Actinobacteria possess an incredible wealth of pathways for production of bioactive compounds. Genetics, biochemistry and biological activity of natural products have most often been studied on a single compound level. Following advances in genome mining, over 50 natural product gene clusters are routinely found in each genome, however the modus operandi of this large arsenal is poorly understood.
After the discovery of TOR signaling, rapamycin has proven an invaluable asset for cell biology research, as well as clinical application. During investigations of the secondary metabolome of its producer, Streptomyces rapamycinicus, we have observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its novel desmethyl analogue. Actinoplanic acids have been left unexplored in their biosynthetic origin after the initial isolation and characterization of their potent and selective Ras farnesyl-protein transferase inhibitor (FTI) activity in early 1990s. The structure of these polyketides contains an extremely rare tricarballylic moiety, presently found only on one other natural product, the fungal toxins fumonisins. In addition, the actinoplanic acid is unique in having one of the tricarballylic groups closing a part of the polyketide backbone into a double-ester lactone.
Supported with the unique structure of these polyketides, we have identified a gene cluster responsible for their biosynthesis through genome mining. Using CRISPR genome editing tools, which we developed for this organism, and the power of bioinformatic functional prediction, we have outlined the actinoplanic acid biosynthetic pathway, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into a natural product. We show that the core polyketide is acylated with tricarballylate by a rare, atypical NRPS-catalyzed ester formation. The proteins involved are phylogenetically distant to other examples of ester-forming NRPS, including those involved in the fumonisin pathway. Strikingly, despite the extremely rare chemistry and similar biosynthetic principles, the two pathways seem to have evolved independently.
Finally, through exploration of the actinoplanic acid pathway, we have observed that actinoplanic acid and rapamycin clusters are without exception co-localized in the genomes of their hosts. This led to discovery of synergistic antifungal activity of actinoplanic acid A and rapamycin. Synergism and contingency in the big arsenal of secondary metabolites have been foreseen for decades, however their teachings are vastly underexploited. Mining for such evolutionary conserved co-harboring of pathways would likely reveal further examples of secondary metabolite sets, attacking multiple targets on the same foe. These can then serve as a guidance for development of new combination therapies.
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