Precursor-Directed Mutational Biosynthesis Facilitates the Functional Assignment of Two Cytochromes P450 in Thiostrepton Biosynthesis

Side-ring-modified thiostrepton (TSR) derivatives that vary in their quinaldic acid (QA) substitution possess more potent biological activities and better pharmaceutical properties than the parent compound. In this work, we sought to introduce fluorine onto C-7' or C-8' of the TSR QA moiety via precursor-directed mutational biosynthesis to obtain new TSR variants. Unexpectedly, instead of the target product, the exogenous chemical feeding of 7-F-QA into the Delta tsrT mutant strain resulted in a unique TSR analog with an incomplete side-ring structure and an unoxidized QA moiety (1). Accordingly, two cytochrome P450 genes, tsrP and tsrR, were in-frame deleted to elucidate the candidate responsible for the monooxidation of the QA moiety in TSR. The unfluorinated analog of compound 1 that was thus isolated from Delta tsrP (2) and the abolishment of TSR production in Delta tsrR revealed not only the biosynthetic logic of the TSR side-ring but also the essential checkpoint in TSR maturation before macro-ring closure.