Potential applications of recombinant bifidobacterial proteins in the food industry, biomedicine, process innovation and glycobiology

Abstract

Bifidobacterial proteins have been widely studied to elucidate the metabolic mechanisms of diet adaptation and survival of Bifidobacteria, among others. The use of heterologous expression systems to obtain proteins in sufficient quantities to be characterized has been essential in these studies. L. lactis and the same Bifidobacterium as expression systems highlight ways to corroborate some of the functions attributed to these proteins. The most studied proteins are enzymes related to carbohydrate metabolism, particularly glycosidases, due to their potential application in the synthesis of neoglycoconjugates, prebiotic neooligosaccharides, and active metabolites as well as their high specificity and efficiency in processing glycoconjugates. In this review, we classified the recombinant bifidobacterial proteins reported to date whose characterization has demonstrated their usefulness or their ability to produce a product of commercial interest for the food industry, biomedicine, process innovation and glycobiology. Future directions for their study are also discussed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-021-00957-1.

Introduction

Bifidobacteria are bacteria resident in the gastrointestinal tracts (GITs) of mammals, birds, and insects (Lugli et al., 2019) that were first isolated in 1900 from the feces of infants (Biavati et al., 2000). Since then, they have been widely studied for their probiotic properties to prevent chronic degenerative and gastrointestinal diseases and modulate agents of the intestinal microbiota (Azad et al., 2018). In addition, as they are natural residents of the gastrointestinal tract and benefit health in multiple ways, there is a growing interest in using genetic modification to strengthen them and develop living therapeutic agents with improved functions for the fight against pathogens and the delivery of prophylactic molecules through the use of bioengineering (Sun et al., 2012; Zuo et al., 2020).

To understand the functions of Bifidobacteria, proteins and enzymes involved in adhesion, colonization, signaling, defense, and metabolism and the transport of carbohydrates, lipids, and amino acids have been studied (Fushinobu, 2010; González-Rodríguez et al., 2013). However, native proteins are sometimes obtained in very low concentrations, are not efficiently purified, or both, making their study difficult. Thus, these proteins have been produced using various expression systems, including those from Escherichia coli, Lactococcus lactis, and even some Bifidobacteria strains (O'Connell Motherway et al., 2008; Sun et al., 2012). These strategies have made it possible to obtain recombinant proteins in sufficient quantities to crystallize, determine their structure, and facilitate their identification and classification. Additionally, structure–function studies have been developed to clarify the catalytic mechanisms of some enzymes (Sato et al., 2017) and improve their properties. Furthermore, some of these proteins have been proposed for various applications.

In this review, we explored the different expression systems used to produce recombinant bifidobacterial proteins while highlighting the contribution of the study of these proteins to understanding their roles in different aspects of metabolism and survival. Finally, we discuss the potential applications of recombinant bifidobacterial proteins in the food industry, biomedicine, industrial process innovation, and glycobiology.

Heterologous expression systems used to produce recombinant bifidobacterial proteins

The first recombinant bifidobacterial proteins produced in E. coli were the β-galactosidases from B. breve YIT 4010 (Iino, 1990; Sako et al., 1999). Since then, to the best of our knowledge, 215 bifidobacterial proteins and some mutant versions have been produced using different expression systems (see Supplementary Material) and various vectors and strains. A total of 83.8% of recombinant proteins expressed and analyzed in this study (254) were produced in the E. coli expression system, while the second most commonly used expression system was L. lactis (11.4%). Less than 2% of proteins were produced in Agrobacterium rhizogenes and the yeasts Saccharomyces cerevisiae and Pichia pastoris, as shown in Fig. 1. In addition, Bifidobacterium longum, B. bifidum, and B. pseudocatenulatum have recently been used to produce bifidobacterial proteins that these strains do not produce naturally. These proteins represent 3.5% of the total, as summarized in Fig. 1.

Fig. 1.

Heterologous expression systems used to produce bifidobacterial recombinant proteins

E. coli

E. coli is the oldest expression system and has been the most widely used to produce bifidobacterial proteins. The pET expression vectors, which are inducible with IPTG, were used to produce 53% of the bifidobacterial proteins expressed in E. coli and analyzed in this review. E. coli BL21 and its derivatives were the expression strains most commonly used (Table 1). Numerous reviews have detailed the drawbacks of producing recombinant proteins in E. coli and the strategies to solve no or low production, inactive or insoluble protein, and other expression issues (Rosano & Ceccarelli, 2014). In 2013, Osman reported an increase in the yield of recombinant bifidobacterial b-galactosidase (BbgIV) with improved solubility and enhanced enzymatic activity by increasing the plasmid copy number and decreasing the growth rate, limiting dissolved oxygen during the exponential phase of fermentation (Osman et al., 2013). The yield of recombinant extracellular solute-binding protein (ESBP) from B. longum KACC 91,563 was increased by optimization of the purification on a larger scale. However, the increase achieved was not sufficient for mass production (Song et al., 2019). Regarding the purification process, the vast majority of bifidobacterial recombinant proteins required a single step purification and were developed using nickel-loaded columns; however, for preparations of recombinant B. longum endo-α-N-acetylgalactosaminidase from larger cultures, cobalt-charged resins were preferred (Hansen et al., 2019). Another strategy used successfully to facilitate the expression and purification of bifidobacterial proteins produced from E. coli has been the cloning of the proteins without the native signal peptide and the transmembrane domains (Chen et al., 2020; Fujita et al., 2011; Garrido et al., 2012; Karav et al., 2016; Komeno et al., 2019; Miwa et al., 2010; Parc et al., 2015; Shimada et al., 2015). In other cases, the enzymatic cleavage of fusion tags from recombinant a-_L-arabinofuranosidase (AfuB-H1) and a-_L-arabinopyranosidase/b-_D-galactosidase (Apy-H1) from B. longum was used to obtain active proteins (Lee et al., 2011).

Table 1.

Vectors and strains used to produce recombinant bifidobacterial proteins in different expression systems

Expression system	Vectors*	Strains*
Escherichia coli	pET28, pET23, pBlueScript KS(-), pCold1,pET101/D-TOPO, pET32, pET22, pET21, pET3, pEXP5-CT/TOPO, pET30, pET24, pET16, pBAD-HisA, p1O-TrC, pET26, pTKNd119, Lambda ZAP II, pGEX-4 T-3, pBES16PR, pET39, pET15, pBlueScript, pBR322, pROEX-HTA, pGR07, pUC18, pDEST17, pTEM-11, pET-Blue1, pET11, pET29, pET303/CT-His, pET36, pEco-T7-c-His, pBT7-N-His, pBAD/Myc-His, pMCSG7, pET-TrxA, pET-TRXA-1a/LIC, pUC19, pEASYBluntE1, pOXO4, pSpeedET, pQE-30, pATE19, pBTaC2, pCR2.1 TOPO, pWSK29, pCX-Kan-P22, pMGS	E. coli BL21 (DE3), E. coli BL21, E. coli BL21 (DE3) pLyss, E. coli BL21 Star, E. coli DH5a, E. coli BL21 (DE3) ΔlacZ, E. coli Rosetta 2 (DE3), E. coli BL21 (DE3) CodonPlus RIL, E. coli Rosetta (DE3), E. coli XL1-Blue MRF, E. coli DH10B, E. coli MT102, E. coli JM109, E. coli B834 (DE3), E. coli MG1655, E. coli MC1061, E. coli ER2566, E. coli T7 Express, E. coli T/ Express GRO, E. coli CodonPlus (DE3)-RIPL, E. coli BL21 (DE3)/pRARE2, E. coli BL21 ΔlacZ (DE3)/pRARE2, E. coli LM1, E. coli Rosetta (DE3) pLacI, E. coli BL21 (DE3) CodonPlus, E. coli C43 (DE3)-RIL, E. coli LMG194, E. coli C43(DE3), E. coli SoluBL21, E. coli TOP10, E. coli C41 (DE3), E. coli RA11r, E. coli Origami B(DE3), E. coli GeneHogs, E. coli M15, E. coli BL21 S1, E. coli Tuner (DE3), E. coli XL1 Blue, E. coli KAM3, E. coli 499ZCS112L, E. coli Tuner (DE3)pLacI, E. coli GR536, E. coli BL21-AI
Lactococcus lactis	pNZ8048, pNZ8150, pUC57, pNZ8148, pNZ8032	L. lactis NZ900, L. lactis MG1363
Bifidobacteria	pBES2, pBLES100, pYBamy59, pGOSBif33	B. bifidum BGN4, B. longum 105-A, B. longum MG1, B. longum NCC2705, B. longum subsp. infantis E18, B. bifidum S17
Pichia pastoris	pPIC9	P. pastoris GS115
Saccharomyces cerevisiae	pRS424	S. cerevisiae L2612
Agrobacterium rhizogenes	pBI121	A. rhizogenes A4

*In order from highest to lowest frequency of use

L. lactis

This gram-positive lactic acid bacteria, generally recognized as safe (GRAS), is desirable for the expression of recombinant proteins due to its probiotic properties, the absence of inclusion bodies and endotoxins, and the development of strategies that favor extracellular secretion and the production of surface proteins (Song et al., 2017). The first bifidobacterial protein expressed in this system was α-L-arabinofuranosidase from B. longum B667 (Margolles & de los Reyes-Gavilán, 2003). The enzyme was efficiently produced using the nisin-inducible expression vector pNZ8048 in L. lactis NZ9000, a derivative of the nisin-negative MG1363 strain modified to induce transcription of two promoters in the nisin gene cluster (Song et al., 2017). This vector and strain have been the most commonly used to produce bifidobacterial proteins in L. lactis. In some cases, the nisin-inducible translational fusion vector pNZ8150 was used with the same strain to produce a β-endogalactanase (GalA) and diverse β-galactosidases from B. breve UCC2003 (O’Connell Motherway et al., 2011, 2013). This vector includes a His-tag-encoding sequence that facilitates protein purification. L. lactis has been used to produce proteins related to carbohydrate metabolism, defense mechanisms, and secondary metabolite biosynthesis (see supplementary material).

Bifidobacteria

Due to the probiotic features of Bifidobacteria, there is a particular interest in genetically modifying them to improve their characteristics and use them as vehicles for vaccines and anticancer agents in the digestive tract (Park et al., 2005). To this end, expression and secretion vectors have been developed for the heterologous expression of proteins, such as the E. coli—Bifidobacterium shuttle vectors pBES2 and pBLES100 used to produce some proteins related to carbohydrate metabolism; in addition, various strains of both B. longum and B. bifidum have been used for the production of recombinant bifidobacterial proteins (see Supplementary Material). Thus, the amylase B of Bifidobacterium adolescentis Int-57 (INT57) was efficiently expressed and secreted in B. longum MG1 (Rhim et al., 2006), the β-galactosidase of B. longum RD47 was expressed in B. bifidum BGN4 (Park et al., 2019), and the α-L-fucosidases AfcA and AfcB from B. bifidum JCM1254 were expressed in B. longum 105-A (Ashida et al., 2009). In all cases, the enzymatic activities produced by the recombinant strains were higher than those obtained from the native strains, or the activity was conferred to a strain that had no intrinsic activity. Other vectors, such as pMGS under the control of the constitutive promoter pGap and pGOSBif33, a shuttle vector employed for the transformation of B. longum and B. bifidum, have been used to produce and characterize lipoproteins and enzymes involved in the activation of the immune system (Gleinser et al., 2012; Guglielmetti et al., 2014). Thus, Bifidobacteria as an expression system has been used to corroborate the roles that specific proteins play in native strains and to explore the possibility of producing strains with improved capacities.

S. cerevisiae and P. pastoris

Yeasts have been little used for the expression of bifidobacterial proteins. Only one bifidobacterial enzyme has been produced in S. cerevisiae, the xylose isomerase from B. longum MG1, using S. cerevisiae strain L2612 and the expression vector pRS424. Using this strategy, a modified strain that ferments xylose was achieved, and the inhibitory effect of xylitol was lower than that of the in vitro enzyme, probably due to the rapid secretion of the intracellular xylitol produced (Ha et al., 2011).

In addition, β-galactosidase from B. animalis ACCC05790 was expressed in P. pastoris GS115 using the vector pPIC9 and different strategies to increase the yield of the enzyme. Fusion to a cherry protein promoted a higher yield; this protein fused to the N-terminal end of some proteins has been used to produce high levels of soluble protein with improved thermal stability (Das et al., 2009; Xu et al., 2019).

A. rhizogenes

The α-L rhamnosidase from B. dentium K13 was produced in ginseng roots using A. rhizogenes A4 and the expression vector pBl121 to increase the production of the ginsenoside Rg1 in the plant (Zhang et al., 2015a). Thus far, this is the only recombinant bifidobacterial protein that has been produced in plants.

Roles of proteins in the metabolism and survival of Bifidobacteria

Carbohydrate metabolism

The use of recombinant proteins varies widely from research involved in understanding their function in vivo to their large-scale production to provide solutions in the biomedical, food, and environmental areas, among others. The most studied bifidobacterial proteins have been those related to the metabolism and transport of carbohydrates. Due to the shortage of digestible carbohydrates in the gastrointestinal tract, the persistence and survival of Bifidobacteria depend significantly on the enzymatic activities that allow them to take advantage of the conditions. Fructose polymers (fructans) from the diet, human milk oligosaccharides (HMOs), mucins, and glycoproteins secreted by mucosal epithelial cells widely distributed in the gastrointestinal tract are used as carbon sources for Bifidobacteria. It is known that not all strains of Bifidobacteria use the same substrates as carbon sources. The expression and characterization of the β-fructofuranosidases of different species (see supplementary material) have shown a correlation between their structural characteristics and the ability to metabolize structurally diverse fructans (Ávila-Fernández et al., 2016). Moreover, the heterologous expression and characterization of some enzymes have demonstrated their specific role in the degradation of HMO and mucins, allowing them to grow selectively and to adapt to specific environments. In 2012, the endoglycosidases from B. longum subsp. infantis ATCC 15,697, B. infantis SC142, and B. longum DJO10A were studied. Two of them had a wide range of activities on complex N-glycans, which could enable the strains to consume a wide range of substrates (Garrido et al., 2012). Enzymes such as exo-α-sialidase (SiaBb2) (Kiyohara et al., 2011), α-L-fucosidase (AfcA) (Katayama et al., 2004), and 1,3-1,4-α-L-fucosidase (AfcB) from B. bifidum (Ashida et al., 2009) are involved in the degradation of sialylated and fucosylated HMOs. SiaBb2 hydrolyzes sialyloligosaccharides, gangliosides, and glycoproteins, contributing to the survival of B. bifidum in the small intestine (Kiyohara et al., 2011). Recently, a gene–phenotype association study involving 217 Bifidobacteria strains and 21 carbohydrates as carbon sources showed species- and strain-specificity in carbohydrate catabolism. The authors highlighted the need to characterize the enzymes involved in species-specific clusters to understand the abilities of some Bifidobacteria strains to utilize specific carbohydrates and to use this knowledge to design new “prebiotic-probiotic” products (Liu et al., 2021), which are highly appreciated as nutritional supplements. Thus, heterologous expression remains a relevant tool for the study of enzymatic functions in carbohydrate metabolism.

Adhesion and intestinal colonization

Among the relevant aspects that promote colonization, survival, and the probiotic effects of Bifidobacteria is adhesion to the intestinal epithelium. In this sense, extracellular proteins play an essential role in host-microbe interactions. The bopA lipoprotein of B. bifidum MIMBb75 expressed in B. bifidum S17 and B. longum E18 increased their ability to adhere to intestinal epithelial cells (Gleinser et al., 2012). The production of the recombinant protein GroEL and the transaldolase Tal identified in extracellular vesicles produced by B. longum NCC2705 allowed to characterize their affinity for mucins, and their immobilization in microbeads increased their permanence in the murine gastrointestinal tract, corroborating that they promote strain adhesion (Nishiyama et al., 2020). Similarly, the transaldolase of B. bifidum A8 expressed in L. lactis promoted bacteria-mucin binding (González-Rodríguez et al., 2012). In addition, the interactions of some bifunctional sugar-lytic enzymes with the host have been identified. Characterization of the recombinant sialidase from B. bifidum ATCC 15,696 (SiaBb2) demonstrated that the enzyme recognizes two different sialylated structures of porcine mucin oligosaccharides, promoting their assimilation by hydrolysis and strain adhesion via specific interactions between the bacteria and the host intestinal epithelium carbohydrates (Nishiyama et al., 2017). Furthermore, recombinant B. lactis BI07 enolase, by interacting with human plasminogen, acquires proteolytic activity and facilitates colonization in the intestinal epithelium. The crucial residues for the interaction were identified by site-directed mutagenesis (Candela et al., 2009). Fimbrial proteins also contribute to adhesion; the polymorphic variants of this protein produced in E. coli showed that the adhesive properties were affected by genetic polymorphisms and were specific to B. longum subsp. longum strains (Suzuki et al., 2016).

Bile tolerance and multidrug resistance

Bifidobacteria are exposed to cytotoxic agents such as bile salts and antibiotics, and they have developed various defense mechanisms to ensure their survival. The use of L. lactis as an expression system has facilitated the study of membrane- and cell surface-associated proteins, both transporters and nontransporters that participate in bile salt tolerance. Thus, some transporters of bile salts of B. breve UCC2003 showed that they increase the resistance and survival of L. lactis to bile salt exposure, probably favoring the extrusion of bile salts from the membrane to the external environment (Ruiz et al., 2012). Furthermore, the heterologous expression of TlyC1, a nonmembrane transporter protein of B. longum BBMN68, also expressed in L. lactis, increased the strain tolerance to bile salts by 45 times (Liu et al., 2014). In addition, the membrane protein BbmR from B. breve UCC2003 expressed in L. lactis conferred moderate resistance to macrolides (Margolles et al., 2005). Thus, the expression of proteins in L. lactis has contributed to elucidating the roles of various proteins in the survival mechanisms of Bifidobacteria.

Potential applications of bifidobacterial recombinant proteins

The bifidobacterial recombinant proteins studied thus far have made it possible to understand their in vivo functions and some differences between strains. In addition, their properties have been evaluated regarding the production of compounds of commercial interest for the development of functional or pharmacologically active foods, the innovation of industrial processes, obtaining biomaterials, and as tools in glycobiology. Endo- and exoglycosidases, pyrophosphorylases, and glycosyltransferases stand out as the most studied for their potential applications. Some of these enzymes have been modified by genetic engineering to increase the yield of the product or change their intrinsic biological activity. Figure 2 describes some of the enzymatic activities and functions of recombinant bifidobacterial proteins assayed for different applications.

Fig. 2.

Enzymatic activities and functions of recombinant bifidobacterial proteins used in the food industry (A), biomedicine (B), innovation of industrial processes (C), and glycobiology (D). Each symbol represents a specific monosaccharide found in nature according to Symbol Nomenclature for Graphical Representation of Glycans (Varki et al., 2015). AgnB (α-N-acetylglucosaminidase), aRham (α-L-rhamnosidase), BbAfcA N423H (1,2-α-L-fucosyntase), BbBgI (β-glucosidase), BbhI (β-N-acetylhexosaminidase), b-Gal (β-galactosidase), BiLA (α-amylase), BiUSP or BLUSP (UDP-sugar pyrophosphorylase), BtAS (amylosucrase), EndoBI (endo-β-N-acetylglucosaminidase), EngBF (endo-α-N-acetylgalactosaminidase), ESBP (extracelular solute-binding protein), GalE (UDP-glucose epimerase), GalT (UDP-glucose-hexose-1-phosphate uridylyltransferase), HypBA2 (β-L-arabinobiosidase), L-AI (L-arabinose isomerase), LNBase (lacto-N-biosidase), NagBb (exo/endo-α-N-acetylgalactosaminidase), NahK_ATCC15697 (N-acetylhexosamine 1-kinase), SP (sucrose phosphorylase)

Food industry

Specific prebiotic oligosaccharides, neoglycoconjugates, and edulcorants are in high demand for functional food development and can be produced by transglycosylation and deglycosylation mediated by endo- and exoglycosidases (Fig. 2A). Thus, recombinant Endo-α-N-acetylgalactosaminidase from B. longum subsp. longum JCM 1217 (EngBF) was used to produce oligosaccharides from a synthetic substrate containing galacto-N-biose (GNB) (Ashida et al., 2010). The α-N-acetylglucosaminidase from B. bifidum JCM 1254 (AngB) could be used to facilitate access by the enzyme EngBF to GNB from glycoconjugates to produce oligosaccharides by the deglycosylation of glycoproteins (Shimada et al., 2015). Moreover, recombinant Exo/endo-α-N-acetylgalactosaminidase from B. bifidum JCM 1254 (NagBb) was used to produce neoglycoconjugates using GalNAca1pNP and serine as substrates (Kiyohara et al., 2012). These products have prebiotic potential since they could be hydrolyzed by Bifidobacteria strains containing enzymes homologous to EngBF and NagBb but not by pathogenic or opportunistic bacteria (Ashida et al., 2010; Kiyohara et al., 2012). The Endo-β-N-acetylglucosaminidases from B. longum subsp. infantis ATCC 15,697 (EndoBI-1) also produced oligosaccharides by deglycosylation but in this case released the total glycans from glycoproteins (Garrido et al., 2012). EndoBI-1 showed activity on a wide range of N-glycans in human milk, and modifying the reaction conditions made it possible to produce selected cohorts of oligosaccharides with prebiotic potential for Bifidobacteria. Therefore, EndoBI-1 may be useful in the dairy industry for the large-scale production of molecules similar to human milk oligosaccharides (HMOs) and glycan-free milk from bovine whey protein concentrate (Cohen et al., 2018; Karav et al., 2015a; Karav et al., 2015b; Karav et al., 2016; Karav et al., 2018; Parc et al., 2015).

The exoglycosidase β-N-acetylhexosaminidase from B. bifidum JCM 1254 (BbhI) was able to produce the trisaccharide GlcNAcβ1-3Lac, which is the main structure of an HMO group, with a high yield (Chen et al., 2016). Meanwhile, various bifidobacterial β-galactosidases produce galactooligosaccharides (GOS) (Yi et al., 2011). However, large-scale GOS production requires process optimization. In this sense, diverse strategies have been implemented; for example, the oxygen solubility was modified in the fermentation process to obtain a high yield of a β-gal from B. bifidum NCIMB 41,171 (BbgIV) in E. coli DH5α (Osman et al., 2013), and the β-gal I and β-gal II from B. breve DSM 20,213 were coexpressed with the GroEL/GroES chaperones to increase their expression (Arreola et al., 2014). Finally, a β-gal from B. longum subsp. longum RD 47 was used to produce a new disaccharide called β-galactosyl fucose (gal-fuc), which was selectively used by probiotic strains, and its effects were better than those of commercial GOS (Oh et al., 2019).

In addition, some bifidobacterial enzymes have been used to produce modified starches by hydrolysis or transglycosylation. The psychrophilic α-amylase from B. longum (BiLA) was used to generate a slowly digestible starch (SDS) via amylopectin hydrolysis (Lee et al., 2016), and 4-α-glucanotransferase from B. longum subsp. longum JCM 1217 (BL-aGTase) produced a thermoreversible gel by elongating the side chains and reducing the molecular weight of potato starch. These gels can be used in the food industry due to their nutritional properties and capacity for thermoreversible freezing (Jeong et al., 2020). Similarly, edulcorants have also been produced by isomerization or transglycosylation. The reaction conditions of amylosucrase from Bifidobacterium thermophilum were modified by increasing the sucrose concentration, favoring isomerization to produce turanose (Choi et al., 2019). In addition, the L-arabinose isomerase from B. longum NRRL B-41409 (L-AI) was used to isomerize D-galactose to D-tagatose, albeit with low yield (Salonen et al., 2012), and the manipulation of the reaction conditions also increased the substrate conversion. Furthermore, the L. lactis strain transformed with L-AI efficiently produced D-tagatose (Salonen et al., 2013). Finally, some enzymes have been used to hydrolyze certain phytochemical compounds and convert them into bioactive compounds that improve the nutritional value, quality, aroma, or composition of fermented products. Specifically, it was demonstrated that the cinnamoyl esterase activity of some Bifidobacteria, such as B. animalis subsp. lactis, can degrade chlorogenic acid and release phenolic acids, which are appreciated in the food industry as antioxidants (Fritsch et al., 2017).

Thus, bifidobacterial enzymes have demonstrated their potential in the production of oligosaccharides, neoglycoconjugates, edulcorants, and diverse bioactive compounds that could be used in the food industry to create new commercial products or improve the characteristics of existing products (Table 2). However, although many of the products have been described as potential prebiotics, their properties need to be evaluated using in vitro or in vivo assays to corroborate and to better describe their prebiotic characteristics.

Table 2.

Potential applications of recombinant bifidobacterial proteins in the food industry

Native strain	Protein	Expression strain	Expression vector	Potential application	Reference
B. longum subsp. longum JCM 1217	Endo-α-N-acetylgalactosaminidase (EngBF)	E. coli BL21 (λDE3)	pET-23d( +)	Synthesis of prebiotic oligosaccharides	Ashida et al. (2010)
B. bifidum JCM 1254	Exo/endo-α-N-acetylgalactosaminidase (NagBb)	E. coli BL21 (DE3) ΔlacZ	pET-23b( +)	Production of prebiotic glycosides containing GalcNAc	Kiyohara et al. (2012)
B. bifidum JCM 1254	α-N-acetylglucosaminidase (AngB)	E. coli BL21 (λDE3) ΔlacZ	pET-23b( +)	Production of prebiotic oligosaccharides from porcine gastric mucin	Shimada et al. (2015)
B. longum subsp. infantis ATCC 15,697	Endo-β-N-acetylglucosaminidase (EndoBI-1)	E. coli BL21*	pEco-T7-cHis	Deglycosylation of glycoproteins from bovine serum for the production of prebiotic glycans	Karav et al. (2015a; 2015b)
B. bifidum JCM 1254	β-N-acetylhexosaminidase (Bbh1)	Escherichia coli BL21 (DE3)	pET-21b( +)	Synthesis of functional glycans	Chen et al. (2016)
B. longum KCTC 3127	α-amylase (BilA)	E. coli MC1061	pTKNd119	Production of slowly digestible starch	Lee et al. (2016)
B. thermophilum ATCC 25,525	Amylosucrase (BtAS)	E. coli BL21-CodonPlus RIL	pBT7-N-His	Production of turanose	Choi et al. (2019)
B. animalis subsp. lactis DSM 10,140	Cinnamoyl esterase	E. coli TOP10	pBAD/Myc-His	Increase of prebiotic products quality by fermentation of cinnamic acids	Fritsch et al. (2017)
B.breve B24	β-galactosidase	E. coli ER2566	pET-36b( +)	Synthesis of prebiotic galactooligosaccharides and proccesing of lactose and milk	Yi et al. (2011)
B. bifidum NCIMB41171	β-galactosidase	E. coli DH5 α	pBluescript KS	Synthesis of prebiotic galactooligosaccharides	Osman et al. (2013)
B. breve DSM 20,213	β-galactosidase (β-gal I)	E. coli T7 Express GRO	pGRO7	Synthesis of prebiotic galactooligosaccharides	Arreola et al. (2014)
	β-galactosidase (β-gal II)
B. longum subsp. longum JCM 1217	4-α-glucanotransferase (BL-α-GTase)	E. coli	pTKNd119	Modification of powdered starch for the production of a heat-reversible gel for food preservation	Jeong et al. (2020)
B. longum subsp. longum RD47	β-galactosidase	E. coli BL21	pCold I	Production of new prebiotics from fucose and lactose	Oh et al. (2019)
B. longum NRRL B-41409	L-arabinose isomerase (L-AI)	L. lactis MG1363	pNZ8032	Production of D-tagatose	Salonen et al. (2012; 2013)

Biomedicine

The production of glycoconjugates and the bioconversion of phytochemical compounds also have applications in biomedicine. Some enzymes have been shown to produce active molecules to develop therapeutic interventions (Assenberg et al., 2013). Such is the case for glycosidases that synthesize neoglycoconjugates and compounds that are difficult to obtain from natural sources (Table 3). The generation of glycoconjugates could favor the stability of proteins with biological activity. The glycosylation of bioactive peptides increases their resistance to proteases and, therefore, the half-life times (t ½) that they remain in the bloodstream. The transglycosylation activity of the endoglycosidase EngBF has been tested to produce various glycoconjugates (Fujita et al., 2005; Suzuki et al., 2009). The endoglycosidase could transfer GNB from donor Galb1,3GalNAca1-pNP to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T, and MUC1a. The latter could be used as an antitumor vaccine to prevent metastasis (Ashida et al., 2010). However, it is necessary to improve the efficiency of this reaction.

Table 3.

Potential applications of recombinant bifidobacterial proteins in biomedicine

Native strain	Protein	Expression strain	Expression Vector	Potential application	Reference
B. longum subsp. longum JCM 1217	Endo-α-N-acetylgalactosaminidase (EngBF)	E. coli BL21 (λDE3)	pET-23d( +)	Synthesis of neoglycoconjugates that could be used as cancer vaccines	Suzuki et al. (2009)
				Synthesis of bioactive glycoconjugates	Ashida et al. (2010)
B. breve ATCC 15,700	β-glucosidase (BbBgl)	E. coli BL21	pEASY-Blunt E1	Production of ginsenoside CK, a pharmacologically active metabolite	Zhang et al. (2019)
B. longum KACC 91,563	Solute-binding proteins (ESBP)	E. coli BL21	pGEXT-4 T-3	Therapeutic protein to decrease the symptoms of food allergy	Kim et al. (2016)
			pET-28a( +)		Song et al. (2019)
		E. coli BL21(DE3) RIPL
B. breve ATCC 15,700	α-L-rhamnosidase	A. rhizogenes A4	pBI121	Production of ginsenoside Rg1, a compound with antitumor, anti-inflammatory, and antidiabetic activity	Zhang et al. (2015a)
		E. coli BL21 (DE3)	pET-28a( +)	Production of isoquercitrin, a flavonoid with antiproliferative, antioxidant, antiallergic, and anti-aging properties	Zhang, et al. (2015b)

Some compounds, such as ginsenosides and flavonoids, produced by plants are highly valued for their therapeutic properties. However, they are challenging to obtain from their natural sources due to their low concentration or the long growth times of the plants that produce them. Therefore, the abilities of some recombinant bifidobacterial enzymes, such as β-glucosidase (BbBgl) and α-L-rhamnosidase, from B. breve ATCC 15,700 to obtain minor metabolites of ginsenosides and flavonoids were explored (Fig. 2B). BbBgI hydrolyzed the glucose residue at position C-3 of ginsenoside Rd to produce F2 and compound K (CK). The increase in the enzymatic activity of BbBgI resulted in the total conversion of ginsenoside Rd to CK. This minor ginsenoside is the primary active metabolite detected in the blood after oral administration of ginsenosides and has antitumor, anti-inflammatory, and antidiabetic activities, among others (Zhang et al., 2019). Similarly, the production of ginsenoside Rg1, a deglycosylated product of ginsenoside Re, the main constituent of ginseng, was explored. In particular, Rg1 exhibits significant anti-inflammatory, antidepressant, and memory effects. Recombinant α-L-rhamnosidase (BbRha) was produced in the hairy roots of Panax ginseng to obtain Rg1. The transgenic roots had higher levels of Rg1, and the concentration of ginsenoside Re decreased. Therefore, the increase in the yield of ginsenoside Rg1 was attributed to the hydrolysis of the rhamnose residue in ginsenoside Re mediated by α-L-rhamnosidase (Zhang et al., 2015a). The same enzyme efficiently produced the flavonoid isoquercitrin from the deglycosylation of the rhamnose residue from rutin. Isoquercitrin has antiproliferative, antioxidant, antiallergic, and antiaging properties. Furthermore, the pharmacological activity of isoquercitrin is considered to be greater than that of rutin (Zhang et al., 2015b).

It should be noted that there are some bifidobacterial proteins that have demonstrated their therapeutic potential per se. Recently, it has been described that B. longum KACC 91,563 secretes an extracellular solute-binding protein (ESBP) via extracellular vesicles. In an animal model, intraperitoneal administration of the recombinant protein ESBP was able to alleviate food allergy symptoms by reducing mast cell numbers in the small intestine and their specific apoptosis without affecting the immune response mediated by T cells (Kim et al., 2016). Although ESBP has therapeutic potential for the treatment of allergies, the study authors recognized that the production of ESBP was insufficient even after testing different conditions for obtaining and purifying the protein and that improvement in the strategies to large-scale production of ESBP is needed for its development as a pharmaceutical product (Song et al., 2019).

The development of in vivo assays to establish the administration route and dose of new products and the optimization for large-scale production of proteins and enzymes and their bioactive products to obtain higher yields in the cases described is required. In addition, the genetic modifications that contribute to improving the specificity and features of the enzymes have not been explored in depth.

Innovation of industrial processes

The development of efficient processes for the production of nucleotide sugars, biomaterials, and biofuels using enzymatic methods is another field of opportunity for bifidobacterial enzymes (Table 4). Nucleotide sugars have previously been synthesized using chemical methods. However, chemical processes are complicated to reproduce and require multiple steps, protection and deprotection strategies, and advanced purification, and the yields of the sugars are low to moderate (Guo et al., 2015; Wang et al., 2018). Large-scale enzymatic synthesis of nucleotide sugars and their derivatives is preferred due to cost-effectiveness. Usually, the most straightforward biosynthetic pathway is used, which involves the formation of monosaccharide-1-phosphate catalyzed by a monosaccharide-1-phosphate kinase followed by nucleotide binding mediated by a nucleotidyltransferase or a pyrophosphorylase (Muthana et al., 2012; Wang et al., 2018). Some of these enzymatic activities are illustrated in Fig. 2C. Recombinant UDP-sugar pyrophosphorylase (USP) from B. longum ATCC 55,813 (BLUSP) has been reported to be efficiently used in a one-pot multienzyme (OPME) system for the large-scale synthesis of UDP-monosaccharides. The enzyme was mixed with an inorganic pyrophosphatase from Pasteurella multocida (PmPpA) and a monosaccharide 1-kinase (N-acetylhexosamine 1-kinase from B. infantis ATCC 15,697 [NahK] or a galactokinase from Streptococcus pneumoniae TIGR4 or E. coli). This system had excellent performance in the formation of UDP-galactose (UDP-Gal), UDP-manofuranose (UDP-ManF), and UDP-ManN3 (Muthana et al., 2012). Likewise, the OPME system was used to synthesize UDP-galacturonic and UDP-glucuronic acids using enzymes such as recombinant galactokinase from B. infantis ATCC 15,697 (BiGalk) or glucuronokinase from Arabidopsis thaliana (AtGlcAK), respectively, in conjunction with other enzymes. This multienzyme system avoids the use of nicotinamide adenine dinucleotide cofactor (NAD+) and can be widely applied to synthesize various molecules since the compounds formed can be used as donor substrates for uranosyltransferases in the production of uronosides. It is worth mentioning that BiGalk was able to catalyze the synthesis of galacturonic acid-1-phosphate (GalA-1-P) in high yield compared to AtGlcAK (Muthana et al., 2015). Recombinant USP from Bifidobacterium infantis 15697 (BiUSP) efficiently catalyzed the formation of UDP-GlcA but not UDP-GalA. Likewise, the specificity of BiUSP was similar to that of BLUSP (Guo et al., 2015).

Table 4.

Potential applications of recombinant bifidobacterial proteins in innovation of industrial processes

Native strain	Protein	Expression strain	Expression vector	Potential application	Reference
B. thermophilum ATCC 25,525	Amylosucrase (BtAS)	E. coli BL21-CodonPlus RIL	pBT7-N-His	Production of biomaterials (linear glucan α- (1,4) type amylose)	Choi et al. (2019)
B. longum subsp. infantis ATCC 15,697	N-acetylhexosamine 1-kinase (NahK)	E. coli BL21 (DE3)	pET-15b	Production of UDP-sugars using one-pot multienzyme (OMPE) systems	Muthana et al., (2012)
B. longum ATCC 55,813	UDP-sugar pyrophosphorylase (BLUSP)
B. longum subsp. infantis ATCC 15,697	Galactokinase (BiGalK)	E. coli BL21 (DE3)	pET-15b	Production of UDP-uronic acids using one-pot multienzyme (OMPE) systems	Muthana et al. (2015)
B. longum ATCC 55,813	UDP-sugar pyrophosphorylase (BLUSP)
	N-acetylhexosamine 1-kinase (NahK)
B. longum subsp. infantis ATCC 15,697	UDP-sugar pyrophosphorylase (BiUSP)	E. coli BL21 (DE3)	pET-22b -	Production of UDP-GlcA from GlcA-1-P	Guo et al. (2015)
	Galactokinase (BiGalK)
B. longum subsp. infantis ATCC 15,697	UDP-sugar pyrophosphorylase (BiUSP)	E. coli BL21		Production of UDP-β-L-arabinose from L-arabinose-1-P by quimioenzymatic methods	Wang et al. (2018)
B. longum subsp. longum JCM1217	Sucrose phosphorylase (SP)	E. coli BL21 (DE3)	pET-30	Enzymatic production of D-Galactosyl-β1 → 4-L-rhamnose	Nakajima et al. (2010)
	UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT)
	UDP-glucose epimerase (GalE)
B. longum MG1	Xilose isomerase (BlXI)	S. cerevisiae L2612	pRS424	Production of biofuel from lignocellulosic biomass	Ha et al. (2011)

Although nucleotide sugars have been produced successfully in OPME systems, some monosaccharides, such as D-xylose and L-arabinose, are not substrates for the kinases used. In such cases, a chemoenzymatic method has been proposed; the monosaccharides D-xylose and L-arabinose are chemically converted to sugar-1-phosphate, and the nucleotide sugars are joined by the enzyme BiUSP or AtUSP. However, unlike AtUSP, BiUSP could not convert D-xylose-1-P to UDP-α-D-xylose but could convert L-arabinose-1-P to UDP-β-L-arabinose (Wang et al., 2018). Finally, the OPME system was used successfully to synthesize the β-galactoside D-galactosyl-β1-4-L-rhamnose (GalRha) using sucrose phosphorylase (SP) from B. longum and 3 more enzymes, achieving a substrate conversion rate of 71% in a large-scale preparation (Nakajima et al., 2010).

Some bifidobacterial enzymes have also been assayed to produce carbohydrate derivatives, analogous to industrial and biofuel applications. Recombinant amylosucrase from B. thermophilum ATCC 25,525 (BtAS) synthesized a linear amylose-like α-glucan from sucrose, which could have a useful application in the formation of biodegradable films for food packaging (Choi et al., 2019). Moreover, the xylose isomerase of B. longum MG1 (BlXI) and that of Bacteroides stercoris HJ-15 (BsXI) were produced in S. cerevisiae to transform xylose from cellulosic biomass into xylulose to be used as a substrate in ethanol production. However, the transformation with BlXI was not functional since the transformed S. cerevisiae strain could not ferment xylose, unlike the BsXI transformed strain. It is worth mentioning that the transformation was confirmed in both cases, but the authors do not mention whether BIXI was produced and was not functional or was not produced (Ha et al., 2011).

In this field, bifidobacterial enzymes used in OPME systems for large-scale synthesis and chemoenzymatic production of UDP-monosaccharides look promising compared to those used for the development of other processes. Access and availability to a greater diversity of nucleotide sugars contribute to having the tools to produce more complex bioactive polymers similar to those of animals and plants and thus, to study their physiological properties.

Glycobiology

Glycosidases recognize the sugar chains of glycoproteins and remove glycosyl residues without damaging them, which is useful in studying the contributions of glycosylations to the biological activity of proteins (Fujita et al., 2011; Katayama et al., 2004). Genetic modification and manipulation of reaction conditions have favored that some glycosidases carry out synthesis reactions, introducing glycosyl residues to various glycans, making them desirable as glycobiology research tools (Table 5).

Table 5.

Potential applications of recombinant bifidobacterial proteins in glycobiology

Native strain	Protein	Expression strain	Expression vector	Potential application	Reference
B. longum subsp. longum JCM 1217	Endo-α-N- acetylgalactosaminidase (EngBF)	E. coli BL21 (λDE3)	pET-23d( +)	Glycan analysis	Fujita et al. (2005)
		E. coli BL21 CodonPlus (DE3)-RIL	pET-28b		Suzuki et al. (2009)
		E. coli B834 (DE3)
B. longum subsp. infantis ATCC 15,796	Endo-β-N-acetylglucosaminidase (EndoBI-1)	E. coli BL21 Star	pET-101/D-TOPO	Proteomic and glycoproteomic analysis	Garrido et al. (2012)
B. longum subsp. longum JCM 1217	β-L-arabinobiosidase (HypBA2)	E. coli BL21 (λDE3)	pET-23d	Study of arabinofuranosides in plant glycoproteins	Fujita et al. (2011)
B. bifidum JCM 1254	1,2-α-L-fucosyntase (BbAfcA N423H mutant)	E. coli BL21 (λDE3)	pET-3a	Tool to introduce the H antigen to diverse glycoproteins	Sugiyama et al. (2016; 2017)
B. longum subsp. longum JCM 1217	Lacto-N-biosidase (LNBase)	E. coli BL21 (DE3)/pRARE2	pET-3a	Glycoconjugates analysis	Sakurama et al. (2013)
			pET-23b

Since practically all the proteins secreted in eukaryotes are glycosylated, the analysis of O- and N-glycan structures is important for biological studies. Interestingly, certain microorganisms, such as Bifidobacteria, have developed various pathways to release O-glycans and N-glycans from different human glycoproteins to use them as a carbon source or alter their biological function (Garrido et al., 2012). Therefore, they have interesting enzymatic activities that can be used as tools in glycobiology (Fig. 2D). As mentioned, EngBF is highly specific for core 1 O-glycans; therefore, its activity can be used to elucidate the presence of O-glycans, determine their function in biological processes and deduce the binding sites on glycoproteins (Fujita et al., 2005). Concerning N-glycosylated proteins, EndoBI-1 was able to deglycosylate glycoproteins such as human lactoferrin, IgA, and IgG, which are practically resistant to many commercial endoglycosidases under native conditions. Therefore, EndoBI-1 can be useful in proteomic and glycoproteomic studies to detect and characterize glycosylated regions in proteins. In particular, glycosylation sites in fucosylated N-glycans (Garrido et al., 2012).

Other glycosidases with potential application in the field of glycobiology are β-L-arabinobiosidase (HypBA2) and lacto-N-biosidase (LBNase) from B. longum subsp. longum. HypBA2 hydrolyzes β-L-arabinofuranoside bonds of hydroxyproline (Hyp), releasing the disaccharide L-arabinofuranose (Araf)-β1,2-Araf from carrot extensin and potato lectin. Therefore, HypBA2 can be used to determine the presence and study the roles of β-L-arabinofuranosides in plant glycoproteins (Fujita et al., 2011). LNBase released GalNAcb1-3-GlcNAc (Lacto-N-biose I) and lactose from lacto-N-tetraose, the main component of HMOs. Consequently, LNBase can serve as a tool in the field of glycomics to examine the structures of glycoconjugates and to distinguish between structures that possess lacto-N-biose I (Sakurama et al., 2013).

Moreover, the mutant protein N423H of 1,2-α-L-fucosidase (AfcA) linked a fucose residue to the terminal galactose of some glycans, synthesizing the H antigen. Furthermore, it introduced the H antigen in O- and N-glycans from fetuin glycoproteins, ganglioside GM1, and xyloglucan oligosaccharide. Glycans containing the H antigen play an essential role in establishing a microbiota-host relationship and preventing diseases related to intestinal dysbiosis. For this reason, the modified AfcA enzyme could serve as a powerful tool to study the function of the H antigen in various proteins (Sugiyama et al., 2016, 2017).

In conclusion, the glycosylation of peptides and proteins and the production of new oligosaccharides and neoglycoconjugates mediated by recombinant bifidobacterial enzymes have been widely reported. However, almost none of the cases described for the different applications mentioned the efficiency of the reactions or whether the production of the enzymes and/or their products on a large scale is considered feasible. A deep exploration of an enzyme’s promiscuous activity against a wide range of nonnatural substrates is required to identify the bifidobacterial enzymes that can be exploited for in vitro synthesis. In this sense, to our knowledge, the genome mining strategy has not been explored in Bifidobacteria in studies identifying enzymes for synthesis and those that may be useful in glycobiology, even though it is known that Bifidobacteria possess the ability to deglycosylate a great diversity of human glycoproteins and that more than 100 genomes of different strains of Bifidobacteria have already been deposited in databases.

Supplementary Information

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Declarations

Conflict of interest

The authors declare that there is no conflict of interest.