Fermentative Production of L-Theanine in Escherichia coli via the Construction of an Adenosine Triphosphate Regeneration System

Abstract

Theanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in Escherichia coli to produce L-theanine. This biosynthesis pathway employs four enzymes to achieve the production of L-theanine. This route involves the co-expression of four functional enzymes: γ-glutamylmethylamide synthetase (GMAS) from Methyloversatilis universalis, polyphosphate kinase (PPK) from E. coli, alanine transaminase from Bacillus subtilis (BsAld), and alanine decarboxylase from Camellia sinensis (CsAlaDC). Polyphosphate kinase from Escherichia coli was overexpressed in E. coli FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to E. coli FD01. A titer of 334 mg/L of L-theanine was produced via engineering strain FD03 in shake flasks. Moreover, glutamine permease from Saccharomyces cereviside (GNP1) was overexpressed in E. coli FD04, and the L-theanine titer increased by 14.7%. Finally, 2.9 g/L of L-theanine was obtained via FD04 in a 1 L bioreactor. In addition, the molecular docking results indicated that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with the surrounding amino acid residues.

L-Theanine an Astounding Amino Acid in Tea, Its Synthesis and Health Benefits: A Review

“…"Microbial production pathways for L-theanine could be broadly classified into two categories: glutamine-mediated and glutamate-mediated pathways. ATP was required to generate energy simultaneously in the glutamate-mediated process, which uses glutamate and ethylamine as precursors" [4]. Yang (2020) evaluated "a Methylovorus mays γ-Glutamylmethylamide synthetase (GMAS) and succeeded in producing 34.49 g/L of Ltheanine required for optimizing both the protein expression and the reaction conditions" [54].…”

“…"By using fed-batch fermentation to introduce GMAS from Paracoccusaminovoransheterologously into a 5-liter bioreactor, scientists were able to reach a high level of L-Theanine synthesis of 70.6 g/L" [4].…”