Pairing comparative genomics with tandem mass-based molecular networking allows to highly efficient discovery of...

Microbial natural products, particularly nonribosomal peptides (NRPs), have attracted significant attention due to their structural diversity and therapeutic potential. Nocardia, a genus of Actinomyces, is an important reservoir for natural products, especially NRPs. However, rediscovery is a significant challenge for mining new specialized metabolites from Nocardia, as well as from other sources. To overcome this challenge, we developed a strategy that combines comparative genomics with tandem mass-based molecular networking, which allows to efficiently discover new NRPs from Nocardia spp.. As a proof of concept, all genomes of Norcardia in NCBI database, including three strains from our lab, were compared with each other to prioritize unique biosynthetic gene clusters (BGCs) in the three in-house Nocardia strains, particularly those containing nonribosomal peptide synthases (NRPSs). Subsequently, the metabolomics data of those three in-house strains were analyzed employing tandem mass-based molecular networking. This led to the identification of a known lipopeptide, nocarjamide (1), and five new congeners (2-6) of nocarjamide, as well as a new decalipopeptide, nocarlipoamide (7), along with nocardimicin, a known compound found in Nocardia. The structure of the new decalipopeptide 7 was further extensively characterized using NMR, MS/MS, Marfey's analysis, and X-ray. In addition, the biosynthesis pathways for 1-7 were proposed through bioinformatics analysis, and thus the gene clusters responsible for biosynthesizing them were confirmed. Our results indicate that this strategy enables prompt dereplication of known compounds, rapid linkage of identified compounds with their biosynthesis gene cluster, and efficient discovery of new compounds.