Monday, July 27, 2009
P110

The chelocardin gene cluster from Amycolatopsis sulphurea

Urska Lesnik1, Peter Raspor2, Iain S. Hunter3, and Hrvoje Petkovic2. (1) Microbiology Dpt., Acies Bio Ltd., Tehnološki park 21, Ljubljana, Slovenia, (2) Chair of Biotechnology, Biotechnical faculty, University of Ljubljana, Jamnikareva 101, Ljubljana, 1000, Slovenia, (3) Royal College, University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, 204 George Street, Glasgow, Scotland

Tetracyclines are a large group of medically-important antibiotics with a common basic structure of four linearly fused six-membered rings. Tetracycline antibiotics are produced by gram-positive bacteria from the genus Actinomyces; some examples are tetracycline, chlortetracycline, oxytetracycine, and demethylchlortetracycline, synthesized by so-called polyketide synthase (PKS) multi-enzyme complexes. A number of potent antibacterial tetracyclines have been generated using a semi-synthetic approach, such as doxycycline, minocycline, and tigecyclin. Tetracyclines were the first broad-spectrum antibiotics, acting at the ribosomal level and interfering with protein synthesis processes in susceptible bacteria. However, a small group of tetracycline analogs are not targeting bacterial ribosomes, thus exhibiting bactericidal activity even against the tetracycline-resistant strains; these analogs include chelocardin, produced by Amycolatopsis sulphurea. The objective of our research was the cloning, sequencing and characterization a gene cluster encoding chelocardin. A genomic library of A. sulphurea was created and screened with the type II PKS probe (KSα), PCRs and restriction analysis. A cosmid VIIC4 was sequenced. The open reading frames were analyzed and putative gene functions were assigned according to the BLASTp analysis. The cloned gene cluster contained 18 genes typical of a Type-II polyketide cluster. Consistently with the chemical structure of the chelocardin, the cluster encodes genes for a typical minimal PKS (KSα, KSβ and ACP), three genes involved in the cyclisation/aromatisation process, two methyltransferases, one aminotransferase, three oxygenases, a ketoreductase, an acyl-CoA ligase, a drug resistance transporter and a transcriptional regulator. From the DNA sequence obtained, the biosynthetic pathway was proposed.

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