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.