The bacterial modular type I polyketide synthases (PKSs) typically furnish nonaromatic lactone and lactam natural products. Here, by the complete in vitro enzymatic production of the polyketide antibiotic pyoluteorin, we describe the biosynthetic mechanism for the construction of an aromatic resorcylic ring by a type I PKS. We find that the pyoluteorin type I PKS does not produce an aromatic product, rather furnishing an alicyclic dihydrophloroglucinol that is later enzymatically dehydrated and aromatized. The aromatizing dehydratase is encoded in the pyoluteorin biosynthetic gene cluster (BGC), and its presence is conserved in other BGCs encoding production of pyrrolic polyketides. Sequence similarity and mutational analysis demonstrates that the overall structure and position of the active site for the aromatizing dehydratase is shared with flavin-dependent halogenases albeit with a loss in ability to perform redox catalysis. We demonstrate that the post-PKS dehydrative aromatization is critical for the antibiotic activity of pyoluteorin.

Antibiosis has been proposed to contribute to the beneficial bacteria-mediated biocontrol against pea Aphanomyces root rot caused by the oomycete pathogen Aphanomyces euteiches. However, the antibiotics required for disease suppression remain unknown. In this study, we found that the wild type strains of Pseudomonas protegens Pf-5 and Pseudomonas fluorescens 2P24, but not their mutants that lack 2,4-diacetylphloroglucinol, strongly inhibited A. euteiches on culture plates. Purified 2,4-diacetylphloroglucinol compound caused extensive hyphal branching and stunted hyphal growth of A. euteiches. Using a GFP-based transcriptional reporter assay, we found that expression of the 2,4-diacetylphloroglucinol biosynthesis gene phlAPf-5 is activated by germinating pea seeds. The 2,4-diacetylphloroglucinol producing Pf-5 derivative, but not its 2,4-diacetylphloroglucinol non-producing mutant, reduced disease severity caused by A. euteiches on pea plants in greenhouse conditions. This is the first report that 2,4-diacetylphloroglucinol produced by strains of Pseudomonas species plays an important role in the biocontrol of pea Aphanomyces root rot.