Biosynthesis of the Klebsiella oxytoca Pathogenicity Factor Tilivalline: Heterologous Expression, in Vitro Biosynthesis, and Inhibitor Development

Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxytoca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a nonenzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Finally, it could be shown that salicylic and acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably by blocking the peptidyl carrier protein ThdA, pointing toward a potential application in combination therapy to prevent or alleviate the symptoms of AAHC.