| Nonactin is a polyether antibiotic and a potent inhibitor of the P170-glycoprotein efflux pump responsible for drug resistance in multiple-drug resistant cancer cell lines. Nonactin is a tetrameric compound containing two molecules of (+)-nonactate and two molecules of (–)-nonactate enantiomers. While the chemistry of polyether antibiotics in general has been well characterized, the genetics and biochemistry responsible for their biosynthesis is virtually unknown. The recent sequence analysis of over 20 kilobases of the nonactin biosynthesis cluster in Streptomyces griseus subsp. griseus ETH A7796 revealed that nonactin is biosynthesized by a unique organization of type II PKS genes. Specifically, two distinct sets of open reading frames with homology to polyketide synthase genes—both arranged in a KAS-α, KAS-β, ketoreductase, ketoreductase order—are present within the cluster. It has been hypothesized that each set of genes is responsible for the synthesis of each enantiomer of nonactate. This work reports the successful gene disruption of one set of KAS-αβ analogues, nonK and nonJ, and shows that the mutant exhibits a 25-fold decrease in overall macrotetrolide production while increasing levels of bishomononactate. The latter result suggests that the genes nonK and nonJ together are possibly responsible for extender unit selection in nonactin biosynthesis. This work also discusses future targeted gene disruption strategies in Streptomyces griseus .; Daunorubicin, and its analogue doxorubicin, are both clinically administered anthracycline antibiotics that have strong biological activity against a wide range of neoplasias. Daunorubicin is generated in small quantities in batch fermentations of Streptomyces sp. strain C5. Previous work, however, has shown that the bioconversion of daunorubicin to doxorubicin—as well as several other anthracycline oxidation reactions in Streptomyces C5—can be accomplished using recombinant, purified DoxA, a cytochrome P450 cloned and overexpressed in the heterologous host, Streptomyces lividans TK24, using an in vitro NADPH-regeneration biochemical assay. This work expands the current knowledge of DoxA biochemistry by showing that DoxA is capable of converting a non-physiological substrate, desacetyladriamycin, to 10-hydroxydesacetyladriamycin. Both the generated product and the catalytic event demonstrate new DoxA biochemistry. |
