© Johan Larsbrink, 2013
ISBN 978-91-7501-834-8
ISSN 1654-2312
TRITA-BIO Report 2013:13
Division of Glycoscience
School of Biotechnology
Royal Institute of Technology
AlbaNova University Centre
SE-106 91 Stockholm
Sweden
Printed at US-AB Universitetsservice
ABSTRACT
The focus of this thesis is a comparative study of approaches in discovery of
carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to,
and degrade all the multitude of carbohydrates found in nature. As such,
when aiming for sustainable methods to degrade plant biomass for the
generation of biofuels, for which there is a strong drive in society,
CAZymes are a natural source of environmentally friendly molecular tools.
In nature, microorganisms are the principal degraders of carbohydrates. Not
only do they degrade plant matter in forests and aquatic habitats, but also
break down the majority of carbohydrates ingested by animals. These
symbiotic microorganisms, known as the microbiota, reside in animal
digestive tracts in immense quantities, where one of the key nutrient sources
is complex carbohydrates. Thus, microorganisms are a plentiful source of
CAZymes, and strategies in the discovery of new enzymes from bacterial
sources have been the basis for the work presented here, combined with
biochemical characterisation of several enzymes.
Novel enzymatic activities for the glycoside hydrolase family 31 have been
described as a result of the initial projects of the thesis. These later evolved
into projects studying bacterial multi-gene systems for the partial or
complete degradation of the heterogeneous plant polysaccharide xyloglucan.
These systems contain, in addition to various hydrolytic CAZymes,
necessary binding-, transport-, and regulatory proteins. The results
presented here show, in detail, how very complex carbohydrates can
efficiently be degraded by bacterial enzymes of industrial relevance.
Keywords: CAZyme discovery, xyloglucan, polysaccharide-utilisation locus,
microbiota, Į-xylosidase, GH31, transglucosidase, human gut
i
SAMMANFATTNING
Den huvudsakliga inriktningen i denna avhandling är jämförelser mellan
olika metoder för att finna nya enzymer som är aktiva på kolhydrater
(CAZymer). Dessa enzymer syntetiserar, binder till, och bryter ned alla de
mängder av kolhydrater som finns i naturen. Det finns ett stort behov i
samhället av hållbara metoder för att bryta ned växtbaserad biomassa för
produktion av biobränslen, och i detta är CAZymer en given källa av
miljövänliga molekylära verktyg.
Mikroorganismer är de främsta nedbrytarna av kolhydrater i naturen. De
utför inte bara dessa processer i skogar och akvatiska miljöer, utan är även
de huvudsakliga nedbrytarna av kolhydrater som konsumeras av djur. Dessa
mikroorganismer återfinns i ofantliga mängder i mag- tarmkanalen, där
komplexa
kolhydrater
är
en
av
de
viktigaste
näringskällorna.
Mikroorganismer är således en rik källa av CAZymer, och strategier i att
finna och karaktärisera nya enzymer från bakterier har varit huvudfokus i
denna avhandling.
Tidigare okända enzymatiska aktiviteter i glykosidhydrolasfamilj 31 har
upptäckts som resultat från de inledande projekten i denna avhandling.
Dessa projekt utvecklades i sin tur till studier av bakteriella system, vilka
inkluderar flera gener, involverade i nedbrytningen av den heterogena
växtpolysackariden xyloglukan. Dessa system innehåller, förutom olika
hydrolaser, proteiner som binder till och transporterar kolhydrater, och även
proteiner som reglerar själva systemen. Resultaten som presenteras visar i
detalj hur väldigt komplexa kolhydrater effektivt kan brytas ned av
industriellt relevanta bakteriella enzymer.
ii
LIST OF PUBLICATIONS
I. Larsbrink J*, Izumi A*, Ibatullin FM, Nakhai A, Gilbert HJ, Davies
GJ, Brumer H (2011). Structural and enzymatic characterization of
DJO\FRVLGHK\GURODVHIDPLO\Į-xylosidase from Cellvibrio
japonicus involved in xyloglucan saccharification. Biochem J.
436(3): 567-80.
II. Cartmell A, McKee LS, Peña MJ, Larsbrink J, Brumer H, Kaneko S,
Ichinose H, Lewis RJ, Viksø-Nielsen A, Gilbert HJ, Marles-Wright
J (2011). The structure and function of an arabinan-specific alpha1,2-arabinofuranosidase identified from screening the activities of
bacterial GH43 glycoside hydrolases. J Biol Chem. 286(17): 1548395.
III. Silipo A, Larsbrink J, Marchetti R, Lanzetta R, Brumer H, Molinaro
A (2012). NMR spectroscopic analysis reveals extensive binding
interactions of complex xylogluco-oligosaccharides with the
Cellvibrio japonicus glycoside hyGURODVHIDPLO\Į-xylosidase.
Chemistry. 18(42): 13395-404.
IV. Larsbrink J*, Izumi A*, Hemsworth GR, Davies GJ, Brumer H (2012).
Structural enzymology of Cellvibrio japonicus Agd31B protein
UHYHDOVĮ-transglucosylase activity in glycoside hydrolase family
31. J Biol Chem. 287(52): 43288-99.
V. Larsbrink J*, Rogers T*, Hemsworth G*, McKee LS, Tauzin A,
Spadiut O, Klinter S, Pudlo NA, Urs K, Koropatkin NM, Creagh
AL, Haynes CA, Kelly AG, Nilsson Cederholm S, Davies GJ,
Martens EC, Brumer H. A discrete genetic locus confers select
Bacteroidetes with a niche capacity for xyloglucan metabolism in
the human gut. Submitted.
VI. Larsbrink J, Lundqvist M, Brumer H. Identification and
characterization of a locus in Cellvibrio japonicus involved in
xylogluco-oligosaccharide saccharification. Manuscript.
VII. Larsbrink J, Brumer H. Screening and activity profiling of the
glycoside hydrolase family 31 members from Bacteroides
thetaiotaomicron reveal novel specificities. Manuscript.
*
Authors contributed equally to the work
iii
THE AUTHOR’S CONTRIBUTION
Publication I: Performed all molecular biology and biochemical
experiments, from cloning, protein production and purification, to kinetic
analyses and bioinformatic analyses. Major writing contributions.
Publication II: Made sequence analyses and alignments for the genes
described, and constructed the phylogenetic tree.
Publication III: Constructed, produced and purified catalytically inert
mutants, as well as wild-type enzyme for the STD-NMR studies. Produced
and purified the GXXG oligosaccharide which was used with wild-type
enzyme.
Publication IV: Performed all molecular biology and biochemical
experiments, from cloning, protein production and purification to kinetic
analyses. Major writing contributions.
Publication V: Was involved in the cloning, recombinant protein
production and purification, biochemical characterisation for all enzymes.
Also extracted xyloglucan from tomato and lettuce and generated
oligosaccharides for biochemical assays. Major writing contributions.
Publication VI: Discovered the putative locus and cloned both hydrolase
genes together with ML. Performed the biochemical characterisation of the
galactosidase and performed the experiments on xyloglucan and xyloglucooligosaccharides for the fucosidase. Performed localisation and qPCR
studies. Wrote the manuscript.
Publication VII: Performed all biochemical experiments and wrote the
manuscript.
iv
LIST OF ABBREVIATIONS
AA
Auxiliary activities
AGP
Arabinogalactan protein
Araf
Arabinofuranose
BACON
Bacteroidetes-associated carbohydrate-binding, often Nterminal
CAZyme
Carbohydrate-active enzyme
CBM
Carbohydrate-binding module
CE
Carbohydrate esterase
D-enzyme
Disproportionation enzyme
Fuc
Fucose
Gal
Galactose
GalNAc
N-acetylgalactosamine
GAX
Glucuronoarabinoxylan
GH
Glycoside hydrolase
Glc
Glucose
GlcA
Glucuronic acid
GRP
Glycine-rich protein
GT
Glycosyl transferase
HGRP
Hydroxyproline-rich glycoprotein
HPAEC-PAD
High
performance
anion-exchange
with pulsed amperometric detection
HTCS
Hybrid two-component system
MALDI-TOF
Matrix-assisted laser desorption/ionisation – Time of
flight mass spectrometry
Man
Mannose
MLG
Mixed-linkage ȕĺĺJOXFDQ
NMR
Nuclear magnetic resonance
PA14
(named after the) Protective antigen of anthrax toxin
PL
Polysaccharide lyase
PNP
para-nitrophenyl/4-nitrophenyl
chromatography
v
PRP
Proline-rich protein
PUL
Polysaccharide utilisation locus
qPCR
Quantitative polymerase chain reaction
RG
Rhamnogalacturonan
SCFA
Short-chain fatty acid
Sus
Starch utilisation system
TLC
Thin-layer chromatography
XyG
Xyloglucan
XyGO
Xylogluco-oligosaccharide
XyGUL
Xyloglucan utilisation locus
Xyl
Xylose
vi
TABLE OF CONTENTS
ABSTRACT ............................................................................................................. I
SAMMANFATTNING ............................................................................................. II
LIST OF PUBLICATIONS ........................................................................................ III
THE AUTHOR’S CONTRIBUTION ........................................................................... IV
LIST OF ABBREVIATIONS....................................................................................... V
TABLE OF CONTENTS .......................................................................................... VII
INTRODUCTION .................................................................................................... 1
BACKGROUND ........................................................................................................... 2
CARBOHYDRATES ....................................................................................................... 3
PLANT POLYSACCHARIDES........................................................................................ 4
STRUCTURAL POLYSACCHARIDES ............................................................................... 5
CELLULOSE ...................................................................................................... 7
HEMICELLULOSES .............................................................................................. 9
PECTIN.......................................................................................................... 15
OTHER PLANT CELL WALL RELATED MOLECULES .......................................................... 15
STRUCTURAL PROTEINS .................................................................................... 16
LIGNIN .......................................................................................................... 16
STORAGE POLYSACCHARIDES .................................................................................. 17
STARCH......................................................................................................... 18
CARBOHYDRATE-ACTIVE ENZYMES ............................................................................... 19
GLYCOSIDE HYDROLASES ....................................................................................... 22
GH MECHANISMS ........................................................................................... 23
CATALYTIC MODE OF ACTION OF GHS.................................................................. 26
CAZYME DISCOVERY................................................................................................. 27
vii
BACTERIAL HABITATS ............................................................................................ 28
THE SOIL BACTERIUM CELLVIBRIO JAPONICUS ............................................................ 28
THE HUMAN GUT BACTEROIDETES SYMBIONTS .......................................................... 30
HUMAN-MICROBE GUT SYMBIOSIS...................................................................... 30
THE STUDIED BACTEROIDES SPECIES .................................................................... 33
POLYSACCHARIDE UTILISATION LOCI AND THE SUS PARADIGM.................................. 34
AIM OF INVESTIGATION ..................................................................................... 37
RESULTS AND DISCUSSION ................................................................................. 41
ENZYME DISCOVERY BY GLYCOSIDE HYDROLASE FAMILY ACTIVITY SCREENING ....................... 42
GH31 ............................................................................................................... 42
CJXYL31A ..................................................................................................... 42
CJAGD31B .................................................................................................... 48
BACTEROIDES THETAIOTAOMICRON GH31 TARGETS .............................................. 51
GH43 ............................................................................................................... 52
ENZYME DISCOVERY FROM GENETIC LOCI ...................................................................... 53
XYLOGLUCAN UTILISATION IN CELLVIBRIO JAPONICUS .................................................. 54
A DISCRETE LOCUS IN BACTEROIDES OVATUS, ENDOWING THE BACTERIUM WITH THE ABILITY
TO UTILISE XYLOGLUCAN ....................................................................................... 58
CONCLUSIONS .................................................................................................... 67
ACKNOWLEDGEMENTS ...................................................................................... 70
REFERENCES ....................................................................................................... 72
viii
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
INTRODUCTION
1
Johan Larsbrink 2013
Background
It is becoming more and more evident that society as a whole is facing a
huge challenge in the future, with rising global temperatures and
diminishing reserves of fossil fuels (Arkema et al. 2013, Himmel et al.
2007, Li et al. 2009). Several approaches have been suggested to alleviate
the possible ramifications of this, and one of them is a shift from our
dependence on fossil-based fuels to renewable alternatives. That goal will
unquestionably involve using plants and plant-derived biomass as a source
of energy (Himmel, et al. 2007, Schubert 2006). However, plants, or more
specifically plant cell walls, are highly recalcitrant to degradation, which is
a key step in harnessing the stored energy. Harsh chemical treatments and
high temperatures are currently required to deconstruct the cell walls using
standard industrial means.
In nature, plant cell walls are nevertheless constantly salvaged in the global
carbon cycle, a feat typically accomplished by microbes at ambient
temperatures and around neutral pH. Thus, understanding of the
fundamental biochemical processes of plant cell wall degradation utilised in
natural systems is essential in developing environmentally friendly methods
to create renewable alternatives to fossil fuels (Li, et al. 2009).
Additionally, understanding the biological processes involved in plant cell
wall degradation may lead not only to sustainable fuels, but also allow for
the generation of new plant-based materials, as well as have an impact on
human health (Flint et al. 2012b, Li, et al. 2009, Schnepp 2013). The reason
for the latter is the obvious: plants, both intact and processed, form the basis
of a healthy diet. However, the human genome does not grant us the ability
to degrade most of the ingested plant cell material, but this feat is instead
performed by the microorganisms residing in our gut, which endows us with
2
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
a huge potential for carbohydrate degradation (El Kaoutari et al. 2013). Due
to the huge complexity of the plant cell wall, finding enzymes with
appropriate properties for carbohydrate degradation and/or modification is
not a trivial task, and involves much screening, both bioinformatically and
biochemically (Li, et al. 2009), and may even require re-design of
individual enzymes to better fit industrial processes (Cobb et al. 2012, Lutz
2010).
Thus, the focus of this thesis is rooted in the need to discover enzymes with
novel or superior qualities, which can expand the toolbox of carbohydrateactive enzymes as well as improve our general knowledge in the field of
carbohydrate degradation and be a stepping stone for future endeavours. To
provide a background for this thesis, the diversity of polymeric structures
present in cell walls, with the main focus on carbohydrates, is presented, as
understanding the architecture of the cell wall is central in finding enzymes
that act upon it. Further, the types of enzymes that are involved in plant cell
wall synthesis and degradation are discussed, followed by descriptions of
the studied bacteria and their habitats, and how studying them may lead to
the discovery of novel enzymes as well as shed light on these intricate
biological systems.
Carbohydrates
Carbohydrates, or sugars, are ubiquitous in nature and are involved in all
major biological processes, from cell communication to metabolism, as well
as being a major constituent of genetic material and micro- and
macrostructures of living (and dead) organisms and tissues (Varki et al.
2009). Carbohydrates also serve as nutrients for organisms from all
kingdoms of life, as building blocks for biosynthesis, and are additionally
used as energy storage molecules for many species.
3
Johan Larsbrink 2013
The simplest carbohydrates are the monosaccharides, since they cannot be
hydrolysed into smaller sugars (Bertozzi et al. 2009). When joined together
by glycosidic bonds, monosaccharides form chains, ranging from simple
oligosaccharides up to complex polysaccharide chains, which can in turn
interact to form intricate hierarchical structures. Examples of these
polysaccharides are chitin, which is the main constituent of the robust
exoskeletons of arthropods and a major part of fungal cell walls (Cummings
et al. 2009, Tiemeyer et al. 2009), the protein-sugar co-polymer
peptidoglycan, which forms the cell walls in bacteria (Esko et al. 2009a),
and the glycoprotein surface layer which is found in the cell envelopes of
archaea (Albers et al. 2011, Esko, et al. 2009a). Animals also synthesise
large amounts of polysaccharides such as the highly complex mucus
polysaccharides lining mucosal membranes, and the heterogeneous sugar
structures found in glycoproteins and proteoglycans (Brockhausen et al.
2009, Esko et al. 2009b, Koropatkin et al. 2012, Stanley et al. 2009).
The majority of carbohydrates on earth are however synthesised by plants,
beginning with photosynthesis.
Plant polysaccharides
Though plant proteins are often heavily glycosylated (Rayon et al. 1998),
the main bulk of carbohydrates synthesised by plants may be classified as
either structural polysaccharides or storage polysaccharides. The structural
polysaccharides are incorporated into the cell walls, which are the main load
bearing structures of plants, while storage polysaccharides can be found
either inside the cell, such as starch and fructans, or in the cell wall, such as
mannans and xyloglucan (Buckeridge et al. 2000).
4
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Structural polysaccharides
In contrast to animal cells, plants are surrounded by a cell wall, similar to
for instance fungi and prokaryotes (Taiz et al. 2010). The cell wall confers
rigidity and robustness to the cell, but is at the same time highly dynamic
and flexible (Carpita et al. 2000). Plant cell walls are the most abundant
reservoir of renewable carbon on the planet, and are as such of major
importance in the global economy. The uses of plant cell walls and their
constituents include familiar products such as lumber, paper, and textiles
(Carpita, et al. 2000, Taiz, et al. 2010), but also more technically advanced
products such as plastics, thickeners, coatings etc. in a vast range of
applications (Baumann et al. 2003, Mishra et al. 2009, Willför et al. 2008).
Plant cell walls are also the main components (excluding water) in fruits and
vegetables, and consequently have a significant impact on human diet and
health (Carpita, et al. 2000).
The cell walls of plants largely consist of different polysaccharides, but also
contain proteins and aromatic compounds (lignin). There are two major
types of cell walls in plants – the primary and the secondary cell walls
(Carpita, et al. 2000). The main load bearing structure of all plant cell walls
is cellulose, which forms fibrillar structures with strong mechanical
properties (Carpita, et al. 2000, Carpita et al. 1993, Taiz, et al. 2010).
The primary cell wall is, as the name implies, the first cell wall created by
growing cells, and governs the shape and size of the cell as well as being
involved
in
defence
against
microbial
attacks
and
in
cell-cell
communication, both within the organism and with other organisms, such as
symbiotic bacteria or fungi. The cellulose fibrils of primary cell walls are
enmeshed in a matrix of non-cellulosic polysaccharides, commonly known
as hemicelluloses or cross-linking glycans, charged polysaccharides known
5
Johan Larsbrink 2013
as pectins, and structural proteins (Figure 1). There are different types of
primary cell walls, depending on the plant species, and they differ mainly in
hemicellulose composition. In type I cell walls, which are present in dicot
plants
and
noncommelinoid
monocots,
xyloglucan
is
the
main
hemicellulose, while in type II walls, present in commelinoid monocots, the
main hemicellulose is glucuronoarabinoxylan. Recently, a type III primary
cell wall was discovered in fern leaves, having mannan as the main
hemicellulose (Silva et al. 2011). However, the separation of primary cell
walls into distinct types, though useful for comparative reasons, may be
misleading, as there is likely a continuum between wall compositions rather
than three definitive types (Fangel et al. 2012, Popper et al. 2011).
In certain cell types, layers of secondary cell wall are constructed inside the
primary cell wall after the cell has adopted its final size and shape (Carpita,
et al. 2000, Taiz, et al. 2010). These secondary cell walls are typically
composed mainly of cellulose, hemicelluloses and lignin, and are much
thicker than the primary cell walls, thus conferring the plant with rigidity
and in some tissues, water impermeability (Carpita, et al. 2000, Doblin et al.
2010). The proportion of cellulose is often much higher in secondary cell
walls compared to primary cell walls, and there are often several layers of
secondary cell wall laid down, with distinct orientations of the cellulose
fibrils, which further strengthens the wall (Figure 1).
6
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Figure 1: Models of plant cell walls. A. Cross-section of a primary cell wall, containing the
load-bearing cellulose fibrils coated with hemicellulose chains, which are enmeshed in a
pectin matrix. Cell wall proteins are exemplified by an extension network. B. The celluloserich secondary cell wall and its different layers, with cellulose fibrils in different orientations.
P – primary cell wall, S1-S3 – layers of secondary cell wall.
Cellulose
The most abundant of all the different plant polysaccharides is cellulose
(Carpita, et al. 2000). It is a homopolymer, consisting exclusively of
ȕ(1ĺ4)-linked glucose moieties, though the repeating unit of cellulose is
often designated as the disaccharide cellobiose, as every other glucose unit
is rotated 180° along the chain axis (Figure 2A). Not only plants synthesise
cellulose, but also bacteria, fungi and oomycetes have the capacity for its
synthesis (Bartnicki-Garcia 1968, Deinema et al. 1971). Cellulose is
synthesised by cellulose synthase enzymes at the surface of the plasma
membrane, where individual cellulose chains bind together through
extensive inter-molecular hydrogen bonds and Van der Waals forces to form
microfibrils, which can consist of thousands of cellulose chains and reach
lengths up to the micrometre scale (Carpita, et al. 2000, Endler et al. 2011).
In plants, the cellulose microfibrils are enmeshed in a hemicellulose
7
Johan Larsbrink 2013
network, which is itself embedded in a pectic matrix, to form a so-called
liquid crystal state (Carpita, et al. 2000). The combination of the matrix
glycans and the cellulose microfibrils leads to a very robust structure, able
to support such immense living structures as the giant sequoias and redwood
trees.
Figure 2: Unbranched glucans of the cell wall. A. Cellulose, consisting of ȕĺ-linked
glucose units, with the repeating unit cellobiose in parenthesis. B. Callose, with ȕĺ
linkages. C: An exemplified stretch of a mixed-linkage ȕĺȕĺ JOXFDQ 0/*
consisting of stretches of cellotriose and cellotetraose, joined together by ȕĺOLQNDJHV
8
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Hemicelluloses
The term hemicellulose was coined in the late 19th century, and defined as
plant cell components possible to extract from plant material by alkaline
solutions (O'Dwyer 1923). To distinguish the non-charged cell wall
polysaccharides from other material extracted from the cell walls, and as the
term was coined when nothing was known about plant polysaccharide
structure or biosynthesis, cross-linking glycans has been suggested as an
alternative term. The word hemicellulose persists as an umbrella term for
these polysaccharides however, and will be used throughout this thesis. The
hemicelluloses are polysaccharides that coat the cellulose microfibrils in the
plant cell wall, and may also further be covalently attached to lignin through
ester bonds (Levasseur et al. 2013). The hemicellulose classification
encompasses several structurally diverse polysaccharides, which can be
found in various amounts in both primary and secondary cell walls,
depending on plant species and types of tissues (Ebringerová et al. 2005).
Callose
A polysaccharide related to cellulose is callose. It also consists solely of
glucose, but in contrast to cellulose, the linkage in callose is ȕĺ (Figure
2B), resulting in helical chains (Carpita, et al. 2000). Callose is not a
general cell wall polysaccharide, but is present in certain cell types, such as
pollen, and is also produced in response to certain microbial attacks, such as
by fungi (Carpita, et al. 2000, Ellinger et al. 2013).
Mixed-linkage glucans
Another major hemicellulose that are mainly found in the Poales order,
which includes the common cereals, are the mixed-linkage ȕĺĺ
glucans (MLGs) (Carpita, et al. 2000, Ebringerová, et al. 2005, Fincher
2009). The MLGs are composed mainly of stretches of cellotriose and
9
Johan Larsbrink 2013
cellotetraose, connected by ȕĺ-linkages in an irregular fashion, making
them soluble in contrast to cellulose which only contains ȕĺ-linkages
(Figure 2C). MLGs, like other hemicelluloses, bind cellulose, and constitute
around 5 – 10 % of the biomass of such commercially important crops as
barley and oat (Ebringerová, et al. 2005). In recent years, the realisation of
the importance of fibres in human diet and their potential in alleviating such
dietary problems as diabetes, colorectal cancer and certain inflammations
have led to much focus on MLGs as wholegrain cereal products are some of
the main sources of dietary fibre in normal diets (Jacobs et al. 2004,
Topping 2007).
Xyloglucan
Xyloglucan (XyG) is the main hemicellulose in primary cell walls in dicot
plants, and is present in all terrestrial plants (Carpita, et al. 2000, Peña et al.
2008, Popper 2008). The amount of XyG can reach 20-25 % of the total
carbohydrate content (dry weight) in some species (Ebringerová, et al.
2005), and will as such be a major influence in a wide variety of plantderived products. In the cell wall, XyG is believed to cross-link the cellulose
microfibrils, thus conferring mechanical strength to the plant structure. The
cross-linking properties of XyG, thus conferring mechanical strength, may
however not be as extensive as most cell wall models have stated in the
past, where long XyG tethers (20 – 40 nm) were thought to connect the
cellulose fibrils (Park et al. 2012). Recent studies have instead indicated that
the main bulk of the XyG chains coat the cellulose fibrils, while only a few
key chains tether cellulose fibrils close together. There may even be
significant intertwining of the XyG and individual cellulose chains in
regions of amorphous non-crystalline cellulose. The high affinity of XyG
toward cellulose has been demonstrated in vitro, and the co-localisation of
XyG and cellulose has been observed in plant cell walls using specific
10
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
antibodies and electron microscopy (Carpita, et al. 2000, Pauly et al. 1999).
XyG is also used as a storage polysaccharide in seeds in certain species,
such as the tamarind tree (Tamarindus indica) and nasturtium (Tropaeolum
majus), providing energy and building materials for the growing embryo
(Buckeridge, et al. 2000, Gidley et al. 1991, Savur et al. 1948).
Structurally, XyG FRQVLVWVRIDOLQHDUȕĺJOXFDQEDFNERQHVXEVWLWXWHG
at regular intervals with xylosyl moieties (Carpita, et al. 2000, Ebringerová,
et al. 2005). The xylosyl side chains are commonly substituted further with
galactosyl, fucosyl and/or arabinofuranosyl units depending on the plant
species and tissue (Figure 3A) (Hoffman et al. 2005, Hsieh et al. 2009). To
more easily describe the various side-chains of XyG, a distinct
nomenclature was developed by (Fry et al. 1993), where G denotes an
unbranched Glc residue, and the most common substitutions are as follows:
X (Xylp-Įĺ-ȕ-Glcp), L (ȕĺ-Galp-Įĺ-Xylp-ȕĺ-Glcp), F
(L-Fucp-Įĺ-Galp-Įĺ-Xylp-ȕĺ-Glcp),
and
S
(L-Araf-
Įĺ-Xylp-Įĺ-ȕ-Glcp. Typically, the XyG chains consist of
repeating patterns of the XXGG or XXXG type, corresponding to a
repeating backbone motif of four glucose residues substituted with two or
three xylose residues, respectively, and carrying various amounts of
additional substitutions (Vincken et al. 1997). The most common type of
XyG in higher land plants is fucogalacto-XyG of the XXXG type such as in
lettuce (Lactuca sativa), carrot (Daucus carota), and soybean (Glycine max)
(Figure 3A, left panel) (Hoffman, et al. 2005). Another important dietary
plant family is the Solanaceae, including potato (Solanum tuberosum),
tomato (Lycopersicon esculentum), and peppers (Capsicum), which are of
the XXGG type, lack fucose and instead contain arabinose (Figure 3A, right
panel).
11
Johan Larsbrink 2013
Figure 3: Structures of common hemicelluloses. Colour code as follows: Glc – blue, Xyl –
orange, Gal – yellow,
L-Fuc
– red, Araf – turquoise, GlcA – purple, Man – green.
A. Xyloglucan oligosaccharide structures from different sources. Fucogalactoxyloglucan of
the XXXG type, as can be obtained from dicot species (left panel), and so-called solanaceous
arabinofuranosylated xyloglucan of the XXGG type, as can be obtained from for example
tomato
and
peppers
(right
panel).
B.
Glucuronoarabinoxylan,
substituted
with
arabinofuranose, glucuronic acid and ferulic ester moieties. The double substitution site at O3 is highlighted by a parenthesis. C. Galactomannan. Pure mannan would lack the galactosyl
decorations. D. Galactoglucomannan.
12
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Other, more rare, patterns also exist, such as fucosyl-lacking XXGGG-based
XyG, which is the case for morning glory (Ipomoea pupurea), basil
(Ocimum basilicum) and plantain (Plantago major) (Hoffman, et al. 2005).
Furthermore, there are many additional substitutions to XyG than the
common L, F and S sidechains, as well as plants creating XyG which
contains both arabinose and fucose, such as oleander (Nerium oleander)
(Hoffman, et al. 2005, Peña, et al. 2008).
XyG is readily available as a by-product from tamarind fruit production in
the form of seeds (kernels) or as tamarind kernel powder, and can be used in
varying applications from food thickeners to drug delivery agents (Mishra,
et al. 2009, Yamatoya et al. 2003). The strong affinity of XyG to cellulose
can also be exploited in biomimetics, to functionalise cellulose surfaces by
adsorption of chemically modified XyG to gain novel properties while
maintaining the strong physical properties of cellulose (Teeri et al. 2007,
Zhou et al. 2005).
Xylan
Xylan is present in the cell walls of both monocots and dicots, but is the
main hemicellulose found in the primary cell walls of grasses, the so-called
type II walls, as well as in the secondary cell walls of hardwood trees
(Carpita, et al. 2000, Carpita, et al. 1993). Xylan is composed of ȕĺlinked xylopyranose moieties, which can be substituted in various ways,
depending on the species (Figure 3B). The type of xylan found in primary
cell walls of commelinoid monocots, glucuronoarabinoxylan (GAX), is
often substituted with Į-L-Araf at the O-3 position, while Įĺ-linked
glucuronic acid decorations and feruloyl moieties at the C-5 position of Į-LAraf moieties are rarer. In contrast, GAX in non-commelinoid monocots and
dicot species may be decorated with Į-L-Araf at both O-2 and O-3. In
13
Johan Larsbrink 2013
secondary cell walls however, arabinose decorations are not found, but
glucuronic acid is still present. Xylan is often a major part of secondary cell
walls, and may constitute up to 30 % of the biomass, making it available in
large amounts in nature (Ebringerová, et al. 2005). Especially important for
human use are the arabinofuranosylated xylans found in common cereals,
where they may constitute approximately 13 % in grain from barley and rye,
and up to 30 % in wheat bran, and as such be a rich source of dietary fibre.
Mannans
There are several types of different polysaccharides containing ȕĺlinked mannan in their backbones: pure mannan, galactomannan,
glucomannan and galactoglucomannan (Carpita, et al. 2000). Pure mannan
is rare, and have only been found in the bulb of the orchid Oncidium (Wang
et al. 2006). Polysaccharides consisting of a pure mannan backbone,
substituted with galactosyl units is called galactomannan, and the level of
substitution affects their solubility in water, where a less substituted
polysaccharide is less soluble, such as the largely unsubstituted mannan in
ivory nut (Phytelephas macrocarpa; Figure 3C) (Buckeridge 2010).
Galactomannans, consisting of a mannan backbone substituted with
galactose in Įĺ-linkages are used as storage polysaccharides in various
plants, such as coffee (Coffea arabica), palm tree (Phoenix dactylifera) and
mimosa (Mimosa scabrella) (Buckeridge 2010, Ebringerová, et al. 2005).
Mannan (or possibly galactomannan with a low level of galactosyl
substitutions) is also the main hemicellulose in the recently described type
III primary cell wall, which was discovered in fern leaves (Silva, et al.
2011). In secondary cell walls, glucomannans and galactoglucomannans are
the major hemicellulose in softwoods, where the backbone may also be
acetylated
(Figure
3D)
(Ebringerová,
et
al.
2005).
Structurally
glucomannans and galactoglucomannans differ from pure mannan and
14
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
galactomannan in that the backbone is made up of alternating stretches of
ȕĺ-linked mannose units and ȕĺ-linked glucose units.
Pectin
Pectin is a class of heterogeneous polysaccharides rich in galacturonic acid,
that classically were defined as material possible to extract from cell walls
by chelators (Carpita, et al. 2000). Pectin is found in both dicot and
monocot primary cell walls, as well as in the middle lamella and to a lesser
extent in woody tissues (Mohnen 2008). In the cell wall, the hemicellulosecoated cellulose fibrils are extruded into the cell wall during synthesis,
where they are embedded in a matrix of pectic polysaccharides (Carpita, et
al. 2000). The most common form of pectin is homogalacturonan, which is
composed of Įĺ-linked galacturonic acid units which can be
methylated at the C-6 position and/or acetylated at O-2 or O-3. More
complex types are the rhamnogalacturonan (RG) I and II, which are highly
branched with a multitude of different monosaccharides and linkages.
Examples of the sidechains of RG I are galactan, arabinan and
arabinogalactan. Pectins are not only a part of the cell walls, but also have
many other roles, such as cell-cell adhesion, signalling and fruit
development. Another common use of pectin is in the food and cosmetics
industries as a gelling agent.
Other plant cell wall related molecules
Polysaccharides, such as the ones described above, are not the only parts of
plant cell walls. Many proteins, which are often heavily glycosylated, fulfil
important roles in the cell walls, and a key factor for the strength of
secondary cell walls are the complex networks created from phenolic
substances called lignin (Carpita, et al. 2000).
15
Johan Larsbrink 2013
Structural proteins
Structural protein networks in the cell walls of plants consist mainly of four
classes of proteins: the hydroxyproline-rich glycoproteins (HGRPs), the
proline-rich proteins (PRPs), the glycine-rich proteins (GRPs) and the
arabinogalactan proteins (AGPs). These proteins are secreted into the cell
wall, and all but the GRPs are glycosylated. One of the more studied
HGRPs is extensin. Extensin forms rod-like polypeptides that are
glycosylated to form a brush-like structure. Extensins form networks in the
cell wall, and are involved for example in defence against attacks by
pathogens, when they are accumulated, and in the formation of the cell plate
during cell division (Lamport et al. 2011). The most heavily glycosylated
structural protein class are the AGPs, which may consist of more than 95 %
carbohydrate, which makes them highly soluble (Carpita, et al. 2000,
Doblin, et al. 2010). Most AGPs have membrane anchors, and may thus be
connected to the cell membrane. AGPs are believed to be involved in root
formation, pollen tube guidance and cell-cell interactions (Doblin, et al.
2010).
Lignin
Lignin is unique to secondary cell walls, and is a key agent in making cell
walls water impermeable and recalcitrant to enzymatic degradation, as well
as making the plant cell wall strong enough to support the tall species we
can observe today (Carpita, et al. 2000, Doblin, et al. 2010, Vanholme et al.
2012). The lignin network is created by radical-reactions joining the basic
monomer building blocks derived of p-hydroxycinnamyl alcohol: pcoumaryl, sinapyl and coniferyl alcohols (Figure 4) (Vanholme, et al. 2012).
The synthesis of lignin begins with oxidation of the monomers to form
monolignol radicals, which then combine through radical-radical couplings,
and also allow the network to combine with polysaccharides. The oxidation
16
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
of lignin monomers is performed by peroxidases and/or laccases that are
encoded by large gene families in plants. The lignin network not only
strengthens the cell walls, and especially for vascular tissues makes them
hydrophobic, but also inhibits cellulases and sterically hinders other
enzymes from attacking the various polysaccharides of the wall (Dixon
2013, Vanholme, et al. 2012). Because of this, many studies are aimed
towards reducing the lignin content or changing the ratio of the monomers
in crops to facilitate saccharification for biofuels, while not adversely
affecting the plants.
Figure 4: The monomers making up the majority of lignin.
Storage polysaccharides
As mentioned previously, hemicelluloses often serve as energy sinks in
various organisms, to be used during nutrient deficiency or as energy
reserves in seeds (Buckeridge 2010). For most plants however, energy
stored as carbohydrates is stored in the form of starch (Zeeman et al. 2010).
17
Johan Larsbrink 2013
Starch
While animals and certain microorganisms store energy in the form of
glycogen, plants use the very similar polysaccharide starch (Elbein 2009,
Wilson et al. 2010, Zeeman, et al. 2010). Both starch and glycogen are, in
common with cellulose, composed purely of glucose, with the difference
lying in the linkages and morphology of the glucan chains. In cellulose, as
described earlier, the ȕ(1ĺ4) linkages give rise to straight polysaccharide
chains. In starch however, the glucan chains are linked in the Įĺ
configuration (Figure 5A), which leads to the formation of helices, and
results in a more soluble polysaccharide. The chains of starch are further
branched by Į(1ĺ6) linkages to form brush-like structures (Figure 5B and
C). One key difference between starch and the hemicellulose storage
polysaccharides is that the former is synthesised within the cells in
specialised compartments called plastids, while the latter are inserted into
the cell wall (Buckeridge 2010, Pérez et al. 2010).
Chemically, the differences between starch and glycogen lie in the amount
of branching, where glycogen is more heavily and continuously branched
than starch. Glycogen particles are also smaller (20-60 nm) than starch
granules (Zeeman, et al. 2010), which can reach diameters of around 200
µm (Pérez, et al. 2010). The amount of branching in starch affects its
digestibility in the gut, and starches with less branching tend to be more
crystalline and are more slowly digested (Gilbert et al. 2013). Starch that is
not fully digested in the small intestine is called resistant starch, and is a
nutrient source for many bacteria in the human distal gut (Flint 2012). The
ingestion of resistant starch can improve colonic health by increasing the
production of short-chain fatty acids by the gut microbiota (Koropatkin, et
al. 2012). Many of the plants utilised by mankind as staple food are either
18
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
seeds or tubers containing predominantly starch, such as maize, potato, rice
and cereals.
Figure 5: Structures of starch. A. The main building block of starch: Įĺ-linked
glucose, exemplified by the trisaccharide maltotriose. B. The branching pattern of starch by
an Įĺ-linkage. C. An example of the brush-like structure adopted by starch, where ovals
represent glucose moieties. Several of these brushes aggregate to form starch granules.
Carbohydrate-active enzymes
As evidenced by the above discussion, the possible structural variations of
carbohydrates are essentially endless, and consequently a large variety of
enzymes capable of acting on these substrates have evolved over the
millennia. As the number of possible carbohydrate structures vastly
outnumbers that of protein folds, proteins acting on carbohydrates have
19
Johan Larsbrink 2013
evolved different specificities for oligo- and polysaccharides as well as
glycoconjugates from a limited number of protein scaffolds. The enzymes
acting on these substrates, collectively known as Carbohydrate Active
enZymes (CAZymes) (Cantarel et al. 2009), have been grouped into distinct
phylogenetic families based on their similarities in primary amino acid
sequences, protein folds, and mechanisms of action. These families can be
found in the CAZy database (www.cazy.org). The database, which currently
contains over 300 protein families (Levasseur, et al. 2013), is continuously
updated as more CAZymes are identified and functionally and/or
structurally characterised. The protein families of CAZy have been clustered
into five different enzyme classes and one class of associated modules, the
carbohydrate binding modules, as follows:
Glycoside Hydrolases (GH) – these enzymes break the bonds between
sugars by hydrolysis. There are to date over 130 GH families,
comprising over 150 000 predicted catalytic modules. Additionally,
some GH enzymes are able to perform transglycosylation reactions.
The GHs are discussed in greater detail below.
Glycosyl Transferases (GT) – the GTs are enzymes which synthesise
all the different di- oligo- and polysaccharides found in nature. They
perform this task by transferring sugar moieties from activated
phospho-sugars; typically nucleotide-sugar donors are used, such as
UDP-Glc for cellulose and callose synthases or UDP-Xyl for xylan
synthases (Campbell et al. 1998, Lairson et al. 2008).
Polysaccharide Lyases (PL) – PL enzymes cleave uronic acidcontaining sugars, such as pectins and many algal polysaccharides. The
mechanism by which they cleave the glycosidic bonds is ȕ-elimination,
20
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
which yields unsaturated hexenuronic acid or ester moiety at the nonreducing end of the polysaccharide (Garron et al. 2010, Lombard et al.
2010).
Carbohydrate Esterases (CE) – several types of carbohydrates are
substituted with acyl moieties, and the enzymes that catalyse the
cleavage of these ester bonds are the CEs. The most common
mechanism of these enzymes is the classical Ser-His-Asp catalytic triad
mechanism employed by serine proteases. (Dodd et al. 2009, Taylor et
al. 2006)
Auxiliary Activities (AA) – the AA classification is a very recent
addition to CAZy (Levasseur, et al. 2013), and comprises enzymes that
break glycosidic bonds by oxidation. The discovery that the proteins of
GH61 and CBM33 were actually metal-dependent polysaccharide
monooxygenases, able to break the backbone of crystalline
polysaccharides, and not GHs or CBMs, respectively, warranted their
re-classification. GH61 and CBM33 were classified as AA families 9
and 10, respectively. Enzymes able to oxidise various positions on
carbohydrates have also been identified: AA3 members can act on C1
and C2, AA7 on C1, and AA9 on C1, C4 and C6. In addition, enzymes
involved in lignin degradation were included in the classification, as
lignin is invariably associated with polysaccharides in plant cell walls.
Carbohydrate Binding Modules (CBM) – CBMs are non-catalytic
proteins with the ability to bind carbohydrates. CBMs are by definition
modules associated with other CAZymes (though exceptions exist),
and function by bringing the catalytic module into close proximity of
its substrate and prolonging the enzyme-carbohydrate interaction,
21
Johan Larsbrink 2013
thereby improving catalysis. CBMs can adopt a number of different
folds, depending on their target substrates, which range from monoand oligosaccharides up to both crystalline and non-crystalline
polysaccharides (Boraston et al. 2004).
The GH enzymes are the most numerous class in CAZy, and when looking
only at carbohydrate degrading enzymes (excluding GTs and CBMs), the
number of GH modules completely dwarfs that of PLs, CEs and AAs
(Cantarel, et al. 2009, Levasseur, et al. 2013). The main focus of this thesis
is the discovery of new GH enzymes with either hydrolytic or
transglycosylating activities, and as such, GH enzymes are discussed in
greater detail below.
Glycoside hydrolases
Glycosidic bonds form some of the most stable polymers on earth. In fact,
cellulose, which consists solely of ȕ(1ĺ4) glucose units, has a half-life of
close to 5 billion years at 25 °C under neutral conditions (Wolfenden et al.
1998). Despite the extraordinary stability of glycosidic bonds, certain
CAZymes are able to enhance the rate of hydrolysis of glycosides by a
factor of up to 1017.
GH enzymes carry out the majority of carbohydrate degradation in nature,
and are consequently responsible for some of the most important
biochemical reactions on a global scale. The number of identified GHs is
ever increasing with more (meta)genomic sequences being released, and
constitute around half the entries of the CAZy database (Cantarel, et al.
2009). As mentioned above, the GHs have been ordered into families based
on amino acid sequence similarity, which correlates with protein folds and
enzymatic mechanisms. A given GH family may be monospecific, i.e.
22
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
having members active on a single substrate, such as GH11 which only
contains xylanases, or polyspecific, containing a wide variety of different
specificities, such as GH5, which contains close to 15 different activities
(Aspeborg et al. 2012). Intensive work by crystallographers has led to about
80 % of the GH families having one or more representative structures
solved to date (Fushinobu et al. 2013).
GH mechanisms
GHs will in general perform their catalytic function following one of two
catalytic routes: either by net inversion or retention of the anomeric
configuration of their respective substrate, as was first described by
Koshland 60 years ago (Koshland 1953). For inverting enzymes, hydrolysis
occurs in a one-step direct displacement reaction of the leaving group with
water (Figure 6A). The catalytic amino acid residues are two carboxylic
acids, typically spaced ~10 Å apart, where one acts as a general acid and the
other as a general base (Davies et al. 1995, McCarter et al. 1994, Zechel et
al. 2000). Also retaining enzymes employ two carboxylic acids, but
hydrolysis is instead performed as a double-displacement reaction,
involving a glycosyl-enzyme intermediate, where one carboxylic acid
residue acts as a nucleophile and leaving group and the other as a general
acid/base (Figure 6B). The catalytic residues of retaining enzymes are also
more closely spaced, at 5.5 Å. The first step of the reaction consists of a
nucleophilic attack on the anomeric carbon by the catalytic nuclephile,
leading to the formation of a glycosyl-enzyme intermediate. In the second
step, the deglycosylation step, the general acid/base residue deprotonates a
water molecule which then displaces the sugar moiety.
23
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Hydrolysis is however not the only possible outcome in the deglycosylation
step, as the glycosyl enzyme can also be intercepted by other acceptor
molecules; often, in these cases, the acceptor molecule is another sugar,
which leads to transglycosylation. In fact, transglycosylation has been
shown, through studies on the reactivation of trapped intermediates, to
always be kinetically favoured over hydrolysis (Withers et al. 1990). How
transglycosylating enzymes are able to exclude water, which is typically at
~55 M, from the reactions has however been a puzzling subject. One
hypothesis is that strict transglycosylases do not allow water molecules to
be positioned in a catalytically favourable manner (leading to deprotonation
and activation) in the active site, but instead the enzymes are perfectly
tailored to activate an acceptor glycoside moiety (Larsbrink et al. 2012).
Exceptions to the classical retaining mechanisms have been discovered in
many GH families. Families 18, 20, 25, 56, 84, and 85, for example, use
substrate-assisted hydrolysis, where no nucleophile is provided by the
enzyme, and instead the N-acetyl group in a substrate such as chitin acts as
an intramolecular nucleophile, forming an oxazoline intermediate, and
typically the intermediate is stabilised by a carboxylate residue (Figure 6C)
(Mark et al. 2001, Terwisscha van Scheltinga et al. 1995). Another
alternative mechanism is displayed by members of families 33 and 34,
which contain sialidases and trans-sialidases, and use a tyrosine instead of a
carboxylic acid as nucleophile (Watts et al. 2003). The nucleophilicity of
the tyrosine is amplified by a neighbouring general base, and a reason for
using a non-charged nucleophile is to avoid charge repulsion, as the
anomeric centre itself contains a negatively charged carboxyl group.
Catalysis in GHs may also be dependent on cofactors, such as NAD+ in
families 4 and 109, and break the glycosidic bond by elimination, yet still
25
Johan Larsbrink 2013
leading to the complete reaction being hydrolysis (Rajan et al. 2004, Yip et
al. 2004).
Catalytic mode of action of GHs
In addition to their mechanisms, GHs are also classified by their mode of
action on carbohydrates; that is, whether they cleave internal bonds in
polysaccharides (endo-acting enzymes), or whether they act on the ends of
poly- or oligosaccharides (exo-acting enzymes) (Davies, et al. 1995). Endoacting enzymes typically have a cleft-like active site, able to recognise, bind
and accommodate a stretch of the target polysaccharide in order to facilitate
hydrolysis, thereby creating breaks in the backbone of the polysaccharide.
The binding of polysaccharide substrates in the active site is achieved by a
number of subsites which bind to individual sugar units along the
polysaccharide chain. The subsites are numbered from the point of
hydrolysis, where monosaccharides toward the non-reducing end are
labelled -1, -2, -3 etc. and consequently monosaccharides toward the
reducing end are labelled +1, +2, +3, etc (Davies et al. 1997). Exo-acting
enzymes on the other hand typically recognise a limited number of
monosaccharide units on their target substrate, and cleave off either a
monosaccharide or a shorter oligosaccharide, thus depolymerising the
substrate from its ends. Most commonly, this is performed at the nonreducing end of the sugar chains, but exceptions to this rule exist, such as a
GH8 xylanase from Bacillus halodurans C-125, which releases xylose from
the reducing-end (Fushinobu et al. 2005, Honda et al. 2004), or the GH7
cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei), which
processively cleaves cellobiose from the reducing end of cellulose chains
(Divne et al. 1998). Certain enzymes, such as the aforementioned
cellobiohydrolase I, are able to prolong the contact with their cognate
substrate and carry out multiple catalytic events, by for example threading
26
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
the substrate through a tunnel in the enzyme, in which the active site
residues are located, such as certain cellulases/cellobiohydrolases (Davies,
et al. 1995, Zou et al. 1999). Enzymes acting in such a manner are called
processive.
CAZyme discovery
The break-down of carbohydrates into their constituent monosaccharides
can be achieved by many means, but using enzymes is regarded as the most
sustainable method, and is one which preserves the basic carbohydrate
structures in contrast to for example thermochemical processes, which can
cause caramelisation and unwanted side-products (Horn et al. 2012). A key
step in designing a biochemical process for biomass saccharification is
finding enzymes with appropriate activities that can efficiently perform this
feat, and there is a long history in the biotechnological field of trying to find
new and better enzymes for biomass conversion (Falch 1991, Mandels et al.
1957). There are many approaches in discovering novel enzymes, like indepth proteomic, metabolic and biochemical studies of efficient biomass
degraders, as in the extensive work done on enzymes from the wood-rotting
fungi Hypocrea jecorina (formerly known as Trichoderma reesei) and
Phanerochete chrysosporium (Dashtban et al. 2009, MacDonald et al. 2012,
Schuster et al. 2010), or by doing metagenomic sequencing to identify all
genes, including CAZymes, of entire environmental samples consisting
whole microbial communities (Li, et al. 2009). As bacteria, or prokaryotes,
are truly ubiquitous in nature, they provide a practically limitless source of
genetic information, and are as such verdant ground for discovering novel
carbohydrate degrading systems (Whitman et al. 1998).
27
Johan Larsbrink 2013
Bacterial habitats
Wherever life is supported on earth, prokaryotes can be found. Not only
have prokaryotes been found in lakes and oceans, forests, and also in
association with other species. Further, they have been found in subsurface
sediments several kilometres deep, subglacial lakes, as well as in such an
unlikely location as the earth’s stratosphere (Shtarkman et al. 2013,
Wainwright et al. 2003, Whitman, et al. 1998). As prokaryotes thrive in all
kinds of environments, they play pivotal roles to the world’s ecosystems and
the build-up and recycling of the molecules of life. A typical cell density of
prokaryotes in aquatic and subsurface environments is in the range of
around a million cells/ml, while cell densities in forest floors ranges
between 106-108 cells/ml (Whitman, et al. 1998). Extremely high prokaryote
cell densities can be found on the surface and the inside of animals,
although this habitat does not constitute a major fraction of prokaryotes
globally.
The enzymes studied in this thesis were acquired from bacteria with high
capabilities for degrading carbohydrates: Cellvibrio japonicus, whose
habitat is the forest floor, and Bacteroides thetaiotaomicron and Bacteroides
ovatus, which are dominant human gut symbionts.
The soil bacterium Cellvibrio japonicus
This bacterium was first isolated in 1952 from Japanese soil, and was
originally named as Pseudomonas fluorescens subsp. cellulosa (Ueda et al.
1952). The bacterium was however not highly similar to the Pseudomonas
genus, and was removed as a member in a revision based on 16S rRNA
sequences in 2000 (Anzai et al. 2000). Later, in 2003, a study revealed that
the bacterium was more closely related to the genus Cellvibrio, and it was
reclassified as a novel species under its current name (Humphry et al. 2003).
28
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Many studies on C. japonicus enzymes had already recognised the
bacterium’s potential for complex carbohydrate degradation (Hazlewood et
al. 1998), and when the complete genome sequence of C. japonicus was
released in 2008, not surprisingly, a vast number of enzymes predicted to be
involved in the degradation of plant cell walls were identified (Deboy et al.
2008).
C. japonicus has become something of a model soil saprophyte organism as
light has been shed on not only its vast CAZyme repertoire and ability to
degrade all major parts of the plant cell wall (Deboy, et al. 2008,
Hazlewood, et al. 1998), but also related processes such as gene regulation
and protein secretion (Emami et al. 2009, Gardner et al. 2010). Especially
noteworthy is the work done on C. japonicus by Prof. Harry Gilbert and coworkers during the years, which has described CAZymes active on a range
of substrates, such as xylan (Beylot et al. 2001, Gilbert et al. 1988), mannan
(Hogg et al. 2003, Hogg et al. 2001), arabinan (Beylot, et al. 2001, McKie
et al. 1997) and cellulose (Gilbert et al. 1987, Hall et al. 1988, Hazlewood
et al. 1992).
The enzymes produced by C. japonicus are, in contrast to the membraneanchored cellulosomes of Clostridia, in many cases secreted into the cell’s
surroundings to degrade plant matter (Hazlewood, et al. 1992), and recently,
the requirement of the type II secretion system for the bacterium to be able
to secrete endo-glucanases, which are enzymes crucial for the
deconstruction of biomass, was demonstrated (Gardner, et al. 2010).
Furthermore, techniques enabling the genetic manipulation of C. japonicus
have been established, and an example of the industrial potential of C.
japonicus was demonstrated by introducing the capacity for ethanol
production into the bacterium (Gardner, et al. 2010, Gardner et al. 2012).
29
Johan Larsbrink 2013
The human gut Bacteroidetes symbionts
The studied human gut symbionts, Bacteroides thetaiotaomicron and B.
ovatus are, as mentioned, dominant species in our intestinal microbiota. In
order to appreciate their success in such a highly competitive habitat, it is
necessary to first give an overview of the forces that shape our internal
ecosystem, as well as point out the potential benefits and negative
consequences a healthy and disturbed microbiota can create, respectively.
Human-microbe gut symbiosis
Although not apparent to the naked eye, each human individual is a
veritable microbe farm. The density of prokaryotic cells found on human
skin is typically around 104/cm2, but the majority of unicellular organisms
are found in the digestive tract (Whitman, et al. 1998). In the small
intestine, the number of microbial cells is comparatively low (~104/ml),
largely due to the low pH and high flow-rate (Berg 1996). In the distal
human gut (colon) however, the numbers of microorganisms may exceed
such immense densities as 1011 bacteria/gram intestinal content, and the
average human harbours roughly 1.5 kg of intestinal biomass (Figure 7)
(Kaper et al. 2005). These trillions of microbes residing in our
gastrointestinal tract actually outnumber somatic human cells by at least a
factor of 10, and evidently contain a plethora of genes that enable the
scavenging of nutrients from the host’s diet (Cantarel et al. 2012, El
Kaoutari, et al. 2013, Martens et al. 2011). The number of genes encoded by
the gut microbiota has in fact been estimated to be 150 times larger than that
encoded by the human genome (Qin et al. 2010), and the multitude of
interactions between the gut microbiota and the human host may warrant the
microbiota being seen as a separate, ‘virtual’, organ in the body (Evans et
al. 2013).
30
Johan Larsbrink 2013
the rapidly increasing volume of (meta)genomic data on the microbiota
(Nelson et al. 2010, Qin, et al. 2010, Xu et al. 2003a), the detailed
molecular
processes
responsible
for
the
degradation
of
dietary
polysaccharides are still not well understood. A deeper understanding of the
carbohydrate utilisation of beneficial gut bacteria might very well lead to
not only novel therapeutic strategies in treating dietary/intestinal disorders,
but may also shed light on how these complex intestinal ecosystems are
created and maintained through an individual’s life.
The dominant phyla of the gut microbiota are species belonging to the
genera of Bacteroides and Firmicutes, and to a lesser degree, Actinobacteria
(Turnbaugh et al. 2009), although at the species level, the relative
proportions of approximately 70 % of the gut microbiota species in any
given individual are specific to the host and are dependent on factors such
as diet (Ley et al. 2006b, Tap et al. 2009, Zoetendal et al. 1998). The
colonisation of the human gut begins at birth, and the composition of
species will change at such key events as transition from milk or baby
formula to solid foods (Koropatkin, et al. 2012). The composition of the
adult gut microbiota can be surprisingly stable, which in some cases, when
there is an abnormal composition of gut symbionts, leads to persistent
digestive problems, such as the severe malnutrition disorder kwashiorkor
(Smith et al. 2013), or other health hazards such as irritable bowel
syndrome, obesity, and colon cancer (Koropatkin, et al. 2012). As an
example, the microbiota of twins and/or between mothers and offspring are
more similar than between unrelated individuals, even if the related
individuals live at locations very distant from each other (Turnbaugh, et al.
2009).
32
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Regarding obesity, studies on twins have revealed that in obese individuals,
the phylogenetic diversity of the gut microbiota is significantly lower than
in lean individuals, which is likely caused by a less diverse diet (Turnbaugh,
et al. 2009). There is also a lower proportion of Bacteroidetes species and a
higher proportion of Actinobacteria, an observation which shows the same
pattern in similar studies on unrelated individuals as well as in mice (Ley et
al. 2005, Ley, et al. 2006b). The gut microbial community furthermore
appears to affect human health in such profound ways as in helping in the
development of the immune system, inhibition of pathogen colonisation,
synthesis of certain vitamins etc. (Xu et al. 2003b).
The administration of broad-spectrum antibiotics sometimes leads to severe
health problems, caused by pathogenic organisms which can establish
themselves when the normal flora has been knocked out. A solution to this,
recently reported in several newspapers, is to introduce a ‘normal’ gut flora
from a healthy individual, by faecal transplants from healthy individuals
(Macconnachie et al. 2009, Persky et al. 2000, Schwan et al. 1984, van
Nood et al. 2013).
The studied Bacteroides species
The studied gut bacteria in this thesis are the closely related Bacteroides
thetaiotaomicron and Bacteroides ovatus. In the distal human gut, species of
the Bacteroidetes phylum are particularly dominant, comprising around
30% of the total population of gut bacteria, and reaching densities of 1010 –
1011 cells per gram of human fecal matter (Ley et al. 2006a). Bacteroides
thetaiotaomicron has served as a culturable and genetically tractable model
species in studies of human-gut/microbial symbiosis. The whole-genome
sequencing of this organism in 2003 was a watershed in the field (Xu, et al.
2003a); currently there is a growing number of genome sequences for
33
Johan Larsbrink 2013
related Bacteroidetes and transcriptomics data are now available for selected
model species (Kim et al. 2011, Martens, et al. 2011, Nelson, et al. 2010,
Xu, et al. 2003a). Particularly striking is the vast number of predicted
glycoside hydrolase and polysaccharide lyase family members in individual
Bacteroidetes genomes, which typically exceeds that of other human gut
bacteria, e.g. representative Firmicutes (Cantarel, et al. 2009, Mahowald et
al. 2009, Xu, et al. 2003a).
B. ovatus is closely related to B. thetaiotaomicron, and the two species
actually share 96.5 % nucleotide sequence identity in their respective 16S
rDNA genes (Martens, et al. 2011). Excluding cellulose, these two species
together share the capability to degrade all major mucosal and plant-derived
polysaccharides that may enter the human gut. While the two bacteria share
some common glycan preferences, a comparison of their carbohydrate
degradative abilities reveals that while B. thetaiotaomicron is adapted to
forage on host glycans and pectins, B. ovatus is a proficient degrader of all
common hemicelluloses. Consequently, these data show how different
bacteria have evolved to not only spatial, but also nutritional niches as
specific as distinct polysaccharides, to thrive in the gut ecosystem.
Polysaccharide Utilisation Loci and the Sus paradigm
Species of Bacteroidetes utilise a complex genetic setup to degrade certain
polysaccharides, called Polysaccharide Utilisation Loci (PULs) (Martens et
al. 2009). These PULs are composed of multiple genes which are coregulated and the transcription of individual PULs is regulated by the
sensing of a corresponding carbohydrate. The existence of PULs was
discovered by studies on the archetypal Starch Utilisation System (Sus) of
B. thetaiotaomicron, which has been described in detail over the past two
decades, and was founded on ground-breaking work by Prof. Abigail
34
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Salyers and co-workers (Cho et al. 2001, D'Elia et al. 1996a, D'Elia et al.
1996b, Reeves et al. 1997, Shipman et al. 1999). The Sus consists of eight
contiguous genes, susRABCDEFG. Starch degradation by the Sus
commences with starch polysaccharide being bound by surface-attached
proteins (SusD, SusE and SusF) and hydrolysed by an endo-amylase (SusG)
into oligosaccharides which are imported into the periplasm by a TonBExbBD complex, using the proton-motive force (Martens, et al. 2009). The
imported oligosaccharides are subsequently hydrolysed into glucose
monosaccharides by periplasmic glycoside hydrolases: the neopullulanase
(SusA) and the Į-glucosidase (SusB). Imported oligosaccharides also bind
to the N-terminal, periplasmic, segment of a hybrid two-component system
(HTCS; SusR) located on the inner membrane, and productive binding
triggers a signal across the membrane into the cytoplasm, which leads to
transcriptional activation of the SusABCDEFG genes. Sensing of the
degradation products from the early actions of the Sus hydrolases thus
informs the bacterium that starch is present in the cell’s surroundings,
allowing a highly specific response in a rapidly shifting environment.
Neither of the binding proteins SusE and SusF, which have been shown
through in vitro experiments to contain multiple carbohydrate binding
modules (CBMs), are required for the bacterium to utilise starch, and
neither are they very homologous to gene products found in other genomes
thus far (Cameron et al. 2012). Similarly, either one of the hydrolases SusA
and SusB can be removed as long as the other remains, as they can both
hydrolyse maltooligosaccharides, albeit with different specificities. The
backbone-cleaving SusG is however indispensable for growth.
The presence of a pair of SusC/D homologous proteins is the signature for
all PULs, and is used to identify novel PULs in the genomes of
Bacteroidetes species (Martens, et al. 2009, Martens, et al. 2011). In
35
Johan Larsbrink 2013
functional PULs, the SusC/D homologous pair is coupled to a variable
number of glycoside hydrolases, reflecting the monosaccharide and linkage
complexity of the targeted polysaccharide. Analogous to the Sus, in other
PULs, the SusC-like proteins import oligosaccharides, aided by SusD-like
proteins which capture specific glycans at the cell surface, while the
hydrolases both cleave the backbone of polysaccharides at the cell surface
and
degrade
imported
oligosaccharides
down
to
the
constituent
monosaccharides in the periplasm. Similarly, SusR-like proteins can be
expected to act as sensors and regulators of respective PULs, specifically
binding to early break-down products of the targeted polysaccharide, thus
enabling the cell to sense and rapidly respond to the different types of
carbohydrates present in the surroundings. An interesting observation is that
in many cases, these bacteria grow faster on poly- and oligosaccharide than
on the monosaccharide constituents, which is likely because of the
adaptation of the SusR-like proteins to bind oligosaccharides, rather than
monosaccharides, which are rapidly consumed in the gut (Martens, et al.
2011).
In B. thetaiotaomicron, 88 putative PULs have been identified, and
including accessory proteins to the SusC/D pair, these PULS make up
almost 20 % of the bacterium’s genome (Martens et al. 2008, Martens, et al.
2011). As a close relative to B. thetaiotaomicron, B. ovatus has also devoted
a substantial part of its genome to PUL structures, and 112 putative PULs
have been identified (Martens, et al. 2011). The large investment in PULs in
these two species, coupled to the dominant status of Bacteroides species in
the human gut, shows how this strategy of carbohydrate sensing and
breakdown has given these species an advantage over other microorganisms
in a highly competitive environment.
36
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
AIM OF INVESTIGATION
37
Johan Larsbrink 2013
The overall scope of this thesis concerns the microbial degradation of
carbohydrates, with a special focus on the hemicellulose xyloglucan (XyG).
The initial goals were to discover novel Į-xylosidases able to hydrolyse the
xyloside substitutions of this polysaccharide, which could potentially open
up routes to new innovative applications where the XyG chains could be
specifically modified to generate value-added materials with new properties,
as well as novel enzyme substrates. The approach was to screen the
members of Glycoside Hydrolase Family 31 (GH31) from the soil
bacterium Cellvibrio japonicus and the human gut bacterium Bacteroides
thetaiotaomicron, as GH31 is the only family which includes Į-xylosidases.
Following this initial scheme, a highly xylogluco-oligosaccharide(XyGO)specific Į-xylosidase from C. japonicus was discovered and characterised
biochemically and structurally. In addition, the screening of enzymes
uncovered from the same bacterium an enzyme with a novel activity for the
family – the CjAgd31B Į(1ĺ4)-glucan transglucosidase. Further, from B.
thetaiotaomicron, several new activities for the family were discovered,
related to carbohydrate metabolism in the human gut.
This initial broad investigation expanded into the detailed study of XyG
utilisation in both the aforementioned C. japonicus as well as a human gut
bacterium closely related to B. thetaiotaomicron, namely Bacteroides
ovatus. Both C. japonicus and B. ovatus possess the ability to utilise XyG as
sole carbon source, and thus harbour the enzymes needed to deconstruct this
complex polysaccharide. The identification of a discrete locus in B. ovatus,
predicted to be involved in XyG degradation, led to a project with the aims
of biochemically characterising all eight predicted hydrolases of the locus,
as well as performing knock-out- and structural studies to fully appreciate
the molecular processes providing this gut symbiont with the ability to
utilise XyG. A similar XyG-related locus was identified in C. japonicus, as
38
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
a development of the C. japonicus Į-xylosidase project, albeit lacking a
number of required enzymatic activities required for full XyG hydrolysis.
The locus instead appears to be geared towards the degradation and import
of XyGOs, which demonstrates how different bacteria have evolved
separate systems to exploit XyG, which is bound to be abundant both in the
human gut and on the forest floor.
39
Johan Larsbrink 2013
40
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
RESULTS AND DISCUSSION
41
Johan Larsbrink 2013
Enzyme discovery by Glycoside Hydrolase family activity
screening
In the discovery of new carbohydrate-active enzymes (CAZymes), several
approaches may be used. For the work presented in Publications I, II, III
(as a follow-up project), IV and VII, whole Glycoside Hydrolase (GH)
family screening was used, comprising cloning, expression and biochemical
characterisation of all members of a particular GH family from selected
bacteria in order to potentially discover novel enzymatic activities or
features. The enzymes studied in these publications come from the soil
bacterium C. japonicus and the human gut symbiont B. thetaiotaomicron,
and the GH families studied were GH31 for Publication I, III, IV and VII,
and GH43 for Publication II.
GH31
The initial intention of the GH31 SURMHFW ZDV WR GLVFRYHU QRYHO Įxylosidases, active on xyloglucan (XyG). GH31 is the only family
FRQWDLQLQJ Į-xylosidases (Cantarel, et al. 2009), and as such the natural
target to find new enzymes with this activity. Previous studies on XyGVSHFLILF Į-xylosidases had shown that *+ Į-xylosidases enzymes are
able to selectively remove the terminal xyloside moiety from the extreme
non-reducing end of xylogluco-oligosaccharides (XyGOs) (Fanutti et al.
1991, Moracci et al. 2000, Sampedro et al. 2001). The main anticipation of
the study was to discover enzymes able to access ‘internal’ xyloside
moieties on XyGOs, which could lead to novel value-added materials as
well as new enzyme substrates.
CjXyl31A
From the three GH31 genes in C. japonicus, DQ Į-xylosidase was indeed
identified, and in Publication I, the characterisation of this highly specific
42
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
XyGO-degrading Į-xylosidase, CjXyl31A, was presented, using both
detailed biochemical and structural analyses, in collaboration with Prof.
Gideon Davies and co-workers. ,QNHHSLQJZLWKSUHYLRXVO\FKDUDFWHULVHGĮxylosidases, the enzyme exclusively hydrolysed the xylose moiety on the
extreme non-reducing end of XyGOs, but was also shown to have a
preference for XyGOs with a backbone length equal or greater than three
glucose residues (Table 1).
Table 1: Activity of CjXyl31A on various substrates.
Substrate
kcat
s–1
Km
mM
p13Į;\O
0.58 ± 0.026
0.39 ± 0.06
p13Į*OF
“–4
5.31 ± 1.0
kcat/Km
s–1 mM–1
ǻǻ*‡
kJ mol-1
1.48
–3
p13Į*DO
N/Dv
N/D
N/D
p13ȕ-L-Arap
N/Dv
N/D
N/D
XXXG
26.7 ± 1.5
0.54 ± 0.073
49.5
XLLG
33.6 ± 3.2
2.6 ± 0.42
13.1
3.30
XX
31.2 ± 1.5
0.72 ± 0.076
44.7
0.25
0.70 ± 0.013
13.4
3.23
XGol
9.39 ± 0.61
X (Iso-
2.94 ± 0.36
55.00 ± 7.4
0.05
17.1
primeverose)
ǻǻ*‡ ZDVFDOFXODWHGXVLQJWKHIRUPXODǻǻ*‡=RTln(kcat/Km[XXXG]/ kcat/Km[XGO])
This observation was attributed to a novel PA14 (named from the protective
antigen of anthrax toxin (Petosa et al. 1997)) domain insert in the Nterminal domain of the enzyme (Figure 8). The N-terminal domains of
GH31 enzymes have previously been presumed to be involved in substrate
recognition (Lovering et al. 2005), but this was the first presented case of a
43
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
and the binding of anti-CjXyl31A antibodies to the fractions was assayed by
the western blot technique. CjXyl31A was identified as a lipoprotein,
enriched in the membrane fractions. The localisation studies, together with
the detailed biochemical data, allowed for a tentative model of the pathway
of XyG degradation by this soil bacterium. In connection to the localisation
studies, it was also shown that C. japonicus secretes significant endoxyloglucanase activity into the growth medium when the carbon source is
XyGOs.
In a follow-up study, Publication III, the interaction between CjXyl31A
and XyGOs was further dissected using advanced NMR techniques, in
collaboration with Prof. Antoni Molinaro and co-workers. The focus of the
study was, specifically, WKHUHDFWLRQRI;;;*ĺ*;;*, performed by the
enzyme. In order to be able to follow the reaction, both the wild-type
enzyme and a catalytically crippled mutant, CjXyl31A_D659A, were
produced, as well as purified GXXG (the reaction product). Thus, the
reaction between wild-type enzyme and XXXG, the interaction between
wild-type
enzyme
and
GXXG,
and
the
interaction
between
CjXyl31A_D659A and XXXG could be monitored. In saturation transfer
difference (STD) NMR, the interaction between a small molecule and a
macromolecule can be studied, such as an oligosaccharide and an enzyme in
this case (Haselhorst et al. 2009). NMR is performed on the free
oligosaccharide, or ligand, as well as the ligand together with the
macromolecule. As macromolecules in general are too bulky to give NMR
signals, only the spectra of the ligands is observed, and by subtracting the
spectra obtained with or without added macromolecule, the parts of the
ligand that interacts with the macromolecule can be identified (Haselhorst,
et al. 2009).
45
Johan Larsbrink 2013
The study revealed how the individual chemical groups of the assayed
oligosaccharides interact with different intensity with the enzyme, and as
expected, the whole length of the oligosaccharides tested did interact with
CjXyl31A and the mutant (Figure 9). The data obtained, together with
molecular modelling, led to a refined model of the CjXyl31A-XXXG
enzyme-substrate complex. Furthermore, for the first time in GH31, the
stereospecificity of a family member could be monitored directly.
46
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
substrate (Figure 10B), and exclusively utilises maltooligosaccharides
and/or starch as donor substrates, with the highest activity observed on
maltotriose. Also, hydrolysis was only observed for the disaccharide
maltose, although very weak even in this case. As acceptor substrates,
CjAgd31B
was
able
to
use
both
Į(1ĺ4)-
and
Į(1ĺ6)-linked
isomaltooligosaccharides.
The nearly complete lack of hydrolysis observed for the enzyme was
investigated by detailed study of the three-dimensional structure of
CjAgd31B, again in collaboration with Prof. Gideon Davies and coworkers. The enzyme apparently circumvents hydrolysis, even in ~55 M
water solutions, by not allowing water molecules with appropriate geometry
or interaction with the catalytic acid/base residue. Instead, only a sugar
moiety can interact favourably with the enzyme’s active site residues, as
seen for the trapped glycosyl-enzyme intermediates. These observations
may explain how other transglycosylases from other families avoid
hydrolysis by similar means.
The action of CjAgd31B was inhibited by the pseudo-saccharide acarbose,
which was also successfully co-crystallised in the active site of the enzyme
in the structural studies. The crystal structure revealed how the enzyme
adopts a classical GH31 fold, with an N-terminal domain, the central (Į/ȕ)8barrel and two C-terminal domains (Figure 11A). The enzyme was also
shown to possess at least four subsites for oligosaccharide recognition (-1 to
+3), comprising a tyrosine clamp at the +2 and +3 subsites for
oligosaccharide binding and recognition, which is a novel observation for
the enzyme family (Figure 11B).
49
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
generate substrates for glycogen phosphorylases. The novel 4-Įglucosyltransferase-activity of CjAgd31B has previously not been described
in the literature for any other enzyme.
Bacteroides thetaiotaomicron GH31 targets
The other aspect of the GH31 screening project concerned the six family
members encoded by the gut bacterium Bacteroides thetaiotaomicron, and
the results from this study are presented in Publication VII. B.
thetaiotaomicron is, as has been described in previous sections, a dominant
member of the human gut microbiota, and has been shown to be an
especially proficient degrader of host glycans and pectins (Martens, et al.
2011). However, B. thetaiotaomicron has, in the time since the GH31
project commenced, been shown to be unable to grow on hemicelluloses,
and as such, the chance of finding Į-xylosidases active on XyG was slim.
Novel activities for the family were nonetheless discovered in the screening
of the enzymes encoded by B. thetaiotaomicron, with Į-galactosidase, Į-NDFHW\OJDODFWRVDPLQLGDVHDQGVWULFWĮĺ-glucosidase activities observed.
The enzyme BT3086 was shown to be an exo-dextranase/Įĺ-linkage
specific glucosidase, active on dextran, which could be expected, as
BT3086 is part of a PUL which is upregulated when B. thetaiotaomicron is
grown on dextran. Enzymes able to hydrolyse Į(1ĺ-linkages are known
in GH31, such as the human sucrose-isomaltase (Heymann et al. 1995, Sim
et al. 2010). However, unlike BT3086, these enzymes are promiscuous and
able to address multiple glucosidic linkages.
BT3085 was active on Gal-Į-PNP, though curiously, hydrolysis was
observed on neither the trisaccharide raffinose (Galp-Įĺ)-GlcpĮĺ-Fru), the disaccharide melibiose (Galp-Įĺ-Glcp) nor conjac
51
Johan Larsbrink 2013
galactomannan polysaccharide. Likely, the enzyme is not active on Įĺlinkages, but instead a substrate might be Įĺ-linked galactosyl units
found in blood group B epitopes (Brockhausen, et al. 2009).
BT3169 hydrolysed the substrate N-acetylgalactosamine-Į-PNP (GalNAcĮ-PNP) with high efficiency. A paucity of available natural substrates,
however, hinders a definitive determination of its substrate(s) in vivo. Likely
targets are, as B. thetaiotaomicron degrades many host glycans, either the
Įĺ-linked galactosyls found in the cores of mucins or the Įĺlinked GalNAc units found in blood group A epitopes (Brockhausen, et al.
2009).
Of the remaining enzymes, BT3299 did not hydrolyse any of the substrates
assayed, while BT0339 and BT3659 only showed activity on Xyl-Į-PNP.
Neither of these Į-xylosidases could address the Įĺ-linked xylosyl
decorations of XyGOs, even though XyG is the predominant source of Įlinked xylose in nature. As B. thetaiotaomicron does not grow on XyG, this
observation is however not illogical, and the enzymes may very well be
active on Į-linked xylose from a yet unknown host glycan or plant-derived
source.
GH43
In an enzyme discovery study (Publication II) related to the work on
GH31, all GH43 enzymes from both C. japonicus and B. thetaiotaomicron
were characterised biochemically, by the group of Prof. Harry Gilbert.
Although the study comprised 47 unique putative enzymes, the one major
finding
of
the
project
was
the
arabinan-specific
Į(1ĺ2)-
arabinofuranosidase, CjAb43A. Biochemical and structural analysis of this
enzyme, and biochemical analysis of the homologous Bt0369, are reported
52
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
in the paper. The remaining enzymes were typical xylan-acting Įarabinofuranosidases, weak endo-xylanases, and endo-arabinanases, with
differing specificities for branched or linear arabinan. A phylogenetic tree
was constructed for all the studied genes, and different clades corresponding
to the different activities could be identified, which allows for future
predictions of GH43 enzymes from other organisms.
Enzyme discovery from genetic loci
During the course of the thesis work, the focus of the field has shifted
rapidly from a focus on individual enzymes or GH families to a more
system oriented approach, especially following studies of the PULs of
Bacteroidetes species. Screening the activities of GH members from certain
bacteria can, as described above, reveal novel activities to the family, but
there is also a risk, as in the case of Publication II, for the yield of novel
activities to be quite meagre. When searching for a particular enzymatic
activity, another possible route is to look at genetic loci of bacteria, as many
bacterial genes are co-expressed in operons or gene clusters (Osbourn et al.
2009). The PULs of Bacteroidetes are especially sophisticated examples of
carbohydrate catabolism-related loci, as they contain not only enzymes for
carbohydrate degradation, but also proteins for the binding, sensing and
transport of carbohydrates as well as PUL regulation proteins (Martens, et
al. 2009, Martens, et al. 2011). Thus, when looking for an enzyme able to
act on a certain linkage in an oligo- or polysaccharide, there is a high chance
of finding it in a PUL predicted to act on said substrate.
53
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
In order to assay the activity of CjAfc95A, fucogalactoxyloglucan from
iceberg lettuce was extracted. Both CjBgl35A and CjAfc95A were active on
and highly specific for XyGOs, and to a lower degree on XyG
polysaccharides, and their modes of action were studied in detail. Both
enzymes were able to fully strip off all of their respective target
monosaccharide substitutions from the substrates, in contrast to the Įxylosidase CjXyl31A.
In order to determine whether the genes of the locus were in fact transcribed
as C. japonicus senses and breaks down XyG, a qPCR study was
undertaken. C. japonicus cultures were grown in minimal media
supplemented with either glucose or XyGOs as carbon source, and total
RNA was extracted and converted into cDNA to be used as qPCR
templates. In the genome of C. japonicus there are three additional predicted
ȕ-galactosidases and one additional Į-L-fucosidase; these genes were
included in the study as reference genes. The four locus genes were all
found to be highly upregulated in the cultures containing XyGOs compared
to the glucose cultures. The reference genes were either not upregulated at
all or at a much lower level than the locus genes, indicating that the locus is
the actual site for transcription of these necessary activities in XyG
metabolism (Figure 13).
By following the fractionation procedure established in Publication I,
proteins were obtained from secreted, periplasmic and intracellular fractions
of C. japonicus grown in minimal media containing either glucose or
XyGOs as carbon source. Substrates for ȕ-galactosidases and Į-Lfucosidases were used to assay the activities in each fraction, and a clear
enrichment of both ȕ-galactosidase and Į-L-fucosidase activities could be
observed in the periplasmic fractions.
55
Johan Larsbrink 2013
A discrete locus in Bacteroides ovatus, endowing the bacterium
with the ability to utilise xyloglucan
As described in previous sections, many strains from the Bacteroidetes
phylum possess PULs, endowing them with the capability to degrade both
simple and complex carbohydrate structures (Martens, et al. 2009, Martens,
et al. 2011). Bacteroides thetaiotaomicron has been a key source of
CAZymes and a useful model for the study of human-gut symbiosis.
However, B. thetaiotaomicron is unable to utilise the various complex
hemicelluloses found in plant cell walls, and instead prefers host glycans
and pectins as nutrient sources (Martens, et al. 2011). Instead, another
member of Bacteroidetes with the ability to grow on all major
hemicelluloses, B. ovatus, has emerged as a potential treasure trove for
CAZyme discovery. In fact, B. ovatus contains even more predicted PULs
than the archetypal B. thetaiotaomicron, and new interesting enzymes and
carbohydrate degradation pathways are bound to be discovered in the near
future.
The ability to utilise xyloglucan (XyG) as a sole carbon source was
demonstrated in B. ovatus through growth studies (Martens, et al. 2011),
which initiated the project presented in Publication V. Through
transcriptomics studies on B. ovatus grown in minimal media supplemented
with XyG, a discrete PUL, endowing B. ovatus with the capability to utilise
XyG (the XyloGlucan Utilisation Locus; XyGUL), was identified. The
scope of the project was to characterise all encoded GH enzymes by
biochemical, genetic and structural studies. Interestingly, the study also
revealed how only a small number of human gut Bacteroides species have
been shown to be able to utilise XyG, despite it being ubiquitous in all land
plants and as such plentiful as dietary fibre in a healthy human diet. Thus, B.
ovatus seems to have adopted a niche in the human gut, in targeting XyG.
58
Johan Larsbrink 2013
Table 2: Summary of the kinetic parameters of the XyGUL hydrolases.
Enzyme
Substrate
BoGH2A
BoGH3A
Gal-ȕ-PNP
Glc-ȕ-PNP
cellobiose
cellotetraose
cellohexaose
GLLG
GXXG
Glc-ȕ-PNP
Gal-ȕ-PNP
cellobiose
cellotetraose
cellohexaose
GLLG
GXXG
XXXG-ȕ-CNP
XLLG-ȕ-CNP
GGGG-ȕ-CNP
Tamarind XG
Lettuce XG
Tomato XG
Tomato XG
Xyl-Į-PNP
BoGH3B
BoGH5A
BoGH9A
BoGH31A
BoGH43A
BoGH43B
kcat
(s-1)
4.0 ± 0.092
65.0 ± 3.81
0.62 ± 0.15
n.d.
n.d.
1.74 ± 0.21
1.57 ± 0.11
3.99 ± 0.31
4.91 ± 0.71
n.d.
n.d.
10.5 ± 0.14
11.1 ± 1.13
0.12 ± 0.025
435.3 ± 25.6
n.d.
n.d.
1.60 ± 0.031
Glc-Į-PNP
(0.071 ± 0.015)
(31.8 ± 5.1)
XXXG
XLLG
Isoprimeverose
L-Araf-Į-PNP
Xyl-ȕ-PNP
Tomato XGOs
Tomato XG
L-Araf-Į-PNP
Tomato XGOs
Tomato XG
32.6 ± 2.1
31.0 ± 1.9
1.78 ± 0.21
0.057 ± 0.001
0.26 ± 0.005
n.d.
0.223 ± 0.046
0.378 ± 0.075
38.1 ± 7.1
0.71 ± 0.03
6.58 ± 0.21
n.d.
Active
6.6 ± 2.3
n.d.
5.0×10-4 ± 8.1×10-5
n.d.
Active
* Units for Km and kcat/Km are given in s-1 mg-1 ml
n.d.: not determined
60
Km
(mM)
0.087 ± 0.0074
Trace activity
Trace activity
2.49 ± 0.42
0.698 ± 0.52
n.d.
n.d.
0.155 ± 0.039
Trace activity
0.68 ± 0.16
0.23 ± 0.060
0.47 ± 0.21
n.d.
n.d.
0.036 ± 0.0027
0.145 ± 0.024
3.59 ± 1.27
0.82 ± 0.17*
n.d.
n.d.
Active
7.7 ± 0.273
kcat/Km
(s-1 mM-1)
46.0
21.3
0.90
0.15
3.41
11.2
2.3
17.3
10.4
0.18
3.34
291.7
76.5
0.034
534.0*
543.1*
501.5*
0.21
0.0018
(0.002165)
146.2
82.0
0.047
0.081
0.039
0.013*
7.6×10-5
0.0024*
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
To further assess the biological function of the XyGUL, knock-out studies
were performed on both individual genes and the entire XyGUL, by Prof.
Eric Martens and co-workers. When the entire XyGUL was knocked out,
ability to grow on XyG was completely abolished, clearly correlating the
existence of the XyGUL with XyG utilisation. Additionally, knocking out
the endo-xyloglucanase BoGH5A resulted in the same phenotype, proving it
to be crucial for the utilisation of XyG polysaccharides. However, the
phenotype could be rescued by feeding the bacteria with XyGOs generated
by recombinantly produced BoGH5A. Also, knocking out the only GH31
enzyme of the XyGUL, BoGH31A, resulted in a phenotype not able to grow
either on polysaccharide or oligosaccharide XyG, showing how the removal
of pendant xyloside substitutions are key in depolymerising XyGOs into
monosaccharides. The rationale behind the severe ǻBoGH31A phenotype is
illuminated by the flowchart of XyG depolymerisation, shown in (Figure
16). An interesting observation regarding the GH31 enzyme, in particular
for this thesis, is that the enzyme has a predicted PA14 domain insert in the
N-terminal domain, analogous to that found in the CjXyl31A Į-xylosidase,
described above. The PA14 domain can thus tentatively be ascribed to the
high specificity for XyGOs also in the B. ovatus enzyme.
61
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Carbohydrate-binding Often N-terminal) domain (Mello et al. 2010),
yielding the first presentation of such a structure in the literature. The
structure of BoGH5A was solved in two conformations, in which the
BACON domain adopted two entirely different orientations. This suggests
that the BACON domain can move rather freely relative to the catalytic
domain. The “Carbohydrate-binding” implied by the name of the BACON
domain warranted analysis, and the domain was shown, by Dr. Alexandra
Tauzin, to not bind any of the tested polysaccharides or proteins to a
significant extent. Furthermore, BoGH5A was shown to be a surfaceexposed lipoprotein attached to the outer membrane at its N-terminus,
analogous to the SusG amylase of the B. thetaiotaomicron Sus (Martens, et
al. 2009, Shipman, et al. 1999), using anti-BoGH5A antibodies. These data
taken together, indicate that the BACON domain acts as a linker domain,
allowing the catalytic domain of BoGH5A freedom of movement at the cell
surface, which is likely a benefit in hydrolysis of bulky polysaccharide
substrates.
63
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
BoGH9A. The oligosaccharides bind to the SusD-like membrane-bound
lipoprotein and are imported into the periplasm by the SusC-like TonBdependent transporter. In the periplasm, the oligosaccharides are degraded
into their monosaccharide constituents by the exo-acting hydrolases to be
further metabolised. The HTCS-like protein, spanning the inner membrane,
binds early degradation products of the endo-xyloglucanases and thereby
triggers the transcription of the entire locus, sans itself.
This work provides detailed insight into the utilisation of such a complex
hemicellulose as XyG by B. ovatus, and how this bacterium has carved out a
nutritional niche in such a highly competitive environment as the human
gut. The dominance of Bacteroides species in the gut indicate that such
sophisticated nutrition acquisition systems as PULs can provide a
significant advantage in a milieu with rapidly changing nutritional
conditions. The insights provided by the study has not only revealed new
CAZymes and shed light on the biology of B. ovatus, but may also be
relevant for future studies regarding prebiotics and human health, as XyG is
such a ubiquitous polysaccharide in human diets.
65
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
CONCLUSIONS
67
Johan Larsbrink 2013
The work presented in this thesis concerns the discovery and
characterisation of Carbohydrate-Active enZymes (CAZymes) from bacteria
living is such varied environments as forests and the human distal gut.
Two main approaches have been employed in the discovery of new
enzymes, with the first being family and species based screening of
Glycoside Hydrolases (GHs). The members of a selected GH family and
bacterial species are in this approach cloned, expressed and screened
biochemically in order to identify enzymes with predicted or, in some cases,
novel activities to the family. The strength of this approach is the chance of
finding unexpected activities, as the CAZyme classification is based solely
on amino acid sequence similarity. Though many GH families are
monospecific, containing a single activity, many are not, and contain a wide
range of activities, where individual enzymes (in general) share basic
mechanisms and folds, but can address various sugars and linkages. A
downside of the approach is however the large amount of screening for
activities that may be required, for discovery of novel activities can in no
way be guaranteed.
The other approach is looking at genetic loci, predicted to encode a cluster
of CAZymes acting on a given polysaccharide. This is especially promising
in Bacteroidetes species, as they have evolved Polysaccharide Utilisation
Loci, containing whole sets of enzymes and accessory proteins required for
the degradation of specific polysaccharides. The SusC/D homologous pairs
that are the signature of a PUL are easily identified by bioinformatical
means, and by looking for predicted activities related to a given
polysaccharide, relevant PULs may be identified. Similar loci or operons are
also known in other bacteria, and can be a route for CAZyme discovery
even in non-Bacteroidetes species. However, not all CAZymes are parts of
68
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
PULs or operons, which is a natural weakness of the approach in the
discovery of novel CAZymes.
In this work, the GH family screening approach has yielded several novel
activities in GH31 – D SUHGRPLQDQW Į-galactosidDVH DQ Įĺ-linkage
specific JOXFRVLGDVH DQ Į-N-acetylgalactosaminidase from Bacteroides
thetaiotaomicron and WKHĮĺ-glucan transglucosidase CjAgd31B from
Cellvibrio
japonicus
as
well
as
an
arabinan-VSHFLILF Įĺ-
arabinofuranosidase in GH43.
The other approach, looking at genetic loci, has led to the description of the
xylogluco-oligosaccharide utilisation locus in C. japonicus, which contains
three GHs and an TonB-dependent transporter. Localisation studies of the
enzymes led to a model of the utilisation of xyloglucan polysaccharides
employed by this efficient plant degrader. A more complete system was
described for the gut symbiont B. ovatus, for which the whole Xyloglucan
Utilisation
Locus
(XyGUL)
was
described.
In-depth
biochemical
characterisation of the eight encoded GHs, coupled to genetic knock-outs
and structural studies of the main endo-xyloglucanase, has revealed, in
detail, how this bacterium can degrade such a complex polysaccharide as
xyloglucan.
The publications collected in this thesis give insight into the discoveries
made using the two enzyme screening approaches discussed. The thesis
adds to the knowledge about CAZyme families, bacterial metabolism of
carbohydrates and human nutrition. We feel that this is a considerable
contribution to the field.
69
Johan Larsbrink 2013
ACKNOWLEDGEMENTS
Of course, this thesis is not the result of the work done solely by me, but has
been influenced by a number of people in an even greater number of ways,
and these people deserve more credit than there is room for here.
First of all, I would like to thank Harry Brumer for welcoming me into the
international community of CAZymologists, through my time in his group.
Even after having moved to Vancouver, advice has never been more than an
e-mail away. I really appreciate the way you’ve trusted me in managing all
my various projects in a largely independent way, as well as the
opportunities to travel all over the world – especially the 6 months in
Canada.
The Brumer group has been quite diverse over the years, and has made the
journey an enjoyable one. First, I’d like to acknowledge the last member of
the Sweden-based team, Lauren; we started at the same time on the same
EU project, and your coming here was a perfect transition in so many ways.
Jens, great travel companion and lab know-it-all. Oliver, fellow initiator of
the Bo project and sorely missed since the return to Austria. Nomchit, you
are missed, as are fellow office-mates Gea (though not part of the Brumer
lab) and Fredrika. Magnus, fantastic diploma student and now a firm friend.
Farid and Sassa, having made great substrates over the years. Gustav, for all
the MS work. The great Vancouver people: Tyler, Alex and Fatemeh. And
all the others, Chunlin, Ana Catarina and the Stefans.
I would like to thank all the collaborators from other labs that I’ve worked
with through the years: Gideon Davies and his structural biology crew –
especially Glyn and Atsushi, Harry Gilbert et. al., NMR specialists Antonio
70
Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
Molinaro and Alba Silipo, Eric Martens and Nicole Koropatkin – I really
appreciated my week at your place!
The glycoscience team and the people on floor 2: Vincent, for keeping an
eye on my development by heading the Glycoscience division; Ela and
Nina, for help with all those practicalities; Cortwa, for comic relief at work;
Francisco and Sara, for taking my gentle slurs in good humour. And of
course all the people on floor two, past and present. Finally I would like to
thank Karl Hult, who encouraged me to go to Japan for my diploma project,
which segued into several enjoyable years of biotech research.
I would also like to thank Clara and Paula, who have been there from the
beginning of my studies at KTH to this day, and Mattias for all those
(work?) lunches, and of course other friends, family and loved ones.
71
Johan Larsbrink 2013
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