Production of l-Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

Abstract

l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number AAY37316) from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt, which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. Our study reports efficient methods to produce l-theanine in the absence of supplemental ethylamine.IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. In this study, we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine.

Keywords: Escherichia coli; alanine decarboxylase; l-theanine; theanine hydrolase; γ-glutamylmethylamide synthetase; ω-transaminase.

FIG 1

Artificial l-theanine biosynthetic pathways mediated by transaminase (TA) or alanine decarboxylase (AlaDC) in E. coli. l-Theanine is synthesized via the TA-utilizing pathway (orange and blue) or the AlaDC-utilizing pathway (green and blue). In the TA pathway, acetaldehyde is supplied by acetyl-CoA via EutE, and subsequently, PpTA catalyzes the transamination reaction that converts acetaldehyde to ethylamine. The amino donor l-alanine (Ala) is regenerated by Bacillus subtilis 168 alanine dehydrogenase (BsAld). In the AlaDC pathway, pyruvate is converted to Ala through the reaction catalyzed by BsAld, and Ala is decarboxylated to produce ethylamine via CsAlaDC. Finally, l-theanine is synthesized from ethylamine and l-glutamate (Glu) via γ-glutamylmethylamide synthetase (GMAS) in both pathways. Genes, their products, and their species of origin are as follows: eutE, acetaldehyde dehydrogenase, E. coli; PpTA, transaminase, Pseudomonas putida; BsAld, alanine dehydrogenase, Bacillus subtilis; CsAlaDC, alanine decarboxylase, Camelia sinensis; GMAS, γ-glutamylmethylamide synthetase; gdhA, glutamate dehydrogenase, E. coli; and gltA, citrate synthase, E. coli. TCA, tricarboxylic acid.

FIG 2

In vitro assay of transaminases derived from Pseudomonas putida KT2440. The rates of conversion of acetaldehyde to ethylamine represent the averages of two biological replicates. Standard deviations between replicates are shown as error bars.

FIG 3

In vitro assay of γ-glutamylmethylamide synthetase homologs. The rates of conversion of ethylamine to l-theanine represent the averages of four biological replicates. Standard deviations between replicates are shown as error bars.

FIG 4

In vitro constitution of the TA pathway. The conversion of acetaldehyde to l-theanine by the combination of TA and GMAS was measured in vitro. The concentrations of l-theanine represent the averages of four biological replicates. Standard deviations between replicates are shown as error bars.

FIG 5

l-Theanine production using the TA pathway in recombinant E. coli W3110S3GK strains harboring the plasmids listed in Table 2. Cells were cultured in TT medium at 30°C for 24 h. Supplemental acetaldehyde and l-alanine were added to the media as required. The yields of l-theanine represent the averages of three biological replicates. Standard deviations between replicates are shown as error bars.

FIG 6

l-Theanine production using the AlaDC pathway in recombinant E. coli W3110S3GK strains harboring the plasmids listed in Table 2. Cells were cultivated in TT medium at 30°C for 24 h. The yields of l-theanine represent the averages of three biological replicates. Standard deviations between replicates are shown as error bars.

FIG 7

Fed-batch fermentation using TEA4 Δggt and TEA6 Δggt. Orange circles represent TEA4 Δggt, and green squares represent TEA6 Δggt. The yields of l-theanine (A), l-alanine (B), and ethylamine (C) and OD₆₆₀ (D) represent the averages of two biological replicates. Standard deviations between replicates are shown as error bars.