Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate–dependent dioxygenase

摘要: Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase−polyketide synthase (NRPS−PKS) system of the trans -acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, well analogs containing terminal carboxylic acid. We provide genetic and biochemical evidence for biosynthesis oxidative decarboxylation rather than previously hypothesized Baeyer–Villiger release postulated to be catalyzed flavin-dependent monooxygenase (FMO) activity internal last module PKS. Inactivation Fe(II)/α-ketoglutarate–dependent dioxygenase gene fr9P led loss congeners, whereas mutant was still able produce all major acid-type compounds. FMO mutants, on other hand, produced both acid an exocyclic methylene instead epoxide, indicating that is involved in epoxidation oxidation. Moreover, recombinant Fr9P enzyme shown catalyze hydroxylation form β-hydroxy acids, which upon FR901464. Finally, third oxygenase encoded biosynthetic cluster, cytochrome P450 Fr9R, assigned 4-hydroxylase based inactivation results. Identification deletion formation allowed us generate strain—the − mutant—that accumulates only analogs, when derivatized increase cell permeability, but chemically more stable.