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 the novel transaminase AAN70747 from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase 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. To our knowledge, this is the first report of 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. Here 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.

Metabolic Engineering of Pseudomonas putida for Fermentative Production of l-Theanine

“…putida. Hagihara et al (2021) described the degradation of theanine in E. coli by a γ-glutamyltranspeptidase, encoded by ggt .…”

“…It has been proposed that MEA is derived from alanine by decarboxylation; however, heterologous expression of the gene for alanine decarboxylase from C. sinensis failed to yield MEA. , Very recently, a synthetic pathway converting acetyl-CoA in two reactions to MEA was embedded in E. coli. To this end, eutE encoding acetaldehyde dehydrogenase from E.…”

“…sinensis failed to yield MEA. , Very recently, a synthetic pathway converting acetyl-CoA in two reactions to MEA was embedded in E. coli. To this end, eutE encoding acetaldehyde dehydrogenase from E. coli and the gene for alanine-dependent ω-transaminase from P.…”

“…Thus, deletion of ansB may prove helpful to improve L-theanine accumulation by P. putida. Hagihara et al (2021) 17 described the degradation of theanine in E. coli by a γ-glutamyltranspeptidase, encoded by ggt. BlastP comparison of its amino acid sequence identified two γ-glutamyltranspeptidase candidates in P. putida KT2440, PP_4659 (sequence identity 44.71%) and PP_3535 (sequence identity 34.67%).…”

“…Subsequently, GMA is converted to ammonium, a nitrogen source for growth, and NMG by the enzyme NMG synthase (NMGS, EC 2.1.1.21, encoded by mgsABC ). Finally, NMG dehydrogenase (EC 1.5.99.5, encoded by mgdABCD ) oxidizes NMG to l -glutamate and formaldehyde, which can be assimilated in the serine cycle in anabolism or is oxidized to CO 2 in catabolism. , Fermentative production of l -theanine based on GMAS has been described by metabolically engineered Escherichia coli and Corynebacterium glutamicum, , but not in pseudomonads.…”