RESUMO
Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone ß-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone ß-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone ß-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.
Assuntos
Oxirredutases do Álcool/metabolismo , Antimicina A/análogos & derivados , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Antimicina A/biossíntese , Antimicina A/metabolismo , Biocatálise , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Alinhamento de Sequência , Streptomyces/enzimologia , Especificidade por Substrato/genéticaRESUMO
The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the â¼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.
Assuntos
Antimicina A/análogos & derivados , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Streptomyces/enzimologia , Streptomyces/genética , Antimicina A/biossíntese , Proteínas de Bactérias/genética , Proteólise , Fator sigma/genética , Estresse FisiológicoRESUMO
Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways.
Assuntos
Actinobacteria/classificação , Actinobacteria/genética , Antimicina A/análogos & derivados , Vias Biossintéticas/genética , Evolução Molecular , Família Multigênica/genética , Filogenia , Actinobacteria/enzimologia , Actinobacteria/metabolismo , Antimicina A/biossíntese , Proteínas de Bactérias/genética , Bases de Dados Genéticas , Genes Bacterianos , Genômica , Hidrolases/genética , Filogeografia , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Reasons for mammalian cell toxicity observed in barley and spring wheat grains were sought. Streptomyces sp. isolates from wheat and barley produced heat-stable methanol-soluble substances which inhibited the motility of exposed porcine spermatozoa used as a toxicity indicator. Several barley isolates produced antimycin A (2 to 5 ng/mg wet wt of biomass), a macrolide antibiotic known to block oxygen utilization in mitochondria. The antimycin-producing isolates were members of the Streptomyces albidoflavus group. In in vitro assays with porcine kidney tubular epithelial cells, the specific toxicity of antimycin A towards mitochondria was higher than that of the mycotoxin enniatin B but lower than that of the mitochondriotoxins cereulide and paenilide, produced by food-related Bacillus cereus and Paenibacillus tundrae, respectively. The toxic wheat isolates, related to Streptomyces sedi, did not produce antimycin A and or any other known toxin. Our results suggest that the presence of toxin-producing streptomycetes in stored cereal grains may pose a thus far unrecognized threat for food and feed safety.
Assuntos
Antimicina A/biossíntese , Grão Comestível/microbiologia , Hordeum/microbiologia , Streptomyces/metabolismo , Triticum/microbiologia , Animais , Linhagem Celular , Microbiologia de Alimentos , Inocuidade dos Alimentos , Masculino , Mitocôndrias/patologia , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/classificação , Streptomyces/isolamento & purificação , SuínosRESUMO
Bioengineering of natural product biosynthesis is a powerful approach to expand the structural diversity of bioactive molecules. However, in polyketide biosynthesis, the modification of polyketide extender units, which form the carbon skeletons, has remained challenging. Herein, we report the rational control of polyketide extender units by the structure-based engineering of a crotonyl-CoA carboxylase/reductase (CCR), in the biosynthesis of antimycin. Site-directed mutagenesis of the CCR enzyme AntE, guided by the crystal structure solved at 1.5â Å resolution, expanded its substrate scope to afford indolylmethylmalonyl-CoA by the V350G mutation. The mutant A182L selectively catalyzed carboxylation over the regular reduction. Furthermore, the combinatorial biosynthesis of heterocycle- and substituted arene-bearing antimycins was achieved by an engineered Streptomyces strain bearing AntE(V350G). These findings deepen our understanding of the molecular mechanisms of the CCRs, which will serve as versatile biocatalysts for the manipulation of building blocks, and set the stage for the rational design of polyketide biosynthesis.
Assuntos
Acil-CoA Desidrogenases/química , Acil-CoA Desidrogenases/metabolismo , Antimicina A/análogos & derivados , Policetídeos/química , Engenharia de Proteínas , Antimicina A/biossíntese , Antimicina A/química , Conformação ProteicaRESUMO
Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.
Assuntos
Antimicina A/análogos & derivados , Produtos Biológicos/metabolismo , Escherichia coli/genética , Salicilatos/metabolismo , Antimicina A/biossíntese , Escherichia coli/metabolismo , Genes Bacterianos/genética , Streptomyces/genéticaRESUMO
The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.
Assuntos
Proteínas de Bactérias , Genes Bacterianos , Peptídeo Sintases , Policetídeo Sintases , Streptomyces , Transferases (Outros Grupos de Fosfato Substituídos) , Antimicina A/análogos & derivados , Antimicina A/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Netropsina/metabolismo , Oligopeptídeos/biossíntese , Oligopeptídeos/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Diversity-oriented biosynthesis of a library of antimycin-like compounds (380 altogether) was accomplished by using multiplex combinatorial biosynthesis. The core strategy depends on the use of combinatorial chemistry at different biosynthetic stages. This approach is applicable for the diversification of polyketides, nonribosomal peptides, and the hybrids that share a similar biosynthetic logic.
Assuntos
Antimicina A/análogos & derivados , Técnicas de Química Combinatória , Variação Genética , Alquilação , Animais , Antimicina A/biossíntese , Antimicina A/farmacologia , Antimicina A/toxicidade , Candida albicans/efeitos dos fármacos , Catálise , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Lactonas/química , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Policetídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
Evolution of natural products, and particularly those resulting from microbial assembly line-like enzymes, such as polyketide (PK) and nonribosomal peptides (NRP), has resulted in a variety of pharmaceutically important and chemically diverse families of molecules. The antimycin-type depsipeptides are one such grouping, with a significant level of diversity and members that have noted activities against key targets governing human cellular apoptosis (e.g. Bcl-xL and GRP78). Chemical variance originates from ring size, with 9-, 12-, 15-, and 18-membered classes, and we show that such distinctions influence their molecular targeting. Further, we present here a systematic interrogation of the chemistry and assembly line evolution of antimycin-type analogues by conducting metabolomic profiling and biosynthetic gene cluster comparative analysis of the depsipeptide assembly lines for each member of the antimycin-group. Natural molecular evolution principles of such studies should assist in artificial re-combinatorializing of PK and NRP assembly lines.
Assuntos
Produtos Biológicos/química , Depsipeptídeos/química , Sequência de Aminoácidos , Antimicina A/análogos & derivados , Antimicina A/biossíntese , Antimicina A/química , Antimicina A/farmacologia , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Análise por Conglomerados , Biologia Computacional/métodos , Depsipeptídeos/biossíntese , Depsipeptídeos/farmacologia , Chaperona BiP do Retículo Endoplasmático , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/químicaRESUMO
The in vivo and in vitro characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl carrier and produces more antimycin analogues with varying C-8 acyloxy moieties.
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Antimicina A/análogos & derivados , Escherichia coli/metabolismo , Antimicina A/biossíntese , Antimicina A/química , Catálise , Escherichia coli/química , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Família Multigênica , Especificidade por SubstratoRESUMO
We describe methods used to isolate and identify antifungal compounds from actinomycete strains associated with the leaf-cutter ant Acromyrmex octospinosus. These ants use antibiotics produced by symbiotic actinomycete bacteria to protect themselves and their fungal cultivar against bacterial and fungal infections. The fungal cultivar serves as the sole food source for the ant colony, which can number up to tens of thousands of individuals. We describe how we isolate bacteria from leaf-cutter ants collected in Trinidad and analyze the antifungal compounds made by two of these strains (Pseudonocardia and Streptomyces spp.), using a combination of genome analysis, mutagenesis, and chemical isolation. These methods should be generalizable to a wide variety of insect-symbiont situations. Although more time consuming than traditional activity-guided fractionation methods, this approach provides a powerful technique for unlocking the complete biosynthetic potential of individual strains and for avoiding the problems of rediscovery of known compounds. We describe the discovery of a novel nystatin compound, named nystatin P1, and identification of the biosynthetic pathway for antimycins, compounds that were first described more than 60 years ago. We also report that disruption of two known antifungal pathways in a single Streptomyces strain has revealed a third, and likely novel, antifungal plus four more pathways with unknown products. This validates our approach, which clearly has the potential to identify numerous new compounds, even from well-characterized actinomycete strains.
Assuntos
Antifúngicos/isolamento & purificação , Formigas/microbiologia , Bioensaio/métodos , Genoma Bacteriano , Genômica/métodos , Streptomyces/isolamento & purificação , Simbiose , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Antimicina A/análogos & derivados , Antimicina A/biossíntese , Antimicina A/química , Antimicina A/isolamento & purificação , Candicidina/biossíntese , Candicidina/química , Candicidina/isolamento & purificação , Candida albicans/efeitos dos fármacos , Cromatografia Líquida/métodos , Clonagem Molecular , Testes de Sensibilidade Microbiana , Família Multigênica , Nistatina/biossíntese , Nistatina/química , Nistatina/isolamento & purificação , Streptomyces/química , Streptomyces/genéticaRESUMO
We herein report comparative analysis of two versions of the biosynthetic gene clusters of antimycins, a natural product family possessing up to 44 distinct entities. The biosynthetic pathway of antimycins is amenable to the high structural variation of the substrates, supported by successes in heterologous expression of the ant cluster and in fluorine incorporation. The latter facilitated the investigation of the structure-activity relationship into the usually invariable 3-formamidosalicylic acid moiety of the molecules.
Assuntos
Antibacterianos/biossíntese , Antimicina A/análogos & derivados , Streptomyces/genética , Antibacterianos/química , Antimicina A/biossíntese , Antimicina A/química , Vias Biossintéticas/genética , Genes Bacterianos , Estrutura Molecular , Streptomyces/química , Streptomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
The volatiles released by several streptomycetes were collected by using a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. The obtained headspace extracts of various species contained blastmycinone, a known degradation product of the fungicidal antibiotic, antimycin A(3b), and several unknown derivatives. The suggested structures of these compounds, based on their mass spectra and GC retention indices, were confirmed by comparison to synthetic reference samples. Additional compounds found in the headspace extracts were butenolides formed from the blastmycinones by elimination of the carboxylic acid moiety. Analysis of a gene knockout mutant in the antimycin biosynthetic gene cluster demonstrated that all blastmycinones and butenolides are formed via the antimycin biosynthetic pathway. The structural variation of the blastmycinones identified here is much larger than within the known antimycins, thus suggesting that several antimycin derivatives remain to be discovered.
Assuntos
4-Butirolactona/análogos & derivados , Antimicina A/análogos & derivados , Streptomyces/química , 4-Butirolactona/análise , 4-Butirolactona/síntese química , Antimicina A/biossíntese , Antimicina A/química , Antimicina A/metabolismo , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Two new antimycin A analogues, antimycin B1 and B2 (1-2), were isolated from a spent broth of a marine-derived bacterium, Streptomyces lusitanus. The structures of 1 and 2 were established on the basis of spectroscopic analyses and chemical methods. The isolated compounds were tested for their anti-bacterial potency. Compound 1 was found to be inactive against the bacteria Bacillus subtilis, Staphyloccocus aureus, and Loktanella hongkongensis. Compound 2 showed antibacterial activities against S. aureus and L. hongkongensis with MIC values of 32.0 and 8.0 µg/mL, respectively.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Streptomyces/metabolismo , Antimicina A/biossíntese , Organismos Aquáticos , Bactérias/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Indicadores e Reagentes , Lactonas/química , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Conformação Molecular , Espectrofotometria Ultravioleta , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Treonina/químicaRESUMO
Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in â¼30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.
Assuntos
Antibacterianos/biossíntese , Formigas/crescimento & desenvolvimento , Streptomyces/metabolismo , Simbiose , Animais , Antibacterianos/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Antimicina A/biossíntese , Antimicina A/farmacologia , Formigas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Candicidina/biossíntese , Candicidina/farmacologia , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Hypocreales/efeitos dos fármacos , Hypocreales/crescimento & desenvolvimento , Espectrometria de Massas/métodos , Mutação , Streptomyces/genética , Streptomyces/fisiologiaRESUMO
AIM: To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. METHODS AND RESULTS: In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. CONCLUSIONS: Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.
Assuntos
Antimicina A/biossíntese , Toxinas Bacterianas/biossíntese , Solanum tuberosum/microbiologia , Streptomyces/metabolismo , Streptomyces/patogenicidade , Animais , Antimicina A/análise , Antimicina A/farmacologia , Toxinas Bacterianas/análise , Toxinas Bacterianas/farmacologia , Cromatografia Líquida de Alta Pressão , Finlândia , Masculino , Espectrometria de Massas , Microbiologia do Solo , Motilidade dos Espermatozoides/efeitos dos fármacos , Suécia , SuínosAssuntos
Antibacterianos/biossíntese , Antimicina A/análogos & derivados , Macrolídeos , Reguladores de Crescimento de Plantas/biossíntese , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Antimicina A/biossíntese , Antimicina A/química , Antimicina A/farmacologia , Candida albicans/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Lactuca/efeitos dos fármacos , Lactuca/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/farmacologia , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria UltravioletaRESUMO
A new antifungal antibiotic was isolated from the fermentation broth of Streptomyces sp. 5140-A1. Degradation studies of the crystalline antibiotic, m.p. 186 approximately 188 degrees C, C21H28O8N2, suggested Piricularia oryzae and less toxicity against killfish than antimycin A--blastmycin antibiotics.
Assuntos
Antimicina A/análogos & derivados , Streptomyces/metabolismo , Animais , Antimicina A/análise , Antimicina A/biossíntese , Antimicina A/toxicidade , Fenômenos Químicos , Físico-Química , Fermentação , Peixes , Fungos/efeitos dos fármacos , Dose Letal Mediana , CamundongosRESUMO
Increase in antimycin A production was achieved through a parallel strain and medium improvement program: a 125-fold augmentation (75 to 9,500 mug/ml) was obtained. The selective system included antimycin A productivity, conidiation, sensitivity to ultraviolet radiation, growth rate and yield, and absence of pigment and actinomycin D production. Among the original strains tested one natural isolate possessed high productivity and several of the above characteristics, and was selected for mutagenesis. Spontaneous and induced variability was then exploited in isolating high-producing strains. The first mutagen used was ultraviolet radiation; it was replaced by ethylenimine when it became no longer efficient in increasing variability. As new, high producers were isolated, the medium was modified to best suit their requirements for still higher productivity. The critical environmental factors were absence of phosphate and organic salts, concentration of the nitrogen source and ratio organic/inorganic nitrogen, ratio ammonium sulfate/calcium carbonate, and addition of slowly utilizable carbon sources, such as lactose and oil; optimum temperature and initial pH were 25 degrees C and 7.0. Aeration/agitation requirements of improved strains were high. Fermentation was characterized by abrupt pH changes which impaired rapid accumulation of the antibiotic. Antimycin A was produced during both the trophophase and idiophase.