Development of a highly efficient and specific l-theanine synthase

Autor:	Yuanyuan Qiu, Guizhi Shi, Dandan Xiong, Shuang-Yan Tang, Jun Yao, Jian-Ming Jin, Yong Tao, Jing Li
Rok vydání:	2020
Předmět:	Glutamate-Cysteine Ligase Mutant Glutamic Acid Protein Engineering Applied Microbiology and Biotechnology Catalysis Industrial Microbiology 03 medical and health sciences chemistry.chemical_compound Adenosine Triphosphate Glutamates Amide Synthases Escherichia coli Ethylamines 030304 developmental biology chemistry.chemical_classification 0303 health sciences ATP synthase biology 030306 microbiology Chemistry Polyphosphate Mutagenesis Substrate (chemistry) General Medicine Directed evolution High-Throughput Screening Assays Enzyme Biochemistry biology.protein Directed Molecular Evolution Ethylamine Biotechnology
Zdroj:	Applied Microbiology and Biotechnology. 104:3417-3431
ISSN:	1432-0614 0175-7598
DOI:	10.1007/s00253-020-10482-6
Popis:	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.
Databáze:	OpenAIRE
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