Use of an inducible promoter for antibiotic production in a heterologous host.
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.
Dangel
Volker
V
Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Westrich
Lucia
L
Smith
Margaret C M
MC
Heide
Lutz
L
Gust
Bertolt
B
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D005818:Genetic Engineering; D009675:Novobiocin / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D009675:Novobiocin