Comparative evaluation of l-theanine synthetases coupled with PPK2 based ATP regeneration under buffer-free and Mn2 + optimized conditions.

l-theanine (γ-glutamylethylamide) is a bioactive amino acid widely valued for its functional and nutraceutical applications. While enzymatic synthesis using γ-glutamylmethylamide synthetase (GMAS) has been extensively studied, the potential of γ-glutamylcysteine synthetase (GCS) as an alternative biocatalyst remains underexplored. In this study, a high-substrate, buffer-free whole-cell conversion system was established for l-theanine production using Escherichia coli expressing either GMAS from Methylovorus mays (MmGMAS) or the GCS from E. coli (ecGCS), with an integrated ATP regeneration mechanism driven by polyphosphate kinase 2 (PPK2). In silico predictions using CatPred, a machine learning-based catalyst prediction tool, indicated that ecGCS exhibits catalytic efficiency comparable to or exceeding that of MmGMAS, which was consistent with the experimental results. Among six PPK2 variants tested, the enzyme from Rhodobacter sphaeroides (PPK2-6) was identified as the most suitable ATP regeneration module, enabling approximately 90% reduction in ATP input while maintaining high l-theanine yields. Both MmGMAS-PPK2-6 and ecGCS-PPK2-6 systems produced 47.9 g/L (34.4%) and 44.5 g/L (31.9%) of l-theanine, respectively, starting from 800 mM substrates. Furthermore, it was confirmed that l-theanine production was not compromised in the absence of exogenous buffers, which may also facilitate downstream processing. This work represents the first demonstration of efficient l-theanine production using native ecGCS under process-relevant conditions, and highlights its potential as a complementary or alternative platform to GMAS-based biosynthesis.