P108: Mutasynthesis of FK506 analogues containing altered starter unit and C21 side chain

FK506 is a 23-membered macrocyclic polyketide with immunosuppressant activity produced by a variety of Streptomyces species, used to prevent rejection of transplanted organs in humans. The C21 allyl moiety and the cyclohexane ring of FK506 are critical for its potent biological activity. In this work, we elucidated the detailed biosynthetic pathway of allylmalonyl-coenzyme A (CoA), from which the FK506 allyl moiety is derived, based on the comparison of three FK506 gene clusters, gene deletion, chemical complementation and biochemical analysis. Several FK506 analogues containing altered side chains were obtained through mutasynthesis by feeding cultures of Streptomyces sp. KCTC 11604BP tcsB (which is involved in the biosynthesis of C21 allyl side chain) deletion mutant with a series of carboxylic acids. In addition, the mutasynthetic approach led to the generation of a new analogue containing a non-natural starter unit by feeding a 3-cyclohexene-1-carboxylic acid to the fkbO (which is responsible for the biosynthesis of the starter unit) deletion mutant strain. By combining non-natural starter unit with the incorporation of an alternative C21 side chain instead of the natural allyl side chain, the mutasynthetic approach can be used to extend the structural diversity of FK506.