Combination of classical bioassay-guided and advanced genomics-driven discovery of novel natural products in Actinomycetes

Julia Piekarski
2020 unpublished
Two Actinomycetes strains, Streptomyces noursei and Amycolatopsis sp. YIM10, were selected to isolate putative new secondary metabolites. Therefore, a combination of the classical bioassay-guided approach and genome mining was used. Streptomyces noursei ATCC 11455 is known to produce an antimicrobial compound additional to nystatin, which is active against Gram+ bacteria and might be a new polyketide. An attempt was made to isolate and purify this substance. MS analysis confirmed the presence
more » ... numerous macrolides. Two already registered molecules (YL 02107Q and CAS 37359-09-4) and potentially new metabolites, related to the antibiotic YL 02107Q, were detected. Genome sequencing revealed a potentially new T1PKS encoded by cluster 14, which might be responsible for the production of YL 02107Q and related compounds. To prove this, an insertional disruption was performed. Analyzing the extract of the resulting knock-out mutant confirmed the absence of antimicrobial activity in bioassay experiments. For Amycolatopsis sp. YIM10, a heterologous expression of cluster 5, which encodes for a putative new NRPS, was performed in Streptomyces coelicolor M1154 and Streptomyces albus Del14. The fermentation in different media and the subsequent analysis of the extracts by analytical HPLC did not yield any new compounds. Based on the results from antiSMASH, three putative regulatory proteins (gntR, asnR and luxR), which are localized within cluster 5, were selected for overexpression. The overexpression was accomplished with gntR in S. albus Del14 harboring cluster 5, but no new metabolites were detected. Nevertheless, an increase of metabolite production was detected in the recombinant strain.
doi:10.25365/thesis.63118 fatcat:67xlt4usdbdwjg4nee76gkju5m