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Xiao, S.; Qian, R.; Hu, S.; Fu, Z.; Bai, T.; Wang, W.; Cheng, J.; Zhang, J. The Synthesis Pathway of L-Theanine in Microorganisms. Encyclopedia. Available online: (accessed on 19 April 2024).
Xiao S, Qian R, Hu S, Fu Z, Bai T, Wang W, et al. The Synthesis Pathway of L-Theanine in Microorganisms. Encyclopedia. Available at: Accessed April 19, 2024.
Xiao, Shujian, Rong Qian, Shunyang Hu, Zhongdan Fu, Ting Bai, Wei Wang, Jie Cheng, Jiamin Zhang. "The Synthesis Pathway of L-Theanine in Microorganisms" Encyclopedia, (accessed April 19, 2024).
Xiao, S., Qian, R., Hu, S., Fu, Z., Bai, T., Wang, W., Cheng, J., & Zhang, J. (2023, June 29). The Synthesis Pathway of L-Theanine in Microorganisms. In Encyclopedia.
Xiao, Shujian, et al. "The Synthesis Pathway of L-Theanine in Microorganisms." Encyclopedia. Web. 29 June, 2023.
The Synthesis Pathway of L-Theanine in Microorganisms

Theanine, a representative non-protein amino acid in tea, is one of the umami components of tea and a major factor in the formation of the unique flavor of tea leaves. In addition to its delicious taste, theanine has a variety of health functions and is used in the food supplement, pharmaceutical, nutraceutical, and cosmetic industries. 

physiological function food supplement microbial synthesis

1. Introduction

To date, there are mainly two biosynthetic routes of L-theanine in microorganisms. One is the glutamate-mediated pathway, which uses glutamate (Glu) as a precursor [1] and requires ATP for energy supply. The other is the glutamine-mediated pathway, which uses glutamine (Gln) as a precursor and is based on the generation of γ-glutamyl transfer reactions [2]. To achieve the microbial synthesis of L-theanine, it is theoretically possible to use TS of the tea tree. However, TS is an enzyme that is easily inactivated and very unstable in vitro in tea tree, is ATP-dependent, and is difficult to isolate and purify as well as to characterize [3]. This characteristic of it dictates the need to study other microbial enzymes applicable to the microbial synthesis of L-theanine. Several microbial enzymes with L-theanine production capacity were identified. They are glutamine synthetase (GS), gamma-glutamine methylamide synthetase (GMAS), gamma-glutamylcysteine synthetase (γ-GCS), gamma-glutamyl transpeptidase (GGT), and L-glutaminase [4][5].

2. Glutamate-Mediated Pathway of L-Theanine in Microorganisms

2.1. Enzyme-Catalyzed Method

The synthases involved in the Glu-mediated pathway of L-theanine are GS, γ-GMAS, and γ-GCS, which catalyze the synthesis of L-theanine using Glu and ethylamine as substrates in the presence of ATP. Tachiki et al. found that GS in bacteria can be used as a biocatalyst for the production of L-glutamine [6]. Subsequently, the first study by the same group of researchers found that the GS of Micrococcus glutamicus ATCC 13032 could synthesize L-theanine when the substrate NH4+ was replaced by ethylamine [7]. This was the first to demonstrate the use of inexpensive L-glutamate and ethylamine, followed by the production of L-theanine by coupling baker’s yeast preparations with GS in bacteria. The GS of Pseudomonas aeruginosa Y-30 was identified and successfully characterized the reactivity to theanine synthesis [8]; then, 29.6 g/L theanine was synthesized by optimizing the reaction system [9]. The isolated GS gene was then ligated into the expression vector pET21a and expressed in E. coli AD494(DE3). The enzyme productivity expressed in this system was 30-times higher than that in P. taetrolens Y-30 and its activity towards ethylamine was 7% higher than that of ammonia [10]. In addition, GS from other different bacterial sources were studied [11][12][13]; they were also consistently identified as having the ability to synthesize theanine, of which, Zhou et al. [11] used Bacillus subtilis-derived GS and 30 g/L yeast cells to catalyze the production of 15.3 g/L theanine from 200 mmol/L glutamate, with a conversion rate of 44%. Zhu et al. [12] constructed an engineered bacterium containing the GS gene of Pseudomonas fluorescens, whose enzyme activity was about 126.64 times that of the starting strain E. coli BL21 [12]. It catalyzed the reaction of sodium L-glutamate and ethylamine hydrochloride to produce 6.2 g/L theanine, and its ability to synthesize theanine was significantly improved compared to the starting strain E. coli BL21 [12]. Although the use of GS in bacteria produces a low level of L-theanine by-products, its low ethylamine reactivity is still unchangeable, which has led researchers to constantly search for catalase enzymes with high reactivity towards ethylamine.
The γ-GCS can make many other types of amino acids, such as alanine glutamylated [1]. Miyake et al. [1] first identified the ability of γ-GCS from E. coli to combine glutamate and ethylamine for the production of theanine. The production of 2.1 g/L theanine was catalyzed by 414 mmol/L glutamate using γ-GCS and its own metabolized ATP; however, the yield was low at 2.9%. It is also worth exploring whether the theanine synthesized here can only be achieved in E. coli [1]. Some researchers have suggested that γ-GCS is expressed at a higher level compared to GS [14]. However, it must be noted that, in addition to the low yield, the formation of the by-product γ-glutamylalanine is also a disadvantage of this theanine production system. Therefore, altering the host glucose metabolic pathway or the substrate specificity of γ-GCS is necessary; however, the latter seems to be better achieved [1]. The crystal structure of γ-GCS in E. coli was determined and the Cys-binding site was identified [15]. Along this line of thought, Yao et al. [14] recently used a random mutagenesis approach for the targeted evolution of E. coli γ-GCS. Mutant γ-GCS13B6 increased the production of L-theanine and the catalytic efficiency of ethylamine by 14.6- and 17.0-fold, respectively, compared with the wild-type enzyme. It catalyzed 200 mmol/L glutamate and ethylamine to produce 30.4 g/L theanine with a conversion rate of 87%.
Kimura et al. [16] first purified approximately 70-fold GMAS from the methyl phage strain Methylo-phage sp. AA-30 in 1992. Studies have shown that the enzyme has maximum activity at pH 7.5 and 40 °C, which binds ethylamine to the γ-amino group of L-glutamate to synthesize L-theanine, demonstrating the broader substrate specificity of GMAS. To investigate this further, Yamamoto et al. [17] selected several strains from about 200 species of methylamine or methanol-assimilating bacteria for the research, and again demonstrated that the amount of theanine formed by GMAS was much greater than that formed by E. coli cells expressing the teatrolens Y-30 GS. The group then went on to investigate the production of L-theanine by GMAS of Methylovorus mays No. 9 through coupled fermentation based on yeast sugars. A total of 110 g/L L-theanine was synthesized in a 100% yield using recombinant GMAS as a catalyst in the optimized mixed system containing 40 g/L yeast cells, 600 mmol/L monosodium glutamate, 600 mmol/L ethylamine hydrochloride, and 30 U/mL GMAS enzyme solution [18].

2.2. Whole-Cell Catalytic Method

Although only inexpensive glutamate and an equivalent amount of ethylamine need to be added, ATP regeneration is still key in relation to the use of GMAS for the industrial production of L-theanine [19]. In addition, microbial conversion is a simpler and more convenient process than enzyme catalysis, which uses intact microbial cells to convert the substrate to L-theanine [20]. The establishment of an ATP regeneration system for PPK after the polyP-fed whole-cell biocatalytic synthesis of L-theanine using inexpensive monosodium glutamate and ethylamine hydrochloride as substrates and the optimum whole-cell catalytic reaction conditions were also determined, and the conversion of monosodium glutamate was 66.34% [2]. However, the examples described above all seem to involve only enzyme-catalyzed and microbial conversion methods for the synthesis of L-theanine, in which, although the cycle time is shorter [21], enzyme preparation and enzymatic reactions need to be conducted in steps, and the catalytic system is complex, cumbersome, and not efficiently recyclable [22]. In contrast, the microbial fermentation method has the advantages of the low cost of substrate raw material, one-step reaction, easily obtained product from the reaction solution, high conversion efficiency, and can be produced in large quantities, which makes it more suitable for scaling up to the industrial production of L-theanine [23].

2.3. Ethanolamine Flow plus Microbial Fermentation Method

Fan et al. [24] attempted to engineer the fermentative production of L-theanine in an industrially safe host, Corynebacterium glutamicum, and is the first example of supplementation with ethylamine to achieve the fermentative production of L-theanine. Compared with C. glutamicum, E. coli is used as a more easily designed host due to its good genetic background [25][26]. By introducing a heterologous GMAS from Paracoccus aminovorans into E. coli, overexpressing natural citrate synthase, introducing glutamate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, as well as optimizing feeding and a series of other operations, the recombinant strain TH11 was able to produce 70.6 g/L of theanine, with a 42% conversion of glucose. This is also the first report on the engineering of a pathway for the production of theanine by fermentation in E. coli [27]. In addition, Benninghaus et al. [28] used Pseudomonas putida KT2440, a fast-growing strain with low nutrient requirements, as a starting strain for the production of theanine. Specifically, the use of a heterologous enzyme from Methylorubrum extorquens DM4 for L-theanine production and the overexpression of the Caulobacter crescentus-derived xylose manipulator xylXABCD using the pEV3 plasmid enabled the strain to utilize xylose as a carbon source. The recombinant strain Thea1 was able to produce 10 g/L of theanine from the recombination of glucose and xylose with a conversion rate of 3.3% to the carbon source when cultured in a batch replenishment bioreactor. It is also capable of producing 17.2 g/L of theanine from glycerol, with a conversion rate of 13% to glycerol. The recombinant strain TheaX was able to produce 21 g/L of theanine from the overlap of glycerol and xylose, with a conversion rate of 3.3% to the carbon source [28]. At present, this is the first L-theanine process to use Pseudomonas aeruginosa and the first to be compatible with the use of various alternative carbon sources [28].

2.4. One-Step Fermentation Method

Ethylamine is an essential starting material for the synthesis of L-theanine [29]; however, its use in the industrial production of L-theanine increases production costs and poses various drawbacks, such as a low boiling point (16.6 °C), toxicity, the need for special equipment for replenishment, which increases the complexity of production, and significant negative impact on human health and the natural environment [30]. In addition, the incomplete metabolism of ethylamine accumulates in high extracellular quantities, limiting the growth of the bacterium and weakening ATP regeneration, thereby inhibiting the synthesis of theanine. This has forced researchers to seek a way to build an efficient ethylamine synthesis pathway in cells to produce L-theanine [13][31]. In tea tree, ethylamine is mainly produced through the decarboxylation of alanine, and no synthetic pathway for ethylamine has been reported in microorganisms [32]. Therefore, researchers began to try to produce L-theanine without adding ethylamine.
Recently, Tabata and Shoto [33] designed an E. coli containing PP_5182, PP_0596, jm49_01725, and RFLU_RS03325 four enzyme genes. These enzymes exist in Pseudomonas and can use acetaldehyde and L-alanine as substrates to produce ethylamine. Finally, the recombinant E. coli was successfully fermented to produce 1.48 g/L of L-theanine. Then, a cell-free protein synthesis system (CFPS) was used to simultaneously overexpress CsAlaDC and PtGS to produce 3.82 mmol/L of theanine using alanine and glutamate as substrates, demonstrating that CsAlaDC can be used for theanine synthesis [13]. In the same year, Hagihara et al. [29] constructed two routes of de novo synthesis from glucose to theanine. One was the AlaDC pathway, which is a plasmid that simultaneously expresses Pseudomonas syringae-derived PsGMAS, CsAlaDC, and B. subtilis 168-derived pyruvate dehydrogenase BsAld in E. coli, and 1.53 g/L of theanine was produced [29]. Another pathway, known as the TA pathway, used plasmids to simultaneously express PsGMAS, BsAld, PpTA8, and the endogenous acetaldehyde dehydrogenase EutE in E. coli. However, the L-theanine yield (>300 mg/L) was lower than the AlaDC pathway [29].

3. Glutamine-Mediated Pathway of L-Theanine in Microorganisms

In the glutamine-mediated pathway, L-glutaminase (GLS) and GGT play a major catalytic role, catalyzing the synthesis of L-theanine from Gln and ethylamine. Unlike the glutamate-mediated synthetic pathway, this pathway does not require ATP [4]. Tachiki et al. [34] first isolated GLS from P. nitroreducens IFO 12694 and found that it can use hydroxylamine, methylamine, and ethylamine as receptor molecules, demonstrating that GLS from Pseudomonas species can synthesize theanine. New research has recently provided an efficient method for the production of theanine by the permeabilization of P. nitroreducens [22]. P. nitroreducens SP.001 cells were treated mainly with 15.5% sucrose solution and permeabilized to obtain a highly active GLS, which produced 85.358 g/L of theanine catalyzed by 1 U/mL of the enzyme, a conversion rate of 66.1% [22]. In addition, GLS from the source fungus Trichoderma koningii could also catalyze the synthesis of theanine. A total of 7.491 g/L of L-theanine was obtained with the addition of 3 mL of enzyme solution, which catalyzed 0.3 mol/L of L-glutamine and 0.9 mol/L of ethylamine; however, this conversion rate was low [21].
GGT and GLS are found in a range of eukaryotes and prokaryotes and can catalyze both γ-glutamyl peptide hydrolysis and transpeptide reactions; however, mostly only E. coli and Bacillus species-derived GGT can be used in the synthesis of theanine [5][21]. The initial demonstration of GGT’s ability to produce L-theanine was the discovery by Suzuji et al. [35] of E. coli K-12-derived EcGGT, with a 60% conversion rate for the synthesis of theanine from Gln and ethylamine catalyzed by the pure enzyme. Jia et al. [36] constructed an E. coli engineered bacterium with 26-times more crude enzyme solution activity under induction than the starting strain; however, Gln apparently did not convert as well as the pure enzyme catalysis. In addition, when using recombinant GGT from Bacillus licheniformis ER-15 for L-theanine biosynthesis, controlled univariate conversions of Gln were approximately 85–87% within 4 h; the immobilization of the recombinant enzyme in calcium alginate could also achieve a similar conversion [37]. Xu et al. [38] used B-FITTER software to analyze all amino acid residues in EcGGT in relation to temperature factors, and screened for a mutant enzyme with significantly improved thermal stability (E387Q). The mutant enzyme catalyzed 120 mmol/L of Gln to produce 18.51 g/L of theanine under 100 W ultrasound conditions, which was 2.61-times the yield of theanine without ultrasound treatment [38]. The recombinant expression of E. coli GGT was enhanced using a small ubiquitin-related modifier (SUMO) fusion technique; the yield of L-theanine was increased to 41 g/L, with a conversion rate of approximately 80% [39]. Bacillus subtilis was significantly superior to E. coli in terms of the secretion of heterologous enzymes and proproteins. By overexpressing the PrsA lipoprotein and improving the mRNA stability of the Bacillus subtilis ggt gene, a yield of L-theanine of 53 g/L and a glutamyl conversion of 74% were achieved in the optimized system [40]. The expression of recombinant ggt in a subspecies of Bacillus glutamicus, followed by the use of a tac promoter with an optimized sequence in the plasmid, significantly increased the activity of the ggt gene, resulting in a high transformation rate of 89.76% [41]. In addition, it was shown that Luteibacter, an endophytic bacterium isolated from tea tree, can also convert Gln and other amino acids into theanine in the absence of ethylamine. Although the yield was only 31.875 μg/L, it also indirectly suggests that there may be another microbial synthesis pathways for theanine production that is not dependent on ethylamine [42].

4. Other Substrate-Mediated Pathways of L-Theanine in Microorganisms

Synthetic pathways mediated by other glutamyl compounds as donors have been shown to exist, and are also capable of producing L-theanine when catalyzed by immobilized cells with GGT activity [43]. These glutamyl compounds include γ-Glutamyl-p-nitroanilide [44][45], glutathione (GSH) [46], glutamic acid γ-methyl ester (GMAE) [43], L-glutamine-Zn(II) (Zn(Gln)2) [47], and γ-L-Glutamylhydrazide [46]. Especially when GMAE is used as a novel substrate, the relative activity of GGT is 85.4%, which is 1.2-times higher than the activity of GGT with Gln as substrate [46]. Moreover, when the ratio of GMAE to ethylamine is 1:12, the conversion rate can reach as high as 96.3% [46].


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