Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces

Knirschová, Renáta; Nováková, Renáta; Mingyar, Erik; Bekeova, Carmen; Homerová, Dagmar; Kormanec, Ján

doi:10.1016/j.mimet.2015.03.017

Cited by 21 publications

(10 citation statements)

References 18 publications

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“…Considering this, we PCR-amplified the putative promoter/operator region upstream of SLIV_09080, and placed it upstream of the bpsA reporter in an integrative plasmid. BpsA is a monomodular nonribosomal peptide synthetase responsible for the biosynthesis of the blue pigment indigoidine, which is easily detectable both visually and spectroscopically . When the resulting putative undecylprodigiosin reporter plasmid pYQS042 (Table S1) was introduced into S. lividans TK24, no indigoidine production was observed in the recombinant strain, suggesting that the SLIV_09075-encoded repressor blocks expression of bpsA .…”

Section: Resultsmentioning

confidence: 99%

Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites

et al. 2017

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Genome mining of actinomycete bacteria aims at the discovery of novel bioactive secondary metabolites that can be developed into drugs. A new repressorbased biosensor to detect activated secondary metabolite biosynthesis gene clusters in Streptomyces was developed. Biosynthetic gene clusters for undecylprodigiosin and coelimycin in the genome of Streptomyces lividans TK24, which encoded TetR-like repressors and appeared to be almost "silent" based on the RNA-seq data, were chosen for the proof-of-principle studies. The bpsA reporter gene for indigoidine synthetase was placed under control of the promotor/operator regions presumed to be controlled by the cluster-associated TetR-like repressors. While the biosensor for undecylprodigiosin turned out to be nonfunctional, the coelimycin biosensor was shown to perform as expected, turning on biosynthesis of indigoidine in response to the concomitant production of coelimycin. The developed reporter system concept can be applied to those cryptic gene clusters that encode metabolite-sensing repressors to speed up discovery of novel bioactive compounds in Streptomyces.

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Section: Resultsmentioning

confidence: 99%

Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites

et al. 2017

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“…Integration of transformed plasmid to specific attachment sites of Streptomyces genome were confirmed by PCR. The gene bpsA encoding a peptide synthetase for the biosynthesis of a blue pigment indigoidine in Streptomyces was used as indicator for double crossover (the color of colonies turned white from blue) after conjugation ( 12 ). Streptomyces spore preparation was obtained by growing on CP6 or soy flour mannitol medium (SFM) plates at 30°C for 4−7 days.…”

Section: Methodsmentioning

confidence: 99%

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

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et al. 2021

Nucleic Acids Research

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DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these ‘mutators’ was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10–5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

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“…271 Alternatively, a reporter system for Streptomyces could be developed, which is responsive to a specific SM for high-throughput screening without using MS. Enzymatic reaction based reporter systems such as GusA have been utilised in Streptomyces, but they need specific substrates for their activity. 272,273 Fluorescence based reporter systems such as sfGFP were reported in a few Streptomyces studies, [274][275][276] but autofluorescence and the diversity of the cell shape were challenges for application to high-throughput screening methods, such as FACS. Thus, an optimised pipeline for pre-treatment and usage of other fluorescent proteins with different excitation wavelengths from auto-fluorescence should be developed.…”

Section: Discovery Of Novel Sms and Linking To Smbgcsmentioning

confidence: 99%

Systems and synthetic biology to elucidate secondary metabolite biosynthetic gene clusters encoded inStreptomycesgenomes

et al. 2021

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Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces

Cited by 21 publications

References 18 publications

Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites

Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

Systems and synthetic biology to elucidate secondary metabolite biosynthetic gene clusters encoded inStreptomycesgenomes

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