Biosynthesis of Indole-3-Acetic Acid in <i>Escherichia coli</i> via Engineered Amidase

Indole-3-acetic acid (IAA) is a vital plant hormone, yet its natural synthesis is insufficient to meet agricultural demand. The indole-3-acetamide (IAM) pathway offers a promising route for microbial IAA production but suffers from inefficient amidase activity. In this study, we identified and engineered an amidase (RsAD) from <i>Rhodococcus</i> sp. through structural analysis, which revealed a narrow substrate channel limiting IAM access, followed by targeted mutagenesis to generate the optimized mutant RsAD-L447A. This mutant exhibited a 3.1-fold increase in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) and enabled complete IAM hydrolysis without the accumulation of intermediates. Coexpression of RsAD-L447A with l-tryptophan monooxygenase established a cascade pathway for IAA synthesis in <i>Escherichia coli</i>. Blocking the competing <i>tnaA</i>-mediated degradation pathway further improved precursor utilization. As a result, IAA production reached 13.3 from 20 g/L l-tryptophan in shake-flask cultures. These findings demonstrate an effective enzyme engineering and metabolic optimization strategy for high-level IAA biosynthesis.