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Glycosylation is one of the most important reactions in living organisms as it results in the formation of glycoconjugates with diverse biological functions. Sugar nucleotides are structurally composed of sugar and nucleoside diphosphate or monophosphate, which are widespread within a variety of biological cells. As glycosyl donors for the transglycosyl reactions catalyzed by Leloir-type glycosyltransferases, sugar nucleotides are essential for the synthesis of glycans and glycoconjugates. However, high costs and limited availability of nucleotide sugars prevent applications of biocatalytic cascades on an industrial scale. Therefore, attentions on synthetic strategies of sugar nucleotides have been increasing to achieve their wide applications in various fields. The 9 common sugar nucleotides in mammals have been fully studied with large-scale synthesis through chemical, enzymatic (chemo-enzymatic) and cell factory strategies. In addition to common sugar nucleotides, many rare sugar nucleotides are present in plants and bacteria. Although unnatural sugar nucleotides cannot be synthesized in organisms, they have great potential in research as substrates for glycosyltransferases in carbohydrate synthesis, as enzyme inhibitors in biochemical studies, and as components of glycoconjugate biosynthesis. Therefore, increasing attention has been paid to explore the efficient synthesis of unnatural sugar nucleotides. Currently, strategies for chemical synthesis of sugar nucleotides have been greatly improved, such as the use of effective catalysts for forming pyrophosphate bonds and the development of entirely new synthesis protocols. Multiple sugar nucleotides, especially unnatural sugar nucleotides, are synthesized chemically. However, chemical synthesis requires tedious protection and deprotection steps, resulting in complex steps, high cost and low yield. In contrast, enzymatic (chemo-enzymatic) and cell factory methods have significant advantages such as high yield, easy operation and easy process scale-up in the preparation of sugar nucleotides. Hence, they are prominent strategies for sugar nucleotide preparation. Herein, the biosynthesis and application of sugar nucleotides are reviewed, mainly focusing on the 9 sugar nucleotides common in mammals. The early strategies for enzymatic synthesis of sugar nucleotides generally used de novo synthesis pathway. With the discoveries of enzymes involved in salvage pathway of sugar nucleotide synthesis and the development of one-pot multienzyme (OPME) method, the synthesis of sugar nucleotides was greatly simplified. Cell factory method employs the microbial living cells as a “processing plant” by engineering their metabolic pathways through genetic engineering technology. The cell factory method has high yield, and has been applied for efficient synthesis of several sugar nucleotides. Moreover, the strategy of gram-scale synthesis of multiple rare sugar nucleotides by cascade reactions from common sugar nucleotides using sugar nucleotides synthases cloned from different sources was illustrated. In recent years, the synthesis cost of sugar nucleotides has been further reduced through various ways, such as regeneration of nucleotides, regeneration of organic cofactors, and application of immobilized enzyme technology. Furthermore, through the continuous improvement of sugar nucleotide purification process, the use of high concentration of multi-enzyme cascade and rapid non-chromatographic purification process, the synthesis of multiple sugar nucleotides and their derivatives from monosaccharides was achieved, which gradually broke the limitations of the existing strategy. With the efficient synthesis of sugar nucleotides, their applications in various fields have been increasingly explored, including the synthesis of glycans and glycoconjugates, biochemical characterization of glycosyltransferases and bioorthogonal labeling strategies, which are of great significance to the research of biochemistry, glycobiology and the development of related pharmaceutical products.
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Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi, of which cycloastragenol-type glycosides are the most typical and major bioactive compounds. This kind of compounds exhibit various biological functions including cardiovascular protective, neuroprotective, etc. Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis, re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides. However, the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps. Herein, guided by transcriptome and phylogenetic analyses, a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus. AmCAS1, the first reported cycloartenol synthase from Astragalus genus, is capable of catalyzing the formation of cycloartenol; AmUGT15, AmUGT14, AmUGT13, and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation, 3-O-glucosylation, 25-O-glucosylation/O-xylosylation and 2'-O-glucosylation of cycloastragenol glycosides, respectively. These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants, also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.
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Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.
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Dendrobium/genética , Reguladores de Crescimento de Plantas , Glicosiltransferases/metabolismo , Perfilação da Expressão Gênica , Micorrizas , Filogenia , Proteínas de Plantas/metabolismoRESUMO
The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca2+, Mn2+ and Co2+) showed inhibitory effect on the activity of UGT74L2, while Mg2+ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the Km value was 29.84 μmol·L-1, the kcat was 0.02 s-1, and the kcat·Km-1 was 572.6 mol-1·s-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.
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Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 μmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.
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Carthamus tinctorius/química , Filogenia , Flavonoides/análise , Glicosídeos/análise , Glicosiltransferases/genética , Anti-Inflamatórios , ChalconasRESUMO
Lu Dangshen is the geoherb in Shanxi Province. The content of Codonopsis pilosula polysaccharides (CPP) in Lu Dangshen is more than that in other Codonopsis Radix from other regions. Glycosyltransferase is the key enzyme for the synthesis of bioactive components, such as CPP and tangshenoside I. Based on the transcriptome data of C. pilosula [Codonopsis pilosula (Franch.) Nannf.] from different producing areas, this study carried out functional annotation of GO and KEGG, conservative domain analysis, phylogenetic tree analysis and expression pattern analysis of glycosyltransferase genes in C. pilosula to provides a theoretical basis for exploring the mechanism of genuineness formation in Lu Dangshen. In this study, 98 glycosyltransferase genes were screened and identified, which belonged to GT family 1, GT family 2, GT family 90 and other families. By GO functional annotation, it was found that most of the glycosyltransferase genes had catalytic activity. Analysis of KEGG functional annotation showed that C. pilosula glycosyltransferase was mainly involved in glycan organism and terpenoid and polyketone metabolism. Among them, conserved domain of 42 glycosyltransferase genes in GT family 1 was [X]-W-[2X]-Q-[3X]-[LH]-[5X]-[FLTHCGWNS]-[2X]-E-[4X]-[GVP]-[4X]-P-[4X]-Q-[2X]-[NAK]. Phylogenetic tree analysis based on the glycosyltransferase sequence in Arabidopsis thaliana showed that C. pilosula glycosyltransferases were mainly located in Arabidopsis thaliana UGT73, 72 and 85 branches. Gene expression pattern analysis showed that expression of CpUGT73AH2 was higher in Lu Dangshen than that in Baitiaodang and could respond to drought and low temperature stress. In conclusion, a glycosyltransferase gene CpUGT73AH2, which is involved in the metabolism of terpenoids and polyketides and can respond to environmental stress, was screened from the C. pilosula glycosyltransferase family 1, which was used to further study the role of C. pilosula glycosyltransferase in Lu Dangshen. It laid a theoretical foundation for further study on the role of C. pilosula glycosyltransferase in the formation of Lu Dangshen.
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@#Nosiheptide is a typical thiopeptide antibiotic displaying potent activity toward various drug-resistant strains of Gram-positive pathogens.Although nosiheptide lacks in vivo activity, and good water-solubility with a series of uncontrollable analogues, which may limit its clinical application, glycosylated analogues may overcome problem of low activity and may improve its druggability.In search of novel glycosylated nosiheptide producers, we applied a genome mining strategy that identified Actinoalloteichus sp.AHMU CJ021 that contains all genes required.However, despite the presence of a predicted glycosyltransferase, glycosylated derivatives of nosiheptide were not detected, after following one strain many compounds (OSMAC) strategy and heterologous expression of a regulatory protein NocP.Nevertheless, nosiheptide produced by this strain was remarkably pure, and further experiments were conducted to improve its production by optimization of the culture medium.Under optimal conditions, 58.73 mg/L nosiheptide was produced, representing an almost 6-fold improvement compared to the original fermentation medium.Therefore, we consider Actinoalloteichus sp.AHMU CJ021 a suitable potential candidate for industrial production of nosiheptide, which provides the basis for solving the problem of nosiheptide structural analogues.
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Triterpenoid saponins are widely used in medicine, health cares, cosmetics, food additives and agriculture because of their unique chemical properties and rich pharmacological activities. UDP-dependent glycosyltransferases (UGTs) are the key enzymes involved in triterpenoid saponin biosynthesis, and play important roles in the diversity of triterpenoid saponin structures and pharmacological activities. This review summarized the UGTs involved in plant triterpenoid saponin biosynthesis based on the sources of UGTs and the types of receptors. Moreover, the application of UGTs in heterologous biosynthesis of triterpenoid saponins based on synthetic biology was also discussed.
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Glicosiltransferases/genética , Plantas , Saponinas/química , TriterpenosRESUMO
In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.
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Sequência de Bases , Clonagem Molecular , Fermentação , Plasmídeos/genética , Sphingomonas/metabolismoRESUMO
Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from Panax ginseng, enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titers of F2 and 3β,20S-Di-O-Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better. Furthermore, the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM, and achieving their de novo biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass, which lays a foundation for drug research and development.
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Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donormolecules to acceptors such as sugars, lipids, proteins, and nucleic acids. Protein glycosylation is one ofthe most important post-translational modifications (PTMs). In recent years, increasing studies haveshown that glycosyltransferases are closely related to the virulence of pathogenic bacteria, and play a keyrole in adhesion, immune evasion, and host colonization. According to the features of three-dimensionalstructure, glycosyltransferases are classified into three groups (GT-A, GT-B and GT-C), among whichGT-A and GT-B folds are more common. Glycosyltransferases, which play a role in bacterial adhesion, adopt the GT-B or GT-C fold and glycosylate the surface proteins of pathogenic bacteria (adhesionproteins, autotransporters, etc.). It plays an important role in the adhesion of pathogenic bacteria, theformation of biofilm, and the virulence mechanisms. Glycosyltransferases take part in bacterial adhesionprocess of infection, and glycosyltransferases belonging to GT-A directly glycosylate host proteins andaffect host signal transduction, protein translation, and immune response. This review discusses thestructure of common pathogenic bacteria glycosyltransferases and the pathogenic mechanisms underlyingthese diseases of glycosylation. One kind of glycosyltransferases mainly modify their surface proteins, suchas the glycosyltransferase for specifically glycosylating high-molecular-weight(HMW) adhesion proteins, glycosyltransferases for glycosylation modification of serine-rich repeat proteins (SRRPs), bacterialautotransporter heptosyltransferase (BAHT) family, and N-linked protein system. The other kinds ofglycosyltransferases modulate host responses by directly modifying host proteins, such as Clostridium largecytotoxin, Legionella glycosyltransferase, and the NleB effector from enterobacteria. This review providesa reference for systematically revealing the pathogenic mechanism of glycosyltransferase in pathogenicbacteria, and contributes scientific knowledge in the development of pathogenic bacteria diagnosis, drugdesign, and vaccine development.
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Objective:To clone the full-length glycosyltransferase genes (<italic>PpUGT</italic>1,<italic>PpUGT</italic>7) related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>,and perform bioinformatics analysis,relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep<sup>®</sup> Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame (ORF) of <italic>PpUGT</italic>1 was 1 827 bp,encoding 608 amino acids,and was predicted as a steroid glycosyltransferase;the ORF of <italic>PpUGT</italic>7 was 1 380 bp,encoding 459 amino acids,and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively,and both of them were hydrophilic proteins,no transmembrane domain,no signal peptides,both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem;the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition,PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs, and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.
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Huang-Qin is a traditional Chinese medicine with antiviral, antioxidant, and anti-inflammatory activities. Its major bioactive compounds are diverse flavone O-glucuronides and glucosides. Although three flavonoid O-glycosyltransferases have been identified from S. baicalensis, this information is not sufficient to elucidate the structural diversity of flavonoid glycosides. In this study, nine glycosyltransferase candidate genes were discovered from S. baicalensis by BLAST analysis and their functions were characterized after heterologous expression. Three new flavone O-glycosyltransferases were able to catalyze the formation of major compounds in S. baicalensis, including baicalin and wogonoside. These enzymes could also utilize exogenous flavones as sugar acceptors. This work further elucidates biosynthetic pathways for Scutellaria flavonoid O-glycosides.
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Type 2 diabetes mellitus( T2 DM) is a common chronic metabolic disease characterized by persistent hyperglycemia and insulin resistance. In pancreatic β-cells,glucose-stimulated insulin secretion( GSIS) plays a pivotal role in maintaining the balance of blood glucose level. Previous studies have shown that geniposide,one of the active components of Gardenia jasminoides,could quickly regulate the absorption and metabolism of glucose,and affect glucose-stimulated insulin secretion in pancreatic β cells,but the specific mechanism needs to be further explored. Emerging evidence indicated that glycosylation of glucose transporter( GLUT) has played a key role in sensing cell microenvironmental changes and regulating glucose homeostasis in eucaryotic cells. In this study,we studied the effects of geniposide on the key molecules of GLUT2 glycosylation in pancreatic β cells. The results showed that geniposide could significantly up-regulate the mRNA and protein levels of Glc NAc T-Ⅳa glycosyltransferase( Gn T-Ⅳa) and galectin-9 but had no signi-ficant effect on the expression of clathrin,and geniposide could distinctively regulate the protein level of Gn T-Ⅳa in a short time( 1 h) under the conditions of low and medium glucose concentrations,but had no significant effect on the protein level of galectin-9. In addition,geniposide could also remarkably affect the protein level of glycosylated GLUT2 in a short-time treatment. The above results suggested that geniposide could quickly regulate the protein level of Gn T-Ⅳa,a key molecule of protein glycosylation in INS-1 rat pancreatic βcells and affect the glycosylation of GLUT2. These findings suggested that the regulation of geniposide on glucose absorption,metabolism and glucose-stimulated insulin secretion might be associated with its efficacy in regulating GLUT2 glycosylation and affecting its distribution on the cell membrane and cytoplasm in pancreatic β cells.
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Animais , Ratos , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glicosilação , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , IridoidesRESUMO
Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.
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Boraginaceae/genética , Ácidos Cafeicos , Cromatografia Líquida , Cinamatos , Clonagem Molecular , Depsídeos , Glicosiltransferases/genética , Filogenia , Espectrometria de Massas em TandemRESUMO
Objective: In order to obtain new glycosyltransferases with highly efficient catalysis, the glycosyltransferases from Carthamus tinctorius which contains diverse types of glycosides were mined. Methods: A new glycosyltransferase gene (UGT88B2) with full length was obtained by PCR and further transformed into Escherichia coli for heterologous expression. The catalytic activity of recombinant UGT88B2 was determined by HPLC-MSn. The structures of representative catalytic products were elucidated by MS and NMR. Results: UGT88B2 exhibited catalytic promiscuity and various patterns in glycosylation of flavonoids with high efficiency. Conclusion: A new glycosyltransferase named UGT88B2 was successfully mined and can be employed as enzymatic tools in glycosylation of flavonoids.
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Objective: With the aim to obtain crocetin glycosyltransferase UGTCs4 in cultured saffron suspension cell, and carry out its bioinformatics and expression mode analysis. Methods: A homologous cloning strategy and 5’ RACE methods were adopted, the full-length cDNA sequence of a crocetin glycosyltransferase, designated UGTCs4 (GeneBank number: KX398932), was obtained. The characteristics of physiochemical properties, structure and function of the deduced UGTCs4 protein were determined using a series of bioinformatics tools. Semi-quantitative PCR was used for gene expression analysis. Results: The results showed that the full length cDNA of UGTCs4 was 1 380 bp in length and encoding a 459 amino acid; UGTCs4 had high identities (83.2%) with UGTCs2 protein from saffron; UTGTCs4 had the same evolutionary tree as UGTCs2. UGTCs4 transcripts were constitutively expressed in the leaves, stems, and roots. UGTCs4 gene could respond to multiple treatments of indoleacetic acid (IAA), abscisic acid (ABA), gibberellin (GA), hydrogen peroxide (H2O2), and methyl jasmonate (MJA), which promoted its transcription. Conclusion: cDNA of crocetin glycosyltransferase was cloned from suspension cells for the first time and the response of UGTCs4 to different inducers was confirmed. Molecular characterization of UGTCs4 will be useful for further functional determination of the gene involving in the crocin biosynthesis and expression regulation.
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Ginsenosides are a series of glycosylated triterpenoids predominantly originated from Panax species with multiple pharmacological activities such as anti-aging, mediatory effect on the immune system and the nervous system. During the biosynthesis of ginsenosides, glycosyltransferases play essential roles by transferring various sugar moieties to the sapogenins in contributing to form structure and bioactivity diversified ginsenosides, which makes them important bioparts for synthetic biology-based production of these valuable ginsenosides. In this review, we summarized the functional elucidated glycosyltransferases responsible for ginsenoside biosynthesis, the advance in the protein engineering of UDP-glycosyltransferases (UGTs) and their application with the aim to provide in-depth understanding on ginsenoside-related UGTs for the production of rare ginsenosides applying synthetic biology-based microbial cell factories in the future.
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OBJECTIVE To investigate the effect of prenatal dexamethasone exposure (PDE) on chondrogenesis of female fetal rats and those of dexamethasone treatment on gene expression of chondrocytes, and to explore the underlying mechanism. METHODS ?In vivo, Wistar female ratswere conceived naturally, and the PDE group was sc given dexamethasone 0.2 mg-kg"1 once a day for 9-20 d after gestation (GD9-20). Pregnant rats were sacrificed on GD20( and cartilage samples of female fetal rats were collected. Tho morphology of cartilege was observed by HE and saffranin O staining. The expressions of aggrecan, O-xylosyltransfefase I (XylT-1), pi, 3-glucuronosyl transferase I [GlcAT-1).,4-galactosyttransterase 7, (GalT-I), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R). protein kinase B {AKT) and activator protein I (AP-1) mRNA in cartilage tissues were examined by realtime quantitative PCR (RT-PCR). The expressions of XylT-I . GlcAT-I . GalT-I . IGF-1. AKT and AP-1 were detected by immunohistochemistry. In vitro, GD20 Wistar female fetal rats were used to extract chondrocytes from fetal knee joints. Dexamethasone (100 and 500 nmol-L"'). IGF-1 (100 ug-L"'). IGF-1 R inhibitor NVP-AEW541 (1 umol-L"), AKT inhibitor MK-2206 (1 pmol-L") and AP-1 inhibitor SR-11302 (1 umol-L∗') were treated for 24 h. The content of glycosaminoglycan (GAG) in the cefl supeRNAtant was detected by DMB staining. The expressions of XylT-1 , GlcAT-1 , GalT-1 , IGF-1 R, AKT and AP-1 mRNA were detected by RT-PCR. The expressions of XylT-I , GlcAT-I and GalT-I protein were detected by Western blotting. RESULTS (J) In vivo the results of HE and saffranin O staining showed that the number of articular chondrocytes of female fetal rats in PDE group was reduced, the arrangement of surface chondrocytes was disorderty. and staining of cartilage matrix became kghter and uneven. The protein and mRNA expression of cartilage, XylT-I , GlcAT-I , GalT-I and proteins related to IGF-1/AKT/AP-1 pathway were reduced in PDE group compared with normal control group (P<0.05, P<0.01). @ In vitro compared with the normal control group, the contcnt of GAG in the supeRNAtant of chondrocytes of female fetal rats was decreased (P<0.05). and the mRNA expressions of XylT-I , GlcAT-I, GalT-I and proteins related with IGF-1/AKT/AP-1 pathways were deceased (P<0.05). Compared with the dexamethasone 500 nmol-L"1 group, IGF-1 100 pg∗L" increased the mRNA expression levels of the above proteins {P<0.05, P<0.01). Compared with the normal control group, the protein and mRNA expressions of XytT-I , GlcAT-I and GalT-| in IGF-1 treatment group were increased, while those in IGF-1R/AKT/AP-1 inhibitor treatment groups were decreased (P<0.05, F<0.01). Compared with the SR-11302 group, there was no significant change in the protein and mRNA expression levete of XylT-I, GlcAT-1 and GalT-I after co-treatment with IGF-1. CONCLUSION PDE induceds chondrodysplasia in female fetal rats, which is related to the insufficiency of cartilage matrix synthesis induced by decreasing expression of glycosyltransferases mediated by low basic activity of IGF-1/AKT/AP-1 pathway.
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Objective: To investigate the expression of glycosyltransferase enzyme 3 (GCNT3) in non-small cell lung cancer (NSCLC) tis-sues and corresponding normal tissues, and to further explore the relationship between GCNT3 expression and clinicopathological fea-tures, overall survival (OS), and progression-free survival (PFS) in patients with NSCLC. Methods: In this study, we used quantitative re-al-time polymerase chain reaction and Western blot to assess the mRNA and protein expression of GCNT3 in paired NSCLC and non-tu-mor tissues. In addition, 164 NSCLC patients were estimated for GCNT3 expression by immunohistochemistry, and the correlation be-tween GCNT3 expression and clinicopathological features was evaluated. Further, the effects of GCNT3 on the proliferation, invasion, and migration abilities of NSCLC cells were studied. Results: The mRNA and protein expression levels of GCNT3 in NSCLC tissues were both significantly higher than those in the corresponding non-tumor tissues. Among the 164 patients with NSCLC, high GCNT3 expres-sion was associated with gender, smoking, histology, pathological stage, and lymph node metastasis. Kaplan-Meier analysis displayed significant differences in OS and PFS among the groups exhibiting differences in GCNT3 expression (P<0.05). The NSCLC patients with increased GCNT3 expression showed poor OS and PFS. A multivariate analysis demonstrated that GCNT3 expression was as an inde-pendent prognostic factor for NSCLC (P<0.05). Cell function experiments showed that the proliferation, invasion, and migration abili-ties of NSCLC cells were significantly attenuated after inhibition of GCNT3 expression (P<0.05). Conclusions: High expression of GCNT3 was associated with unfavorable OS and PFS in patients with NSCLC; GCNT3 might, therefore, act as a prognostic biomarker for NSCLC.