Glycosyltransferases and non-alcoholic fatty liver disease

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.

Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

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.

Glycosyltransferases:Mining,engineering and applications in biosynthesis of glycosylated plant natural products

UDP-Glycosyltransferases(UGTs)catalyze the transfer of nucleotide-activated sugars to specific acceptors,among which the GT1 family enzymes are well-known for their function in biosynthesis of natural product glycosides.Elucidating GT function represents necessary step in metabolic engineering of aglycone glycosylation to produce drug leads,cosmetics,nutrients and sweeteners.In this review,we systematically summarize the phylogenetic distribution and catalytic diversity of plant GTs.We also discuss recent progress in the identifi-cation of novel GT candidates for synthesis of plant natural products(PNPs)using multi-omics technology and deep learning predicted models.We also highlight recent advances in rational design and directed evolution engineering strategies for new or improved GT functions.Finally,we cover recent breakthroughs in the appli-cation of GTs for microbial biosynthesis of some representative glycosylated PNPs,including flavonoid glycosides(fisetin 3-O-glycosides,astragalin,scutellarein 7-O-glucoside),terpenoid glycosides(rebaudioside A,ginseno-sides)and polyketide glycosides(salidroside,polydatin).

Glycoside-specific metabolomics combined with precursor isotopic labeling for characterizing plant glycosyltransferases

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.

Advance in glycosyltransferases, the important bioparts for production of diversified ginsenosides

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.

Enzymatic biosynthesis of benzylisoquinoline alkaloid glycosides via promiscuous glycosyltransferases from Carthamus tinctorius

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.

Unraveling the serial glycosylation in the biosynthesis of steroidal saponins in the medicinal plant Paris polyphylla and their antifungal action

Sugar-sugar glycosyltransferases play important roles in constructing complex and bioactive saponins.Here,we characterized a series of UDP-glycosyltransferases responsible for biosynthesizing the branched sugar chain of bioactive steroidal saponins from a widely known medicinal plant Paris polyphylla var.yunnanensis.Among them,a 2'-O-rhamnosyltransferase and three 6'-O-glucosyltrasferases catalyzed a cascade of glycosylation to produce steroidal diglycosides and triglycosides,respectively.These UDP-glycosyltransferases showed astonishing substrate promiscuity,resulting in the generation of a panel of 24 terpenoid glycosides including 15 previously undescribed compounds.A mutant library containing 44 variants was constructed based on the identification of critical residues by molecular docking simulations and protein model alignments,and a mutant UGT91AH1^(Y187A)with increased catalytic efficiency was obtained.The steroidal saponins exhibited remarkable antifungal activity against four widespread strains of human pathogenic fungi attributed to ergosterol-dependent damage of fungal cell membranes,and 2'-O-rhamnosylation appeared to correlate with strong antifungal effects.The findings elucidated the biosynthetic machinery for their production of steroidal saponins and revealed their potential as new antifungal agents.

Characterization of C_(30) carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1

Methylobacterium species,the representative bacteria distributed in phyllosphere region of plants,often synthesize carotenoids to resist harmful UV radiations.Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C_(30) backbone.However,its exact structure remains unknown.In the present study,the carotenoid produced by M.extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid(1),a glycosylated C_(30) carotenoid.Furthermore,the genes related to the C_(30)carotenoid synthesis were investigated.Squalene,the precursor of the C_(30) carotenoid,is synthesized by the co-occurrence of META1p1815,META1p1816 and META1p1817.Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1.By using gene deletion and gene complementation experiments,the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1.In conclusion,the structure and biosynthetic genes of carotenoid 1 produced by M.extorquens AM1 were firstly characterized in this work,which shed lights on engineering M.extorquens AM1 for producing carotenoid 1 in high yield.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases(UGTs)mediate the glycosylation of plant metabolites,thereby altering their physicochemical properties and bioactivities.Plants possess numerous UGT genes,with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics,but the biological function and molecular basis of these phenomena are not fully understood.The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition(Ki)at 4 mM scopoletin,whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 mM.We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs.A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reducedscopoletin substrate inhibition,whereas its monolignolgly cosylation activity was almost unaffected.Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation,leading to enhanced promiscuity,which was accompanied by substrate inhibition.Studies of 3D structures identified open and closed UGT conformers,allowing visualization of the dynamics of conformational changes that occur during catalysis.Previously postulated substrate access tunnels likely serve as drainage channels.The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release.Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions.The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Dietary fiber in plant cell walls-the healthy carbohydrates

Dietary fiber(DF)is one of the major classes of nutrients for humans.It is widely distributed in the edible parts of natural plants,with the cell wall being the main DF-containing structure.DF content varies significantly in different plant species and organs,and the processing procedure can have a dramatic effect on the DF composition of plant-based foods.Given the considerable nutritional value of DF,a deeper understanding of DF in food plants,including its composition and biosynthesis,is fundamental to the establishment of a daily intake reference of DF and is also critical to molecular breeding programs for modifying DF content.In the past decades,plant cell wall biology has seen dramatic progress,and such knowledge is of great potential to be translated into DF-related food science research and may provide future research directions for improving the health benefits of food crops.In this review,to spark interdisciplinary discussions between food science researchers and plant cell wall biologists,we focus on a specific category of DF--cell wall carbohydrates.We first summarize the content and composition of carbohydrate DF in various plant-based foods,and then discuss the structure and biosynthesis mechanism of each carbohydrate DF category,in particular the respective biosynthetic enzymes.Health impacts of DF are highlighted,and finally,future directions of DF research are also briefly outlined.

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase(SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase(SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides(ASGs). Initially,an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3β-and 17β-hydroxyl steroids. Unexpectedly,in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA,specifically catalyzing the acetylations of sugar moieties of steroid 17β-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-β-glucosides(AT-17β-Gs) and acetylated estradiol-17-O-β-glucosides(AE-17β-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides(SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 30-acetylated testosterone17-O-β-glucoside towards seven human tumor cell lines were thus observable.

A UV-B-responsive glycosyltransferase,OsUGT706C2,modulates flavonoid metabolism in rice

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.

UGT88B2: A promiscuous O-glycosyltransferase from Carthamus tinctorius

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.

Structural Insights into the CatalyticMechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

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.

O-glycosylation in liver cancer:Clinical associations and potential mechanisms

Liver cancer can be an aggressive disease,and is highly prevalent in Asia and Africa.However,its currently approved therapeutic strategies are far from satisfactory.Recent progress in genomic,proteomic and glycomic profiling technologies have enabled the identification of biomarkers that significantly correlate with clinical outcomes.Many biomarkers are related to O-glycosylation of glycoproteins,which belong to an important but less-explored field of liver cancer biology.Here,we review these clinical studies and discuss potential underlying mechanisms.

Discovery and Characterization of a Novel Method for Effective Improvement of Cyclodextrin Yield and Product Specificity

Cyclodextrins(CDs) are widely used in food,phamiaceuticals, drug delivery, and chemical industries and in agriculture and environmental engineering. To improve the yield and selectivity of CDs, this work presented a tacile, scalable and efficient enzymatic synthesis of β-CD from starch using β-cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19) from Bacillus cereus. First, we found that the pretreatment of starch dramatically influenced CDs yield that was related to the structure and molecular weight of the substrate starch. Second, alcohol solvents influenced the yield and product selectivity of CDs;tertiary alcohols enhanced CDs yield(from 54.95% to 68.21%) and secondary alcohols increased the product selectivity(β-CD/γ-CD changed from 6.25 to 8.05). Fluorescence quenching analysis showed that the binding constants and entropy of the solvents influenced the yield and product selectivity, respectively. In conclusion, the results demonstrate that this study provides a promising method for the industrial production of β-CD.

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins.However,plants produce complex N-glycans bearingβ1,2-xylose and coreα1,3-fucose residues,which are absent in mammals.The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy.In this study,we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L.cv Bright Yellow 2(BY2),which is a well-established cell line widely used for the expression of therapeutic proteins.The expression of the endogenous xylosyltranferase(XylT)and fucosyltransferase(FucT)was downregulated by using RNA interference(RNAi)strategy.The xylosylated and core fucosylated N-glycans were significantly,but not completely,reduced in the glycoengineered lines.However,these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype.Therefore,this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.