Discovery of actinomycin L, a new member of the actinomycin family of antibiotics

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L 1 and L 2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L 1 and L 2 are diastereomers, and the structure of actinomycin L 2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X 2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X 2 producer Streptomyces antibioticus , which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X 2 and for anthranilamide. Actinomycins L 1 and L 2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.