A transposon-based strategy to identify the regulatory gene network responsible for landomycin E biosynthesis

Horbal, Lilya; Fedorenko, Victor; Bechthold, Andreas; Luzhetskyy, Andriy

doi:10.1111/1574-6968.12117

Cited by 16 publications

(9 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, the combination of the described approach with transposon mutagenesis enables simple and rapid identification of mutated loci, eliminating the need for genome re-sequencing 32 . The obvious bottleneck of the transposon technique, the ability to generate only inactivation mutants, can be easily overcome with a transposon carrying strong constitutive promoters at the ends of the transposable construct 40 .…”

Section: Discussionmentioning

confidence: 99%

Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074

Ahmed

Rebets

Tokovenko

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

A large majority of genome-encrypted chemical diversity in actinobacteria remains to be discovered, which is related to the low level of secondary metabolism genes expression. Here, we report the application of a reporter-guided screening strategy to activate cryptic polycyclic tetramate macrolactam gene clusters in Streptomyces albus J1074. The analysis of the S. albus transcriptome revealed an overall low level of secondary metabolism genes transcription. Combined with transposon mutagenesis, reporter-guided screening resulted in the selection of two S. albus strains with altered secondary metabolites production. Transposon insertion in the most prominent strain, S. albus ATGSal2P2::TN14, was mapped to the XNR_3174 gene encoding an unclassified transcriptional regulator. The mutant strain was found to produce the avenolide-like compound butenolide 4. The deletion of the gene encoding a putative acyl-CoA oxidase, an orthologue of the Streptomyces avermitilis avenolide biosynthesis enzyme, in the S. albus XNR_3174 mutant caused silencing of secondary metabolism. The homologues of XNR_3174 and the butenolide biosynthesis genes were found in the genomes of multiple Streptomyces species. This result leads us to believe that the discovered regulatory elements comprise a new condition-dependent system that controls secondary metabolism in actinobacteria and can be manipulated to activate cryptic biosynthetic pathways.

show abstract

Section: Discussionmentioning

confidence: 99%

Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074

Ahmed

Rebets

Tokovenko

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Instead, Tn5-and mariner-based transposons were used to mutagenize genomic libraries of S. coelicolor in Escherichia coli, and the mutated alleles were reintroduced into Streptomyces via homologous recombination (30)(31)(32). Recently, a codon-optimized hyperactive Tn5-based transposition system (33,34) was developed and shown to efficiently and randomly mutagenize Streptomyces genomes in vivo. However, the temperature-sensitive replicative vector used to deliver the transposon had to be cured by a temperature upshift.…”

mentioning

confidence: 99%

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Wang

Chater

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genomewide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.KEYWORDS genome-wide, transposition mutagenesis, Streptomyces coelicolor, antibiotic biosynthesis, prodiginine

show abstract

“…Some members of the GntR family have previously been reported as repressors of secondary metabolite biosynthesis in actinomycetes. For example, MtdA represses nucleoside antibiotic A201A [ 12 ], SCO3269 represses actinorhodin and undecylprodigiosin [ 13 ], and ptnR1 represses platensimycin [ 14 ]. Thus, in order to activate the expression of related silent secondary metabolic gene clusters, we disrupted orf-1741 by replacing it with a kanamycin-resistance gene ( neo ).…”

Section: Resultsmentioning

confidence: 99%

Mycemycins A–E, New Dibenzoxazepinones Isolated from Two Different Streptomycetes

et al. 2015

View full text Add to dashboard Cite

Five new dibenzoxazepinone derivatives, mycemycins A–E (1–5), were isolated from the ethanol extracts of mycelia of two different streptomycetes. 1 and 2 were isolated from an acidic red soil-derived strain, Streptomyces sp. FXJ1.235, and 3–5 from a gntR gene-disrupted deep-sea strain named Streptomyces olivaceus FXJ8.012Δ1741. The structures of mycemycins were elucidated by a combination of spectroscopic analyses, including 1D- and 2D-NMR techniques.

show abstract

A transposon-based strategy to identify the regulatory gene network responsible for landomycin E biosynthesis

Cited by 16 publications

References 24 publications

Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074

Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Mycemycins A–E, New Dibenzoxazepinones Isolated from Two Different Streptomycetes

Contact Info

Product

Resources

About