Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin

Background Mithramycin 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. Results Expression 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. Conclusions We 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.


Additional file 4. Generation of gene cassette plasmids for 4DMPC
A cloning strategy was developed to allow the expression of different gene cassettes involved in early stages of MTM biosynthesis. The different genes (mtmPKS, mtmL, mtmQ, mtmX, mtmY, mtmOII, mtmTI y mtmTII) were amplified flanked by unique restriction sites using the oligonucleotides shown in Table S3. To facilitate the following cloning steps, the PCR products were individually subcloned into the vector pCR-blunt to generate plasmids pbluntPKS, pbluntL, pbluntQ, pbluntX, pbluntY, pbluntOII, pbluntTI and pbluntTII, respectively. The shuttle bifunctional E. coli-Streptomyces vector pEM4 was used to express genes under the erythromycin resistance gene promoter (permE*), as follows: pDZPKS1: a SpeI-EcoRI fragment containing mtmP, mtmK and mtmS was subcloned from pbluntPKS into the XbaI and EcoRI sites of pEM4, generating pDZPKS. Then, to facilitate following cloning steps and allow the expression of further genes, a second permE* promoter was introduced downstream of mtmS. To do this, the HindIII-EcoRI (blunt ended) fragment containing mtmP mtmK, mtmS and the permE* promoter was subcloned from pDZPKS into the HindIII (blunt ended) site of pEM4 upstream of its permE* promoter.
pDZPKSQ: mtmQ was rescued as a NheI-XbaI DNA fragment from pbluntQ and subcloned in the right orientation, into the XbaI site of pDZPKS1. pDZPKS5: first, mtmL was isolated as a SpeI-XbaI DNA fragment from pbluntL and subcloned in the right orientation, into the XbaI site of pDZPKS1 generating pDZPKS2. Then, mtmQ was rescued as a NheI-XbaI DNA fragment from pbluntQ and subcloned in the right orientation, into the XbaI site of pDZPKS2 generating pDZPKS3. Afterwards, mtmX was digested as a Spe-XbaI DNA fragment from pbluntX and subcloned in the right orientation, into the same sites of pDZPKS3 generating pDZPKS4. Finally, mtmY was rescued as HpaI-XbaI from plasmid pbluntY and subcloned in the same restriction sites pDZPKS4 generating the final construct pDZPKS5. pDZPKS8: mtmY was isolated from the plasmid pbluntY as a SpeI-XbaI DNA fragment and subcloned in the same sites of pDZPKS3.
pDZPKS9: mtmQ and mtmY were isolated as a NheI-XbaI DNA fragment from pDZPKS8 and subcloned in the right orientation, into the XbaI site of pDZPKS1.
pDZPKS10: mtmTI was isolated as a SpeI-XbaI DNA fragment from pbluntTI and subcloned in the right orientation, into the XbaI site of pDZPKS9. pDZPKS11: mtmX was isolated from pbluntX as a SpeI-XbaI DNA fragment and subcloned in the right orientation, into the site XbaI of pDZPKS9. pDZPKS12: First, mtmOII was isolated as a PacI-XbaI DNA fragment from pbluntOII and subcloned in the same restriction sites of pDZPKS5, generating pDZPKS6. Then, mtmTI was rescued as a SpeI-XbaI DNA fragment from pbluntTI and subcloned in the right orientation, into the XbaI site of pDZPKS6 generating pDZPKS7. Afterwards, mtmTII was rescued as a SpeI-XbaI DNA fragment from pbluntTII and subcloned in the right orientation, into the site XbaI of pDZPKS7 generating pDZPKS14. Finally, mtmQ; mtmX; mtmY; mtmOII; mtmTI and mtmTII were rescued as a NheI-XbaI DNA fragment from pDZPKS14 and subcloned in the right orientation, into the XbaI site of pDZPKS1 generating the final construct pDZPKS12.
pDZPKS13: mtmTII was isolated as a SpeI-XbaI fragment from the vector pbluntTII and subcloned into a XbaI site of pbluntOII to generate pbluntOIITII. Then, mtmOII and mtmTII were isolated as a SpeI-XbaI DNA fragment and subcloned in the site XbaI of pDZPKS11 generating pDZPKS13.
pDZPKS15: mtmOII was isolated as a BamHI-EcoRI DNA fragment from the vector pbluntOII and subcloned in the same site of pEM4AT. pDZPKS21: mtmTI was digested as a EcoRI DNA fragment from pblunTI and subcloned in the right orientation, into the same site of pDZPKS15 generating pDZPKS19. Then, mtmTII was rescued as a BamHI-NheI fragment from pblunTII and subcloned in the sites BamHI-SpeI of pDZPSK19. Table S3. Primers used in this work.