Complete elucidation of the late steps of bafilomycin biosynthesis in Streptomyces lohii.

Bafilomycins are an important subgroup of polyketides with diverse biological activities and possible applications as specific inhibitors of vacuolar H+-ATPase. However, the general toxicity and structural complexity of bafilomycins present formidable challenges to drug design via chemical modification, prompting interests in improving bafilomycin activities via biosynthetic approaches. Two bafilomycin biosynthetic gene clusters have been identified, but their post-polyketide synthase (PKS) tailoring steps for structural diversification and bioactivity improvement remain largely unknown. In this study, the post-PKS tailoring pathway from bafilomycin A1 (1)→C1 (2)→B1 (3) in the marine microorganism Streptomyces lohii was elucidated for the first time by in vivo gene inactivation and in vitro biochemical characterization. We found that fumarate is first adenylated by a novel fumarate adenylyltransferase Orf3. Then, the fumaryl transferase Orf2 is responsible for transferring the fumarate moiety from fumaryl-AMP to the 21-hydroxyl group of 1 to generate 2. Last, the ATP-dependent amide synthetase BafY catalyzes the condensation of 2 and 2-amino-3-hydroxycyclopent-2-enone (C5N) produced by the 5-aminolevulinic acid synthase BafZ and the acyl-CoA ligase BafX, giving rise to the final product 3. The elucidation of fumarate incorporation mechanism represents the first paradigm for biosynthesis of natural products containing the fumarate moiety. Moreover, the bafilomycin post-PKS tailoring pathway features an interesting cross-talk between primary and secondary metabolisms for natural product biosynthesis. Taken together, this work provides significant insights into bafilomycin biosynthesis to inform future pharmacological development of these compounds.