High-Concentration Production of <scp>l-</scp>Theanine from <scp>l-</scp>Glutamate Derived from Fermentation Using Novel GMAS and ATP Regeneration with Co-Immobilized Hybrid Nanoflower

l-Theanine, a nonprotein amino acid predominantly found in green tea, is renowned for its unique flavor and diverse physiological benefits, including cognitive enhancement and stress reduction. Traditional extraction methods for l-theanine are costly and inefficient, prompting the exploration of enzymatic synthesis as a more viable alternative. This study presents the development of a highly efficient enzymatic production of l-theanine using glutamate derived from fermentation through a series of systematic approaches. Our work incorporates novel enzyme screening, the use of new metal cofactor, and the enhancement of ATP regeneration efficiency through innovative approaches, along with hybrid nanoflower immobilization, each representing a new perspective in the optimization process. Through a homology-based screening of 1,000 GMAS candidates, M3GMAS from Methylobacillus sp. MM3 was identified as exhibiting the highest l-theanine productivity, significantly surpassing that of the previously reported MmGMAS (1.78-fold increase). Coupling M3GMAS with the PPK2-III enzyme from Erysipelotrichaceae bacterium significantly improved ATP regeneration, achieving a 70% conversion rate with 350 mM l-theanine production under optimized conditions. Further enhancements were attained by co-immobilizing these enzymes in inorganic hybrid nanoflowers (CoHNF), which mitigated substrate and product inhibition, leading to an impressive yield of 630 mM l-theanine at high substrate concentrations. This work demonstrates a highly efficient and scalable method for l-theanine production, integrating advanced biotechnological processes with traditional biomass-derived substrates.