RESUMO
Tuberculosis is an infectious disease of global concern. Members of the diazaquinomycin (DAQ) class of natural products have shown potent and selective activity against drug-resistant Mycobacterium tuberculosis. However, poor solubility has prevented further development of this compound class. Understanding DAQ biosynthesis may provide a viable route for the generation of derivatives with improved properties. We have sequenced the genomes of two actinomycete bacteria that produce distinct DAQ derivatives. While software tools for automated biosynthetic gene cluster (BGC) prediction failed to detect DAQ BGCs, comparative genomics using MAUVE alignment led to the identification of putative BGCs in the marine Streptomyces sp. F001 and in the freshwater Micromonospora sp. B006. Deletion of the identified daq BGC in strain B006 using CRISPR-Cas9 genome editing abolished DAQ production, providing experimental evidence for BGC assignment. A complete model for DAQ biosynthesis is proposed based on the genes identified. Insufficient knowledge of natural product biosynthesis is one of the major challenges of productive genome mining approaches. The results reported here fill a gap in knowledge regarding the genetic basis for the biosynthesis of DAQ antibiotics. Moreover, identification of the daq BGC shall enable future generations of improved derivatives using biosynthetic methods.
Assuntos
Actinobacteria/genética , Equinomicina/análogos & derivados , Água Doce/microbiologia , Genes Bacterianos , Família Multigênica , Água do Mar/microbiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Equinomicina/biossíntese , Equinomicina/química , Deleção de GenesRESUMO
Chemical probes were devised and evaluated for the capture of biosynthetic intermediates involved in the bio-assembly of the nonribosomal peptide echinomycin. Putative intermediate peptide species were isolated and characterised, providing fresh insights into pathway substrate flexibility and paving the way for novel chemoenzymatic approaches towards unnatural peptides.
Assuntos
Equinomicina/biossíntese , Sondas Moleculares/análise , Equinomicina/química , Sondas Moleculares/química , Estrutura MolecularRESUMO
Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.
Assuntos
Dissulfetos/química , Equinomicina/biossíntese , Catálise , Cristalografia por Raios X , Equinomicina/química , Homocisteína/biossíntese , Homocisteína/química , Ligação de Hidrogênio , Metionina/química , Metionina/metabolismo , Metiltransferases/metabolismo , Estrutura Terciária de Proteína , Quinoxalinas/químicaRESUMO
Echinomycin is an antitumor antibiotic secondary metabolite isolated from streptomycetes, whose core structure is biosynthesized by nonribosomal peptide synthetase (NRPS). The echinomycin biosynthetic pathway was successfully reconstituted in Escherichia coli. NRPS often contains a thioesterase domain at its C terminus for cyclorelease of the elongating peptide chain. Those thioesterase domains were shown to exhibit significant substrate tolerance. More recently, an oxidoreductase Ecm17, which forms the disulfide bridge in triostin A, was characterized. Surprisingly, an unrelated disulfide-forming enzyme GliT for gliotoxin biosynthesis was also able to catalyze the same reaction, providing another example of broad substrate specificity in secondary metabolite biosynthetic enzymes. Those promiscuous catalysts can be a valuable tool in generating diversity in natural products analogs we can produce heterologously.
Assuntos
Equinomicina/biossíntese , Biocatálise , Vias Biossintéticas , Ciclização , Dissulfetos/química , Dissulfetos/metabolismo , Equinomicina/química , Sulfito Redutase (NADPH)/metabolismoRESUMO
Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on ß-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) ß-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.
Assuntos
Equinomicina/biossíntese , Streptomyces/metabolismo , 5-Hidroxitriptofano/metabolismo , Vias Biossintéticas , Clonagem Molecular , Ativação Enzimática , Genes Bacterianos , Hidroxilação , Família Multigênica , Mutação , Fases de Leitura Aberta , Peptídeo Sintases/metabolismo , Filogenia , Quinoxalinas/metabolismo , Streptomyces/genética , Especificidade por Substrato , Triptofano Oxigenase/classificação , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismoRESUMO
Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-[6'-(2)H]-3-hydroxy-L-kynurenine, and functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate 3-hydroxy-L-kynurenine.
Assuntos
Antibióticos Antineoplásicos/biossíntese , Equinomicina/análogos & derivados , Escherichia coli/metabolismo , Ácido Cinurênico/análogos & derivados , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Peptídeo Sintases/metabolismo , Quinoxalinas/metabolismo , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Equinomicina/biossíntese , Escherichia coli/genética , Genes Bacterianos , Ácido Cinurênico/metabolismo , Cinurenina/biossíntese , Espectroscopia de Ressonância Magnética , Família Multigênica , Rotação Ocular , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Fatores Biológicos/biossíntese , Depsipeptídeos/biossíntese , Equinomicina/análogos & derivados , Escherichia coli/metabolismo , Engenharia Genética , Fatores Biológicos/química , Fatores Biológicos/isolamento & purificação , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Equinomicina/biossíntese , Equinomicina/química , Equinomicina/isolamento & purificação , Escherichia coli/química , Escherichia coli/enzimologia , Metiltransferases/genética , Metiltransferases/metabolismo , Conformação MolecularRESUMO
Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.
Assuntos
Antibacterianos/biossíntese , Equinomicina/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Vias Biossintéticas , Equinomicina/análogos & derivados , Equinomicina/farmacologia , Proteínas de Escherichia coli/genética , Genes Bacterianos , Família Multigênica , Streptomyces/genéticaRESUMO
Echinomycin, a bis-intercalator antitumor cyclic peptide, is biosynthesized by a unique nonribosomal peptide synthetase (NRPS). Successful heterologous expression of the whole gene cluster of echinomycin in Escherichia coli let us to investigate a further application of echinomycin NRPS. To construct a cyclic peptide library, our approach through both chemoenzymatic and rational genetic engineering has been successfully demonstrated. These achievements provided the further support that E. coli-based system can serve as a flexible yet robust platform for producing complex natural products and their analogs.
Assuntos
Vias Biossintéticas , Equinomicina/biossíntese , Equinomicina/química , Engenharia Genética/métodos , Equinomicina/metabolismo , Escherichia coli/genética , Expressão Gênica , Peptídeo Sintases/metabolismoRESUMO
Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase-long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal-forming enzyme responsible for the triostin A-to-echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful re-analysis for metabolites from previously characterized natural product producers with the current technologies.
Assuntos
Antibacterianos/biossíntese , Equinomicina/biossíntese , Família Multigênica/genética , Streptomyces/metabolismo , Antibacterianos/química , Sequência de Bases , Vias Biossintéticas , Clonagem Molecular , Equinomicina/química , Biblioteca Genômica , Conformação Molecular , Dados de Sequência Molecular , Quinoxalinas/química , Quinoxalinas/metabolismo , Análise de Sequência de DNA , Streptomyces/genéticaAssuntos
Bactérias/genética , Bactérias/metabolismo , Produtos Biológicos/biossíntese , Desenho de Fármacos , Compostos Organofosforados , Antibióticos Antineoplásicos , Biologia , Fenômenos Químicos , Química , Técnicas de Química Combinatória , Equinomicina/biossíntese , Escherichia coli/metabolismo , Engenharia Genética/métodos , Lactonas , Macrolídeos , Família Multigênica , Policetídeo Sintases , PironasRESUMO
[reaction: see text] Little is known about how quinoxaline-2-carboxylic acid (QC) is synthesized in nature. On the basis of analysis of echinomycin biosynthetic gene clusters as well as feeding experiments with labeled precursors, we have proposed a biosynthetic pathway to QC and identified the (2S,3S)-beta-hydroxytryptophan as a key intermediate.
Assuntos
5-Hidroxitriptofano/química , Equinomicina/biossíntese , Quinoxalinas/metabolismo , Streptomyces , Equinomicina/química , Estrutura Molecular , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismoAssuntos
Artemisininas/metabolismo , Equinomicina/biossíntese , Engenharia Genética , Monensin/biossíntese , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Artemisininas/química , Ciclização , Equinomicina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Hemiterpenos/metabolismo , Monensin/química , Compostos Organofosforados/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Policetídeo Sintases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
An actinomycete strain designated as Actinomadura sp. INA 654 was isolated from a chernozem soil sample in the Voronezh Region by the soil sample treatment with millimetric waves (EHF band). The strain produced an antibiotic complex of 2 components, named A-654-I and A-654-II. Investigation of their physico-chemical properties showed that A-654-I was identical to echinomycin, a heteropeptide lactone of the quinoxaline group with antitumor activity, while A-654-II proved to be likely a new natural compound. Production of echinomycin by a representative of the Actinomadura genus was detected for the first time. Up to now, only representatives of the Streptomyces genus were known to produce echinomycin.
Assuntos
Actinobacteria/metabolismo , Antibacterianos/biossíntese , Equinomicina/biossíntese , Actinobacteria/isolamento & purificação , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Bactérias/efeitos dos fármacos , Equinomicina/química , Equinomicina/farmacologia , Testes de Sensibilidade Microbiana , Federação Russa , Microbiologia do SoloRESUMO
An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.
Assuntos
Antibacterianos/biossíntese , Antimaláricos/metabolismo , Proteaceae/microbiologia , Streptomyces/metabolismo , Antibacterianos/análise , Antibacterianos/química , Cromatografia Líquida de Alta Pressão , Equinomicina/análise , Equinomicina/biossíntese , Equinomicina/química , Inibidores da Síntese de Ácido Nucleico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Quinaldopeptin, a new type of quinomycin antibiotic, was isolated from the culture of Streptoverticillium album strain Q132-6. The antibiotic exhibited strong in vitro antimicrobial and cytotoxic activity and significantly prolonged the survival time of mice inoculated with a murine tumor. Quinaldopeptin is a symmetric cyclic peptide linked only by peptide bonds and differs from known antibiotics of the quinomycin family by the lack of ester linkage.
Assuntos
Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Equinomicina/farmacologia , Leucemia P388/tratamento farmacológico , Leucemia Experimental/tratamento farmacológico , Quinoxalinas/farmacologia , Streptomycetaceae/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibacterianos/uso terapêutico , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/uso terapêutico , Bactérias/efeitos dos fármacos , Equinomicina/análogos & derivados , Equinomicina/análise , Equinomicina/biossíntese , Equinomicina/isolamento & purificação , Equinomicina/uso terapêutico , Fungos/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Análise Espectral , Streptomycetaceae/classificação , Streptomycetaceae/ultraestrutura , Células Tumorais CultivadasRESUMO
Antibiotic nonproducing variants of Streptomyces lasaliensis NRRL 3382R, which makes the polyether antibiotic lasalocid A (Las) and the quinoxaline antibiotic echinomycin (Ech), arose at a frequency of 3-11% after treatment with three different mutagens or regeneration of protoplasts compared with a spontaneous frequency of less than 0.1%. Cosynthesis of lasalocid A was not observed upon testing a large number of Las- mutants in different pair-wise combinations, nor did these mutants accumulate probable intermediates of lasalocid A biosynthesis. These results suggest that loss of the las genes or their expression is induced at a high frequency by mutagenic treatments. In fusions of protoplasts of a strain with the las+ ech+ spo+ nic-1 rif-3 markers with strains bearing the Las- LasS Ech- Bld- (or spo+) str-1 markers, Las+ Ech+ Spo+ StrR progeny were produced at a 61-89% frequency compared with a 1-9% frequency of StrR antibiotic producing progeny with the nic-1 or rif-3 genotypes. The more frequent restoration of antibiotic production than prototrophy or rifampicin sensitivity indicates that these antibiotic characters did not behave as normal chromosomal markers. Therefore the genetic instability might be due to the involvement of a plasmid in antibiotic production. The apparent lack of infectious transfer of the Las+ character to Las- parents in conjugal matings between the few strains tested and no correlation between the presence of a large plasmid, pKSL, and lasalocid A production in several strains of S. lasaliensis do not favor the latter hypothesis, but they do not conclusively disprove it. Consequently, we suggest that a plasmid or another mobile genetic element is controlling antibiotic production in S. lasaliensis.
Assuntos
Equinomicina/biossíntese , Lasalocida/biossíntese , Quinoxalinas/biossíntese , Streptomyces/genética , 4-Butirolactona/análogos & derivados , Conjugação Genética , Genes , Genes Bacterianos , Substâncias de Crescimento/fisiologia , Lasalocida/genética , Mutação , Plasmídeos , Protoplastos , Recombinação Genética , Streptomyces/metabolismoRESUMO
In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized by Streptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.
Assuntos
Equinomicina/biossíntese , Quinoxalinas/biossíntese , Streptomyces/metabolismo , Equinomicina/isolamento & purificação , Mutação , Streptomyces/citologia , Streptomyces/genética , TemperaturaRESUMO
Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.