P450 Catalyzed Tailoring Steps in Three Polyketide Biosynthetic Pathways

Many P450s are involved in the biosynthesis of polyketides which are structurally a very diverse family of natural products. The gene clusters of three anticancer polyketides, iso-migrastatin from Streptomyces platensis NRRL 18993,  lactimidomycin from Streptomyces amphibiosporus R310-104 and leinamycin from Streptomyces atroolivaceus S-140, had been identified by us before. There are four P450s, MgsK, LtmK, LnmA and LnmZ, in these three biosynthetic pathways. To characterize the functions of these P450s, we carry out gene inactivation to generate mutant strains using lambda-Red-mediated PCR-targeting mutagenesis strategy, and isolate intermediates accumulated in the mutants using a combination of chromatographic and spectroscopic methods. Results show the four P450s catalyze quite different reactions in three biosynthetic pathways. MgsK is a hydroxylase oxidizing C-8 of iso-migrastatin, LtmK is an unusual P450 dehydrogenase generating double bond at C-8/9 of lactimidomycin, LnmA and LnmZ are hydroxylases oxidizing C-8 and C-2' of leinamycin, respectively. Moreover, engineering the tailoring steps between iso-migrastatin and lactimidomycin biosynthetic pathways by introducing mgsk or mgsIJK gene into ΔltmK mutant gives two new “non-natural ” products, which can not be produced in neither of the two pathways and show potent cytotoxicity against several cancer cell lines. These results show the diverse functions of P450s in polyketide biosynthesis. Current ongoing in vitro experiments and structural elucidations of these P450s will highlight the mechanisms of their substrate recognizations and catalytic reactions.