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.

中文翻译：

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开发高效，特异的L-茶氨酸合酶。

以L-谷氨酸和乙胺为底物，将大肠杆菌中的L-谷氨酰半胱氨酸合成酶（γ-GCS）催化从L-谷氨酸和L-半胱氨酸形成L-谷氨酰半胱氨酸，将其工程化为L-茶氨酸合酶。开发了一种使用96孔板的高通量筛选方法，以评估L-茶氨酸合成反应。同时应用了位点饱和诱变和随机诱变。经过三轮定向进化，与野生型酶相比，性能最佳的突变酶13B6的L-茶氨酸产量和乙胺催化效率分别提高了14.6和17.0倍。此外，13B6对原始底物L-半胱氨酸的比活性下降到野生型酶的约14.6％。从而，通过定向进化成功地将γ-GCS酶转换为特定的L-茶氨酸合酶。此外，引入了一种基于多磷酸盐激酶的ATP再生系统，该系统催化磷酸盐从多磷酸盐向ADP的转移，从而降低了ATP的消耗水平和L-茶氨酸的合成成本。突变体13B6在2小时内最终产生的L-茶氨酸产量达到30.4±0.3 g / L，转化率为87.1％，具有很大的工业应用潜力。