{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:49:58Z","timestamp":1790182198352,"version":"4.1.0"},"reference-count":35,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"publisher","award":["2022YFC2104900"],"award-info":[{"award-number":["2022YFC2104900"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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. \u03b3-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.<\/jats:p>","DOI":"10.3390\/molecules31091476","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T10:34:42Z","timestamp":1777458882000},"page":"1476","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Antibiotic-Free Expression of \u03b3-Glutamyl Transpeptidase in Bacillus subtilis and Process Optimization for L-Theanine Separation and Purification"],"prefix":"10.3390","volume":"31","author":[{"given":"Ran","family":"Zhang","sequence":"first","affiliation":[{"name":"School of Food Science and Technology, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0638-6456","authenticated-orcid":false,"given":"Bo","family":"Jiang","sequence":"additional","affiliation":[{"name":"School of Food Science and Technology, Jiangnan University, Wuxi 214122, China"},{"name":"International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziqun","family":"Xiao","sequence":"additional","affiliation":[{"name":"School of Food Science and Technology, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longbei","family":"Xiang","sequence":"additional","affiliation":[{"name":"School of Food Science and Technology, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1080\/10408398.2018.1552245","article-title":"Understanding different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea (Camellia sinensis) provides new insights into the safe and effective alteration of tea flavor and function","volume":"60","author":"Yu","year":"2020","journal-title":"Crit. 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