Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers.
Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.
Savic
Miloje
M
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. Box 23, 11 010, Belgrade, Serbia and Montenegro.
Vasiljevic
Branka
B
D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial; D018942:Macrolides / Q000032:analysis / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D048630:Polyketide Synthases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D018942:Macrolides; D048630:Polyketide Synthases