Deoxysugar Moieties Research Articles

Bioprospecting potentials of moderately halophilic bacteria and the isolation of squalene producers from Kuwait sabkha.

Sabkhas in Kuwait are unique hypersaline marine environments under-explored for bacterial community composition and bioprospecting. The 16S rRNA sequence analysis of 46 isolates with distinct morphology from two Kuwait sabkhas recovered 11 genera. Phylum Firmicutes dominated these isolates, and Bacillus (32.6%) was recovered as the dominant genera, followed by Halococcus (17.4%). These isolates were moderately halophilic, and some of them showed tolerance and growth at extreme levels of salt (20%), pH (5 and/or 11), and temperature (55 °C). A higher percentage of isolates harbored protease (63.0), followed by DNase (41.3), amylase (41.3), and lipase (32.6). Selected isolates showed antimicrobial activity against E. faecalis and isolated Halomonas shengliensis, and Idiomarina piscisalsi harbored gene coding fordNDP-glucose 4,6-dehydratase (Glu 1), indicating their potential to produce biomolecules with deoxysugar moieties. Palmitic acid or oleic acid was the dominant fatty acid, and seven isolates had some polyunsaturated fatty acids (linolenic or γ-linolenic acid). Interestingly, six isolates belonging to Planococcus and Oceanobacillus genus produced squalene, a bioactive isoprenoid molecule. Their content increased 30-50% in the presence of Terbinafine. The potential bioactivities and extreme growth conditions make this untapped bacterial diversity a promising candidate for future bioprospecting studies.

A chiral template-driven synthesis of a 3′-deoxy-3′-18F-fluorothymidine precursor

An asymmetric synthesis of a 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) precursor has been developed wherein the deoxysugar moiety was synthesized using a novel Ga-mediated allylation of (R)-2,3-cyclohexylideneglyceraldehyde as the key step. The synthesis deviates significantly from the previous syntheses of the 18F-FLT precursors wherein the expensive starting material, thymidine was used.

One-Pot Combinatorial Biosynthesis of Glycosylated Anthracyclines by Cocultivation of Streptomyces Strains Producing Aglycones and Nucleotide Deoxysugars.

Anthracyclines, such as doxorubicin, are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Cocultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that coexpress a substrate-flexible glycosyltransferase led to the generation of 16 aklavinone or ε-rhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on cocultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development.

New insights into paulomycin biosynthesis pathway in Streptomyces albus J1074 and generation of novel derivatives by combinatorial biosynthesis

BackgroundStreptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.ResultsInactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.ConclusionsThe paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure–activity relationships.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0452-4) contains supplementary material, which is available to authorized users.

Inactivation and identification of three genes encoding glycosyltransferase required for biosynthesis of nogalamycin.

Nogalamycin is an anthracycline antitumor antibiotic, consisting of the aromatic aglycone attached with a nogalose and a nogalamine. At present, the biosynthesis pathway of nogalamycin, especially the glycosylation mechanism of the two deoxysugar moieties, had still not been extensively investigated in vivo. In this study, we inactivated the three glycotransferase genes in the nogalamycin-produced strain, and investigated the function of these genes by analyzing the metabolites profiles in the fermentation broth. The in-frame deletion of snogD and disruption of snogE abolished the production of nogalamycin completely, indicating that the gene products of snogD and snogE are essential to the biosynthesis of nogalamycin. On the other hand, in-frame deletion of snogZ does not abolish the production of nogalamycin, but production yield was reduced to 28% of the wild type, implying that snogZ gene may involved in the activation of other glycotransferases in nogalamycin biosynthesis. This study laid the foundation of modification of nogalamycin biosynthesis/production by genetic engineering methods.

Spectrophotometric Investigations of Macrolide Antibiotics: A Brief Review

Macrolides, one of the most commonly used class of antibiotics, are a group of drugs produced by Streptomyces species. They belong to the polyketide class of natural products. Their activity is due to the presence of a large macrolide lactone ring with deoxy sugar moieties. They are protein synthesis inhibitors and broad-spectrum antibiotics, active against both gram-positive and gram-negative bacteria. Different analytical techniques have been reported for the determination of macrolides such as chromatographic methods, flow injection methods, spectrofluorometric methods, spectrophotometric methods, and capillary electrophoresis methods. Among these methods, spectrophotometric methods are sensitive and cost effective for the analysis of various antibiotics in pharmaceutical formulations as well as biological samples. This article reviews different spectrophotometric methods for the determination of macrolide antibiotics.

Bioconversion of deoxysugar moieties to the biosynthetic intermediates of daunorubicin in an engineered strain of Streptomyces coeruleobidus.

Daunorubicin (DNR) is a representative anthracycline with anti-tumor bioactivity. Its convergent biosynthetic pathway has promoted the research on pursuing novel anthracyclines by combinatorial biosynthesis. SnoaL is a special polyketide cyclase that catalyzes the closure of nogalonic acid methyl ester with the C9-S stereochemistry. In this study, the gene cluster of DNR was cloned, and snoaL was integrated into the DNR biosynthetic pathway for the substitution of dnrD in Streptomyces coeruleobidus DM, which resulted in the production of epi-aklaviketone. The biosynthetic pathway of NDP-4-deacetyl-L-chromose B was then expressed in the engineered strain, which led to the production of corresponding glycosylated anthracycline compounds. Finally, the bioactivities of these engineering strains were evaluated.

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics

BackgroundThe macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases. MethodsThe inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction. ResultsWhen individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT≥1μM. The TBT-driven enzyme desensitization was reversed by DTE. ConclusionsThe macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT. General significanceThe findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.

Biosynthesis of tetronate antibiotics: A growing family of natural products with broad biological activities

Tetronate antibiotics, a growing family of natural products featuring a characteristic tetronic acid moiety, are of importance and of particular interest for their typical structures, especially the spirotetronate structure, and corresponding versatile biological activities. Considerable efforts have persistently performed since the first tetronate was isolated, to elucidate the biosynthesis of natural tetronate products, by isotope-labeled feeding experiments, genetical characterization of biosynthetic gene clusters, and biochemical reconstitution of key enzymatic catalyzed reactions. Accordingly, the biosynthesis of spirotetronates has been gradually determined, including biosynthesis of a polyketide-derived backbone for spirotetronate aglycone, incorporation of a glycerol-derived three-carbon unit into tetronic acid moiety, formation of mature aglycone via Diels-Alder-like reaction, and decorations of aglycone with various deoxysugar moieties. In this paper, the biosynthetic investigations of natural tetronates are well documented and a common biosynthetic route for this group of natural products is summarized accordingly.

Chemobiosynthesis of New Antimalarial Macrolides

We have synthesized new derivatives of the macrolide antibiotics erythromycin and azithromycin. Novel deoxysugar moieties were attached to these standard antibiotics by biotransformation using a heterologous host. The resulting compounds were tested against several standard laboratory and clinically isolated bacterial strains. In addition, they were also tested in vitro against standard and drug-resistant strains of human malaria parasites (Plasmodium falciparum) and the liver stages of the rodent malaria parasite (Plasmodium berghei). Antibacterial activity of modified erythromycin and azithromycin showed no improvement over the unmodified macrolides, but the modified compounds showed a 10-fold increase in effectiveness after a short-term exposure against blood stages of malaria. The new compounds also remained active against azithromycin-resistant strains of P. falciparum and inhibited growth of liver-stage parasites at concentrations similar to those used for primaquine. Our findings show that malaria parasites have two distinct responses to macrolide antibiotics, one reflecting the prokaryotic origin of the apicoplast and a second, as-yet uncharacterized response that we attribute to the eukaryotic nature of the parasite. This is the first report for macrolides that target two different functions in the Plasmodium parasites.

Two bifunctional enzymes, MTM GIV and MTM C, involved in the biosynthesis of deoxysugar moieties of the antitumor antibiotic mithramycin

: Mithramycin is an aureolic acid-type anticancer agent consisting of a polyketide core that is decorated with disaccharide and trisaccharide units. Previous studies have shown that two gene products, MtmGIV (a putative glycosyltransferase) and MtmC (a putative C-methyltransferase), are indispensible for the assembly of the complex trisaccharide component. However, their exact functions remained unclear. Using in vitro reconstitution, these two enzymes are now shown to both have bifunctional activities and hence dual roles in the biosynthesis of mithramycin. MtmC is demonstrated to function as both a 4-ketoreductase and 3-C-methyltransferase, with each activity representing a branching point from TDP-4-keto-D-olivose to yield TDP-D-olivose and TDP-D-mycarose, respectively. Both sugars then serve as donor substrates for MtmGIV, a bifunctional glycosyltransferase that incorporates the first sugar (D-olivose) and the last (D-mycarose) into two distinct acceptor substrates to yield the trisaccharide component of mithramycin. The discovery of these dual enzyme functions explains two previously missing activities that are essential for mithramycin biosynthesis that were not readily predicted from bioinformatic analysis of the gene products.

Influence of 2′‐Deoxy Sugar Moiety on Excited‐State Protonation Equilibrium of Adenine and Adenosine with Acridine inside SDS Micelles: A Time‐Resolved Study with Quantum Chemical Calculations

The protonation dynamics of the DNA base adenine (Ade) and its nucleoside 2'-deoxyadenosine (d-Ade) are investigated by monitoring the deprotonation kinetics of an N-heterocyclic DNA intercalator, acridine (Acr), in the confined environment of sodium dodecyl sulfate (SDS) micelles. Protonation of acridine (AcrH(+)) occurs at the hydrophilic interface and this species remains in dynamic equilibrium with its deprotonated counterpart (Acr) inside the hydrophobic core of SDS micelles. Quenching of the fluorescence of AcrH(+)* at 478 nm is observed after addition of Ade and d-Ade with Stern-Volmer constant (K(SV)) 298 and 75 M(-1), respectively, with a concomitant increment in Acr* at 425 nm. Time-resolved fluorescence studies reveal quenching in the lifetime of AcrH(+)*. The relative amplitude of AcrH(+)* decreases from 0.97 to 0.51 and 0.97 to 0.89 with equimolar addition of Ade and d-Ade, respectively. These observations are explained by excited-state proton transfer (ESPT) from AcrH(+)* to the bases. The reduced K(SV) value and negligible change in the relative amplitudes of AcrH(+)* with d-Ade infer that ESPT is hindered substantially by the presence of a 2'-deoxy sugar unit. Transient time-resolved absorption spectra of Acr reflect that Ade reduces the absorbance of (3)AcrH(+)*; however, d-Ade keeps it unaltered for more than a time delay of 2 μs. The optimized geometries calculated by quantum chemical methods reflect deprotonation of AcrH(+)* with protonation at the N1 position of Ade, while it remains protonated with d-Ade. The hindered ESPT between AcrH(+)* and d-Ade singles out the significance of the 2'-deoxy sugar moiety in controlling the deprotonation kinetics.

Identification of Late‐Stage Glycosylation Steps in the Biosynthetic Pathway of the Anthracycline Nogalamycin

Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C-C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C-C bond intact (nogalamycin R) or by 2-deoxyfucose without the C-C bond (nogalamycin F). In addition, a monoglycosylated intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the cluster-snogZ-appears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

Caprazamycins are antimycobacterials produced by Streptomyces sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in Streptomyces coelicolor M512 with pRHAM, containing all the required genes for dTDP-l-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the l-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An l-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of cpzDIII. The l-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of cpzDI and cpzDV in the l-rhamnose subcluster confirmed that four genes, cpzDII, cpzDIII, cpzDIV, and cpzDVI, are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.

Biological Activities of Novel Gyrase Inhibitors of the Aminocoumarin Class

Thirty-one aminocoumarin antibiotics derived from mutasynthesis experiments were investigated for their biological activities. Their inhibitory activities toward Escherichia coli DNA gyrase were determined in two different in vitro assays: an ATPase assay and a DNA supercoiling assay. The assays gave a similar rank order of the activities of the compounds tested, although the absolute 50% inhibitory concentrations (IC(50)s) obtained in each assay were different. To confirm that the compounds also acted as gyrase inhibitors in vivo, reporter gene assays were carried out with E. coli by using gyrA and sulA promoter fusions with the luxCDABE operon. A strong induction of both promoters was observed for those compounds that showed gyrase inhibitory activity in the biochemical assays. Compounds carrying analogs of the prenylated benzoyl moiety (ring A) of clorobiocin that were structurally very different showed high levels of activity both in the biochemical assay and in the reporter gene assay, indicating that the structure of this moiety can be varied considerably without a loss of affinity for bacterial gyrase. The experimentally determined IC(50)s were compared to the binding energies calculated in silico, which indicated that a shift of the pyrrole carboxylic acid moiety from the O-3'' to the O-2'' position of the deoxysugar moiety has a significant impact on the binding mode of the compounds. The aminocoumarin compounds were also investigated for their MICs against different bacterial pathogens. Several compounds showed high levels of activity against staphylococci, including a methicillin-resistant Staphylococcus aureus strain. However, they showed only poor activities against gram-negative strains.

Improved Mutasynthetic Approaches for the Production of Modified Aminocoumarin Antibiotics

This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1H-NMR spectroscopy.

Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1H-NMR and mass spectroscopy and their antibacterial activity was assayed.

Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes

Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.