One-Pot Synthesis of β-Alanine from Fumaric Acid via an Efficient Dual-Enzyme Cascade Biotransformation

As the only naturally occurring β-amino acid, β-alanine has important application prospects in many fields. Driven by the huge demand, biosynthesis is becoming more and more popular as a potential alternative to the chemical synthesis of β-alanine. Although the direct pathway from L-aspartic acid to β-alanine, catalyzed by L-aspartic acid-α-decarboxylase (PanD), is ideal for β-alanine synthesis, it is hindered by the high cost of the substrate and limited economic viability. In this work, a cell-free dual enzyme cascade system based on methylaspartate lyase (EcMAL) and panD was constructed to safely and efficiently synthesize β-alanine using fumarate as a substrate. Taking the previously engineered EcMAL as the target, CgPanD was finally screened as the best candidate through gene mining, sequence alignment, and enzyme property analysis. Finally, under the optimal conditions of 35 °C, pH 8.0, and EcMAL: CgPanD concentration ratio of 1:5, the yield of β-alanine reached 80% theoretical yield within 120 min. This study provides a potential strategy for the biosynthesis of β-alanine, paving the way for future industrial-scale production.