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Biotechnological routes for transglutaminase production: Recent achievements, perspectives and limits
Accepted Manuscript Biotechnological Routes for Transglutaminase Production: Recent Achievements, Perspectives and Limits Limin Wang, Bo Yu, Ruixuan Wang, Jianchun Xie PII:
S0924-2244(17)30755-0
DOI:
10.1016/j.tifs.2018.09.015
Reference:
TIFS 2322
To appear in:
Trends in Food Science & Technology
Received Date: 23 November 2017 Revised Date:
17 July 2018
Accepted Date: 11 September 2018
Please cite this article as: Wang, L., Yu, B., Wang, R., Xie, J., Biotechnological Routes for Transglutaminase Production: Recent Achievements, Perspectives and Limits, Trends in Food Science & Technology (2018), doi: https://doi.org/10.1016/j.tifs.2018.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Biotechnological Routes for Transglutaminase Production:
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Recent Achievements, Perspectives and Limits
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Limin Wang1, 2*, Bo Yu2, Ruixuan Wang3, Jianchun Xie1
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Technology & Business University (BTBU), Beijing 100048, PR China
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Beijing Advanced Innovation Center for Food Nutrition and Human Health，Beijing
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering,
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Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
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School of Business, George Washington University, Washington DC 20052, USA
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* Corresponding authors.
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E-mails: [email protected] (L. Wang)
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Phone/Fax: +86-10-64806132
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Abstract
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Background
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Transglutaminase (TGase) belongs to the transferases family that catalyses the
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formation of isopeptide bonds between proteins. Its cross-linking properties are
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extensively used in food industry. Nowadays, TGase isolation from Streptomyces sp.
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is used in almost all industrial branches. The complexity of current procedure prompt
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scientists to develop an efficient and easy-to-use system for the “green” production of
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TGase.
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Scope and approach
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This article reviews the application of biotechnological routes in transglutaminase
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(TGase) production. The nature sources and industrial production of TGase were
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discussed in this review. Furthermore, the potential of biotechnological routes for
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TGase production was highlighted. Metabolic engineering of microorganisms is
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exemplified for biotechnological synthesis of TGase. The drawbacks as well as
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improvements of the production of TGase via biotechnological routes were also
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discussed.
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Key findings and conclusions
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Biotechnological routes provide the possible measures to heterologous expression of
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TGase. After decades of efforts, the expression of recombinant TGase has significant
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improvements. Further research and development towards cost-effective production
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may resulted in a more affordable product that could be exploited for a broad range of
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applications.
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Keywords
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Transglutaminase; food industry; metabolic engineering; biotechnological routes
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1. Introduction Protein-glutamine: amine γ-glutamyltransferase, known as transglutaminase
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(TGase, EC 2.3.2.13), is a member of transferases family. It catalyzes the acyl transfer
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of glutamine residues in γ-carboxamides and primary amines. TGase also catalyzes
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the deamidation of glutamine residues when amine substrates is absent. During this
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reaction, water molecules are used as acyl acceptors (Kieliszek & Misiewicz, 2014;
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Santhi, Kalaikannan, Malairaj, & Arun Prabhu, 2017). TGase has a wide application
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in food industries due to its remarkable role to form cross-links within protein
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molecule, which makes the changes in protein functionalities, solubility, emulsifying
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capacity, foaming properties and gelation (Figure 1) (Martins et al., 2014). For
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example, TGase is used in various meat products because it may enhance the texture,
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rigidity and gel strength of meat products, and improve the functional and textual
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properties (Santhi et al., 2017). It also could form stable and covalent links between
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proteins, so it has a profound role in all types of food proteins, such as fish, tofu, jelly
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and flours (Martins et al., 2014). Furthermore, TGase has the ability to improve
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textural properties of protein gel-based dairy products (Zhang et al., 2012b). This
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enzyme is recognized as GRAS (Generally Recognized as Safe), which is a safe
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substance for human ingestion (Gaspar & de Góes-Favoni, 2015). Notably, the
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application of TGase is not limited to food processing. It has wide applications
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outside the food areas, such as biomedical engineering, material science, textiles and
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leather processing (Zhu & Tramper, 2008). TGase is used in tissue engineering due to
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the ability of the construction of proteinaceous scaffold (Long, Ma, Xiao, Ren, &
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Yang,), and it is also used to alleviate the negative effects of proteolytic wool
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treatment in wool processing (Cortez, Bonner, & Griffin, 2004). Nature TGase was firstly found in animal tissues, and extraction from animal
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tissues was the commercial way until the end of the eighteenth century. However, this
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process is intrinsically complicated and rare source resulted in an extremely high price
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(Zhu & Tramper, 2008). Fermentation is a mild method and has been practiced
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worldwide for a long time. The first microbial TGase was found in the fermentation
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broth of Streptoverticillium sp. in 1989 and TGase of microbial origin became the
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commercial way since then (Kieliszek & Misiewicz, 2014). However, the expensive
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ingredients required for Streptoverticillium sp. growth prompted researchers to found
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more cost-effective production. The attempts at TGase production by genetically
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modified strains are an example of such efforts (Javitt, Ben-Barak-Zelas,
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Jerabek-Willemsen, & Fishman, 2017). In this review, we will focus on
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biotechnological routes based on TGase production. The drawbacks as well as
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improvements of the production of TGase via biotechnological routes were also
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discussed.
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2. Natural sources of TGase To date, many comprehensive reviews have discussed the nature source of TGase
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(Cortez, Bonner, & Griffin, 2004; Del Duca, Verderio, Serafini-Fracassini, Iorio, &
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Cai, 2014; Gaspar & de Góes-Favoni, 2015; Kieliszek & Misiewicz, 2014). As
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discussed above, nature TGase presents in all life domains including plants, mammals, 5
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antibacterial immune reactions and photosynthesis (Kieliszek & Misiewicz, 2014).
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Animal TGase was discovered in guinea pig’s liver in 1973. Although animal TGase
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has the problem of rare source and high prices, it plays an essential role in the market
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of TGase. Extraction from animal tissues was the only form arriving at the market by
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the end of the eighteenth century (Martins et al. 2014). In general, animal TGase was
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extracted commercially from the blood of cattle and swine. However, this source of
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TGase is rarely used in food industry. One reason is that animal TGase is detrimental
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to the appearance of the product (Yokoyama, Nio, & Kikuchi, 2004). The other is that
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animal TGase is calcium (Ca2+)-dependent. Because most food proteins, such as
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caseins, soybean globulins, are easily precipitated by Ca2+, animal TGase could not be
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used in most foods (Cui, Du, Zhang, Liu, & Chen, 2007). Plant TGase presents in the
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cell wall. It is involved in the post-translational modification of proteins, stabilization
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of the matrix and modulation of the integrin-fibronectin receptor interaction (Del
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Duca, Verderio, Serafini-Fracassini, Iorio, & Cai, 2014). Compared with animal
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TGase, plant TGase was sensitive to light (Kieliszek & Misiewicz, 2014).
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Interestingly, eight active forms of TGase are identified in one plant and those forms
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of TGase show multiple functions (Aloisi, Cai, Serafini-Fracassini, & Del Duca,
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2016). The enzymatic properties and functions of plant TGase have been less studied.
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Because plant TGase expression is regulated by light and other factors related to
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photosynthesis and photoprotection, it is difficult to obtain the purified enzyme.
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However,
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it
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plant
TGase
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(Serafini-Fracassini & Del Duca, 2008; Martins et al. 2014). Microbial TGase could be found in the cell wall of fungi. For example, in Candida
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and Saccharomyces, TGase forms cross-link connection of structural glycoproteins. In
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Phytophthora, TGase involves in pathogenicity. While in the alga Chlamydomonas,
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TGase links polyamines to glycoproteins (Del et al., 2014; Ruiz-Herrera, Iranzo,
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Elorza, Sentandreu, & Mormeneo, 1995). The first purified TGase from
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microorganisms was identified in Streptomyces mobaraensis (Hiroyasu et al., 1989).
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Since then, many microbial strains that produce TGase were discovered (Liu et al.,
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2011). As shown in Table 1, several strains were proven to have the ability to produce
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TGase. Most of them are Streptomyces sp. strains and TGases derived from wild-type
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Streptomyces sp. have been commercialized. At present, S. mobaraense is the
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commonly used strain (H-Kittikun, Bourneow, & Benjakul, 2012). In contrast to
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animal TGase, microbial TGases have some advantages, such as they have low
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molecular weight (~40 KDa) and are stable at a wide range of pH values (from 4.5 to
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8.0) (Lin, Chao, Liu, & Chu, 2004). The isoelectric point is 9 and the optimal pH of
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reaction is 6~7. The optimal temperature of reaction is 55°C and maintains full
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activity for 10 min at 40°C (Hiroyasu et al., 1989). Most importantly, this enzyme is
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Ca2+-independent, which is clearly different from animal TGase and plant TGase. This
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property is very useful in modifying food proteins (Hiroyasu et al., 1989; Yokoyama,
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Nio, & Kikuchi, 2004).
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Furthermore, microbial TGases do not need complex extraction processes, which
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provided energy and economical savings. At same time, microbial TGases also have 7
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the culture medium for Streptomyces sp., which are not suitable for large-scale
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production (Kieliszek & Misiewicz, 2014). Whereas, it is difficult to produce the
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improved variant TGase with tailored properties using microbial fermentation (Javitt
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et al., 2017). Thanks to the establishment of trans-genesis procedures, gene transfer
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became possible. TGase production using genetic modified strains have shed light on
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its applications. Several expression systems, including Corynebacterium sp.,
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Escherichia coli, etc, have been set up (Table 1). Compared with the conventional
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fermentation with wild strains, TGase production by genetic modified strain is more
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efficient for the simple nutrition required and high productivity (Yokoyama, Nio, &
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Kikuchi, 2004). Strategies for recombinant expression of foreign proteins have been
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used for a long history and biotechnological routes is considered a “green” and safe
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method in contrast to the chemical one (Gao, Ma, & Xu, 2011).
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3. Biotechnological production of microbial TGase
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3.1. Recombinant TGase expression using E. coli as a host
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E. coli is a widespread used strain since it can be grown and cultured easily and
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inexpensively. It has been intensively investigated and easy molecular biological
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manipulations have been set up (Liu et al., 2011a). The structure of Streptomyces
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TGase is well recognized, and most studies focus on the expression of Streptomyces
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TGase in E. coli. Streptomyces TGase consists of two parts: the pro-peptide domain
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and the active domain (Fig. 2A). Microbial TGase is secreted as inactive pro-TGase 8
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and becomes active after deletion of pro-peptide by proteases (Liu, Wang, Du, &
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Chen, 2016). Since pro-peptide is essential for the correct folding and E. coli does not
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contain
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overexpression in E. coli is how to obtain the active enzyme. This was demonstrated
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by Takehana et al. (1994), who expressed S. mobaraensis TGase in E. coli, but this
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strategy resulted in a low-level protein expression. The authors concluded that TGase
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is synthesized as an inactive zymogen (pro-TGase) in wild-type strains (Takehana et
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N-terminal domain is the key region for the secretion of TGase (Yang, Pan, & Lin,
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2009). This result was confirmed by Liu et al. (2011a), who added pelB signal peptide
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to the N-terminal of pro-peptide from S. hygroscopicus. This strategy resulted in the
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secretion of pro-TGase E. coli (Fig. 2B). After addition of protease, pro-TGase
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directly transformed into an active form, with an enzymatic activity of 4.5 U/mL (Liu
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et al., 2011a). Later, same research group reported a novel co-expression method for
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the direct production of active TGase in E. coli (Fig. 2B). The order of TGase
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encoding genes were changed. The fusion of pelB signal peptide and pro-peptide was
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prior expressed, then followed by the fusion expression of pelB signal peptide and
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TGase. Soluble TGase was expressed in E. coli without protease addition (Liu et al.,
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2011b). More recently, this approach was validated by Javitt et al. (2017), who
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constructed a constitutive expression system of TGase in E. coli (Fig. 2C). The active
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form of TGase was obtained without addition of protease. The enzymatic activities of
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wild-type and heterologous TGase had the same characterization (Javitt et al., 2017).
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TGase, many studies have tried to find the key site for the active expression (Chen et
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al., 2013; Liu et al., 2011a). Site-directed mutagenesis provides good insights into the
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roles of amino acid residues. The residues of Tyr12, Asn27, Asn30 and Arg32 are
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associated with the interaction with TGase region via hydrogen bonds. Mutations of
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above 4 residues resulted in the complete inhibition of TGase secretion (Chen et al.,
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2013). Rickert et al. (2015) carried out experiments combining the alanine-scan of
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pro-domain with site-directed mutations to identify the key sites for soluble S.
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mobarensis TGase expression in E. coli. Results showed that the pro-domain residues
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of Tyr14, Asp20, Ile24, and Asn25 were the most important residues for achieving
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active and soluble TGase. Those results may guide the engineering of pro-peptide
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domain to produce TGase in E. coli with excellent solubility and fully active form.
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Strong correlations were found between N-terminal domain and active expression
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of TGase, and recombinant expression of TGase in E. coli has resulted in some
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success by changing the gene order of N-terminal region. Same strategies could be
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adopted in other hosts. However, there are some problems for the heterologous
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expression of TGase, such as the low yield compared with the wild-type strain.
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Integrated on-column activation and His6 purification online have been used to
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improve the yield of TGase. This procedure contributed to 89% active TGase of the
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total pro-TGase. Under high-density cultivation, soluble pro-TGase was expressed up
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to about 8,000 U/l, which has potential to be used in TGase production on a
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commercial scale (Yang, Pan, & Lin, 2009). Furthermore, different fermentation
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modes were carried out to increase the yield, such as the fed-batch fermentation.
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Under fed-batch condition, the specific activity of S. mobaraensis TGase in E. coli
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reached 1386 U/g dry mass (Sommer, Volk, & Pietzsch, 2011). Studies on heterologous expression of TGase using E. coli as a host have provided
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some invaluable results. However, E. coli is not approved to be used in food
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processing for the consideration of GRAS status. Whereas, for other applications,
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such as the areas of biomedical engineering, material science and leather processing,
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E. coli production system is an economically feasible alternative (Martins et al., 2014;
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Sommer, Volk, & Pietzsch, 2011).
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3.2. Recombinant TGase expression using S. lividans as a host Although the expression system of S. lividans is not as powerful as that of E. coli, S.
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lividans has made a great contribution. For example, S. lividans was used as a host for
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several kinds of sugars, biomass-degrading enzymes and industrially useful
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compounds (Noda, Miyazaki, Tanaka, Ogino, & Kondo, 2012). Notably, S. lividans is
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capable to convert the inactive pro-TGase into active form with its endogenous
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protease (Liu, Wang, Du, & Chen, 2016). So, it has some advantages compared with
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other hosts.
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One technical challenge for recombinant TGase expression in S. lividans is to
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obtain an active form. S. ladakanum TGase was expressed in S. lividans.
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Consequently, incorrectly secreted TGase was obtained in S. lividans (Lin et al.,
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2004). Noda et al. (2012) described a method for S. cinnamoneus expression in S. 11
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of S. cinnamoneus, combing with the pro-domain of S. cinnamoneus TGase and the
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sequence encoding mature TGase was fused together. This strategy provided 360
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mg/L TGase (Noda et al., 2012). Taguchi et al. (2002) introduced an approach to
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co-expressing the encoding genes of S. albogriseolus proteases and the encoding
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genes of S. cinnamoneus TGase. As a consequent, less than 1 U/mL TGase was
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produced (Taguchi, Arakawa, Yokoyama, Takehana, Takagi, & Momose, 2002).
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Another challenge for recombinant TGases expression in S. lividans is to increase
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the yield. Several microbial TGases, such as S. mobaraense, S. ladakanum, S.
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platensis, have been expressed in S. lividans, but the yields were no higher than 2.22
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U/mL (Liu, Wang, Du, & Chen, 2016). Liu et al. (2016) employed promoter
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engineering and codon optimization to produce active S. hygroscopicus TGase in S.
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lividans. The encoding genes of native promoter was partially deleted to analyze the
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differences of TGase expression. Furthermore, the gene sequence of TGase ORF
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(open reading frame) was optimized to improve the expression. As a result, the yield
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of TGase increased from 1.8 U/mL to 5.73 U/mL (Liu, Wang, Du, & Chen, 2016).
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Till now, TGase produced by wild-type Streptomyces sp. is the main commercial way.
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After decades of development, the activity of TGase reached 6.0 U/mL (Zhang, Zhu,
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& Chen, 2010). The recombinant TGase produced by S. lividans has reached 5.73
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U/mL, which was comparable with the wild strain. However, such yield is still low.
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Accurate control of this process is recommended to improve recombinant TGase
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production at large scale.
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3.3. Recombinant TGase expression using C. glutamicum as a host C. glutamicum is a gram-positive, nonsporulating bacterium. It is an industrial
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workhorse with a long history. The most widely known for C. glutamicum is the role
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in the production of a variety of amino acids and other useful materials, such as
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L-glutamic acid, L-serine, poly (3-hydroxybutyrate) (Freudl, 2017; Kikuchi, Date,
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Yokoyama, Umezawa, & Matsui, 2003). Most importantly, C. glutamicum is safety to
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human and possesses an enormous potential for the secretory production of
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heterologous proteins, which make it have wide applications in food industry.
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Kikuchi et al. (2003) reported a method for the secretion of S. mobaraense
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pro-TGase in C. glutamicum. The signal peptide of a cell surface protein derived from
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C. glutamicum was used and inactive pro-TGase was converted to active form after
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addition of protease. As a consequent, the amount of TGase accumulated to 142 mg/L
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(Kikuchi et al., 2003). Because Phe-Arg-Ala-Pro residues were added to the
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N-terminal amino acid sequence, the sequence of secreted enzyme differed from that
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of the native one. Later, a preferred SAM-P45 cleavage site was introduced into the
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C-terminus of the pro-region, and the native-type TGase was successfully secreted by
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C. glutamicum, with an activity of 132 mg/L (Date, Yokoyama, Umezawa, Matsui, &
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Kikuchi, 2003). Date et al. (2004) utilized the chimeric pro-region consisting of S.
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mobaraensis and TGase from S. cinnamoneus to express TGase in C. glutamicum. As
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a result, the amount of TGase secreted by the strain carrying the chimeric pro-region
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is higher than that using the native one (Date, Yokoyama, Umezawa, Matsui, &
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Kikuchi, 2004). This study demonstrated that, similar with other hosts mentioned in
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this review, the pro-region of TGase is important for active expression. Like other members of Corynebacterianeae sp., C. glutamicum possesses an outer
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membrane-like structure composed of mycolic acids and other complex glycolipids.
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The structure has profound impacts on the efficient permeability for hydrophilic
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molecules (Richter, Hänel, & Hilliger, 1985). To date, it comes to conclude that C.
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glutamicum contains two major protein export pathways. One is the general secretion,
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which transports extracytosolic bacterial proteins. The other is the alternative twin
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arginine translocation pathway, which transports fully folded proteins (Freudl, 2017).
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Because protein transportation across outer membrane is a bottleneck in the
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heterologous expression of TGase, C. glutamicum is more suitable for the expression
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host for TGase than other hosts are. Till now, the yield of TGase expressed in C.
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glutamicum is low. Some strategies, such as signal peptide variation, promoter
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optimization, could be developed to improve the yield of secreted TGase.
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4. Concluding remarks and outlook
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TGase has wide uses in food industry. Commercial TGase (trade mark Activa™) is
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produced by fermentation of S. mobaraensis as extracellular protein. However, only
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1% protein exists in Activa™ and 99% Maltodextrin is added to stabilize TGase
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(Sommer et al., 2011). One drawback of current commercial process is the presence
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of proteases, which can hydrolyze the target proteins that are intended to be
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cross-linked. The other drawback is the complex fermentation procedure. Thus, it is 14
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necessary to develop an efficient and easy-to-use system for the “green” production of
285
TGase. Biotechnological routes provide the possible measures to heterologous expression
287
of TGase. After decades of efforts, the expression of recombinant TGase has
288
significant improvements: from the formation of inclusion bodies, low recovery yields,
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and non-active enzyme to the soluble forms. However, many problems need to be
290
solved for its realization in industrial level. For example, since there are no
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appropriate TGase-activating system in E. coli or C. glutamicum, recombinant
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pro-TGase is difficult for activation in these hosts. Although active TGase could be
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obtained in Streptomyces system, the yields of TGase are only close to those of wild
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strain.
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So, efforts on bioprocess engineering and genetic engineering for the improvement
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of TGase are desired in the near future. With the development of industrial level
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resolution, the biotechnological routes for TGase production would be an
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economically feasible alternative and will accelerate the application of TGase.
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Conflict of Interest
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All the authors declare no competing interests.
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Author Agreement/Declaration
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All the authors have seen and approved the final version of the manuscript being
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submitted. They warrant that the article is the authors' original work, hasn't received 15
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prior publication and isn't under consideration for publication elsewhere.
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Acknowledgements
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This work was supported by grants from the Beijing Advanced Innovation Center for
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Food Nutrition and Human Health, Beijing Technology & Business University
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(BTBU) (20171013), and the National Natural Science Foundation of China
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(31670045).
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mobaraensis DSM 40587 in high salt and effect of enzymatic cross-linking of yak
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milk proteins on functional properties of stirred yogurt. Journal of Dairy Science,
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Procedure
Yield
Reference
Euphausia superba
Purified from whole cell
54 (U/mg)
Streptomyces lividans
Fermentation
Streptomyces lividans Providencia sp. C1112
Genetic modification Fermentation
1.23–2.22 (U/mL) 5.73 (U/mL) 1.8 (U/mL)
Zhang, He, & Simpson, 2017 Lin et al., 2004
Streptoverticillium mobaraense Streptomyces mobaraensis Streptomyces lividans Streptomyces platensis
Fermentation
1.6 (U/mL)
Fermentation
4.3 (U/mL)
Genetic modification Fermentation
360 (mg/L) 2.3 (U/mL)
Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Yarrowia lipolytica
Genetic modification
132 (mg/L)
Noda et al., 2012 Yeo, Yoon, Lee, & Kim, 2009 Date et al., 2003
Genetic modification
142 (mg/L)
Kikuchi et al., 2003
Genetic modification
863-881 (mg/L) 7.8 (U/mL)
Date et al., 2004
Escherichia coli Escherichia coli
Genetic modification Genetic modification
Escherichia coli Escherichia coli Pichia pastoris
Genetic modification Genetic modification Genetic modification
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Genetic modification
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4.5 (U/mL) 18.2-22 (U/mg) 1 (U/mL) 30-75 (mg/L) 0.9 U/mg
Liu, Wang, Wang, Madzak, Du, & Chen, 2015 Liu et al., 2011a Liu et al., 2011b Javitt et al., 2017 Rickert et al., 2015 Li et al., 2014
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Figure Legends:
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Figure 1
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Transglutaminase applications in food industry.
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Figure 2
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Genetic organization of Streptomyces TGase.
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A, Genetic organization of TGase in Streptomyces sp.. B, Genetic organization of
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active TGase from Streptomyces in E. coli (by reference Liu et al., 2011b). C, Genetic
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organization of active TGase from Streptomyces in E. coli (by reference Javitt et al.,
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2017).
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Highlights TGase’s cross-linking properties are extensively used in food industry and outside
Natural sources and commercial production of TGase.
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the food area.
Biotechnological routes for TGase production using E. coli, S. lividans, and C.
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glutamicum as hosts.
The drawbacks as well as improvements of the production of TGase via
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biotechnological routes.
S0924-2244(17)30755-0
DOI:
10.1016/j.tifs.2018.09.015
Reference:
TIFS 2322
To appear in:
Trends in Food Science & Technology
Received Date: 23 November 2017 Revised Date:
17 July 2018
Accepted Date: 11 September 2018
Please cite this article as: Wang, L., Yu, B., Wang, R., Xie, J., Biotechnological Routes for Transglutaminase Production: Recent Achievements, Perspectives and Limits, Trends in Food Science & Technology (2018), doi: https://doi.org/10.1016/j.tifs.2018.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Biotechnological Routes for Transglutaminase Production:
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Recent Achievements, Perspectives and Limits
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Limin Wang1, 2*, Bo Yu2, Ruixuan Wang3, Jianchun Xie1
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1
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Technology & Business University (BTBU), Beijing 100048, PR China
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Beijing Advanced Innovation Center for Food Nutrition and Human Health，Beijing
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering,
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Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
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School of Business, George Washington University, Washington DC 20052, USA
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* Corresponding authors.
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E-mails: [email protected] (L. Wang)
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Phone/Fax: +86-10-64806132
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Abstract
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Background
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Transglutaminase (TGase) belongs to the transferases family that catalyses the
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formation of isopeptide bonds between proteins. Its cross-linking properties are
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extensively used in food industry. Nowadays, TGase isolation from Streptomyces sp.
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is used in almost all industrial branches. The complexity of current procedure prompt
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scientists to develop an efficient and easy-to-use system for the “green” production of
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TGase.
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Scope and approach
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This article reviews the application of biotechnological routes in transglutaminase
27
(TGase) production. The nature sources and industrial production of TGase were
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discussed in this review. Furthermore, the potential of biotechnological routes for
29
TGase production was highlighted. Metabolic engineering of microorganisms is
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exemplified for biotechnological synthesis of TGase. The drawbacks as well as
31
improvements of the production of TGase via biotechnological routes were also
32
discussed.
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Key findings and conclusions
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Biotechnological routes provide the possible measures to heterologous expression of
35
TGase. After decades of efforts, the expression of recombinant TGase has significant
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improvements. Further research and development towards cost-effective production
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may resulted in a more affordable product that could be exploited for a broad range of
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applications.
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Keywords
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Transglutaminase; food industry; metabolic engineering; biotechnological routes
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1. Introduction Protein-glutamine: amine γ-glutamyltransferase, known as transglutaminase
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(TGase, EC 2.3.2.13), is a member of transferases family. It catalyzes the acyl transfer
45
of glutamine residues in γ-carboxamides and primary amines. TGase also catalyzes
46
the deamidation of glutamine residues when amine substrates is absent. During this
47
reaction, water molecules are used as acyl acceptors (Kieliszek & Misiewicz, 2014;
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Santhi, Kalaikannan, Malairaj, & Arun Prabhu, 2017). TGase has a wide application
49
in food industries due to its remarkable role to form cross-links within protein
50
molecule, which makes the changes in protein functionalities, solubility, emulsifying
51
capacity, foaming properties and gelation (Figure 1) (Martins et al., 2014). For
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example, TGase is used in various meat products because it may enhance the texture,
53
rigidity and gel strength of meat products, and improve the functional and textual
54
properties (Santhi et al., 2017). It also could form stable and covalent links between
55
proteins, so it has a profound role in all types of food proteins, such as fish, tofu, jelly
56
and flours (Martins et al., 2014). Furthermore, TGase has the ability to improve
57
textural properties of protein gel-based dairy products (Zhang et al., 2012b). This
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enzyme is recognized as GRAS (Generally Recognized as Safe), which is a safe
59
substance for human ingestion (Gaspar & de Góes-Favoni, 2015). Notably, the
60
application of TGase is not limited to food processing. It has wide applications
61
outside the food areas, such as biomedical engineering, material science, textiles and
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leather processing (Zhu & Tramper, 2008). TGase is used in tissue engineering due to
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the ability of the construction of proteinaceous scaffold (Long, Ma, Xiao, Ren, &
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Yang,), and it is also used to alleviate the negative effects of proteolytic wool
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treatment in wool processing (Cortez, Bonner, & Griffin, 2004). Nature TGase was firstly found in animal tissues, and extraction from animal
67
tissues was the commercial way until the end of the eighteenth century. However, this
68
process is intrinsically complicated and rare source resulted in an extremely high price
69
(Zhu & Tramper, 2008). Fermentation is a mild method and has been practiced
70
worldwide for a long time. The first microbial TGase was found in the fermentation
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broth of Streptoverticillium sp. in 1989 and TGase of microbial origin became the
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commercial way since then (Kieliszek & Misiewicz, 2014). However, the expensive
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ingredients required for Streptoverticillium sp. growth prompted researchers to found
74
more cost-effective production. The attempts at TGase production by genetically
75
modified strains are an example of such efforts (Javitt, Ben-Barak-Zelas,
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Jerabek-Willemsen, & Fishman, 2017). In this review, we will focus on
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biotechnological routes based on TGase production. The drawbacks as well as
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improvements of the production of TGase via biotechnological routes were also
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discussed.
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2. Natural sources of TGase To date, many comprehensive reviews have discussed the nature source of TGase
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(Cortez, Bonner, & Griffin, 2004; Del Duca, Verderio, Serafini-Fracassini, Iorio, &
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Cai, 2014; Gaspar & de Góes-Favoni, 2015; Kieliszek & Misiewicz, 2014). As
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discussed above, nature TGase presents in all life domains including plants, mammals, 5
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antibacterial immune reactions and photosynthesis (Kieliszek & Misiewicz, 2014).
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Animal TGase was discovered in guinea pig’s liver in 1973. Although animal TGase
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has the problem of rare source and high prices, it plays an essential role in the market
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of TGase. Extraction from animal tissues was the only form arriving at the market by
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the end of the eighteenth century (Martins et al. 2014). In general, animal TGase was
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extracted commercially from the blood of cattle and swine. However, this source of
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TGase is rarely used in food industry. One reason is that animal TGase is detrimental
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to the appearance of the product (Yokoyama, Nio, & Kikuchi, 2004). The other is that
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animal TGase is calcium (Ca2+)-dependent. Because most food proteins, such as
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caseins, soybean globulins, are easily precipitated by Ca2+, animal TGase could not be
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used in most foods (Cui, Du, Zhang, Liu, & Chen, 2007). Plant TGase presents in the
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cell wall. It is involved in the post-translational modification of proteins, stabilization
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of the matrix and modulation of the integrin-fibronectin receptor interaction (Del
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Duca, Verderio, Serafini-Fracassini, Iorio, & Cai, 2014). Compared with animal
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TGase, plant TGase was sensitive to light (Kieliszek & Misiewicz, 2014).
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Interestingly, eight active forms of TGase are identified in one plant and those forms
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of TGase show multiple functions (Aloisi, Cai, Serafini-Fracassini, & Del Duca,
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2016). The enzymatic properties and functions of plant TGase have been less studied.
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Because plant TGase expression is regulated by light and other factors related to
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photosynthesis and photoprotection, it is difficult to obtain the purified enzyme.
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However,
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it
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plant
TGase
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Ca2+-dependent
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(Serafini-Fracassini & Del Duca, 2008; Martins et al. 2014). Microbial TGase could be found in the cell wall of fungi. For example, in Candida
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and Saccharomyces, TGase forms cross-link connection of structural glycoproteins. In
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Phytophthora, TGase involves in pathogenicity. While in the alga Chlamydomonas,
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TGase links polyamines to glycoproteins (Del et al., 2014; Ruiz-Herrera, Iranzo,
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Elorza, Sentandreu, & Mormeneo, 1995). The first purified TGase from
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microorganisms was identified in Streptomyces mobaraensis (Hiroyasu et al., 1989).
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Since then, many microbial strains that produce TGase were discovered (Liu et al.,
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2011). As shown in Table 1, several strains were proven to have the ability to produce
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TGase. Most of them are Streptomyces sp. strains and TGases derived from wild-type
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Streptomyces sp. have been commercialized. At present, S. mobaraense is the
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commonly used strain (H-Kittikun, Bourneow, & Benjakul, 2012). In contrast to
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animal TGase, microbial TGases have some advantages, such as they have low
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molecular weight (~40 KDa) and are stable at a wide range of pH values (from 4.5 to
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8.0) (Lin, Chao, Liu, & Chu, 2004). The isoelectric point is 9 and the optimal pH of
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reaction is 6~7. The optimal temperature of reaction is 55°C and maintains full
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activity for 10 min at 40°C (Hiroyasu et al., 1989). Most importantly, this enzyme is
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Ca2+-independent, which is clearly different from animal TGase and plant TGase. This
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property is very useful in modifying food proteins (Hiroyasu et al., 1989; Yokoyama,
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Nio, & Kikuchi, 2004).
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Furthermore, microbial TGases do not need complex extraction processes, which
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provided energy and economical savings. At same time, microbial TGases also have 7
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the culture medium for Streptomyces sp., which are not suitable for large-scale
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production (Kieliszek & Misiewicz, 2014). Whereas, it is difficult to produce the
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improved variant TGase with tailored properties using microbial fermentation (Javitt
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et al., 2017). Thanks to the establishment of trans-genesis procedures, gene transfer
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became possible. TGase production using genetic modified strains have shed light on
136
its applications. Several expression systems, including Corynebacterium sp.,
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Escherichia coli, etc, have been set up (Table 1). Compared with the conventional
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fermentation with wild strains, TGase production by genetic modified strain is more
139
efficient for the simple nutrition required and high productivity (Yokoyama, Nio, &
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Kikuchi, 2004). Strategies for recombinant expression of foreign proteins have been
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used for a long history and biotechnological routes is considered a “green” and safe
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method in contrast to the chemical one (Gao, Ma, & Xu, 2011).
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3. Biotechnological production of microbial TGase
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3.1. Recombinant TGase expression using E. coli as a host
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E. coli is a widespread used strain since it can be grown and cultured easily and
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inexpensively. It has been intensively investigated and easy molecular biological
148
manipulations have been set up (Liu et al., 2011a). The structure of Streptomyces
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TGase is well recognized, and most studies focus on the expression of Streptomyces
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TGase in E. coli. Streptomyces TGase consists of two parts: the pro-peptide domain
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and the active domain (Fig. 2A). Microbial TGase is secreted as inactive pro-TGase 8
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and becomes active after deletion of pro-peptide by proteases (Liu, Wang, Du, &
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Chen, 2016). Since pro-peptide is essential for the correct folding and E. coli does not
154
contain
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overexpression in E. coli is how to obtain the active enzyme. This was demonstrated
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by Takehana et al. (1994), who expressed S. mobaraensis TGase in E. coli, but this
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strategy resulted in a low-level protein expression. The authors concluded that TGase
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is synthesized as an inactive zymogen (pro-TGase) in wild-type strains (Takehana et
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N-terminal domain is the key region for the secretion of TGase (Yang, Pan, & Lin,
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2009). This result was confirmed by Liu et al. (2011a), who added pelB signal peptide
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to the N-terminal of pro-peptide from S. hygroscopicus. This strategy resulted in the
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secretion of pro-TGase E. coli (Fig. 2B). After addition of protease, pro-TGase
164
directly transformed into an active form, with an enzymatic activity of 4.5 U/mL (Liu
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et al., 2011a). Later, same research group reported a novel co-expression method for
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the direct production of active TGase in E. coli (Fig. 2B). The order of TGase
167
encoding genes were changed. The fusion of pelB signal peptide and pro-peptide was
168
prior expressed, then followed by the fusion expression of pelB signal peptide and
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TGase. Soluble TGase was expressed in E. coli without protease addition (Liu et al.,
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2011b). More recently, this approach was validated by Javitt et al. (2017), who
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constructed a constitutive expression system of TGase in E. coli (Fig. 2C). The active
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form of TGase was obtained without addition of protease. The enzymatic activities of
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wild-type and heterologous TGase had the same characterization (Javitt et al., 2017).
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TGase, many studies have tried to find the key site for the active expression (Chen et
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al., 2013; Liu et al., 2011a). Site-directed mutagenesis provides good insights into the
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roles of amino acid residues. The residues of Tyr12, Asn27, Asn30 and Arg32 are
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associated with the interaction with TGase region via hydrogen bonds. Mutations of
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above 4 residues resulted in the complete inhibition of TGase secretion (Chen et al.,
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2013). Rickert et al. (2015) carried out experiments combining the alanine-scan of
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pro-domain with site-directed mutations to identify the key sites for soluble S.
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mobarensis TGase expression in E. coli. Results showed that the pro-domain residues
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of Tyr14, Asp20, Ile24, and Asn25 were the most important residues for achieving
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active and soluble TGase. Those results may guide the engineering of pro-peptide
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domain to produce TGase in E. coli with excellent solubility and fully active form.
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Strong correlations were found between N-terminal domain and active expression
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of TGase, and recombinant expression of TGase in E. coli has resulted in some
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success by changing the gene order of N-terminal region. Same strategies could be
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adopted in other hosts. However, there are some problems for the heterologous
190
expression of TGase, such as the low yield compared with the wild-type strain.
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Integrated on-column activation and His6 purification online have been used to
192
improve the yield of TGase. This procedure contributed to 89% active TGase of the
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total pro-TGase. Under high-density cultivation, soluble pro-TGase was expressed up
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to about 8,000 U/l, which has potential to be used in TGase production on a
195
commercial scale (Yang, Pan, & Lin, 2009). Furthermore, different fermentation
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modes were carried out to increase the yield, such as the fed-batch fermentation.
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Under fed-batch condition, the specific activity of S. mobaraensis TGase in E. coli
198
reached 1386 U/g dry mass (Sommer, Volk, & Pietzsch, 2011). Studies on heterologous expression of TGase using E. coli as a host have provided
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some invaluable results. However, E. coli is not approved to be used in food
201
processing for the consideration of GRAS status. Whereas, for other applications,
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such as the areas of biomedical engineering, material science and leather processing,
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E. coli production system is an economically feasible alternative (Martins et al., 2014;
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Sommer, Volk, & Pietzsch, 2011).
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3.2. Recombinant TGase expression using S. lividans as a host Although the expression system of S. lividans is not as powerful as that of E. coli, S.
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lividans has made a great contribution. For example, S. lividans was used as a host for
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several kinds of sugars, biomass-degrading enzymes and industrially useful
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compounds (Noda, Miyazaki, Tanaka, Ogino, & Kondo, 2012). Notably, S. lividans is
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capable to convert the inactive pro-TGase into active form with its endogenous
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protease (Liu, Wang, Du, & Chen, 2016). So, it has some advantages compared with
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other hosts.
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One technical challenge for recombinant TGase expression in S. lividans is to
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obtain an active form. S. ladakanum TGase was expressed in S. lividans.
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Consequently, incorrectly secreted TGase was obtained in S. lividans (Lin et al.,
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2004). Noda et al. (2012) described a method for S. cinnamoneus expression in S. 11
ACCEPTED MANUSCRIPT lividans. In that study, the signal peptide sequence encoding the phospholipase D gene
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of S. cinnamoneus, combing with the pro-domain of S. cinnamoneus TGase and the
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sequence encoding mature TGase was fused together. This strategy provided 360
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mg/L TGase (Noda et al., 2012). Taguchi et al. (2002) introduced an approach to
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co-expressing the encoding genes of S. albogriseolus proteases and the encoding
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genes of S. cinnamoneus TGase. As a consequent, less than 1 U/mL TGase was
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produced (Taguchi, Arakawa, Yokoyama, Takehana, Takagi, & Momose, 2002).
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Another challenge for recombinant TGases expression in S. lividans is to increase
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the yield. Several microbial TGases, such as S. mobaraense, S. ladakanum, S.
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platensis, have been expressed in S. lividans, but the yields were no higher than 2.22
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U/mL (Liu, Wang, Du, & Chen, 2016). Liu et al. (2016) employed promoter
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engineering and codon optimization to produce active S. hygroscopicus TGase in S.
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lividans. The encoding genes of native promoter was partially deleted to analyze the
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differences of TGase expression. Furthermore, the gene sequence of TGase ORF
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(open reading frame) was optimized to improve the expression. As a result, the yield
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of TGase increased from 1.8 U/mL to 5.73 U/mL (Liu, Wang, Du, & Chen, 2016).
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Till now, TGase produced by wild-type Streptomyces sp. is the main commercial way.
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After decades of development, the activity of TGase reached 6.0 U/mL (Zhang, Zhu,
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& Chen, 2010). The recombinant TGase produced by S. lividans has reached 5.73
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U/mL, which was comparable with the wild strain. However, such yield is still low.
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Accurate control of this process is recommended to improve recombinant TGase
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production at large scale.
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3.3. Recombinant TGase expression using C. glutamicum as a host C. glutamicum is a gram-positive, nonsporulating bacterium. It is an industrial
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workhorse with a long history. The most widely known for C. glutamicum is the role
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in the production of a variety of amino acids and other useful materials, such as
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L-glutamic acid, L-serine, poly (3-hydroxybutyrate) (Freudl, 2017; Kikuchi, Date,
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Yokoyama, Umezawa, & Matsui, 2003). Most importantly, C. glutamicum is safety to
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human and possesses an enormous potential for the secretory production of
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heterologous proteins, which make it have wide applications in food industry.
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Kikuchi et al. (2003) reported a method for the secretion of S. mobaraense
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pro-TGase in C. glutamicum. The signal peptide of a cell surface protein derived from
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C. glutamicum was used and inactive pro-TGase was converted to active form after
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addition of protease. As a consequent, the amount of TGase accumulated to 142 mg/L
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(Kikuchi et al., 2003). Because Phe-Arg-Ala-Pro residues were added to the
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N-terminal amino acid sequence, the sequence of secreted enzyme differed from that
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of the native one. Later, a preferred SAM-P45 cleavage site was introduced into the
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C-terminus of the pro-region, and the native-type TGase was successfully secreted by
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C. glutamicum, with an activity of 132 mg/L (Date, Yokoyama, Umezawa, Matsui, &
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Kikuchi, 2003). Date et al. (2004) utilized the chimeric pro-region consisting of S.
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mobaraensis and TGase from S. cinnamoneus to express TGase in C. glutamicum. As
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a result, the amount of TGase secreted by the strain carrying the chimeric pro-region
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is higher than that using the native one (Date, Yokoyama, Umezawa, Matsui, &
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Kikuchi, 2004). This study demonstrated that, similar with other hosts mentioned in
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this review, the pro-region of TGase is important for active expression. Like other members of Corynebacterianeae sp., C. glutamicum possesses an outer
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membrane-like structure composed of mycolic acids and other complex glycolipids.
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The structure has profound impacts on the efficient permeability for hydrophilic
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molecules (Richter, Hänel, & Hilliger, 1985). To date, it comes to conclude that C.
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glutamicum contains two major protein export pathways. One is the general secretion,
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which transports extracytosolic bacterial proteins. The other is the alternative twin
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arginine translocation pathway, which transports fully folded proteins (Freudl, 2017).
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Because protein transportation across outer membrane is a bottleneck in the
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heterologous expression of TGase, C. glutamicum is more suitable for the expression
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host for TGase than other hosts are. Till now, the yield of TGase expressed in C.
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glutamicum is low. Some strategies, such as signal peptide variation, promoter
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optimization, could be developed to improve the yield of secreted TGase.
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4. Concluding remarks and outlook
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TGase has wide uses in food industry. Commercial TGase (trade mark Activa™) is
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produced by fermentation of S. mobaraensis as extracellular protein. However, only
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1% protein exists in Activa™ and 99% Maltodextrin is added to stabilize TGase
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(Sommer et al., 2011). One drawback of current commercial process is the presence
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of proteases, which can hydrolyze the target proteins that are intended to be
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cross-linked. The other drawback is the complex fermentation procedure. Thus, it is 14
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necessary to develop an efficient and easy-to-use system for the “green” production of
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TGase. Biotechnological routes provide the possible measures to heterologous expression
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of TGase. After decades of efforts, the expression of recombinant TGase has
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significant improvements: from the formation of inclusion bodies, low recovery yields,
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and non-active enzyme to the soluble forms. However, many problems need to be
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solved for its realization in industrial level. For example, since there are no
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appropriate TGase-activating system in E. coli or C. glutamicum, recombinant
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pro-TGase is difficult for activation in these hosts. Although active TGase could be
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obtained in Streptomyces system, the yields of TGase are only close to those of wild
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strain.
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So, efforts on bioprocess engineering and genetic engineering for the improvement
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of TGase are desired in the near future. With the development of industrial level
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resolution, the biotechnological routes for TGase production would be an
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economically feasible alternative and will accelerate the application of TGase.
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Conflict of Interest
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All the authors declare no competing interests.
302 303
Author Agreement/Declaration
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All the authors have seen and approved the final version of the manuscript being
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submitted. They warrant that the article is the authors' original work, hasn't received 15
ACCEPTED MANUSCRIPT 306
prior publication and isn't under consideration for publication elsewhere.
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Acknowledgements
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This work was supported by grants from the Beijing Advanced Innovation Center for
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Food Nutrition and Human Health, Beijing Technology & Business University
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(BTBU) (20171013), and the National Natural Science Foundation of China
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(31670045).
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Procedure
Yield
Reference
Euphausia superba
Purified from whole cell
54 (U/mg)
Streptomyces lividans
Fermentation
Streptomyces lividans Providencia sp. C1112
Genetic modification Fermentation
1.23–2.22 (U/mL) 5.73 (U/mL) 1.8 (U/mL)
Zhang, He, & Simpson, 2017 Lin et al., 2004
Streptoverticillium mobaraense Streptomyces mobaraensis Streptomyces lividans Streptomyces platensis
Fermentation
1.6 (U/mL)
Fermentation
4.3 (U/mL)
Genetic modification Fermentation
360 (mg/L) 2.3 (U/mL)
Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Yarrowia lipolytica
Genetic modification
132 (mg/L)
Noda et al., 2012 Yeo, Yoon, Lee, & Kim, 2009 Date et al., 2003
Genetic modification
142 (mg/L)
Kikuchi et al., 2003
Genetic modification
863-881 (mg/L) 7.8 (U/mL)
Date et al., 2004
Escherichia coli Escherichia coli
Genetic modification Genetic modification
Escherichia coli Escherichia coli Pichia pastoris
Genetic modification Genetic modification Genetic modification
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Genetic modification
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4.5 (U/mL) 18.2-22 (U/mg) 1 (U/mL) 30-75 (mg/L) 0.9 U/mg
Liu, Wang, Wang, Madzak, Du, & Chen, 2015 Liu et al., 2011a Liu et al., 2011b Javitt et al., 2017 Rickert et al., 2015 Li et al., 2014
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Figure Legends:
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Figure 1
446
Transglutaminase applications in food industry.
447
Figure 2
449
Genetic organization of Streptomyces TGase.
450
A, Genetic organization of TGase in Streptomyces sp.. B, Genetic organization of
451
active TGase from Streptomyces in E. coli (by reference Liu et al., 2011b). C, Genetic
452
organization of active TGase from Streptomyces in E. coli (by reference Javitt et al.,
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2017).
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Highlights TGase’s cross-linking properties are extensively used in food industry and outside
Natural sources and commercial production of TGase.
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the food area.
Biotechnological routes for TGase production using E. coli, S. lividans, and C.
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glutamicum as hosts.
The drawbacks as well as improvements of the production of TGase via
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biotechnological routes.