Antibiotic-Free Expression of γ-Glutamyl Transpeptidase in Bacillus subtilis and Process Optimization for L-Theanine Separation and Purification.

L-theanine is a characteristic non-proteinogenic amino acid found in tea leaves and has attracted considerable attention because of its diverse physiological activity and broad application prospects. γ-glutamyl transpeptidase (GGT) can catalyze the synthesis of L-theanine from L-glutamine and ethylamine without ATP consumption, highlighting its advantages for enzymatic production. In this study, a complete process was established for L-theanine production. Through screening of single and dual promoters, the optimal expression combination, PyxiE-PspoVG, was identified. Furthermore, by integrating the dal selection marker, an antibiotic-free engineered strain was developed. After flask-level optimization, the GGT activity reached 27.32 U/mL and further increased to 127.37 U/mL in 3 L fed-batch fermentation. Using the fermentation broth as the biocatalyst, fed-batch conversion of 0.6 M L-glutamine and 2 M ethylamine yielded 0.52 M (91.44 g/L) L-theanine within 24 h. Further integration of ceramic membrane filtration, ultrafiltration, nanofiltration, electrodialysis, activated-carbon decolorization and ethanol crystallization afforded a final product purity of 95.6%. This study offers a useful reference for large-scale L-theanine production.