ATP-regenerating System Research Articles

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system

Backgroundβ-Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide coenzymes such as NAD+ and NADP+, which are widely applied in industrial biocatalysis especially involving cofactor-dependent oxidoreductases. Moreover, NMN is a promising candidate for medical uses since it is considered to be beneficial for improving health of aged people who usually suffer from an insufficient level of NAD+. To date, various methods have been developed for the synthesis of NMN. Chemical phosphorylation of nicotinamide riboside (NR) to NMN depends on excessive phosphine oxychloride and delicate temperature control, while fermentation of NMN is limited by low product titers, making it unsuitable for industrial-scale NMN production. As a result, the more efficient synthesis process of NMN is still challenging.AimThis work attempted to construct an eco-friendly and cost-effective biocatalytic process for transforming the chemically synthesized NR into the highly value-added NMN.ResultsA new nicotinamide riboside kinase (Klm-NRK) was identified from Kluyveromyces marxianus. The specific activity of purified Klm-NRK was 7.9 U·mg–1 protein, ranking the highest record among the reported NRKs. The optimal pH of Klm-NRK was 7.0 in potassium phosphate buffer. The purified Klm-NRK retained a half activity after 7.29 h at 50 °C. The catalytic efficiencies (kcat/KM) toward ATP and nicotinamide riboside (NR) were 57.4 s−1·mM−1 and 84.4 s−1·mM−1, respectively. In the presence of an external ATP regeneration system (AcK/AcP), as much as 100 g·L–1 of NR could be completely phosphorylated to NMN in 8 h with Klm-NRK, achieving a molar isolation yield of 84.2% and a space–time yield of 281 g·L−1·day−1. These inspiring results indicated that Klm-NRK is a promising biocatalyst which provides an efficient approach for the bio-manufacturing of NMN in a high titer.Graphical

Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System

Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously.

Characterization of polyphosphate kinases for the synthesis of GSH with ATP regeneration from AMP

Polyphosphate kinase (PPK) is important for industrial processes involving ATP regeneration. While a variety of methods have been reported for regenerating ATP from ADP, few have explored enzyme catalyzed ATP regeneration from cheaper and stable AMP. In this work, PPKs from different sources were expressed and their catalytic activity were tested at different reaction temperatures, reaction pH and with different polyphosphate (polyPn) types. The ATP regeneration system for glutathione (GSH) synthesis was established using a single PPK capable of phosphorylating AMP to synthesize ATP from AMP and short chain polyPn. GSH yield was obtained using adenosine mono-, di- and triphosphates, which confirmed the flexibility of our constructed ATP regeneration system coupled with GSH synthesis via bifunctional GSH synthase. Finally, optimization of the GSH synthesis yielded conversion value above 80 %. Overall, these results illustrate that PPK is suitable for a broader range of substrates than previously expected, and has great untapped potential for applications involving ATP regeneration.

Electrocatalytic CO2 fixation by regenerating reduced cofactor NADH during Calvin Cycle using glassy carbon electrode

In this study, an enzymatic pathway has been developed to replicate the Calvin Cycle by creating the individual steps of the carbon cycle in a bioreactor. The technology known as “artificial photosynthesis” converts CO2 emissions into a variety of intermediates that serve as precursors to high-value products. CO2, light, water, and electricity were used as feedstock. An electrochemical reactor was also studied for the regeneration of active NADH operating at constant electrode potential. Initially, a batch electrochemical reactor containing 80 mL of 0.2 mM NAD+ in Tris-buffer (pH 7.40) was used to evaluate the electrode material operating at normal temperature and pressure. The results showed that the cathode is highly electrocatalytically efficient and selective to regenerate 97.45±0.8% of NADH from NAD+ at electrode potential of -2.3 V vs. mercury standard electrode (MSE). The NADH regeneration system was then integrated with ATP regeneration system and bioreactor containing Ribulose bisphosphate carboxylase/oxygenase (RuBisCO). NADH was regenerated successfully during the process electrochemically and then was used by the enzymatic reaction to produce triose phosphate and 3-Phosphoglycerate (3GPA).

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light‐driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS‐g‐PEO and diblock copolymer PBd‐PEO. Activity of more than 90 % in lipid/polymer‐based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long‐term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer‐based hybrids.

Development of a highly efficient and specific l-theanine synthase

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3g/L in 2h, with a conversion rate of 87.1%, which has great potential for industrial applications.

ATP Regeneration System in Chemoenzymatic Amide Bond Formation with Thermophilic CoA Ligase

AbstractCoA ligases are enzymes catalyzing the ATP‐dependent addition of coenzyme A to carboxylic acids in two steps through an adenylate intermediate. This intermediate can be diverted by a nucleophilic non enzymatic addition of amine to get the corresponding amide for synthetic purposes. To this end, we selected thermophilic CoA ligases to study the conversion of various carboxylic acids into their amide counterparts. To limit the use of ATP, we implemented an ATP regeneration system combining polyphosphate kinase 2 (PPK2 Class III) and inorganic pyrophosphatase. Suitability of this system was illustrated by the lab‐scale chemoenzymatic synthesis of N‐methylbutyrylamide in 77 % yield using low enzyme loading and 5 % molar ATP.

Abstract A092: Multiplexed measurement of DNA repair capacity for tumor heterogeneity

Abstract Purpose of Study: DNA damage has been shown to have a strong link to the origin and propagation of many cancers. Specific damages to different DNA repair pathways within the cell spell accumulations of DNA damaging events and alterations in normal biochemical activities of the cell. More recently, these biochemical differences have begun to be explored and have demonstrated potential evidence supporting a heterogeneous picture of tumor and cancer pathology. The goal of this project focused upon developing a cellular assay to measure the DNA repair capacities of cancer cells and to apply it at the level of an individual cell. Methods Used: Bulk Hap1 cellular lysate was prepared and incubated with the assay and repair activities were recovered from DNA substrates. Amplification and ILLUMINA sequencing was then used to detail repair capacities. Bioinformatic pipelines systematically compared differences in repair profiles across tested conditions in the optimization of the assay with ATP regeneration and chemotherapy pre-treated cells. Future direction will include the combination of a microfluidic platform for single cell analysis. Summary of Results: The use of a creatine phosphokinase (CPK) ATP regeneration system demonstrated increased capture across all repair pathways as compared to a control. Nucleotide Excision Repair (NER) and mismatch repair (MMR) saw significant increases in repair activities not previously seen without the regeneration system. Temozolomide pretreated cells demonstrated increased repair activities in Direct Reversal repair of methylated adducts as compared to Hydrogen Peroxide pretreated cells and positive controls. Conclusions: The addition of an ATP regeneration system significantly increased capture of repair events by increasing local concentrations of ATP to support cellular enzyme activities. Increases in repair activates were seen across all pathways tested, indicating reproducible results. DNA damaging pretreatments indicated predictable up-regulations in certain DNA repair pathways matching the type of DNA damage event generated. Citation Format: Lakotah Hardie, Amanda Richer, Jay Hesselberth. Multiplexed measurement of DNA repair capacity for tumor heterogeneity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A092. doi:10.1158/1535-7163.TARG-19-A092

Immobilization of a polyphosphate kinase 2 by coordinative self-assembly of his-tagged units with metal-organic frameworks and its application in ATP regeneration from AMP

Self-assembly of the functional units onto the surface of nanoparticles is a powerful approach to generate functional nanosystems. In this work, we first expressed a recombinant class III polyphosphate kinase 2 (ArPPK2) with his-tag. It is able to synthesize ATP from AMP by a single enzyme, simplifying two-step reaction of ATP regeneration from AMP. Then we chose the Fe-based metal-organic frameworks (MOF)s as carriers to produce the enzyme-MOF biocomposite, based on the interaction between the his-tags and coordinatively unsaturated metal sites present on the external surface of MOFs by a self-assembly process. It was found that ArPPK2@MIL-101-NH2@Fe3O4-COOH exhibited better reusability than other candidates during cycle analysis, preserving 70.1% of initial activity after reusing thirteen times, and also retained high storage stability. The optimum pH of the enzyme-MOF biocomposite was increased from 8.0 to 9.0 and the optimum temperature was increased from 30℃ to 45℃. Compared to free ArPPK2, the enzyme-MOF biocomposite showed increased thermal and pH stability. In addition, we successfully constructed an ATP regeneration system from AMP using the enzyme-MOF biocomposite, coupled with amide bond formation catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). The immobilized ArPPK2 will provide a promising route for ATP regeneration from AMP in industrial processes. And the generation of the enzyme-MOF biocomposite by the self-assembly approach can be extended to efficiently immobilize other recombinant his-tagged enzymes.

Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5′-phosphate and ATP

Cadaverine, also known as 1,5-pentanediamine, is an important platform chemical with a wide range of applications and can be produced either by fermentation or bioconversion. Bioconversion of cadaverine from l-lysine is the preferred method because of its many benefits, including rapid reaction time and an easy downstream process. In our previous study, we replaced pyridoxal-5-phosphate (PLP) with pyridoxal kinase (PdxY) along with pyridoxal (PL) because it could achieve 80% conversion with 0.4 M of l-lysine in 6 h. However, conversion was sharply decreased in the presence of high concentrations of l-lysine (i.e., 1 M), resulting in less than 40% conversion after several hours. In this study, we introduced an ATP regeneration system using polyphosphate kinase (ppk) into systems containing cadaverine decarboxylase (CadA) and PdxY for a sufficient supply of PLP, which resulted in enhanced cadaverine production. In addition, to improve transport efficiency, the use of surfactants was tested. We found that membrane permeabilization via hexadecyltrimethylammonium bromide (CTAB) increased the yield of cadaverine in the presence of high concentrations of l-lysine. By combining these two strategies, the ppk system and addition of CTAB, we enhanced cadaverine production up to 100% with 1 M of l-lysine over the course of 6 h.

High Conversion of D-Fructose into D-Allulose by Enzymes Coupling with an ATP Regeneration System.

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2mM ATP, 5mM polyphosphate, 20mM D-fructose, and 20mMMg2+ when incubated at 50°C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.

Production of active manganese peroxidase in Escherichia coli by co-expression of chaperones and in vitro maturation by ATP-dependent chaperone release

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn2+ to Mn3+, a diffusible and strong non-specific oxidant capable of attacking bulky phenolic substrates. Therefore, MnP is indispensable in the polymer and paper industries. Previous attempts of MnP expression in Escherichia coli resulted in the formation of inclusion bodies which required invitro refolding. Aiming to investigate the bacterial production of MnP, we have revealed an interesting mechanism underlying chaperone-assisted maturation of this enzyme to its active form. Since we previously found that invitro expression of MnP in E.coli system depends on disulfide bond isomerase DsbC, we chose SHuffle T7 Express, an E. coli constitutively expressing DsbC, as the host for invivo expression of MnP. Initially, only a low amount of the enzyme was present in the soluble fraction, with no detectable peroxidase activity. Co-expression of MnP with different chaperone revealed that DnaK, DnaJ, and GrpE contributed the most to the solubility improvement, however, remained in a complex with the MnP, preventing the enzyme to assume its active conformation. We resolved this by invitro maturation, involving incubation of the MnP-chaperone complex with hemin, ATP, and ATP regeneration system. While ATP enables the chaperones to finish the refolding cycle and release the MnP in its correctly folded form, hemin supports the formation of the holo-enzyme with fully recovered peroxidase activity. We believe that the findings of this paper will serve as an important clue for establishing the bacterial production of fungal peroxidases in the future.

Effects of ATP regeneration systems on the yields and solubilities of cell-free synthesized proteins

We compared the abilities of several ATP regeneration systems to support the production of soluble proteins in a cell-free synthesis system. While changes in the ATP level in the reaction mixture were variable depending on the ATP regeneration system employed, the yields and solubilities of synthesized proteins did not always correlate with the ATP level. Instead, the efficiency of cell-free protein synthesis was substantially affected by changes in the pH of the reaction mixture. These results suggest that measures to maintain the pH of the reaction mixture should be considered when choosing an energy source for cell-free protein synthesis.

Cdc48/p97 segregase is modulated by cyclin‐dependent kinase to determine cyclin fate during G1 progression

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short-lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1-cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk-target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1-cyclin levels and activity. Cdk-dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear.

Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration

An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87mM of l-Trp-l-Pro was successfully synthesized from AMP after 72h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2mM l-Trp-l-Pro. When the coupling reaction was applied to whole-cell reactions in E.coli BL21(DE3) pepQ-putA-, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7mM of l-Trp-l-Pro was produced after 24h with the supplementation of 10mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only l-Trp-l-Pro, but also various other l-Xaa-l-Pro (Xaa=Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.

Chemoenzymatic synthesis of 3'-phosphoadenosine-5'-phosphosulfate coupling with an ATP regeneration system.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a commonly used anticoagulant drug. Previously, using ATP as the substrate, we had developed a one-pot synthesis to prepare PAPS with 47% ATP conversion efficiency. During the reaction, 47% of ATP was converted into the by-product, ADP. Here, to increase the conversion ratio of ATP to PAPS, an ATP regeneration system was developed to couple with PAPS synthesis. In the ATP regeneration system, the chemical compound, monopotassium phosphoenolpyruvate (PEP-K+), was synthesized and used as the phospho-donor. By using 3-bromopyruvic acid as the starting material, the total yield of PEP-K+ synthesis was over 50% at low cost. Then, the enzyme PykA from Escherichia coli was overexpressed, purified, and used to convert the by-product ADP into ATP. When coupled the ATP regeneration system with PAPS synthesis, the higher ratio of PEP-K+ to ADP was associated with higher ATP conversion efficiency. By using the ATP regeneration system, the conversion ratio of ATP to PAPS was increased to 98% as determined by PAMN-HPLC analysis, and 5g of PAPS was produced in 1L of the reaction mixture. Furthermore, the chemoenzymatic synthesized PAPS was purified and freeze-dried without observed decomposition. However, the powdery PAPS was more unstable than the PAPS sodium salt in aqueous solution at ambient temperature. This developed chemoenzymatic approach of PAPS production will contribute to the synthesis of heparin, in which PAPS is necessary as the individual sulfo-donor.

Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production.

To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production. We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24h, the concentration of CDP and CTP reached 3.8±0.2 and 6.9±0.3mMl-1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system. A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

ATP is dispensable for both miRNA- and Smaug-mediated deadenylation reactions.

MicroRNAs (miRNAs), as well as the RNA-binding protein Smaug, recruit the CCR4-NOT deadenylase complex for shortening of the poly(A) tail. It has been believed that ATP is required for deadenylation induced by miRNAs or Smaug, based on the fact that the deadenylation reaction is blocked by ATP depletion. However, when isolated, neither of the two deadenylases in the CCR4-NOT complex requires ATP by itself. Thus, it remains unknown why ATP is required for deadenylation by ribonucleoprotein complexes like miRNAs and Smaug. Herein we found that, in the absence of the ATP-regenerating system, ATP is rapidly consumed into AMP, a strong deadenylase inhibitor, in Drosophila cell lysate. Importantly, hydrolysis of AMP was sufficient to reactivate deadenylation by miRNAs or Smaug, suggesting that AMP accumulation, rather than ATP depletion, caused the inhibition of the deadenylation reaction. Our results indicate that ATP is dispensable for deadenylation induced by miRNAs or Smaug and emphasize caution in the use of ATP depletion methods.

Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.