The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli

Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli . In this study, the pro-TGase from Streptomyces hygroscopicus was efﬁciently secreted by E. coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efﬁciently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the ﬁrst six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efﬁcient secretion and solubility in E. coli .


Introduction
Transglutaminase (EC 2.3.2.13, TGase) catalyzes crosslinking between the c-carboxyamide group in glutamine residues (acyl donors) and a variety of primary amines (acyl acceptors) in many proteins . In the absence of primary amines, water can act as an acyl acceptor, which results in the deamidation of glutamine residues . Multifunctional TGases are widely found in mammals (Schmid et al., 2011), plants (Carvajal et al., 2011), and microorganisms . The first microbial TGase was discovered in Streptomyces mobaraensis (Ando et al., 1989). Subsequently, many new microbial strains that produce TGase were identified (Zhang et al., 2010). Streptomyces TGase has been widely used in the food industry to improve the functional properties of food products . Recent studies have suggested that TGase-mediated cross-linking also has great potential for tissue engineering, textiles and leather processing, biotechnological tools, and other non-food applications (Zhu & Tramper, 2008). Thus, it is desirable to develop an efficient and easy-to-use expression system for the production and modification of TGase.
To date, attempts have been made to express TGase in Streptomyces lividans (Lin et al., 2004(Lin et al., , 2006, Escherichia coli (Marx et al., 2007;Yu et al., 2008;Yang et al., 2009), Corynebacterium glutamicum (Date et al., , 2004Kikuchi et al., 2003), and methylotropic yeasts (Yurimoto et al., 2004). As a screening platform for directed evolution, E. coli has particular advantages over other expression systems because of its simple cell culture and ease of molecular biological manipulations. Because Streptomyces TGase is synthesized as an inactive zymogen (pro-TGase) in wild-type strains (Pasternack et al., 1998;Zhang et al., 2008a), three strategies have been used for the expression of microbial TGase in E. coli: (i) the direct expression of mature TGase fused or not fused to an additional peptide; (ii) the expression of pro-TGase followed by processing to mature TGase in vitro; and (iii) the co-expression of pro-TGase with the activation protease. The first strategy often leads to a low-level of protein expression or the formation of S. mobaraensis TGase in inclusion bodies (Takehana et al., 1994;Kawai et al., 1997). The second strategy produces a large amount of soluble pro-TGase (Marx et al., 2007) that can be converted into an active TGase in vitro by adding exogenous proteases (Marx et al., 2008). In the third strategy, the active TGase is produced by combining pro-TGase expression and its activation in vivo (Zhao et al., 2010). However, all three strategies only result in the intracellular production of the TGase or the pro-TGase even in the presence of a signal peptide (Takehana et al., 1994;Marx et al., 2007;Yang et al., 2009).
In contrast to intracellular production, the efficient secretion of TGase or pro-TGase is considerably more cost-effective for the recovery and purification of the protein in E. coli because it does not require a cell disruption step (Mergulhao et al., 2005). In addition, secretion of the enzyme will benefit the rapid and high throughput screening of mutant libraries for desired catalytic properties. In this study, the pro-TGase from S. hygroscopicus was successfully secreted in E. coli using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was directly transformed into an active form after the addition of dispase to the culture supernatant of the recombinant strain. This is the first report of pro-TGase secretion by E. coli. In addition, we identified the residues in the pro-region of S. hygroscopicus TGase that affect the solubility and secretion of TGase in E. coli.

Cloning of the TGase gene containing flanking regions
Cloning of the TGase gene containing flanking regions from S. hygroscopicus was performed in two steps. First, the pro-TGase gene was cloned from S. hygroscopicus genomic DNA by PCR using TG-NcoI and TG-BamHI primers ( Table 1) that were designed based on the conserved terminal sequence of pro-TGases from Streptomyces platensis, Streptomyces cinnamoneus, and Streptomyces fradiae (GenBank accession nos. AY555726, AB085698, and DQ432028). The target PCR product was inserted into the NcoI-BamHI sites of pET-22b + and was sequenced. Secondly, based on the sequence of the pro-TGase gene, an inverse PCR (Ochman et al., 1988) was performed to amplify the flanking regions of the cloned pro-TGase gene. Streptomyces hygroscopicus genomic DNA was digested with PstI. The digested DNA was circularized and served as the inverse PCR template. The inverse PCR primers ITG1 and ITG2 (Table 1) were designed based on the sequence of the cloned pro-TGase gene. The PCR product containing the flanking regions of the pro-TGase gene was cloned and sequenced. Assembling the gene sequences of the pro-TGase and its flanking regions generated a TGaserelated fragment that was named tgh (Fig. 1a).

Sequence analysis
The signal peptide sequence prediction was performed on the SIGNALP 3.0 Server (http://www.cbs.dtu.dk/services/ SignalP/). The promoter region sequence was predicted by BDGP (http://www.fruitfly.org/seq_tools/promoter.html). Homology searches, alignments, and other basic analyses of the nucleotide sequence were completed using VECTOR NTI Advance 11.0 (Invitrogen, Beijing, China). A sequence-based homology model of S. hygroscopicus pro-TGase protein was constructed using the web-based SWISS-MODEL Workspace. The model was viewed, and figures were prepared using PYMOL (DeLano Scientific, San Carlos, CA).

Construction of plasmids encoding pro-TGase containing a signal peptide
To construct the plasmid encoding pro-TGase containing the pelB signal peptide, the pro-TGase gene was amplified from S. hygroscopicus genomic DNA using the primer pair PTG1 and PTG2 (Table 1). To construct the plasmid encoding pro-TGase with its endogenous signal peptide, the complete open reading frame (ORF) of the TGase gene was amplified from S. hygroscopicus genomic DNA using the primer pair ORFTG1 and ORFTG2 (Table 1). Each amplified PCR product was cloned into the NcoI-XhoI sites of pET-22b + to produce pBB1-1010 and pBB1-1020, respectively.

Construction of plasmids encoding pro-TGase with N-terminal deletions
Each gene fragment of pro-TGase containing an N-terminal deletion was amplified from pBB1-1020 by PCR using a specific forward primer and a constant reverse primer (TG2) ( Table 1). For the deletion of the first six N-terminal amino acids in the pro-region, TG7 (Table 1) was used as a forward primer. For further deletions in the pro-region, TG17, TG23, TG33, and TG58 were used as the forward primers (Table 1). The resulting PCR products were inserted into the NcoI-XhoI sites of pET-22b + to produce pBB1-1011, pBB1-1012, pBB1-1013, pBB1-1014, and pBB1-1015, respectively (Fig. 2a).

Protein expression in E. coli
Pro-TGase and its derivatives were expressed in E. coli BL21(DE3). A seed culture of each recombinant strain was prepared by growing cells in Luria-Bertani medium containing ampicillin (100 lg mL À1 ) at 37°C for 12 h. The seed culture was inoculated into Terrific Broth medium containing ampicillin (100 lg mL À1 ) and cultivated at 37°C until the optical density at 600 nm reached 1.0-1.5. Isopropyl-b-D-thiogalactopyranoside was added to a final concentration of 0.4 mM. After incubation for 40 h at 20-37°C, the cells and its culture supernatant were separated by centrifugation. Cells (1 OD 600 nm unit) were sonicated in 100 lL Tris-HCl buffer (pH 8) and centrifuged. The supernatant of the sonicated cells is the intracellular soluble fraction. The cell debris from the centrifugation step was resuspended in the Tris-HCl buffer containing 1% and corresponds to the intracellular insoluble fraction.

Pro-TGase activation in vitro
The pro-TGase activation by dispase (Worthington, Lakewood, NJ) was performed as previously described (Marx et al., 2008) with the following modification. Instead of activation in the specific buffer, the activation here was initiated by directly adding dispase solution (Marx et al., 2008) to the culture supernatant of each recombinant E. coli strain.

Protein analysis
Purification of pro-TGase and TGase from S. hygroscopicus and pro-TGase from the recombinant strains was performed as previously described (Zhang et al., 2008b). Tests of TGase activity, protein content, and SDS-PAGE were conducted as previously described (Zhang et al., 2008b). Amino acid sequencing of the TGase N-terminal was performed by Shanghai Gene Core Biotechnologies Co., Ltd.

Sequence analysis of the TGase gene from S. hygroscopicus
The TGase gene with flanking regions, here named tgh, was cloned using two PCR procedures (see Materials and methods). The TGase ORF encoding 418 amino acids (TGase precursor) was identified in the tgh sequence (Fig. 1a). The TGase precursor sequence was highly homologous to sequences from other Streptomyces species (Table 2). N-terminal sequencing (pro-TGase: ASGGDG; TGase: DAADE) revealed that the TGase precursor could be divided into three regions: a pre-region, a pro-region, and the mature TGase (Fig. 1a). The mature TGase shared high identity (over 79%) with TGases from other Streptomyces species (Table 2). Although the conservation of the pro-region was lower than that of the mature TGase (Table 2), several highly conserved amino acids were found in the pro-region of the TGases from different Streptomyces species (Fig. 1b).
The pre-region of the TGase ORF exhibited approximately 34-72% identity with TGases from other Streptomyces species (Table 2). SIGNALP 3.0 analysis indicated that the pre-region displayed strong characteristics of a signal peptide. Putative regulation elements neighboring the TGase ORF were identified (Fig. 1a). A putative promoter region was found upstream of the TGase ORF (Fig. 1a), and this region was well conserved in the upstream sequence of TGase genes from different Streptomyces species (Fig. 1c). The importance of this region was confirmed by the observation that an N-terminal deletion in this region of the Streptoverticillium ladakanum TGase gene resulted in reduced expression in S. lividans (Lin et al., 2004). Unexpectedly, a 468-bp ORF was found upstream of the putative promoter (Fig. 1a).
NCBI BLAST analysis showed that the amino acid sequence of this ORF was more than 80% homologous with that of the IS110 family of transposases from Streptomyces avermitilis MA-4680 and Streptomyces ghanaensis ATCC14672, suggesting that this ORF might encode a transposase.

Secretion of pro-TGase in E. coli
To secrete pro-TGase in E. coli, pBB1-1010 (containing the pelB signal peptide gene) and pBB1-1020 (containing the TGase signal peptide gene) (Fig. 2a) were used to express pro-TGase. When induced with isopropyl-b-Dthiogalactopyranoside at 20°C, SDS-PAGE analysis (Fig. 2b) and N-terminal amino acid sequencing determined that the two recombinant strains secreted distinct forms of pro-TGase. This is the first report of pro-TGase secretion by E. coli. Subsequently, the effect of induction temperature on pro-TGase secretion was examined. As shown in Fig. 2b, the band corresponding to secreted pro-TGase was significantly reduced when the cells were induced at 25°C, and no target protein band was detected at higher induction temperatures. In all cases, the ability of the TGase signal peptide to mediate pro-TGase secretion was lower than that of the pelB signal peptide (Fig. 2b), and neither signal peptide resulted in significant intracellular pro-TGase accumulation when induced at 20°C (Fig. 2c). These results suggest that both the TGase signal peptide and the pelB signal peptide can mediate the pro-TGase secretion in E. coli and are correctly processed.

Pro-TGase activation in vitro
Dispase activates S. mobaraensis pro-TGase when incubated in a Tris-HCl buffer at pH 8 (Marx et al., 2007). To study the activation efficiency of pro-TGase in culture supernatants, the dispase solution was added directly to the culture supernatant of E. coli expressing pBB1-1010 or pBB1-1020. SDS-PAGE analysis showed that the pro-TGase secreted by E. coli expressing pBB1-1010 was rapidly transformed (within 30 min) into a smaller protein with a molecular weight corresponding to that of the mature TGase (37.8 kDa), and TGase activity increased during the process (Fig. 2d,e). In addition, the intensity of the band corresponding to TGase and the TGase activity remained constant (approximately 4.5 U mL À1 ) in the later stages of activation (Fig. 2d,e). As expected, activation of the pro-TGase secreted by E. coli expressing pBB1-1020 showed a similar trend (data not shown). These results demonstrate that the secreted pro-TGase is directly activated by dispase and is not continuously degraded. The precursor is composed of a pre-region, a pro-region, and the mature TGase.

Functional analysis of the TGase pro-region
It has been reported that the N-terminal pro-region of thermophilic subtilase greatly influences the secretion of its zymogen in E. coli (Fang et al., 2010). To elucidate the role of the TGase pro-region during pro-TGase secretion, N-terminal deletion mutants within the TGase pro-region were constructed. Each deletion was designed to remove a conserved part of the pro-region of TGase as determined by the alignment of sequences from different Streptomyces strains (Fig. 1b). When the first six N-terminal amino acids of pro-TGase were removed, the secretion of the corresponding pro-TGase derivative decreased (Fig. 3b), and intracellular accumulation of the soluble pro-TGase derivative was observed (Fig. 3c). After removal of the first 16 N-terminal amino acids of the pro-region, neither extracellular ( Fig. 3b) nor intracellular soluble (Fig. 3c) pro-TGase derivatives were detected. However, an insoluble pro-TGase derivative was present (Fig. 3d). Further deletion of amino acids at the N-terminal of pro-TGase produced only insoluble pro-TGase derivatives (Fig. 3d).
These results show that the pro-region of TGase is essential for TGase secretion and solubility in E. coli.

Discussion
Without disruption of cells, the efficient secretion of TGase in E. coli would undoubtedly simplify the recovery of the enzyme and the screening of mutants for directed evolution. In this study, S. hygroscopicus pro-TGase was efficiently secreted in E. coli using the TGase signal peptide or the pelB signal peptide. After activation in the culture supernatant, the yield of secreted TGase was 4.5 U mL À1 , which is three times the amount of the TGase produced intracellularly (Marx et al., 2007). However, the S. mobaraensis pro-TGase that is fused to the pelB signal peptide failed to be secreted in E. coli (Marx et al., 2007;Yang et al., 2009). It has been reported that export of the glycolytic enzyme in E. coli was enhanced by modifying its N-terminal residues (Tian & Bernstein, 2009). Additionally, the pelB-mediated secretion of the precursor of a thermophilic subtilase in E. coli increased threefold after a mutation of its pro-region (Fang et al., 2010). These findings suggest that the N-terminal proregion greatly influences protein secretion mediated by signal peptides in E. coli. Notably, the amino acid sequence homology between the pro-regions of TGases from S. mobaraensis and S. hygroscopicus is low (45.6%), whereas their mature regions shared a 79.2% homology. The pro-TGase from S. hygroscopicus may have a secretion-competent pro-region that is different from that of the pro-TGase from S. mobaraensis. The N-terminal deletions performed in this study preliminarily identified the residues in the pro-region that affect pro-TGase solubility and secretion. It was shown that the first six amino acids have an impact on pro-TGase secretion, and the next 10 residues are responsible for soluble expression. In general, a protein goes through a series of three steps before its secretion in E. coli: translocation across the cytoplasmic membrane, signal peptide cleavage in the periplasm, and translocation across the outer membrane (Mergulhao et al., 2005). Following the removal of the first six amino acids of the pro-region, TGase activity was detected in the periplasm but not in the cytoplasm after dispase treatment (data not shown), suggesting that the intracellular pro-TGase derivative (Fig. 3c) produced by the deletion was exported into the periplasm. Accordingly, the first six amino acids of the pro-region may affect pro-TGase secretion by improving its translocation across the outer membrane of E. coli. The next 10 residues (amino acids 7-16) in the proregion contain five conserved residues (serine11, tyro-sine12, alanine13, glutamic acid14, and threonine15) (Fig. 1b), and deletion of the 10 residues resulted in an insoluble pro-TGase derivative (Fig. 3d). Structural modeling of the pro-TGase showed that the five conserved residues constitute the first a-helix of the pro-region and that tyrosine12 interacts with asparagine362 and aspara-gine334 in the mature region through a hydrogen bond (Fig. 4). Similar interactions between the pro-region and the mature region were also identified in the recently published crystal structure of pro-TGase from S. mobaraensis (Yang et al., 2011). During the maturation of the alpha-lytic protease precursor, the N-terminal pro-region folds into a stable structure, which acts as a scaffold for packing of the mature region into a native structure (Chen & Inouye, 2008). Therefore, it is possible that the a-helix of the pro-region assists TGase folding through a hydrogen bond interaction, and the absence of this assistance leads to the production of an insoluble pro-TGase derivative.

Asn334
Asp362 Fig. 4. The interactions between the pro-region and mature region of pro-TGase from Streptomyces hygroscopicus. The a-helix is marked by yellow within the TGase pro-region. The hydrogen bonds are indicated by the dashed line.