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J. Biol. Chem., Vol. 280, Issue 45, 37455-37460, November 11, 2005

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Ablation of the otcC Gene Encoding a Post-polyketide Hydroxylase from the Oxytetracyline Biosynthetic Pathway in Streptomyces rimosus Results in Novel Polyketides with Altered Chain Length^*

Nataa Peri-Concha, Branko Borovika¶, Paul F. Long, Daslav Hranueli¶1, Peter G. Waterman2, and Iain S. Hunter3

From the Department of Bioscience, University of Strathclyde, Glasgow G1 1QW, Scotland, United Kingdom, ^¶PLIVA, Research & Development Ltd., Biotechnology, 10000 Zagreb, Croatia, and the School of Pharmacy, University of London, London WC1N 1AX, United Kingdom

Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC "minimal" polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex.

Received for publication, March 23, 2005 , and in revised form, September 6, 2005.

^* This work was supported by the Wellcome Trust Grant 051550/Z/97 (to I. S. H. and D. H.) and by Grant 0058008 (to D. H.) from the Ministry of Science, Education and Sport, Republic of Croatia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY916128 [GenBank] .

¹ Present address: University of Zagreb, Faculty of Food Technology and Biotechnology, 10000 Zagreb, Croatia.

² Present address: The Centre for Phytochemistry, Southern Cross University, Lismore NSW 2480, Australia.

³ To whom correspondence should be addressed: Dept. of Bioscience, Royal College, University of Strathclyde, Glasgow G1 1QW, Scotland, UK. Tel.: 44-141-548-4111; Fax: 44-141-548-4124; E-mail: i.s.hunter{at}strath.ac.uk.

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