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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.

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Sakaguchi and Shishido (6) cloned in Escherichia coil a 4.5-kbp HindIII DNA fragment with the tetracycline-resistance (Tcr) determinant (named tetBS908) from Bacillus subtilis GSY908 chromosome using a shuttle plasmid vector pLS353 (4). In this isolation, a 5.2-kbp HindIII fragment was also obtained, which was an insertion derivative of the 4.5-kbp fragment (1). It carried E. coil transposable element IS1 (8) flanked by 8-bp duplications of the target sequence, AAAATTTG, just upstream from tetBS908 (Figs. l and 2) (1). The plasmid pLS353 with the 5.2-kbp HindIII fragment was named pTBS 1 ( Fig. 1) (1, 6). During a competent cell transformation of B. subtilis, IS1 was found to be deleted from pTBS 1 (see Figs. 1 and 2) with a high frequency.Two types of Tcr transformants were obtained by the competent cell transformation of B, subtilis RM 125 (9), which maintain either the pTBS 1 parent or particular deletion derivative. Nucleotide sequence analysis revealed that the deletion derivative lacks the IS1 element and one of the 8-bp target sequences for IS1 insertion (1) (Fig. 2). No other deletion occurred in the plasmid. We examined whether the IS1 element deleted from pTBS 1 had transposed into host chromosomal DNA. The Tcr transformant was treated with ethidium bromide to obtain plasmidless Tcs derivatives. The chromosomal DNAs were then isolated from the Tcs derivatives, digested with HindIII, and subjected to Southern blot hybridization (7) with a digoxigenin-labeled probe (the 0.6-kbp PvuII-Tth111I fragment of ISI element; part (a) of Fig. 2). No hybridization signal was obtained