Novel hybrid polyketide compounds produced by genetic engineering of the oxytetracycline biosynthetic pathway

The whole oxytetracycline (OTC) gene cluster was cloned from the strain Str eptomyces rimosus R6. Its restriction map is indistinguishable from that of the strain S. rimosus M15883, which means that results from the two strain s can probably be combined. Constructions to induce gene disruptions of otc D1 and otcC were undertaken within the chromosome of S. rimosus R6. OtcD1 i s thought to be involved in the folding, cyclization and aromatization of t he hypothetical nonaketide intermediate in OTC biosynthesis (pre-polyketide ), whereas OtcC hydroxylates the completed tetracyclic nucleus at C-6 (post -polyketide). The disrupted strains no longer produced OTC. OtcC::gmr produ ced three novel compounds, while otcD1::ermE produced a number of compounds , not detectable in the wild-type strain. These novel compounds contained 9 , 15 and 17 C-atoms in their backbones instead of 19, as with OTC. The disr upted strains did not have the expected DNA structures, but carried DNA amp lifications and deletions. This is probably due to the fact that the otc cl uster is located within an amplifiable unit of DNA (AUD) which undergoes fr equent spontaneous DNA amplifications and deletions. Spontaneous mutants (C lass II) that have deleted the otc cluster are potential hosts for expressi ng new hybrid polyketide clusters. However, it was possible to isolate deri vatives (Class IIR) that produce an antibiotic activity. It was shown that this compound was oxytetracycline and that the Class II mutants themselves also produce very small amounts of oxytetracycline.