Search Results (16)

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, can rapidly degrade crystalline cellulose through direct cell-to-substrate contact. Most of its cellulases are secreted by the Type IX secretion system (T9SS) and anchored to the cell surface. Our previous study proved that the C-terminal domain (CTD) of the T9SS substrate cellulase Cel9A is glycosylated in C. hutchinsonii. However, its glycosylation mechanism has remained elusive. In this study, we found that chu_3394, which encodes UDP-glucose 6-dehydrogenase (Ugd), was important for the glycosylation of large amounts of periplasmic and outer membrane proteins in C. hutchinsonii. The contents of mannose, glucose, galactose, and xylose were detected to be reduced in the glycoproteins of the ∆ugd mutant compared to that of wild-type. They might be essential monosaccharides that contribute to the structure and function of glycans attached to proteins in C. hutchinsonii. The depletion of mannose, glucose, galactose, and xylose indicates a decrease in glycosylation modifications in the ∆ugd mutant strain. Then, we found that the deletion of ugd resulted in weakened glycosylation modification of the recombinant green fluorescent protein-tagged CTD of Cel9A. Additionally, the outer-membrane localization of Cel9A was affected in the mutant. Besides this, Ugd was also important for the synthesis of O-antigen of lipopolysaccharide (LPS). Thus, Ugd was involved in the synthesis of glycans in both glycoproteins and LPS in C. hutchinsonii. Moreover, the deletion of ugd affected the cellulose degradation, cell motility, and stress resistance of C. hutchinsonii. Full article

Background: Glioblastoma (GBM) uses Glut3 and/or Glut14 and the Leloir pathway to catabolize D-Galactose (Gal). UDP-4-deoxy-4-fluorogalactose (UDP-4DFG) is a potent inhibitor of the two key enzymes, UDP-galactose-4-epimerase (GALE) and UDP-Glucose 6-dehydrogenase (UGDH), involved in Gal metabolism and in glycan synthesis. The Gal antimetabolite 4-deoxy-4-fluorogalactose (4DFG) is a good substrate for Glut3/Glut14 and acts as a potent glioma chemotherapeutic. Methods: Primary GBM cell cultures were used to examine toxicity and alterations in glycan composition via lectin binding in fixed cells and by Western blots. Toxicity/efficacy in vivo data was performed in mouse flank and intracranial models. The effect of 4DFG on D-glucose (Glc) metabolism in GBM cells was assessed by using ¹³C NMR-based tracer studies. Results: 4DFG is moderately potent against GBM cells (IC₅₀: 125–300 µM). GBM glycosylation is disrupted by 4DFG. Survival analysis in an intracranial mouse model showed that treatment with 4DFG (6 × 25 mg/kg of 4DFG, intravenously) improved outcomes by three-fold (p < 0.01). Metabolic flux analysis revealed that both glycolytic and mitochondrial metabolic fluxes of [U-¹³C]Glc were significantly decreased in the presence of 4DFG in GBM cells. Conclusion: A functional Gal-scavenging pathway in GBM allows Gal-based antimetabolites to act as chemotherapeutics. 4DFG is metabolized by GBM in vitro and in vivo, is lethal to GBM tumors, and is well tolerated in mice. Full article

Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (Ips typographus). Despite these defences, the ESBB infests Norway spruce, causing significant ecological damage by exploiting weakened trees and using pheromones for aggregation. However, the mechanism of sensing and resistance towards host allelochemicals in ESBB is poorly understood. We hypothesised that the exposure of ESBB to spruce allelochemicals, especially monoterpenes, leads to an upsurge in the important detoxification genes like P450s, GSTs, UGTs, and transporters, and at the same time, genes responsible for development must be compromised. The current study demonstrates that exposure to monoterpenes like R-limonene and sabiene effectively elevated detoxification enzyme activities. The differential gene expression (DGE) analysis revealed 294 differentially expressed (DE) detoxification genes in response to R-limonene and 426 DE detoxification genes in response to sabiene treatments, with 209 common genes between the treatments. Amongst these, genes from the cytochrome P450 family 4 and 6 genes (CP4 and CP6), esterases, glutathione S-transferases family 1 (GSTT1), UDP-glucuronosyltransferase 2B genes (UDB), and glucose synthesis-related dehydrogenases were highly upregulated. We further validated 19 genes using RT-qPCR. Additionally, we observed similar high expression levels of detoxification genes across different monoterpene treatments, including myrcene and α-pinene, suggesting a conserved detoxification mechanism in ESBB, which demands further investigation. These findings highlight the potential for molecular target-based beetle management strategies targeting these key detoxification genes. Full article

Floridoside is a galactosyl–glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed Grateloupia imbricata. In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of G. imbricata and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of galactose-1-phosphate uridyltransferase (GALT) as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through α-galactosidase gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low GALT transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, α-galactosidase modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of G3PD, MGDGS, and DGDGS gene expression in G. imbricata treated with MEJA supports lipid remodeling, as high levels of transcripts for MGDGS and DGDGS provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages—namely, fertile, fertilized, and fertile—under 65 psu salinity conditions. Low transcripts for α-galactosidase and high for G3PD are reported in infertile and fertilized thalli, which is the opposite to high transcripts for α-galactosidase and low for G3PD encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for MGDGS and DGDGS. It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides. Full article

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG), a rare and innovative compound in licorice, exhibits high-potency sweetness and improved physiological activities. However, low amounts of GAMG from plants cannot meet the demands of growing markets. In this study, an efficient one-pot multienzyme cascade reaction for GAMG biosynthesis was constructed using a coupled catalysis of glycosyltransferase and uridine 5′-diphosphate (UDP) glucuronic acid (GlcA) regeneration system. The Glycyrrhiza uralensis glycosyltransferase UGT73F15 was expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of UGT73F15 were found to be pH 7.5 and 35 °C. The catalytic efficiency (k_cat/K_m) for glycyrrhetic acid (GA) was 2.14 min⁻¹ mM⁻¹ when using UDP-GlcA as sugar donor. To regenerate costly UDP-GlcA, the one-pot multienzyme cascade reaction including UGT73F15, sucrose synthase, UDP-glucose dehydrogenase, and lactate dehydrogenase was adopted to synthesize GAMG from GA on the basis of the UDP-GlcA regeneration system. By optimizing the cascade reaction conditions, the GAMG production successfully achieved 226.38 mg/L. Our study developed an economical and efficient one-pot multienzyme cascade method for facile synthesis of GAMG and other bioactive glucuronosides. Full article

Nucleotide sugar-dependent glycosyltransferases (UGTs) are critical to the homeostasis of endogenous metabolites and the detoxification of xenobiotics. Their impact on the cell metabolome remains unknown. Cellular metabolic changes resulting from human UGT expression were profiled by untargeted metabolomics. The abundant UGT1A1 and UGT2B7 were studied as UGT prototypes along with their alternative (alt.) splicing-derived isoforms displaying structural differences. Nineteen biochemical routes were modified, beyond known UGT substrates. Significant variations in glycolysis and pyrimidine pathways, and precursors of the co-substrate UDP-glucuronic acid were observed. Bioactive lipids such as arachidonic acid and endocannabinoids were highly enriched by up to 13.3-fold (p < 0.01) in cells expressing the canonical enzymes. Alt. UGT2B7 induced drastic and unique metabolic perturbations, including higher glucose (18-fold) levels and tricarboxylic acid cycle (TCA) cycle metabolites and abrogated the effects of the UGT2B7 canonical enzyme when co-expressed. UGT1A1 proteins promoted the accumulation of branched-chain amino acids (BCAA) and TCA metabolites upstream of the mitochondrial oxoglutarate dehydrogenase complex (OGDC). Alt. UGT1A1 exacerbated these changes, likely through its interaction with the OGDC component oxoglutarate dehydrogenase-like (OGDHL). This study expands the breadth of biochemical pathways associated with UGT expression and establishes extensive connectivity between UGT enzymes, alt. proteins and other metabolic processes. Full article

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells’ survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes. Full article

Sourdough is a fermentation culture which is formed following metabolic activities of a multiple bacterial and fungal species on raw dough. However, little is known about the mechanism of interaction among different species involved in fermentation. In this study, Lactiplantibacillus plantarum Sx3 and Saccharomyces cerevisiae Sq7 were selected. Protein changes in sourdough, fermented with single culture (either Sx3 or Sq7) and mixed culture (both Sx3 and Sq7), were evaluated by proteomics. The results show that carbohydrate metabolism in mixed-culture-based sourdough is the most important metabolic pathway. A greater abundance of L-lactate dehydrogenase and UDP-glucose 4-epimerase that contribute to the quality of sourdough were observed in mixed-culture-based sourdough than those produced by a single culture. Calreticulin, enolase, seryl-tRNA synthetase, ribosomal protein L23, ribosomal protein L16, and ribosomal protein L5 that are needed for the stability of proteins were increased in mixed-culture-based sourdough. The abundance of some compounds which play an important role in enhancing the nutritional characteristics and flavour of sourdough (citrate synthase, aldehyde dehydrogenase, pyruvate decarboxylase, pyruvate dehydrogenase E1 and acetyl-CoA) was decreased. In summary, this approach provided new insights into the interaction between L. plantarum and S. cerevisiae in sourdough, which may serve as a base for further research into the detailed mechanism. Full article

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications. Full article

The bioenergetics of the vast majority of terrestrial mammals evolved to consuming glucose (Glc) for energy production under regular atmosphere (about 21% oxygen). However, some vertebrate species, such as aquatic turtles, seals, naked mole rat, and blind mole rat, Spalax, have adjusted their homeostasis to continuous function under severe hypoxic environment. The exploration of hypoxia-tolerant species metabolic strategies provides a better understanding of the adaptation to hypoxia. In this study, we compared Glc homeostasis in primary Spalax and rat skin cells under normoxic and hypoxic conditions. We used the targeted-metabolomics approach, utilizing liquid chromatography and mass spectrometry (LC-MS) to track the fate of heavy Glc carbons (¹³C₆ Glc), as well as other methodologies to assist the interpretation of the metabolic landscape, such as bioenergetics profiling, Western blotting, and gene expression analysis. The metabolic profile was recorded under steady-state (after 24 h) of the experiment. Glc-originated carbons were unequally distributed between the cytosolic and mitochondrial domains in Spalax cells compared to the rat. The cytosolic domain is dominant apparently due to the hypoxia-inducible factor-1 alpha (HIF-1α) mastering, since its level is higher under normoxia and hypoxia in Spalax cells. Consumed Glc in Spalax cells is utilized for the pentose phosphate pathway maintaining the NADPH pool, and is finally harbored as glutathione (GSH) and UDP-GlcNAc. The cytosolic domain in Spalax cells works in the semi-uncoupled mode that limits the consumed Glc-derived carbons flux to the tricarboxylic acid (TCA) cycle and reduces pyruvate delivery; however, it maintains the NAD⁺ pool via lactate dehydrogenase upregulation. Both normoxic and hypoxic mitochondrial homeostasis of Glc-originated carbons in Spalax are characterized by their massive cataplerotic flux along with the axis αKG→Glu→Pro→hydroxyproline (HPro). The product of collagen degradation, HPro, as well as free Pro are apparently involved in the bioenergetics of Spalax under both normoxia and hypoxia. The upregulation of 2-hydroxyglutarate production detected in Spalax cells may be involved in modulating the levels of HIF-1α. Collectively, these data suggest that Spalax cells utilize similar metabolic frame for both normoxia and hypoxia, where glucose metabolism is switched from oxidative pathways (conversion of pyruvate to Acetyl-CoA and further TCA cycle processes) to (i) pentose phosphate pathway, (ii) lactate production, and (iii) cataplerotic pathways leading to hexosamine, GSH, and HPro production. Full article

UDP-glucose-dehydrogenase (UGDH) synthesizes UDP-glucuronic acid. It is involved in epirubicin detoxification and hyaluronan synthesis. This work aimed to evaluate the effect of UGDH knockdown on epirubicin response and hyaluronan metabolism in MDA-MB-231 breast cancer cells. Additionally, the aim was to determine UGDH as a possible prognosis marker in breast cancer. We studied UGDH expression in tumors and adjacent tissue from breast cancer patients. The prognostic value of UGDH was studied using a public Kaplan–Meier plotter. MDA-MB-231 cells were knocked-down for UGDH and treated with epirubicin. Epirubicin-accumulation and apoptosis were analyzed by flow cytometry. Hyaluronan-coated matrix and metabolism were determined. Autophagic-LC3-II was studied by Western blot and confocal microscopy. Epirubicin accumulation increased and apoptosis decreased during UGDH knockdown. Hyaluronan-coated matrix increased and a positive modulation of autophagy was detected. Higher levels of UGDH were correlated with worse prognosis in triple-negative breast cancer patients that received chemotherapy. High expression of UGDH was found in tumoral tissue from HER2^--patients. However, UGDH knockdown contributes to epirubicin resistance, which might be associated with increases in the expression, deposition and catabolism of hyaluronan. The results obtained allowed us to propose UGDH as a new prognostic marker in breast cancer, positively associated with development of epirubicin resistance and modulation of extracellular matrix. Full article

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by β-1,4 and β-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors’ ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production. Full article

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae. Full article

Hyaluronic Acid (HA) is a biopolymer composed by the monomers Glucuronic Acid (GlcUA) and N-Acetyl Glucosamine (GlcNAc). It has a broad range of applications in the field of medicine, being marketed between USD 1000–5000/kg. Its primary sources include extraction of animal tissue and fermentation using pathogenic bacteria. However, in both cases, extensive purification protocols are required to prevent toxin contamination. In this study, aiming at creating a safe HA producing microorganism, the generally regarded as safe (GRAS) yeast Kluyveroymyces lactis is utilized. Initially, the hasB (UDP-Glucose dehydrogenase) gene from Xenopus laevis (xlhasB) is inserted. After that, four strains are constructed harboring different hasA (HA Synthase) genes, three of humans (hshasA1, hshasA2, and hshasA3) and one with the bacteria Pasteurella multocida (pmhasA). Transcript values analysis confirms the presence of hasA genes only in three strains. HA production is verified by scanning electron microscopy in the strain containing the pmHAS isoform. The pmHAS strain is grown in a 1.3 l bioreactor operating in a batch mode, the maximum HA levels are 1.89 g/L with a molecular weight of 2.097 MDa. This is the first study that reports HA production in K. lactis and it has the highest HA titers reported among yeast. Full article

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions. Full article