TY - JOUR

T1 - Design and utility of oligonucleotide gene probes for fungal polyketide synthases

AU - Nicholson, Thomas P.

AU - Rudd, Brian A.M.

AU - Dawson, Mike

AU - Lazarus, Colin M.

AU - Simpson, Thomas J.

AU - Cox, Russell J.

N1 - Funding information: We are grateful to Christine K Farinelli for generous help with experiments and discussion. We thank Dianne Allen for experimental help. We thank the investigators who provided bacterial strains for the experiments. This work was supported by the Anna Fuller Fund Graduate Fellowship in Molecular Oncology to Z.Z.Z., by National Institutes of Health Grant CA52127 to J.M.E., and by a Howard Hughes Medical Institute Research Resources Program for Medical Schools Award to the University of Massachusetts Medical School.

PY - 2001/1/1

Y1 - 2001/1/1

N2 - Background: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. Results: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as β-ketoacylsynthases (KS), β-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments 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 for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.

AB - Background: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. Results: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as β-ketoacylsynthases (KS), β-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments 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 for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.

KW - C-Methyltransf erase

KW - Fungus

KW - Mycotoxin

KW - Polyketide synthase

KW - Squalestatin

KW - β-Ketoacylsynthase

UR - http://www.scopus.com/inward/record.url?scp=0035060868&partnerID=8YFLogxK

U2 - 10.1016/S1074-5521(00)90064-4

DO - 10.1016/S1074-5521(00)90064-4

M3 - Article

AN - SCOPUS:0035060868

VL - 8

SP - 157

EP - 178

JO - Chemistry and Biology

JF - Chemistry and Biology

SN - 1074-5521

IS - 2

ER -