Glycans-by-design: Engineering bacteria for the biosynthesis of complex glycans and glycoconjugates

REVIEW

Glycans-By-Design: Engineering Bacteria for theBiosynthesis of Complex Glycans andGlycoconjugates

Judith H. Merritt,1 Anne A. Ollis,2 Adam C. Fisher,1 Matthew P. DeLisa2

1Glycobia, Inc., Ithaca, New York2School of Chemical and Biomolecular Engineering, Cornell University, 254 Olin Hall,

Ithaca, New York 14853; telephone: 607-254-8560; fax: 607-255-9166;

e-mail: [email protected]

ABSTRACT: There is an urgent need for new tools thatenable better understanding of the structure, recognition,metabolism, and biosynthesis of glycans as well as theproduction of biologically important glycans and glycocon-jugates. With the discovery of glycoprotein synthesis inbacteria and functional transfer of glycosylation pathwaysbetween species, Escherichia coli cells have become a tractablehost for both understanding glycosylation and the underly-ing glycan code of living cells as well as for expressingglycoprotein therapeutics and vaccines. Here, we reviewrecent efforts to harness natural biological pathways andengineer synthetic designer pathways in bacteria for makingcomplex glycans and conjugating these to lipids and pro-teins. The result of these efforts has been a veritable trans-formation of bacteria into living factories for scalable,bottom-up production of complex glycoconjugates bydesign.

Biotechnol. Bioeng. 2013;xxx: xxx–xxx.

� 2013 Wiley Periodicals, Inc.

KEYWORDS: glycosylation; glycolipids; glycoproteins; gly-come; glycoengineering; glycosyltransferase; oligosacchar-yltransferase; lipopolysaccharides; sugar nucleotides;Escherichia coli

Introduction

Carbohydrates or saccharides represent a vast source ofbiological diversity across all domains of life. The entire com-plement of saccharides (monosaccharides, oligosaccharides,

polysaccharides, and their modifications) found either freeor conjugated to other biomolecules (such as proteins,lipids, and nucleic acids) of an organism is known as theglycome. Glycans are polysaccharides or oligosaccharidesthat can exist in a free form or as attachments to otherbiomolecules. As conjugates, glycans add an additional layerof information to biomolecules and have diverse biologicalroles such as regulating protein structure and function andaltering cell surface properties (Hitchen and Dell, 2006).

The process whereby a glycan (i.e., a glycosyl donor) isenzymatically attached to a hydroxyl or other functionalgroup of another biomolecule (i.e., a glycosyl acceptor) isreferred to as glycosylation. When attached to proteins,glycans can influence folding, stability, molecular interac-tions, and quality control (Helenius and Aebi, 2001;Helenius and Aebi, 2004; Schwarz and Aebi, 2011; Varki,1993). The significance of glycosylation is perhaps bestillustrated by the fact that approximately 70% of therapeuticproteins either approved by European and US regulatoryagencies or in clinical and preclinical development areglycoproteins (Sethuraman and Stadheim, 2006). Two of themost common types of glycosylation associated withtherapeutic glycoproteins are N-linked, in which glycansare covalently attached to the amide nitrogen of asparagineresidues, and O-linked, in which glycans are typicallyattached to the hydroxy oxygen of serine or threonineresidues.

N- and O-linked glycans impact several parameters thatdirectly affect the therapeutic efficacy of glycoproteins inhumans. For example, they are known to improve thepharmacokinetics of glycoproteins such as erythropoietin(EPO; Macdougall, 2002), to target therapeutic proteins tospecific cells or tissues such as in the case of glucocer-ebrosidase (Friedman et al., 1999), and to modulate thebiological activity through interactions with specificreceptors such as in the case of immunoglobulin G (IgG)molecules (Rothman et al., 1989). Conversely, the incorrectstructure or attachment site (on a protein backbone) ofglycans can adversely affect pharmacokinetics and in some

Correspondence to: M. P. DeLisa

Contract grant sponsor: National Science Foundation

Contract grant number: CBET-1159581

Contract grant sponsor: National Institutes of Health

Contract grant number: 2R44GM088905-02

Received 17 December 2012; Revision received 5 February 2013;

Accepted 22 February 2013

Accepted manuscript online xx Month 2012;

Article first published online in Wiley Online Library

(wileyonlinelibrary.wiley.com).

DOI 10.1002/bit.24885

� 2013 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2013 1

cases lead to immunogenic responses (LaTemple et al.,1999). For these reasons, expression systems used to producetherapeutic glycoproteins must be carefully chosen basedon their ability to synthesize human-compatible glycanstructures.

Mammalian expression systems, such as Chinese hamsterovary (CHO) cell lines, are currently the preferred host forproducing therapeutic glycoproteins; however, severalalternative glycoprotein expression hosts are currently beingexplored including, but not limited to, plant and insect cells,yeast, and bacteria. The potential of bacteria to produceglycoprotein therapeutics and vaccines is a recent develop-ment (Feldman et al., 2005; Ihssen et al., 2010; Valderrama-Rincon et al., 2012) and the focus of this review (Fig. 1);however, the ability of these organisms to produce naturaland engineered glycans and glycoconjugates has long been

known. Bacteria produce a large repertoire of glycans as partof their lipopolysaccharide (LPS), capsular polysaccharide(CPS), and antibiotic glycosides. For example, some bacteriaproduce surface-attached CPSs that surround the cell withhigh molecular weight glycans. Approximately 80 capsularserotypes have been identified in Escherichia coli alone(Orskov et al., 1977). In pathogens, CPSs are knownvirulence factors and function in protecting the cell from thehost immune system (Domenico et al., 1994; O’Riordan andLee, 2004; Whitfield, 2006). The symbiotic gut microorgan-ism Bacteriodes fragilis is particularly adept at CPS synthesisand is able to vary its surface by expressing eight distinctpolysaccharides (Krinos et al., 2001). One polysaccharide inparticular is an unusual zwitterionic structure that aids indevelopment of the host immune system (Mazmanian et al.,2005).

Figure 1. Production of human therapeutics and vaccines in glycoengineered E. coli. Pathways for assembling pathogen-specific O-polysaccharide antigens and

‘‘humanized’’ N-glycans and their site-specific transfer to target secretory proteins have been described recently (Feldman et al., 2005; Ihssen et al., 2010; Valderrama-Rincon

et al., 2012). Several glycosyltransferases sequentially add the sugars to a lipid carrier (1 and 2). The lipid-linked O-antigens or N-glycans are flipped into the periplasm (3). In the

O-antigen pathway, the O-antigen subunits are polymerized (4). An oligosaccharyltransferase transfers the O-antigens or N-glycans to Asn residues of the acceptor protein (5),

which is translocated into the periplasm (6).

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The first bacterial glycoproteins were discovered in thesurface layers (S-layers) of Clostridium species (Sleytr, 1975).Glycosylation of proteins in the bacterial S-layers involvesthe attachment of long glycan repeats in O-linkages toserine, threonine, or even tyrosine residues (Zarschler et al.,2010). This process is unlike eukaryotic O-linked proteinglycosylation, more closely resembling bacterial LPSsynthesis. Various O-linked glycoproteins have beendiscovered in the flagella of bacteria (for a recent reviewsee, Nothaft and Szymanski, 2010). However, only recentstudies have shown general pathways of O-linked glycosyla-tion capable of modifying multiple bacterial proteins(Faridmoayer et al., 2007; Gebhart et al., 2012; Iwashkiwet al., 2012b; Ku et al., 2009; Vik et al., 2009).

Just over 10 years ago, evidence of a bona fide N-linkedprotein glycosylation pathway was found in the Gram-negative pathogenic bacterium Campylobacter jejuni(Szymanski et al., 1999). Importantly, it was shown thatthis N-linked protein glycosylation pathway was transferableto other bacterial species, such as E. coli lacking nativeglycosylation pathways (Linton et al., 2002; Wacker et al.,2002; Young et al., 2002). Overall, there are manycommonalities shared by bacterial and mammalian proteinglycosylation pathways (Dell et al., 2010). However,bacterial glycoprotein synthesis systems also share somesimilarity with bacterial LPS synthesis including the abilityto utilize some of the same glycan substrates (Hug andFeldman, 2011).

With the knowledge that bacteria can produce bothN-linked and O-linked glycoproteins, there has been arenewed interest in engineering bacteria for the synthesis ofglycans and glycoconjugates. Although the manipulationof glycans in eukaryotic cells has been the subject ofmuch study (Elliott et al., 2003), the field of bacterial

glycoengineering—particularly focused on the synthesis ofdesigned glycans in E. coli—is still relatively young and is thefocus of this review.

Bacterial Glycoengineering

Compared to other biological information carriers that codefor cellular processes, monosaccharides far exceed bases ofnucleic acids, and amino acids of proteins (Gabius, 2000;Gupta et al., 2010; Turnbull and Field, 2007). This is becausesaccharides are unique in their enormous capacity forcomplexity and diversity due to the many potential linkages,stereo-isomerism, and frequent branching that can be madebetween monosaccharide components. Owing to thisdiversity, the information flow from genome to glycomeincreases exponentially (Fig. 2). Further complicatingmatters is the fact that the synthesis of oligo- andpolysaccharides is technically complex as it is not a templatedriven process. Instead, it is driven by biosyntheticglycosylation pathways, governed by the availability ofcarbohydrate substrates and the expression and activitylevels of enzymes and sugar-nucleotide transporters. Forthese reasons, there are advantages to using a biologicalsystem to control glycan synthesis. Indeed, in their report tothe National Academies, the committee on assessing theimportance and impact of glycomics and glycosciencesconcluded that a complete understanding of glycans andtheir many important roles can only be realized through thedevelopment of new tools including the metabolicglycoengineering of microorganisms (Walt et al., 2012).

Genetic and metabolically engineered bacteria haveproven to be effective systems for the production of freeoligo- or polysaccharides. Such glycoengineered bacteriaavoid the need for purified enzymes or use of protective

Figure 2. Complexity and diversity of the glycome. The information flow from genome to glycome increases exponentially (diagram not drawn to scale). Glycans amplify the

genomic information content of cells and their proteins, providing functional diversity originating from only a single gene. Genome, transcriptome, and proteome are governed by

template-driven processes using unidirectional (nucleotide base pairs indicated using single letter codes 30 ! 50) or bidirectional building blocks (amino acids indicated by standardsingle-letter codes N! C, C!N). In contrast, glycans are generated by complex biosynthetic glycosylation pathways in a non template-driven manner, governed by availability of

carbohydrate substrates, expression and activity levels of enzymes and sugar–nucleotide transporters. The monosaccharide building blocks (shown using standard abbreviations)

of mammalian glycans are combined in a variety of configurations and exhibit linkage and stereo-isomerism and frequent branching (adapted from Gupta et al., 2010).

Merritt et al.: Glycans-By-Design 3

Biotechnology and Bioengineering

groups to control regio-specificity during chemical synthesis(Perugino et al., 2004). The engineered bacterial culturesbecome a ‘‘living factory’’ and production can be scaledvolumetrically (Ruffing and Chen, 2006). Many oligosac-charides have been successfully produced in metabolicallyengineered E. coli including Lewis X tetrasaccharides(Dumon et al., 2006), the nonsulfated HNK-1 carbohydrate(Yavuz et al., 2008), globotriose and globotetraose (Antoineet al., 2005), human milk oligosaccharides (Priem et al.,2002), chitinbiose (Cottaz and Samain, 2005), and thecarbohydrate portions of the gangliosides GM1 and GM2(Antoine et al., 2003).

Although the processes described above are closely relatedand represent important engineering advances, we make animportant distinction here between the synthesis of freeglycans versus the coupled synthesis and conjugation ofglycans. In general, coupled glycan synthesis and conjuga-tion requires: (i) a pool of nucleotide sugars to serve assubstrates; (ii) one or more glycosyltransferase enzymes tocatalyze assembly of the desired stereospecific glycan; and(iii) a biomolecule to serve as the foundation for glycanassembly. Glycans can be assembled directly on the desiredbiomolecule, or they can be transferred to the targetbiomolecule as a final step (as in the case of proteinglycosylation). There has recently been much work towardproducing glycoproteins in bacteria. Here, we will focus onthe synthesis of glycans for in vivo conjugation to a moleculeof interest, with particular focus on recombinant glycopro-teins in E. coli.

Engineering Glycosyl Donors

The first step in glycan synthesis is the generation of thenucleotide sugars required as glycosyl donors for mostglycosyl transferases. Enzymatic production of nucleotidesugars in vitro may be impractical because their synthesissometimes requires the activities of multiple enzymesworking consecutively. Bacterial glycan synthesis typicallyutilizes nucleotide sugars that are synthesized completelyor partially in vivo from precursors supplied in themedium. Here, we focus on seven major monosaccharidesthat are common in organisms ranging from bacteria tohumans: glucose (Glc), galactose (Gal), N-acetylglucosamine(GlcNAc),N-acetylgalactosamine (GalNAc), mannose (Man),fucose (Fuc), and N-acetylneuraminic acid (NeuNAc or sialicacid). Of course, there are many other monosaccharidesproduced naturally by bacteria that may be of future interestfor glycoengineering such as glucuronic acid (GlcA).However, other sugars like GlcA can be enzymaticallyderived from the seven major monosaccharide buildingblocks (i.e., from the activity of glucose dehydrogenase inthe case of GlcA; Schiller et al., 1976).

To be useful as glycosyl donors, monosaccharides areconverted to nucleotide sugars, which are the activatedforms of monosaccharides. The nucleotide sugars UDP-Glcand UDP-GlcNAc are present in E. coli as these are requiredfor housekeeping functions including cell wall and LPS

biosynthesis (Samuel and Reeves, 2003). UDP-Gal, alsopresent in laboratory E. coli, is formed directly fromUDP-Glc by UDP-glucose epimerase (GalE) or by galactose-1-phosphate uridyltransferase (GalU). Similarly, UDP-GalNAc may be formed directly from UDP-GlcNAc byUDP-GlcNAc epimerase (Gne; Paton and Paton, 1999).Other nucleotide sugar donors are more difficult tosynthesize. GDP-mannose is an intermediate of E. coli colanicacid synthesis and is formed from mannose-1-phosphate andGTP by mannose-1-phosphate guanylyltransferase (ManC;Stevenson et al., 1996). GDP-fucose may then be made fromGDP-mannose through the action of GDP-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (WcaG; Andrianopoulos et al., 1998;Stevenson et al., 1996). CMP-NeuNAc is formed from CTPand NeuNAc (which can be provided in the media orproduced in vivo) by CMP-NeuNAc synthase (NeuA; Priemet al., 2002). All of these nucleotide sugars can, in theory, besynthesized from glucose (Fig. 3).

Production is complicated by the fact that while nativepathways might exist for generation of the desirednucleotide sugars, sometimes the biosynthesis pathway isnot constitutively expressed in E. coli. The manB, manC,gmd, and wcaG genes for GDP-fucose formation in thecolanic acid pathway must be induced, such as throughoverexpression of the pathway activating protein RcsA,or expressed ectopically (Dumon et al., 2001, 2006).Accumulation of a desired nucleotide sugar may beenhanced in some cases by deleting genes required fornon-essential competing pathways. For example, deletingthe wcaJ gene required for the first step of colanic acidsynthesis promotes availability of GDP-fucose (Dumon etal., 2001). Alternatively microbial coupling approaches,wherein multiple strains are combined to provide differentnecessary components, have been successfully applied togenerate nucleotide sugars (Ruffing and Chen, 2006).

In eukaryotes, nucleotide sugars are synthesized in thecytostol (except CMP-NeuNAc which is synthesized in thenucleus) and transported into the Golgi by nucleotide sugartransporters (for a recent review see (Liu et al., 2010). Inbacteria, nucleotide sugars are typically synthesized andremain in the cytoplasm, except in cases where they areobtained directly from a host (Severi et al., 2007). Thismeans that all bacterial glycans must be assembled in thecytoplasmic environment or else nucleotide sugar trans-porters must be expressed to allow glycan assemblyextracytoplasmically (e.g., in the periplasm). While a recentreport indicates that nucleotide sugar transporters can betargeted to the E. coli inner membrane (Tiralongo andMaggioni, 2011), it remains unclear if this strategy will beeffective in the context of bacterial glycoengineering.

Engineering Glycans and Glycolipids

The second step in glycan synthesis is the concomitant orstepwise expression of glycosyltransferases that catalyze


the assembly of glycans directly on a lipid carrier (e.g.,undecaprenyl pyrophosphate [Und-PP] in E. coli) or aprotein. Because these enzymes can be derived from a widevariety of organisms and native biological processes, theremay be technical hurdles in achieving functional products.These challenges include: (i) proper folding and localizationof the desired enzyme(s) at sufficient levels; (ii) obtaining aspecific glycosyltransferase activity towards the requisitesubstrate molecule; and (iii) developing a reasonable assayto determine success or failure. Furthermore, strategies forengineering glycans must be designed to assemble anoligosaccharide in a conformation compatible with conju-gation to a desired biomolecule. Common destinations forengineered glycans include Und-PP in the inner membrane,

lipid A core oligosaccharide on the cell surface, or peptide/protein acceptors expressed in a subcellular compartment(e.g., cytoplasm, periplasm). It is known that Und-PP-linked glycans can also be released as free oligosaccharidesas has been observed with the C. jejuni N-linked proteinglycosylation pathway (Nothaft et al., 2009); hence, theremay be alternative fates for engineered glycans besidesconjugation to target biomolecules. Nonetheless, glycolipidbiosynthesis impacts many important applications includ-ing: (i) the design of CPS- and LPS-based vaccines wherebacterial glycolipids are the desired end product; and (ii) thegeneration of glycoproteins where bacterial glycolipids serveas a key intermediate as discussed in more detail below.Because the synthesis pathways for many bacterial

Figure 3. Bacterial sugar-nucleotide synthesis. The sugar-nucleotide precursors for common monosaccharides found in human glycoproteins can be produced in E. coli

using bacterially derived genes. UDP-Glc, UDP-Gal, and UDP-GlcNAc are all typically available as precursors in laboratory E. coli. Therefore, expression of only one or three

additional genes can be used to provide UDP-GalNAc or CMP-NeuNAc, respectively. GDP-Fuc is synthesized in E. coli as part of the colanic acid synthesis pathway. Accumulation

of GDP-Man or GDP-Fuc may be enhanced by expressing additional copies of the native biosynthetic pathway, and/or by mutating competing pathways for example, deleting the

gmd gene encoding the GDP-mannose-4,6-dehydratase to prevent degradation of GDP-Man.



glycolipids diverge from a single starting point, controllingbacterial glycolipid biosynthesis is crucial for generatingglycans that will eventually become part of various productsincluding CPS, LPS, and glycoproteins.

To understand how bacterial glycolipids can be exploited,it is instructive to look at their native biosynthesis pathways.All bacteria naturally synthesize numerous glycolipids thathave specific functions and cellular destinations. Theseinclude serotype-specific structures that may vary substan-tially within a species such as CPS and LPS, as well as morebroadly conserved features such as the enterobacterialcommon antigen (ECA), a polymer of amino sugars linkedto a glycophospholipid common to most Enterobacteriaceaeand restricted to this family (Kuhn et al., 1988). Manybacterial glycolipids are displayed on the cell surface wherethey often play an important role in mediating interactionsbetween the cell and its environment. For example, Gram-negative bacteria such as E. coli display LPS as a majorcomponent of the outer envelope. LPS is composed of ahydrophobic lipid A (endotoxin) component that serves asthe membrane anchor, a core oligosaccharide linked to lipidA, and an O-polysaccharide antigen (O-antigen) extendingoutward from the cell (Fig. 4). The O-antigen portion ofLPS often exhibits significant variability within a speciesand is thus useful in identifying strains. There are reportedly193 individual O-antigens synthesized by Vibrio cholera, 20O-antigens made by Pseudomonas aeruginosa, 54 O-antigensmade by Salmonella enterica, and 181 O-antigens synthe-sized by E. coli serotypes (Samuel and Reeves, 2003; Stenutzet al., 2006). The lipid-linked oligosaccharides (LLOs)destined for the O-antigen and core oligosaccharide aresynthesized on Und-PP or lipid A, respectively (Fig. 4a).These LLOs are independently translocated from the

cytoplasmic to the periplasmic face of the inner membraneby two inner membrane lipid flippases called Wzx (for Und-PP-linked O-antigen) and MsbA (for lipid A-linked coreoligosaccharides). Once flipped, O-antigens are transferredfrom Und-PP to a specific residue on the lipid A core bythe enzyme WaaL, thereby forming an LPS molecule. Thecompletely assembled LPS is then shuttled to the cell surfacewith the carbohydrate portion facing outward from the cellsurface (Fig. 4b). LPS synthesis has previously been reviewedin detail (Raetz and Whitfield, 2002). Importantly, thenative LPS pathway provides a mechanism for the assemblyand cell surface display of oligosaccharides in E. coli and hasbecome an attractive target for glycoengineering as discussedbelow.

Several groups have utilized E. coli lipid A as a base forengineering glycans. By making specific mutations totruncate the core saccharide, attachment of the native O-antigen is prevented (Heinrichs et al., 1998) and a newlyexposed sugar is made available for further modification.The terminal glucose of the core saccharide can then serve asa substrate for attachment of additional sugars, which aresubsequently displayed on the cell surface. Glycan structuresmade in this manner are summarized in Table I. Thisapproach has been used to decorate the E. coli cell surfacewith a number of glycans of clinical relevance withpotentially interesting implications. For instance, theE. coli outer envelope can be used to display and clustertherapeutic glycans, and the bacterium itself could beharnessed to act as a self-perpetuating delivery system. Inaddition, non-pathogenic glycoengineered bacteria canprovide a useful tool for studying glycans of interest outsideof their native context or for recreating molecular mimicrymechanisms (i.e., the display of carbohydrates that resemble

Figure 4. LPS biosynthesis and transport. a: O-antigen subunits are translocated across the inner membrane by Wzx, where they are polymerized by Wzy (chain length

determined by Wzz) and ligated by WaaL onto complete Core-Lipid A molecules (which are translocated separately by MsbA; Wang and Quinn, 2010). b: Completed LPS molecules

are transported across the periplasm and outer membrane by the proteins LptA, B, C, D, E, F, and G (Ruiz et al., 2009).


those of human cells and tissues) such as those used byHelicobacter pylori and Neisseria meningitidis (Moran et al.,1996; Tsai, 2001).

Paton and coworkers applied this strategy to designa potential therapeutic for Shiga-toxin (Stx) producingbacteria such as Shigella dysentariae and E. coli O157:H7.Taking advantage of the fact that some pathogenic bacterianaturally synthesize human-like glycans, the glycosyltrans-ferases LgtC and LgtE from Neisseria were expressed in anE. coli R1 waaOmutant resulting in a strain with a proposedlipooligosaccharide (LOS) structure terminating inGala(1,4)Galb(1,4)Glca (Paton et al., 2000). This glycanclosely resembles the human receptor for Stx:Gala(1,4)Galb(1,4)Glc-ceramide (globotriaosylceramide,GB3; Lingwood, 1996). A globotetraose (GB4)-like glycanresembling the receptor for an Stx variant (Stx2e) couldsimilarly be made with the expression of two additionalproteins, namely the GalNAc transferase LgtD and theUDP-GalNAc-4-epimerase Gne (Paton et al., 2001).Whenadministered orally, these glycoengineered E. coli werefound to protect mice against challenge with Shiga-toxigenicE. coli (STEC) suggesting that an effective molecular mimicof the toxin binding site had been created (Paton et al.,2000).

The same host strain of E. coli R1 was also engineered toexpress cell-surface glycans known to bind cholera toxin(Ctx) or the heat labile enterotoxin (LT) with the intention

of making potential treatments or probiotics. The GM1glycan is the intestinal receptor for Ctx, which is responsiblefor the copious diarrhea that is a hallmark of cholera. LT, animportant virulence factor of enterotoxigenic E. coli (ETEC)that causes traveler’s diarrhea, can also bind GM1 inaddition to alternate glycans including GM2 or lacto-N-neotetraose (LNnT; Angstrom et al., 1994; Angstrom et al.,2000). E. coli waaO mutant strains having truncated coresaccharides were used as a scaffold for the creation of hybridLOS structures displaying the terminal structure of the GM1or GM2 gangliosides, or LNnT. The gne gene from E. coliO113 was also included to generate the necessary UDP-GalNAc needed in the production of the GM2 mimic. Inboth cases, the toxins were found to bind the surface of therespective glycoengineered E. coli, and administration of thetoxin-binding E. coli provided a protective effect in animalmodels of the respective diseases (Focareta et al., 2006; Patonet al., 2005).

The lipid A core saccharides of E. coli K-12 and S. entericasv. Typhimurium have been similarly modified to allow forectopic glycan display. A terminal glucose residue is madeavailable for glycan assembly by mutating waaI and waaBin E. coli K-12 or waaO and waaB in S. Typhimurium. ThenanA gene encoding the sialic acid aldolase NanA was alsodeleted in both of these strains to prevent the degradation ofsialic acid that was provided in the medium. In these strainbackgrounds, expression of the CMP-sialic acid synthetase

Table I. Engineered glycans assembled on truncated lipid A core oligosaccharide.

Glycan Proposed recombinant structure Host organism Glycosyl-transferases Refs.

GB3 Gala(1,4)Galb(1,4) Glca(1,3) [Hepa(1,7)] Hepa(1,3)

Hepa(1,5) [Kdoa(2,4)] Kdoa-LipidA

E. coli R1: CWG308 LgtE, LgtC Paton et al.

(2000)

GB4 GalNAcb(1,3)Gala(1,4)Gal b(1,4) Glca(1,3)

[Hepa(1,7)] Hepa(1,3) Hepa(1,5) [Kdoa(2,4)]

Kdoa-LipidA

E. coli R1: CWG308 LgtE, LgtC, LgtD Paton et al.

(2001)

GM1 GalNAcb(1,3)GalNAcb(1,4)NeuNAca(2,3)Galb(1,4)

Glca(1,3)[Hepa(1,7)] Hepa(1,3) Hepa(1,5)

[Kdoa(2,4)] Kdoa-LipidA

E. coli R1: CWG308 CgtB, CgtA,

LgtE, CstII

Focareta et al.

(2006)

GM2 GalNAcb(1,4)NeuNAca(2,3)Galb(1,4) Glca(1,3)


Kdoa-LipidA

E. coli R1: CWG308 CgtA, LgtE, CstII Paton et al.

(2005)

GM3 NeuNAca(2,3)Galb(1,4) Glca(1,3) [Hepa(1,7)]

Hepa(1,3) Hepa(1,5) [Kdoa(2,4)] Kdoa-LipidA

E. coli K-12: LPS-1;

S. enterica sv.

Typhimurium: SK122

Lst, LgtE Ilg et al.

(2010)

Lacto-N-neotetraose Galb(1,4)GlcNAcb(1,3)Galb(1,4) Glca(1,3)[Hepa(1,7)] Hepa(1,3) Hepa(1,5) [Kdoa(2,4)

Kdoa-LipidA

E. coli R1: CWG308 LgtB, LgtA, LgtE Paton et al.

(2005)

H type II Fuca(1,2)Galb(1,4)GlcNAcb(1,3) Galb(1,4) Glca(1,3)

[Hepa(1,7)]Hepa(1,3) Hepa(1,5) [Kdoa(2,4)]

Kdoa-LipidA

E. coli K-12: LPS-1 LgtE, LgtA,

FutC, LgtB

Yavuz et al.

(2011)

Lewis X Galb(1,4)Fuca(1,3)GlcNAcb(1,3) Galb(1,4) Glca(1,3)


Kdoa-LipidA


S. enterica sv.

Typhimurium: SK122

LgtE, LgtA,

LgtB, FutA

Yavuz et al.

(2011);

Mally et al.

(2012)

Lewis Y Fuca(1,2) Galb(1,4)Fuca(1,3) GlcNAcb(1,3)Galb(1,4)

Glca(1,3) [Hepa(1,7)] Hepa(1,3) Hepa(1,5)

[Kdoa(2,4)] Kdoa-LipidA

E. coli K-12: LPS-1 LgtE, LgtA,

FutC, LgtB, FutA

Yavuz et al.

(2011)

type-2 N-acetyllactosamine

(LacNAc)

Galb(1,4)GlcNAcb(1,3)Galb(1,4) Glca(1,3)

[Hepa(1,7)]Hepa(1,3) Hepa(1,5) [Kdoa(2,4)]

Kdoa-LipidA


S. enterica sv.

Typhimurium: SK122

LgtA, LgtB Mally et al.

(2012)



SiaB, and the glycosyltransferases LgtE (b1,4-galactosyl-transferase), and Lst (a2,3-sialyltransferase) allowed fordisplay of the GM3 glycan on the truncated lipid A core (Ilget al., 2010).

With a few modifications to this system, fucosylatedglycans were also expressed on the E. coli K-12 cell surface.The wcaJ gene was mutated to prevent colanic acid synthesisthereby allowing for cellular accumulation of the requiredGDP-fucose. Building on the a-glucose in the lipid A core,expression of the glycosyltransferases LgtA, LgtB, and LgtEwere used to assemble the previously described LNnT glycan(Paton et al., 2005). Addition of H. pylori FutC transferredan a1,2-fucose to the terminal galactose generating a mimicof the blood group H type II glycan (Yavuz et al., 2011).Co-expressing the glycosyltransferases necessary to synthe-size the LNnt glycan along with the H. pylori a1,3-fucosyltransferase FutA led to the appearance of a Lewis Xglycan mimic. However, multiple glycoforms were presentwhich was attributed to the low specificity of FutA. WhenFutC was also co-expressed in the Lewis X strain, someproduction of a Lewis Y-like glycan was detectable with aLewis Y-specific antibody; however, this glycan did notappear to be the major species present (Yavuz et al., 2011).Recently polymeric Galb(1,4)GlcNAc (LacNAc), a type-2N-acetyllactosamine common in mammalian N-linked gly-cans, was also built on the truncated lipid A core throughco-expression of LgtA and LgtB in both E. coli K-12 and S.enterica sv. Typhimurium (Mally et al., 2012).

A second strategy exists for engineering glycans foreventual display on the lipid A core saccharide. Thisapproach highjacks the pathway for making O-antigens,which are assembled separately on the lipid Und-PP, flippedinto the periplasm by Wzx, and subsequently ligated tothe lipid A core saccharide by WaaL (Fig. 4a). Whileoligosaccharides made in this way are also ultimatelydisplayed on the cell surface from the lipid A core, theprocess whereby these sugars are assembled and transferredonto lipid A is mechanistically distinct from the processesdescribed above. It is noteworthy that many typicallaboratory E. coli K-12 strains have lost the ability tosynthesize a full O-antigen and instead, a single GlcNAcresidue is transferred from Und-PP to the lipid A coresaccharide (Stevenson et al., 1994). This GlcNAc is aconvenient site for glycoengineering.

Taking advantage of the truncated LPS in E. coli K-12,researchers have used it as a tool to study other glycosyl-transferases outside of their native context. Plasmid DNAencoding LOS synthesis genes from Haemophilus influenzawas used to transform an E. coliK-12 strain (Abu Kwaik et al.,1991), and the resulting LPS was examined by massspectrometry (Phillips et al., 2000). Analysis revealed ahybrid LPS structure in which the E. coli lipid A core hadbeen modified with oligosaccharides from H. influenza.Several different chimeric LPS structure were generatedin this way with the following proposed structures: Gal(1,3)GlcNAc; Gal(1,4)GlcNAc(1,3)Gal(1,3)GlcNAc; GlcNAc/Gal(1,6)Gal(1,4)GlcNAc(1,3)Gal(1,3)GlcNAc (Phillips et al.,

2000). This approach could easily be extended to makenumerous hybrid LPS structures simply by transforming E.coli cells with genes encoding glycosyltransferases derivedfrom the LOS or O-antigen pathways of other bacteria.Owing to the diversity and number of glycosyltransferases innature, the spectrum of unique glycans that could beproduced biosynthetically would be staggering. Across E.coli strains alone, there are predicted to be 272 glycosyl-transferases associated with O-antigen synthesis (Lundborget al., 2010). These enzymes attach 23 sugars in specificcombinations to assemble O-antigens numbered O1through O181 (Stenutz et al., 2006). Thus, bacteria mayserve as a rich source of glycosyltransferases for makingdiverse prokaryotic and eukaryotic glycan structures.

Engineering Glycan Attachment to Proteins

In contrast to the widespread occurrence of bacterial glycanlayers such as CPS and LPS, the incidence of glycoproteinsin bacteria is much less prevalent. Nonetheless, it is nowfirmly established that a growing number of bacteria harborgeneral pathways for N- and O-linked protein glycosylation,both of which are capable of modifying multiple substrateproteins (Nothaft and Szymanski, 2010). As mentionedabove, a bona fide N-glycosylation pathway was discoveredin C. jejuni (Szymanski et al., 1999; Fig. 5a) and subsequentlytransferred to E. coli, endowing the latter organism with theability to produce N-linked glycoproteins (Linton et al.,2002; Wacker et al., 2002; Young et al., 2002). Formation ofnatural and heterologous N-glycoproteins in C. jejuni andE. coli, respectively, is catalyzed by the bacterial oligo-saccharyltransferase (OST), named PglB, which transfersUnd-PP-linked glycans to specific asparagine residues insecretory proteins (Fig. 5b). PglB is similar in sequence andoverall structure to STT3, the catalytic subunit of theeukaryotic OST, and both are essential for N-glycosylationin prokaryotes and eukaryotes, respectively (Wacker et al.,2002; Yan and Lennarz, 2002a,b). In fact, the entire bacterialglycosylation pathway is strikingly similar to the earlystages of the eukaryotic glycosylation process (Weerapanaand Imperiali, 2006; Fig. 5b). There are several notabledifferences, however, including: (i) the structures of thetransferred glycans, specifically a GalNAc5GlcBac heptasac-charide in C. jejuni (Jervis et al., 2012) and high mannosein eukaryotes; and (ii) the fact that the eukaryotic OST isa heterooligomeric complex that involves nine differentsubunits including STT3 (Knauer and Lehle, 1999), whereasthe bacterial OST constitutes a single subunit (Lizak et al.,2011b).

Since these pioneering studies, E. coli cells expressingC. jejuni PglB have been used for producing a wide variety ofglycoproteins. Initial work demonstrated the glycosylationof native C. jejuni glycoproteins such as AcrA (Wacker et al.,2002). More recently, eukaryotic proteins such as antibodyfragments (scFvs), immunoglobulin G (IgG) Fc domains,and green fluorescent protein (GFP) have also beensuccessfully glycosylated by PglB in E. coli (Fisher et al.,


Figure 5. Bacterial N-linked glycosylation. a: The 17-kb pgl locus of C. jejuni encodes the N-linked glycosylation machinery and has been fully reconstituted in E. coli.

b: Comparison of N-linked glycosylation in prokaryotes (left) and eukaryotes (right). In both systems, several glycosyltransferases synthesize the glycan by sequential addition of

nucleotide-activated sugars on a lipid carrier on the cytoplasmic face of the inner membrane. Once assembled, a flippase transfers the LLOs across the membrane where the OST

catalyzes the transfer to Asn residues of periplasmic or ER substrate proteins. PglB is a single-subunit, integral membrane protein that is homologous to the catalytic subunit of the

eukaryotic OST STT3 (note that PglB and STT3 complex are not drawn to scale). Whereas eukaryotes and archaea use an N-X-S/T acceptor sequence (where X is any amino acid

but Pro), PglB requires an extended motif that includes an Asp or Glu residue in the �2 position (D/E-X�1-N-Xþ1-S/T, where X�1 and Xþ1 can be any amino acid except Pro). PglB

can transfer sugars co-translationally like eukaryotes and also post-translationally to locally flexible structures in folded proteins. c: Schematic of the synthetic pathway for

synthesis of a trimannosyl core glycan and transfer to acceptor sites in target proteins (Valderrama-Rincon et al., 2012).



2011; Lizak et al., 2011a; Schwarz et al., 2010). Even thoughglycan attachment occurs in the periplasm, target proteinsdo not require extensive or permanent residence in theperiplasmic space to be glycosylated. For example, proteinsdestined for the outer membrane (e.g., E. coli OmpX),secreted into the medium as soluble proteins (e.g., E. coliYebF), or released from the cell in membrane vesicles (e.g.,E. coli ClyA) can all be glycosylated prior to export from theperiplasm of glycoengineered E. coli (Fisher et al., 2011).

Bacterial glycoengineering is potentiated by the abilityof PglB to transfer diverse oligosaccharide structures,provided that the glycans are assembled on the Und-PPcarrier (Fig. 1). For example, expression of C. jejuni PglB inE. coli has been exploited to create novelN-glycan structurescontaining distinct O-antigens from E. coli, Pseudomonasaeruginosa, Yersinia enterocolitica, and Shigella dysenteriaetype 1 (Feldman et al., 2005; Ihssen et al., 2010; Iwashkiwet al., 2012a). Motivated by the ability of PglB to transferdiverse Und-PP-linked glycans, we recently assembled thefirst eukaryotic protein glycosylation pathway in E. coli(Valderrama-Rincon et al., 2012; Fig. 5c). This involvedbottom-up engineering of a synthetic pathway foreukaryotic glycan biosynthesis involving four eukaryoticglycosyltransferases, including the yeast uridine diphos-phate-N-acetylglucosamine transferases Alg13 and Alg14and the yeast mannosyltransferases Alg1 and Alg2. Together,these enzymes produced Und-PP-linked Man3GlcNAc2glycans. Combining this pathway with C. jejuni PglBenabled glycosylation of specific asparagine residues intarget proteins including an scFv, the Fc domain of an IgG,and a variant of human growth hormone (hGH).Interestingly, the ability to transfer eukaryotic trimannosylchitobiose glycans could also be accomplished with C. lariPglB (Valderrama-Rincon et al., 2012), suggesting that therelaxed specificity toward the glycan structure may be ageneral feature of different PglB homologs. In support ofthis notion, PglB from Desulfovibrio desulfuricans wasobserved to transfer its endogenous glycan and the C. jejuniheptasaccharide glycan, which are thought to be structurallydistinct (Ielmini and Feldman, 2011). However, unlike withC. jejuni PglB, D. desulfuricans PglB transferred Und-PP-linked O-antigens inefficiently or not at all. Hence, somePglB homologs may have more stringent glycan specificitythan C. jejuni PglB, preferring short over long glycan chains.Clearly, more work needs to be done to fully understand therules governing bacterial OST function because, so far, onlya small number of bacterial OSTs have been characterizedin the context of a similarly small number of structurallydistinct glycans.

In an ideal system for scalable glycoprotein production,diverse glycan synthesis pathways reconstituted in E. coliwould allow for complete glycoprotein modification in asingle in vivo step. However, an alternative two-stageglycosylation strategy for making eukaryotic glycoproteinshas been reported (Schwarz et al., 2010). In this method,the key GlcNAc-Asn linkage is produced in vivo usinga modified C. jejuni glycosylation pathway. Following

purification of the GlcNAc-containing glycoproteins, thebacterial portion of the glycan is trimmed and enzymaticallyremodeled in vitro to yield a eukaryoticN-glycan. While thiscombined approach may be slower and more difficult toscale than an entirely in vivo approach, it enables productionof complex glycan structures that have not yet beenassembled in bacteria. For example, this method has beenused to create glycoproteins modified with a high mannose-type glycan (Man9GlcNAc2), a bi-antennary complex-typeglycan, and the Lewis X (Lex) antigen, a Galb(1,4)Fuca(1,3)GlcNAc-containing glycan of interest for immunother-apeutics. In the case of Man9GlcNAc2 glycans, E. coli wereused to produce target glycoproteins with a modifiedC. jejuni (GalNAc)5 glycan attached through a GlcNAc-Asnlinkage (Schwarz et al., 2010), the same linking sugar that isfound in eukaryotic glycoproteins. Subsequent in vitroenzymatic hydrolysis of the bacterial GalNAc residuesand chemoenzymatic extension resulted in site-specificattachment of a mammalian glycan. For glycoproteinsmodified with the Lex antigen, a tetrasaccharide ofGalb(1,4)GlcNAcb(1,3)Galb1,3)GlcNAc was produced inE. coli harboring the lsgC-F gene cluster from H. influenzafor lipooligosaccharide synthesis. The resulting glycan wastransferred to AcrA by C. jejuni PglB. The terminal sugars ofthis glycan only require addition of a1–3 linked fucose tocomplete the Lex antigen. This final step was completed by invitro fucosylation of the purified glycoprotein using theH. pylori fucosyltransferase FucT (Hug et al., 2011).

With the engineering of several artificial proteinglycosylation pathways in E. coli (Hug et al., 2011;Valderrama-Rincon et al., 2012; Wacker et al., 2002), andthe potential for many more ‘‘designer’’ glycosylationsystems, comes a growing need for tools that enable enzyme,pathway, and strain optimization. Metabolic engineering ofglycosylation-competent E. coli has been used to improveglycosylation efficiency. For example, overexpressing po-tential rate-limiting glycosyltransferases (e.g., E. coli WecA)and codon optimization of heterologous genes (e.g., C.jejuni pglB) have been reported to improve glycosylationefficiency of bacterial glycoproteins by approximatelytwofold (Pandhal et al., 2012). However, no significantdifferences were observed following an increased expressionof BacA, which was thought to increase availability of thelipid-linked donor oligosaccharides. A more sophisticatedmetabolic engineering strategy based on high-throughputproteomics and probability-based metabolic networkanalysis identified target genes that increase glycosylationof the model glycoprotein AcrA (Pandhal et al., 2011).Specifically, enhancing flux through the glyoxylate cycle viaoverexpression of isocitratelyase (ICL) resulted in a 24%increase in AcrA glycosylation efficiency. Changing themode of protein translocation may also influence the extentof glycosylation. Indeed, clear differences in glycosylationefficiency of AcrA were observed depending on whether theprotein was directed to the Sec, twin-arginine translocation(Tat), or signal-recognition particle (SRP) pathway fortranslocation into the periplasm (Fisher et al., 2011). Even


simple changes in growth medium and culture conditionscan influence the extent of AcrA glycosylation in E. coli(Ihssen et al., 2010), demonstrating the potential merit ofmore extensive studies on the effects of glycoproteinproduction in response to physiological changes of theglycoengineered bacteria. The availability of systems forglycan display, either on the surface of bacteria or onfilamentous phage particles (so-called glycophage display;Celik et al., 2010; Durr et al., 2010; Fisher et al., 2011),should open the door to enzyme-, pathway-, and/orgenome-scale optimization of glycosylation-competentE. coli.

A current obstacle to the production of humanbiotherapeutic proteins in glycoengineered E. coli is themore stringent acceptor site specificity that has beenobserved for C. jejuni PglB and some of its homologs.Whereas the eukaryotic OST is specific for acceptor sitescomprised of N-X-S/T (where X is any amino acid exceptproline), the bacterial OST follows the ‘‘minus two rule’’(Kowarik et al., 2006). This rule establishes that PglBrequires a negatively charged amino acid at position minustwo from the acceptor Asn residue. Hence, bacterialglycosylation involves an extended motif of D/E-X1-N-X2-S/T (where X1 and X2 are any residues except proline). Theminus two rule may be the result of a conserved arginineresidue of the OST, which, based on R331 of C. lari PglB,appears to form a salt bridge with the negatively chargedresidue in the �2 position of the acceptor site (Lizak et al.,2011b). Interestingly, eukaryotic OSTs, which do notrequire an acidic residue in the acceptor motif, contain aconserved aspartic acid in the OST itself, corresponding tothe bacterial R331 (Lizak et al., 2011b). Regardless, becauseC. jejuni PglB obeys this rule, glycosylation of eukaryoticglycoproteins in bacteria typically requires modification ofthe native acceptor site(s) to include an acidic residue at the�2 position (Fisher et al., 2011; Schwarz et al., 2010;Valderrama-Rincon et al., 2012). Sequence changes tobiotherapeutic proteins, however, have to be carefullyconsidered to avoid unwanted changes in protein activity orspecificity.

While C. jejuni PglB is currently the preferred OST forbacterial glycoprotein production, this is largely becauseonly a small number of OSTs have been functionallycharacterized in E. coli. Even with a small sample size, asurprising amount of variation among bacterial OSTs hasbeen observed upon expression in E. coli. For example,C. lari PglB, which exhibits 56% sequence identity toC. jejuni PglB, glycosylated an acceptor site in C. jejuniAcrA that lacked the canonical �2 acidic residue, albeitinefficiently (Schwarz et al., 2011b). C. lari PglB alsomodified the native E. coli protein PotD, which is notdetected as a substrate for C. jejuni PglB. Along similar lines,D. desulfuricans PglB preferentially glycosylates an acceptorsite in AcrA that does not possess a �2 acidic residue(Ielmini and Feldman, 2011). Similar relaxed acceptor sitespecificity may also exist for the D. gigas OST. This view isbased on the crystal structure of D. gigas HmcA, which is

glycosylated at an acceptor site—T259AN261GT263—thatdoes not have a negatively charged amino acid in the �2position relative to the Asn. Even OSTs that follow theminus two rule, such as one of the Helicobacter pullorumPglB homologs, can still give rise to different glycosylationpatterns compared to C. jejuni PglB (Jervis et al., 2010).These few examples show that natural variations in theprimary sequence of PglB are sufficient to alter the activity ofthis enzyme towards target proteins, either expanding orrestricting acceptor site specificity. Moreover, with a three-dimensional structure for C. lari PglB now available (Lizaket al., 2011b), structure-guided rational design and directedevolution can be implemented for the isolation of mutationsthat alter or improve bacterial OST function (Ihssen et al.,2012).

Other Modes of Protein Glycosylation in Bacteria

Thus far, the majority of studies on N-linked glycosylationin bacteria have focused on the transfer of preassembledglycans from a lipid carrier to periplasmic acceptor proteinsin a process catalyzed by a membrane-bound OST.However, a novel N-linked protein glycosylation systemthat functions in the cytoplasm was described recently(Schwarz et al., 2011a). This pathway involves a soluble,cytoplasmic N-glycosyltransferase that uses nucleotide-activated monosaccharides as donors to modify peptidesand proteins. In one notable example, the HMW1Chomolog from A. pleuropnemoniae was observed to transferglucose to asparagine residues within the same N-X-S/Tacceptor motif used by eukaryotic and some prokaryoticOSTs. Despite the similar acceptor site specificity aseukaryotic OST, the HMW1C homolog is structurallyunrelated and apparently uses a different catalytic mecha-nism. Interestingly, the APP7_1697 gene from A. pleur-opnemoniae encodes a polymerizing a6GlcT that modifiesthe product of the HMW1C homolog with the addition ofup to six glucose units (Schwarz et al., 2011a). However,whether this pathway can be functionally reconstitutedin the cytoplasm of E. coli for scalable production ofglycoproteins has not been demonstrated.

The attachment of GalNAc to cytoplasmic proteins inE. coli has been achieved by expressing a GalNAc-T enzymethat is involved in the first step of human, mucin-typeO-glycosylation (Henderson et al., 2011). The resultingglycoproteins bear a GalNAc that can be further function-alized by in vitro chemical modification. The advantage ofthis approach is the ability to control the attachment site(s)for further bioconjugation (e.g., PEGylation), which canreduce product heterogeneity and minimize the loss inactivity that often accompanies unspecific bioconjugationmethods. The site-specificity of conjugation is controlled bythe known or introduced glycosylation sites in the targetprotein and the attached glycan, which is defined by theglycosyltransferase pathways expressed in the bacteria.Methods commonly used to label glycans can then be



employed to attach other conjugates to the glycoprotein.The hydroxyl groups of the glycan are first oxidized, whichfunctionalizes the sugar with aldehyde groups. Sincealdehydes are uncommon in proteins, they can serve asunique sites for chemical conjugation. While treatment withperiodate is common, enzymatic oxidation by galactoseoxidase (GAO) is thought to result in more homogenousreaction products due to less promiscuous reactivity.Oxidized glycans at defined sites in proteins can then becovalently modified in reactions involving hydroxylamine orhydrazide-functionalized molecules. Bioconjugation of asingle aGalNAc linked to a serine or threonine residue in anO-linked acceptor peptide fused to a human Fab antibodyfragment was accomplished using GAO oxidation followedby an aniline catalyzed reaction with aminooxy-polyethyl-ene glycol (AO-PEG; Henderson et al., 2011). The resultingglyco-PEGylated Fab was modified at a single site with a highdegree of conversion from the starting glycoproteins(Henderson et al., 2011), highlighting the potential ofglycoengineered bacteria for making bioconjugates.

Certain bacteria perform OST-dependent O-linkedglycosylation of pilins in a mechanism similar to N-linkedglycosylation whereby the OST transfers the glycan en blocfrom the Und-PP carrier onto target proteins in theperiplasm (for a recent review see, Nothaft and Szymanski,2010). This is distinct from the eukaryotic O-linkedglycosylation discussed above whereby nucleotide-activateddonors are directly transferred to proteins. At the heart ofbacterial O-glycosylation is an OST for which there isno eukaryotic equivalent. Examples include the PglLhomologs from Burkholderia thailandensis, N. meningitidis,and V. cholera, PilO from P. aeruginosa, and PglA from N.gonorrhoeae and Francisella tularensis. These enzymes havebeen shown to catalyze glycan attachment to naturalO-glycoprotein targets, namely pilins, in a reconstitutedE. coli system (Aas et al., 2007; Egge-Jacobsen et al., 2011;Faridmoayer et al., 2007; Gebhart et al., 2012). Similar toPglB, the OSTs PglL, PilO, and PglA exhibit relaxed glycanspecificity as evidenced by their ability to transfer non-nativeglycans to target proteins in glycoengineered E. coli(Faridmoayer et al., 2007, 2008; Gebhart et al., 2012).Differences in glycan specificities, however, were alsoapparent when the transfer of E. coli O7 antigen wasexamined. PilO was limited to transfer of two O7 antigensubunits, whereas N. meningitidis PglL transferred long andshort chains of polymerized O7 antigen as well as theP. aeruginosa O11 antigen (Faridmoayer et al., 2007). Themonosaccharide diNAcBac was also successfully attached toacceptor proteins by all PglL homologs (Gebhart et al.,2012). It should be pointed out, however, that this relaxedglycan specificity is not immediately useful because of thelimited number of glycoprotein targets available formodification and the highly variable glycosylation efficiencyassociated with different glycan/OST/target protein combi-nations. For example, relatively efficient transfer of theC. jejuni glycan and the diNAcBac monosaccharide could beachieved, but not with the same acceptor proteins (Gebhart

et al., 2012). Nonetheless, the unique characteristics ofbacterial O-linked glycosylation make this system apromising tool for glycoengineering novel glycan-basedvaccines and therapeutics, much like its N-linkedcounterpart.

Conclusions

Bacterial glycoengineering has experienced a rise inpopularity following the discovery of bacterial N-glycosyla-tion systems with the ability to generate non-native glycansand conjugate these to recombinantly expressed proteins.Because of the facile genetic manipulation in E. coli and lowassociated costs, there is a continued opportunity to explorethe synthesis of novel glycans in bacteria that may havebiological or medical relevance. This is expected to result insimple and cost effective strategies for production ofchemically defined glycans and glycoproteins at largerscales: a development that will benefit the greater scientificcommunity by providing low cost materials in sufficientquantities for research.

The expansion of glycoprotein engineering in bacteria hasthe potential to provide several important advances inglycobiology including the ability to tightly regulate theglycosylation profile including site-specific glycosylationand glycan homogeneity, development of tools to betterunderstand the role of glycosylation in protein function, andcapacity to explore the biological role of novel glycans.Expression and glycosylation of recombinant proteins inE. coli while maintaining strict control of the glycosylationprofile will also have important repercussions in manu-facturing. Eventually, it may become possible to shiftproduction of certain glycoproteins from manufacture ineukaryotic cell lines to bacteria. Compared with traditionalglycoprotein production platforms, a bacterial system mayprovide added benefits including rapid growth, relativelylow cost culture, and reduced risk of viral contamination.The recently published findings from the committee onassessing the importance and impact of glycomics andglycosciences reported that, ‘‘Glycans are directly involvedin the pathophysiology of every major disease’’ (Walt et al.,2012). We believe that advancing bacterial glycoengineeringcould provide research reagents, generate diagnostic tools,and manufacture therapeutic agents to directly impactunderstanding and treatment of these diseases.

This material is based upon work supported by the National Science

Foundation under Grant No. CBET-1159581 (to M.P.D.) and by the

National Institutes of Health under Grant No. 2R44GM088905-02

(to Glycobia and M.P.D.).

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Glycans-by-design: Engineering bacteria for the biosynthesis of complex glycans and glycoconjugates