A non‑natural shortcut biosynthetic route enables de novo microbial production of 3‑hydroxyanthranilic and quinolinic acids.

3‑Hydroxyanthranilic acid (3‑HAA) and quinolinic acid (QA) are central metabolites of the kynurenine pathway with broad biomedical and industrial relevance, yet their microbial production remains limited by long pathways and rate-limiting enzymes. Here, we report the rational design and validation of a non‑natural shortcut pathway enabling de novo biosynthesis of 3‑HAA and QA in Escherichia coli. The pathway begins from anthranilic acid (AA), a chorismate‑derived intermediate that can be efficiently overproduced. Regioselective 3-hydroxylation of AA was achieved using an FAD-dependent monooxygenase HpaH from Geobacillus thermodenitrificans, supported by a dedicated FAD reductase (GTNG_3158) essential for efficient cofactor regeneration. The resulting 3‑HAA was subsequently converted to QA through oxidative ring cleavage by NbaC from Pseudomonas fluorescens, followed by spontaneous cyclization. Through enzyme expression tuning, precursor availability enhancement, and fermentation optimization, the engineered strains produced 2.17 g/L 3‑HAA and 2.04 g/L QA from glucose in shake flasks, representing the highest reported microbial 3‑HAA titer and demonstrating the efficient extension of the artificial pathway to QA production. This work establishes a concise and productive biosynthetic strategy, enabling sustainable and high‑level microbial production of 3-HAA‑derived compounds.