could be used as diagnostic and prognostic indicators, providing therapeutic targets for new diseases. © 2020 Society of Chemical Industry.

Developing a Cell-Free Extract Reaction (CFER) System in Clostridium thermocellum to Identify Metabolic Limitations to Ethanol Production

The cellulolytic bacterium Clostridium thermocellum is a promising candidate for lignocellulosic biofuel production, however ethanol titer needs to be improved for commercialization. To understand the factors limiting ethanol titer in C. thermocellum, we developed a cell-free extract reaction (CFER) system. We demonstrated that 15 mM cellobiose could be converted, in vitro, to 25 mM ethanol, and that this reaction functions both at thermophilic (55C) and mesophilic (37C) temperatures. Although the yield was similar to that produced by whole cells, the rate was much slower (~0.5 vs 12 mM/h). In order to reliably quantify metabolites, rapid CFER quenching is necessary. Among the methods tested, filtration with a 3 kDa molecular weight cutoff filter proved to be the most effective. Metabolomic analysis revealed high levels of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) in the CFER, identifying potential rate limiting enzymes downstream of F6P. NADH was also found to accumulate in the CFER, suggesting NADH recycling rate-limiting. We used two complementary strategies to identify enzymes that limit metabolic flux, including feeding different substrates and supplementing with exogenous enzymes. In the enzyme addition experiment, the largest improvement was observed with the addition of yeast alcohol dehydrogenase (ADH), indicating a limitation at that reaction. The development of a CFER system for C. thermocellum, combined with detailed measurements of intermediate metabolites, allowed us to directly observe the metabolism of this organism, and suggested several potential metabolic engineering interventions for increasing ethanol titer. This demonstrates a technique that may be of general use for metabolic engineering in non-model organisms.

Characteristics of a fructose 6-phosphate 4-epimerase from Caldilinea aerophila DSM 14535 and its application for biosynthesis of tagatose

Tagatose is a rare hexoketose with potential health benefits. Here, an enzyme, GatZ subunit ofd-tagatose-1,6-bisphosphate aldolase, was characterized. GatZ is involved in a multi-enzyme cascade reaction system that can produce tagatose from maltodextrin. It showed maximum activity at 70 °C and a pH 8.0, and required supplementation with 5 mM Mg2+ to achieve the highest catalytic activity. The Km and Vmax values of GatZ using fructose 6-phosphate as substrate were 5.66 mM and 0.0329 mmol/L min, respectively. An in vitro multi-enzyme system containing GatZ was constructed, and 1.75 g/L tagatose was produced from 5 g/L maltodextrin after 10 h. This biosystem could potentially enrich the application of C4 epimerases in rare sugar bioproduction.

Dehydration during storage affects carbohydrate metabolism and the accumulation of non-sucrose carbohydrates in postharvest sugarbeet roots

Sugarbeet roots produced in cold climates are stored in large piles prior to processing and are cooled by ambient winter air. These storage conditions dehydrate roots, causing root respiration rate and sucrose loss in storage to increase. Dehydration likely alters carbohydrate metabolism due to a greater demand for respiratory substrates and may affect the accumulation of non-sucrose carbohydrates that impede processing. However, effects of dehydration on carbohydrate metabolism of postharvest sugarbeet roots have not been studied. Research, therefore, was conducted to determine the effect of mild and severe dehydration on concentrations of the non-sucrose carbohydrates, glucose, fructose, and raffinose, their biosynthetic precursors, and the concentrations of metabolites that are intermediates in respiratory sucrose catabolism through glycolysis and the TCA cycle. Roots were stored at 85 and 40% relative humidity for up to 28 d to generate mildly and severely dehydrated roots typical of roots in the interior and exterior of storage piles. Severe dehydration was associated with reductions in raffinose, galactinol, glucose 6-phosphate, fructose 6-phosphate, phosphoenolpyruvate, and pyruvate concentrations, and increases in inositol and malate concentrations, while mild dehydration was associated with increased raffinose concentration. Concentrations of other glycolytic and TCA cycle intermediates, as well as the invert sugars, glucose and fructose, were largely unaffected by dehydration. From these results, it is concluded that raffinose accumulation and biosynthesis is highly sensitive to root dehydration and that carbohydrate metabolism through glycolysis is altered by dehydration, presumably to provide increased substrates for respiration. Overall, this research provides new insight into the impact of environmental conditions within storage piles on raffinose accumulation and the mechanisms by which dehydration accelerates sucrose loss in stored sugarbeet roots. • Raffinose accumulates in roots stored under mildly dehydrating conditions. • Raffinose content declines in roots stored under severely dehydrating conditions. • Severe dehydration alters flux through glycolysis to support elevated respiration. • Invert sugar concentration is not significantly altered by dehydration in storage.

Methanol production by reversed methylotrophy constructed in Escherichia coli.

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle

BackgroundThe Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus.ResultsThe Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts.ConclusionsWhile it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.