Unlocking Microbial Access to Amino-substituted 4-Hydroxycoumarin Derivatives via Enzymatic Promiscuity and Metabolic Engineering.

Coumarins and their derivatives possess diverse bioactivities and broad applications in pharmaceuticals, food additives, and materials science. For example, amino-functionalized coumarin derivatives hold significant promise for the development of novel functional materials and drug discovery. However, the microbial biosynthesis of these compounds remains underexplored. In this study, we characterized the substrate flexibility of the type III polyketide synthase PqsD from Pseudomonas aeruginosa in Pseudomonas putida KT2440, showing its ability to convert various para- and meta-substituted salicylic acids into novel coumarin derivatives. Leveraging the substrate promiscuity of PqsD, we established a novel microbial route to produce 6-amino-4-hydroxycoumarin (6A4HC). Furthermore, we developed a new de novo biosynthetic route for its direct precursor, 5-aminosalicylic acid (5ASA), which is an FDA-approved pharmaceutical compound. This work expands the enzymatic toolbox for coumarin biosynthesis and highlights microbial platforms as sustainable alternatives for generating structurally diverse and functionalized coumarins.