The Biosynthetic Mechanism For The Polyether Antibiotic Salinomycin

Salinomycin, a polyether antibiotic, is produced by Streptomycesalbus. It is widely used as an anticoccidial and growth-promoting agent inanimal husbandry. Recently, salinomycin has also been identified as anagent to kill epithelial cancer stem cells. However, the biosynthesis ofsalinomycin have not been studied, except the feeding experiments withisotope-labeled precursors.The previously reported4.5-kb region was proven to be irrelevant tosalinomycin biosynthesis by deletion of this portion. Therefore, a strategywith degenerate epoxidase-specific PCR primers was developed to clonethe polyether biosynthetic gene clusters. A putative salinomycin-specificepoxidase gene slnC was cloned from the producer S. albus XM211usingthis strategy. The targeted replacement of slnC and subsequenttrans-complementation proved its involvement in salinomycinbiosynthesis. A127-kb region was sequenced, including genes encodingtype I PKS (slnA1-slnA9), epoxidase, epoxide hydrolase, regulator andtransporter. The domain structure of distributed modules of salinomycinPKS were also deduced. Intriguingly, the lack of dehydratase (DH)domains in modules14and6implies the presence of hydroxyl groups at C3and C19, which does not match the predicted double bonds at C2-C3and C18-C19of salinomycin. This discrepancy suggests the possibilitythat the required dehydration reactions may be catalyzed by discretedehydratases after polyketide chain assembly. There is no putativedehydratase gene, however, in the cloned region.Within the salinomycin gene cluster, immediately downstream ofslnDI resides slnM. In silico analysis reveals that SlnM shows moderatehomology to TcmP in tetracenomycin biosynthesis, which catalyzesmethylation of a terminal carboxyl group. However, there is nomethylated salinomycin detected in XM211fermentation extract throughHPLC-MS analysis. Through gene replacement andtrans-complementation, the involvement of slnM in salinomycinbiosynthesis was confirmed. Besides, slnM mutant accumulates a novelcompound1. The NMR data indicated C18-C19bond saturation and thepresence of a C19hydroxyl group in1. Different from our expectation,no tricyclic spiroacetal was found in1, instead, a hydroxyl group ispresent at C13. Thus, it was very interesting to study the mechanism andtiming of the formation of the tricyclic spiroacetal. Feeding of1to JCY16(ΔslnC) or JCY34(ΔslnA9), both of which abolished the production ofsalinomycin but harbored the complete slnM gene, resulted in therestoration of salinomycin, suggesting that1is a possible intermediate ofsalinomycin biosynthesis. Heterologously expressed SlnM catalyzes the conversion of1tosalinomycin. This result suggests SlnM is responsible for the last reactionin salinomycin biosynthesis. To test whether S-adenosyl-methionine(SAM) serves as a methyl donor, we monitored the concentrations ofSAM and S-adenosyl-L-homocysteine (SAH) in the reaction mixture.Unexpectedly, neither consumption of SAM nor accumulation of SAHoccurred in the reaction mixture as detected by HPLC. Furthermore,sinefungin, a methyltransferase inhibitor and an SAM analogue unable todonate a methyl group, was found to stimulate the activity of SlnM ascomparable to SAM. In additional, the carboxyl group of1was proven tobe essential since esterified compound1could not be catalyzed by SlnM.Taking all this information into account, we have proposed a mechanismfor SlnM-catalyzed reaction. With a positive charge at neutral pH, SAMor sinefungin stabilizes the conformation of compound1similar tosalinomycin by electrostatic interactions in SlnM, which helps thedehydration of C18-C19and subsequent formation of the tricyclicspiroacetal. The biochemical characterization of SlnM as aSAM-dependent enzyme not only provides new insight into the catalyticroles of SAM, but also reveals an unprecedented mechanism forpolyketide double bond formation.In order to gain insight into the salinomycin biosynthetic mechanism,other12gene replacements were conducted. Among these genes,5genes were found to be essential for salinomycin biosynthesis and possiblyresponsible for polyketide chain release (slnBI), modification (slnF),oxidative cyclization (slnBII, slnBIII) and regulation (slnR). Moreover,5genes were identified as relevant to salinomycin biosynthesis andputatively involved in removal of aberrant extender units (slnDII),precursor supply (orf11, orf12) and regulation (orf15, orf16). Differentfrom other reported polyether gene clusters, salinomycin biosyntheticgene cluster contains three epoxide hydrolase genes: slnBI, slnBII andslnBIII. Gene replacements suggest that SlnBII and SlnBIII areresponsible for epoxide-opening cascades while SlnBI is involved inpolyketide chain release.The data presented here expand our understanding of polyetherbiosynthetic mechanisms and pave the way for targeted engineering ofsalinomycin bioactivity and productivity.