(235f) Characterization of Novel Tetracycline Family Tailoring Enzymes for Engineered Biosynthesis

In our previous work, we have sequenced and analyzed the gene cluster responsible for producing SF2575, a tetracycline family natural product with anticancer properties, from native host Streptomyces sp. SF2575. Chemically, tetracyclines share in common their characteristic linear four-ringed structure, heavily oxidized upper periphery and C-2 amide group. Commercially available tetracyclines such as doxycycline and recently tigecycline are produced semisynthetically due to the structural complexity of the tetracycline core which renders de novo synthesis difficult and makes biosynthesis an attractive option both for production and potential engineering of these compounds. In addition to the traditional tetracycline features, SF2575 has further tailoring which makes its structure even more complex including a salicylic acid substitution at C-4 and a C-glycosylation at C-9, which is itself acylated with an angelic acid moity. These features not only add new chemical diversity to the tetracycline family but are also vital to the bioactivity of SF2575 as a potent cytotoxic compound. Recently we have verified the early biosynthetic steps leading to common tetracycline intermediates including 6-methylpretetramid and have focused efforts on characterizing key tailoring enzymes.

In this study, the enzymes responsible for the attachment of the salicylic acid moity to the A ring have been identified and characterized. These include an ATP dependent salicyl-CoA ligase SsfL1, and a GDSL family acyltransferase SsfX3. SsfX3 has shown broad substrate promiscuity and has been shown to accept a wide array of chemically diverse substrates leading to the isolation of novel tetracycline compounds. Interestingly, while SsfL1 has homology to enzymes from many natural product pathways which contain an aromatic acid ligand similar to salicylic acid, SsfX3 has only a single homolog from a natural product pathway found in the NCBI protein database, and the function of this homolog is yet to be identified. This study sheds light on these unusual tetracycline tailoring enzymes and is a promising step toward using this pathway to generate novel tetracycline analogs with potent anticancer activity.