A Comprehensive Review of l-Theanine Production in Escherichia coli: The Recent Progress, Metabolic Engineering Strategies, and Future Prospects.

l-theanine is a nonproteinogenic amino acid widely utilized as a food additive and cosmetic ingredient, with promising potential in medical applications. Owing to its distinctive properties and well-established safety profile across the food, cosmetics, health supplement, and pharmaceutical sectors, global demand for l-theanine has been rising rapidly. Among available production methods, microbial fermentation, recognized for its environmental sustainability, has emerged as the preferred approach, gradually supplanting traditional techniques, such as plant extraction and chemical synthesis. Recent advances, particularly the development of ethylamine-free biosynthetic pathways and the successful de novo biosynthesis of l-theanine in Escherichia coli, a well-established microbial chassis with rapid growth and versatile genetic engineering tools, have opened new avenues for industrial-scale production. This review highlights the biosynthetic pathways of l-theanine and outlines targeted metabolic engineering strategies to enhance its yield. These include the identification and overexpression of key biosynthetic enzymes, enhancement of ATP regeneration, construction of endogenous ethylamine biosynthetic routes, redirection of metabolic flux toward l-theanine production, and suppression of competing pathways. Furthermore, current limitations in strain optimization are discussed along with perspectives on future directions for developing E. coli as a robust microbial cell factory for l-theanine production.