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- Title
Development of a highly efficient and specific l-theanine synthase.
- Authors
Yao, Jun; Li, Jing; Xiong, Dandan; Qiu, Yuanyuan; Shi, Guizhi; Jin, Jian-Ming; Tao, Yong; Tang, Shuang-Yan
- Abstract
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.
- Subjects
INDUSTRIAL capacity; ESCHERICHIA coli; CYSTEINE; KINASES; MUTAGENESIS
- Publication
Applied Microbiology & Biotechnology, 2020, Vol 104, Issue 8, p3417
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-020-10482-6