Mining of actinomycete genomes for the discovery of new natural products

Biotechnological potential of natural products from microorganisms are receiving more attention for discovery of novel bioactive compounds. Terrestrial and marine actinomycetes are an important source for the discovery of novel bioactive compounds. Therefore, a new method genome mining is a good strategy for a natural product discovery. There are different strategies for genome mining. One of them is PCR based genome mining. Degenerate primers are used in order to defined biosynthetic gene clusters. The other is MS guided genome mining. The present study was designed to assess the genetic potential of rhizosphere streptomyces strains by amplifying adenylation and ketosynthase domains of NRPS and PKS-I genes, respectively. Using degenerate PCR and sequencing of cloned products, NRPSs were detected in all (sixty-five) isolates. PKS-I genes were successfully amplified from 80% of the isolates. Based on the BLASTX analysis, most of the NRPS (34%) and PKS (56%) fragments showed below 70% identity to their closest relatives. The results reveal that there is a great diversity in NRPS A domains in those habitats and the majority of domains cloned in this study are probably involved in different biosynthetic pathways. According to the bioinformatic analysis of NRPS genes, different A domains were determined in the obtained sequences, but in some cases their substrate specifity failed to be predicted. This means that these domains can recognize novel amino acids as substrate. The obligate marine actinomycete genus Salinispora is model microorganism for natural product research. All Salinispora strains selected already had whole genome sequence data available and their natural product gene clusters were evaluated with MS guided genome mining. The draft genome sequences of all Salinispora strains were analyzed by bioinformatic tools for the MS-guided genome mining. These tools were used to determine the presence of biosynthetic gene clusters such as PKS, NPRS, terpene, indole, lantibiotic, siderophore so on. Molecular networks were generated when extracts of Salinispora strains were analyzed via LC/MS/MS. Molecular networking of the data resulted in dereplication of lomaiviticin C, 7-OH-staurosporine, staurosporine and cyanosporaside B. As a result of molecular networking analysis, totally 1347 MS/MS data were compared with one another in the network. Finally, we determined specific MS/MS and bioinformatic data for S. pacifica CNY703, S. tropica CNT250 and S. arenicola CNY011 and CNY299. Both chemical and bioinformatic data showed that a glycosylated natural product was found in S. pacifica CNY703 culture broth and reached NRPS-PKS-I glycosylated natural product gene clusters in S. pacifica CNY703 genome. Structural elucidation of a compound which is product of NRPS-PKS-I gene clusters is going on. The major bioactive metabolite produced by Streptomyces sp. CAH29 was extracted, purified and identified as tetrangomycin. This known anthraquinone exhibited antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, methicillin resistant Staphylococcus aureus (MRSA) and Candida albicans with inhibition zones of 14, 10, 12 and 8 mm, respectively. Tetrangomycin showed moderate free DPPH radical scavenging activity and potent cytotoxic activity against cell lines LNCaP (human prostate adenocarcinoma cell) and A549 (human alveolar adenocarcinoma cell). Docking results demonstrate that tetrangomycin has a similar mode of action. As a result of this analysis, it was determined that the binding energy of tetrangomycin and positive control dipotassium (2-oxo-2 - {[3- (3-phenoxyphenyl) propyl] amino} ethyl phosphonate (830) to the dehidrosqual synthase CrtM enzyme of MRSA was found to be close to each other. Therefore, this result suggest that tetrangomycin has the potential to be used as an anti-MRSA agent.