FOLDING OF THE POLYKETIDE CHAIN IS NOT DICTATED BY MINIMAL POLYKETIDESYNTHASE IN THE BIOSYNTHESIS OF MITHRAMYCIN AND ANTHRACYCLINE

Background: Mithramycin, nogalamycin and aclacinomycins are aromatic p olyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionat e (for aklavinone) is used as a starter unit and nine acetates are use d as extender units. The assembly of building blocks is catalyzed by t he minimal polyketide synthase (PKS). Further steps include regiospeci fic reductions (if any) and cyclization. In the biosynthesis of mithra mycin, however, ketoreduction is omitted and the regiospecificity of t he first cyclization differs from that of anthracycline antibiotics (e .g. nogalamycin and aclacinomycins). These significant differences pro vide a convenient means to analyze the determinants for the regiospeci ficity of the first cyclization step. Results: In order to analyze a p ossible role of the minimal PKS in the regiospecificity of the first c yclization in polyketide biosynthesis, we expressed the mtm locus, whi ch includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which nor mally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient str ain generated mithramycin production, Auramycins, instead of aclacinom ycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of sta rter unit. We also describe structural analysis of the compounds accum ulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. Conclusions: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cycli zation of the biosynthetic pathway for aromatic polyketides.