Development of a highly efficient and specific l -theanine synthase

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli , which catalyzes the formation of l -glutamylcysteine from l -glutamic acid and l -cysteine, was engineered into an l -theanine synthase using l -glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the l -theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in l -theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, l -cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific l -theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of l -theanine synthesis. The final l -theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.