[Objective] The aim was to study the effects of over-expressed sm-Ngt1,a glycosyltransferase gene induced by both methyl jasmonate and salicylic acid from tobacco,on the plant height of rice.[Method] The binary expres...[Objective] The aim was to study the effects of over-expressed sm-Ngt1,a glycosyltransferase gene induced by both methyl jasmonate and salicylic acid from tobacco,on the plant height of rice.[Method] The binary expression vector of the sm-Ngt1 gene was constructed and transferred to matured embryo of indica rice YTB with the method of Agrobacterium infection.The height of the positive transgenic plants was measured.[Result] 117 positive transgenic plants with sm-Ngt1 were obtained.The results showed that the rice plants dwarfed with different degrees after transferring the sm-Ngt1 gene,the height of 37% of transgenic plants is 71.4±9.8 cm,27% is 65.1±4.6 cm,and the average height of YTB（CK） is 130.0±4.3 cm.[Conclusion] These results aid a foundafion for further study on function of sm-Ngt1.展开更多
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 sy...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.展开更多
Enzymatic glycosylation catalyzed by glycosyltransferases (GTs) has great potential in creating diverse novel and bioactive glycosides. Herein, three new GTs (UGT84 A33, UGT71 AE1 and UGT90 A14) from Carthamus tinctor...Enzymatic glycosylation catalyzed by glycosyltransferases (GTs) has great potential in creating diverse novel and bioactive glycosides. Herein, three new GTs (UGT84 A33, UGT71 AE1 and UGT90 A14) from Carthamus tinctorius exhibited robust catalytic promiscuity to benzylisoquinoline alkaloids, and were used as enzymatic tools in glycosylation of bioactive benzylisoquinoline alkaloids. Seven novel benzylisoquinoline alkaloids O-glycosides were synthesized with high efficiency. These studies indicate the significant potential of promiscuous GTs in synthesis of benzylisoquinoline alkaloids glycosides for drug discovery.展开更多
Non-alcoholic fatty liver disease(NAFLD) is the most common form of chronic liver disease and its incidence is increasing worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not...Non-alcoholic fatty liver disease(NAFLD) is the most common form of chronic liver disease and its incidence is increasing worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not fully understood. Glycosyltransferases(GTs) are a diverse class of enzymes involved in catalyzing the transfer of one or multiple sugar residues to a wide range of acceptor molecules. GTs mediate a wide range of functions from structure and storage to signaling, and play a key role in many fundamental biological processes. Therefore, it is anticipated that GTs have a role in the pathogenesis of NAFLD. In this article, we present an overview of the basic information on NAFLD, particularly GTs and glycosylation modification of certain molecules and their association with NAFLD pathogenesis. In addition, the effects and mechanisms of some GTs in the development of NAFLD are summarized.展开更多
KD （Keshan disease） is an endemic cardiomyopathy occurring only in China. Its pathogenesis is unclear till now. In the study, gene expression profiles of the PBMC （peripheral blood mononuclear cell） derived respec...KD （Keshan disease） is an endemic cardiomyopathy occurring only in China. Its pathogenesis is unclear till now. In the study, gene expression profiles of the PBMC （peripheral blood mononuclear cell） derived respectively from KD patients and healthy in KD areas were compared. Total RNA was isolated, amplified, labeled and hybridized to Agilent 4 ~ 44 K Whole Human Genome Oligonucleotide Microarray. Significant canonical pathways were analyzed by IPA （ingenuity pathway analysis） to identify differently expressed genes and pathways involved in the cardiovascular system development and function. Quantitative RT-PCR was applied to further validate our microarray results. Eighty-three up-regulated （ratios 〉 2.0） and nine down-regulated glycosyltransferase genes （ratios 〈 0.5） in PBMC in KD patients were detected by significance analysis of microarrays. Two significant canonical pathways from glycosyltransferase gene expression profiles were screened by IPA. The results of qRT-PCR show that four up-regulated （BMP 1/7/10 and FGF 18） and one down-regulated （BMP2） genes are consistent with those in microarray experiment, confirming the validity of the microarray data. Based on the results of the study, it is suggested that bone morphogenetic proteins and fibroblast growth factors might play an important role in the pathogenesis of KD. This further helps us to understand the pathogenesis of KD, as well as dilated cardiomyopathy.展开更多
UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their p...UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their physiological significance in plants remains largely unknown. Here, we reveal the functionsand mechanisms of two Arabidopsis UGT genes, UGT73C3 and UGT73C4, which are strongly induced byPseudomonas syringae pv. tomato (Pst) DC3000. Overexpression of these genes significantly enhancedplant immune response, whereas their loss of function in double mutants led to increased sensitivity topathogen infections. However, single mutants showed no obvious alteration in pathogen resistance. Tofurther investigate the regulatory mechanisms of UGT73C3/C4 in plant immunity, we conducted compre-hensive secondary metabolome analyses and glycoside quantification. Overexpression lines accumulatedhigher levels of pinoresinol diglucosides than wild-type plants, both before and after Pst DC3000 treatment,whereas double mutants accumulated lower levels. Furthermore, in vitro and in vivo experiments demon-strated that UGT73C3 and UGT73C4 can glycosylate pinoresinol to form pinoresinol monoglucoside anddiglucoside. Moreover, pinoresinol glycosylation promotes the plant immune response by increasing reac-tive oxygen species production and callose deposition. Additionally, the transcription factor HB34 wasfound to activate UGT73C3 and UGT73C4 transcription and play a key role in plant immunity. Overall,this study reveals a novel pathway in which UGT73C3/C4-mediated pinoresinol glycosylation, regulatedby HB34, enhances the plant immune response.展开更多
Objective:To characterize a glycosyltransferase(RpUGT1)from Rheum palmatum and investigate its specificity toward flavonoid compounds.Methods:The RpUGT1 was expressed in Escherichia coli and screened for catalytic act...Objective:To characterize a glycosyltransferase(RpUGT1)from Rheum palmatum and investigate its specificity toward flavonoid compounds.Methods:The RpUGT1 was expressed in Escherichia coli and screened for catalytic activity against a range of flavonoid substrates using a high-throughput HPLC assay method.Mass spectrometry(MS)and nuclear magnetic resonance(NMR)were used to determine the structure of the product.Homology modeling,molecular docking analyses and site-directed mutagenesis studies were conducted to identify key residues responsible for its function.Results:The recombinant RpUGT1 protein exhibited catalytic activity towards various flavonoids.Notably,RpUGT1 catalyzed the glycosylation of isorhamnetin to form 3-O-glucoside and kaempferol to form 7-O-glucoside,utilizing uridine diphosphate(UDP)glucose as the sugar donor.The homology modeling and molecular docking analyses identified key residues responsible for its activity.Subsequent sitedirected mutagenesis studies highlighted the crucial role of K307 in catalysis.Conclusion:These discoveries offer valuable perspectives on the role of the UGT family and establish a groundwork for forthcoming research on the synthesis of flavonoids in plants.展开更多
Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphospha...Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphosphate-dependent glycosyltransferases(UGTs).These enzymes not only enhance the physicochemical properties of natural products but also markedly increase their biological activity.This review provides an in-depth exploration of the characteristic features of UGTs and their diverse applications in the glycosylation modification of natural products,encompassing terpenoids,flavonoids,alkaloids,phenolic acids,and amide compounds.Special emphasis has been placed on the latest strategies in UGTs’protein engineering and high-throughput screening methods,which provides insights into the study of UGTs’protein engineering and facilitate their application in the fields of food and medicine.展开更多
Objective:Pancreatic ductal adenocarcinoma(PDAC)is one of the most challenging malignancies in the digestive system.Immune evasion is a crucial factor leading to poor prognosis in patients.This study aims to investiga...Objective:Pancreatic ductal adenocarcinoma(PDAC)is one of the most challenging malignancies in the digestive system.Immune evasion is a crucial factor leading to poor prognosis in patients.This study aims to investigate the molecular mechanisms of immune evasion in PDAC cells.Methods:We used The Cancer Genome Atlas(TCGA),and Genotype-Tissue Expression Project(GTEx),Gene Expression Omnibus(GEO)databases,and immunohistochemistry to analyze the expression patterns of Beta-1,3-N-acetylglucosaminyltransferase 3(B3GNT3)in normal pancreatic tissues and tumor tissues.We explored the biological pathways involving B3GNT3 through RNA-sequencing(RNA-seq)and gene ontology(GO)enrichment analysis.The correlation between B3GNT3 and major histocompatibility complex class I(MHC-I)levels was examined using flow cytometry.In addition,by establishing a mouse PDAC orthotopic model,we investigated the relationship between B3GNT3,PDAC progression,and CD8+T cell function.Results:B3GNT3 mRNA levels are significantly higher in tumor samples compared with normal tissue,and its expression is negatively correlated with patient prognosis.RNA-seq analysis suggests B3GNT3 may impact MHC-I mediated antigen presentation.Knockdown of B3GNT3 increases MHC-I levels on tumor cells,while overexpression decreases them.In a mouse PDAC model,B3GNT3 knockdown inhibits tumor growth and increases CD8+T cell infiltration.Conclusions:The expression level of B3GNT3 in PDAC cells is negatively correlated with MHC-I levels,thereby affecting the ability of CD8+T cells to recognize tumor cells and exert their cytotoxic function,making tumor cells more prone to immune evasion.展开更多
Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose(G-G-R)sugar chain attached at the C-28 position of the pentacyclic triterpene skeleto...Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose(G-G-R)sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton.Until recently,the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive.In this study,we identified two novel glycoside glycosyltransferases(GGTs)that can sequentially install the sugar chain’s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside,two representative G-G-R sugar chain-containing triterpenoid saponins produced by Centella asiatica.Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase.We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families,suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order.Moreover,expression patterns of the two GGTs in pierced leaves of C.asiatica were found to be correlated with the production of asiaticoside and madecassoside,implying their involvement in host defense against herbivores and pathogens.Our work sheds light on the biosynthesis and evolution of complex saponin sugars,paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.展开更多
Glycosylation is an important post-modification reaction in plant secondary metabolism,and contributes to structural diversity of bioactive natural products.In plants,glycosylation is usually catalyzed by UDP-glycosyl...Glycosylation is an important post-modification reaction in plant secondary metabolism,and contributes to structural diversity of bioactive natural products.In plants,glycosylation is usually catalyzed by UDP-glycosyltransferases.Flavonoid 2′-O-glycosides are rare glycosides.However,no UGTs have been reported,thus far,to specifically catalyze 2′-O-glycosylation of flavonoids.In this work,UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase.It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds.Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2.The crystal structure of UGT71AP2(2.15Å)was solved,and mechanisms of its regio-selectivity was interpreted by pKa calculations,molecular docking,MD simulation,MM/GBSA binding free energy,QM/MM,and hydrogen‒deuterium exchange mass spectrometry analysis.Through structure-guided rational design,we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity(the ratio of 7-O-glycosylation byproducts decreased from 48%to 4%)and catalytic efficiency of 2′-O-glycosylation(kcat/Km,0.23 L/(s·μmol),12-fold higher than the native).Moreover,UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity,and is a dual function glycosyltransferase.This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.展开更多
Lactic acid bacteria(LAB)exopolysaccharides(EPS)reveal high safety and multiple activities,and are typical postbiotics produced by LAB during fermentation.In this paper,6583 articles on LAB-EPS from Web of Science and...Lactic acid bacteria(LAB)exopolysaccharides(EPS)reveal high safety and multiple activities,and are typical postbiotics produced by LAB during fermentation.In this paper,6583 articles on LAB-EPS from Web of Science and Elsevier databases were retrieved,and 236 articles related to this review were screened.The EPS from 90 LAB strains were summarized in terms of their extraction methods,yield,molecular weight,monosaccharide composition,glycosidic bond configuration and the structural and activity relationships(SARs).However,there exist great challenges as for the low yield and high cost in EPS production.Therefore,this review further elaborated the mechanism of EPS secretion,the anabolic pathway of EPS,the structure and mechanism of key enzymes involving in EPS synthesis process,the prospect of gene regulation for EPS secretion,and proposed the engineering strategies for increasing EPS yield or tailored EPS design in recent years.In addition,CRISPR/Cas9 gene editing technology was also discussed in the production control of EPS in LAB.Finally,the engineering strategy of increasing EPS yield in recent years was proposed.This work might provide important theoretical support for the production and application of LAB-based EPS.展开更多
Plants emit an overabundance of volatile compounds, which act in their producers either as appreciated attractants to lure beneficial animals or as repellent toxins to deter pests in a species-specific and concentrati...Plants emit an overabundance of volatile compounds, which act in their producers either as appreciated attractants to lure beneficial animals or as repellent toxins to deter pests in a species-specific and concentration-dependent manner. Plants have evolved solutions to provide sufficient volatiles without poisoning themselves. Uridine-diphosphate sugar-dependent glycosyltransferases （UGTs） acting on vola-tiles is one important part of this sophisticated system, which balances the levels of bioactive metabolites and prepares them for cellular and long-distance transport and storage but enables the remobilization of disarmed toxins for the benefit of plant protection. This review provides an overview of the research history of glycosidically bound volatiles （GBVs）, a relatively new group of plant secondary metabolites, and discusses the role of UGTs in the production of GBVs for plant protection.展开更多
Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides a...Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides and their underlying genetic basis. In this study, we carried out metabolic profiling of tricin-lignan-glycosides and identified a major quantitative gene annotated as a UDPdependent glycosyltransferase OsUGT706C2 by metabolite-based genome-wide association analysis. The putative flavonoid glycosyltransferase OsUGT706C2 was characterized as a flavonoid 7-O-glycosyltransferas in vitro and in vivo. Although the in vitro enzyme activity of OsUGT706C2 was similar to that of OsUGT706D1, the expression pattern and induced expression profile of OsUGT706C2 were very different from those of OsUGT706D1. Besides, OsUGT706C2 was specifically induced by UV-B. Constitutive expression of OsUGT706C2 in rice may modulate phenylpropanoid metabolism at both the transcript and metabolite levels. Furthermore, overexpressing OsUGT706C2 can enhance UV-B tolerance by promoting ROS scavenging in rice. Our findings might make it possible to use the glycosyltransferase OsUGT706C2 for crop improvement with respect to UVB adaptation and/or flavonoid accumulation, which may contribute to stable yield.展开更多
Glycosylation by uridine diphosphate-dependent glycosyltransferases(UGTs)in plants contributes to the complexity and diversity of secondary metabolites.UGTs are generally promiscuous in their use of acceptors,making i...Glycosylation by uridine diphosphate-dependent glycosyltransferases(UGTs)in plants contributes to the complexity and diversity of secondary metabolites.UGTs are generally promiscuous in their use of acceptors,making it challenging to reveal the function of UGTs in vivo.Here,we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis.We revisited the UGT72E cluster,which has been reported to catalyze the glycosylation of monolignols.Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90%compared with that from traditional untargeted metabolomics analysis.In addition to the two previously reported monolignol glycosides,a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant,22 of which were phenylalanine-derived glycosides,including 5-OH coniferyl alcohol-derived and lignan-derived glycosides,as confirmed by isotopic tracing of[^(13)C_(6)]-phenylalanine precursor.Our method revealed that UGT72Es could use coumarins as substrates,and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions.Using the newly developed method,the function of UGT78D2 was also evaluated.These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes,the complexity of which have been highly underestimated.展开更多
Glycosyltransferases （GTs; EC 2.4.x.y） constitute a large group of enzymes that form glycosidic bonds through transfer of sugars from activated donor molecules to acceptor molecules. GTs are critical to the biosynth...Glycosyltransferases （GTs; EC 2.4.x.y） constitute a large group of enzymes that form glycosidic bonds through transfer of sugars from activated donor molecules to acceptor molecules. GTs are critical to the biosynthesis of plant cell walls, among other diverse functions. Based on the Carbohydrate-Active enZymes （CAZy） database and sequence similarity.searches, we have identified 609 potential GT genes （loci） corresponding to 769 transcripts （gene models） in rice （Oryza sativa）, the reference monocotyledonous species. Using domain composition and sequence similarity, these rice GTs were classified into 40 CAZy families plus an additional unknown class. We found that two Pfam domains of unknown function, PF04577 and PF04646, are associated with GT families GT61 and GT31, respectively. To facilitate functional analysis of this important and large gene family, we created a phylogenomic Rice GT Database （http：//ricephylogenomics. ucdavis.edu/cellwalls/gtJ）. Through the database, several classes of functional genomic data, including mutant lines and gene expression data, can be displayed for each rice GT in the context of a phylogenetic tree, allowing for comparative analysis both within and between GT families. Comprehensive digital expression analysis of public gene expression data revealed that most （-80%） rice GTs are expressed. Based on analysis with Inparanoid, we identified 282 ‘rice-diverged＇ GTs that lack orthologs in sequenced dicots （Arabidopsis thaliana, Populus tricocarpa, Medicago truncatula, and Ricinus communis）. Combining these analyses, we identified 33 rice-diverged GT genes （45 gene models） that are highly expressed in above-ground, vegetative tissues. From the literature and this analysis, 21 of these loci are excellent targets for functional examination toward understanding and manipulating grass cell wall qualities. Study of the remainder may reveal aspects of hormone and protein metabolism that are critical for rice biology. This list of 33 genes and the Rice GT Database will facilitate the study of GTs and cell wall synthesis in rice and other plants.展开更多
Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly un...Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL 1.1 and PdGATL 1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT-PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL 1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATLI.1 and PdGATL1.2 are functional orthologs of PARVUS/ GATL1 and can play a role in xylan synthesis, but may also have role（s） in the synthesis of other wall polymers.展开更多
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 vario...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.展开更多
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 glycosyltran...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.展开更多
Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76...Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.展开更多
基金Supported by National Science Foundation of China(30871318)Key Research Project of Ministry of Education,PRC(210265)~~
文摘[Objective] The aim was to study the effects of over-expressed sm-Ngt1,a glycosyltransferase gene induced by both methyl jasmonate and salicylic acid from tobacco,on the plant height of rice.[Method] The binary expression vector of the sm-Ngt1 gene was constructed and transferred to matured embryo of indica rice YTB with the method of Agrobacterium infection.The height of the positive transgenic plants was measured.[Result] 117 positive transgenic plants with sm-Ngt1 were obtained.The results showed that the rice plants dwarfed with different degrees after transferring the sm-Ngt1 gene,the height of 37% of transgenic plants is 71.4±9.8 cm,27% is 65.1±4.6 cm,and the average height of YTB（CK） is 130.0±4.3 cm.[Conclusion] These results aid a foundafion for further study on function of sm-Ngt1.
基金supported by the National Natural Science Foundation of China (Nos. 81673540,81530096,81573581,and 81920108033)the Natural Science Foundation of Shanghai (No. 16ZR1434100)。
文摘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.
基金financially supported by the National Natural Science Foundation of China (No. 81573317)CAMS Innovation Fund for Medical Sciences(Nos. CIFMS-2016-I2M-3-012 and CIFMS-2016-I2M-2-002)
文摘Enzymatic glycosylation catalyzed by glycosyltransferases (GTs) has great potential in creating diverse novel and bioactive glycosides. Herein, three new GTs (UGT84 A33, UGT71 AE1 and UGT90 A14) from Carthamus tinctorius exhibited robust catalytic promiscuity to benzylisoquinoline alkaloids, and were used as enzymatic tools in glycosylation of bioactive benzylisoquinoline alkaloids. Seven novel benzylisoquinoline alkaloids O-glycosides were synthesized with high efficiency. These studies indicate the significant potential of promiscuous GTs in synthesis of benzylisoquinoline alkaloids glycosides for drug discovery.
基金Supported by The National Natural Science Foundation of China No.81041017Beijing Municipal Natural Science Foundation+1 种基金No.7112032Beijing Municipal Laboratory for Liver Protection and Regulation of Regeneration,and Scientific Research Common Program of Beijing Municipal Commission of Education No.KM201510025017
文摘Non-alcoholic fatty liver disease(NAFLD) is the most common form of chronic liver disease and its incidence is increasing worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not fully understood. Glycosyltransferases(GTs) are a diverse class of enzymes involved in catalyzing the transfer of one or multiple sugar residues to a wide range of acceptor molecules. GTs mediate a wide range of functions from structure and storage to signaling, and play a key role in many fundamental biological processes. Therefore, it is anticipated that GTs have a role in the pathogenesis of NAFLD. In this article, we present an overview of the basic information on NAFLD, particularly GTs and glycosylation modification of certain molecules and their association with NAFLD pathogenesis. In addition, the effects and mechanisms of some GTs in the development of NAFLD are summarized.
文摘KD （Keshan disease） is an endemic cardiomyopathy occurring only in China. Its pathogenesis is unclear till now. In the study, gene expression profiles of the PBMC （peripheral blood mononuclear cell） derived respectively from KD patients and healthy in KD areas were compared. Total RNA was isolated, amplified, labeled and hybridized to Agilent 4 ~ 44 K Whole Human Genome Oligonucleotide Microarray. Significant canonical pathways were analyzed by IPA （ingenuity pathway analysis） to identify differently expressed genes and pathways involved in the cardiovascular system development and function. Quantitative RT-PCR was applied to further validate our microarray results. Eighty-three up-regulated （ratios 〉 2.0） and nine down-regulated glycosyltransferase genes （ratios 〈 0.5） in PBMC in KD patients were detected by significance analysis of microarrays. Two significant canonical pathways from glycosyltransferase gene expression profiles were screened by IPA. The results of qRT-PCR show that four up-regulated （BMP 1/7/10 and FGF 18） and one down-regulated （BMP2） genes are consistent with those in microarray experiment, confirming the validity of the microarray data. Based on the results of the study, it is suggested that bone morphogenetic proteins and fibroblast growth factors might play an important role in the pathogenesis of KD. This further helps us to understand the pathogenesis of KD, as well as dilated cardiomyopathy.
文摘UDP-glycosyltransferases (UGTs) constitute the largest glycosyltransferase family in the plant kingdom,regulating many metabolic processes by transferring sugar moieties onto various small molecules. How-ever, their physiological significance in plants remains largely unknown. Here, we reveal the functionsand mechanisms of two Arabidopsis UGT genes, UGT73C3 and UGT73C4, which are strongly induced byPseudomonas syringae pv. tomato (Pst) DC3000. Overexpression of these genes significantly enhancedplant immune response, whereas their loss of function in double mutants led to increased sensitivity topathogen infections. However, single mutants showed no obvious alteration in pathogen resistance. Tofurther investigate the regulatory mechanisms of UGT73C3/C4 in plant immunity, we conducted compre-hensive secondary metabolome analyses and glycoside quantification. Overexpression lines accumulatedhigher levels of pinoresinol diglucosides than wild-type plants, both before and after Pst DC3000 treatment,whereas double mutants accumulated lower levels. Furthermore, in vitro and in vivo experiments demon-strated that UGT73C3 and UGT73C4 can glycosylate pinoresinol to form pinoresinol monoglucoside anddiglucoside. Moreover, pinoresinol glycosylation promotes the plant immune response by increasing reac-tive oxygen species production and callose deposition. Additionally, the transcription factor HB34 wasfound to activate UGT73C3 and UGT73C4 transcription and play a key role in plant immunity. Overall,this study reveals a novel pathway in which UGT73C3/C4-mediated pinoresinol glycosylation, regulatedby HB34, enhances the plant immune response.
基金financially supported by National Natural Science Foundation(No.82204567,8220142901,82104334)Hebei Natural Science Foundation(No.H2023201005)+1 种基金Shaanxi Provincial Key Industry Innovation Chain(Group)Project(No.2021ZDLSF04-01)China University Student Innovation and Entrepreneurship Training Program Project(No.202310075016)。
文摘Objective:To characterize a glycosyltransferase(RpUGT1)from Rheum palmatum and investigate its specificity toward flavonoid compounds.Methods:The RpUGT1 was expressed in Escherichia coli and screened for catalytic activity against a range of flavonoid substrates using a high-throughput HPLC assay method.Mass spectrometry(MS)and nuclear magnetic resonance(NMR)were used to determine the structure of the product.Homology modeling,molecular docking analyses and site-directed mutagenesis studies were conducted to identify key residues responsible for its function.Results:The recombinant RpUGT1 protein exhibited catalytic activity towards various flavonoids.Notably,RpUGT1 catalyzed the glycosylation of isorhamnetin to form 3-O-glucoside and kaempferol to form 7-O-glucoside,utilizing uridine diphosphate(UDP)glucose as the sugar donor.The homology modeling and molecular docking analyses identified key residues responsible for its activity.Subsequent sitedirected mutagenesis studies highlighted the crucial role of K307 in catalysis.Conclusion:These discoveries offer valuable perspectives on the role of the UGT family and establish a groundwork for forthcoming research on the synthesis of flavonoids in plants.
基金supported by the National Natural Science Foundation of China(No.32172192)the Zhejiang Provincial Natural Science Foundation of China(No.ZCLQ24C2001).
文摘Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphosphate-dependent glycosyltransferases(UGTs).These enzymes not only enhance the physicochemical properties of natural products but also markedly increase their biological activity.This review provides an in-depth exploration of the characteristic features of UGTs and their diverse applications in the glycosylation modification of natural products,encompassing terpenoids,flavonoids,alkaloids,phenolic acids,and amide compounds.Special emphasis has been placed on the latest strategies in UGTs’protein engineering and high-throughput screening methods,which provides insights into the study of UGTs’protein engineering and facilitate their application in the fields of food and medicine.
基金supported by the Scientific Research Foundation of SUMHS(No.SSF-23-15-002 to XX).
文摘Objective:Pancreatic ductal adenocarcinoma(PDAC)is one of the most challenging malignancies in the digestive system.Immune evasion is a crucial factor leading to poor prognosis in patients.This study aims to investigate the molecular mechanisms of immune evasion in PDAC cells.Methods:We used The Cancer Genome Atlas(TCGA),and Genotype-Tissue Expression Project(GTEx),Gene Expression Omnibus(GEO)databases,and immunohistochemistry to analyze the expression patterns of Beta-1,3-N-acetylglucosaminyltransferase 3(B3GNT3)in normal pancreatic tissues and tumor tissues.We explored the biological pathways involving B3GNT3 through RNA-sequencing(RNA-seq)and gene ontology(GO)enrichment analysis.The correlation between B3GNT3 and major histocompatibility complex class I(MHC-I)levels was examined using flow cytometry.In addition,by establishing a mouse PDAC orthotopic model,we investigated the relationship between B3GNT3,PDAC progression,and CD8+T cell function.Results:B3GNT3 mRNA levels are significantly higher in tumor samples compared with normal tissue,and its expression is negatively correlated with patient prognosis.RNA-seq analysis suggests B3GNT3 may impact MHC-I mediated antigen presentation.Knockdown of B3GNT3 increases MHC-I levels on tumor cells,while overexpression decreases them.In a mouse PDAC model,B3GNT3 knockdown inhibits tumor growth and increases CD8+T cell infiltration.Conclusions:The expression level of B3GNT3 in PDAC cells is negatively correlated with MHC-I levels,thereby affecting the ability of CD8+T cells to recognize tumor cells and exert their cytotoxic function,making tumor cells more prone to immune evasion.
基金supported by the Guangxi Science and Technology Base and Talent Special Project(AD23026030)the Guiding Funds of Central Government for Supporting the Development of the Local Science and Technology(ZY23055032)+2 种基金the National Natural Science Foundation of China(22277095)to Y.YResearch in the Z.L.group is supported by a Key Project at the Central Government level:the Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources(2060302)the National Technologies Key R&D Program of China(2022YFC2602000)。
文摘Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose(G-G-R)sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton.Until recently,the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive.In this study,we identified two novel glycoside glycosyltransferases(GGTs)that can sequentially install the sugar chain’s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside,two representative G-G-R sugar chain-containing triterpenoid saponins produced by Centella asiatica.Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase.We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families,suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order.Moreover,expression patterns of the two GGTs in pierced leaves of C.asiatica were found to be correlated with the production of asiaticoside and madecassoside,implying their involvement in host defense against herbivores and pathogens.Our work sheds light on the biosynthesis and evolution of complex saponin sugars,paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.
基金supported by the National Key Research and Development Program of China(No.2023YFA0914100)China National Postdoctoral Program for Innovation Talents(No.BX20220022)+2 种基金National Natural Science Foundation of China(No.82304326)Natural Science Foundation of Anhui Province(No.2008085MC92,China)the National Supercomputer Center(SNIC2022-3-34)at Linköping University(Sweden).
文摘Glycosylation is an important post-modification reaction in plant secondary metabolism,and contributes to structural diversity of bioactive natural products.In plants,glycosylation is usually catalyzed by UDP-glycosyltransferases.Flavonoid 2′-O-glycosides are rare glycosides.However,no UGTs have been reported,thus far,to specifically catalyze 2′-O-glycosylation of flavonoids.In this work,UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase.It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds.Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2.The crystal structure of UGT71AP2(2.15Å)was solved,and mechanisms of its regio-selectivity was interpreted by pKa calculations,molecular docking,MD simulation,MM/GBSA binding free energy,QM/MM,and hydrogen‒deuterium exchange mass spectrometry analysis.Through structure-guided rational design,we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity(the ratio of 7-O-glycosylation byproducts decreased from 48%to 4%)and catalytic efficiency of 2′-O-glycosylation(kcat/Km,0.23 L/(s·μmol),12-fold higher than the native).Moreover,UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity,and is a dual function glycosyltransferase.This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.
基金supported by the Natural Science Foundation of Heilongjiang Province(LH2021C075)Basic Research Business Expenses and Research Projects of Provincial Higher Education Institutions in Heilongjiang Province(2022-KYYWF-1077).
文摘Lactic acid bacteria(LAB)exopolysaccharides(EPS)reveal high safety and multiple activities,and are typical postbiotics produced by LAB during fermentation.In this paper,6583 articles on LAB-EPS from Web of Science and Elsevier databases were retrieved,and 236 articles related to this review were screened.The EPS from 90 LAB strains were summarized in terms of their extraction methods,yield,molecular weight,monosaccharide composition,glycosidic bond configuration and the structural and activity relationships(SARs).However,there exist great challenges as for the low yield and high cost in EPS production.Therefore,this review further elaborated the mechanism of EPS secretion,the anabolic pathway of EPS,the structure and mechanism of key enzymes involving in EPS synthesis process,the prospect of gene regulation for EPS secretion,and proposed the engineering strategies for increasing EPS yield or tailored EPS design in recent years.In addition,CRISPR/Cas9 gene editing technology was also discussed in the production control of EPS in LAB.Finally,the engineering strategy of increasing EPS yield in recent years was proposed.This work might provide important theoretical support for the production and application of LAB-based EPS.
基金This work was supported by the Deutsche Forschungsgemeinschaft DFG （SCHW634/32-1） and National Natural Science Foundation of China （grant numbers 31870678）.
文摘Plants emit an overabundance of volatile compounds, which act in their producers either as appreciated attractants to lure beneficial animals or as repellent toxins to deter pests in a species-specific and concentration-dependent manner. Plants have evolved solutions to provide sufficient volatiles without poisoning themselves. Uridine-diphosphate sugar-dependent glycosyltransferases （UGTs） acting on vola-tiles is one important part of this sophisticated system, which balances the levels of bioactive metabolites and prepares them for cellular and long-distance transport and storage but enables the remobilization of disarmed toxins for the benefit of plant protection. This review provides an overview of the research history of glycosidically bound volatiles （GBVs）, a relatively new group of plant secondary metabolites, and discusses the role of UGTs in the production of GBVs for plant protection.
基金supported by the Foundation for the Major Science and Technology Program of Ningxia Hui Autonomous Region (2016BZ06)the State Key Program of National Natural Science Foundation of China (31530052)+2 种基金the National Science Fund for Distinguished Young Scholars (31625021)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31821005)the Hainan University Startup Fund (KYQD(ZR)1866) to JL。
文摘Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides and their underlying genetic basis. In this study, we carried out metabolic profiling of tricin-lignan-glycosides and identified a major quantitative gene annotated as a UDPdependent glycosyltransferase OsUGT706C2 by metabolite-based genome-wide association analysis. The putative flavonoid glycosyltransferase OsUGT706C2 was characterized as a flavonoid 7-O-glycosyltransferas in vitro and in vivo. Although the in vitro enzyme activity of OsUGT706C2 was similar to that of OsUGT706D1, the expression pattern and induced expression profile of OsUGT706C2 were very different from those of OsUGT706D1. Besides, OsUGT706C2 was specifically induced by UV-B. Constitutive expression of OsUGT706C2 in rice may modulate phenylpropanoid metabolism at both the transcript and metabolite levels. Furthermore, overexpressing OsUGT706C2 can enhance UV-B tolerance by promoting ROS scavenging in rice. Our findings might make it possible to use the glycosyltransferase OsUGT706C2 for crop improvement with respect to UVB adaptation and/or flavonoid accumulation, which may contribute to stable yield.
基金the National Key R&D Program of China(2019YFA0903900)National Natural Science Foundation of China(grant number 31870273)+2 种基金Guangdong Provincial Key Laboratory of Synthetic Genomics(2019B030301006)Shenzhen Key Laboratory of Synthetic Genomics(ZDSYS201802061806209)Shenzhen Instituteof Synthetic Biology Scientific Research Program(ZTXM20190007).
文摘Glycosylation by uridine diphosphate-dependent glycosyltransferases(UGTs)in plants contributes to the complexity and diversity of secondary metabolites.UGTs are generally promiscuous in their use of acceptors,making it challenging to reveal the function of UGTs in vivo.Here,we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis.We revisited the UGT72E cluster,which has been reported to catalyze the glycosylation of monolignols.Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90%compared with that from traditional untargeted metabolomics analysis.In addition to the two previously reported monolignol glycosides,a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant,22 of which were phenylalanine-derived glycosides,including 5-OH coniferyl alcohol-derived and lignan-derived glycosides,as confirmed by isotopic tracing of[^(13)C_(6)]-phenylalanine precursor.Our method revealed that UGT72Es could use coumarins as substrates,and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions.Using the newly developed method,the function of UGT78D2 was also evaluated.These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes,the complexity of which have been highly underestimated.
文摘Glycosyltransferases （GTs; EC 2.4.x.y） constitute a large group of enzymes that form glycosidic bonds through transfer of sugars from activated donor molecules to acceptor molecules. GTs are critical to the biosynthesis of plant cell walls, among other diverse functions. Based on the Carbohydrate-Active enZymes （CAZy） database and sequence similarity.searches, we have identified 609 potential GT genes （loci） corresponding to 769 transcripts （gene models） in rice （Oryza sativa）, the reference monocotyledonous species. Using domain composition and sequence similarity, these rice GTs were classified into 40 CAZy families plus an additional unknown class. We found that two Pfam domains of unknown function, PF04577 and PF04646, are associated with GT families GT61 and GT31, respectively. To facilitate functional analysis of this important and large gene family, we created a phylogenomic Rice GT Database （http：//ricephylogenomics. ucdavis.edu/cellwalls/gtJ）. Through the database, several classes of functional genomic data, including mutant lines and gene expression data, can be displayed for each rice GT in the context of a phylogenetic tree, allowing for comparative analysis both within and between GT families. Comprehensive digital expression analysis of public gene expression data revealed that most （-80%） rice GTs are expressed. Based on analysis with Inparanoid, we identified 282 ‘rice-diverged＇ GTs that lack orthologs in sequenced dicots （Arabidopsis thaliana, Populus tricocarpa, Medicago truncatula, and Ricinus communis）. Combining these analyses, we identified 33 rice-diverged GT genes （45 gene models） that are highly expressed in above-ground, vegetative tissues. From the literature and this analysis, 21 of these loci are excellent targets for functional examination toward understanding and manipulating grass cell wall qualities. Study of the remainder may reveal aspects of hormone and protein metabolism that are critical for rice biology. This list of 33 genes and the Rice GT Database will facilitate the study of GTs and cell wall synthesis in rice and other plants.
文摘Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL 1.1 and PdGATL 1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT-PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL 1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATLI.1 and PdGATL1.2 are functional orthologs of PARVUS/ GATL1 and can play a role in xylan synthesis, but may also have role（s） in the synthesis of other wall polymers.
基金supported by the National Key Research and Development Program of China(2020YFA0908000)CAMS Innovation fund for Medical Sciences(CIFMS,No.2021-I2M-1029,China)Beijing Key Laboratory of non-Clinical Drug Metabolism and PK/PD Study(Z141102004414062)。
文摘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.
基金Tfinancially supported by the National Natural Science Foundation of China(No.81573317)。
文摘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.
基金This work was financially supported by the National Key R&D Program of China(2018YFA0900600)the Strategic Priority Research Program "Molecular Mechanism of Plant Growth and Development" of CAS(XDB27020202,XDB27020103)+5 种基金the National Natural Science Foundation of China(31700263,31670099,31700261)grants from the Shanghai Science and Technology Commission(19XD1424500)J.L.is supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of SciencesThis work was also financially supported by the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science(no.ZSYS-016)the International Partnership Program of Chinese Academy of Science(no.153D31KYSB20170121)the National Key Laboratory of Plant Molecular Genetics,SIPPI,CAS.
文摘Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.
