RESUMEN

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 µM and 2.0 ± 0.1 µM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 µM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.

Asunto(s)

Antibacterianos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oligomicinas/biosíntesis , Streptomyces/metabolismo , Antibacterianos/química , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Cartilla de ADN , Modelos Moleculares , Oligomicinas/química , Reacción en Cadena de la Polimerasa , Streptomyces/enzimología

RESUMEN

σ(25) is an extracytoplasmic function (ECF) σ factor in the bacterium Streptomyces avermitilis that plays a differential regulatory role in avermectin and oligomycin biosynthesis. Gene deletion, complementation, and overexpression experiments showed that σ(25) inhibited avermectin production but promoted oligomycin production. σ(25) indirectly inhibited avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activated oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. The divergently transcribed genes smrAB are located upstream of sig25 and encode a putative two-component system (TCS). σ(25) was found to initiate its own transcription, and its expression was directly activated by SmrA. The precise SmrA-binding sites in the region upstream of sig25 were determined by DNase I footprinting assays and identified two direct repeat sequences CTGTGA-n5-CTGTGA, suggesting that SmrA regulates sig25 transcription by binding to these direct repeats. The deletion of smrAB had the similar effect on avermectin and oligomycin A production to the deletion of sig25, indicating that σ(25) and SmrAB function similarly in the regulation of antibiotic production. These findings helpfully clarify the regulation of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in S. avermitilis.

Asunto(s)

Antibacterianos/biosíntesis , Regulación Bacteriana de la Expresión Génica , Ivermectina/análogos & derivados , Oligomicinas/biosíntesis , Factor sigma/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Sitios de Unión , Huella de ADN , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Ivermectina/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética

RESUMEN

A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.

Asunto(s)

Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Actinas/genética , Antraquinonas , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retroalimentación Fisiológica , Orden Génico , Proteínas del Helminto/genética , Ivermectina/análogos & derivados , Datos de Secuencia Molecular , Mutación , Oligomicinas/biosíntesis , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal

RESUMEN

The function of the regulatory protein AveR in Streptomyces avermitilis was examined. An aveR deletion mutant abolished avermectin production and produced more oligomycin, and its phenotype was complemented by a single copy of the aveR gene. Removal of the C-terminal HTH domain of AveR abolished avermectin biosynthesis, indicating the importance of HTH domain for AveR function. Promoter titration and promoter probe assays suggested that the transcription of aveA1, encoding polypeptide AVES1 of avermectin PKS, was activated by AveR. Chromatin immunoprecipitation (ChIP) assay showed that the predicted promoter regions of both the ave cluster and the olm cluster were target sites of AveR, and the DNA-binding activity of AveR was dependent on its HTH domain. RT-PCR analysis revealed that the transcriptions of ave structural genes were dependent on AveR, but that of olm structural genes and putative pathway-specific regulatory genes increased in the aveR mutants. Consistent with these observations, overexpression of aveR successfully increased avermectin production. These results indicated that aveR encodes a pathway-specific activator essential for avermectin biosynthesis and it also negatively affects oligomycin biosynthesis.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Ivermectina/análogos & derivados , Oligomicinas/biosíntesis , Proteínas Represoras/metabolismo , Streptomyces/genética , Transactivadores/metabolismo , Inmunoprecipitación de Cromatina , Eliminación de Gen , Secuencias Hélice-Giro-Hélice/genética , Ivermectina/metabolismo , Familia de Multigenes , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Streptomyces/metabolismo , Transactivadores/genética

RESUMEN

Certain streptomycin resistance mutations (i.e., rpsL and rsmG) result in the overproduction of antibiotics in various actinomycetes. Moreover, rpsL rsmG double-mutant strains show a further increase in antibiotic production. rpsL but not rsmG mutations result in a marked enhancement of oligomycin production in Streptomyces avermitilis and erythromycin production in Saccharopolyspora erythraea, accompanied by increased transcription of a key developmental regulator gene, bldD, in the latter organism.

Asunto(s)

Antibacterianos/biosíntesis , Farmacorresistencia Bacteriana , Proteínas Ribosómicas/genética , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Eritromicina/biosíntesis , Mutación del Sistema de Lectura , Proteínas Mutantes/genética , Mutación Missense , Oligomicinas/biosíntesis

RESUMEN

A new actinomycete strain, isolated from soil in China, strongly inhibited in vitro proliferation of human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The strain, designated L033, was identified as a strain of Streptomyces avermitilis based on cultural property, morphology, carbon source utilization, 16s rRNA gene analysis, and DNA-DNA relatedness studies. The anticancer component from L033 was purified to homogeneity by preparative positive-phase high-performance liquid chromatography and crystallization. Nuclear magnetic resonance and mass spectrometric analysis showed that this compound had the same structure as oligomycin A. Different with other reported naturally occurring strains of S. avermitilis, L033 produced high quantity of oligomycin A (maximal 1,461 microg/ml). Therefore, L033 was considered of great potential as an industrial oligomycin-A-producing strain.

Asunto(s)

Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Oligomicinas/biosíntesis , Oligomicinas/farmacología , Streptomyces/clasificación , Streptomyces/metabolismo , Antineoplásicos/aislamiento & purificación , Metabolismo de los Hidratos de Carbono , Línea Celular Tumoral , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligomicinas/aislamiento & purificación , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Microbiología del Suelo , Streptomyces/genética , Streptomyces/aislamiento & purificación

RESUMEN

Oligomycin and its analogues, produced by Streptomyces avermitilis and other actinomycetes, are of interest for their potent and selective biological activities. PCR-mediated gene replacement, targeting bkdF, one of avermectin's starter unit encoding genes in S. avermitilis, was performed to yield an oligomycin producer, BIB0423. The engineered strain produced oligomycin A at 2.3 mg/ml compared to the wild type strain at 0.1 mg/ml. This resulting mutant was genetically stable and should be useful for the industrial production of oligomycin.

Asunto(s)

Regulación Bacteriana de la Expresión Génica/genética , Ivermectina/análogos & derivados , Oligomicinas/biosíntesis , Streptomyces/metabolismo , Ingeniería Genética/métodos , Vectores Genéticos/genética , Microbiología Industrial/métodos , Ivermectina/metabolismo , Reacción en Cadena de la Polimerasa , Streptomyces/genética

RESUMEN

Target screening of natural immunosuppressors resulted in isolation of a strain of Streptomyces griseolus (No. 182) producing a complex of antifungal antibiotics. The strain proved to be an aerobe with the growth temperature of 26 to 28 degrees C. Morphological features and physiological properties of the strain were studied. Scanning electron microscopy revealed smooth, oval spores 1.10-1.25 mu in size. The findings showed that the strain belonged to Streptomyces griseolus. Unlike the previously described organisms producing the oligomycin complex the new strain formed straight or twisted sporophores and did not produce melanoid pigment or soluble pigment when grown on the Gauze mineral agar medium No. 1. The procedures for biosynthesis and chemical recovery of the antibiotic complex from the mycelium are described. The complex was shown to include 3 components at a ratio of 80:15:5 identified as oligomycins A, B and C respectively. The oligomycin complex was highly active against Aspergillus niger 137, Tolypocladium inflatum, Fusarium ocsisporum, Curvularia lunata 645 and Trichoderma alba F-32 (MIC 0.1-1.0 mcg/ml). The activity against yeast and bacterial cultures was observed only when the doses were higher than 100 mcg/ml.

Asunto(s)

Antifúngicos/biosíntesis , Oligomicinas/biosíntesis , Streptomyces , Antifúngicos/farmacología , Fermentación , Microscopía Electrónica , Oligomicinas/farmacología , Streptomyces/clasificación , Streptomyces/aislamiento & purificación , Streptomyces/metabolismo

RESUMEN

In the programme for screening antibiotics with antifungal and immunosuppressing activity a culture of Streptomyces griseolus 182 was isolated. The culture produced a complex of oligomycin structure antibiotics. Individual components of the complex were isolated by HPLC. The complex was shown to contain three components at a ratio of 80:15:5. The findings were compared with the physico-chemical characteristics of the described antibiotics. On the basis of the analysis it was concluded that fraction 2 of the antibiotic complex 182 could be oligomycin A. A detailed comparative investigation of oligomycin A and component 2 with HPLC, FMR, IR spectroscopy and mass spectrometry revealed their identity. The other two components were shown to be oligomycins B and C.

Asunto(s)

Oligomicinas/biosíntesis , Oligomicinas/química , Streptomyces/metabolismo , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Oligomicinas/análisis , Espectrofotometría Infrarroja

RESUMEN

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.

Asunto(s)

Antihelmínticos/metabolismo , Ivermectina/análogos & derivados , Mutagénesis Insercional/métodos , Oligomicinas/biosíntesis , Streptomyces/genética , Cromosomas Bacterianos , Elementos Transponibles de ADN/genética , Ivermectina/metabolismo , Plásmidos/genética , Recombinación Genética , Homología de Secuencia de Ácido Nucleico

RESUMEN

Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity). However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains. A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before. All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium. Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used. In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton. The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production. No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

Asunto(s)

Antibacterianos/biosíntesis , Antinematodos/metabolismo , Macrólidos , Plásmidos , Streptomyces/genética , Antibacterianos/química , Regulación Bacteriana de la Expresión Génica , Estructura Molecular , Oligomicinas/biosíntesis , Pigmentos Biológicos/biosíntesis , Replicón , Streptomyces/metabolismo , Transformación Bacteriana