REvIEWS

Carbohydrate-​active enzymes
(CAZymes) in the gut microbiome
Jacob F. Wardman 1,2,4, Rajneesh K. Bains2,3,4, Peter Rahfeld2,3 and
Stephen G. Withers1,2,3 ✉
Abstract | The 1013–1014 microorganisms present in the human gut (collectively known as the human
gut microbiota) dedicate substantial percentages of their genomes to the degradation and uptake
of carbohydrates, indicating the importance of this class of molecules. Carbohydrates function
not only as a carbon source for these bacteria but also as a means of attachment to the host, and
a barrier to infection of the host. In this Review, we focus on the diversity of carbohydrate-​active
enzymes (CAZymes), how gut microorganisms use them for carbohydrate degradation, the
different chemical mechanisms of these CAZymes and the roles that these microorganisms and
their CAZymes have in human health and disease. We also highlight examples of how enzymes
from this treasure trove have been used in manipulation of the microbiota for improved health
and treatment of disease, in remodelling the glycans on biopharmaceuticals and in the potential
production of universal O-​type donor blood.

Short-​chain fatty acids

Fatty acids containing up to six
carbon atoms (predominantly
acetate, propionate and
butyrate in the human gut)
that can be produced by
gut microorganisms during
fermentation.
1
Department of Biochemistry
and Molecular Biology,
University of British
Columbia, Vancouver,
British Columbia, Canada.
2
Michael Smith Laboratories,
University of British
Columbia, Vancouver,
British Columbia, Canada.
3
Department of Chemistry,
University of British
Columbia, Vancouver,
British Columbia, Canada.
4
These authors contributed
equally: Jacob F. Wardman,
Rajneesh K. Bains.
✉e-​mail: withers@chem.ubc.ca
https://doi.org/10.1038/
s41579-022-00712-1
Humans have an intimate relationship with their micro­
biota. The community of 1013–1014 bacteria in our gut1 is
well known for its many roles in human health and phys­
iology. These roles range from immune system develop­
ment2 and nutrient acquisition3 to drug metabolism4 and
modulation of host behaviour5. Accordingly, there has
been increased interest in understanding and manipu­
lating the gut microbiota. The global market for human
microbiota therapies, profiling and diagnostics was esti­
mated to be around US $275–400 million in 2019 and is
expected to rise to US $750–1,500 million by 2024 (ref.6).
Carbohydrates have an important role in shaping the
microbiota. The gut is a competitive environment for
microorganisms, and the ability to degrade carbohy­
drates (whether found in food or derived from the host
or other members of the human gut microbiota) provides
a competitive advantage to certain bacteria3. Microbial
carbohydrate-​degrading activities provide substantial
benefits to the host as well. Humans lack most of the
enzymes necessary to degrade dietary carbohydrates7.
Instead, we outsource these enzymatic needs to our res­
ident gut bacteria3,7. Although the bacteria degrade these
carbohydrates to fuel their own needs, in return the host
receives energy, typically up to 10% of the bodily energy
budget8, in the form of short-​chain fatty acids. Short-​chain
fatty acids also have anti-​inflammatory activities, can
modulate epigenetic remodelling and influence host
metabolism9. For the most part, this interplay between
carbohydrates, the microbiota and human health pro­
ceeds smoothly and quietly, but occasionally it becomes
evident. An example of this being the flatulence brought
on by excessive anaerobic fermentation of dietary oli­
gosaccharides present in legumes, brassicas and dairy
products by gas-​producing bacteria of the colon10.
The enzymes that act on carbohydrates — carbohydrate-
active enzymes (CAZymes) — operate on a diverse range
of substrates. Several distinct enzyme classes and asso­
ciated modules are included in the CAZy database and
the associated CAZypedia11, as summarized in Fig. 1.
CAZymes involved in carbohydrate degradation include
the glycoside hydrolases, polysaccharide lyases and carbo­
hydrate esterases12–14. Additionally, auxiliary activity
enzymes perform various redox transformations, which
in many cases are degradative in nature. The classes of
enzymes, and the chemical mechanisms used, are highly
dependent upon the structure of the carbohydrate sub­
strate to be cleaved. A further limitation imposed by
the anaerobic environment of the gut and the oxygen
depen­dence of many auxiliary activity enzymes is that
auxiliary activity enzymes do not seem to be widely
encoded in the human gut microbiome11,15, although
exceptions do exist16. Meanwhile, the primary enzymes
involved in carbo­hydrate synthesis are classified as gly­
cosyltransferases17. Often, CAZymes are appended with
carbohydrate-binding modules (CBMs), which have
an important role in enhancing catalytic activity of the
appended CAZyme but contain no catalytic activity of
their own18.
Because the evolution and acquisition of particular
CAZymes provide certain bacteria with competitive
advantages, bacteria of the human gut dedicate large
proportions of their genomes towards CAZymes7,19.

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Glycoside hydrolases Glycosyltransferases

O O
O O OR1 + R2 R2
OR + H2O OH R1
ROH Donor Acceptor

R = Monosaccharide, oligosacccharide,
polysaccharide or aglycone R1 (donor) R2 (acceptor)
Exo-glycoside
hydrolase
Reducing end O
Nucleoside Glycan assembly
O O O O O O P O
O O O O O OH or lipid
–
O
n
Non-reducing end
Endo-glycoside hydrolase n = 1 or 2 Protein glycosylation

O
Polysaccharide lyases GlcNAc Man
NH
O

–
Glycolipids OH
O
O–
O
O
RO O O Carbohydrate binding modules
OR′ OR′
ROH
CBM Associated with CAZymes

R′
Directs CAZymes
–
O2C
–
O2C
–
O2C to substrate
O O O O O
O O OH
O O O O
O O O O O O
O O O O O OH

R
Point of cleavage CBM binds to specific
carbohydrate motif

Carbohydrate esterases Auxiliary activities

OH O Various redox
XCOR XH OH Galactose OH transformations
O O O oxidase
X = NH or O OH
O
OH
catalysed by
HO HO
H2O RCOOH O2 H2O2
auxiliary activity
OH OH enzymes

Fig. 1 | overview of the cAZyme classes and associated modules.
Glycoside hydrolases catalyse hydrolysis of glycosidic linkages within a
wide range of carbohydrate substrates and can be classified as either
exo-​glycoside hydrolases or endo-​glycoside hydrolases depending
upon where they cleave within the carbohydrate substrate. Glycosyl­
transferases form glycosidic linkages using activated donors to transfer
sugars onto specific acceptors such as proteins, lipids or other glycans.
Polysaccharide lyases utilize a distinct mechanism to break down
polysaccharides containing uronic acids. Glycos­a minoglycans are
among the most frequently encountered substrates of polysaccharide
lyases in the human gut microbiota. Carbohydrate-​binding modules
(CBMs) are domains without catalytic activity that are often appended
to carbohydrate-​active enzymes (CAZymes). CBMs bind a range of
carbohydrate motifs of varying sizes and compositions and aid in directing
the associated CAZyme to the substrate. Carbohydrate esterases catalyse
de-​esterification of various carbohydrate substrates. Auxiliary activity
enzymes encompass a large class of redox active enzymes that function
upon carbohydrates.

Glycoside hydrolases are the largest and best-​studied class
of CAZymes responsible for carbohydrate degradation
and will be the primary focus of this Review11. Despite
their vast substrate scope, diverse 3D structures and evo­
lutionary origins, the majority of glycoside hydrolases
use one of the two chemical mechanisms that were first
proposed by Koshland almost 70 years ago based on con­
temporary understandings of acid-​catalysed glycoside
hydrolysis20 (Box 1). However, there are some intriguing
adaptations of these mechanisms to match peculiarities of
substrate structures, many of which are described later7,13.
The sheer abundance and diversity of the CAZymes
within the gut microbiome thus makes the gut an appeal­
ing starting point in CAZyme discovery for a range of
applications7,21. Metagenomic sequencing has revealed
that the human gut microbiome as a whole has one of the
highest densities of glycoside hydrolases per kilobase out
of all environments on earth21. Accordingly, gut residents
such as Bacteroides thetaiotaomicron and Bacteroides
ovatus dedicate remarkable proportions of their genomes
to glycoside hydrolases and polysaccharide lyases (~6%
of all genes encoded, whereas most organisms dedi­
cate 1–3% of their genes to CAZymes)7,19. In particular,
CAZymes involved in the degradation of dietary poly­
saccharides and human glycans are likely to be especially
abundant7. Using this as a resource, enzymes involved
in the preparation of high-​value biopharmaceuticals22,23,
and even blood type remodelling24,25, have been identified
from the gut microbiome.
This Review will focus on essentials of the enzymol­
ogy of CAZymes, common themes in bacterial carbo­
hydrate degradation (with a particular focus on the

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well-​studied Bacteroidetes phylum) and the physiologi­
cal roles of these enzymes in the deconstruction of car­
bohydrates by the human gut microbiota. Moreover, by
discussing the chemical mechanisms of these CAZymes,
as well as their applications, we aim to provide a unique
perspective into the field. Such work will highlight the
importance of connecting our understandings of funda­
mental glycobiology of the human gut microbiota with
our knowledge of CAZyme mechanisms to yield further
advances in medicine and biotechnology.
Food, drugs and the microbiota
Diet and the microbiota
Across cultures and dietary habits, microbial compo­
sition and the associated consortiums of CAZymes
within the human gut are specialized towards dietary
polysaccharides26,27. Moreover, relationships between
Box 1 | The classical Koshland mechanisms
The glycoside linkages found in starch and cellulose are among the most stable linkages
found in nature166. Fortunately, nature has done an exquisite job at evolving glycoside
hydrolases to hydrolyse such linkages. In a now seminal paper, Daniel Koshland Jr
proposed that glycoside hydrolases could follow two mechanisms on the basis of whether
the stereochemistry at the anomeric centre is retained or inverted upon hydrolysis20.
As shown in the figure (panel a), cleavage of the glycosidic linkage can be achieved via
a two-​step double displacement mechanism with net retention of stereochemistry12,167.
The first step implicates two catalytic residues (typically Asp or Glu) acting in a concerted
manner. One residue acts as the nucleophile, attacking at the anomeric centre, whereas
the other donates a proton to the departing leaving group. Next, the acid or base residue
deprotonates water as it attacks the glycosyl enzyme and releases the sugar product12,167.
Alternatively, glycoside hydrolases can proceed through a single displacement
mechanism with inversion of stereochemistry (see the figure, panel b)12,167. One carboxylic
acid residue protonates the glycosidic oxygen whereas the other residue assists
the deprotonation of a water nucleophile for direct displacement of the anomeric
substituent12,167. All steps proceed via oxocarbenium ion-​like transition states. Although
glycoside hydrolases largely follow the mechanisms described above, deviations have
been reported, several of which are described in the text13. For interested readers,
CAZypedia provides clear and useful descriptions of the mechanisms, their history and
other concepts related to carbohydrate-​active enzymes (CAZymes) and carbohydrates168.
a interesting example of this phenomenon being the
Catalytic Catalytic
acid/base acid/base
O
O
–
O rides commonly consumed in East Asian cultures) by
O
O ROH
H
O H O
O H
O acquired via transposon-​mediated horizontal gene trans­
O OH
R fer from marine bacteria that were probably consumed
–
O O
O O –
O O
diet and the microbiome composition and functional­
ities are also present across animal species26,28. As pro­
posed by Freter et al. in 1983, a large but finite number
of ecological niches are available to microorganisms
in the gut29. One of the methods by which microor­
ganisms establish themselves in the gut is through
evolution of CAZymes capable of degrading specific
polysaccharides3. Accordingly, different types of bacteria
have differing CAZyme contents7. Generally in humans,
diets richer in plant-​based carbohydrates enrich for
Prevotella spp., whereas Bacteroides spp. are more abun­
dant with diets in which fewer dietary carbohydrates
reach the gut30–34. However, species-​level and strain-​level
differences in genome content as well as other dietary
factors also affect microbial abundance in the gut35,36.
Introduction of different dietary carbohydrates drives
changes to the microbiome and the associated CAZyme
composition on various time scales. For example, within
a day of diet modification, small changes in resident
microbial diversity and metabolism are observed, along
with the introduction of metabolically active bacteria
from the new food sources35. The Hadza hunter–gatherers
undergo seasonal changes in their diet as they alternate
between a meat-​rich diet in the dry season and a honey
and berry-​rich diet during the wet season34. These die­
tary changes result in changes in both the microbiome
composition (for example, decline of Prevotella spp.
during the wet season34) and in associated CAZyme
abundance (for example, an increased abundance of
animal-​derived sialic acid (Neu5Gc)-​cleaving GH33
family members during the dry season37). It should
be noted that, in comparison, adults in industrialized
nations often have relatively stable microbiomes27,38. This
stability has been proposed to be driven by a typically
consistent (and often fibre-​poor) diet resulting in the
extinction of other­wise mutable bacterial populations
accompanied by an overall loss of bacterial diversity27,39.
As an adaptation to longer-​term dietary habits, sym­
bionts can also undergo genome-​level changes, an
Catalytic acquisition of CAZymes involved in the degradation
acid/base
of porphyran and agarose (present in algal polysaccha­
OH gut symbionts40–42. These CAZymes were most likely
with the algae40,41.

Dietary fibres
Catalytic nucleophile Catalytic nucleophile Catalytic nucleophile Gut bacteria have evolved many methods for the degra­
dation of carbohydrates from the diet. These carbohy­
drates — commonly referred to as dietary fibre — are
b Catalytic acid Catalytic acid
generally plant-​derived, escape host degradation and are
O O instead acted on by the human gut microbiota32. Starch
O ROH O–
is one of the best studied fibres in terms of its role in the
H
O O gut microbiota and is the only polysaccharide for which
O
R Homo sapiens produces its own degradative enzymes7.
OH
O H However, depending on the preparation of the food and
H
the properties of the starch, between 2 and 50% of it can
–
O O HO O escape host degradation and will be passed on to the gut
microbiota43. Numerous different pathways for starch
Catalytic base Catalytic base degradation and uptake in various gut microorganisms

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Cross-​feeding
have been characterized (recently reviewed by Cerqueira
A phenomenon in which et al.43) but one of the best known is that of the starch uti­
microorganisms utilize lization system (Sus) in B. thetaiotaomicron43. Elucidation
metabolites and degradation of the eight-​gene polysaccharide utilization locus (PUL),
products derived from other
microorganisms.
which itself was the first PUL elucidated, was initiated by
Anderson and Salyers in 1989 (refs44,45) (Box 2).
Outer membrane vesicles The seminal work on Sus provided a general model
Vesicles with distinct protein for PULs, which has been extended to others through­
cargo that are derived from
out the Bacteroidetes phylum46. This method of degra­
the outer membrane of
Gram-​negative bacteria
dation has been described as ‘selfish’ in some instances
and are released into the as it minimizes the distribution of nutrients to neigh­
environment. bouring gut microorganisms47. In general, degradation
of polysaccharides is initiated by extracellular cleav­
age of the polysaccharide and is carried out by surface
endo-​glycoside hydrolases. This produces large oli­
gosaccharides that are then rapidly transported to the
periplasm where the oligosaccharide is broken down
into simple monosaccharides and disaccharides. This
process effectively excludes other bacteria from taking
in any released carbohydrates47. In other examples, the
degradation products will not be rapidly transported
into the periplasm and can instead be shared with other
bacteria as described below48–51.
The selfish model was first noted by Cuskin et al. for the
degradation of yeast α-​mannan by B. thetaiotaomicron47.
Yeast-​containing fermented foods have been part of
human diets for centuries; thus, it is of little wonder that
gut bacteria such as B. thetaiotaomicron have evolved
ways to digest the yeast cell wall47. α-​Mannan degradation
is initiated by BT3862, a surface endo-​α-1,2-​mannosidase
(GH99) responsible for liberating Man-​α-1,3-​Man
disaccharides47 (Fig. 2). A novel substrate-​assisted catalytic
mechanism proceeding through an epoxide intermediate
Box 2 | Susc and SusD: defining polysaccharide utilization loci
Polysaccharide utilization loci (PUL) are genomic loci that encode the carbohydrate-​
active enzymes (CAZymes), glycan binding proteins, transporters, and sensors and
regulators required for binding, cleavage and import of carbohydrates169. Within
the loci, SusC, an outer membrane TonB-​dependent transporter, and SusD, a surface
glycan-​binding protein, are of particular interest. Both SusC and SusD are critical to PUL
functionality, and removal of either leads to a strong reduction of PUL functionality. Thus,
the presence of these two genes is a canonical hallmark for Gram-​negative PULs. Recent
structural characterization has shown that SusC and SusD associate in the membrane.
In what has been termed the ‘pedal bin’ model (see the figure), SusD acts as a flexible
hinged lid over SusC in the absence of substrate, exposing binding sites in SusC170. Once
substrate binds inside SusC, the SusD lid closes and substrate is allowed to pass through
SusC into the periplasm in a TonB-​dependent manner170. Recently, the definition of a PUL
has been expanded to include similar gene clusters in Gram-​positive bacteria that lack
a SusC/D pair. These, along with many other aspects of carbohydrate degradation and
uptake, are discussed in greater detail in the recent review by Briggs et al.54.
Substrate
SusD
Outer membrane
SusC
Inner membrane
TonB
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was recently proposed for the GH99 family and supported
by elegant mechanistic studies52. Briefly, a carboxylate in
the active site deprotonates the mannose 2-​hydroxyl con­
comitantly with nucleophilic attack at the anomeric centre
by the alcohol, generating a short-​lived 1,2-​anhydro sugar.
This intermediate is then hydrolysed by attack of water at
the anomeric centre with general base catalysis provided
by an active-​site glutamate residue52 (Fig. 2). Two surface
endo-​α-1,6-​mannanases (GH76) perform further degra­
dation to produce oligosaccharides that are then trans­
ported into the periplasm for further metabolism47. In the
periplasm, the degradation of the imported oligosaccha­
rides is carried out by an array of glycoside hydrolases
with varying specificity. Complete degradation of the
phosphorylated branches requires mannose-6 phos­
phatases (BT2630 or BT3783) to remove the 6-​phosphate
linkages, thereby allowing further degradation by BT3774
(GH38), which is unable to accommodate the charged
6-​phosphate linkage in the –1 subsite.
It must be noted that not all carbohydrate degradation
in the human gut microbiota is selfish. In fact, complex
carbohydrate cross-​feeding networks seem to be com­
mon in the microbiota. In many instances, co-​culturing
of bacteria that initiate carbohydrate degradation (often
referred to as keystone degraders) can enable growth of
strains that would otherwise not be able to utilize the
carbohydrate (secondary degraders)48–51. In numerous
cases, it seems that secondary degraders just lack the sur­
face endo-​glycoside hydrolases that are responsible for
initial cleavage, as exogenous addition of these enzymes
or integration of the required genes enables carbohydrate
utilization50,51,53. In a notable example of cooperation
through cross-​feeding, B. ovatus produces extracellu­
lar inulin-​degrading enzymes despite not utilizing the
degradation products (and, in fact, under certain cir­
cumstances exhibiting a loss of fitness upon production
of these glycoside hydrolases)49. Rather, the production
of these enzymes seems to aid in growth of secondary
degraders of inulin such as Bacteroides vulgatus. In turn,
B. vulgatus provides a competitive edge to B. ovatus
through an as yet unclear mechanism49. Similar rela­
tionships have since been identified between numerous
different bacteria54. This network of interactions is fur­
ther expanded by long-​range carbohydrate degradation
mediated by outer membrane vesicles50,53. Bacteroides spp.
have been shown to package certain CAZymes into outer
membrane vesicles and release them into the extracel­
lular milieu — enabling other members of the human
gut microbiota to access a communal pool of utilizable
carbohydrates50,53.
The degradation of carbohydrates of higher complex­
ity necessitates larger PULs containing a broader array
of enzyme classes. One of the most complex examples
concerns the degradation of rhamnogalacturonan II
(RG-​II), a common polysaccharide in pectin55. There
are a total of 21 distinct glycosidic linkages in RG-​II
with numerous ester modifications, pyranose and fura­
nose sugars, as well as l-​sugars and d-​sugars55. B. theta­
iotaomicron carries out near complete degradation
of this carbohydrate (leaving a single glycosidic link­
age uncleaved) using numerous CAZymes contained
across three PULs. Perhaps surprisingly, degradation
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α1,3 α1,3 α1,3

P Phosphate
α1,3 α1,3
α1,3
Man
α1,2
α1,2
α1 α1,3 GlcNAc
α1,3 α1,3
α1,2 6 P
α1,2 α1,3 α1
α1,6
α1,2 α1,2 P
α1,6 α1,2 α1,2 6 α1,3 α1,3
α1,2 α1,2
α1,6
α1,6 α1,3 α1,2
α1,2 α1,2
α1,6 α1,6 α1,2 α1,3 α1,6
Surface endo-α-1,6-
α1,3 α1,6 Surface endo-α-1,2- α1,2 α1,3 mannosidases α1,3
α1,6
β1,4 mannosidases α1,6
BT2623 GH76
BT3862 GH99 α1,3 α1,6 BT3792 GH76 α1,3
β1,4 α1,3
β1,4 α1,3
α1,3 α1
β1,4 P
6
α1,2 α1,2
α1,6 α1,2
α1,6
α1,6

Outer membrane

SusC
Periplasm +SusD
α1,3
Proposed GH99 mechanism
α1,3
α1,2 α1,2 α1,3
Enzyme Enzyme Enzyme α-1,2/3-Mannosidases (GH92)
H α-1,6-Endo-mannanases (GH76) α1,6
α1,6
O O– O O H O O– α-1,2-Mannosidase (GH38) α1
O
HO HO HO OH α-1,6-Mannosidases (GH76 and GH125) 6 P
O +H2O
HO HO OO
HO O α-1,3-Mannosidases (GH92) α1,2
Man Mannose-6-phosphatases
-ROH Man Man α1,6 α1,2

OR α1,3
H OH α1,6
O
O O H O O– O O H
H
Enzyme Enzyme Enzyme

Fig. 2 | Mechanism of α-mannan digestion by B. thetaiotaomicron. Overview of degradation carried out by Bacteroides
thetaiotaomicron first described by Cuskin et al.47. Briefly, several cell surface-​localized carbohydrate-​active enzymes
(CAZymes) initiate degradation of α-​mannan at the outer membrane. SusC/D-​like proteins then import oligosaccharides
into the periplasm. Following transportation into the periplasm, various enzymes carry out complete degradation. For
clarity, enzymes are represented as Pac-​Man shapes, and those drawn attached to the membrane are surface-​associated
lipoproteins. Insert: proposed chemical mechanism by which the glycoside hydrolase GH99 family carries out the first step
in α-​mannan degradation.

is accomplished with a single enzyme specific for each
glycosidic linkage cleaved, highlighting the exquisite
regioselectivity and stereoselectivity of these enzymes55.
Homologues of one of the RG-​II PULs are present in
several bacteria and likely confer RG-​II metabolism55.
Indeed, it seems that the presence of homologous PULs
for many carbohydrates can be used as a relatively reli­
able indicator of glycan degradation capabilities48,56,57.
Moreover, the presence of homologous PULs in differ­
ent bacteria likely provides a level of functional stability
and resilience to the human gut microbiota, as even if a
certain glycan degrader is lost, another is able to carry
out the same role58.
The presence of a PUL also suggests that there is
a glycan that is capable of being degraded59. Recently,
Lapébie et al. extended this concept to estimate the total
number of carbohydrate structures present in nature as
being a few thousand59 (in contrast to the theoretical
1012 structures possible for hexasaccharides as originally
calculated by Laine60). Lapébie et al. largely did this by
surveying the number of distinct PULs sequenced to
date59. However, there are additional complexities to this
‘one PUL, one glycan’ paradigm. Increasingly complex
carbohydrates often require additional co-​regulated
gene clusters outside a central PUL, known as CAZyme
clusters61, in order to be completely degraded62. In other
cases, different components of a polysaccharide will be
targeted by different PULs48,57. In such instances, certain
organisms can contain only some of these PULs and it
is likely that further carbohydrate degradation is only
achieved through cross-​feeding48.
Host glycan degradation
Mucin O-​glycans. Coating the lumen of the gut, at the
interface between host and microorganism, is a layer of
mucus. The primary constituents of this mucosal lin­
ing are a family of proteins known as mucins, which are
densely glycosylated with O-​glycans, and lesser amounts
of N-​glycans63. From the human perspective, the thick
mucus layer functions as a barrier against bacterial

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infection64. From the bacterial perspective, it functions
as an attachment point to maintain their position in
the flowing gastrointestinal tract65,66 and as a nutrient
source67–69, especially during times of limited dietary
carbohydrates70,71. Indeed, there is a poorly understood
interplay between the microbiota and the host centred
on the mucus layer such that, in a somewhat paradoxi­
cal manner, mucus degradation by symbionts such
as Akkermansia muciniphila and B. thetaiotaomicron
induces host synthesis of mucus and thickens the
mucosal lining72–74.
The complexity of mucosal carbohydrate structures,
with numerous different monosaccharide components,
glycosidic linkages and modifications, likely is an impor­
tant barrier to bacterial penetration of the mucus71,75,76.
Nonetheless, some bacteria do achieve this, typically
through a large repertoire of enzymes spread across
many glycoside hydrolase families67, as well as acces­
sory enzymes such as sulfatases77 (Fig. 3a,b). By contrast,
dietary polysaccharides such as starch that evolved to
be easily degradable are less complex and require fewer
enzymes for coordinated degradation43,71. Even special­
ized mucin degraders often target relatively few struc­
tures, perhaps to limit the energetic costs of genome
maintenance and enzyme expression71. Interestingly,
the N-​glycans and O-​glycans of mucin share certain
structural motifs that lend themselves to being broken
down by the same enzymes, and to induction of the
same PULs68. Work by Crouch et al. identified GH16
endo-​β-​galactosidases in B. thetaiotaomicron that cleave
the poly-​N-​acetyllactosamine (Galβ1–4GlcNAcβ1–3)
structures, which are common to O-​glycans, N-​glycans
and keratan sulfate78 (Fig. 3a). These GH16 family mem­
bers can accommodate many of the modifications and
decorations present on these diverse glycan structures.
It seems likely that only after initial cleavage of these
extended decorations and transport across the outer
membrane does a consortium of periplasmic enzymes
then remove these modifications78.
Recently, there has been increased interest in a
class of enzymes known as O-​glycoproteases. As the
name suggests, these proteases have a strict specific­
ity for O-​glycosylated proteins79 with requirements for
both the substrate glycan structure and the amino acid
sequence79–82. O-​Glycoproteases have varying specifici­
ties, with some having higher activities towards mucin
domains (often referred to as mucinases), whereas others
are active against mucins as well as less densely glyco­
sylated proteins79,80,82. Recent structural characteriza­
tion by Pluvinage et al. has shown how the multidomain
structure of the O-​glycoprotease ZmpB from Clostridium
perfringens is specialized for mucin degradation83. In
particular, the CBMs of ZmpB appear to be spaced such
that they would almost perfectly match the periodicity
of heavily glycosylated domains of mucin (which them­
selves have only recently been observed84) while placing
the catalytic domain near the more exposed regions of
the mucin substrate83. O-​Glycoproteases have also been
identified in PULs and likely have a role in nutrient
acquisition85. In such cases, cleavage of mucin at the
cell surface is carried out by an O-​glycoprotease rather
than an endo-​g lycoside hydrolase, producing large
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glycopeptides from mucins for subsequent uptake and
metabolism (Fig. 3b).
N-​Glycans. Addition of N-​linked glycans is one of the
most common post-​translational modifications found
on extracellular and cell-​surface proteins86. The ubi­
quity of N-​glycans on host epithelial surfaces likely
provides a nutritional advantage for microorganisms
that can degrade and metabolize these glycans. Work by
Briliūtė et al. revealed the extensive enzymatic machin­
ery that Bacteroides spp. use for the degradation of
complex N-​glycans68. In response to being grown on an
N-​glycoprotein as a sole carbon source, B. thetaiotao­
micron upregulates 19 CAZymes from different families
across 6 loci. This extensive machinery provides access
to many structurally diverse complex N-​glycans68.
As highlighted in Fig. 3c, the initial extracellular
degradation of N-​glycans involves an array of glycoside
hydrolases, including endo-​β-​N-​acetylglucosaminidases
(ENGases), along with an extracellular sialidase and an
endo-​glycoside hydrolase from the newly established
GH163 family68. ENGases, primarily from the GH18 and
GH85 families, are responsible for the initial release of
the glycan through cleavage within the chitobiose core
(GlcNAc-​β1–4GlcNAc) of the core sugar sequence87.
ENGases follow a substrate-​assisted mechanism (Fig. 3c),
which departs from the canonical Koshland mecha­
nism described in Box 1. Briefly, the 2-​acetamide acts
as the catalytic nucleophile to form the key glycosyl
oxazoline intermediate which is then hydrolysed with
overall net retention of stereochemistry88,89. ENGases
are often quite specific. For example, BT3987 from
B. thetaiotaomicron cleaves within the chitobiose core
of oligomannose-​type N-​glycan but remains inactive
against complex N-​glycans90.
Degradation of the core tetrasaccharide and other
N-​glycan-​derived oligosaccharides occurs upon trans­
port into the periplasm and cytoplasm. In the periplasm,
an array of CAZymes, sulfatases and esterases further
degrade the oligosaccharide into monosaccharides and
disaccharides, which can then be transported into the
cytoplasm47,68. Recent efforts have shed light on cyto­
plasmic degradation of N-​glycan-​derived disaccha­
rides. For example, in B. thetaiotaomicron, a GH130
glycoside phosphorylase (BT1033) cleaves the Manβ-1,
4-GlcNAc disaccharide produced upon degradation of
the N-​glycan core tetrasaccharide to generate α-​Man-1-
phosphate (α-​Man1P) and GlcNAc91. This strategy,
which seems widespread amongst Bacteroidetes, enables
α-​Man1P to enter glycolysis while avoiding the expendi­
ture of ATP through use of a hexokinase91. An alternative
strategy, applied by Actinobacteria in various environ­
ments including the gut, utilizes a GH5 (subfamily 18)
to hydrolyse the same linkage92,93.
Glycosaminoglycans. Epithelial cell-​derived glycosamino­
glycans represent a reliable source of complex carbohy­
drates for the human gut microbiota (Fig. 3d). PULHep from
B. thetaiotaomicron encodes several genes required for the
degradation of heparan sulfate, along with the structur­
ally related heparin. PULHep contains four polysaccharide
lyases, a sulfamidase and a GH88 Δ4,5-​glucuronidase in
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 Reviews

a α1,2
c d
Gal Neu5Ac GlcN
β1,3 6S
α1,3 β1,4 α2,6 α2,6
β1,4 β1,3 α1 Man GalNAc GlcA
β1,4 β1,4
6S β1,3 Fuc GlcNAc IdoA
α2,3 β1,4 β1,4 β1,3 β1,4 β1,3 α1 α1,3 α1,6
β1,2
4,5-Uronic acid
β1,2
α2,3
Sialidase (GH33) β1,3
β1,4
Endo-galactosidase (GH16) α1 α1,3 α1,6 Sialidase (GH33) 6S 2S 6S 6S
α1,4 α1,4 α1,4 β1,4 α1,4 β1,4 α1,4 β1,4
6S β1,4
α1,3 Endo-glucosaminidases
α2,3 β1,3 β1,4 β1,6 β1,4 (GH18) NS NS
Endo-GlcNAcase α2,6
α1,2 β1,4 (GH163) β1,4 Polysaccharide lyase
β1,3
α1,3 β1,4 β1,3
α1,6 (PL12)
β1,4
6S β1,4 β1
β1,3 β1,4
Protein 6S 2S 6S
β1,4 α1,4 α1,4

SusC
+SusD Periplasm
α1,2 SusC
α1,3 β1,4 β1,3 +SusD Polysaccharide lyase
β1,3 6S β1,4 β1,3
(PL12 + PL13 + PL15)
Glucuronyl β1,4
Exo-hexosaminidases α1,3
α1,6
(GH20) hydrolase (GH88)
b α2,3 β1,3 Sulfatases 6S 2S 6S
α1 Sialidase (GH33) β1,4 β1,4 α1,4 α1,4
α1,3 α1,6
α2,3 β1,3 α1
Galactosidase (GH2)
β1,3 α1 β1,4 Fucosidase (GH29)
α2,6 Polysaccharide lyase mechanism
α2,3 β1,4 β1,3 α1 β1,4
A NG A NG
α1 β1,4
β1,3 Mucinase
β1,4 H
α2,3 H
β1,3 α1
O O –
–
O –
O O
Substrate-assisted mechanism (GH18, GH20)
O
Enzyme Enzyme Enzyme O O O
O
OR OR
O O H O O– O OH H
B– HB
+H2O H
O O O
OR O OH
-ROH –
NG
N O H HN O A
N O
H
α2,3 β1,3 α1 O –
O O
– – OH
O O H O O O O
α2,3
β1,3
β1,3 O
α1
β1,3 α1 Enzyme HB
Enzyme Enzyme
OR

e HN
O HN
O HN O
N –
O2C
O– O H N –
O 2C
O CO2– H N O–
O O
H O O OH
O H OR O
O
O H2O OH
OR CO2– ROH
O H2N
H2N
H2N

Fig. 3 | Host glycan degradation by bacteroidetes. a,b | Glycoside hydrolase GH16 family endo-​galactosidases (part a)
or mucinases (part b) can initiate mucin digestion by generating large oligosaccharides or glycopeptides for uptake and
further depolymerization. c | An array of carbohydrate-​active enzymes (CAZymes) from Bacteroides thetaiotaomicron
catalyse the break down of complex N-​glycans (top). Substrate-​assisted mechanism used by GH18 and GH20 families
(bottom). d | Degradation of the glycosaminoglycan heparan sulfate (top). Mechanism used by polysaccharide
lyases (bottom). e | Simplified mechanism utilized by GH88 and GH105 families in the processing of 4,5-​unsaturated
oligosaccharides produced by polysaccharide lyases. A, general acid catalytic residue; B, general base catalytic residue;
NG, neutralizing group.

addition to a co-​regulated O-​sulfatase to carry out the of uronic acids facilitates a stepwise E1cb mechanism
E1cb mechanism
A two-​step chemical reaction depolymerization of heparin and heparan sulfate94. involving deprotonation at C-5 and cleavage of the bond
in which an acidic proton is Degradation of heparin and heparan sulfate into oligosac­ from C-4 to the adjoining sugar via elimination, leading
abstracted by a base to form charides is initiated by a surface-​localized polysaccharide to the formation of a 4,5-​unsaturated oligosaccharide
a stabilized carbanion, followed lyase PL12 (BT4662) which exhibits preferential cleav­ product95 (Fig. 3d).
by elimination of a leaving
group as the carbanion lone
age at low sulfation regions46. Polysaccharide lyases have Following transport of the released oligosaccharides
pair moves to form a π-​bond a distinct mechanism compared with glycoside hydro­ into the periplasm, three other polysaccharide lyases
with the adjacent atoms. lases. The presence of a carboxylic acid at the C-5 position of varying specificity, in addition to several sulfatases,

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Reviews
Xenobiotics
Molecules that are not
produced or expected to
be found within an organism
(for example, drugs).
Endobiotics
Molecules that are produced
or are expected to be
found within an organism
(for example, hormones).
generate 4,5-​unsaturated disaccharides, which them­ xenobiotics and endobiotics, the human glycosyltrans­
selves are substrates for a glucuronyl hydrolase (BT4658) ferase 1 (GT1) UDP-​glucuronyltransferase covalently
from the GH88 family94,96. BT4658 cleaves the linkage attaches a β-​glucuronic acid moiety to available OH, NH
between 4,5-​unsaturated uronic acid, and GlcNAc or SH (and even activated CH) functionalities on small
(or GlcN), generating monosaccharide products that molecules via a single SN2 displacement17,100,101, as shown
are further metabolized, thus resulting in complete deg­ in Fig. 4. These glucuronides are then excreted into the
radation of target glycosaminoglycans by commensal gastrointestinal tract for disposal100. However, certain gut
bacteria. Enzymes of the GH88 family (and also GH105) bacteria can use these glucuronides as a carbon source
employ a non-​Koshland mechanism involving hydration by use of β-​glucuronidases (GUSs) (primarily of CAZy
of the double bond and spontaneous decomposition of family GH2) to release the glucuronic acid, thereby reac­
the resultant hemiketal resulting in glycosidic bond tivating the compound. In numerous cases this reacti­
cleavage13 (Fig. 3e). B. thetaiotaomicron utilizes a simi­ vation has been shown to lead to toxic effects, with a
lar strategy for the metabolism of chondroitin sulfate, notable example being the severe diarrhoea brought on
dermatan sulfate and hyaluronic acid97–99. by cancer treatment using irinotecan102,103.
Structural characterization and activity profiling
Glucuronidation: linking CAZymes and drug of GUS enzymes in the gut microbiota have revealed
metabolism distinct active site structures that have clear effects on
It is becoming increasingly evident that the gut micro­ activity and substrate scope. In particular, the pres­
biome can have important roles in drug metabolism4. In ence or absence of two specific loops within the GUS
order to inactivate and increase the solubility of certain active site can result in enzymes that are specific for
R–X Excretion and/or
reabsorption
R–X
R–X
O–
O– H O–
UDP O–
O O– O
O X R O O
HO O O Microbial GUS HO
HO O HO X R OH
HO X R HO
HO HO OH
HO
O O OH +
HO
P R–X
–
O UMP Inactivated
with ↑ solubility Reactivated
with ↓ solubility
Where X=O, N, S or other suitable nucleophile
b OH
H H
O– O–
O O H
O O H O–
HO O O HO N
HO HO O O
N HO
HO HO O
N HO
N OH
O
Nicotine glucuronide Oestradiol-17-glucuronide
SN-38 glucuronide
OH O
Cl O N
F O
O O N
F N
H S
F N N N
H – O O O
F O –
O O OH
OH
O O
HOOH HO
OH
Regorafenib glucuronide Sulfinpyrazone glucuronide
Fig. 4 | glucuronidation of small molecules and reactivation by bacterial gUSs. a | In the liver, human
UDP-​glucuronyltransferase inactivates small molecules through glucuronidation. In the large intestine, microbial
β-​glucuronidases (GUSs) can reactivate these compounds. b | Selection of molecules known to be glucuronidated.
Note that SN-38 is the active metabolite of the anticancer drug irinotecan.
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either small nonpolar molecules102,104,105 or glucuronic
acid-​containing glycosaminoglycans 104,106. Interest
now lies in the identification of the particular enzymes
that reactivate troublesome conjugated drugs and
the development of specific inhibitors, which could
be co-​administered with those drugs to selectively
shut down their reactivation, thereby mitigating side
effects. The differing active site structures of these
GUS enzymes offers promise in the design of selective
inhibitors102,103,105,107.
Disease and the microbiota
Most pathogens cannot compete with resident microbi­
ota for their carbohydrate food source, and so are effec­
tively excluded from the gut under normal conditions.
Consequently, disruptions to the gut ecosystem seem to
have an important role in establishment of pathogenic
bacteria. For example, antibiotic treatment disrupts
cross-​feeding networks between mucin degraders and
non-​mucin degraders and allows the proliferation of
pathogenic bacteria such as Salmonella typhimurium and
Clostridioides difficile108. In such cases, pathogens that
lack the enzymes for host glycan degradation will scav­
enge free monosaccharides released by the residents108,109.
Introduction of new niches can also drive dysbiosis. For
instance, trehalose is a disaccharide (Glcα1–1αGlc) that
has only been introduced into the western diet on a large
scale in the past 20 years110,111. Paralleling its introduction
has been an increased incidence of C. difficile infection
and the evolution of mechanisms for enhanced trehalose
utilization by epidemic strains110,111.
Whereas most commensal bacteria are relegated to
the outer mucosal layers, during infection pathogens
must often cross the mucosal barrier. Towards this end,
bacterial67,112 and viral113 pathogens express glycoside
hydrolases that break down mucosal glycans in the gut
and airways. Often assisting in this process are micro­
bial adhesins, lectins and CBMs that endow bacteria
with the ability to attach to the mucosa, and thereby
localize the degradative CAZymes114. Microorganisms
can also take advantage of host-​expressed proteins with
lectin activities115. In that regard, humans secrete trefoil
factor proteins, which bind a GlcNAc-​α1,4-​Gal disaccha­
ride present on mucins115. This binding helps to oligo­
merize mucin proteins, increasing the viscoelasticity
of the mucus. Helicobacter pylori has evolved a similar
Glc-​α1,4-​Gal disaccharide epitope, which is bound by
trefoil factors and which likely, in turn, aids the binding
of H. pylori to gut mucins116. Many bacteria also express
CAZymes that cleave this epitope117, thereby effectively
abrogating trefoil binding and likely weakening the
mucosal barrier115.
Many CAZymes are involved in evasion of the host
immune response during infection. Perhaps the best
known examples of this are the glycosyltransferases that
synthesize host glycan-​mimicking structures to help
the pathogen evade detection as non-​self118. In parti­
cular, the presence of sialic acid on pathogens often
helps trick the immune system into complacency118.
Enzymatic breakdown of molecules involved in the
immune response is another strategy used by patho­
gens. For example, O-​glycoproteases in Pseudomonas
NATURe RevIeWS | Microbiology
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Reviews
aeruginosa, Acinetobacter nosocomialis M2 and other
pathogens promote infection through degradation
of glycoproteins involved in immune function81,119,120.
Likewise, during infection, the GH18 ENGase from
Streptococcus pyogenes cleaves N-​glycans from host IgG
molecules, thereby decreasing the affinity of IgG for the
Fc receptor and promoting survival of S. pyogenes121,122.
The degradation of N-​glycans in serum for use as an
energy source has also been shown to fuel Bacteroides
fragilis during infection123.
Applications of CAZymes
Bacterial glycosylation systems for biotherapeutic
production
Glycosyltransferases from the human gut microbiota
are valuable tools in glycobiology. Compared with their
eukaryotic counterparts, bacterial glycosyltransferases
are often easier to express in Escherichia coli and do not
have such strict substrate requirements. Furthermore,
as many bacteria synthesize polysaccharides that mimic
human glycosylation (to escape immunity)118, bacterial
glycosyltransferases can be used for the synthesis of
glycoproteins with eukaryotic glycosylation patterns.
As an example, bacterial sialyltransferases have enabled
production of therapeutic glycoproteins with prolonged
half-​life124,125.
Bacterial oligosaccharyltransferases (OSTs) are a
class of enzyme with interesting biomedical applica­
tions. Physiologically, these enzymes are responsible
for the en bloc transfer of lipid-​linked oligosaccharides
to extracellular proteins in numerous pathogens 126.
Intriguingly, the OST that generates N-​linked glycans in
Campylobacter jejuni recognizes a motif similar to that
of eukaryotic OSTs and has a similar fold127. However,
in contrast to their far more complex, multisubunit
eukaryotic counterparts, bacterial OSTs are remarkably
promiscuous and can transfer oligosaccharides with
minimal recognition requirements (often requiring
just an acetamide group at the C-2 position) to protein
acceptors126. In nature, the requisite glycosyltransferases
for synthesis of the lipid-​linked oligosaccharide donor
often occur as gene clusters, which can be transferred
into E. coli with the appropriate OST to enable synthesis
of glycoproteins carrying oligosaccharides that are nor­
mally found in lipopolysaccharides128. Such systems have
been engineered for the development and production of
glycoconjugate vaccines129,130. Through the introduction
of non-​natural gene clusters, these same OSTs can also
transfer non-​bacterial glycans of choice. Interestingly,
this has enabled production of human-​like N-​linked
and O-​linked glycans in E. coli131,132. Further work is
needed to improve the yield and substrate scope of these
enzymes (in many cases they only act on highly flexi­
ble regions of proteins)126 if they are to be viable means
of production of therapeutic glycoproteins in E. coli.
Indeed, it seems likely that mixing both eukaryotic and
prokaryotic glycosyltransferases within glycosylation
pathways will provide an optimal route to production
of glycoprotein therapeutics131,133. For further details,
interested readers are directed to the in-​depth review
by Kightlinger et al. on recent advances in glycoprotein
synthesis126.
Reviews
Antibody-​dependent
cellular toxicity
An immune response in which
binding of antibodies to a cell
surface results in the lysis of
that cell by effector cells of the
immune system.
Synthesis of human milk oligosaccharides for
microbiota manipulation
Given the key roles of the gut microbiota in human
health, it is of little surprise that there has been great
interest in the development of ways to manipulate it.
Towards this end, approaches using dietary intervention,
drugs and prebiotics have been developed. The intimate
link between carbohydrate metabolism and microbiome
composition makes carbohydrate supplementation an
obvious choice3,134,135.
Bifidobacteria are naturally enriched in the infant
gut microbiota. Human breast milk contains a diverse
array of glycan structures collectively known as human
milk oligosaccharides (HMOs)136. Humans lack the
enzymes necessary for HMO degradation. Rather,
HMOs enrich for beneficial microorganisms, primarily
bifidobacteria, that are capable of their degradation136–138.
Thus, HMOs have been suggested as an additive to
infant formula to enrich the microbiota of bottle-​fed
infants. However, unlike many dietary polysaccha­
rides, HMOs cannot be readily obtained from natu­
ral sources at an industrial scale. The most successful
method for the commercial production of HMOs, such
as 2′-​fucosyllactose (Fuc-​α1,2-​Gal-​β1,4-​Glc), has been
through incorporation of glycosyltransferases (and other
enzymes) from gut bacteria (commonly H. pylori) into
workhorse microorganisms for large-​scale production
by fermentation139. 2′-​Fucosyllactose produced in this
manner is now incorporated into numerous infant milk
formulas worldwide139.
Complementary methods to fermentation for HMO
synthesis include in vitro approaches, which repurpose
glycoside hydrolases from gut bacteria for glycan synthe­
sis rather than degradation. An excellent example of this
is the kilogram-​scale synthesis of lacto-​N-​biose (LNB;
Gal-​β1,3-​GlcNAc) from sucrose and free GlcNAc using
a glycoside phosphorylase from Bifidobacterium bifidum
in a multienzyme reaction23.
More recent work has highlighted the potential for
glycosynthase technology to be used for industrial-​scale
synthesis of HMOs. The original glycosynthase concept
was introduced by Withers and co-​workers in 1998
(ref.140). The basic principle relies on the mutation of
a catalytic residue of the glycoside hydrolase such that
it cannot degrade its natural substrate (Fig. 5a,b). When
this inactivated mutant is presented with an activated
glycosyl donor of opposite anomeric configuration to
that of its natural substrate (and thereby mimicking the
covalent glycosyl enzyme intermediate of retaining gly­
coside hydrolases), these are accepted as substrates in
the glycosylation reaction and can form oligosaccharides
and other glycoconjugates22. One of the key benefits of
using glycosynthases over glycosyltransferases, the nat­
ural enzymes for glycan synthesis, is that they do not
require the use of expensive nucleotide sugar substrates.
Many variations of glycosynthases have been developed
over the years, but the basic principle remains the same
(for a comprehensive review see Danby and Withers22).
Using glycosynthases, the Nidetzky group has devised
synthetic routes for production of lacto-​N -​triose
(GlcNAc-​β1,3-​Gal-​β1,4-​Glc) and lacto-​N-​tetraose
(Gal-​β1,3-​GlcNAc-​β1,3-​Gal-​β1,4-​Glc) with good yields
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and selectivity141–143 (Fig. 5c). Their approach largely relies
on mutants of GH20 enzymes from B. bifidum, which
normally catalyse the breakdown of HMOs141–143. These
enzymes can use the readily prepared oxazoline deriva­
tives of GlcNAc and LNB as glycan donors144 and lactose
as an inexpensive glycan acceptor.
Glycan remodelling with bacterial enzymes
The ability to generate homogeneous, well-​defined gly­
coforms of antibodies and other therapeutic proteins
is highly desirable for many pharmaceutical appli­
cations as it enables evaluation of the impact of spe­
cific glycan structures on biopharmaceutical efficacy,
safety and function145. For example, removal of the
core fucose or replacement of this core fucose (equiv­
alent to 6-​deoxy-​l-​galactose) with 6-​azido-​l-​fucose
or l-​galactose on N-​glycans present on monoclonal
antibody therapeutics leads to desirable increases
in antibody-​dependent cellular toxicity146. Such results
highlight the importance of glycan remodelling efforts.
One approach for generating defined glycoforms
involves in vitro remodelling of the glycoprotein using
a wild-​type ENGase (GH18 or GH85) and its respec­
tive glycosynthase mutants with activated substrates
for selective installation of N-​glycan structures. First,
deglycosylation of the glycoprotein is performed using
wild-​type ENGases, leaving a GlcNAc handle on the
target protein22. ENGase glycosynthase mutants can
then be used for the attachment of N-​glycan structures
to this stub using activated oxazoline donors, which
are readily generated from free oligosaccharides22,144
(Fig. 5b,d). Bacterial endoglycosidases from the GH18
and GH85 families have been particularly useful in
glycan remodelling147 and have been used to generate
defined glycoforms of well-​known therapeutic anti­
bodies, including the blockbuster drugs Herceptin and
rituximab148–150. Currently, this approach is largely lim­
ited to the modification of a single glycoform on one site
or multiple sites with the same glycoform149,150. However,
recent work conducted by Wang and colleagues beauti­
fully demonstrated how the glycan structures at Fc and
Fab domains could be introduced differentially by tak­
ing advantage of the highly selective nature of ENGases
and of an α-1,6-​fucosidase from Lactobacillus casei151.
In this way, the authors generated an engineered glyco­
form of cetuximab with eightfold higher efficacy than
the non-​engineered version. Similar approaches have
also been used to edit glycans on cell surfaces152. Another
approach towards selectivity is through introduction of
N-​linked GlcNAc and (pseudo-​eukaryotic) Glc handles
into proteins using bacterial glycosyltransferases153–155.
Use of these introduced handles along with ENGase
glycosynthase technology has opened the door to
site-​s elective and structure-​s elective installation of
N-​glycans153–155. Clearly, this technology is in its early
phases and an expanded toolkit of ENGases and other
enzymes is needed for biopharmaceutical remodelling
to enable access to more synthetically complex glycan
structures and provide site selectivity.
The human gut microbiome is an ideal place to
search for endo-​glycoside hydrolases against host gly­
cans to be used in glycan remodelling, as it contains
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 Reviews

a Catalytic Catalytic b Catalytic Catalytic

acid/base acid/base acid/base acid/base
O – O O O
F
O– OH O– OH
O H O O H O
O O R
O R O R
R H N+ HN
F O
O

CH3 CH3 CH3 CH3

Mutated catalytic Mutated catalytic Mutated oxazolinium Mutated oxazolinium

nucleophile nucleophile stabilizer stabilizer

c
OH HO OH OH HO OH
OH OH OH
HO
HO O O O O
O O O HO O O
HO O O
HO O
HN OH HO OH OH HO OH
O OH HN OH
OH O

Lacto-N-triose Lacto-N-tetraose

Wild-type ENGase Mutant ENGase

N O

e
OH OH OH
HO HO HO
O O O
OH OH
HO AcHN H AcHN HO
O O ROH O H2O O OH NADH
O NADH R AcHN O
HO NAD+ HO
NAD+
AcHN AcHN
O OH
R

Type O red blood cell

β1,3 β1
α1,2
GH109
Red blood cell

α1,3 β1,3 β1

GH36 galactosaminidase
α1,2
GalNAc
Type A red deacetylase
blood cell α1,3 β1,3 β1

α1,2
GalNAc GlcNAc Gal Galactosamine

Fig. 5 | Applications of enzymes from the gut microbiome. a,b | Glycosynthase concept initially put forward by Withers
and colleagues (part a) and adaptation for enzymes following a substrate-​assisted mechanism (part b). c | Human milk
oligosaccharides (HMOs) produced using bifidobacterial enzymes following the glycosynthase concept. d | General
scheme for N-​glycan remodelling using endo-​β-​N-​acetylglucosaminidases (ENGases). e | Two recent methods for use
of carbohydrate-​active enzymes (CAZymes) to generate type O blood from type A. Insert: overview of the mechanism
utilized by the glycoside hydrolase GH109 family for cleavage of the terminal GalNAc from the A antigen.

a vast reservoir of bacterial genetic diversity7. The ten­ of novel endo-​glycoside hydrolases extremely probable.
dency of gut bacteria to cleave and import whole oligo­ Indeed, recent work has highlighted the potential for
saccharides prior to degradation makes the discovery discovery of endo-​glycoside hydrolases from the human

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gut microbiome that are active on host glycans such as
mucins68,78,156. Of particular note has been the discovery
of enzymes that degrade the repeating LacNAc struc­
tures common in host glycans, as well as enzymes that
cleave the intact sialyl-​T antigen (a ubiquitous O-​glycan)
from proteins and cell surfaces78,156.
Universal blood production
Blood transfusion is an indispensable part of the
health-​care system, saving many thousands of lives annu­
ally, but must be performed with very careful attention to
the ABO blood types of both donor and recipient157,158.
The basis of the ABO blood group system revolves
around a set of related carbohydrates attached to glyco­
proteins and lipids on the surface of red blood cells. The
basic structure is that of the H antigen (which confers
type O blood), with type B and A antigens being derived
from this structure by decoration with α-​galactose or
α-​N-​acetylgalactosamine, respectively. The type O cells
are non-​antigenic for the vast majority of people and can
be donated to people with type A, B or AB as well as type
O blood, and are thus known as the universal donor type.
As a consequence, type O blood is particularly important
in emergency situations and is often in short supply159.
The use of glycosidases to remove the α-​galactose or
α-​N-​acetylgalactosamine residues of type B and A blood
to produce universal donor type O blood was first pro­
posed by Goldstein et al. in 1982 and was shown to be a
useful strategy for conversion of type B to type O in initial
campaigns160. However, those studies used an inefficient
α-​galactosidase from green coffee beans that required
unfavourable conditions and enormous amounts of
enzyme for successful blood group conversion160. A sub­
stantial advance came in 2007 when Liu et al. discovered
the GH109 and GH110 families, which act on the A and
B antigen, respectively24. These enzymes function at much
lower enzyme loadings and under more favourable con­
ditions than the coffee bean α-​galactosidase24. Curiously,
GH109s are proposed to follow a non-​conventional
reaction mechanism similar to that first demonstrated
for enzymes of the GH4 family161. In this mechanism,
the enzyme uses a tightly bound NAD+ cofactor to carry
out a sequence of chemical steps (oxidation, elimination,
addition and reduction) to achieve net hydrolysis of the
terminal GalNAc24, as shown in Fig. 5e.
To identify even better suited enzymes, we recently
screened the human gut microbiome for enzymes capa­
ble of blood type degradation. The mucus layer of the
human gut contains glycan structures including the A,
B and H antigens162. Thus, it seemed likely that bacte­
ria inhabiting the gut may produce enzymes capable of
blood antigen degradation67. This effort yielded a pair
of highly active CAZymes — a carbohydrate esterase
and a GH36 galactosaminidase from the bacterium
Flavonifractor plautii — that work in tandem to remove
the terminal N-​acetylgalactosamine of the A antigen25
(Fig. 5e). This enzyme pair outcompetes any previously
known A antigen cleaving enzyme, and potentially pro­
vides a more feasible method of production of universal
donor blood25. Results such as this further highlight the
wealth of valuable enzyme activities present within
the human gut microbiome and portends the many yet
to be uncovered.
Concluding remarks
Knowledge of the diversity of CAZymes present in the
human gut microbiota will advance our understanding
of the intimate relationship between us and our micro­
bial selves and provide new insight into enzymes that
can be exploited for various purposes. However, despite
efforts thus far, we have only begun to scrape the surface
of CAZyme diversity within the human gut microbiota.
Further research into how carbohydrates are partitioned
within the gut microbiota will provide insights into how
dietary carbohydrates may affect our overall health.
Although there is an abundance of sequence information
about gut bacteria, we still do not know the functions
of most of the genes present. Future efforts could use
ultra-​high-​throughput functional screening approaches
to catalogue such enzymatic activities 163–165. Such
enzymes, and their engineered glycosynthetic mutants,
could prove useful for tailoring the glycosylation of bio­
pharmaceuticals for improved efficacy and even advance
our understanding of the fundamental roles of glycans.
Published online xx xx xxxx

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Acknowledgements
The authors were supported by funding from the Canadian
Institutes of Health Research (CIHR) and the Natural Sciences
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Reviews
and Engineering Research Council of Canada (NSERC) during
the writing of this Review.
Author contributions
All authors contributed to all aspects of the article.
Competing interests
P.R. and S.G.W. are co-founders of a company to develop
enzymes for conversion of blood type. The research underly-
ing that is mentioned in this Review. J.F.W. and R.K.B. declare
no competing interests.
Peer review information
Nature Reviews Microbiology thanks David Bolam, Bernard
Henrissat and Nicole Koropatkin for their contribution to the
peer review of this work.
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