Coupling of the engineered DNA 'mutator' to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces
| Autor: | Jörn Kalinowski, Yi-Qian Sun, Anna Stich, Christian Rückert, Martin Zehl, Tobias Busche, Olga N. Sekurova, Sergey B. Zotchev |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
DNA Replication
dnaE AcademicSubjects/SCI00010 DNA polymerase Mutagenesis (molecular biology technique) Biosensing Techniques Streptomyces Gene Expression Regulation Enzymologic dnaQ 03 medical and health sciences chemistry.chemical_compound Gene cluster Escherichia coli Genetics Gene Silencing DNA Polymerase III 030304 developmental biology 0303 health sciences biology 030306 microbiology Escherichia coli Proteins DNA replication DNA Mycobacterium tuberculosis biology.organism_classification Anti-Bacterial Agents chemistry Biochemistry biology.protein Synthetic Biology and Bioengineering |
| Zdroj: | Nucleic Acids Research (NAR) Nucleic Acids Research |
| Popis: | 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 alpha 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. © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. |
| Databáze: | OpenAIRE |
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