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

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