Development of a highly efficient and specific L-theanine synthase

Area of Science:

• Biocatalysis and enzyme engineering
• Metabolic engineering
• Synthetic biology

Background:

• γ-Glutamylcysteine synthetase (γ-GCS) naturally synthesizes L-glutamylcysteine.
• Engineering γ-GCS for L-theanine synthesis offers a novel biocatalytic route.
• Directed evolution is a powerful tool for enzyme optimization.

Purpose of the Study:

• To engineer γ-GCS from Escherichia coli into an efficient L-theanine synthase.
• To develop a high-throughput screening method for L-theanine synthesis.
• To improve L-theanine production and catalytic efficiency using directed evolution.

Main Methods:

• Site-saturation and random mutagenesis were employed for enzyme evolution.
• A 96-well plate high-throughput screening assay was established.
• An ATP-regeneration system using polyphosphate kinases was integrated.

Main Results:

• Directed evolution yielded mutant 13B6 with 14.6-fold higher L-theanine production.
• Catalytic efficiency for ethylamine increased 17.0-fold in mutant 13B6.
• Mutant 13B6 achieved 30.4 g/L L-theanine production with 87.1% conversion in 2 hours.

Conclusions:

• γ-GCS was successfully engineered into a specific L-theanine synthase.
• The developed biocatalytic process demonstrates high efficiency and industrial potential.
• Enzyme engineering coupled with an ATP-regeneration system significantly reduces L-theanine synthesis costs.