Skip to main content

Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae

Abstract

Transglutaminase (TG) catalyzes the formation of cross-links between proteins. TG from Streptoverticillium mobaraense (SmTG) is used widely in food, cosmetic, biomaterial and medical industries. SmTG is occasionally supplied as a mixture with the activator peptide glutathione. Currently, glutathione is industrially produced using a budding yeast, Saccharomyces cerevisiae, because of its intracellular high content of glutathione. In this study, active SmTG was produced together with glutathione in S. cerevisiae. SmTG extracted from S. cerevisiae expressing SmTG showed cross-linking activity when BSA and sodium caseinate were substrates. The cross-linking activity of SmTG increased proportionally as the concentration of added glutathione increased. Furthermore, SmTG was prepared by extracting SmTG from an engineered S. cerevisiae whose glutathione synthetic pathway was enhanced. The SmTG solution showed higher activity when compared with a SmTG solution prepared from a S. cerevisiae strain without enhanced glutathione production. This result indicates that a high content of intracellular glutathione further enhances active SmTG production in S. cerevisiae. S. cerevisiae co-producing SmTG and a higher content of glutathione has the potential to supply a ready-to-use industrial active TG solution.

Key points

  • Streptoverticillium mobaraense transglutaminase (SmTG) is used widely in the food industry.

  • SmTG is activated by the peptide glutathione.

  • Active SmTG was produced in an engineered high glutathione-producing S. cerevisiae.

Introduction

Transglutaminase (TG; EC 2.3.2.13) is an acyl transferase that forms both intra- and inter-molecular isopeptide bonds in and between proteins. TG catalyzes an acyl transfer reaction between the γ-carboxyamide group of glutamine residues and the γ-amino group of lysine residues to form ε-(γ-glutamyl)lysine isopeptide bonds. Such reactions facilitate the polymerization of proteins (Folk 1980; Lorand and Conrad 1984; Kashiwagi et al. 2002).

TG is used widely in the food, chemical, medical and cosmetic industries because of its broad cross-linking activity (Ikura et al. 1981; Kurth and Rogers 1984; Savoca et al. 2018). In particular, TG is often used widely in the food industry to avoid chemical modification processes to link proteins (Kuraishi et al. 1997; Gerrard et al. 1998; Abou-Soliman et al. 2017), which often give rise to unwanted side reactions and require high-pressure/temperature conditions and chemical solvents (Miwa 2020). TG is distributed widely in nature and has been identified in bacteria and mammals. However, when focusing on the industrial use of TG, microbial transglutaminase produced by S. mobaraense (SmTG) is a major choice because of various advantages that include Ca2+ independence, higher reaction rates, thermostability, wider substrate specificity for acyl donors and a smaller molecular size when compared with other TGs (Ando et al. 1989; Nonaka et al. 1989; Kanaji et al. 1993; Miwa 2020).

The addition of glutathione has been reported to activate SmTG (Bönisch et al. 2007; Miwa 2020). Glutathione is an anti-oxidant tripeptide that is distributed widely among animals, plants and microorganisms, playing an important role in the protection of cells against oxidation and the maintenance of enzymes and other cellular components in the reduced state (Schmacht et al. 2017). Currently, industrial production of glutathione uses the budding yeast, Saccharomyces cerevisiae, because of its high intracellular glutathione content (Li et al. 2004). SmTG mixed with yeast extract containing glutathione is commercially available (Bönisch et al. 2007; Miwa 2020). The mechanism of glutathione activating SmTG remains unresolved, although Bönisch et al. (2007) demonstrated that reduction of an unknown inhibitor(s) present in milk by glutathione improved crosslinking of casein (Bönisch et al. 2007). Therefore, production of both SmTG and glutathione using S. cerevisiae expressing SmTG should be a promising, simple one-pot process to obtain highly active SmTG. In S. cerevisiae, glutathione biosynthesis is continuously catalyzed by glutathione synthase I (Gsh1) and glutathione synthase II (Gsh2) from three precursor amino acids (Li et al. 2004; Schmacht et al. 2017). We previously developed an engineered S. cerevisiae strain (GCI strain) whose glutathione biosynthesis was improved by overexpression of Gsh1 and Gsh2 (Hara et al. 2012).

Several studies have reported heterologous expression of SmTG in microorganisms such as Escherichia coli (Salis et al. 2015), Corynebacterium glutamicum (Itaya and Kikuchi 2008) and Pichia pastoris (Özçelik et al. 2019), whereas the glutathione-producing strain S. cerevisiae has never been used as a host strain. In the current study, S. cerevisiae was evaluated as a host strain to produce SmTG. Moreover, the produced SmTG solution was active when expressed in the engineered S. cerevisiae GCI strain. The results demonstrate the potential of S. cerevisiae as a host strain to produce industrial levels of active SmTG.

Materials and methods

Strain and media

Escherichia coli DH5α cells were used for recombinant DNA manipulation. S. cerevisiae BY4741 strain [MATα his3Δ1 ura3Δ0 leu2Δ0 met15Δ0] and the previously engineered glutathione-hyper producing yeast (GCI strain) (Hara et al. 2015) that overexpresses the GSH1 and GSH2 genes were used.

Escherichia coli transformants were grown in Luria–Bertani medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) supplemented with 100 µg/mL ampicillin. Yeast transformants were grown in rich yeast-extract peptone dextrose (YPD) medium (10 g/L yeast extract, 20 g/L bacto-peptone, 20 g/L glucose) supplemented with or without 0.5 µg/mL aureobasidin A (Takara Bio, Shiga, Japan), and in selective synthetic dextrose (SD) medium (6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose) supplemented with appropriate concentrations of amino acids and nucleobases.

Plasmid and yeast transformation

The gene encoding TG from S. mobaraense (SmTG) is deposited in the Gene bank with accession No. AF531437.1. The nucleotide sequence encoding SmTG was codon optimized for production in S. cerevisiae (SmTG; DDBJ accession No. LC583746) by Eurofins genomics (Tokyo, Japan). The synthesized DNA encoding SmTG was digested with SalI/BglII and inserted into the SalI/BglII sites of pGK425 (Ishii et al. 2009) to give the expression vector pGK425-TG. The S. cerevisiae BY4741 strain and GCI strain were transformed with pGK425-TG using a lithium acetate method (Ito et al. 1983; Gietz et al. 1992) to yield the BY4741-TG and GCI-TG strains, respectively.

Preparation of S. mobaraense transglutaminase

Yeast transformants were pre-cultured in 5 mL of YPD at 30 °C and 200 rpm overnight. This pre-culture was inoculated into 20 mL of SD medium in the presence of appropriate concentrations of amino acids and nucleobases for the BY4741-TG strain and additionally 0.5 µg/mL aureobasidin A for the GCI-TG strain. The inoculation volumes were adjusted to an initial OD600 of 0.15 in 200 mL baffled Erlenmeyer flasks and cultured at 200 rpm in a shaking incubator (BR-43FL, TAITEC, Saitama, Japan) at 30 °C.

For preparation of the Y×SmTG (SmTG extracted from S. cerevisiae expressing SmTG) solution, the supernatant from Y mL of a 24 h cell culture was discarded after centrifugation at 3000×g for 5 min at 4 °C. The collected cells were suspended in 300 µL ice-cold TE buffer (100 mM Tris–HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid) and disrupted by a cell disruptor (Shake Master NEO, Bio Medical Sciences, Tokyo, Japan) with zirconia beads at 1500 rpm for 10 min. The cell debris were removed by centrifugation at 16,000×g for 10 min at 4 °C. The supernatants (SmTG solution) were stored at − 80 °C until use.

Cross-linking assay of SmTG

For the BSA cross-linking assay of SmTG at different reaction times, 40 µL of the reaction buffer BD [1.25 mg/mL BSA, 50 µM dithiothreitol (DTT)] and 10 µL of the 1×SmTG solution in 100 mM Tris–HCl (pH 8.0) were mixed and incubated for 0, 30, 60 and 120 min at 50 °C. For the BSA cross-linking assay with different volumes of SmTG solutions, 40 µL of the reaction buffer BD and 10 µL of 1×, 5×, or 10×SmTG solution in 100 mM Tris–HCl (pH 8.0) were mixed and incubated for 120 min at 50 °C.

Because BSA required DTT as a reducing agent for cross-lining, the caseinate was used for cross-linking assay to evaluate the presence of several concentrations of glutathione as the other reducing agent for unknown inhibitor(s) (Bönisch et al. 2007). One hundred and sixty microliters of the reaction buffer containing 0.625 mg/mL sodium caseinate with 0–3.2 mM glutathione and 40 µL of the 1×SmTG solution prepared from the BY4741-TG strain were mixed and incubated overnight at 50 °C. Forty microliters of the reaction mixture was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Sixty microliters of the reaction mixture was ultra-filtered by using Amicon ultrafiltration units (Merck KGaA, Darmstadt, Germany) to remove proteins whose molecular weights were under 100 kDa, and the concentrations of the cross-linked higher molecular weight proteins (> 100 kDa) were measured by staining with Coomassie Brilliant Blue (CBB) solution (Nacalai-Tesque, Kyoto, Japan).

Cross-linking activity of SmTG extracted from BY4741-TG and GCI-TG strains was measured by using caseinate as the substrate. Forty microliters of 0.625 mg/mL caseinate and 10 µL of 12×SmTG solution in 100 mM Tris–HCl (pH 8.0) were mixed and incubated overnight at 50 °C. After the cross-linking reaction, 10 µL of the reaction mixture was suspended into an equal volume of sample buffer for SDS-PAGE (Nacalai-Tesque) analysis, incubated for 3 min at 100 °C, loaded onto a 12% SDS-PAGE gel, and the gel was stained by CBB stain One Super (Nacalai-Tesque). Relative level of protein was calculated from the intensity of each protein band using Image J software (https://imagej.nih.gov/ij/).

Measurement of SmTG activity

Activity of SmTGs was measured by incorporation of the fluorescent amine, dansyl cadaverine [N-(5-aminopentyl)-5-dimethylamino-1-naphthalenesulfonamide, MDC] (Sigma-Aldrich, Missouri, USA), into N,N-dimethyl casein according to the fluorescence method reported (Liu et al. 2014). Twenty microliters of a 12×SmTG solution extracted from either the BY4741-TG or GCI-TG strain was mixed with 460 µL of the mixture (50 mM Tris–HCl (pH 8.0), 0.2% N,N-dimethyl casein, 13 µM MDC). The assay mixture was incubated for 30 min at 37 °C and the reaction terminated by adding (NH4)2SO4 to a final concentration of 42 mM. The fluorescence intensity was measured by a microplate microplate reader Synergy HTX (BioTek, Vermont, USA) using excitation and emission wavelengths of 350 and 500 nm, respectively.

Analytical methods

The cell concentration was determined by measuring the OD600 using a Gene Quant1300 spectrometer (GE Healthcare Life Sciences, Buckinghamshire, UK). The glutathione concentration was measured via a 5,5′-dithiobis (2-nitorobenzonic acid)-glutathione reductase recycling assay, as described previously (Hara et al. 2009).

Results

Heterologous expression of SmTG in S. cerevisiae

The gene encoding SmTG was codon optimized for expression in S. cerevisiae and transformed into the S. cerevisiae BY4741 strain. Cell growth of BY4741 expressing SmTG (BY4741-TG strain) was similar to that of the control strain. A solution of SmTG was obtained by disrupting the BY4741-TG strain expressing SmTG after cultivation for 24 h. Intracellular expression of SmTG was confirmed by SDS-PAGE analysis by the presence of a band of ~ 37 kDa (Fig. 1). Cross-linking activity of the SmTG solution extracted from the BY4741-TG strain was also confirmed using BSA as a substrate. A smeared band was observed when incubating BSA with SmTG, which represented polymerized BSA and the intensity of this smeared band was proportional to the reaction time (Fig. 1). Moreover, more BSA polymerized as the concentration of SmTG was increased (Additional file 1: Fig. S1).

Fig. 1
figure1

Cross-linking assay using BSA as a substrate after incubation with a SmTG solution extracted from the BY4741-TG strain for several reaction times. Lane 1, molecular size marker; lane 2, BSA; lanes 3, 4, 5 and 6, 1×SmTG (+BSA) for 0, 30, 60 and 120 min, respectively; lane 7, 1×SmTG (−BSA)

Effect of glutathione addition on cross-linking activity of SmTG

Glutathione was added to the SmTG solution extracted from the BY4741-TG strain to investigate the affect of glutathione on cross-linking activation of SmTG (Fig. 2 and Additional file 1: Fig. S2). In general, milk proteins are good substrates for SmTG. In particular, caseins, major milk proteins, are a favorable substrate for SmTG to cross-link because they are non-globular and have limited tertiary structure, which makes them readily accessible to enzymes. Among them, both α-casein and κ-casein have two cysteines and have been reported to play an important role in the formation of cross-links (Fox et al. 1998). Proteins that have molecular weights less than 100 kDa were removed by ultrafiltration, and the remaining proteins were quantified to determine the level of caseinate cross-linking by SmTG. As shown in Fig. 2, proteins with molecular weights above 100 kDa increased as the glutathione concentration increased from 0.2 to 0.8 mM (Fig. 2). The concentration of proteins with molecular weights greater than 100 kDa in the presence of 0.8 mM glutathione was 1.5-fold higher when compared with the results obtained in the absence of glutathione. Polymerization was saturated at concentrations above 0.8 mM glutathione (Fig. 2).

Fig. 2
figure2

Effect of reduced glutathione (GSH) addition on polymerization of caseinate by SmTG. The concentration of proteins whose molecular weights were more than 100 kDa were detected after polymerization of caseinate by 0.2×SmTG extracted from the BY4741-TG strain in the presence of different GSH concentrations

Heterologous overexpression of SmTG in a S. cerevisiae strain producing high amounts of glutathione

We previously constructed the S. cerevisiae GCI strain, which produces higher levels of intracellular glutathione when compared with the parental S. cerevisiae strain (Hara et al. 2012). From the viewpoint of enhancing SmTG activity, S. cerevisiae host strain producing enhanced levels of glutathione for expression of SmTG is an attractive approach to enhance SmTG activity. Therefore, in this study, the gene encoding SmTG was overexpressed in the GCI strain. Cell growth and glutathione production were compared between the GCI and BY4741 strains when overexpression of SmTG was induced (Additional file 1: Fig. S3). The cell growth of both strains was similar (Additional file 1: Fig. S3a). In contrast, glutathione content increased drastically in the GCI-TG strain when compared with that of the BY4741-TG strain (Additional file 1: Fig. S3b). The BY4741-TG strain and GCI-TG strain produced 0.0017 mM and 0.16 mM glutathione, respectively, after cultivation for 24 h, although the expression levels of SmTG were the same between the both strains (Additional file 1: Fig. S3c). As shown in Fig. 2, addition of 1.6–3.2 mM glutathione showed saturated activity enhancement. Therefore, the 12×SmTG solution extracted from the GCI-TG strain contatined saturated 1.92 mM glutathione. The 12×SmTG solution extracted from the BY4741-TG strain containing less than 0.2 mM was used as an almost non-enhancement control (Fig. 2). The enhancement of SmTG activity extracted from the BY4741-TG strain and GCI-TG strain were compared (Fig. 3). The enhanced activity of SmTG extracted from the GCI-TG strain was 1.4-fold higher when compared with that of the BY4741-TG strain. This result indicates that activity of SmTG expressed in the GCI strain was enhanced because of the higher glutathione content present when compared with that of the BY4741 strain.

Fig. 3
figure3

SmTG activity measured by the incorporation of MDC into N,N-dimethylcasein. 12×SmTG extracted from BY4741-TG and GCI-TG strains were used for cross-linking reactions. The values are means and the error bars show the SD (n = 5). Statistically significant differences are indicated by an asterisk (p = 0.023)

Finally, the cross-linking activity of SmTG extracted from that GCI-TG strain was evaluated using caseinate as a substrate. As shown in Fig. 4a, the intensities of higher molecular-weight bands increased when using the SmTG solution extracted from the GCI-TG strain rather than that from the BY4741-TG strain. The yields of polymerized α-caseins were estimated to be 44% and 51%, because the residual α-caseins were calculated to be 56% and 49% from the BY4741-TG strain and GCI-TG strain, respectively. These results indicate that SmTG extracted from the GCI-TG strain showed enhanced cross-linking activity of α-casein because of the higher content of glutathione present in the GCI-TG strain when compared with that of the BY4741-TG strain. Proteins that have molecular weights less than 100 kDa were removed by ultrafiltration, and the remaining proteins were quantified to determine the level of caseinate cross-linking by SmTG extracted from the BY4741-TG strain and the GCI-TG strain. As shown in Fig. 4c, the concentration of proteins with molecular weights greater than 100 kDa obtained from the GCI-TG strain was 1.3-fold higher when compared with the result obtained from the BY4741-TG strain.

Fig. 4
figure4

Polymerization of caseinate by SmTG extracted from BY4741-TG and GCI-TG strains. Each sample volume loaded was 20 µL (a) and 5 µL (b). a Lane 1, protein molecular size marker; lane 2, caseinate control; lane 3, polymerized caseins by 12×SmTG extracted from the BY4741-TG strain; lane 4, polymerized caseins by 12×SmTG extracted from the GCI-TG strain; lane 5, BY4741-TG extract; lane 6, GCI-TG extract. b Expanded region of the SDS-PAGE in (a boxed region). Ratios of cross-linked α-casein were calculated from the intensity of each α-casein band and represented as relative values (%). c The concentration of proteins whose molecular weights were more than 100 kDa, were detected after polymerization of caseinate by 12×SmTG extracted from the BY4741-TG and GCI-TG strain

Discussion

TG is used frequently as an industrial enzyme, especially in the food industry, and reports describing its use in the food industry are numerous (Miwa 2020). The biggest advantage of TG from S. mobaraense (SmTG) is its suitability in a variety of applications. In this study, SmTG expressed in S. cerevisiae was confirmed to have polymerizing activity. Furthermore, SmTG extracted from the higher glutathione producing S. cerevisiae, which overexpresses GSH1 and GSH2 together with SmTG, also showed activity. The cell growth of both BY4741-TG and GCI-TG strains decreased after 24 h cultivation (Additional file 1: Fig. S3a). This decrease in cell growth is hypothesized to be cell damage caused by polymerization of various intracellular proteins required for survival. However, the reduction in growth rate was small and may not pose as an issue when producing SmTG in S. cerevisiae. Polymerization activity of SmTG increased in the presence of glutathione concentrations up to 1.6 mM when caseinate was used as the substrate (Fig. 2). The cross-linking assay was carried out using 12×SmTG extracted from the GCI-TG strain, which contained 1.92 mM glutathione after cultivation for 24 h. The crosslinking activity using 12×SmTG extracted from the GCI-TG strain was enhanced by 1.4-fold when compared with the 12×SmTG extracted from the BY4741-TG strain (Fig. 3). This fold increase is similar to the value (1.5-fold) observed when glutathione was added externally at concentrations above 0.8 mM (Fig. 2). Thus, the production of active SmTG because of the higher levels glutathione produced in the engineered S. cerevisiae is advantageous for cross-linking activity by SmTG.

Miwa (2020) reported that crosslinking of pasteurized milk was enhanced in the presence of 0.02–0.2 mM glutathione (Miwa 2020). Mixtures of SmTG and glutathione were supplied from food companies (Bönisch et al. 2007). In the current study, we showed the potential of obtaining a mixture of SmTG and glutathione produced simultaneously by overexpression of SmTG in an engineered S. cerevisiae strain producing high levels of glutathione.

Glutathione is industrially produced by fermentation of S. cerevisiae. In this study, we succeeded in integrating the production of SmTG and glutathione using S. cerevisiae as a host strain. This simple strategy for production of active SmTG in S. cerevisiae should reduce production costs and waste as both TG and GSH will be available from one source.

Availability of data and materials

All data and materials are available.

References

  1. Abou-Soliman NHI, Sakr SS, Awad S (2017) Physico-chemical, microstructural and rheological properties of camel-milk yogurt as enhanced by microbial transglutaminase. J Food Sci Technol 54:1616–1627

    CAS  Article  Google Scholar 

  2. Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, Tanaka H, Motoki M (1989) Purification and characteristics of a novel transglutaminase derived from microorganism. Agric Biol Chem 53:2613–2617

    CAS  Google Scholar 

  3. Bönisch MP, Lauber S, Kulozik U (2007) Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition. Int Dairy J 17:3–11

    Article  Google Scholar 

  4. Folk JE (1980) Transglutataminases. Ann Rev Biochem 49:517–531

    CAS  Article  Google Scholar 

  5. Fox PF, Uniacke-Lowe T, McSweeney PLH, O’Mahony JA (1998) Dairy chemistry and biochemistry. Blackie Academic & Professional, Glasgow

    Google Scholar 

  6. Gerrard J, Fayle S, Wilson A, Newberry M, Ross M, Kavale S (1998) Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase. J Food Sci 63:472–475

    CAS  Article  Google Scholar 

  7. Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425

    CAS  Article  Google Scholar 

  8. Hara KY, Shimodate N, Hirokawa Y, Ito M, Baba T, Mori H, Mori H (2009) Glutathione production by efficient ATP-regenerating Escherichia coli mutants. FEMS Microbiol Lett 297:217–224

    CAS  Article  Google Scholar 

  9. Hara KY, Kiriyama K, Inagaki A, Nakayama H, Kondo A (2012) Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 94:1313–1319

    CAS  Article  Google Scholar 

  10. Hara KY, Aoki N, Kobayshi J, Kiriyama K, Nishida K, Araki M, Kondo A (2015) Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 99:9771–9778

    CAS  Article  Google Scholar 

  11. Ikura K, Yoshikawa M, Sasaki R, Chiba H (1981) Incorporation of amino acids into food proteins by transglutaminase. Agric Biol Chem 45:2587–2592

    CAS  Google Scholar 

  12. Ishii J, Izawa K, Matsumura S, Wakamura K, Tanino T, Tanaka T, Ogino C, Fukuda H, Kondo A (2009) A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast. J Biochem 145:701–708

    CAS  Article  Google Scholar 

  13. Itaya H, Kikuchi Y (2008) Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol 78:621–625

    CAS  Article  Google Scholar 

  14. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    CAS  Article  Google Scholar 

  15. Kanaji T, Ozaki H, Takao T, Kawajiri H, Ide H, Motoki M, Shimonishi Y (1993) Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. J Biol Chem 268:11565–11572

    CAS  Article  Google Scholar 

  16. Kashiwagi T, Yokoyama KI, Ishikawa K, Ono K, Ejima D, Matsui H, Suzuki E (2002) Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biol Chem 277:44252–44260

    CAS  Article  Google Scholar 

  17. Kuraishi C, Sakamoto J, Yamazaki K, Susa Y, Kuhara C, Soeda T (1997) Production of restructured meat using microbial transglutaminase without salt or cooking. J Food Sci 62:488–490

    CAS  Article  Google Scholar 

  18. Kurth L, Rogers P (1984) Transglutaminase catalyzed cross-linking of myosin to soya protein, casein and gluten. J Food Sci 49:573–576

    CAS  Article  Google Scholar 

  19. Li Y, Wei G, Chen J (2004) Glutathione: a review on biotechnological production. Appl Microbiol Biotechnol 66:233–242

    CAS  Article  Google Scholar 

  20. Liu Y, Lin S, Zhang X, Liu X, Wang J, Lu F (2014) A novel approach for improving the yield of Bacillus subtilis transglutaminase in heterologous strains. J Ind Microbiol Biotechnol 41:1227–1235

    CAS  Article  Google Scholar 

  21. Lorand L, Conrad SM (1984) Transglutaminases. Mol Cell Biochem 58:9–35

    CAS  Article  Google Scholar 

  22. Miwa N (2020) Innovation in the food industry using microbial transglutaminase: keys to success and future prospects. Anal Biochem 597:113638–113646

    CAS  Article  Google Scholar 

  23. Nonaka M, Tanaka H, Okiyama A, Motoki M, Ando H, Umeda K, Matsuura A (1989) Polymerization of several proteins by Ca2-independent transglutaminase derived from microorganisms. Agric Biol Chem 53:2619–2623

    CAS  Google Scholar 

  24. Özçelik AT, Ersöz F, İnan M (2019) Extracellular production of the recombinant bacterial transglutaminase in Pichia pastoris. Protein Expr Purif 159:83–90

    Article  Google Scholar 

  25. Salis B, Spinetti S, Scaramuzza S, Bossi M, Jotti GS, Tonon G, Crobu D, Schrepfer R (2015) High-level expression of a recombinant active microbial transglutaminase in Escherichia coli. BMC Biotechnol 15:84

    Article  Google Scholar 

  26. Savoca MP, Tonoli E, Atobatele AG, Verderio EAM (2018) Biocatalysis by transglutaminases: a review of biotechnological applications. Micromachines 9:562–585

    Article  Google Scholar 

  27. Schmacht M, Lorenz E, Senz M (2017) Review microbial production of glutathione. World J Microbiol Biotechnol 33:106–119

    Article  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. J. Ishii (Kobe University) for providing us with pGK425.

Funding

This study was supported by the Special Coordination Funds for Promoting Science and Technology, MEXT, Japan, Grant-in-Aid for Scientific Research (C) 16K00616, and the JST-Mirai Program Grant Number JPMJMI17EJ.

Author information

Affiliations

Authors

Contributions

All authors designed the study and wrote the manuscript. YHH and YM conducted the experiments. KYH advised on the project and was involved in revising the manuscript. KYH coordinated this study, contributed to the major funding support and managed the project. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Kiyotaka Y. Hara.

Ethics declarations

Ethics approval and consent to participate

This manuscript does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

All authors consent the publication.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Cross-linking assay using BSA as a substrate after incubation for 120 min with several volumes of SmTG solution extracted from the BY4741-TG strain. Lane 1, molecular size marker; lane 2, BSA only; lanes 3, 4 and 5, 1×, 5× and 10×SmTG (+BSA), respectively; lanes 6, 7 and 8, 1×, 5× and 10×SmTG (−BSA), respectively. Figure S2. Effect of adding reduced glutathione to polymerization of caseinate by SmTG. SDS-PAGE profiles show the protein pattern of caseinate after incubation with SmTG. 1×SmTG extracted from the BY4741-TG strain was used. Lane 1, molecular size marker; lane 2, caseinate only; lanes 3, 4, 5, 6, 7 and 8, caseinate, SmTG and 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mM glutathione, respectively; lane 9, SmTG only. The polymerized caseins (molecular weight > 100 kDa) presented in the top panel were recovered and measured as shown in Fig. 2. Figure S3. Growth and glutathione production of S. cerevisiae expressing SmTG. (a) Time course of cell growth. Solid and dotted lines represent the BY4741-TG and GCI-TG strains, respectively. (b) Glutathione concentrations in 1×SmTG solutions extracted from the BY4741-TG strain (black) and GCI-TG strain (white). (c) Expression level of SmTG after cultivation for 24 h. lane 1, BY4741-TG strain; lane 2, GCI-TG strain. Expression level of SmTG was calculated from the intensity of each SmTG band and represented as relative values (%). The values are means and the error bars show the SD (n = 3).

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hirono-Hara, Y., Yui, M. & Hara, K.Y. Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae. AMB Expr 11, 13 (2021). https://doi.org/10.1186/s13568-020-01176-3

Download citation

Keywords

  • Transglutaminase
  • Glutathione
  • Streptoverticillium mobaraense
  • Yeast
  • Saccharomyces cerevisiae
  • Coproduction