Sustainable co‐production of L‐theanine via an integrated biocatalytic system exploiting atrazine degradation

L-Theanine is a health-beneficial tea-specific amino acid. This amino acid is synthesized through the catalytic action of L-theanine synthase or its isoenzymes, utilizing ethylamine and L-glutamic acid as primary substrates. As the rate-limiting substance, traditional ethylamine synthesis relies on tea root metabolism, and exploring new synthetic pathways can boost L-theanine production and explore environmental application values. Self-developed Tongji_Software was used to predict a potential pathway for ethylamine synthesis. A cell-free protein synthesis (CFPS) system was adopted for verification, and genome engineering was performed to construct a recombinant strain. According to the software predictions, atrazine chlorohydrolase (AtzA) and hydroxyatrazine deethylaminase (AtzB) could convert the herbicide atrazine into ethylamine. CFPS based on Pseudomonas knackmussii (P. knackmussii) produced 88.5 μM L-theanine within a 24-h period, while the engineered strain P. knackmussii-AtzAB yielded 445.7 μM with the conversion rate of 44.6% over 84 h of fermentation. In summary, we revealed a novel pathway for ethylamine synthesis, which can promote L-theanine accumulation in tea trees and degrade environmental pollutants.