Investigation of the antibiotic biosynthetic potential and phylogenetic diversity of Indonesian actinomycetes

Abstract

Actinomycetes are known as a treasure trove for bioactive compounds as they are the origin of about two-thirds of all antibiotics in clinical use. Unique habitats such as marine and unexplored habitats are considered promising sources for sampling new actinomycetes with a high potential to produce new natural products. Indonesia is one of the countries with the most extensive mangrove marine ecosystems and a vast diversity of microbial species. Thus, exploration of Indonesian samples for actinomycetes may lead to the discovery of new antibiotics. About 422 strains of actinomycetes were isolated from marine sediments of Bali and Lombok Island and soil samples from Enggano Island. The nine most bioactive strains have been prioritized for further drug screening approaches. The nine strains were cultivated on different solid and liquid media using the OSMAC cultivation strategy to assess their biosynthetic capacity to produce natural compounds. A combination of genome mining and mass spectrometry-based (MS) molecular network analysis was applied to identify potential new substances from the nine strains. Several biosynthetic products encoded by the gene clusters of the nine strains were identified, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Additionally, sixteen putative biosynthesis products and numerous biosynthetic gene clusters were found, which could not be associated with any known compound or biosynthetic gene cluster, respectively, indicating that the nine Indonesian strains have the potential to produce new secondary metabolites. Furthermore, a regulator-driven strategy was applied to activate (silent) gene clusters in the Indonesian strains. As an example, the SARP-type activator PapR2 was expressed in the Indonesian strain isolate SHP 22-7 to unlock its chemical diversity. Thereby it could be shown that PapR2 expression activates the biosynthesis of the nucleoside antibiotic plicacetin in SHP 22-7. The increased transcription of the plicacetin cluster was detected by RT-PCR analysis and production of the compound was confirmed by HPLC-MS analysis. Overall, the methodologies described here represent promising strategies for acquiring new natural products from well-known bacterial sources. In addition, the reported unidentified compounds provide a basis for the further characterization and development of these compounds as drug candidates.