Formicamycin biosynthesis involves a unique reductive ring contraction

Abstract

Fasamycin natural products are biosynthetic precursors of the formicamycins. Both groups of compounds are polyketide natural products that exhibit potent antibacterial activity despite displaying different three-dimensional topologies. We show here that transformation of fasamycin into formicamycin metabolites requires two gene products and occurs via a novel two-step ring expansion-ring contraction pathway. Deletion of forX, encoding a flavin dependent monooxygenase, abolished formicamycin production and leads to accumulation of fasamycin E. Deletion of the adjacent gene forY, encoding a flavin dependent oxidoreductase, also abolished formicamycin biosynthesis and led to the accumulation of new lactone metabolites that represent Baeyer-Villiger oxidation products of the fasamycins. These results identify ForX as a Baeyer-Villiger monooxygenase capable of dearomatizing ring C of the fasamycins. Through in vivo cross feeding and biomimetic semi-synthesis experiments we showed that these lactone products represent biosynthetic intermediates that are reduced to formicamycins in a unique reductive ring contraction reaction catalyzed by ForY.

Fig. 1. Chemical skeletons of fasamycins (1–3) and formicamycins (4–16) isolated from S. formicae KY5, and Baeyer–Villiger lactone intermediates (17–21) isolated from the S. formicae ΔforY mutant in this study (for detailed substituent variations including halogenation or O-methylation, see Fig. S1†). The 2D NMR structure determination of 18 is shown by COSY (bold), NOESY (red double-head arrow), and selected HMBC (blue single-head arrow) correlations respectively. R_1–4 = H or Cl; R₅ = H or CH₃.

Fig. 2. Mutational analysis of the tailoring enzyme encoding genes involved in formicamycin biosynthesis. Deletion of forX abolishes the production of formicamycins but leads to accumulation of fasamycin E (3). Deletion of forY abolishes the production of formicamycins and leads to accumulation of six biosynthetic intermediates (17–22). Reconstituted HPLC traces (UV = 285 nm) showing: (a) S. formicae wild type; (b) S. formicae ΔforV (for details see ref. 7); (c) S. formicae ΔforX; (d) S. formicae ΔforY.

Fig. 3. Comparison of the fasamycin/formicamycin biosynthetic gene clusters (top) and the alternate pathways for each step of the proposed two-step ring expansion-contraction mechanism of formicamycin biosynthesis from fasamycin E (3) (bottom).

Fig. 4. Biomimetic reduction of the biosynthetic intermediate 19 and isolated products. (b) HPLC-UV (285 nm) trace of the initial reaction products from (a); (c) UV spectra of the reaction substrate (19), two main products of interest (23 and 24), plus formicamycin B (5) as a standard for comparison.

Fig. 5. Reconstituted UPLC-UV (285 nm) analysis for the isolated reduction products 23 (a), 24 (b), and a mixture of the isolated products (c) indicating the diastereomeric composition of these samples.