Engineered Escherichia coli Nissle 1917 for the High Level Biosynthesis of γ-Aminobutyric Acid

γ-Aminobutyric acid (GABA), a vital bioactive component, is biosynthesized via the decarboxylation of L-glutamate (L-Glu) catalyzed by glutamate decarboxylase (GAD). However, the GADs from various sources commonly suffer from low thermal stability, which hampers their industrial applications. In this work, four ancestral sequences of GAD (Anc19, Anc20, Anc28, and Anc30) were designed via an ancestral sequence reconstruction (ASR) approach. Thereafter, the genes were synthesized and heterologously expressed in the probiotic Escherichia coli strain Nissle 1917 (EcN). Among all variants tested, Anc28 exhibited the highest catalytic performance. The Km and kcat values were determined to be 26.80 mM and 57.41 s−1, respectively, yielding a catalytic efficiency (kcat/Km) of 2.14 s−1mM−1, which was 2.71-fold higher than that of the wild-type enzyme. Meanwhile, compared with the wild-type GAD, Anc28 exhibited a 6.74 °C increase in T5015 and a 4.1-fold extension in t1/2 at 60 °C. Furthermore, the GABA synthesis system using dormant Escherichia coli Nissle (T7)/pET28a-gadBAnc28 cells as the biocatalyst and pure water as a sole medium was also constructed. Upon completion of the 4 h reaction, the GABA titer reached 307.53 g/L with a conversion ratio of 99.36%. The resulting engineered strains were successfully employed for the efficient biosynthesis of GABA.