Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI , oleN2 and oleR ). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus . The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI , oleR and oleD genes were expressed in Streptomyces lividans . OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.     

Functional analysis of desVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation

The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.     

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.     

多烯大环内酯类抗生素生物合成基因簇中调控因子的研究进展

多烯大环内酯类抗生素具有良好的抗真菌活性,广泛应用于医疗卫生、食品加工和农业生产领域。随着高通量测序技术和生物信息学技术的发展,越来越多的链霉菌抗生素生物合成基因簇被发现和鉴定,调控因子作为生物合成基因簇中的重要组成部分,在庞大复杂的调控网络中起着至关重要的作用。本文总结了链霉菌中重要的调控因子类型,综述了多烯大环内酯类抗生素生物合成基因簇中调控因子的生物学功能、结合位点、作用机制等研究进展,并展望了后续研究工作。     

Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic.

A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently.     

A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.     

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native d-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as d-quinovosyl-10-deoxymethynolide, l-rhamnosyl-10-deoxymethynolide, l-olivosyl-10-deoxymethynolide, and d-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with l-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar d-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.     

Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase

Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.     

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics

BACKGROUND: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci. RESULTS: The glucosyltransferase, GtfB, that transfers the glucose residue from UDP-glucose to the 4-OH-Phegly4 residue of the vancomycin aglycone, initiating the glycosylation pathway in chloroeremomycin maturation, has been crystallized, and its structure has been determined by X-ray analysis at 1.8 A resolution. The enzyme has a two-domain structure, with a deep interdomain cleft identified as the likely site of UDP-glucose binding. A hydrophobic patch on the surface of the N-terminal domain is proposed to be the binding site of the aglycone substrate. Mutagenesis has revealed Asp332 as the best candidate for the general base in the glucosyltransfer reaction. CONCLUSIONS: The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.     

Functional analysis of OleY L-oleandrosyl 3-O-methyltransferase of the oleandomycin biosynthetic pathway in Streptomyces antibioticus

Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.     

Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism, Streptomyces antibioticus.

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.     

Identification and characterization of a new exopolysaccharide biosynthesis gene cluster from Streptomyces

We report the identification and characterization of the ste (Streptomyces eps) gene cluster of Streptomyces sp. 139 required for exopolysaccharide (EPS) biosynthesis. This report is the first genetic work on polysaccharide production in Streptomyces. To investigate the gene cluster involved in exopolysaccharide 139A biosynthesis, degenerate primers were designed to polymerase chain reaction amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in exopolysaccharide biosynthesis. Screening of a genomic library of Streptomyces sp. 139 with this polymerase chain reaction product as probe allowed the isolation of a ste gene cluster containing 22 open reading frames similar to polysaccharide biosynthesis genes of other bacterial species. Involvement of the ste gene cluster in exopolysaccharide biosynthesis was confirmed by disrupting the priming glycosyltransferase gene in Streptomyces sp. 139 to generate non-exopolysaccharide-producing mutants.     

Effects and mechanisms of action of polyene macrolide antibiotic nystatin on Babesia gibsoni in vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 μg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Carminic acid, a non-competitive inhibitor of kidney UDP-glucose:galactosylhydroxylysine-collagen glucosyltransferase

1. UDP-glucose:galactosylhydroxylsine-collagen glucosyltransferase was purified 12-fold from rat kidney. 2. An assay using calf-skin gelatin as substrate showed time- and enzyme-dependent incorporation; KmS for UDP-glucose and gelatin were 16-7 microM and 4.5 mg/ml, respectively. 3. Column chromatography of the alkaline hydrolysate of reaction product on Dowex 50W-4X(H+) showed that 84% of the radioactivity was in the glycosylgalactosylhydroxylsine peak. 4. Carminic acid inhibited collagen glycosyltransferase; a dose-dependent study showed a two-stage inhibition and kinetic analysis by double-reciprocal plots at varying UDP-glucose concentrations revealed a non-competitive mode of inhibition.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Enhanced production of heterologous macrolide aglycones by fed-batch cultivation of Streptomyces coelicolor

A media development program for the enhanced production of macrolide aglycones by Streptomyces coelicolor is described. Shake flask studies utilizing a yeast extract and a bakers' yeast increased production by 200% and 80%, respectively. However, ammonia generation and high pH were identified as potential problems in these enriched media. Studies in pH-controlled fermentors revealed that production stage pH significantly affects macrolide titers, with low pH (5.5) being more productive than high pH (6.5). Implementation of glucose feeding in shake flask cultures reduced ammonia generation and controlled production stage pH, resulting in significantly enhanced productivities. The combined effects of media supplementation and glucose feeding resulted in a three to five-fold overall improvement in total macrolide aglycone titers, and is the first reported high-level (>1 g/l) production of recombinant polyketides in a heterologous host. Journal of Industrial Microbiology & Biotechnology (2002) 28, 297–301 DOI: 10.1038/sj/jim/7000246 Received 06 August 2001/ Accepted in revised form 26 January 2002     

The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular- and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.     

A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens.

The Streptomyces mycarofaciens mdmB gene encodes a 3-O-acyltransferase that catalyzes the addition of acetyl and propionyl groups to position 3 of the lactone ring in 16-member macrolide antibiotics like midecamycin and spiramycin. A putative O-methyltransferase gene (mdmC) is immediately downstream of mdmB, and both of these genes are closely linked to the mdmA midecamycin resistance gene.