The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.     

Engineering deoxysugar biosynthetic pathways from antibiotic-producing microorganisms. A tool to produce novel glycosylated bioactive compounds

A plasmid (pLN2) was generated in which genes involved in the biosynthesis of L-oleandrose in the oleandomycin producer Streptomyces antibioticus ATCC11891 were cloned. pLN2 was used to direct the biosynthesis of different deoxysugars by exchanging and/or adding genes from other antibiotic biosynthetic clusters. Transfer of the synthesized deoxysugars to the tetracenomycin C aglycon, 8-demethyl-tetracenomycin C, through the use of the "sugar flexible" glycosyltransferase ElmGT, validated the system. Several pLN2 derivatives were constructed by replacement of the oleU 4-ketoreductase gene by different 4-ketoreductase genes. Some of them, such as EryBIV and UrdR, reduced the keto group of the 4-keto intermediates, generating L-olivosyl and D-olivosyl derivatives, respectively. The system was also used to generate an L-rhamnosyl derivative (through a two-gene deletion) and an L-rhodinosyl derivative (through a combination of a gene replacement and a gene addition).     

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.     

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.     

Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin

BACKGROUND: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.     

A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of epsilon-rhodomycinone to rhodomycin D

BACKGROUND: The biological activity of many microbial products requires the presence of one or more deoxysugar molecules attached to agylcone. This is especially prevalent among polyketides and is an important reason that the antitumor anthracycline antibiotics are avid DNA-binding drugs. The ability to make different deoxyaminosugars and attach them to the same or different aglycones in vivo would facilitate the synthesis of new anthracyclines and the quest for antitumor drugs. This is feasible using the numerous bacterial genes for deoxysugar biosynthesis that are now available. RESULTS: Production of thymidine diphospho (TDP)-L-daunosamine (dnm), the aminodeoxysugar present in the anthracycline antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), and its attachment to epsilon-rhodomycinone to generate rhodomycin D has been achieved by bioconversion with a strain of Streptomyces lividans that bears two plasmids. One contained the Streptomyces peucetius dnmJVUZTQS genes plus dnmW (previously named dpsH and considered to be a polyketide cyclase gene), dnrH, which is not required for the formation of rhodomycin D, and dnrI, a regulatory gene required for expression of the dnm and drr genes. The other plasmid had genes encoding glucose-1-phosphate thymidylyltransferase and TDP-glucose-4,6-dehydratase (dnmL and dnmM, respectively, or mtmDE, their homologs from Streptomyces agrillaceus) plus the drrAB DNR/DXR resistance genes. CONCLUSIONS: The high-yielding glycosylation of the aromatic polyketide epsilon-rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for L-daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.     

The neocarzinostatin biosynthetic gene cluster from Streptomyces carzinostaticus ATCC 15944 involving two iterative type I polyketide synthases

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.     

Characterisation of Streptomyces spheroides NovW and revision of its functional assignment to a dTDP-6-deoxy-D-xylo-4-hexulose 3-epimerase

Characterisation of recombinant Streptomyces spheroides NovW in vitro suggests that it is not a kinetically competent dual action dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase, but possesses only significant 3-epimerase activity.     

Synthesis,bioactivities and 3D-QSAR of novel avermectin B2a aglycon derivatives

Fourteen avermectin B2 a aglycon derivatives were designed and synthesized after removing the oleandrose disaccharide of avermectin B2 a.Their structures were characterized by’H NMR,13 C NMR,HMRS.Preliminary bioassays indicated that these compounds exhibited good insecticidal activity against diamondback moth at 200 mg/L,with mortality no less than 90%.Compounds 10 b,12 a,12 c,17 demonstrated good acaricidal activity against the adult mites,larvae,and good inhibition rate of hatching to mite eggs of Tetranychus cinnabarinus.Compounds 5,10 b,10 c exhibited excellent fungicidal activity against fourteen fungal pathogens in vitro.3 D-QSAR analysis showed that the fungicidal activity of avermectin B2 a aglycon derivatives would be increased when a negatively charged and bulky group was introduced at 13-position,which will be instructive for the further modification of avermectin B2 aaglycon.     

Ketopremithramycins and ketomithramycins, four new aureolic acid-type compounds obtained upon inactivation of two genes involved in the biosynthesis of the deoxysugar moieties of the antitumor drug mithramycin by Streptomyces argillaceus, reveal novel insights into post-PKS tailoring steps of the mithramycin biosynthetic pathway

Mithramycin is an aureolic acid-type antimicrobial and antitumor agent produced by Streptomyces argillaceus. Modifying post-polyketide synthase (PKS) tailoring enzymes involved in the production of mithramycin is an effective way of gaining further information regarding the late steps of its biosynthetic pathway. In addition, new "unnatural" natural products of the aureolic acid-type class are likely to be produced. The role of two such post-PKS tailoring enzymes, encoded by mtmC and mtmTIII, was investigated, and four novel aureolic acid class drugs, two premithramycin-type molecules and two mithramycin derivatives, were isolated from mutant strains constructed by insertional gene inactivation of either of these two genes. From data bank comparisons, the corresponding proteins MtmC and MtmTIII were believed to act as a C-methyltransferase involved in the production of the D-mycarose (sugar E) of mithramycin and as a ketoreductase seemingly involved in the biosynthesis of the mithramycin aglycon, respectively. However, gene inactivation and analysis of the accumulated products revealed that both genes encode enzymes participating in the biosynthesis of the D-mycarose building block. Furthermore, the inactivation of MtmC seems to affect the ketoreductase responsible for 4-ketoreduction of sugar C, a D-olivose. Instead of obtaining premithramycin and mithramycin derivatives with a modified E-sugar upon inactivation of mtmC, compounds were obtained that completely lack the E-sugar moiety and that possess an unexpected 4-ketosugar moiety instead of the D-olivose at the beginning of the lower deoxysaccharide chain. The inactivation of mtmTIII led to the accumulation of 4E-ketomithramycin, showing that this ketoreductase is responsible for the 4-ketoreduction of the D-mycarose moiety. The new compounds of the mutant strains, 4A-ketopremithramycin A2, 4A-keto-9-demethylpremithramycin A2, 4C-keto-demycarosylmithramycin, and 4E-ketomithramycin, indicate surprising substrate flexibility of post-PKS enzymes of the mithramycin biosynthetic pathway. Although the glycosyltransferase responsible for the attachment of D-mycarose cannot transfer the unmethylated sugar to the existing lower disaccharide chain, it can transfer the 4-ketoform of sugar E. In addition, the glycosyltransferase MtmGIV, which is responsible for the linkage of sugar C, is also able to transfer an activated 4-ketosugar. The oxygenase MtmOIV, normally responsible for the oxidative cleavage of the tetracyclic premithramycin B into the tricyclic immediate precursor of mithramycin, can act on a substrate analogue with a modified or even incomplete trisaccharide chain. The same is true for glycosyltransferases MtmGI and MtmGII, both of which partake in the formation and attachment of the A-B disaccharide in mithramycin.     

The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives

Rebeccamycin, a halogenated natural product of the indolocarbazole family, is produced by Saccharothrix aerocolonigenes ATCC39243. Several rebeccamycin analogues, which target DNA topoisomerase I or II, have already entered clinical trials as anticancer drugs. Using as a probe an internal fragment of ngt, a Saccharothrix aerocolonigenes gene encoding an indolocarbazole N-glycosyltransferase, we isolated a DNA region that directed the biosynthesis of rebeccamycin when introduced into Streptomyces albus. Sequence analysis of 25.6 kb revealed genes for indolocarbazole core formation, halogenation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. Heterologous expression of subsets of these genes resulted in production of deschloro-rebeccamycin, 4'-demethyldeschloro-rebeccamycin, and deschloro-rebeccamycin aglycone. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and set the stage for the generation of novel indolocarbazole analogues by genetic engineering.     

A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of -rhodomycinone to rhodomycin D

Background: The biological activity of many microbial products requires the presence of one or more deoxysugar molecules attached to agylcone. This is especially prevalent among polyketides and is an important reason that the antitumor anthracycline antibiotics are avid DNA-binding drugs. The ability to make different deoxyaminosugars and attach them to the same or different aglycones in vivo would facilitate the synthesis of new anthracyclines and the quest for antitumor drugs. This is feasible using the numerous bacterial genes for deoxysugar biosynthesis that are now available. Results: Production of thymidine diphospho (TDP)- -daunosamine (dnm), the aminodeoxysugar present in the anthracycline antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), and its attachment to -rhodomycinone to generate rhodomycin D has been achieved by bioconversion with a strain of Streptomyces lividans that bears two plasmids. One contained the Streptomyces peucetius dnmJVUZTQS genes plus dnmW (previously named dpsH and considered to be a polyketide cyclase gene), dnrH, which is not required for the formation of rhodomycin D, and dnrl, a regulatory gene required for expression of the dnm and drr genes. The other plasmid had genes encoding glucose-1-phosphate thymidylyltransferase and TDP-glucose-4,6-dehydratase (dnmL and dnmM, respectively, or mtmDE, their homologs from Streptomyces agrillaceus) plus the drrAB DNR/DXR resistance genes. Conclusions: The high-yielding glycosylation of the aromatic polyketide -rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for -daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.     

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.     

Benzodiazepine biosynthesis in Streptomyces refuineus

Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A lambda-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a "dehydroproline acrylamide" moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin.     

Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.     

Biosynthesis of the antitumor agent chartreusin involves the oxidative rearrangement of an anthracyclic polyketide

Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.     

Biosynthesis of TDP-l-mycarose: the specificity of a single enzyme governs the outcome of the pathway

The biosynthetic pathway for l-mycarose, a 2,6-dideoxy-3-methylhexose found in the antibiotic tylosin produced by Streptomyces fradiae, has been studied with only two genes in the gene cluster, tylK and tylC2, remaining to be characterized. To verify the postulated functions of these two genes and to establish the sequence of events in the pathway, functional analyses of the proteins encoded by tylK and tylC2 were carried out. The results clearly show that TylK functions as the 5-epimerase and TylC2 functions as the 4-ketoreductase. However, both enzymes exhibit relaxed substrate specificity such that the production of TDP-l-mycarose as the sole product of the pathway depends on the absolute specificity of TylC3, a methyltransferase involved in an early step in the pathway. Exploitation of the promiscuous substrate specificity in the late steps of the mycarose pathway could produce new analogues of tylosin as well as other antibiotics containing this sugar.     

Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars

The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A. kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-l-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase, a conserved set of enzymes involved in formation, attachment, and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar d-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-d-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitrosugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.     

Characterisation of anthocyanidins by electrospray ionisation and collision-induced dissociation tandem mass spectrometry.

The present study describes the use of electrospray ionisation mass spectrometry, in combination with collision-induced dissociation (CID) and tandem mass spectrometry, for the structural characterisation of anthocyanidins and their O-glycosides. The high-energy CID spectra of [M-Cl](+) ions of the free aglycones show characteristic fragmentation pathways, which provide useful information about the substitution pattern in the A- and B-rings of each compound. The major fragmentation observed in the high-energy CID spectra of [M-Cl](+) ions of anthocyanins involves loss of the mono- or disaccharide units resulting in ions containing only the aglycone moiety. From the spectral data, the identity of the aglycone can be established as well as the number and the class of monosaccharide units in the O-glycosides.     

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.