전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Research article


References

  1. Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. 2004. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In Vivo 18: 55-62.
    Pubmed
  2. Turkozu D, Sanlier N. 2017. L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety. Crit. Rev. Food Sci. Nutr. 57: 1681-1687.
    Pubmed CrossRef
  3. Saeed M, Naveed M, Arif M, Kakar MU, Manzoor R, Abd El-Hack ME, et al. 2017. Green tea (Camellia sinensis) and l-theanine: Medicinal values and beneficial applications in humans-A comprehensive review. Biomed. Pharmacother. 95: 1260-1275.
    Pubmed CrossRef
  4. Adhikary R, Mandal V. 2017. L-theanine: A potential multifaceted natural bioactive amide as health supplement. Asian Pac. J. Trop. Biomed. 7: 842-848.
    CrossRef
  5. Vuong QV, Stathopoulos CE, Golding JB, Nguyen MH, Roach PD. 2011. Optimum conditions for the water extraction of L-theanine from green tea. J. Sep. Sci. 34: 2468-2474.
    Pubmed CrossRef
  6. Vuong QV, Bowyer MC, Roach PD. 2011. L-Theanine: properties, synthesis and isolation from tea. J. Sci. Food Agric. 91: 1931-1939.
    Pubmed CrossRef
  7. Kawagishi H, Sugiyama K. 1992. Facile and large-scale synthesis of L-Theanine. Biosci. Biotechnol. Biochem. 56: 689.
    Pubmed CrossRef
  8. Shuai Y, Zhang T, Jiang B, Mu W. 2010. Development of efficient enzymatic production of theanine by gamma-glutamyltranspeptidase from a newly isolated strain of Bacillus subtilis, SK11.004. J. Sci. Food Agric. 90: 2563-2567.
    Pubmed CrossRef
  9. Tachiki T, Yamada T, Mizuno K, Ueda M, Shiode J, Fukami H. 1998. gamma-glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO 12694 and their application for the syntheses of theanine and gamma-glutamylmethylamide. Biosci. Biotechnol. Biochem. 62: 1279-1283.
    Pubmed CrossRef
  10. Mu W, Zhang T, Jiang B. 2015. An overview of biological production of L-theanine. Biotechnol. Adv. 33: 335-342.
    Pubmed CrossRef
  11. Chen X, Su L, Wu D, Wu J. 2014. Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of ltheanine. Process Biochem. 49: 1429-1439.
    CrossRef
  12. Pu H, Wang Q, Zhu F, Cao X, Xin Y, Luo L, et al. 2013. Cloning, expression of glutaminase from Pseudomonas nitroreducens and application to theanine synthesis. Biocatal. Biotransform. 31: 1-7.
    CrossRef
  13. Sharma E, Joshi R, Gulati A. 2018. l-Theanine: An astounding sui generis integrant in tea. Food Chem. 242: 601-610.
    Pubmed CrossRef
  14. Liu S, Li Y, Zhu J. 2016. Enzymatic production of l-theanine by γ-glutamylmethylamide synthetase coupling with an ATP regeneration system based on polyphosphate kinase. Process Biochem. 51: 1458-1463.
    CrossRef
  15. Berke W, Schüz HJ, Wandrey C, Morr M, Denda G, Kula MR. 1988. Continuous regeneration of ATP in enzyme membrane reactor for enzymatic syntheses. Biotechnol. Bioeng. 32: 130-139.
    Pubmed CrossRef
  16. Endo T, Koizumi S. 2001. Microbial conversion with cofactor regeneration using genetically engineered bacteria. Adv. Synth. Catal. 343: 521-526.
    CrossRef
  17. Moon Y-M, Yang SY, Choi TR, Jung H-R, Song H-S, hoon Han Y, et al. 2019. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP. Enzyme Microb. Technol. 127: 58-64.
    Pubmed CrossRef
  18. Wei LL, Goux WJ. 1992. ATP cofactor regeneration via the glycolytic pathway. Bioorg. Chem. 20: 62-66.
    CrossRef
  19. Sato M, Masuda Y, Kirimura K, Kino K. 2007. Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for D-amino acid dipeptide synthesis. J. Biosci. Bioeng. 103: 179-184.
    Pubmed CrossRef
  20. Kameda A, Shiba T, Kawazoe Y, Satoh Y, Ihara Y, Munekata M, et al. 2001. A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase. J. Biosci. Bioeng. 91: 557-563.
    Pubmed CrossRef
  21. Yan B, Ding Q, Ou L, Zou Z. 2014. Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system. World J. Microbiol. Biotechnol. 30: 1123-1128.
    Pubmed CrossRef
  22. Resnick SM, Zehnder AJ. 2000. In vitro ATP regeneration from polyphosphate and AMP by polyphosphate: AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A. Appl. Environ. Microbiol. 66: 2045-2051.
    Pubmed PMC CrossRef
  23. Wakisaka S, Ohshima Y, Ogawa M, Tochikura T, Tachiki T. 1998. Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast. Appl. Environ. Microbiol. 64: 2952-2957.
    Pubmed PMC CrossRef
  24. Horinouchi N, Sakai T, Kawano T, Matsumoto S, Sasaki M, Hibi M, et al. 2012. Construction of microbial platform for an energyrequiring bioprocess: practical 2'-deoxyribonucleoside production involving a C− C coupling reaction with high energy substrates. Microb. Cell Fact. 11: 82.
    Pubmed PMC CrossRef
  25. Matsuno R, Asada M, Nakanishi K, Kamikubo T. 1982. ATP Regeneration by Enzymes of Alcohol Fermentation and Kinases of Yeast and its Computer Simulation, pp. 351-352. In: Enzyme Engineering. Springer.
    CrossRef
  26. Horinouchi N, Ogawa J, Kawano T, Sakai T, Saito K, Matsumoto S, et al. 2006. Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli. Biosci. Biotechnol. Biochem. 70: 1371-1378.
    Pubmed CrossRef
  27. Yamamoto S, Wakayama M, Tachiki T. 2005. Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells. Biosci. Biotechnol. Biochem. 69: 784-789.
    Pubmed CrossRef
  28. Lin J-P, Tian J, You J-F, Jin Z-H, Xu Z-N, Cen P-L. 2004. An effective strategy for the co-production of S-adenosyl-L-methionine and glutathione by fed-batch fermentation. Biochem. Eng. J. 21: 19-25.
    CrossRef
  29. Bhatia SK, Bhatia RK, Yang Y-H. 2016. Biosynthesis of polyesters and polyamide building blocks using microbial fermentation and biotransformation. Rev. Environ. Sci. Bio/Technol. 15: 639-663.
    CrossRef
  30. Yang S-Y, Choi T-R, Jung H-R, Park Y-L, Han Y-H, Song H-S, et al. 2020. Development of glutaric acid production consortium system with α-ketoglutaric acid regeneration by glutamate oxidase in Escherichia coli. Enzyme Microb. Technol. 133: 109446.
    Pubmed CrossRef
  31. Moon Y-M, Gurav R, Kim J, Hong Y-G, Bhatia SK, Jung H-R, et al. 2018. Whole-cell immobilization of engineered Escherichia coli JY001 with barium-alginate for itaconic acid production. Biotechnol. Bioprocess Eng. 23: 442-447.
    CrossRef
  32. Bhatia SK, Shim Y-H, Jeon J-M, Brigham CJ, Kim Y-H, Kim H-J, et al. 2015. Starch based polyhydroxybutyrate production in engineered Escherichia coli. Bioprocess Biosys. Eng. 38: 1479-1484.
    Pubmed CrossRef
  33. Bhatia SK, Kim S-H, Yoon J-J, Yang Y-H. 2017. Current status and strategies for second generation biofuel production using microbial systems. Energy Convers. Manage 148: 1142-1156.
    CrossRef
  34. Alaiz M, Navarro JL, Girón J, Vioque E. 1992. Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. J. Chromatogr. 591: 181-186.
    Pubmed CrossRef
  35. Kim J, Seo H-M, Bhatia SK, Song H-S, Kim J-H, Jeon J-M, et al. 2017. Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli. Sci. Rep. 7: 39768.
    Pubmed PMC CrossRef
  36. Griffiths MW, Muir DD. 1978. Properties of a thermostable β‐galactosidase from a thermophilic Bacillus: Comparison of the enzyme activity of whole cells, purified enzyme and immobilised whole cells. J. Sci. Food Agric. 29: 753-761.
    Pubmed CrossRef
  37. Buchachenko AL, Kuznetsov DA. 2008. Magnetic field affects enzymatic ATP synthesis. J. Am. Chem. Soc. 130: 12868-12869.
    Pubmed CrossRef
  38. Moon Y-M, Yang SY, Choi TR, Jung H-R, Song H-S, hoon Han Y, et al. 2019. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP. Enzyme Microb. Technol. 127: 58-64.
    Pubmed CrossRef
  39. Taylor RG, Walker DC, McInnes R. 1993. E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.  Nucleic Acids Res. 21: 1677.
    Pubmed PMC CrossRef
  40. Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11: 753.
    Pubmed PMC CrossRef
  41. Lee SG, Lee JO, Yi JK, Kim BG. 2002. Production of cytidine 5'‐monophosphate N‐acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst. Biotechnol. Bioeng. 80: 516-524.
    Pubmed CrossRef
  42. Jakoby M, Ngouoto-Nkili C-E, Burkovski A. 1999. Construction and application of new Corynebacterium glutamicum vectors. Biotechnol. Tech. 13: 437-441.
    CrossRef

Related articles in JMB

More Related Articles

Article

Research article

J. Microbiol. Biotechnol. 2020; 30(5): 785-792

Published online May 28, 2020 https://doi.org/10.4014/jmb.1910.10044

Copyright © The Korean Society for Microbiology and Biotechnology.

Production of L-Theanine Using Escherichia coli Whole-Cell Overexpressing
γ-Glutamylmethylamide Synthetase with Baker’s Yeast

Soo-Yeon Yang 1, Yeong-Hoon Han 1, Ye-Lim Park 1, Jun-Young Park 1, So-young No 1, Daham Jeong 2, Saerom Park 1, Hyung Yeon Park 1, Wooseong Kim 3, Seung-Oh Seo 4* and Yung-Hun Yang 1, 5*

1Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea
2Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea
3College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University Seoul 03760,
Republic of Korea
4Department of Food Science and Nutrition, The Catholic University of Korea, Bucheon 14662, Republic of Korea
5Institute for Ubiquitous Information Technology and Applications, Konkuk University, Seoul 05029, Republic of Korea

Received: October 21, 2019; Accepted: February 21, 2020

Abstract

L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50oC, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Baker’s yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.

Keywords: 5L-theanine, whole-cell biocatalyst, baker&rsquo,s yeast, ATP regeneration

Introduction

Tea is widely consumed worldwide because of its delicate flavor and physiological benefits because it contains many components such as catechins, caffeine, vitamins, L-theanine, and mineral elements [1]. Among them, L-theanine, also known as γ-glutamylethylamide, is naturally found in Japanese green tea leaves and contributes to the quality of green tea [2]. The physiological activities of L-theanine include effects as remarkable relaxation in humans, enhancing immunity, and decreasing anti-oxidative stress and diseases [3]. Accordingly, the demand for L-theanine is increasing annually and large scale-production methods are necessary to supply L-theanine to the food and pharmaceutical industry [4].

Many methods for the synthesis of L-theanine have been investigated extensively such as extraction from tea leaves [5, 6], chemical synthesis [7], and enzymatic synthesis [8, 9]. Direct extraction has numerous limitations for large-scale production such as its high cost, time-consuming steps, and demanding operational processes. Additionally, the chemically synthesized product is a racemic mixture of the L-form and D-form, thereby reducing product purity and requiring additional purification. [10]. Thus, biological synthesis of L-theanine has attracted much attention recently.

The enzymes involved in L-theanine production include γ-glutamyltranspeptidase (GGT), L-glutaminase, L-glutamine synthetase (GS), or γ-glutamylmethylamide synthetase (GMAS). Under overexpression of GGT in E. coli followed by purification, 156 mM L-theanine was produced using glutamine and ethylamine as substrates [11]. Additionally, production of 126 mM L-theanine was achieved using purified L-glutaminase from Pseudomonas nitroreducens through a γ-glutamyl transfer reaction of glutamine [12]. However, conversion yields remain low because GGT and L-glutaminase tend to hydrolyze L-glutamine to other γ-glutamyl derivatives [13]. Liu et al. produced 254 mM L-theanine using GMAS from Methylovorus mays No. 9 strain by glutamate and ethylamine through the hydrolysis of ATP. Compared to other enzymes, GMAS shows greater potential for producing high concentrations of L-theanine [14].

Many synthetases require high ATP concentrations during catalysis to ensure high titers of various products [15, 16]. Therefore, systems for ATP regeneration have been investigated [17-19]. These methods use a variety of enzymes capable of synthesizing ATP, such as polyphosphate kinase, acetate kinase, and pyruvate kinase [20, 21]. However, the use of pyruvate kinase and acetate kinase is limited by the high cost of phosphoryl donors and their product inhibition activity. Additionally, both systems possess broad substrate specificities, which can regenerate other types of nucleosides and 2’-deoxynucleoside triphosphates, such as GTP, UTP, and CTP [22]. The system using polyphosphate kinase shows low activity towards specific substrates, so that it causes poor availabilities. As an alternative to ATP regeneration, the use of baker’s yeast has been considered in many studies [23-25]. Sugar-fermentative systems can effectively produce ATP as an energy source from AMP or ADP, effectively [26]. Therefore, the excessive consumption of ATP can be overcome by taking advantage of the energy transfer system of baker’s yeast in an ATP-dependent reaction [27, 28].

In this study, we developed a whole-cell biotransformation system for production of L-theanine. GMAS from M. mays No. 9 was overexpressed in E. coli BW25113 and was used for the synthesis of L-theanine using glutamate and ethylamine as substrates. Compared to conventional usage of purified enzymes, this whole-cell system can mitigate complex purification processes and decrease operational costs to enhance economic feasibility. Through this result, the cell cultivation and reaction conditions were optimized to achieve a high titer of L-theanine. Additionally, we found that ATP and different phosphate sources affected the reaction. To our knowledge, this is the first application of whole-cell overexpressed by GMAS for L-theanine production.

Materials and Methods

Reagents

Sodium L-glutamate monohydrate (>99%) and ethylamine hydrochloride (>98%) were purchased from Sigma-Aldrich Co. (USA). L-Theanine (>98%) and Adenosine 5’-triphosphate disodium salt hydrate were obtained from Tokyo Chemical Industry Co. (Japan). Baker’s yeast was purchased from ACH Food Companies, Inc. (Oakbrook Terrace, USA). Tris hydrochloride (>99%), Tris-base (>99.9%), sodium borate decahydrate (>99%), isopropyl-β-d-thiogalactopyranoside (>99%), and other medium components were obtained from Biosesang Co. (Korea). Diethyl ethoxymethylenemalonate (DEEMM) was purchased from Fluka Co. (Japan) for the derivatization reaction.

Bacterial Strains, Plasmids, and Media

E. coli DH5α strain was used as the host for gene cloning. Based on the sequence of GMAS from Methylovorus mays No. 9, GMAS was codon-optimized and synthesized by Cosmogenetech (Korea) and utilized as the PCR template for gene amplification. Amplified genes were inserted into pET24ma (constructed by Dr. David Sourdive, Pasteur Institute, France) containing a p15A replication origin, pXMJ19, and pMAL vectors using restriction enzymes and T4-ligase. The constructed plasmid was transformed into competent E. coli cells to prepare the whole-cell biocatalyst [29]. All bacterial strains and plasmids are provided in Table 1. All E. coli strains were pre-cultured in 5 ml lysogeny broth (LB) medium containing 10 g/l tryptone, 5 g/l yeast extract, and 5 g/l sodium chloride with antibiotics by inoculating a single colony from an agar plate. Cells were cultured in a shaking incubator (Han‐Beak Science Co., Korea) at 37°C with 200 rpm. The pre-culture was inoculated into 50 ml of main culture medium containing antibiotics in a 250-ml baffled Erlenmeyer flask and incubated at 37°C with shaking [30, 31]. Upon reaching an OD600 of 0.6–0.7, the cultures were induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) at 0.5 mM. The cultures were then incubated at 25°C with shaking for 16 h. Cultures were harvested by centrifugation at 5,500 rpm for 10 min at 4°C, and the cell pellet was washed with deionized water [32]. The cell suspension was stored at 4oC until further use and referred to as whole-cell.

Table 1 . List of bacterial strains and plasmids..

Strain/plasmidRelevant informationReference
Bacterial strains
E. coli DH5αF- φ 80lacZ M15 endA recA hsdR(rk-mk-) supE thi gyrA relA Δ (lacZYAargF)U169[39]
E. coli BL21(DE3)F- ompT hsdSB(rB-mB-) gal dcmNovagen
E. coli BW25113F' λ- Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), lambda-, rph-1, Δ(rhaD_rhaB)568, hsdR514CGSC
E. coli BW25113(DE3)λDE3 lysogen of BW25113This study
E. coli ShufflefhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1cDsbC (Specr, lacIq) ΔtrxB sulA11 R(mcr-73::miniTn10– TetS)2 [dcm] R(zgb-210::Tn10 --TetS) endA1 Δgor Δ(mcrC-mrr)114::IS10[40]
E. coli Star TM (DE3)F- ompT rne131 lon hsdSB (rB-mB-) gal dcm λ(DE3)Invitrogen
B. subtilis str. 168ATCC
C. glutamicum ATCC13032ATCC
P. putida KT2440ATCC
Baker’s yeast
Plasmid
pET24maKmr.1 MCS site with T7 promoter, lac operator, RBS. P15A replicon. Copy number: 10–12[41]
pCDF duet 1Specr.2 MCS site with T7 promoter, lac operator, RBS. CloDF13 replicon. Copy number: 20–40Novagen
pRSF duet 1Kmr.2 MCS site with T7 promoter, lac operator, RBS. RSF1030 replicon (NTP1). Copy number: >100Novagen
pACYC duet 1Cmr.2 MCS site with T7 promoter, lac operator, RBS. P15A replicon. Copy number: 10–12Novagen
pMALAmpr. MCS site with tac promoter, lac operator, RBS, ColE1 replicon. Copy number: 20Novagen
pXMJ19Cmr. MCS site with tac promoter, lac operator, pBL1 and pUC replicon[42]
pET24ma::gmasgmas of Methylovorus mays No. 9 inserted in pET24maThis study
pCDF duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pCDF duet 1This study
pRSF duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pRSF duet 1This study
pACYC duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pACYCThis study
pMAL::gmasgmas of Methylovorus mays No. 9 inserted in pMALThis study
pXMJ19::gmasgmas of Methylovorus mays No. 9 inserted in pXMJ19This study


Whole-Cell Reaction

GMAS activity was evaluated using the whole-cell as a catalyst. The reaction was performed with 100 mM glutamate, 100 mM ethylamine, 200 mM glucose, 60 mM ATP, 5 mM MnCl2, 10 mM MgCl2, 300 mM potassium phosphate and 40 mg/ml baker’s yeast. Each parameter was varied to identify the overall optimal reaction conditions. The initial pH was adjusted to 7 with 5 N NaOH. Each parameter was varied to identify the optimal reaction conditions. The reaction solution was incubated at 50oC for 24 h in a shaking mixer at 1,200 rpm. The reaction was stopped by heating at 90°C for 5 min. The reaction solution was then diluted to a suitable concentration for high-performance liquid chromatography (HPLC, YL-9100; Korea) analysis [33].

Derivatization and HPLC Analysis

Amine derivatives were prepared in a mixture of 300 μl borate buffer (50 mM, pH 9), 100 μl methanol, 47 μl distilled water, 50 μl target sample, and 3 μl DEEMM [34]. The derivatization reaction was performed at 70oC for 2 h to derivatize glutamate, L-theanine, and ethylamine. Analysis by HPLC was performed after derivatization of the reaction products at a UV-absorbance of 284 nm. Chromatographic separation was conducted using a reverse-phase C18 column (ZORBAX SB-C18 column, 4.6 × 250 mm, 5 μm particle size; Agilent Technologies, USA) and the column temperature was maintained at 35°C. The mobile phase was composed of 100% acetonitrile (solvent A) and 25 mM sodium acetate buffer pH 4.8 (solvent B). The flow rate was maintained at 1 ml/min and the composition of solvent A to B (A:B, v/v) was changed with the following gradient program: 0 min (20:80), 2 min (25:75), 32 min (60:40), 37 min (20:80), 40 min (20:80).

Results and Discussion

Optimization of Protein Expression Conditions and Cultivation Medium

To obtain a high level of protein expression, GMAS activities in the whole-cell reaction were investigated using various vectors with different copy numbers in several E. coli strains (Fig. 1A). pET24ma (copy numbers 10–12), pXMJ19 (copy number 10-20), and pMAL (copy number 20) were evaluated as GMAS overexpression vectors. The results were indicated as relative conversion, which regards the highest value as 100% conversion. Results showed that all strains containing the pET24ma::gmas exhibited the highest activities. The pMAL::gmas only exhibited activity in the BL21 strain. When comparing GMAS overexpression using pET24ma and pXMJ19, GMAS overexpression with pET24ma showed slightly higher activities in all expression strains. Additionally, evaluation of the pCDF::gmas, pACYC::gmas, and pRSF::gmas, revealed no activities. To explain these results, SDS-PAGE was performed to see GMAS expression level. However, the expression levels from the three kinds of vectors were much lower than those from vectors that showed activity (data not shown). Therefore, we decided to use BW25113 harboring pET24ma::gmas in further experiments.

Figure 1. Optimization of expression and cultivation conditions. GMAS activities were compared in several expression strains containing vectors with different copy numbers for producing L-theanine (A). Cultivation medium were tested by comparing cell dry weight (B) and relative conversion of substrates (C). The results were indicated as relative conversion, which regards the highest value as 100% conversion.

After selecting the expression vector and strains, we tested various expression media because it can affect cell growth and expression levels [35]. Several media were compared, such as terrific broth (TB), 2xYT, and super broth (SB), which contain higher nitrogen levels than LB media, which is commonly used for E. coli cultivation. The results revealed that TB showed the greatest influence on cell growth after 24 h of culture (Fig. 1B). The cell dry weight (CDW) by TB was the 2.5, 1.6 and 1.3-fold higher than those grown in LB, 2xYT, and SB, respectively. However, the cell activity after cultivation in TB was slightly lower than that in LB and 2xYT (Fig. 1C). However, it is inefficient to use cells with a low growth rate but slightly higher activities from an industrial perspective. Therefore, TB was utilized to prepare the biocatalyst.

Optimization of Whole-Cell Reaction with Temperature, pH, and Metal ions

To increase the conversion yields of the whole-cell reaction, reaction conditions considered as key factors were optimized. Reaction temperatures ranging from 20–80°C were tested to determine the optimal temperature (Fig. 2A). The highest activity was observed at 50°C, which differs from a previous report that showed an optimal temperature of 37°C [14]. This may be because our study used a whole-cell as biocatalyst rather than purified enzyme. A whole-cell bioconversion system generally possesses advantages such as robustness against harsh reaction conditions and higher stability and storability than purified enzymes [36]. The optimal pH was also studied to enhance production of L-theanine (Fig. 2B). Various initial pH values were tested from 5.5 to 11, which were adjusted by adding Tris-HCl buffer and NaOH solution. The activity increased gradually until pH 7. However, the activity started to decrease from pH 7 and no activity observed at pH 10 and 11. In further experiments, the reaction temperature and pH were adjusted to 50°C and 7, respectively, as the optimal conditions.

Figure 2. Optimization of whole-cell reaction conditions. Optimal temperature was evaluated from 20°C to 80°C (A) and optimal initial pH was evaluated from 5.5 to 11 (B). Various metal ions were investigated (C) and optimal MnCl2 was found (D).

In the ATP-dependent reactions, metal ions are often necessary as cofactors for the main enzyme for effective synthesis of ATP [37, 38]. Thus, many types of metal ions were investigated to identify the most effective factor during the whole-cell reaction (Fig. 2C). Among these metal ions, Mn2+, Co2+, Mg2+, Zn2+, and Ca2+ were found to influence the reaction. Particularly, the reaction with Mn2+ could produce 2.4-fold higher L-theanine than that with Ca2+. However, activities were not observed when Ba2+ and Cu2+ were utilized. To determine the optimal concentration of Mn2+, MnCl2 was fed into the reactant to the desired concentration (Fig. 2D). As a result, the reaction containing 5 mM MnCl2 could produce the largest amount of L-theanine. Additionally, 10 mM MgCl2 had positive effect in L-theanine production when we fixed ATP and MnCl2 concentrations (Fig. S1). Thus, 5 mM MnCl2 and 10mM MgCl2 were used in further experiments.

Effect of Co-Factors on Reaction

To confirm the effects of ATP on the GMAS enzyme, different concentrations of ATP were utilized for L-theanine production (Fig. 3A). The substrate level was 100 mM and pH was pH 7. No conversion was observed in the absence of ATP and glutamate also remained as initial substrates. As the concentration increased, L-theanine was also increased gradually. Approximately 80 mM L-theanine was achieved with 80% conversion when more than 60 mM ATP was used. These data indicate that GMAS is an ATP-dependent enzyme.

Figure 3. The effect of different ATP concentration on the production of L-theanine by the GMAS enzyme. Optimal ATP concentration was determined by detecting glutamate and L-theanine (A) and the effects of ATP, ADP, and AMP. All components were tested at various concentrations (B).

We also tested other kinds of energy sources such as ADP and AMP for comparison with ATP in L-theanine production (Fig. 3B). AMP or ADP can be used as energy source because we introduced an ATP regeneration system. Therefore, the source most effective for L-theanine production was evaluated. When these factors were compared to the control which contained no sources of ATP, the conversion yields were greater than control in all parameters. The use of AMP resulted in lower conversion yields than the other sources at all concentrations. Thus, we used ATP considering its lower cost, although ADP and ATP showed similar conversion yields.

Effects of Baker’s Yeast and Phosphate for ATP Regeneration

Various strains were cultivated with the whole-cell reaction for energy transfer (Fig. 4A). Then, we added 200 mM glucose for cell growth. Among them, baker’s yeast was the most effective for ATP synthesis, which leaded to the highest production of L-theanine. The relative conversion of E. coli, Pseudomonas putida, and Bacillus subtilis were nearly half of that from baker’s yeast. Corynebacterium glutamicum showed slightly higher than those of activity with 53% relative conversion.

Figure 4. Effect of baker’s yeast for ATP regeneration. Various strains were cultivated during the reactions for ATP regeneration (A). Activities were checked in comparison between control reaction and reaction containing baker’s yeast depending on rpm rates (B). The effects of phosphate sources were examined by E. coli whole-cell and baker’s yeast using polyphosphate (C) and potassium phosphate (D).

To clarify the effects of baker’s yeast on ATP regeneration, whole-cell reactions were compared after adding baker’s yeast (Fig. 4B). Approximately 3.8-fold higher L-theanine levels were obtained in the static reaction. When we increased the reaction speed to 1,200 rpm, 4.3-fold higher L-theanine was produced, which was more efficient than in the static reaction. Thus, ATP regeneration shows better results when the baker’s yeast is added for respiration than for fermentation.

In the reaction using sugar fermentative methods with baker’s yeast, phosphate sources are necessary to enhance production. Therefore, polyphosphate and potassium phosphate were tested (Figs. 4C and 4D). Additionally, some treatments were carried out to evaluate cell permeability, which facilitates the transport of substrates and product. Cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene sorbitan monooleate (Tween 80), and sonicated whole-cell (cell extract) were compared to the whole-cell system. Among them, the cell extract showed low L-theanine production. Additionally, activities using the whole-cell were similar to those of the whole-cell treated with CTAB and Tween 80. However, polyphosphate slightly influenced conversion yields compared to the control, which showed a 1.8-fold higher value in the presence of 10 mM polyphosphate than the control. To enhance the titers, additional phosphatases are needed to convert the polymers into monomers. In contrast, potassium phosphate was greatly effective for increased the production of L-theanine. Conversion yields were approximately 4-fold higher than those of the control in the presence of 300 mM potassium phosphate. When we tested sodium phosphate, L-theanine production was lower than that after potassium phosphate addition (Fig. S2).

Enhanced L-Theanine Production by Repetitive Use and Additional GMAS Expression

To examine the reusability of GMAS enzyme for the repetitive use of the whole-cell, the cell was recycled 3 times (Fig. 5A). The L-theanine concentration after each cycle and the accumulated L-theanine were monitored. The reactants containing cells were centrifuged after 24 h, and then the reacted medium was replaced with fresh substrate medium. After the first cycle, 161 mM L-theanine was recorded from 200 mM substrates. The produced L-theanine concentration was decreased by 0.51- and 0.26-fold in the second and third cycles, respectively. The total amount of L-theanine was obtained until 286.3 mM in 3 cycles.

Figure 5. Enhanced production by repeating GMAS whole-cell for accumulation of L-theanine (A) and overexpressing additional gmas gene (B).

Cells harboring pXMJ19::gmas exhibited activities as high as those of BW25113 (DE3) harboring pET24ma::gmas according to previous data and the repetitive use of the whole-cells. Therefore, the additional vector was inserted into BW25113 (DE3) harboring pET24ma::gmas. When the substrate concentration was 200 mM, approximately 140 mM L-theanine was obtained with 70% conversion (Fig. 5B). After pXMJ19::gmas was introduced, production increased to 178 mM with 89% conversion while substrate consumption also increased. Accordingly, we found that additional GMAS expression affects positively to enhance L-theanine production.

Finally, the time-dependence of the reaction was carried out with BW25113 (DE3) overexpressing pXMJ19::gmas and pET24ma::gmas. The reaction was performed with 300 mM glutamate, 300 mM ethylamine, 200 mM glucose, 60 mM ATP, 5 mM MnCl2, 10 mM MgCl2, 300 mM potassium phosphate and 40 mg/ml baker’s yeast (Fig. 6). Because most substrates were converted to L-theanine in the previous data (Fig. 5B), we increased the substrate level to 300 mM. Glutamate and ethylamine as the substrates started to be consumed after 3 h. After the reaction was complete, approximately 100 mM glutamate and 148 mM ethylamine remained. 198 mM L-theanine could be achieved within 12 h We conclude L-theanine can be produced by 16.5 mmol/l/h productivity.

Figure 6. L-theanine production monitoring by time-dependent manner under optimized concentration of substrates and co-factor.

Many studies have been conducted to evaluate the use of various enzymes for L-theanine production. L-Theanine exhibits effects such as anti-tumor, relaxation, and enhanced immunity. Previous reports show that among the enzymes used in L-theanine synthesis, GMAS shows potential for application in industrial production. However, the process of GMAS enzyme purification is difficult, costly, and time-consuming. In contrast, the whole-cell is easy to handle and does not require complicated procedures for its preparation. Therefore, the whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.

To construct a whole-cell system for L-theanine, protein expression and the reaction conditions were optimized. Additionally, the effect of baker’s yeast in ATP regeneration was clarified. Additional expression was also efficient for enhancing L-theanine production. To our knowledge, this is the first application of a whole-cell reaction of GMAS to develop an L-theanine production system.

Supplemental Material

Acknowledgments

This work was supported by Research Program to solve social issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234) and National Research Foundation of Korea (NRF) (NRF-2017R1D1A1B03033594, NRF-2019M3E6A1103979 and NRF-2019R1F1A1058805). In addition, this work was also supported by the polar academic program (PAP, PE18900). This paper was also supported by Konkuk University Researcher Fund in 2019. Consulting service from the Microbial Carbohydrate Resource Bank (MCRB, Korea) was kindly appreciated.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Optimization of expression and cultivation conditions. GMAS activities were compared in several expression strains containing vectors with different copy numbers for producing L-theanine (A). Cultivation medium were tested by comparing cell dry weight (B) and relative conversion of substrates (C). The results were indicated as relative conversion, which regards the highest value as 100% conversion.
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Fig 2.

Figure 2.Optimization of whole-cell reaction conditions. Optimal temperature was evaluated from 20°C to 80°C (A) and optimal initial pH was evaluated from 5.5 to 11 (B). Various metal ions were investigated (C) and optimal MnCl2 was found (D).
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Fig 3.

Figure 3.The effect of different ATP concentration on the production of L-theanine by the GMAS enzyme. Optimal ATP concentration was determined by detecting glutamate and L-theanine (A) and the effects of ATP, ADP, and AMP. All components were tested at various concentrations (B).
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Fig 4.

Figure 4.Effect of baker’s yeast for ATP regeneration. Various strains were cultivated during the reactions for ATP regeneration (A). Activities were checked in comparison between control reaction and reaction containing baker’s yeast depending on rpm rates (B). The effects of phosphate sources were examined by E. coli whole-cell and baker’s yeast using polyphosphate (C) and potassium phosphate (D).
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Fig 5.

Figure 5.Enhanced production by repeating GMAS whole-cell for accumulation of L-theanine (A) and overexpressing additional gmas gene (B).
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Fig 6.

Figure 6.L-theanine production monitoring by time-dependent manner under optimized concentration of substrates and co-factor.
Journal of Microbiology and Biotechnology 2020; 30: 785-792https://doi.org/10.4014/jmb.1910.10044

Table 1 . List of bacterial strains and plasmids..

Strain/plasmidRelevant informationReference
Bacterial strains
E. coli DH5αF- φ 80lacZ M15 endA recA hsdR(rk-mk-) supE thi gyrA relA Δ (lacZYAargF)U169[39]
E. coli BL21(DE3)F- ompT hsdSB(rB-mB-) gal dcmNovagen
E. coli BW25113F' λ- Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), lambda-, rph-1, Δ(rhaD_rhaB)568, hsdR514CGSC
E. coli BW25113(DE3)λDE3 lysogen of BW25113This study
E. coli ShufflefhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1cDsbC (Specr, lacIq) ΔtrxB sulA11 R(mcr-73::miniTn10– TetS)2 [dcm] R(zgb-210::Tn10 --TetS) endA1 Δgor Δ(mcrC-mrr)114::IS10[40]
E. coli Star TM (DE3)F- ompT rne131 lon hsdSB (rB-mB-) gal dcm λ(DE3)Invitrogen
B. subtilis str. 168ATCC
C. glutamicum ATCC13032ATCC
P. putida KT2440ATCC
Baker’s yeast
Plasmid
pET24maKmr.1 MCS site with T7 promoter, lac operator, RBS. P15A replicon. Copy number: 10–12[41]
pCDF duet 1Specr.2 MCS site with T7 promoter, lac operator, RBS. CloDF13 replicon. Copy number: 20–40Novagen
pRSF duet 1Kmr.2 MCS site with T7 promoter, lac operator, RBS. RSF1030 replicon (NTP1). Copy number: >100Novagen
pACYC duet 1Cmr.2 MCS site with T7 promoter, lac operator, RBS. P15A replicon. Copy number: 10–12Novagen
pMALAmpr. MCS site with tac promoter, lac operator, RBS, ColE1 replicon. Copy number: 20Novagen
pXMJ19Cmr. MCS site with tac promoter, lac operator, pBL1 and pUC replicon[42]
pET24ma::gmasgmas of Methylovorus mays No. 9 inserted in pET24maThis study
pCDF duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pCDF duet 1This study
pRSF duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pRSF duet 1This study
pACYC duet 1::gmasgmas of Methylovorus mays No. 9 inserted in pACYCThis study
pMAL::gmasgmas of Methylovorus mays No. 9 inserted in pMALThis study
pXMJ19::gmasgmas of Methylovorus mays No. 9 inserted in pXMJ19This study

References

  1. Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. 2004. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In Vivo 18: 55-62.
    Pubmed
  2. Turkozu D, Sanlier N. 2017. L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety. Crit. Rev. Food Sci. Nutr. 57: 1681-1687.
    Pubmed CrossRef
  3. Saeed M, Naveed M, Arif M, Kakar MU, Manzoor R, Abd El-Hack ME, et al. 2017. Green tea (Camellia sinensis) and l-theanine: Medicinal values and beneficial applications in humans-A comprehensive review. Biomed. Pharmacother. 95: 1260-1275.
    Pubmed CrossRef
  4. Adhikary R, Mandal V. 2017. L-theanine: A potential multifaceted natural bioactive amide as health supplement. Asian Pac. J. Trop. Biomed. 7: 842-848.
    CrossRef
  5. Vuong QV, Stathopoulos CE, Golding JB, Nguyen MH, Roach PD. 2011. Optimum conditions for the water extraction of L-theanine from green tea. J. Sep. Sci. 34: 2468-2474.
    Pubmed CrossRef
  6. Vuong QV, Bowyer MC, Roach PD. 2011. L-Theanine: properties, synthesis and isolation from tea. J. Sci. Food Agric. 91: 1931-1939.
    Pubmed CrossRef
  7. Kawagishi H, Sugiyama K. 1992. Facile and large-scale synthesis of L-Theanine. Biosci. Biotechnol. Biochem. 56: 689.
    Pubmed CrossRef
  8. Shuai Y, Zhang T, Jiang B, Mu W. 2010. Development of efficient enzymatic production of theanine by gamma-glutamyltranspeptidase from a newly isolated strain of Bacillus subtilis, SK11.004. J. Sci. Food Agric. 90: 2563-2567.
    Pubmed CrossRef
  9. Tachiki T, Yamada T, Mizuno K, Ueda M, Shiode J, Fukami H. 1998. gamma-glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO 12694 and their application for the syntheses of theanine and gamma-glutamylmethylamide. Biosci. Biotechnol. Biochem. 62: 1279-1283.
    Pubmed CrossRef
  10. Mu W, Zhang T, Jiang B. 2015. An overview of biological production of L-theanine. Biotechnol. Adv. 33: 335-342.
    Pubmed CrossRef
  11. Chen X, Su L, Wu D, Wu J. 2014. Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of ltheanine. Process Biochem. 49: 1429-1439.
    CrossRef
  12. Pu H, Wang Q, Zhu F, Cao X, Xin Y, Luo L, et al. 2013. Cloning, expression of glutaminase from Pseudomonas nitroreducens and application to theanine synthesis. Biocatal. Biotransform. 31: 1-7.
    CrossRef
  13. Sharma E, Joshi R, Gulati A. 2018. l-Theanine: An astounding sui generis integrant in tea. Food Chem. 242: 601-610.
    Pubmed CrossRef
  14. Liu S, Li Y, Zhu J. 2016. Enzymatic production of l-theanine by γ-glutamylmethylamide synthetase coupling with an ATP regeneration system based on polyphosphate kinase. Process Biochem. 51: 1458-1463.
    CrossRef
  15. Berke W, Schüz HJ, Wandrey C, Morr M, Denda G, Kula MR. 1988. Continuous regeneration of ATP in enzyme membrane reactor for enzymatic syntheses. Biotechnol. Bioeng. 32: 130-139.
    Pubmed CrossRef
  16. Endo T, Koizumi S. 2001. Microbial conversion with cofactor regeneration using genetically engineered bacteria. Adv. Synth. Catal. 343: 521-526.
    CrossRef
  17. Moon Y-M, Yang SY, Choi TR, Jung H-R, Song H-S, hoon Han Y, et al. 2019. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP. Enzyme Microb. Technol. 127: 58-64.
    Pubmed CrossRef
  18. Wei LL, Goux WJ. 1992. ATP cofactor regeneration via the glycolytic pathway. Bioorg. Chem. 20: 62-66.
    CrossRef
  19. Sato M, Masuda Y, Kirimura K, Kino K. 2007. Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for D-amino acid dipeptide synthesis. J. Biosci. Bioeng. 103: 179-184.
    Pubmed CrossRef
  20. Kameda A, Shiba T, Kawazoe Y, Satoh Y, Ihara Y, Munekata M, et al. 2001. A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase. J. Biosci. Bioeng. 91: 557-563.
    Pubmed CrossRef
  21. Yan B, Ding Q, Ou L, Zou Z. 2014. Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system. World J. Microbiol. Biotechnol. 30: 1123-1128.
    Pubmed CrossRef
  22. Resnick SM, Zehnder AJ. 2000. In vitro ATP regeneration from polyphosphate and AMP by polyphosphate: AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A. Appl. Environ. Microbiol. 66: 2045-2051.
    Pubmed KoreaMed CrossRef
  23. Wakisaka S, Ohshima Y, Ogawa M, Tochikura T, Tachiki T. 1998. Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast. Appl. Environ. Microbiol. 64: 2952-2957.
    Pubmed KoreaMed CrossRef
  24. Horinouchi N, Sakai T, Kawano T, Matsumoto S, Sasaki M, Hibi M, et al. 2012. Construction of microbial platform for an energyrequiring bioprocess: practical 2'-deoxyribonucleoside production involving a C− C coupling reaction with high energy substrates. Microb. Cell Fact. 11: 82.
    Pubmed KoreaMed CrossRef
  25. Matsuno R, Asada M, Nakanishi K, Kamikubo T. 1982. ATP Regeneration by Enzymes of Alcohol Fermentation and Kinases of Yeast and its Computer Simulation, pp. 351-352. In: Enzyme Engineering. Springer.
    CrossRef
  26. Horinouchi N, Ogawa J, Kawano T, Sakai T, Saito K, Matsumoto S, et al. 2006. Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli. Biosci. Biotechnol. Biochem. 70: 1371-1378.
    Pubmed CrossRef
  27. Yamamoto S, Wakayama M, Tachiki T. 2005. Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells. Biosci. Biotechnol. Biochem. 69: 784-789.
    Pubmed CrossRef
  28. Lin J-P, Tian J, You J-F, Jin Z-H, Xu Z-N, Cen P-L. 2004. An effective strategy for the co-production of S-adenosyl-L-methionine and glutathione by fed-batch fermentation. Biochem. Eng. J. 21: 19-25.
    CrossRef
  29. Bhatia SK, Bhatia RK, Yang Y-H. 2016. Biosynthesis of polyesters and polyamide building blocks using microbial fermentation and biotransformation. Rev. Environ. Sci. Bio/Technol. 15: 639-663.
    CrossRef
  30. Yang S-Y, Choi T-R, Jung H-R, Park Y-L, Han Y-H, Song H-S, et al. 2020. Development of glutaric acid production consortium system with α-ketoglutaric acid regeneration by glutamate oxidase in Escherichia coli. Enzyme Microb. Technol. 133: 109446.
    Pubmed CrossRef
  31. Moon Y-M, Gurav R, Kim J, Hong Y-G, Bhatia SK, Jung H-R, et al. 2018. Whole-cell immobilization of engineered Escherichia coli JY001 with barium-alginate for itaconic acid production. Biotechnol. Bioprocess Eng. 23: 442-447.
    CrossRef
  32. Bhatia SK, Shim Y-H, Jeon J-M, Brigham CJ, Kim Y-H, Kim H-J, et al. 2015. Starch based polyhydroxybutyrate production in engineered Escherichia coli. Bioprocess Biosys. Eng. 38: 1479-1484.
    Pubmed CrossRef
  33. Bhatia SK, Kim S-H, Yoon J-J, Yang Y-H. 2017. Current status and strategies for second generation biofuel production using microbial systems. Energy Convers. Manage 148: 1142-1156.
    CrossRef
  34. Alaiz M, Navarro JL, Girón J, Vioque E. 1992. Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. J. Chromatogr. 591: 181-186.
    Pubmed CrossRef
  35. Kim J, Seo H-M, Bhatia SK, Song H-S, Kim J-H, Jeon J-M, et al. 2017. Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli. Sci. Rep. 7: 39768.
    Pubmed KoreaMed CrossRef
  36. Griffiths MW, Muir DD. 1978. Properties of a thermostable β‐galactosidase from a thermophilic Bacillus: Comparison of the enzyme activity of whole cells, purified enzyme and immobilised whole cells. J. Sci. Food Agric. 29: 753-761.
    Pubmed CrossRef
  37. Buchachenko AL, Kuznetsov DA. 2008. Magnetic field affects enzymatic ATP synthesis. J. Am. Chem. Soc. 130: 12868-12869.
    Pubmed CrossRef
  38. Moon Y-M, Yang SY, Choi TR, Jung H-R, Song H-S, hoon Han Y, et al. 2019. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP. Enzyme Microb. Technol. 127: 58-64.
    Pubmed CrossRef
  39. Taylor RG, Walker DC, McInnes R. 1993. E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.  Nucleic Acids Res. 21: 1677.
    Pubmed KoreaMed CrossRef
  40. Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. 2012. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11: 753.
    Pubmed KoreaMed CrossRef
  41. Lee SG, Lee JO, Yi JK, Kim BG. 2002. Production of cytidine 5'‐monophosphate N‐acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst. Biotechnol. Bioeng. 80: 516-524.
    Pubmed CrossRef
  42. Jakoby M, Ngouoto-Nkili C-E, Burkovski A. 1999. Construction and application of new Corynebacterium glutamicum vectors. Biotechnol. Tech. 13: 437-441.
    CrossRef