New from old: discovery of the novel antibiotic actinomycin L in Streptomyces sp. MBT27

ABSTRACT Streptomycetes are major producers of bioactive natural products, including the majority of the antibiotics. While much if the low-hanging fruit has been discovered, it is predited that less than 5% of the chemical space has been mined. Here, we describe the novel actinomycins L 1 and L 2 , which are produced by Streptomyces sp. MBT27. The molecules were discovered via metabolic analysis combined with molecular networking of cultures grown with different combinations of carbon sources. Actinomycins L 1 and L 2 are diastereoisomers, and the structure of actinomycin L 2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via a unique spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X 2, prior to the condensation of the actinomycin halves. Feeding anthranilamide to cultures of Streptomyces antibioticus , which has the same biosynthetic gene cluster as Streptomyces sp. MBT27 but only produces actinomycin X 2 , resulted in the production of actinomycin L. This shows that actinomycin L results from joining two distinct metabolic pathways, 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. IMPORTANCE Actinomycin was the first antibiotic discovered in an actinobacterium by Selman Waksman and colleagues, as early as 1940. This period essentially marks the start of the ‘golden era’ of antibiotic discovery. Over time, emerging antimicrobial resistance (AMR) and the declining success rate of antibiotic discovery resulted in the current antibiotic crisis. We surprisingly discovered that under some growth conditions, Streptomyces sp. MBT27 can produce actinomycins that are significantly different from those that have been published so far. The impact of this work is not only that we have discovered a novel molecule with very interesting chemical modifications in one of the oldest antibiotics ever described, but also that this requires the combined action of primary and secondary metabolic pathways, namely the biosynthesis of anthranilamide and of actinomycin X 2 , respectively. The implication of the discovery is that even the most well-studied families of natural products may still have surprises in store for us.