Molecular Biology of Polyketide Biosynthesis

Daslav Hranueli^1,2*, Nataša Perić^2,3, Branko Borovička^1,3, Stjepan Bogdan¹, John Cullum4, Peter G. Waterman³** and Iain S. Hunter<sup>3

1</sup>PLIVA d.d., Research and Development, Prilaz baruna Filipovića 25, HR-10000 Zagreb, Croatia
²University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, P.O.B. 625, HR-10000 Zagreb, Croatia
³University of Strathclyde, Department of Pharmaceutical Sciences, 204 George Street, Glasgow G1 1XW, Scotland
⁴Universität Kaiserslautern, LB Genetik, Postfach 3049, D-67653 Kaiserslautern, Germany

Article history:
Received February 8, 2001
Accepted June 20, 2001

Key words:
polyketides, biosynthetic pathway engineering, Streptomyces rimosus, oxytetracycline

Summary:
Streptomyces species and related genera synthesize a large number of secondary metabolites, many of which are biologically active. Amongst them, polyketides is the largest class. Polyketides are a structurally diverse family of natural products with a broad range of biological activities. The formation of polyketides is very similar to the biosynthesis of long chain fatty acids – both in the enzymatic reactions that take place and the enzyme proteins that are involved. During the last decade many polyketide gene-clusters have been cloned and sequenced. DNA sequencing has shown that the clusters have substantial homology suggesting that they originated from a common ancestor. This similarity has resulted in the development of combinatorial biology techniques to create novel chemical entities. Two approaches have been used: targeted manipulation, e.g. disruption and, often, replacement of certain genes involved in the biosynthetic pathway, and the random approach, e.g. »gene shuffling«. A targeted approach has been used to generate several novel scaffolds by manipulation of the S. rimosus oxytetracycline gene-cluster. Genes encoding ketosynthase,
α and β, ketoreductase, cyclase/aromatase and C-6 hydroxylase were disrupted to construct four recombinant strains. Thin layer chromatography and high pressure liquid chromatography of extracts from fermentation broths of all four recombinants showed that they produce about 20 potentially novel metabolites, 12 of which have been characterised chemically. In this review, data on the disruption and replacement of the otcC gene will be described in more detail.

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