Abstract
BackgroundMithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear.ResultsExpression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps.ConclusionsWe established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.
Highlights
Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus
Delimiting the set of genes required for 4‐demethyl‐premithramycinone biosynthesis The mtm biosynthesis gene cluster (BGC) contains thirty-four genes (Fig. 1a), some of which have been associated with the biosynthesis of 4DMPC [11, 12]: mtmP (β-ketoacyl synthase α), mtmK (β-ketoacyl synthase β) and mtmS, b a c
In vivo reconstitution of the 4‐demethyl‐premithramycinone biosynthetic pathway Based on the above results, we aimed to reconstitute the biosynthesis pathway for 4DMPC in vivo to determine the minimal set of genes required and the order of biosynthetic steps
Summary
Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. This has allowed the generation of new MTM analogs through combinatorial biosynthesis strategies [12] Some of these new analogs showed higher antitumor activity and/or lower toxicity than the parental compound [13,14,15,16], and were shown to affect the Ewing sarcoma xenograft growth and triple negative breast cancer growth in mice [17, 18]. These promising activities have led to the development of these compounds as potential new anticancer drugs [19, 20], which requires sufficient amounts for preclinical and clinical trials
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