Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays

• Published in: Enzyme and microbial technology Vol. 180; p. 110481
• Main Authors: Fan, Chao, Qi, Jiakun, Cong, Yunhan, Zhang, Chunzhi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2024
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; biosynthesis; Carbon-Nitrogen Ligases; catalytic activity; directed evolution; Directed Molecular Evolution; Disulfide bridge; disulfides; enzyme activity; Enzyme Stability; enzymes; Glutamates - biosynthesis; Glutamates - metabolism; half life; Kinetics; L-theanine; Methylovorus mays; Peptide Synthases - chemistry; Peptide Synthases - genetics; Peptide Synthases - metabolism; Semi-rational design; Site-directed mutations; technology; Temperature; Thermostability; γ-glutamylmethylamide synthetase; Thermostability; Site-directed mutations; L-theanine; γ-glutamylmethylamide synthetase; Disulfide bridge; Semi-rational design
• ISSN: 0141-0229; 1879-0909; 1879-0909
• DOI: 10.1016/j.enzmictec.2024.110481

The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process. [Display omitted] •The designed potential disulfide bond was introduced into the structure of GMAS and played an active role.•The variants of GMAS were obtained with obviously improved thermal stability.•Fed-batch catalysis strategy resulted in the highest titer and productivity of L-theanine production till now.