Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering

L-Theanine is a multifunctional nonprotein amino acid found naturally in tea leaves. It has been developed as a commercial product for a wide range of applications in the food, pharmaceutical, and healthcare industries. However, L-theanine production catalyzed by γ-glutamyl transpeptidase (GGT) is limited by the low catalytic efficiency and specificity of this class of enzymes. Here, we developed a strategy for cavity topology engineering (CTE) based on the cavity geometry of GGT from B. subtilis 168 (CGMCC 1.1390) to obtain an enzyme with high catalytic activity and applied it to the synthesis of L-theanine. Three potential mutation sites, M97, Y418, and V555, were identified using the internal cavity as a probe, and residues G, A, V, F, Y, and Q, which may affect the shape of the cavity, were obtained directly by computer statistical analysis without energy calculations. Finally, 35 mutants were obtained. The optimal mutant Y418F/M97Q showed a 4.8-fold improvement in catalytic activity and a 25.6-fold increase in catalytic efficiency. The recombinant enzyme Y418F/M97Q exhibited a high space-time productivity of 15.4 g L -1 h -1 by whole-cell synthesis in a 5 L bioreactor, which was one of the highest concentrations reported so far at 92.4 g L -1 . Overall, this strategy is expected to enhance the enzymatic activity associated with the synthesis of L-theanine and its derivatives. Key points • Cavity topology engineering was used to modify the GGT for L-theanine biocatalysis. • The catalytic efficiency of GGT was increased by 25.6-fold. • Highest productivity of L-theanine reached 15.4 g L -1 h -1 (92.4 g L -1 ) in a 5 L bioreactor.