Integrated Approaches for Marine Actinomycete Biodiscovery

Abstract

This PhD project examined integrated approaches for marine actinomycete biodiscovery. A library of 120 actinomycete strains derived from the three Australian ascidians, Symplegma rubra, Aplidium solidium and Polyclinum vasculosum, was established in order to access new chemical diversity of underexplored marine ecological niches for natural product drug discovery. Specifically, the genera Streptomyces and Micromonospora were highly diverse and abundantly present, while fewer Nocardia and Rhodococcus and only one Streptosporangium representative were isolated. Only two isolates occurred in all three ascidians indicating speciesspecificity of actinomycetes in the ascidian. LC-MS/MS profiling of extracts obtained from the ascidians and their actinomycete associates revealed many overlapping ions between hosts and cultured isolates indicating that these compounds were likely to be synthesised by the microbial associates. Laboratory cultures of the actinomycetes displayed even more diverse metabolomes than those of their ascidian hosts; thus making ascidian-associated actinomycetes an excellent target for biodiscovery. Reisolation of already known compounds is a major obstacle to natural product drug discovery; therefore, sophisticated dereplication approaches have to be employed in the early stages of discovery. To facilitate the dereplication, a new strain prioritisation approach using HSQC-TOCSY NMR spectra together with anti-plasmodial activity data was developed. This allowed for prioritisation of microbial strains that are more likely to produce diverse polyketide or peptide natural products. This approach was further validated through dereplication of monoand co-culture extracts of four different microbial strains. HSQC-TOCSY NMR profiles of the extracts clearly showed co-cultivation induced changes in the microbial metabolomes. Streptomyces sp. (USC-16018) was selected based on the strain prioritisation approach and large-scale fermentations led to the isolation of a new ansamycin polyketide, herbimycin G with weak anti-plasmodial activity (77 % at 40 μM), as well as the known compounds elaiophylin, and the four diketiopiperazines, Cyclo-L-Pro-L-Leu, Cyclo-L-Pro-L-Phe, Cyclo-L-Pro-L-Val and Cyclo-L-Pro-L-Tyr. The crude NMR profile of the sediment-derived Streptomyces sp. (USC- 636) revealed the presence of indole-associated signals and three tryptophan containing diketopiperazines, naseseazine A – C, were isolated. The new compound naseseazine C exhibited moderate anti-plasmodial activity (IC50 3.52 μM). Overall, this thesis highlights the immense chemical diversity of actinomycetes from marine sources, as well as the importance of sophisticated dereplication approaches and provides an alternative new solution for strain selection that has led to the discovery of new bioactive natural products with potential as new drug leads.