De Novo Biosynthesis of Kynurenic Acid Derivative-Based Antagonist Drug Collection in E. coli

Among heterocycles, quinoline scaffolds are privileged for their numerous biological activities in the pharmaceutical field and have garnered significant attention in the development of novel and potent therapeutic agents. Kynurenic acid (KYNA), also known as 4-hydroxyquinolin-2-carboxylic acid, is a key metabolite of l-tryptophan (l-Trp), which exhibits diverse therapeutic implications in neurodegenerative diseases and certain inflammations. Currently, KYNA and its derivatives are commercially available through chemical synthesis that relies on rigorous reaction conditions and fairly toxic solvents. Therefore, the construction of microbial cell factories offers a promising alternative for the sustainable production of these medicinal moieties. However, no microbial biosynthetic route has been reported so far. In this study, we establish a one-pot, three-enzyme platform in Escherichia coli for the diversified biosynthesis of KYNA analogues. As a result, 13 KYNA derivatives with diverse substituent groups (e.g., F, Cl, Br, CH3, and OCH3) can be rapidly assembled in a predictable manner with 21–85% isolated yields. And our approach enables translating the reaction parameters from Eppendorf tubes to the 1 L scale, affording 7-Cl-KYNA with 907 mg/L. Moreover, the finding that the enzymatic steps mediated by kynurenine formamidase and kynurenine aminotransferase can be reversed in order undoubtedly provides fresh and exciting cognitive horizons for the l-Trp metabolic pathway. Additionally, targeted engineering of kynurenine aminotransferase (R20G mutant) achieved a 2.5-fold improvement in solubility and a 2.8-fold enhancement in catalytic efficiency, laying a foundation for future structural biological studies, drug-targeting research, and industrial applications. Overall, this is the first report on the microbial production of KYNA and its derivatives and provides an environmentally friendly method for sustainable scale-up synthesis via E. coli.