Biomimetic total synthesis and paired omics identify an intermolecular Diels-Alder reaction as the key step in lugdunomycin biosynthesis

Microbial natural products are the basis of the majority of the clinical drugs, such as antibiotics, anticancer agents, antifungals and immunosuppressants. The sharp rise in antimicrobial resistance (AMR) requires new ways to replenish the drug-discovery pipelines, whereby the discovery of truly novel structural scaffolds is a major challenge. Lugdunomycin is a highly rearranged angucycline polyketide produced by Streptomyces sp. QL37, but the later stages of its biosynthesis so far remained elusive. Here, using combined biomimetic chemical synthesis, computational methods, genomics and mutational analysis, we shed important new light on the biosynthesis of a molecule with highly complex chemical scaffold. We show that lugdunomycin is formed by a rare intermolecular Diels-Alder reaction, with elmonin as a masked diene and iso-maleimycin as dienophile. The biosynthesis of the two substrates is encoded by distinct biosynthetic gene clusters (BGCs), whereby elmonin is specified by an angucycline BGC, while the biosynthesis of iso-maleimycin is encoded by a BGC for a β-lactone-like compound. Biomimetic total synthesis of lugdunomycin showed that the Diels-Alder reaction leads to the production of a diastereomer of lugdunomycin as the main product in vitro. The diastereomeric ratio of the in vitro Diels-Alder reaction shifted towards lugdunomycin in the presence of proteinaceous material, suggesting that the in vivo Diels-Alder reaction is templated. The requirement of distinct biosynthetic pathways and complex chemical reactions indicate the challenges we face in discovering new chemical space.