| Tetracycline antibiotics are one of the most well known classes of natural products and have been produced commercially for nearly 60 years. Recent biosynthetic studies over the last decade have elucidated many of the enzymatic steps and solved several of the longstanding mysteries of tetracycline biosynthesis, however several steps remain to be fully elucidated. SF2575 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards a broad range of cancer cell lines. In addition to the heavily oxidized 2-naphthacenecarboxamide carbon skeleton characteristic of the tetracycline family, SF2575 displays a number of unique structural modifications. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogs.;This study reports the identification, sequence and functional analysis of the ssf biosynthetic gene cluster responsible for the production of SF2575. The gene cluster was found to contain 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features are found in the gene cluster. Using a combination of genetic knockouts, stepwise heterologous reconstitution and in vitro enzyme assays, many of the enzymatic steps in the biosynthesis of SF2575 have been identified. Results led to new insights into tetracycline cyclization and tailoring enzymes. In addition the enzymes responsible for the C-4 acylation of salicylic acid were identified and characterized. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity towards substituted benzoic acids which enabled the preparation of analogs of a late stage SF2575 intermediate for structure activity relationships. In addition, the crystal structure of SsfX3 was solved which revealed an intriguing bidomain architecture containing an N-terminal carbohydrate binding module-like beta-sandwich domain and a C-terminal atypical alpha/beta hydrolase domain. Mutational analysis of active site residues revealed important residues for substrate binding and catalysis. Characterization of the SF2575 biosynthetic pathway has therefore furthered our understanding of tetracycline biosynthesis, led to the identification of new tetracycline tailoring enzymes, enabled the biosynthesis of unnatural analogs, and sets the stage for further metabolic engineering of tetracycline pathways toward the generation of novel small molecule therapeutics. |
