Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae

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.


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 crosslinking 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 Open Access *Correspondence: k-hara@u-shizuoka-ken.ac.jp † Yoko Hirono-Hara and Miyuu Yui contributed equally to this work 2 Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Full list of author information is available at the end of the article 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 Ca 2+ 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.

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.

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 OD 600 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 ultrafiltered 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 ://image j.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 (NH 4 ) 2 SO 4 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 OD 600 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).

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 Fig. 1 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) 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).

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).

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. 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 crosslinking 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.

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 . 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 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 crosslinking 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.