Induction of secondary metabolism across actinobacterial genera

Author: Rio Risandiansyah

Risandiansyah, Rio, 2016 Induction of secondary metabolism across actinobacterial genera, Flinders University, School of Medicine

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Current research in drug discovery suggests that co-culture of actinobacteria would result in the activation of “cryptic” genes in the production of novel secondary metabolites. In an effort to study co-culture, 388 unique combinations of actinobacteria were screened for interactivity (defined as changes in morphology, pigment production, sporulation and antibiotic activity), in which 45 possible interactions were discovered. Among these interactions, Pseudonocardia sp. PIP 161 was found to interact with at least 17 other Streptomyces species, in which an increase in antibiotic activity and sporulation was observed.

HPLC-DAD analysis, however, revealed that co-culture with Pseudonocardia sp. PIP 161 had varying effects on the induction of secondary metabolism in different Streptomyces species. While both co-cultured Streptomyces spp. showed an increase in antibiotic production, Streptomyces sp. EUC 63 showed an increase in the production of most, if not all, compounds, with up to a 7 fold increase in the production of an enterobactin-like compound. Furthermore, the interaction between Pseudonocardia sp. PIP 161 and Streptomyces spp. were found to occur via a signal from a secreted chemical, which was producible in axenic conditions of Pseudonocardia sp. PIP 161 in both liquid and solid medium.

As enterobactin was known as a potent siderophore, co-culture studies were undertaken in conditions of varying iron levels, and whether an increase in iron levels would mimic the changes observed during co-culture with Pseudonocardia sp. PIP 161. However, while iron was observed to play an essential role in the formation of aerial mycelia and sporulation, the production of antibiotics and pigments were found, in this case, to be more dependent on chemical signals from Pseudonocardia sp. PIP 161 compared to iron levels. Therefore, efforts were made to isolate the signal producing chemical, in order to be used as an inducer for secondary metabolite production.

Pseudonocardia sp. PIP 161 was then subcultured and tested for inducing activity in several Streptomyces spp from broth eluates. The extraction of these compounds followed by tandem MS/MS reveals a major chemical compound from Pseudonocardia sp. PIP 161 to be a diketopiperazine, cyclo (His-Pro), as well as at least one other diketopiperazine. Although previously detected from mammals and plant pathogens, this is the first report of cyclo (His-Pro) being detected in endophytic actinobacteria. Induction with cyclo (His-Pro) significantly increased antibiotic activity in Streptomyces sp. EUC 63 and increased blue pigment production from Streptomyces sp. SC 36. However, cyclo (His-Pro) induction alone fails to completely mimic the morphological and metabolic changes observed in co-culture with Pseudonocardia sp. PIP 161.

Therefore, based on these findings, it was concluded that the interactivity of Pseudonocardia sp. PIP 161 was in part mediated by diketopiperazines, and provides preliminary evidence of the role of diketopiperazines as cell signalling compounds in actinobacteria. Continuation of this study may show the potential of diketopiperazine induction in awakening previously unexpressed genes from actinobacteria, and thus be used as inducers in drug discovery projects.

Keywords: Drug Discovery, Co-culture, Interaction, Actinobacteria, Actinomycetes, Pseudonocardia, Diketopiperazines, Antibiotic, Secondary Metabolism

Subject: Medical Biochemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2016
School: School of Medicine
Supervisor: Prof. Christopher M.M Franco