Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization

Area of Science:

• Metabolomics
• Natural Product Discovery
• Fungal Biotechnology

Background:

• Filamentous fungi produce valuable bioactive compounds like antibiotics and statins.
• Linking fungal genes to their metabolites is crucial for future bio-production.
• Stable isotope labeling offers a method to trace metabolite biosynthesis.

Purpose of the Study:

• To investigate the utility of stable isotope labeled amino acids (SILAAs) in identifying fungal metabolites.
• To couple gene-metabolite information for the production of novel bioactive compounds.
• To study the biosynthesis of the cyclic tetrapeptide nidulanin A in Aspergillus nidulans.

Main Methods:

• Cultivation of Aspergillus nidulans with SILAAs.
• Analysis using Ultra-High-Performance Liquid Chromatography-Time of Flight Mass Spectrometry (UHPLC-TOFMS).
• Tandem Mass Spectrometry (MS/MS) for molecular networking and data analysis.

Main Results:

• SILAAs were successfully incorporated into nidulanin A, confirming their utility for metabolite tracing.
• Molecular networking revealed known and unknown SILAA-labeled compounds, including fungisporin.
• Novel analogues of nidulanin A and fungisporin were detected, originating from the same nonribosomal peptide synthase.

Conclusions:

• The combination of SILAA and molecular networking is effective for automated detection of related nonribosomal peptides.
• Isotope labeling facilitates peptide sequencing, providing insights into bioactive compound biosynthesis.
• This approach enhances the discovery and characterization of fungal-derived bioactive compounds.