Design and utility of oligonucleotide gene probes for fungal polyketide synthases

Background: Recent advances in the molecular biology of polyketide biosynth esis have allowed the engineering of polyketide synthases and the biologica l ('combinatorial') synthesis of novel polyketides. Additional structural d iversity in these compounds could be expected if more diverse polyketide sy nthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the struct ure and function of fungal PKS complexes. Relatively few fungal synthases h ave been investigated, perhaps because of a lack of suitable genetic techni ques available for the isolation and manipulation of gene clusters from div erse hosts. We set out to devise a general method for the detection of spec ific PKS genes from fungi. Results: We examined sequence data from known fungal and bacterial polyketi de synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conser vation. Using individual domains such as beta -ketoacylsynthases (KS), beta -ketoreductases (KR) and methyltransferases (MeT) we determined specific s hort (ca 7 amino acid) sequences showing high conservation for particular f unctional domains (e.g. fungal KR domains involved in producing partially r educed metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching th ese regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing specie s. Products obtained from these reactions were sequenced and shown to be fr agments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA. Conclusions: A number of sequences are presented which have high utility fo r the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, pa rtially reduced or highly reduced KS domains). We have also developed prime rs suitable for amplifying segments of fungal genes encoding polyketide C-m ethyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potentia l as aids for the eventual cloning of new fungal PKS gene clusters. (C) 200 1 Elsevier Science Ltd. All rights reserved.