|Abstract or Summary
- The discovery of new antibiotics reached its peak during the golden era of antibiotics in the mid 20th century and continued to thrive until the 1980s. However, after more than 50 years of fruitful exploration and exploitation of microbial natural products the discovery rate of new antibiotics has dropped drastically in recent decades. On the other hand, known compounds were re-discovered again and again, possibly from related producing organisms, making natural products discovery an expensive and time-consuming endeavor. Therefore, in recent years, efforts for discovering new natural products have been redirected towards organisms from unique and untapped areas of the globe with remarkable biodiversity potential and/or extreme environment, such as deep-sea vents, caves, and other unusual ecosystems. This dissertation describes our efforts to discover new bioactive natural products from microorganisms isolated from a unique black water ecosystem in Indonesia.
Indonesia is recognized as a major center in the world for biodiversity and is one of the countries with the highest level of endemic species and microorganisms
that live in its various ecosystems. Among them are the black-water rivers, which flow in the steamy jungles of islands along the Indonesian archipelago, providing unique environments for its flora and fauna. In collaboration with scientists at the Indonesian Center for Biodiversity and Biotechnology (ICBB), we investigated bioactive secondary metabolites of microorganisms from the soil samples of a black water ecosystem on Borneo Island, Indonesia. 16S rRNA sequence analysis was used for the identification of the bacteria, and from 141 bacterial strains investigated, 77% were Streptomyces, 6% were non-Streptomyces actinomycetes, and 17% were non-actinomycete strains. About one third of the strains were screened for their bioactive secondary metabolites production. The EtOAc and n-BuOH extracts of the culture broths of the bacteria were subjected to antibacterial and antifungal assays, as well as mass spectrometry analysis. More than half of the screened Streptomyces (18 out of 24) showed activities against one or more pathogenic microorganisms, whereas only 2 out of 14 of the non-actinomycete strains showed antibacterial activities. Furthermore, chemical screening of 9 non-Streptomyces actinomycetes revealed that 3 strains, all of which are Amycolatopsis spp., produce the anticancer agents the apoptolidins.
Chemical investigation of the strain Streptomyces albiflaviniger ICBB 9297 delivered four new elaiophylin-like macrolides and five known elaiophylins. The new compounds have macrocyclic skeletons distinct from those of the known elaiophylins, in which one or both of the dimeric polyketide chains contain(s) an additional pendant methyl group. However, they showed comparable antibacterial activity to elaiophylin against Staphylococcus aureus. Interestingly, those with the additional
pendant methyl group on only one of the polyketide chains showed significantly increased activity against Mycobacterium smegmatis, whereas the one with two additional methyl groups and the known elaiophylin analogues showed no activity. The production of the new analogues suggests that the last acyltrasferase (AT) domain of the elaiophylin polyketide synthases has relaxed substrate specificity. Bioinfomatic analysis of the conserved active site residues within the AT domains revealed an unusual amino acid sequence of the active site motif in the AT7 domain.
Investigations of the apoptolidin producer Amycolatopsis sp. ICBB 8242 led to the discovery of two succinylated apoptolidin analogues and linear apoptolidin A. The two succinylated compounds can inhibit the proliferation and viability of human H292 and HeLa cells. However, in contrast to apoptolidin A, they do not inhibit cellular respiration in H292 cells. This seemingly contradictory results may be due to an alternative biological mechanism of action or due to degradation of succinyl-apoptolidin A to apoptolidin A in the cell viability assay. This hydrolysis may not occur during the real time analysis of cellular respiration, which was performed in a relatively short experimental time. The production of succinylated apoptolidins by Amycolatopsis sp. ICBB 8242 suggests that succinylation may play a role in self-resistance and/or as an export mechanism.