Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli

Liu Fengmin; Zhang Heng; Zhang Xiangjun; Wei Xiaobo; Liu Huiyan; Fang Haitian

doi:10.1002/elsc.202200064

Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli

Eng Life Sci. 2023 Mar 9;23(4):e2200064. doi: 10.1002/elsc.202200064. eCollection 2023 Apr.

Authors

Liu Fengmin¹, Zhang Heng¹, Zhang Xiangjun¹, Wei Xiaobo¹, Liu Huiyan¹, Fang Haitian¹

Affiliation

¹ School of Food and Wine Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control Ningxia University Yinchuan China.

Abstract

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K_m and V_max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.

Keywords: glutamate decarboxylase; site‐directed mutation; whole cell transformation; γ‐aminobutyric acid.