L-theanine is a unique amino acid that is naturally found in tea plants, especially in green tea [1], [2]. Owing to its unique flavor, often described as umami, and various therapeutic effects, such as promoting relaxation, reducing blood pressure, enhancing antitumor activity, providing neuroprotection, and improving learning ability, it is increasingly being employed as a flavor enhancer in healthy food items [3], [4].

L-theanine can be produced by either extraction from tea plants (Camellia sinensis) [5], chemical synthesis, or enzymatic synthesis [6], [7]. However, the extraction method is a time-consuming and cost-ineffective process, and chemical synthesis involves the formation of a racemic mixture of L- and D-forms, making it unsuitable for use as a food additive [6], [8]. Despite the difficulty in achieving industrial mass production, enzymatic synthesis offers advantages, such as short reaction times and high purity of the L-form, making it an attractive option for the production of L-theanine [4], [9], [10].

L-theanine production using enzymatic synthesis has been studied using various enzymes, such as γ-glutamyltranspeptidase (GGT) [11], [12], L-glutaminase [13], L-glutamine synthetase (GS) [14], and γ-glutamylmethylamide synthetase (GMAS)[8], [15], [16]. While GGT and L-glutaminase have shown promise in L-theanine production, the conversion yields remained low due to their tendency to hydrolyze L-glutamine to other γ-glutamyl derivatives, and the reaction needed large excess of ethylamine [4], [10]. GS and GMAS can efficiently convert L-glutamic acid and ethylamine into L-theanine using a more cost-effective substrate, resulting in fewer byproducts [4], [17]. Notably, GMAS exhibited higher reactivity towards ethylamine than GS, making it a promising candidate for efficient L-theanine production [18]. Particularly, GMAS from Methylovorus mays No. 9 has been shown to be highly efficient in producing L-theanine [16], [18].

However, the biosynthetic pathway catalyzed by GMAS is highly dependent on ATP, which is not suitable for commercial use, due to its high price and low stability [19], [20], [21]. Therefore, to avoid the direct use of ATP in theanine production using GMAS, alternative strategies have been developed, such as fermentative production from glucose [22], [23], coupling with baker’s yeast fermentation [16], [24], and enzymatic regeneration of ATP from other substrates [25], [26]. Notably, the regeneration of ATP using polyphosphate kinase (PPK2), which contained subclasses PPK2-Ⅰ, PPK2-Ⅱ, and PPK2-Ⅲ, is a promising strategy for ATP supply owing to its short reaction time and high conversion rate (Table 1) [25], [26], and has also been applied to synthetic studies of various substances (Table 2) [27], [28], [29], [30], [31], [32], [33], [34], [35], [36]. PPK2 in classes Ⅰ and Ⅲ facilitated the ATP regeneration by transferring phosphate groups from polyphosphate to ADP, however, class Ⅱ specifically catalyzed the phosphorylation of AMP to ADP [37]. Among these, Especially, PPK2 from Corynebacterium glutamicum in class Ⅰ was known to efficiently utilize the short chain length of polyphosphate to regenerate ATP from ADP [27], [38].

Previously, the feasibility of using a whole-cell biotransformation system for l-theanine production was identified [16]. Compared to the conventional use of purified enzymes, the whole cell system could simplify complex purification processes and render the biocatalyst easier to handle, ultimately leading to reduced operational costs [39], [40], [41]. However, the use of whole cell biocatalysts for high concentration reaction still presented challenges in terms of reusability and stability, and separating them from a reaction mixture for reuse could be cumbersome [39], [42], [43]. Considering immobilization of whole cells could not only enhance the stability and reusability of biocatalysts, but also facilitate the separation process [42], [43], [44], [45], immobilization could be an answer for enhancement of whole cell system due to its simplicity, low cost, easy availability, and good biocompatibility[42], [43]. As a result, in this study, whole cells expressing both gmas and ppk2 were immobilized using the alginate entrapment method. Theanine was successfully produced by immobilizing whole cells in the form of alginate beads. Since the beads possessed an ATP regeneration function, efficient production of theanine was achieved using just a small amount of ATP. In addition, reusability of the biocatalysts was enhanced after immobilization, thereby enabling a long storage period while maintaining their high activity. Considering the feasibility of an immobilization strategy equipped with ATP regeneration, the current study aimed to suggest a reasonable strategy for efficient theanine production.