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Novel Hybrid Polyketide Compounds Produced by Genetic Engineering of the Oxytetracycline Biosynthetic Pathway

D. Hranueli,1 Nataša Perić,1,3* H. Petković,3** G. Biuković,1,2 Zora Toman,1 Jasenka Pigac,1,3 B. Borovička,1,3 Anita Bago,1 I. Crnolatac,1 Tereza Maršić,1,4 Lihong Zhou,3 S. Matošić,4 P.G. Waterman,3 J. Cullum2 and I.S. Hunter3


1
PLIVA d.d., Research Institute, Anti-infective Research, HR-10000 Zagreb, Croatia

2 LB Genetik, Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany
3 Department of Pharmaceutical Sciences, University of Strathclyde, 204 George Street, Glasgow G1 1XW, U. K.
4Faculty of Food Technology and Biotechnology, University of Zagreb, HR-10000 Zagreb, Croatia

Article history:

Received June 16, 1998
Accepted February 15, 1999

Key words:

Streptomyces rimosus, oxytetracycline, polyketides, biosynthetic pathway engineering

Summary:

The whole oxytetracycline (OTC) gene cluster was cloned from the strain Streptomyces rimosus R6. Its restriction map is indistinguishable from that of the strain S. rimosus M15883, which means that results from the two strains can probably be combined. Constructions to induce gene disruptions of otcD1 and otcC were undertaken within the chromosome of S. rimosus R6. OtcD1 is thought to be involved in the folding, cyclization and aromatization of the 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 produced 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 disrupted strains did not have the expected DNA structures, but carried DNA amplifications and deletions. This is probably due to the fact that the otc cluster is located within an amplifiable unit of DNA (AUD) which undergoes frequent spontaneous DNA amplifications and deletions. Spontaneous mutants (Class II) that have deleted the otc cluster are potential hosts for expressing new hybrid polyketide clusters. However, it was possible to isolate derivatives (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.

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