Development of the ability to produce secondary metabolites in Streptomyces through the acquisition of erythromycin resistance

Imai, Yu; Fujiwara, Tatsuya; Ochi, Kozo; Hosaka, Takeshi

doi:10.1038/ja.2012.16

Cited by 20 publications

(12 citation statements)

References 15 publications

(3 reference statements)

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“…It is also notable that the rpoB mutations were effective in enhancing actinomycin production in S. antibioticus but not S. parvulus, indicating the strain dependency of the rpoB mutation effect. Similarly, erythromycin resistance (ery) mutations were effective in enhancing actinomycin production in S. parvulus but not S. antibioticus, although the reason for this difference remains unknown (35). The genetic regulation of streptomycin biosynthesis has been studied in detail in relation to A-factor (28).…”

Section: Discussionmentioning

confidence: 99%

Activation and Products of the Cryptic Secondary Metabolite Biosynthetic Gene Clusters by Rifampin Resistance ( rpoB ) Mutations in Actinomycetes

Tanaka

Kasahara²,

Hirose³

et al. 2013

J Bacteriol

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A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization. Actinomycetes produce a variety of natural products that are of major importance in the pharmaceutical industry. More than 50% of all anti-infective and anticancer compounds developed over the past 25 years have been natural products or derivatives thereof (1). Discovery of novel antibiotics and strain improvement for overproduction are important in applied microbiology research, especially in the production of clinically important antibiotics as well as antibiotics important in veterinary medicine and agriculture. There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. That is, genome sequencing projects have revealed many biosynthetic gene clusters for the production of unknown secondary metabolites. For example, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, and Saccharopolyspora erythraea are each known to produce three to five secondary metabolites but actually possess Ͼ20 clusters that encode known or predicted biosynthetic pathways for secondary metabolites (2-5). Exploitation of such genetic potential in actinomycetes may lead to the isolation of new biologically active compounds (6-8). We recently described a new method to increase antibiotic production in bacteria by modulating ribosomal components (ribosomal proteins or rRNA), i.e., by introducing mutations conferring drug resistance, as many antibiotics target the ribosome (9-11). This new approach, called "ribosome engineering" (12, 13), has several advantages, including the ability to screen for drug resistance mutations by simple selection on drugcontaining plates, even if the mutation frequency is extremely low (e.g., Ͻ10 Ϫ10 ), and the ability to select for mutations without prior genetic information. Hence, this method requires no induced mutagenesis. Interestingly, the introduction of several drug resistance mutations has a cumulative effect on antibiotic production (14-16).In addition to enhancement of antibiotic production, we have demon...

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Section: Discussionmentioning

confidence: 99%

Activation and Products of the Cryptic Secondary Metabolite Biosynthetic Gene Clusters by Rifampin Resistance ( rpoB ) Mutations in Actinomycetes

Tanaka

Kasahara²,

Hirose³

et al. 2013

J Bacteriol

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“…Actinorhodin (ACT) production on MR5 agar was directly assessed by blue color intensity. The production of ACT and undecylprodigiosin (RED) in liquid culture with MR5 medium was determined as described previously (2,25). A bioassay to detect antibacterial compound produced by S. lividans was performed as follows.…”

Section: Methodsmentioning

confidence: 99%

“…We have alternatively developed a novel method for activating poorly expressed Streptomyces genes to produce antibiotics via a mutation that confers resistance to drugs by targeting RNA polymerase or ribosomes, such as rifampin, streptomycin, gentamicin, and erythromycin (25)(26)(27)(28)(29). This method, a so-called ribosome engineering approach, led to the discovery of a novel antibiotic, piperidamycin, produced by Streptomyces sp.…”

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confidence: 99%

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp

Imai

Sato

Tanaka

et al. 2015

Appl Environ Microbiol

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e Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the bluepigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F o F 1 ATP synthase ␣ and ␤ subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed. Streptomyces is the largest genus of Gram-positive filamentous actinomycetes, and members of this genus produce abundant amounts of numerous bioactive metabolites, including antitumor agents, immunosuppressants, and antibiotics in particular (1, 2). Whole-genome sequencing studies of Streptomyces strains have shown that each species could produce many more secondary metabolites than were expected. For example, Streptomyces coelicolor A3(2), S. avermitilis MA-4680, and S. griseus IFO 13350 each produce several secondary metabolites, although they have more than 20 gene clusters that can encode a number of known or predicted biosynthetic pathways for secondary metabolites (3-5). This indicates that the vast majority of secondary metabolites remain unexpressed or barely expressed under standard laboratory conditions. Thus, there is considerable interest in exploring practical means to induce this genetic potential in streptomycetes, which could result in the isolation of novel bioactive secondary metabolites.Recent developments in new methodologies, including physiological and genetic engineering approaches, have opened the door for the discovery of novel secondary metabolites by activating cryptic biosynthetic pathways in streptomycetes (6-13). The improvements and modifications of ...

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“…These include erythromycin and gentamicin, both of which target the ribosome (Chai et al 2012;Imai et al 2012). While further studies with these antibiotics are necessary, initial mechanistic experiments indicate that their mode of induction is different from those of streptomycin and rifampicin.…”

Section: Mechanismmentioning

confidence: 99%

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules

Okada¹,

Seyedsayamdost²

2016

FEMS Microbiology Reviews

170

150

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One sentence summary: Microbial secondary metabolites represent a significant source of potential drug leads; however, the majority of the corresponding biosynthetic genes are not expressed under normal laboratory conditions. In this review, we assess the capacity of exogenous small molecules, especially antibiotics, to activate these silent gene clusters. Editor: Aimee Shen ABSTRACTNatural products have traditionally served as a dominant source of therapeutic agents. They are produced by dedicated biosynthetic gene clusters that assemble complex, bioactive molecules from simple precursors. Recent genome sequencing efforts coupled with advances in bioinformatics indicate that the majority of biosynthetic gene clusters are not expressed under normal laboratory conditions. Termed 'silent' or 'cryptic', these gene clusters represent a treasure trove for discovery of novel small molecules, their regulatory circuits and their biosynthetic pathways. In this review, we assess the capacity of exogenous small molecules in activating silent secondary metabolite gene clusters. Several approaches that have been developed are presented, including coculture techniques, ribosome engineering, chromatin remodeling and high-throughput elicitor screens. The rationale, applications and mechanisms attendant to each are discussed. Some general conclusions can be drawn from our analysis: exogenous small molecules comprise a productive avenue for the discovery of cryptic metabolites. Specifically, growth-inhibitory molecules, in some cases clinically used antibiotics, serve as effective inducers of silent biosynthetic gene clusters, suggesting that old antibiotics may be used to find new ones. The involvement of natural antibiotics in modulating secondary metabolism at subinhibitory concentrations suggests that they represent part of the microbial vocabulary through which inter-and intraspecies interactions are mediated.

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Development of the ability to produce secondary metabolites in Streptomyces through the acquisition of erythromycin resistance

Cited by 20 publications

References 15 publications

Activation and Products of the Cryptic Secondary Metabolite Biosynthetic Gene Clusters by Rifampin Resistance ( rpoB ) Mutations in Actinomycetes

Activation and Products of the Cryptic Secondary Metabolite Biosynthetic Gene Clusters by Rifampin Resistance ( rpoB ) Mutations in Actinomycetes

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules

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