GlycosideAntibiotic analogs are widely used antibacterial and anti-tumor compounds in clinic.These compounds are catalyzed by glycosyltransferases in vivo, and glycosylation is usually carried out in antibioticsbiosynthesisThe position, type and quantity of glycosyl groups have a great influence on the activity of glycoside antibiotics.This paper reviews the types, functions, characteristics and applications of glycosyltransferases incombinatorial biosynthesisThe application and research prospect of.
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Glycosyltransferase of antibiotics glycosylation of antibiotic glycosides
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Recent advances in antibiotic glycosyltransferases ABSTRACT Glycoside antibiotics, a category of compounds widely used clinically for anti?bacterial and anti?cancer, are catalyzed by antibiotic glycosyltransferases (Gtfs) in vivo. The sugar moieties are transferred to the corresponding aglycon by Gtfs, often work at very late stages of biosynthesis of antibiotics. The position, type and number of sugar moieties incorporated to the antibiotics have great impact on its bioactivity. This article provides an overview of the categories, functions, characteristics of Gtfs, their applications in combinatorial biosynthesis, and the prospects for research.
KEY WORDS Antibiotic glycosyltransferase; Glycoside antibiotics; Glycosylation
antibioticGlycosideIt is mainly used for antibacterial and antitumour, in antibioticsbiosynthesisMany codes have been found in the gene clusterGlycosylThe gene of transferases [1], but people'sSpecificityandCatalytic mechanismNot much.Glycosyl and differentLigandsOfbinding energyGreatly increasednatural productIn terms of function, these sugar components usually participate in themolecular recognition, affecting compoundsbiological activity[2]。At present, with the widespread use of antibiotics,Drug resistant bacteriaIt is also increasing year by year, and it is urgent to find new antibiotics to fight against it.adoptGlycosylationIt is a promising way to increase the types of antibiotics and change the activity of antibiotics. Exploring the production mechanism of glycoside antibiotics and the catalytic characteristics of glycosyltransferase is expected to lay the foundation for the discovery and modification of new active antibiotics.
1 Glycoside antibiotics
Many glycoside antibiotics have experienced stereo andRegional selectivityGlycosylation in thisBiological reactionThe glycosyltransferase catalyzes the glycosyl group in its structure tomonosaccharide、disaccharideandoligosaccharideThe form of chain is combined intoLigandsSo as to form a specific CN?And O?Glycosides (Fig. 1).GlycosidicBiosynthetic pathwayGlycosylation is usually the last step of post modification, such asvancomycin(vancomycin)、Tekoplanin(teicoplanin)sugar chainAre introduced at the end of biosynthesis [3].Fig.1 The C?, N? and O?glycoside antibiotics
twoThe Main Functions of Glycosyl in Glycoside Antibiotics
The role and significance of glycosylation are mainly reflected in the following three aspects: first, increase theWater solubility。HexoseDerivative binding to antibioticsGlycoside ligandAbove, antibioticHydrophilicityIt is beneficial to the exertion of drug effect, and the typical N?Acetylglucosamine(N?acetyl?Glucosamine, GlcNAc) and remoraninemannoseChain;Second, it is conducive to secretion.A40926Mannosyltransferase(mannosyltransfer) specifically recognize mannosyl?PP?As a sugar donor, C55 facilitates the secretion of glycosylated antibiotics [6].Third, glycosylation isProducing bacteriumOne ofSelf protection mechanism。stayZhutaomicinDuring the production of oleandomycin, the glycosyltransferase OleD makes the intermediate antibioticGlycosylation, causing it to be short in the cellInactivationAfter the secretion of antibioticsGlycosidaseHydrolysis removes sugar groups and restores their activity [7].This mechanism is available inMacrolide antibioticsMgtA [8] is a common enzyme with similar functions.
3 Glycosyltransferase
Glycosyltransferases catalyze the connection of activated sugars to differentReceptor molecule, such as proteinnucleic acid、oligosaccharide, grease andSmall moleculeThe glycosylation products have many biological functions.In different Gtfs families, there is a class of antibioticsbiosynthesisThe related Gtfs, whose function is to glycosylate antibiotics in the late stage of biosynthesis, regulate the activity of antibiotics by changing the position, type and quantity of sugars.
With the biosynthesis of antibioticsgene clusterIn-depth research fromActinomycetesMore than 100 glycosyltransferases genes related to antibiotic biosynthesis have been isolated in. Sequence analysis shows that the proteins encoded by these genes belong to the glycosyltransferases family.Most glycosyltransferase genes haveC-endIs rich inglycine(glycine? Rich), which also exists in UDP?Sugar and UDP?Glucuronosyltransferase.choiceGenBankRegistered inRepresentativeness119 glycosyltransferase sequences were systematically analyzed with PAUP 4.0 software, using the nearest neighbor methodBuild systemEvolutionary tree(Fig.2)。PhylogeneticAnalysis resultsIt indicates that the glycosyltransferase genegenetic relationshipThe biological function of EryC Ⅲ and MegC Ⅲ can not be accurately inferred [9]amino acidIt has 83.4% consistency at the horizontal level and can recognize the same ligand. UrdGT1b and UrdGT1c show highHomology(There are 91% identical amino acids and only a few different amino acids in 31 amino acid regions), but they transport different hexoses. UrdGT1c transfers L?Oleander (L? Rhodinose), UrdGT1b transfers D?Olivose [10].Catalytic C?C、C?N and C?OGlycosidic bondThe formed glycosyltransferase has no obvious difference in gene sequence and amino acid level, such as Asm25(AmphotericinamideCatalytic C?Gtfs formed by N-glycosides) [9, 11], UrdGT2 (UdaMycomycinCatalytic C?Gtfs formed by C-glycosides) [12], GtfB (Gtfs catalyzing the formation of C-O glycosides in the post modification of vancomycin biosynthesis) [13].
In the post modification process of vancomycin, the function of Fig. 2 A dendrogram of different antioxidant glycosyltransfers of the glycosyltransferase GtfB is to integrate UDP?glucoseInGlucosylTransfer to the vancomycin frameworkcrystal structureThe results showed that the enzyme has twoDomain, the ditch in the middle area may contain UDP?Glucose binding region [13].The structure determination of glycosyltransferase will help us to further study its catalytic specificity.
The two significant characteristics of Gtfs are: firstbiosynthesisThe process finally works, which makes itcombinatorial biosynthesisThe elucidation of the substrate structure specificity can lay a foundation for the discovery of new structures and new functional compounds.In the Gtfs catalyzed glycosyl transfer reaction, C1 of hexose passes throughphosphoric acidnucleosideIs activated by dephosphorylation, so as to capture the glycosyl substrate at the electrophilic C1 position, and then through nucleophilic attackGlycoside ligandHydroxyl group binding.Second, most glycosyltransferases can catalyze the binding of activated hexose to the hydroxyl group of the glycoside ligand substrate to form C?O glycosides, with only a few exceptions, such as in derivatives of rebeccamycin [14] and urdamycin [13], there are sugar groups passing through C?N and C?Compounds with C-bond and glycosidic ligand.YMG of amphotericinPlate cultureThe fermentation product was isolated to obtain an amphotericin amide N glycoside compound, and the sugar group in its structure passed throughAmide bondN on is linked to the glycoside ligand skeleton [11].In the biosynthesis of urdamycin, the nucleophilic C atom andPhenolsThe hydroxyl group ofAdjacencyCause C?C glycoside bond formation [12], but catalyzes C?N and C?The specificity of C-formed glycosyltransferases has not been further studied.
3.1 Application research of Gtfs
(1) In vivo research of Gtfs is mainly carried out by genetic methods, one of which is through plasmid mediated genesAlternate hostTDP of bacteria?DeoxyhexosesynthetaseThe mutants were used as cell factories for the transformation of antibiotics.These studies include engineeringdaunomycin(Daunomycin)[15] Approach forPicamycin(pikromycin) producer S.venezuleae and urdamycin producer S.fradiae?EpimerismWith different sugar unitsAnthracycline antibiotics[16,17]。
The other way is in different hostsEctopic expressionGtfs, examples of this include the expression of oleGII gene in the host S.erythraea to produce 3?O?rhamnosyl?6?DEB, expression of tylM2 gene produce 5?O?desosaminyl(tylactone)[18]。
Research shows that Gtfs has substrate adaptability to different sugar molecules. Salas' research team has created a co expression system, which is derived from ellioramycinbiosynthesisgene clusterThe glycosyl synthesis gene cassette (Cassette) and the glycosyltransferase gene elMGT were successfully expressed together in [19].Although the broad-spectrum nature of many glycosyltransferase substrates has been proved, there are still many reports on the existence of specific glycosyltransferase genes, such as UrdGT2 [20] in C. cyanogeneus and NovM [21] from S. spheroid NCIMB11891.Undoubtedly, with the discovery of more Gtfs coding genes and the in-depth study of the mechanism of action, the strategies and modes of glycosylation of antibiotics in vivo will also increase.Therefore, establish diversified microbial factories to produce different glycosyl modifiednatural product, such as expressing glycosylatedPolyketone(PK), non ribosomal peptide (NRP) and hybrid PK/NRP antibiotics can provide the possibility for screening new active compounds.
(2) In vitro study of Gtfs In vitro study of Gtfs depends on active Gtfs, diversifiedGlycoside ligandAnd glycosyl donors.Because the content of antibiotic glycosyltransferase in vivo is very low and the heterologous expression operation is relatively simple, researchers often use heterologousExpressionIn microorganisms such asE.coliOr heterologous expression in S.livans [22].There have been several successful examples, for example, NovM was found and cloned in S.spheroids for the first time, and expressed and purified in E.coli in its active form [21].Through chemical synthesis andBiological enzymeCatalytic method to generate TDP?D?Glucose is possible [23].In comparison, glycosidic ligands are the easiest to obtain, and their production can be controlled by the degradation of parent antibiotics. For example, the corresponding glycosidic ligands are easily obtained from vancomycin and teicoplanin, and novobionic acid can be obtained fromAcid catalysisNeomycinFrom the reaction.In addition, different ligands can also be obtained by total synthesis or partial modification.For example, chemical synthesis of daunomycin and carminomycin(Erythromycin)AndBleomycin(bleomycin) derivatives [23~25].
The application of genetic methods to produce new polyketide and polypeptide compounds has attracted increasing attentionbiosynthesisGlycosylationIts compounds are as complex as polyketide and polypeptide, but compared with the complexity of polyketide and polypeptide synthetase, the enzyme that catalyzes deoxygose production and its reaction mechanism are relatively conservative, so the recombination of glycosylated compounds has more practical significance [25].
Spain's Salas Research Group has established a successfulGene cloningAnd expression systems used to produce activated deoxygenated sugars,Target genebe inOperonDownstream of, viaPromoterCan be controlled inStreptomycesExpressed in.Integrate the glycosyltransferase gene oleGII in Streptomyces albus, and introduce the synthetic L?After the plasmid of oleandroseerythromycinlactoneB (erythronolide B), while elmGT gene is integrated into biosynthetic L?The plasmid pOLV of L olivose can successfully produce tetracenomycin C [26, 27].
The structure of staurosporine compounds is composed of a sugar molecule and a heterocycleindoleThe composition of the carbazole unit is difficult to obtain by chemical means. By co expressing the biosynthetic genes of rebeccamycin and other staurosporine compounds in one organism, about 30 staurosporine derivatives were isolated and identified [28].adoptgeneticsMethods It is difficult to change the specificity of Gtfs. Salas research group co expressed glycosylbiosynthesisThe biosynthetic genes of gene box, Gtfs gene (staN and staG) and staurosporine have successfully replaced the natural glycosyl groups attached to indole carbazole groups, laying the foundation for the application of glycosyltransferase in recombinant biosynthesis, proving that recombinant biosynthesis is more beneficial than pharmaceutical chemistry in the production of new glycosylation products [16].
4.1 Use of producing bacteria as cell factory
Using the microorganism producing glycoside compounds as a cell factoryCoding areaBy insertingAntibiotic resistanceMark or delete some genes for knockout to produce mutants and introduce themExogenousGlycosyltransferase gene, which makes use of activated sugar molecules and microorganisms in cellssecondary metabolismSynthesis of new antibiotics from intermediates of.This method has been used in many production strains such as erythromycin [29]Pucamycin(plicamycin)、urdamycin、Jiumycin(methylmycin) [29] andPicamycin(pikro? Mycin).
Another strategy to promote the binding of different sugar units to ligands is to produce similarbiological activityCompoundOrganismGtfs is heterologously expressed on the host, which is provided as a cell factorynucleosideActiveSugar source。Ligand skeletons can be synthesized by the host or controlled by theChromosome geneandforeign geneTo synthesize or feed with compounds.A new glycosyl A47934 derivative was formed by expressing the gtrE gene from vancomycin producing strain A. orientalis in Streptomyces S. toyocaensis, which does not produce the glycosyl polypeptide A47934 [17].
Gtfs transports different sugars to specific C atoms of the ligand skeleton, andgenetic engineeringIt is more meaningful to change the position of sugar on the skeleton by the method ofAntineoplastic drugs[30]。
4.2 Use non producing bacteria as cell factory
In the transformation experiment of recombinant strain, the host does not produceLigandsSkeleton, it is necessary to integrate a plasmid with ligand skeleton biosynthesis gene to the host or add the ligand toculture mediumMedium, viaBiotransformationDecorate.Sugar molecules are provided by transferring one or more plasmids containing necessary genes. Now some plasmids that can synthesize different deoxyhexaose have been developed as an important tool [31].
Research prospect of glycosyltransferase
Recently, onGlycopeptideGtfscrystalThe structural determination shows that this kind of Gtfs family hasTwo in commonOfDomain。NDP?Sugar bound to C?End,Glycoside ligandCombined to N?End (AGV/GtfB, DVV/GtfA) [13].This bifid structure is only connected by two peptides, suggesting that it is possible to mix and match their respective domains [32, 33].Therefore,DNA shufflingOr related enzymesDirected evolutionIt can construct the seemingly absurd Gtfs, change its substrate specificity for hexose units and ligands, greatly improve the structural diversity of glycoside compounds, and lay the foundation for screening new active glycoside antibiotics.
In conclusion, there are three requirements for studying the diversity of glycosylation modes in biosynthesis.
(1) To establish a glycosyltransferase library and produce new antibiotic glycosides through recombinant biosynthesis, it is necessary to establish ageneSo that it can be applied in industry.With more gene clustersSequencingThe number of Gtfs will also increase year by year. It is found that N?Or C?The Gtfs region of the terminal is used to construct a hybrid catalytic system, change the recognition of ligands and deoxyglycans, and find new deoxyglycans for specific deoxyglycansbinding site[34]。
(2) Establish a library of ligands These compounds include simple amino coumarin scaffolds, non ribosomal peptides (NRP), and aromatic polyketone ligands [25].However, similarEnzyme catalysisConduct C?N,C?C-glycosylation needs further research.
(3) To establish a compound library of sugar donors, sugar donors should include many UDP or TDP activated sugars and deoxygenated sugars.The addition of natural deoxygenated sugar to the ligand endows the ligand with new activity.Ammonia of deoxysaccharideFormylChemistry exists in many types of compounds such as polyketide (novobiocin), non ribosomal peptide (teicoplanin) and other antibiotics, so all TDP?D?And TDP?L?Deoxyhexose derivatives are worth preparing in the library [32, 33].
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So far, many antibiotics have been foundbiosynthesisRelated glycosyltransferases, but the existing research is not in-depthBiological significance, glycosyltransferase structure andFunctional relationship, catalytic mechanism, activation form of glycosides andcombinatorial biosynthesisThe application of.Shen Yuemao's research team found that glycosylation was related to the growth conditions of microorganismsPlate cultureThe glycosylation products produced atliquid cultureBut cannot be detected.The similarities and differences between glycosyltransferases that catalyze the formation of three different types of glycosides in terms of their mechanism of action and substrate selectivity depend on the elucidation of the advanced structure of different types of glycosyltransferases. There is still a long way to go before they are practical and industrialized.
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