C H A P T E R
6
Databases for Bioenergy-Related Enzymes
Yanbin Yin
Department of Biological Sciences, Northern Illinois University, DeKalb, IL, USA
email: yyin@niu.edu
O U T L I N E
Plant Biomass
Bioenergy-Related Enzymes and Regulation
Databases and Web Servers
CAZy Database
CAT and dbCAN
FOLy Database
PLANT BIOMASS
The major components of plant biomass are
carbohydrate-rich cell walls, composed of different bio-
polymers such as polysaccharides and lignins as well
as some minor wall structural glycoproteins (Somerville
et al., 2004). Biomass used for biofuel production is pri-
marily derived from secondary cell walls. For example,
wood cells from poplar trees contain a thin layer of pri-
mary cell walls and multiple layers of much thicker and
tougher secondary cell walls. All plant cells of different
tissues have primary cell walls while only in developed
cells (stopped growing) secondary cell walls appear
(Cosgrove, 2005). The chemical compositions in primary
and secondary cell walls differ significantly (Mohnen
et al., 2008). The primary cell wall contains no lignins
and the polysaccharides include celluloses, hemicellu-
loses (primarily xyloglucans and mannans in dicots
and xylans in monocots) and pectins. However, in the
secondary cell walls, there are higher percentage of cellu-
loses, different hemicellulosic polysaccharides (primar-
ily xylans in both dicots and monocots) and lignins. For
example, wood secondary cell walls contain 35e50%
celluloses, 25e30% hemicelluloses (mostly xylans) and
15e30% lignins (Himmel et al., 2007).
Bioenergy Research: Advances and Applications
http://dx.doi.org/10.1016/B978-0-444-59561-4.00006-1
95 Purdue Cell Wall Genomics and UC-Riverside Cell
Wall Navigator Databases 102
96
Plant Coexpression Network Databases: PlaNet and
98 ATTED 102
98
Future Perspectives 103
101
102 References 103
Note that unlike celluloses, hemicelluloses and
pectins both refer to a collection of complex polysaccha-
rides mostly with side chains. Hemicelluloses contain
four major groups: xyloglucans, mannans, xylans and
mixed-linkage glucans, while pectins contain three ma-
jor groups: galacturonans, rhamnogalacturonan I and
rhamnogalaturonan II. Each of the groups of hemicellu-
loses and pectins do not refer to a single type of polysac-
charides; they often refer to polysaccharides with the
same backbone structure (sugars and linkages) while
with different side chains. Due to this reason, all
these biopolymers are cross-linked and interwoven
(Somerville et al., 2004; Himmel et al., 2007) to form
very complex and heterogeneous cell wall structures.
Particularly celluloses are wrapped by hemicelluloses
and buried in a lignin network and not accessible to
enzymes so that the degradation efficiency is very low
if no costly chemical pretreatment is applied.
Although celluloses are simple polymer of glucose
linked by beta-1,4,-glucosidic bond, the complexity of
chemical compositions of hemicelluloses and pectins is
remarkably high (Somerville et al., 2004). The reasons
are as follows: (1) there are 14 different monosacchar-
aides (sugar units) found in hemicelluloses and pectins
(Pauly and Keegstra, 2008b); (2) the possible glycosidic
95
Copyright Ó 2014 Elsevier B.V. All rights reserved.
96 6. DATABASES FOR BIOENERGY-RELATED ENZYMES
linkages formed between two sugars are extremely
diverse as theoretically they can be connected between
any hydroxyl group of two sugars and (3) sugars in
the polysaccharides can be further modified by, e.g.
methylation, acetylation or esterification.
Lignins, however, are complex heterogeneous
phenolic polymers and chemically very distinct from
polysaccharides. They are formed by three major
monomers: hydroxyphenyl (H), guaiacyl (G) and
syringyl (S) units, which are derived from coumaryl
alcohol, coniferyl alcohol and sinapyl alcohol, res-
pectively (Boerjan et al., 2003). All the biopolymers
in plant cell walls are cross-linked and interwoven
(Somerville et al., 2004; Himmel et al., 2007) to form
very complex and heterogeneous structures, which is
believed to enable cell walls recalcitrant to enzymatic
degradation.
Besides, cell wall compositions and structures also
vary from tissue to tissue. The reason is because the
cell wall biosynthetic enzymes are differentially regu-
lated and expressed in different tissues. Furthermore,
different plants, especially those of distant evolutionary
clades, have very distinct cell wall biopolymer composi-
tions. For example, grasses generally have significantly
higher percentage of xylans than trees (Pauly and
Keegstra, 2008a).
For biofuel production, polysaccharides especially
celluloses are favored as their degradation releases
fermentable sugars. Lignins, however, are phenolic
polymers and chemically distinct from polysaccharides,
not giving rise to sugars and meant to be removed in the
biofuel production. In order to develop transgenic
plants with modified cell wall compositions (i.e. higher
cellulose and lower lignin content), we need a better un-
derstanding of how plant cell wall polysaccharides and
lignins are synthesized.
BIOENERGY-RELATED ENZYMES
AND REGULATION
Because of the overwhelming complexity of cell
wall polymers in terms of their chemical compositions,
linkages and structures, plant biomass formation and
microbial degradation involve a surprisingly large num-
ber of genes in plant and microbes, respectively. For
example, presumably every different glycosidic bond in
the polysaccharides will be formed using a different
enzyme. It is estimated that w10% of genes in Arabidopsis
genome are involved in cell wall synthesis and modifi-
cation (Yong et al., 2005), which account for w2000
genes encoding enzymes for sugar and lignin precursor
synthesis, polysaccharide and lignin synthesis and
modification, lignin-polysaccharide cross-linking, tran-
scription factors (TFs)and signaling proteins, etc.
The most important enzymes are clearly those
involved in polysaccharide synthesis and lignin synthe-
sis. To form polysaccharides, glycosyltransferases (GTs)
take the activated sugar donors, nucleoside diphosphate
sugars (NDP-sugars), as the substrates to build glyco-
sidic bonds between two sugars. Except for celluloses,
other cell wall polysaccharides are mostly synthesized
in Golgi apparatus, where GTs, NDP-sugar biosynthetic
enzymes and sugar transporters are located and work
together. Glycoside hydrolases (GHs), on the other
hand, are used to break glycosidic bonds through hydro-
lysis reactions to release sugars from polysaccharides. In
plants this is often used to modify existing polysaccha-
rides, e.g. when plant cells are growing, while in mi-
crobes GHs are the most critical enzymes degrading
plant biomass. Clearly not all GH and GT enzymes are
involved in cell wall polysaccharide metabolism, as
many of them are involved in metabolism of storage
polysaccharides, glycoproteins, glycolipids and other
glycol-conjugates that are not relevant to plant cell walls.
All GT and GH enzymes are categorized by the CAZy
(Carbohydrate Active enZyme) database (CAZyDB)
(Cantarel et al., 2009), which provides a general classifi-
cation scheme for all carbohydrate active enzymes
(CAZymes) and is widely accepted by the carbohydrate
research community. So far there are a limited number of
enzymes biochemically or genetically characterized to
be involved in plant cell wall synthesis or modification,
many of which belong to some large GH and GT fam-
ilies. For example, the GT2 family is known to include
cellulose synthases and some hemicellulose backbone
synthases (Lerouxel et al., 2006), such as mannan syn-
thases (Dhugga et al., 2004; Liepman et al., 2005), puta-
tive xyloglucan synthases (Cocuron et al., 2007), and
mixed linkage glucan synthases (Burton et al., 2006).
With respect to the synthesis of xylan, the most abun-
dant hemicellulose, proteins of GT43, GT47 and GT8
are likely to be involved (Zhong et al., 2005; Brown
et al., 2007; Lee et al., 2007; Pena et al., 2007; Persson
et al., 2007; York and O’Neill, 2008; Brown et al., 2009;
Wu et al., 2009).
Some of these known cell wall-related CAZyme fam-
ilies are included in Purdue’s Cell Wall genomics data-
base (Yong et al., 2005) and UC-Riverside’s (UCR) Cell
Wall Navigator database (Girke et al., 2004), and more
families are discussed in the literature or to be character-
ized in terms of their roles in biomass-related polysac-
charide formation and degradation. For example, a
few recent papers (Scheller and Ulvskov, 2010; Driouich
et al., 2012) and a book (Ulvskov, 2011) updated our
knowledge about the GT family members involved in
cell wall synthesis: GT2, 8, 31, 34, 37, 43, 47, 61, 64, 75,
77, while there must be more GT families not included
and to be identified as cell wall related (CWR), e.g.
GT92 (Liwanag et al., 2012).
 BIOENERGY-RELATED ENZYMES AND REGULATION
Lignins are complex heterogeneous polymers with
lots of aromatic rings. The monolignol synthesis
pathway that starts from phenylalanine to synthesize
G, S and H units has been relatively well known, with
about 10 gene families characterized encoding most of
the enzymes in the pathway (Humphreys and Chapple,
2002; Boerjan et al., 2003; Vanholme et al., 2008; Xu et al.,
2009; Zhong and Ye, 2009; Li and Chapple, 2010; Weng
and Chapple, 2010; Carpita, 2012). All these lignin
synthesis-related enzymes have been extensively
reviewed in the literature and are included in Purdue’s
Cell Wall genomics database. Transporting the units to
the outside of the cell and assembling them into lignin
polymers are less understood but some candidate trans-
porters and two major enzyme families, peroxidase and
laccase, are suggested (McCaig et al., 2005; Liu et al.,
2011; Zhang et al., 2011; Alejandro et al., 2012; Carpita,
2012; Handford et al., 2012; Sibout and Hofte, 2012).
As with all other metabolic pathways, biomass forma-
tion and degradation are also under strict regulation.
However, compared to enzymatic activities, regulatory
mechanism is even more difficult to elucidate. In plants,
only a handful of TFs are known to regulate cell wall
synthesis. The most studied process is the regulation
of lignin biosynthesis (Zhong and Ye, 2009; Zhong
et al., 2010; Zhao and Dixon, 2011; Wang and Dixon,
2012). TF families NAC, WRKY, and MYB among a
few others have been shown to directly or indirectly con-
trol the monolignol synthesis. Some of the TF family
members are global regulators that regulate the entire
secondary cell wall synthesis, including the synthesis
of celluloses and xylans, suggesting that the different
biopolymers in biomass are not synthesized indepen-
dently but in a coordinated way. On the other hand,
genetically modifying the regulation of cell wall biosyn-
thesis represents a very promising way to improve the
desired traits of bioenergy crops. For example, Wang
et al. showed that a mutation found in a WRKY TF could
rewire the regulatory network of secondary cell wall
synthesis and improve 50% of the biomass production
in Arabidopsis (Wang et al., 2010). Similarly, micro ribo-
nucleic acids (miRNAs) are also excellent targets for
controlling the regulation of cell wall synthesis (Fu
et al., 2012), which is less discussed in the literature.
Clearly looking for novel transcription regulators, either
TFs and miRNAs, and further building the regulatory
network of cell wall synthesis is the ultimate goal for
the elucidation of the mechanism of biomass formation.
Recently a few plant journals published special issues
on plant cell wall researches: Plant Physiology (McCann
and Rose, 2010), Current Opinion in Plant Biology
(Pauly and Keegstra, 2008b), Frontiers in Plant Science
(Debolt and Estevez, 2012) and Molecular Plant (has a
cell wall biology category). Particularly, a number of
review articles published in these special issues and a
97
few book chapters (Table 6.1) gave overviews of latest
progress in a specific area of cell wall research and are
very useful for pointing to the original research papers
reporting the characterization of specific CWR genes.
In terms of degradation, cell wall polysaccharides are
degraded by microbial GHs and other CAZymes that
are defined and categorized in CAZyDB. Lignins are
mostly degraded by microbes too particularly by certain
fungi (Dashtban et al., 2009). Enzymes involved in the
degradation include fungal laccases and peroxidases,
which are categorized in the FOLy (fungal oxidative
enzymes) database (Levasseur et al., 2008). Note that
these two families are not restricted to fungi. Instead
they both belong to large protein families having many
homologs in various organisms such as plants, animals
and bacteria, bearing slightly different biochemical
activities (Welinder, 1992). As mentioned above, these
enzyme families are also used for lignin polymerization
TABLE 6.1 Selected Publications for CWR Genes
Category Publications
Lignins (Humphreys and Chapple, 2002; Boerjan et al.,
2003; Vanholme et al., 2008; Xu et al., 2009; Zhong
and Ye, 2009; Li and Chapple, 2010; Weng and
Chapple, 2010; Carpita, 2012)
Celluloses (Somerville, 2006; Joshi and Mansfield, 2007; Gu
and Somerville, 2010; Endler and Persson, 2011;
Lei et al., 2012)
Xylans (York and O’Neill, 2008; Carpita, 2012; Doering
et al., 2012)
Hemicelluloses (Sandhu et al., 2009; Scheller and Ulvskov, 2010;
Carpita, 2012; Driouich et al., 2012)
Pectins (Mohnen, 2008; Harholt et al., 2010; Driouich
et al., 2012)
NDP-sugars (Reiter and; Vanzin, 2001; Seifert, 2004; Reiter,
2008; Bar-Peled and O’Neill, 2011)
TFs* (Zhong and Ye, 2009; Zhong et al., 2010; Zhao and
Dixon, 2011; Carpita, 2012; Wang and Dixon,
2012)
Transporters (Liu et al., 2011; Zhang et al., 2011; Alejandro
et al., 2012; Carpita, 2012; Handford et al., 2012;
Sibout and Hofte, 2012)
miRNAs (Sun; Sun et al.; Fu et al., 2012)
GTs* (Yokoyama and Nishitani, 2004; Scheller and
Ulvskov, 2010; Driouich et al., 2012; Harholt et al.,
2012)
Other CAZymes (Yokoyama and Nishitani, 2004; Minic, 2008)
DUF* families (Carpita, 2012; Hansen et al., 2012; Harholt et al.,
2012)
Bioinformatics (Egelund et al., 2004; Yong et al., 2005; Penning
et al., 2009; Michel et al., 2010)
* TF, transcription factor; GT, glycosyltranferase; DUF, domain of undefined function.
98 6. DATABASES FOR BIOENERGY-RELATED ENZYMES
in plants. There is also increasing evidence to show that
such enzymes are also used for lignin degradation in
bacteria (Claus, 2003; Li et al., 2009; Bugg et al., 2011a,
2011b).
Notably many cell wall biosynthesis-related gene
families are also rooted in bacteria (Royo et al., 2000;
Nobles and Brown, 2004; Emiliani et al., 2009; Yin
et al., 2009; Weng and Chapple, 2010; Yin et al., 2010,
2011; Popper et al., 2011). In other words, although car-
bohydrate and lignin-rich plant cell walls are almost
unique to plants, the biosynthetic machinery has
evolved from ancient gene families that were already
present in early prokaryotes. On the degradation side,
microbes are responsible for breaking down biomass,
while plants also contain homologs of many microbial
degrading enzymes such GHs and peroxidases. Obvi-
ously plants also inherited these enzymes for different
purposes: modify existing polysaccharide or complete
the lignification process.
Similarly, less is known about the regulation of
enzymes for the polysaccharide and lignin degradation
in microbes than the enzymes themselves. As opposed
to plants, microbes involved in biomass degradation
are more taxonomically distributed spanning from
eukaryotic fungi (e.g. Neurospora crassa) to prokaryotic
bacteria (Clostridium thermocellum). As a result, the regu-
lation systems in these divergent organisms are often
not very conserved, e.g. many of the TFs found in fungi
are not present in bacteria and vice versa. Furthermore,
there are numerous model microbes used for bioenergy
research and the transcription regulators regulating
cellulases, hemicellulases or ligninases are highly
dispersed in the literature, e.g. (Aro et al., 2005; Portnoy
et al., 2011; Coradetti et al., 2012; Sun et al., 2012). All
these make the curation and annotation of the regulators
and targeting cis elements to be very difficult. Recently,
global gene expression data (e.g. microarray) and other
omics data have been generated to help study the regu-
lation of biomass degradation (Nataf et al., 2010; Raman
et al., 2011; Riederer et al., 2011; Yang et al., 2012), which
represents the future trend of understanding biofuel
production at the systems biology level. Similar to regu-
lators for cell wall synthesis, there is a lack of web-based
bioinformatics databases to include the regulatory genes
for bioenergy-related degradation enzymes.
DATABASES AND WEB SERVERS
Our knowledge about cell wall biosynthesis and
biodegradation has steadily increased in the past
decades, although there is still a long way to go to fully
understand these extremely complex processes. So far
our knowledge is at best fragmented and characterized
genes are often from various organisms and dispersed
in the literature. Therefore, dedicated, centralized
and frequently updated databases of bioenergy-related
genes are crucial to guide the development of transgenic
biofuel crops and the annotation of newly sequenced
metagenomes/genomes to look for novel enzymes.
Like other biology research areas, bioenergy research
has also been benefiting from bioinformatics. Table 6.2
provides a list of bioinformatics databases and web-
based resources developed specifically for bioenergy-
related enzymes as well as some general bioinformatics
web resources that are valuable for bioenergy research.
Here we offer a summary of a few selected resources
that are particularly useful.
CAZy Database
For bioenergy research, CAZymes are obviously the
most important enzymes. The CAZyDB team started
to classify and annotate CAZyme proteins from Gen-
Bank, UniProt and PDB to protein families since the
early 1990s. It is the original database that defined
over 300 CAZyme protein families throughout the past
20 years and the most comprehensive database
providing high-quality manual annotation by extracting
associated knowledge from the literature (Cantarel et al.,
2009). Its Web site regularly updates every few weeks,
mainly by assigning new proteins in public databases
to existing CAZyme families by sequence similarity
search or creating new families if there are new bio-
chemically characterized CAZyme proteins (supported
by published papers) that do not belong to existing
CAZyme families. Sometimes the functional annotation
information (e.g. known activities) for some families is
also updated if relevant literature came out.
Currently the database comprises five classes of pro-
tein families: in addition to GHs and GTs, there are three
other classes, carbohydrate esterases (CEs), polysaccha-
ride lyases (PLs) and carbohydrate binding modules
(CBMs). As aforementioned, GTs are used for building
polysaccharides or glycolconjugates, while GH for
breaking them. CE and PL are also used for breaking car-
bohydrate molecules while using different mechanisms
or cutting different chemical bonds. CBMs, as indicated
by names, are structural modules used for recognizing
and binding different sugars. Currently the five classes
contain 94 GT families, 131 GH families, 16 CE families,
22 PL families and 64 CBM families. Each class also has
an unclassified family, meaning proteins are annotated
to belong to a certain class but are not able to be assigned
to any existing families in that class. Each family is
named with the class name followed by a sequential
number, e.g. GT2. Note such name does not indicate
any biochemical activity of each family. The reason is
that these families are defined solely based on sequence
similarity: there are many cases that one family contains
 DATABASES AND WEB SERVERS 99
TABLE 6.2 Bioenergy-Related Databases

Database Description References

General CAZyDB General carbohydrate active enzyme database; a classification (Cantarel et al., 2009)
scheme with five classes (GH, GT, CE, PL and CBM) and over 300
families; supported by the biochemical literature; links to proteins in
GenBank, UniProt and structures in PDB; subfamilies for selected
families; Enzyme commission annotation and biochemically curated
function annotation
CAT CAZyme analysis toolkit; allow CAZyme annotation of user (Park et al., 2010)
submitted data using BLAST (basic local alignment search tool) and
Pfam-based search
dbCAN Web server for automated CAZyme annotation; allow submission of (Yin et al., 2012)
predicted protein sequences from newly sequenced genomes and
metagenomes and return a table and graphical diagram to show the
matched CAZyme domains; users could also download HMMs
representing all CAZyme domains to perform annotation locally by
running HMMER3 (hmmer.org)

Plant biomass Survey of databases A paper reviewed nine public databases (Cao et al., 2010)
formation for cell wall
synthesis
XTH World Xyloglucosyl transferase gene nomenclature; gene structure and
literature in Arabidopsis, rice and tomato
MAIZEWALL Cell wall gene catalogue, expression data and literature in maize (Guillaumie et al., 2007)
coreCarb PlaNet family tool; Arabidopsis CAZyme proteins; sequence, (Mutwil et al., 2009)
expression, regulon (coexpressed genes), phylogeny data
GolgiP Web server for prediction of Golgi localized proteins (Chou et al., 2010)
Purdue cell wall General classification scheme for plant cell wall synthesis; with (Yong et al., 2005)
genomics mutants and spectrotype information; also including lignin
synthesis and modification and NDP-sugar synthesis genes and
signaling proteins etc.; phylogeny for gene families in Arabidopsis,
rice, maize and sorghum; literature
UC-Riverside cell Similar classification scheme for plant cell wall synthesis proteins to (Girke et al., 2004)
wall navigator Purdue cell wall database but not including lignin-related and
signaling proteins; including sequence, literature and microarray
expression data; primarily for Arabidopsis and rice, but also generally
from UniProt

Stanford cellulose Designed for the CesA (cellulose synthase) superfamily and (Richmond and
homologs; deprecated web site Somerville, 2000)
Rice GT Part of the rice phylogenomics database; rice GT protein phylogeny, (Cao et al., 2008)
sequence, expression, mutants, ortholog, BLAST
Csl families Web site supplemental to (Yin et al., 2009); protein sequences, (Yin et al., 2009)
alignments and phylogeny of the CesA superfamily in fully
sequenced plant and algal genomes
pDAWG CAZymes in fully sequenced plant and algal genomes based on (Mao et al., 2009)
search against CAZyDB; phylogeny, predicted subcellular
localization and proteineprotein interaction data; BLAST

PPI for cell wall Proteineprotein interaction graphs for cell wall-related proteins in (Zhou et al., 2010a)
Arabidopsis

PlaNet General coexpression database for seven plant organisms and (Mutwil et al., 2011)
comparison among them; highest reciprocal rank based
coexpression and clustering using Heuristic Cluster Chiseling
Algorithm (HCCA, Mutwil et al.); queried gene-centered display of
coexpression network

(Continued)
100 6. DATABASES FOR BIOENERGY-RELATED ENZYMES

TABLE 6.2 Bioenergy-Related Databasesdcont’d

Database Description References

Cell wall Biclustering coexpression analysis of cell wall-related genes from (Wang et al., 2012)
coexpression Purdue cell wall genomics database; coexpression modules and
database graphs generated using Cytoscape (Shannon et al., 2003); cis-
regulatory elements identified in promoter regions of genes of a
same module
ATTED General coexpression database and predicted cis-regulatory (Obayashi et al., 2009)
elements for Arabidopsis and rice; mutual rank based; also identified
conserved coexpression links and referred to proteineprotein
interaction data
Biomass GAS db Glycosyl hydrolase AnnotationS (GAS) database; GH data identified (Zhou et al., 2010b)
degradation from UniProt and JGI metagenomes based on CAZyDB and Pfam
search; featured with the graphical domain diagrams and
comparison between two selected bacteria
FOLyDB Fungal enzymes for lignin degradation; 10 families of lignin oxidases (Levasseur et al., 2008)
and auxiliary enzymes; proteins from GenBank, UniProt and PDB
PeroxiBase General peroxidase database including peroxidases (EC 1.11.1.x) (Fawal et al., 2012)
from over 1000 organisms; lignin-related peroxidases are a subset of
the database
LccED General laccase database and their homologs in the multicopper (Sirim et al., 2011)
oxidase superfamily
Misc Biofuel feedstock Database of 54 plant organisms with sequenced genomes or (Childs et al., 2012)
genomic significant amount of EST (expressed sequence tag) data; integrated
resource (BFGR) data including expression, ortholog and paralog, pathway
prediction, and functional information

BESC-KB Knowledgebase for the Bioenergy Science Center of DOE; a web (Syed et al., 2012)
portal to a number computational tools and databases dedicated for
bioenergy research and developed within the center
Pathway-genome Populus trichocarpa metabolic pathways generated automatically (Nag et al., 2012)
database of poplar through the Pathway Tool; currently the NDP-sugar biosynthetic
pathways were manually curated by experts
JGI IMG/M Joint Genome Institute’s integrated microbial genomes and (Markowitz et al., 2012)
metagenomes web site
Phytozome JGI’s plant genome web site; currently most sequenced plant (Goodstein et al., 2012)
genomes are available in this web site

proteins characterized with different biochemical activ-
ities. Recent efforts from the CAZyDB team suggest
that further classification of family into subfamilies
could be useful as subfamily may contain proteins
with the same activity (Stam et al., 2006; Lombard
et al., 2010; Aspeborg et al., 2012).
CAZyDB’s annotation also evolved in the past 20
years. Among the 327 CAZyme families as of December
2012, there are 10 depreciated families; they were
created during the life course of CAZyDB but later
were deleted since they were shown not related to carbo-
hydrate metabolism or due to some other reasons. How-
ever, to keep the existing nomenclature system
unchanged, these family names remain in the system
but indicated to be deleted on the Web pages for these
families. Other examples include CE10 family, whose
Web page was not updated since 2002 because after
the family was created it was shown that most CE10
family members do not take carbohydrates as substrate;
CBM33 was thought to be a carbohydrate active binding
module but later shown likely to be an oxidase family.
For a decade, CAZyDB provides an HTML page for
each family to list member proteins and associated func-
tional information. In recent updates, CAZyDB added a
Web page for each genome, providing a list of GenBank
protein accession numbers of that genome together with
the CAZyme family assignment for each protein, which
is termed “CAZyome” of an organism. So far, there are
almost 2400 genomes spanning from eukaryotes to
prokaryotes and viruses annotated in CAZyDB. It is
said that such genome-scale annotation of CAZyme
proteins is done semiautomatically (Coutinho and
 DATABASES AND WEB SERVERS
Henrissat, 2011). A backend automated domain module-
based search is performed first and then manual curation
will be conducted to remove false positives or include
false negatives. Obviously this process is rather accurate
and of high quality but time consuming because it is
done manually and requires expert knowledge. Indeed
such genome annotation can only be done by the collab-
oration with the CAZyDB team, which is often invisible
to and out of the control of the users, e.g. people who
sequenced the genome. Over the past 10 years, the
CAZyDB team has done expert CAZyme annotation
for dozens of genome sequencing projects that led to a
lot of collaborative genome annotation papers.
CAT and dbCAN
With more and more bioenergy-related genomes of
plants and microbes as well as environmental metage-
nomes sequenced, there is an urgent need for automated
CAZyme annotation. Although such annotation will not
reach a quality as accurate as the expert annotation from
CAZyDB, it is expected to be much faster and users can
control the annotation at their will. Moreover, nowadays
all newly sequenced genomes are relying on generic
protein domain/family databases such as Pfam (Finn
et al., 2006), InterPro (Hunter et al., 2009), and conserved
domain database (CDD, Marchler-Bauer et al., 2009) for
automatic genome annotation. Clearly annotation from
these databases is often too general and too far from
the exact function; the precisely actual function still
needs to be determined by experimental approaches.
However, most genome annotators are still interested
in such genome-scale annotation, as it can give them a
quick summary about what the genome encodes, how
large the gene families are and how that compares to
other genomes.
In fact, even CAZyDB’s manual annotation (assign
proteins to existing CAZyme families) on newly
sequenced genomes is unlikely to be 100% correct.
Considering every new genome contains a high percent-
age of proteins that are not experimentally studied, the
manual curation is still largely based upon additional
bioinformatics analysis such as BLAST search against
public protein sequence databases (e.g. UniProt (Bairoch
et al., 2005)) and domain databases (e.g. Pfam) and
inspection of top matches.
With these in mind, automated CAZyme annotation is
still very useful, e.g. particularly for a quick and general
overview of how many CAZymes and what CAZymes a
newly sequenced genome has. Using the annotated
CAZyme proteins and classification scheme in CAZyDB
as the foundation, two bioinformatics efforts have
been published since 2010, both supporting automated
CAZyme annotation, given a protein sequence dataset
predicted from a genome/metagenome. The CAZyme
101
Analysis Toolkit (CAT) (Park et al., 2010) allows a BLAST
search against CAZyme proteins annotated by CAZyDB
and also a Pfam domain-based search. The simple BLAST
search suffers from the inability to accurately annotate
the prevalent multidomain CAZyme proteins. The
Pfam domain-based search can solve this problem.
These Pfam domains are either given by CAZyDB in
the CAZyme family Web pages or identified to corre-
spond to CAZyme family using an association rule built
by CAT. However, there are only 142 (46%) of over 300
CAZyme families linked to Pfam domains by CAZyDB.
In fact, many of the Pfam domains were originally
created after CAZyDB.
We recently developed dbCAN (Yin et al., 2012) to
define a signature domain model for all CAZyme fam-
ilies. Aside from the 142 CAZyme families annotated
with a Pfam domain, we managed to associate other
CAZyme families to functional domains in a broader
and general protein domain database CDD . This way,
we were able to find a CDD domain for 248 CAZyme
families. For the remaining families, we performed a
literature curation by reading relevant biochemical
papers that are linked to these families by CAZyDB. In
the end, we extracted the domain regions in all the mem-
ber proteins annotated in CAZyDB and built a multiple
sequence alignment (MSA) for each of the CAZyme fam-
ily. These MSAs were further processed and represented
by hidden Markov models (HMMs), statistical models
widely used in the bioinformatics field to represent pro-
tein sequence alignments, e.g. by Pfam.
As of June 2012, dbCAN has 320 HMMs representing
317 CAZyme families and three cellulosome modules.
We provide all these HMMs freely to the public so that
they can run domain-based tool hmmscan of the
HMMER 3.0 package (hmmer.org) to annotate their
genomes/metagenomes in a local computer, exactly the
way that people perform Pfam, InterPro or CDD annota-
tion. To help users who do not know how to run hmmscan
on a Linux PC, we offer the web server (http://csbl.bmb.
uga.edu/dbCAN/annotate.php) so that people can sub-
mit their sequences for annotation on the web. The 320
CAZyme family-specific HMMs are our key contribution
to the carbohydrate research community and ideally
should be included in the general protein domain/family
database such as Pfam in the future.
In addition to the Web server, dbCAN also provides a
database where precomputed CAZyme homologs in a
number of protein databases are showed on the Web.
Particularly, starting from the 320 dbCAN HMMs, we
scanned public metagenome datasets such as NCBI-
env-nr, CAMERA (Seshadri et al., 2007), JGI metagenomes
(Markowitz et al., 2012), human gut metagenomes
(Meta-HIT) (Qin et al., 2010) and cow rumen metage-
nomes (Hess et al., 2011) as well as plant (Goodstein
et al., 2012), bacterial and fungal genomes. Tests on
102 6. DATABASES FOR BIOENERGY-RELATED ENZYMES
Arabidopsis thaliana (plant) and C. thermocellum (bacteria)
using CAZyDB as the positive set suggest that the auto-
mated CAZyme annotation achieved a fairly good accu-
racy (A. thaliana: sensitivity ¼ 96.3%, precision ¼ 78.8%
and average ¼ 87.6%; C. thermocellum: sensitivity ¼ 99.3%
and precision ¼ 89.4%). Particularly the sensitivity is over
95% for both organisms, meaning dbCAN annotation
tends not to lose true CAZyme proteins.
FOLy Database
Inspired by CAZyDB, Levasseur et al. developed a
new database named FOLy, for the classification of ligni-
nases in fungi (Levasseur et al., 2008), as these enzymes
are critical for breaking down lignins in the biomass but
are not included in CAZyDB. Similar to CAZyDB,
FOLyDB started from biochemically characterized pro-
teins or structures to recruit homologs from GenBank,
UniProt and PDB databases. Based on sequence similar-
ity, three lignin oxidase families and seven lignin deg-
rading auxiliary enzyme families were created, each
containing biochemically characterized proteins together
with their sequence homologs. Similarly, FOLyDB is
featured with expert manual curation of continuingly
published literature to include more characterized pro-
teins in order to create new families and populate the
database. Like CAZyDB, it is not designed for automated
genome annotation but BLAST and Pfam domain-based
search against annotated proteins in FOLyDB has been
widely used to annotate newly sequenced genomes for
ligninases.
Purdue Cell Wall Genomics and UC-Riverside
Cell Wall Navigator Databases
Unlike the above general protein family databases,
Purdue’s Cell Wall Genomics (Yong et al., 2005) and
UCR’s Cell Wall Navigator (Girke et al., 2004) databases
are specifically designed for plant cell wall biosynthesis.
As opposed to sequence similarity-based classification,
both databases categorize proteins based on their physi-
ological roles in cell wall synthesis. In UCR’s database,
there are five categories: monosaccharide synthesis,
polysaccharide synthesis, reassembly, structural proteins
and glycoprotein synthesis, basically all centered on car-
bohydrate molecules in cell walls. However, Purdue’s
database is even broader with six categories: pathways
for substrate generation, polysaccharide synthases,
secretion and targeting pathways, assembly/architec-
ture and growth, differentiation and secondary wall
formation and signaling and response pathways. Partic-
ularly, Purdue’s database also includes lignin synthesis
and polymerization proteins as well as signaling proteins
involved in cell wall synthesis. Both databases also pro-
vide references and the literature associated with each
category to support their classification. Proteins of the
two databases are primarily from model organisms
such as Arabidopsis and rice. However, it is easy to use
the sequences as query to search for their homologs and
even orthologs in other plants. The protein accessions
and classification complied by the two databases serve
as an excellent overview of the current achievement
made by the entire plant cell wall community in terms
of our latest understanding of cell wall synthesis.
Plant Coexpression Network Databases: PlaNet
and ATTED
As we mentioned earlier, regulation of biomass for-
mation and degradation is also extremely important.
Studying the regulation has been benefited a lot from
coexpression analysis of microarray data and recently
on high-throughput RNA sequencing data. There are
numerous tools, Web based or stand alone, allowing
for coexpression analysis with user-submitted genes as
query or by general browsing. Many online tools even
offer prebuilt coexpression networks, with nodes in the
network graphs representing genes and edges repre-
senting coexpression relationships. These coexpression
networks are very informative and insightful, in terms
of suggesting candidate genes involving in the same
metabolic pathways or potential regulators of genes of
interest to the users (Ruprecht and Persson, 2012).
Although there are many such tools available, in plant
cell wall field PlaNet (Mutwil et al., 2011) family tools
(coreCarb (Mutwil et al., 2009), AraNet, GeneCat) stand
out as they were developed by researchers of the cell
wall field, have a Web-based interface and have been
shown to be effective in suggesting new genes for cell
wall synthesis (Mutwil et al., 2009; Ruprecht et al., 2011).
PlaNet placed query genes in network graphs of three
levels: (1) the coexpressed node vicinity network, con-
taining the query gene and genes coexpressed two steps
away and the links among them; (2) a larger coexpres-
sion cluster containing the query gene and genes coex-
pressed, which resulted from running a heuristic
clustering algorithm; and (3) the largest meta-network
with nodes now representing all coexpression clusters
instead of individual genes. PlaNet also has a module
called NetworkComparer, allowing a comparative anal-
ysis of gene expression networks across seven plant or-
ganisms. Such comparative coexpression analysis has
recently become very popular as it can help deal with
missing data in a single species, reduce false positives
identified as coexpressed in a single species, and enable
to study the conservation of coexpression network from
an evolution perspective (Movahedi et al., 2012).
An earlier tool ATTED-II (Obayashi et al., 2009) is also
well known, which was developed by plant biologists
since 2003 as a database for Arabidopsis tissue-specific
 REFERENCES
(ATTED) expression. Compared to PlaNet, ATTED-II
provides richer annotation and a lot of useful links to
external resources in the query gene browse page. It
also has a nicer network graph with less genes (top
certain amount of genes for better visualization) and
gene function information (biologically meaningful
gene names) and labeled TFs. Besides, ATTED-II also
predicted cis-regulatory elements in the upstream re-
gions of coexpression genes. However, ATTED-II
currently includes only two plants: Arabidopsis and
rice, and the gene locus page is only available for
Arabidopsis.
FUTURE PERSPECTIVES
As a perspective for the future development of
bioenergy-related databases, we ask: what do we
need from newly developed databases? Nucleic Acids
Research publishes a prestigious annual Database Spe-
cial Issue since 20 years ago. Most databases published
there for a particular class of proteins such as plant
TFs (Guo et al., 2008), peroxidases (Fawal et al., 2012),
transporters (Saier et al., 2006) and peptidases (Rawlings
et al., 2012), all provide the following data or functional-
ities: (1) a general classification of targeted protein
families, manually collected references, a list of charac-
terized proteins curated from the literature and/or pre-
dicted member proteins; (2) secondary data derived
from further in-depth bioinformatics analysis, such
as computer-based functional annotation (e.g. Gene
Ontology or protein domain annotation), sequence
alignment, phylogenetic trees, predicted protein struc-
tures etc.; (3) simple Web-based BLAST search against
the sequence database and text search using keywords;
(4) long-term maintenance to update regularly with
new data; and (5) plenty of documentation such as
help, FAQ or tutorial pages. These could be considered
as criteria for a good protein family database.
Although the plant biomass formation-related data-
bases listed in Table 6.2 are all very useful, none of
them have sufficiently integrated various functional
omics data. Biologists working on one model plant often
want to take advantage of these data to study their inter-
ested genes, e.g. investigate fully sequenced plant ge-
nomes to look for orthologs, or transcriptome data
(microarray and RNA-seq) for expression profiles or
look for coexpressed genes and go to the upstream re-
gions for candidate cis-regulatory motifs; all these ana-
lyses have to be done using individual bioinformatics
tools or servers, which often requires expert knowledge
to run or to interpret the results. In addition, many of the
databases are outdated and none of them have included
all CWR genes. For example, Purdue’s database is an
excellent resource, but it does not include many of the
103
newly characterized CWR genes such as TF family
NAC, WRKY, MYB members shown to control lignin
synthesis; many of the newly characterized CAZyme
families such as GT43, 61, 75, transporters for NDP-
sugars and monolignols; miRNAs; DUF (domain of un-
known function) families etc. It includes neither much
annotation data nor any search functionalities.
Therefore, the future plant CWR gene databases
should aim to include all experimentally characterized
CWR genes from any organisms, associated sequences
and functional descriptions collected from the published
literature, e.g. those listed in Table 6.1. Such character-
ized gene list could be highly useful for annotating
sequenced bioenergy plants such as switchgrass, poplar,
maize, sorghum and Eucalyptus grandis. The CWR gene
repertories for these organisms will be highly valuable
for the bioenergy research community as people are
trying to select candidate CWR genes to knock down
or overexpress for developing transgenic plants in these
model organisms. Gene families for CWR genes and
other extensive secondary bioinformatics data should
also be included in the databases, particularly phylog-
eny (used to identify orthologs from homologs), pre-
dicted cis-regulatory element, conserved coexpression
network modules of known CWR genes, expression
profiling, coexpressed gene list including noncoding
RNAs, genomic location, gene neighborhood, epige-
nomics, proteineprotein interactions, structures, subcel-
lular locations, single-nucleotide polymorphism, indels,
etc. Similar databases should also be developed for plant
CAZymes and include the above bioinformatics-derived
data types. The reason is that CAZyDB now only
covered 2 (A. thaliana and Oryza sativa) of over 40
sequenced plant and green algal genomes, not to
mention there are more incomplete genomes and tran-
scriptomes (ESTs and RNA-seq data).
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