Improved Mutasynthetic Approaches for the Production of Modified Aminocoumarin Antibiotics

Abstract

This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1H-NMR spectroscopy.