An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identifica tion of all such gene clusters in a genome. The approach involves sequencin g small fragments of a random genomic DNA library containing one or more mo dular PKS gene clusters, and identifying which fragments emanate from PKS g enes. Knowing the approximate sizes of the genome and the target gene clust er, one can predict the the frequency that a PKS gene fragment will be pres ent in the library sequenced. Computer simulations of the approach were app lied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clus ters in the Bacillus subtilus genome. The approach was then used to identif y PKS gene fragments in a strain of Sorangium cellulosum that produces epot hilone. In addition to identifying fragments of the epothilone gene cluster , we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrat ing that the approach is also applicable for identification of these modula r gene clusters. (C) 2000 Elsevier Science B.V. All rights reserved.