Omics-guided bioactive natural products discovery from Streptomyces

Streptomyces, a captivating and diverse genus within the Actinobacteria phylum, holds a prominent place in the field of natural product discovery. They are a rich source of bioactive compounds, such as antibiotics, anticancer agents, and immunosuppressants. More than 70% of the clinical antibiotics are derived from this valuable genus. In recent years, advanced analytical techniques in Mass Spectrometry (MS) and genome mining methods have accelerated the process of drug discovery in different microbes, while a large number of secondary metabolites in Streptomyces remain largely unexplored.

My PhD project aims at discovering unknown bioactive secondary metabolites from Streptomyces, especially those found in soil. I have employed a mix of targeted and untargeted metabolomics, MS, Nuclear Magnetic Resonance spectroscopy (NMR) and cheminformatics to explore and characterize the structures of new metabolites, analyze the biosynthesis, and potential mode of actions. Firstly, using genome mining method, it revealed that Streptomyces rapamycinicus is a producer of various unknown metabolites. An untargeted LCMS analysis approach was employed to analyze novel secondary metabolites. Initial a one strain-many compounds (OSMAC) approach followed by large scale fermentation, metabolomics and activity-guided chemical isolation, with dereplication approach revealed five previously described and four novel ansamycin analogues (hygrocins W–Z) from Streptomyces rapamycinicus (Paper 1). I carried out electronic circular Dichroism (ECD) calculations to elucidate the stereospecific configuration. Furthermore, the biosynthesis was confirmed through CRISPR-BEST gene editing. Additionally, known PKS alchivemycin A, rapamycins, cuevaenes, and finally two new hybrid PKS-NRPs related to nyuzenamides were also isolated from the same strain.

Secondly, I have discovered new pepticinnamin E analogues (pepticinnamins N–P) from a Streptomyces mutant. Streptomyces mirabilis P8-A2 was a producer of a class of new azoxy compounds azodyrecins. Mutation of the azodyrecin biosynthesis gene cluster led to the activation of a silent pepticinnamin biosynthetic gene cluster. Non-ribosomal peptides pepticinnamins were isolated and identified. The chirality was elucidated through Marfey´s method. The study was also extended to identify the structural diversity of cytotoxic pepticinnamins (Paper-2).

Thirdly, I investigated the soil metabolites from Streptomyces sp. P82B18, isolated from the Dyrhaven park from Danish soil. Through co-cultivation, it revealed that, the strain exhibited antifungal activities against a series of fungal strains. Using the metabolomics approach, four new lysolipin derivatives (lysolipins J–M) were discovered. They belong to the family of polycyclic xanthones, several other analogues were identified by molecular networking, UV patterns and MSMS requiring additional work for purification and structural elucidation. Using MALDI imaging, those lysolipins appeared to be responsible for the antifungal activities, in vitro antifungal assays further confirmed this assumption Moreover, the lysolipins exhibited strong cytotoxicity effects, at nanomolar level, however, the mechanism underlying is not fully understood. Thus, a systematic molecular docking approach using different scoring functions was employed to try and understand the mode of action. The best docking results were obtained with Topoisomerase IIb of all the tested proteins, further in vitro assays are required to confirm obtained results (Paper-3).

Lastly, simulation of interactions between bacteria in shared environments was studied through co-cultivation of two marine bacteria Pseudoalteromonas luteoviolacea and Vibrio coralliilyticus, known for their proficiency in antibiotic secondary metabolite production. To analyze the resulting chemical diversity, we employ advanced analytical methods, including comparative untargeted metabolomics, HPLC-DAD-HRMS-based molecular networking, and manual dereplication. Results showed the upregulation of violacein production alongside the production of secondary metabolites not observed in the monocultures including Morimide C and new analogues and downregulation of others.

Overall, the research of this PhD project has discovered several classes of bioactive secondary metabolites from Streptomyces, revealed the bioactivity potential of isolated compounds, and highlighted the chemical diversity of Streptomyces sp. using different analytical approaches. This has included targeted and untargeted mass spectrometry analyses, molecular docking, and molecular networking strategies. The applied approaches and obtained results have contributed new insights into the mass-spectrometry-based discovery of secondary metabolites.