Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering

Zhang, Zehua; Long, Mengfei; Zheng, Nan; Deng, Yu; Wang, Qiong; Osire, Tolbert; Xia, Xiaole

doi:10.1007/s00253-023-12544-x

Cited by 3 publications

(1 citation statement)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fan et al [31] achieved a high L-theanine production of 70.6 g/L by In 2020, the market output value of L-theanine surpassed USD 50 million [21]. The theanine market is expected to be worth more than USD 60 million by 2025 [22]. At present, due to the limited sources of theanine, the many extraction steps, the low extraction rate, and the high cost, the application range of theanine is greatly limited [19].…”

Section: Introductionmentioning

confidence: 99%

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

Cao,

Hu,

et al. 2023

Fermentation

View full text Add to dashboard Cite

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.

show abstract