Use of an inducible promoter for antibiotic production in a heterologous host

• Published in: Applied microbiology and biotechnology Vol. 87; no. 1; pp. 261 - 269
• Main Authors: Dangel, Volker, Westrich, Lucia, Smith, Margaret C. M, Heide, Lutz, Gust, Bertolt
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.06.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Anti-Bacterial Agents - biosynthesis; Antibiotics; Applied Genetics and Molecular Biotechnology; Binding sites; Biological and medical sciences; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Chemical reactions; E coli; Fundamental and applied biological sciences. Psychology; Genes; Genetic Engineering; Inducible promoter; Life Sciences; Medical research; Microbial Genetics and Genomics; Microbiology; novobiocin; Novobiocin - biosynthesis; Plasmids; Promoter Regions, Genetic; Ribonucleic acid; RNA; Streptococcus infections; Streptomyces; Streptomyces coelicolor; Streptomyces coelicolor - genetics; Streptomyces coelicolor - metabolism; Studies; Streptomyces; Genetic engineering; Novobiocin; Inducible promoter; Actinomycetales; Antibiotic; Streptomycetaceae; Production; Bacteria; Actinomycetes
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2435-4

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L⁻¹ of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L⁻¹). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.