PCR Screening for Aromatic Polyketide Producers

We have previously reported the isolation of 180 marine bacteria from 15 sponge samples that were collected from South China Sea [19 (link)]. We have extracted all strains’ genomic DNAs and discovered nonribosomal peptides (bacillibactin and bacillomycin D analogues) by using a PCR screening method [19 (link)]. In this study, the forward primer (5′-GGCAGCGGITTCGGCGGITTCCAG-3′) and the reverse primer (5′-CGITGTTIACIGCGTAGAACCAGGCG-3′), designed from the conserved sequences of KS_α and KS_β in the biosynthesis of tetracenomycin, daunorubicin, actinorhodin and fredericamycin [20 (link)], were used in the PCR screening for aromatic polyketide producers. A 20 μL PCR system consisting of 10 µL Easy Taq Polymerase (Beijing TransGen Biotech, Beijing, China), 2 µL of forward and reverse primer mixture (each for 10 µM), 1 µL genomic DNA (50–100 ng), and 7 µL sterilized water, were used. The PCR program was performed with an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min, followed by incubation at 72 °C for 10 min. In total, 167 strains were screened and the PCR products were analyzed by agarose gel electrophoresis, recovered with a gel purification kit (Beijing TransGen Biotech, Beijing, China) and sequenced to afford 12 “positive” strains.