Streptomyces produce a wide variety of bioactive substances, including the anthracycline anticancer polyketides. Anthracyclines are potent topoisomerase II poisons that inhibit DNA replication in human cancers. Because of their cardiotoxicity, anthracyclines are restricted in their clinical use. However, engineering new anthracycline analogs could decrease cardiotoxicity and could result in more potent antiproliferative activity. Anthracyclines are biosynthesized by a polyketide synthase and are consecutively aromatized, cyclized, and oxidatively modified to afford a tetracyclic anthracycline core. The post polyketide synthase (post-PKS) modification steps include glycosylations, methylations, and oxygenations that generate biological activity. Our team has developed a Streptomyces metabolic engineering platform for interrogation of post-PKS enzymes. Engineering of several constructs for the production of oxidatively modified polyketides was performed using S. coelicolor M1152 delta matAB strains engineered for production of aklavinone, auramycinone, 9-epi-aklavinone, and nogalamycinone. First, we engineered in the aklavinone 11-hydroxylase genes dnrF or rdmE the strains to produce 11-hydroxylated anthracyclinones. Secondly, we engineered in the kstA15/kstA16 or snoaL2/snoaW cassettes for 1-hydroxylation. Thirdly, we engineered in rdmE+rdmC+rdmB to generate 10,11-hydroxylated anthracyclinones. We engineered in a four gene cassette from the kosinostatin pathway consisting of kstA15+kstA16+kstA10+kstA11 cassette to rearrange the alcohol from the 1-postion to the 4-position to generate idarubicin-like derivatives. These proof-of-concept results provide the basis for a comprehensive biosynthetic platform used for synthesis of designer anthracyclines.

Nora Schwartz

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