Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.
Pimaricin is an important antifungal antibiotic for antifungal therapy and prevention of mould contamination in the food industry. In this study, three new pimaricin derivatives, 12-decarboxy-12-methyl pimaricin (1), 4,5-desepoxy-12-decarboxy-12-methyl pimaricin (2), and 2-hydro-3-hydroxy-4,5-desepoxy-12-decarboxy-12-methyl pimaricin (3), were generated through the inactivation of P450 monooxygenase gene scnG in Streptomyces chattanoogensis L10. Compared with pimaricin, 1 displayed a twofold increase in antifungal activity against Candida albicans ATCC 14053 and a 4.5-fold decrease in cytotoxicity with erythrocytes, and 2 had comparable antifungal activity and reduced cytotoxicity, whereas 3 showed nearly no antifungal and hemolytic activities. Genetic and biochemical analyses proved that 1 is converted from 2 by P450 monooxygenase ScnD. Therefore, the overexpression of scnD in scnG-null strain eliminated the accumulation of 2 and improved the yield of 1 by 20 %. Conversely, scnG/scnD double mutation abolished the production of 1 and improved the yield of 2 to 2.3-fold. These results indicate that the pimaricin derivatives with improved pharmacological properties obtained by genetic engineering can be further developed into antifungal agents for potential clinical application.
Salinomycin is a widely used polyether coccidiostat and was recently found to have antitumor activities. However, the mechanism of its biosynthesis remained largely speculative until now. Reported herein is the identification of an unprecedented function of SlnM, homologous to O‐methyltransferases, by correlating its activity with the formation of the Δ18,19 double bond and bis(spiroacetal). Detailed in vivo and in vitro investigations revealed that SlnM, using positively charged S‐adenosylmethionine (SAM) or sinefungin as the cofactor, catalyzed the spirocyclization‐coupled dehydration of C19 in a highly atypical fashion to yield salinomycin.
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