PCR-Based Genomic Screening For Conserved Ⅰ-PKS And NRPS Gene To Discover Natural Products From Actinomycetes

Many important bioactive natural products were synthesized by the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, the conserved ketosynthase gene, methyl-malonyl-CoA transferase gene in the PKS, and the conserved adenylation domain gene in the NRPS can be used as target marker gene for the rapid identification of polyketide or polypeptide producing strains from large strain collections.In this paper, the primers I-PKS-K1F/I-PKS-M6R. NRPS-A3F/NRPS-A7R were used for screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) producing strains from50actinomycetes.23polyketide synthase gene positive strains were obtained. Meanwhile, nonribosomal peptide synthetase (NRPS) were detected by PCR amplification in the polyketide synthase gene-positive strains,17strains show both positive PKS and NRPS gene. In addition, the antitumor activity of this17strains" metabolites was detected by MTT method. The strains1044and1128show high activity to the tumor cells. Furthmore, strain1044was chosen for genome walking, The results exhibited a full KS and AT domains in the module. This strategy can be applied efficiently to hunting for polyketide metabolite gene clusters in actinomycetes without giving much consideration to their bioactive status. Meanwhile, the1044KS-AT domails were used to dispute the polyketide synthase in the stain, that the metabolites from the wide type and the mutant strain is obviously different is helpful for identify the products synthesized by the polyketide synthasc.With the molecular biology methods combining traditional taxonomy,2(1044,1128) out of17positive strains were preliminarily characterized to species level. Then,HPLC-MS/MS and NMR techniques were used to analyse the metabolites of this two positive strains screened above. UPLC-MS/MS and iron fragment show that S.parvulus strain1044produce the polypeptide compound actinomycin D. NMR spectrum of the1128-1of S.diasiatochromogenes strain1128produce the compound of polyketide Aurodox.