Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

UDP-glucose pyrophosphorylase as a target for regulating carbon flux distribution and antioxidant capacity in Phaeodactylum tricornutum.

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme for polysaccharide synthesis, and its role in plants and bacteria is well established; however, its functions in unicellular microalgae remain ill-defined. Here, we perform bioinformatics, subcellular localization as well as in vitro and in vivo analyses to elucidate the functions of two UGPs (UGP1 and UGP2) in the model microalga Phaeodactylum tricornutum. Despite differences in amino acid sequence, substrate specificity, and subcellular localization between UGP1 and UGP2, both enzymes can efficiently increase the production of chrysolaminarin (Chrl) or lipids by regulating carbon flux distribution without impairing growth and photosynthesis in transgenic strains. Productivity evaluation indicate that UGP1 play a bigger role in regulating Chrl and lipid production than UGP2. In addition, UGP1 enhance antioxidant capacity, whereas UGP2 is involved in sulfoquinovosyldiacylglycerol (SQDG) synthesis in P. tricornutum. Taken together, the present results suggest that ideal microalgal strains can be developed for the industrial production of Chrl or lipids and lay the foundation for the development of methods to improve oxidative stress tolerance in diatoms.

Single-mutations at the galactose-binding site of enzymes GalK, GalU, and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-d-galactose and azido-functionalized Gb3 analogs.

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson-Fabry disease and an elevated level of deacylated Gb3 is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1, 4ßGal1, and 4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of galactokinase, UTP-glucose-1-phosphate uridylyltransferase, and α-1,4-galactosyltransferase LgtC, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes galactokinase/UTP-glucose-1-phosphate uridylyltransferase generate UDP-6-azido-6-deoxy-d-galactose, which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the 3 enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy-d-galactose-1-phosphate, UDP-6-azido-6-deoxy-d-galactose, and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V, and LgtC-Q187S, respectively, is 3-6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6-azido-6-deoxy-d-galactose with ~90% conversion yields, and products azido-globotriose and lyso-AzGb3 with substrate conversion of up to 70%. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series.

Exploring Redox Modulation of Plant UDP-Glucose Pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus.

Herein, two genes (LBA0625 and LBA1719) encoding UGPases (UDP-glucose pyrophosphorylase) in Lactobacillus acidophilus (L. acidophilus) were successfully transformed into Escherichia coli BL21 (DE3) to construct recombinant overexpressing strains (E-0625, E-1719) to investigate the biological characteristics of UGPase-0625 and UGPase-1719. The active sites, polysaccharide yield, and anti-freeze-drying stress of L. acidophilus ATCC4356 were also detected. UGPase-0625 and UGPase-1719 belong to the nucleotidyltransferase of stable hydrophilic proteins; contain 300 and 294 amino acids, respectively; and have 20 conserved active sites by prediction. Αlpha-helixes and random coils were the main secondary structures, which constituted the main skeleton of UGPases. The optimal mixture for the high catalytic activity of the two UGPases included 0.5 mM UDP-Glu (uridine diphosphate glucose) and Mg2+ at 37 °C, pH 10.0. By comparing the UGPase activities of the mutant strains with the original recombinant strains, A10, L130, and L263 were determined as the active sites of UGPase-0625 (P < 0.01) and A11, L130, and L263 were determined as the active sites of UGPase-1719 (P < 0.01). In addition, UGPase overexpression could increase the production of polysaccharides and the survival rates of recombinant bacteria after freeze-drying. This is the first study to determine the enzymatic properties, active sites, and structural simulation of UGPases from L. acidophilus, providing in-depth understanding of the biological characteristics of UGPases in lactic acid bacteria.Key points• We detected the biological characteristics of UGPases encoded by LBA0625 and LBA1719.• We identified UGPase-0625 and UGPase-1719 active sites.• UGPase overexpression elevates polysaccharide levels and post-freeze-drying survival.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

GLYCOGEN ACCUMULATION IN TRICHOMONAS IS DRIVEN BY THE AVAILABILITY OF EXTRACELLULAR GLUCOSE.

The parasitic protist Trichomonas vaginalis is the causative agent of trichomoniasis, a highly prevalent sexually transmitted infection. The organism is known to accumulate substantial deposits of the polysaccharide glycogen, which is believed to serve as a store of carbon and energy that can be tapped during periods of nutrient limitation. Such nutrient limitation is likely to occur when T. vaginalis is transmitted between hosts, implying that glycogen may play an important role in the lifecycle of the parasite. Both T. vaginalis glycogen synthase and glycogen phosphorylase, key enzymes of glycogen synthesis and degradation, respectively, have been cloned and characterized, and neither enzyme is subject to the post-translational controls found in other, well-characterized eukaryotic systems. Thus, it is unclear how glycogen metabolism is regulated in this organism. Here we use a glucose limitation/re-feeding protocol to show that the activities of key enzymes of glycogen synthesis do not increase during re-feeding when glycogen synthesis is stimulated. Rather, a simple model appears to operate with glycogen storage being driven by the extracellular glucose concentration.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth.

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Inactivation of GalU Leads to a Cell Wall-Associated Polysaccharide Defect That Reduces the Susceptibility of Enterococcus faecalis to Bacteriolytic Agents.

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose-1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal amino acid (aa) region 288 to 298 of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1 Interestingly, these mutants had reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalisIMPORTANCE Cell wall-associated polysaccharides of bacteria are involved in various physiological characteristics. Recent studies demonstrated that the cell wall-associated polysaccharide of Enterococcus faecalis is required for susceptibility to bactericidal antibiotic agents. Here, we demonstrated that a galU mutation resulted in resistance to the enterococcal lytic bacteriocin Bac41. The galU homologue is reported to be essential for the biosynthesis of species-specific cell wall-associated polysaccharides in other Firmicutes In E. faecalis, the galU mutant lost the E. faecalis-specific cell wall-associated polysaccharide EPA (enterococcal polysaccharide antigen). The mutant also displayed reduced susceptibility to antibacterial agents and an abnormal cell morphology. We demonstrated here that galU was essential for EPA biosynthesis in E. faecalis, and EPA production might underlie susceptibility to lytic bacteriocin and antibiotic agents by undefined mechanisms.

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.

BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumour associated with a high mortality rate and poor prognosis worldwide. Uridine diphosphate-glucose pyrophosphorylase 2 (UGP2), a key enzyme in glycogen biosynthesis, has been reported to be associated with the occurrence and development of various cancer types. However, its diagnostic value and prognostic value in HCC remain unclear. The present study observed that UGP2 expression was significantly downregulated at both the mRNA and protein levels in HCC tissues. Receiver operating characteristic (ROC) curve analysis revealed that UGP2 may be an indicator for the diagnosis of HCC. In addition, Kaplan-Meier and Cox regression multivariate analyses indicated that UGP2 is an independent prognostic factor of overall survival (OS) in patients with HCC. Furthermore, gene set enrichment analysis (GSEA) suggested that gene sets negatively correlated with the survival of HCC patients were enriched in the group with low UGP2 expression levels. More importantly, a significant correlation was identified between low UGP2 expression and fatty acid metabolism. In summary, the present study demonstrates that UGP2 may contribute to the progression of HCC, indicating a potential therapeutic target for HCC patients.

Involvement of UDP-glucose pyrophosphorylase from Verticillium dahliae in cell morphogenesis, stress responses, and host infection.

UDP-glucose pyrophosphorylase (UGP, EC 2.7.7.9) is an essential enzyme involved in carbohydrate metabolism. In Saccharomyces cerevisiae and other fungi, the UGP gene is indispensable for normal cell development, polysaccharide synthesis, and stress response. However, the function of the UGP homolog in plant pathogenic fungi has been rarely explored during pathogenesis. In this study, we characterize a UGP homolog named VdUGP from Verticillium dahliae, a soil-borne fungus that causes plant vascular wilt. In comparison with wild-type strain V07DF2 and complementation strains, the VdUGP knocked down mutant 24C9 exhibited sensitivity to sodium dodecyl sulfate (perturbing membrane integrity) and high sodium chloride concentration (high osmotic pressure stress). More than 25 % of the conidia of the mutant developed into short and swollen hypha and formed hyperbranching and compact colonies. The mutant exhibited decreased virulence on cotton and tobacco seedlings. Further investigation determined that the germination of the mutant spores was significantly delayed compared with the wild-type strain on the host roots. RNA-seq analysis revealed that a considerable number of genes encoding secreted proteins and carbohydrate-active enzymes were significantly downregulated in the mutant at an early stage of infection compared with those of the wild-type strain. RNA-seq data indicated that mutation affected many Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways both in the pathogen and in the inoculated plants at the infection stage. These alterations of the mutant in cultural phenotypes, virulence, and gene expression profiles clearly indicated that VdUGP played important roles in fungal cell morphogenesis, stress responses, and host infection.

Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

Biosynthesis pathway of indole-3-acetyl-myo-inositol during development of maize (Zea mays L.) seeds.

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-ß-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-ß-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.

Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP-RoGalU1 and RoGalU2.

Uridine-5'-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism-Rhodococcus opacus 1CP-were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the Vmax was determined to be 177 U mg-1 (uridine-5'-triphosphate (UTP)) and Km to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.

Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG.

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.

Comparative proteomic analysis revealed the metabolic mechanism of excessive exopolysaccharide synthesis by Bacillus mucilaginosus under CaCO3 addition.

The metabolic mechanism of excessive exopolysaccharide (BMPS) synthesis by Bacillus mucilaginosus CGMCC5766 under CaCO3 addition was investigated. Under CaCO3 (5 g/L), the maximum BMPS concentration reached 28.4 g/L, which was 11.2 folds higher than that of the control. Proteomics was then used to analyze the proteins with substantial differences expressed by B. mucilaginosus with and without CaCO3 addition. The proteomic results revealed that the enzymes related to the central metabolic pathway, amino acid biosynthesis, and nucleotide metabolism were depressed. By contrast, the UDP-glucose pyrophosphorylase involved in BMPS biosynthesis was overexpressed and converted metabolic flux from the biomass accumulation to the biosynthesis of BMPS. This research provides a new and widened perspective into understanding the mechanism of BMPS biosynthesis and applying theoretical and practical significance for the improvement of BMPS production from B. mucilaginosus.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.