Engineered Glutamate Decarboxylase with Expanded Substrate Scope for One-Step Synthesis of β-Alanine and Aminopropanols

In recent years, the demand for β-alanine, 3-aminopropanol (3-AP), and 1-aminopropan-2-ol (MIPA) has increased because of their applications in pharmaceuticals, food additives, and polymers. The industrial production of these high-value amines relies on polluting chemical methods. To address this, we engineered Escherichia coli glutamate decarboxylase (GadB) via directed evolution to enable one-step enzymatic synthesis of all three compounds from renewable substrates (l-aspartate, l-homoserine, and l-threonine). A growth-coupled high-throughput screening platform identified the D86E mutant, which exhibits broad substrate promiscuity and enhanced kinetics: a 46-fold and 12.3-fold increase in kcat (26.36 s–1, 121.89 s–1) for β-alanine and 3-AP synthesis versus native decarboxylases, and activity toward first-time one-step MIPA production. Molecular dynamics simulations revealed that altered hydrogen-bonding interactions between the mutation site (E86) and substrates underpin the shifted specificity. In whole-cell biocatalysis, D86E achieved near-quantitative conversion (250 mM) to β-alanine (15.99 g/L) and 3-AP (13.51 g/L) within 12 h at neutral pH, with the MIPA yield reaching 8.48 g/L, demonstrating industrial feasibility. This work advances enzyme engineering for multisubstrate catalysis and provides a sustainable route for high-value chemical production.