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COVER ARTICLE
Timothy D. H. Bugg et al.
Pathways for degradation of lignin in
bacteria and fungi 0265-0568(2011)28:12;1-D
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Cite this: Nat. Prod. Rep., 2011, 28, 1883

www.rsc.org/npr REVIEW
Pathways for degradation of lignin in bacteria and fungi
Timothy D. H. Bugg,* Mark Ahmad, Elizabeth M. Hardiman and Rahman Rahmanpour
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J

Received 6th May 2011

DOI: 10.1039/c1np00042j

Covering: up to 2011

Lignin is a heterogeneous aromatic polymer found as 10–35% of lignocellulose, found in plant cell
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walls. The bio-conversion of plant lignocellulose to glucose is an important part of second generation
biofuel production, but the resistance of lignin to breakdown is a major obstacle in this process, hence
there is considerable interest in the microbial breakdown of lignin. White-rot fungi are known to break
down lignin with the aid of extracellular peroxidase and laccase enzymes. There are also reports of
bacteria that can degrade lignin, and recent work indicates that bacterial lignin breakdown may be
more significant than previously thought. The review will discuss the enzymes for lignin breakdown in
fungi and bacteria, and the catabolic pathways for breakdown of the b-aryl ether, biphenyl and other
components of lignin in bacteria and fungi. The review will also discuss small molecule phenolic
breakdown products from lignin that have been identified from lignin-degrading microbes, and
includes a bioinformatic analysis of the occurrence of known lignin-degradation pathways in Gram-
positive and Gram-negative bacteria.

1 Introduction to lignin and sub-structures 1 Introduction to lignin and sub-structures

2 Enzymes implicated in lignin breakdown in fungi and
bacteria Modern society is highly reliant on the utilisation of oil, coal and
2.1 White-rot and brown-rot fungi for lignin breakdown gas deposits for provision of energy and petrochemicals.
2.2 Extracellular fungal lignin and Mn peroxidases However, the dwindling supplies of oil and gas, coupled with the
2.3 Involvement of extracellular laccases in lignin greenhouse gas emissions of coal-based power stations and oil-
degradation based fuel utilisation, has led to a realisation that society must
2.4 Use of Fenton chemistry in brown-rot fungi turn to renewables for production of energy and chemicals, and
2.5 Bacterial lignin degraders and emerging enzymology hence there is considerable interest in the utilisation of plant
3 Small molecule products from lignin metabolism biomass for production of bioenergy and renewable chemicals.1
4 Catabolic pathways for breakdown of lignin Bioethanol has been produced since the 1970’s in Brazil on
components a large scale, from processing of sugar cane, via extraction of
4.1 b-Aryl ether degradation pathways sucrose, conversion to glucose, and fermentation to ethanol,
4.2 Biphenyl degradation pathways followed by distillation.2 Biodiesel can also be obtained from
4.3 Diarylpropane degradation pathways plant-based oils, via base-catalysed methanolysis, to form fatty
4.4 Degradation of phenylcoumarane and pinoresinol acid methyl esters. These ‘‘first-generation’’ biofuel technologies
lignin components have attracted criticism from environmental organisations, due
4.5 Bacterial degradation of ferulic acid to the large commitment of land needed for biofuel production,
4.6 Oxidative cleavage of protocatechuic acid and the consequent shortage of land for food production, the
5 Occurrence of lignin degradation pathways in Gram- ‘‘food vs. fuel debate’’. A solution to this dilemma is to use the
positive and Gram-negative bacteria non-food part of a plant crop for bioenergy production, by
6 Conclusions conversion of plant lignocellulose into biofuels, known as
7 Acknowledgements ‘‘second-generation’’ or cellulosic bioethanol production, as
8 References shown in Fig. 1.1
Lignocellulose is the structural material used to make plant
cell walls, and is therefore the main component of plant biomass.
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, Lignocellulose consists of three main components: cellulose,
U.K. E-mail: T.D.Bugg@warwick.ac.uk; Tel: +44-2476-573018 hemi-cellulose, and lignin.2,3 Cellulose, which accounts for

This journal is ª The Royal Society of Chemistry 2011 Nat. Prod. Rep., 2011, 28, 1883–1896 | 1883
 30–50% dry weight of lignocellulose, is a polysaccharide
composed of b-1,4-linked D-glucose units. Cellulose is used for
paper and cardboard manufacture, and can be converted via the
action of cellulase enzymes into glucose, for bioethanol
production. Hemi-celluloses consist of other polysaccharides,
principally xylans and mannans, which are closely associated
with the cellulose filaments, and chemically linked with lignin.
The major hemicellulose in hardwoods is xylan (15–30% dry
weight), a polysaccharide composed of b-1,4-linked D-xylose
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
units, which can be substituted with other monosaccharide units;
whereas softwood hemicellulose contains mainly gal-
actoglucomannan (15–20% dry weight), a polysaccharide
composed of b-1,4-linked D-glucose and D-galactose units.
Tim Bugg is Professor of Bio-
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logical Chemistry at the
University of Warwick.
Following his PhD studies with
Dr C. Abell at the University of
Cambridge, he spent two years
as a SERC/NATO postdoctoral
research fellow in the laboratory
of Prof. C. T. Walsh at Harvard
Medical School. He began his
academic career in 1991 as
a lecturer in organic chemistry
at the University of South-
Timothy D:H: Bugg ampton, before moving to War-
wick in 1999. His research
interests are in the study of
enzyme mechanisms, particularly enzymes involved in the bacterial
degradation of aromatic compounds. He has been studying
enzymes of bacterial lignin degradation with the co-authors since
2007. He also enjoys volleyball, cycling, and playing the violin.
Mark Ahmad is a Research
Fellow at the University of
Warwick, working in the group
of Dr Andrew Clark on the
synthesis of sustainable mate-
rials from lignin. Having gradu-
ated with an MChem degree in
Chemistry with Medicinal
Chemistry at the University of
Warwick, Mark studied for
a PhD in Professor Bugg’s
research group, studying the
enzymology of bacterial degra-
Mark Ahmad dation of lignin, completing in
2010.
1884 | Nat. Prod. Rep., 2011, 28, 1883–1896
Lignin is a complex aromatic heteropolymer, composed of
phenylpropanoid aryl-C3 units, linked together via a variety
of ether and C–C bonds. Lignin accounts for 15–30% dry weight
of lignocellulose, in which it forms a matrix that is closely
associated with the cellulose filaments, and is covalently attached
to hemicelluloses. Lignin is formed by radical polymerisation of
guaiacyl (G) units from precursor coniferyl alcohol, syringyl (S)
units from precursor sinapyl alcohol, and p-hydroxyphenyl (H)
units from precursor p-coumaryl alcohol.4 The ratio of G:S:H
units varies from species to species, but softwoods are generally
G type lignins, containing mainly G units, while hardwoods are
generally GS-type lignins, containing mixtures of G and S units,
and grass lignins are G type lignins containing a higher
Elizabeth Hardiman works as
a post-doctoral researcher in the
Department of Chemistry at the
University of Warwick. She
completed a BTech in Biotech-
nology at the University of
Auckland, Auckland, New Zea-
land in 2001. Her PhD was
obtained at Macquarie Univer-
sity in Sydney, Australia under
the supervision of Prof. Peter
Bergquist. Her PhD work
involved the directed evolution of
Elizabeth M: Hardiman a thermophilic b-glucosidase
using flow cytometry as a tool
for screening enzyme activity.
Currently she is investigating the role of bacterial enzymes in lignin
degradation and modification.
Rahman Rahmanpour is
a Masters graduate in
Biochemistry and Biophysics
from Tarbiat Modares Univer-
sity (TMU) of Tehran, Iran.
Having obtained the first rank in
the MSc national entrance exam
in Iran, he was awarded the
opportunity to study both
Biochemistry and Biophysics.
He graduated with Honours in
June 2008. He worked on his
Masters thesis under supervision
Rahman Rahmanpour of Dr Bathaie, studying the
effects of glycation on H1
structure and its interaction with
DNA. At present he is a visiting researcher in Professor Bugg’s
research group at the University of Warwick, working on purifi-
cation and characterization of novel enzymes responsible for lignin
degradation from bacteria. Recently, he has been awarded a PhD
scholarship from the University of Warwick.
This journal is ª The Royal Society of Chemistry 2011

Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
Fig. 1 Biofuel production scheme.
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proportion of H units.4 In Fig. 2 are shown the major types of
linkages found in lignin, which can be analysed structurally used
13
C solid state NMR spectroscopy.5 The most common linkage is
the b-aryl ether, containing an ether linkage to another aryl unit
at C-2 of the C3 chain, which is found as 45–50% of units in
softwoods, and 60% in hardwoods. The next most common
linkage in softwoods is the biphenyl linkage, containing an aryl–
aryl C–C bond, which is found as 20–25% units in softwoods, but
only 3–9% in hardwoods. The diaryl propane contains a C–C
bond at C-2 of the C3 chain to a second aryl ring, while the diaryl
ether contains an ether linkage between two aryl rings. Hetero-
cyclic linkages are also found: the phenylcoumarane linkage
contains a dihydrofuran ring fused to an aryl unit; the pinor-
esinol contains a linkage formed by two fused tetrahydrofuran
Fig. 2 Structural units found in lignin. A. Structures of G, S, and H
monomeric units. B. Structures of chemical linkages found in lignin
(shown as G units). SW, softwood; HW, hardwood.
This journal is ª The Royal Society of Chemistry 2011
rings; and the spirodienone linkage contains a more complex
tetrahydrofuran-aryl linkage.
The formation of each of these linkages can be rationalised by
radical dimerisation or polymerisation reactions from cinnamyl
alcohol precursors.6,7 Single electron oxidation of the cinnamyl
alcohol precursor by plant peroxidase and laccase enzymes
generates a phenoxy radical, which has radical character on the
phenolic oxygen, on the adjacent aromatic carbon, and at the b-
position of the C3 chain, and hence reaction at each of these sites
can generate the linkages observed (Fig. 3).
The ether and C–C linkages present in lignin are not suscep-
tible to hydrolytic attack, and therefore, lignin is highly resistant
to breakdown. The encasement of cellulose filaments in lignin
provides a physical barrier to lignocellulose breakdown, which is
an obstacle to second-generation biofuel production. Current
methods for cellulosic bioethanol production use a physical pre-
treatment step, such as steam explosion, to liberate cellulose for
conversion to glucose monomers,8 which is energy-intensive.
Lignin also represents a potentially valuable source of renewable
aromatic chemicals.9 Hence there is interest in chemical and
biological methods for lignin breakdown, which might be used to
aid lignocellulose breakdown for biofuel production, or to
generate aromatic chemicals from lignin, and hence form the
basis of a ‘‘biorefinery’’.1 For the latter application, there is
a need to understand the biochemical pathways used for lignin
breakdown. This review will summarise the biochemical basis for
lignin breakdown, and what is known about the downstream
pathways for lignin breakdown.
2 Enzymes implicated in lignin breakdown in fungi
and bacteria
2.1 White-rot and brown-rot fungi for lignin breakdown
Lignin modification and degradation has been most extensively
studied in basidiomycetes, in which a number of enzymes and
mechanisms involved in lignin attack have been elucidated.10,11
White-rot basidiomycetes (notably Phanerochaete chrys-
osporium) are the most frequent wood-rotting organisms,
because of their ability to degrade lignin, hemicelluloses, and
cellulose, often giving rise to cellulose-enriched white material.
Brown-rot fungi grow mainly on softwoods and represent only
Fig. 3 Formation of linkages found in lignin via radical intermediates.
Radical electron density is also found at C-1 of the oxidised CA unit.
Nat. Prod. Rep., 2011, 28, 1883–1896 | 1885
 7% of wood-rotting basidiomycetes. They can also degrade wood
polysaccharides after only a partial modification of lignin, which
results in a brown material consisting of oxidized lignin, which
represents a potential source of aromatic compounds for the
stable organic matter fraction in forest soils.12 Lignin is degraded
to a lesser extent by brown-rot fungi, via a different mechanism
to white-rot fungi,13 which will be discussed in Section 2.4.
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
2.2 Extracellular fungal lignin and Mn peroxidases
White-rot fungi produce several types of extracellular oxidative
enzymes (oxidoreductases) that are involved in the degradation
of lignin content in a plant cell wall. These include laccases and
high redox potential ligninolytic peroxidases (lignin peroxidase,
manganese peroxidase, and versatile peroxidase). The onset of
their production is associated with secondary metabolism
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conditions in response to nutrient depletion.14 Lignin peroxidase
(LiP) and manganese peroxidase (MnP) were discovered in the
mid-1980s in the white-rot basidiomycete Phanerochaete chrys-
osporium. LiP degrades non-phenolic lignin units (up to 90% of
the polymer), whereas MnP generates Mn3+, which acts as
a diffusible oxidizer on phenolic or non-phenolic lignin units via
lipid peroxidation reactions. More recently, versatile peroxidase
(VP) has been described in Pleurotus and other fungi as a third
type of ligninolytic peroxidase that combines the catalytic
properties of LiP, MnP and plant/microbial peroxidases.12
P. chrysosporium possesses over a dozen different peroxidase
genes, and it is the most thoroughly investigated white-rot
basidiomycete. It has 16 candidate genes corresponding to lignin
degradation; 10 LiP enzymes, 5 MnP enzymes and 1 NoP (novel
peroxidase).15 A family of 10 structurally related genes desig-
nated lipA through to lipJ encodes LiP. The reason for the
multiplicity of lip genes is unclear, although differences in the
oxidation–reduction potential among LiP isoenzymes have been
observed.16 The crystal structures of both LiP and MnP from P.
chrysosporium have been determined,17,18 and were found to be
structurally related to each other and to other peroxidases,
indicative of divergent evolution.19 These peroxidases share
a general tertiary folding and helical topography, they are
globular proteins formed by 11–12 a-helices predominantly in
two domains, with a central cavity containing the heme group. In
order to stabilize the protein structure, LiP contains four Cys
disulfide bridges, and MnP contains a fifth bridge in its
C-terminal region, and two Ca2+ binding sites.20
The catalytic cycle of both peroxidases is similar to other
peroxidase enzymes, in which the resting state of the enzyme
contains ferric heme, which reacts with hydrogen peroxide to
form a compound I oxo-ferryl intermediate (two-electron
oxidized form), and subsequently a compound II intermediate
(one-electron oxidized form).12,20 However, two aspects in their
molecular structure differentiate ligninolytic peroxidases from
other peroxidases: first, a heme environment that confers high
redox potential to the oxo-ferryl complex (due to the position of
N3 of the side-chain of the proximal histidine residue, increasing
its electron deficiency and increasing the redox potential of the
oxo-ferryl complex); secondly, the presence of binding sites for
oxidation of their specific substrates, including non-phenolic
aromatics in the cases of LiP, and Mn2+ in the case of MnP.12,20
1886 | Nat. Prod. Rep., 2011, 28, 1883–1896
Ferriperoxidase + H2O2 / Compound I + H2O (1)
Compound I + AH2 / Compound II + AH (2)
Compound II + AH2 / Ferriperoxidase + AH + H2O (3)
Manganese peroxidase (MnP) is a heme-containing glyco-
protein which was first discovered in P. chrysosporium. MnP is
often produced in multiple forms, and up to 11 different iso-
forms have been described in one fungal strain, C. subvermispora,
which differ in their isolectric points.21 The Mn peroxidases
catalyze the oxidation of complexed Mn2+ to Mn3+,which in turn
can oxidize a large number of phenolic substrates. Mn3+ is widely
accepted as a diffusible oxidant, able to oxidize secondary
substrates at a distance away from the active site of MnP.19
White-rot fungi secrete oxalic acid and other organic acids that
form stable Mn3+ chelates, which act as stable diffusing oxidizers
of phenolic compounds and dyes.20
The catalytic cycle of MnP resembles those of other heme
peroxidases, such as horseradish peroxidase (HRP) and lignin
peroxidase, involving reaction of the native ferric enzyme with
hydrogen peroxide to form a Compound I oxo-ferryl interme-
diate, which then reacts with Mn2+ to form Compound II and
Mn3+. The active site consists of a proximal histidine ligand (His-
173) hydrogen-bonded to an aspartic acid residue (Asp-242) and
a distal side peroxidase-binding pocket comprising catalytic
histidine (His-46), which acts as a proton donor to generate
compound I, and a nearby arginine (Arg-42) residue that is
thought to stabilise compound I.20,21 The reaction of P. chrys-
osporium MnP with b-aryl ether lignin model compounds has
been studied in detail, and found to proceed via a phenoxy
radical intermediate, followed by Ca–Cb bond cleavage, and also
alkyl-phenyl bond cleavage reactions.22 Rate constants for
oxidation of Mn2+ by either MnP compound I or compound II
have been determined by stopped flow kinetic methods.23
At least six different lignin peroxidase isozymes (H1, H2, H6,
H7, H8, and H10) have been detected in nitrogen-limited cultures
of P. chrysosporium, which are all glycosylated, and all isozymes
except for isozyme H1 are phosphorylated on their N-linked
carbohydrate moiety in the form of mannose 6-phosphate.24 Due
to its high redox potential, LiP can also oxidise nonphenolic
methoxy-substituted lignin subunits. The catalytic cycle of LiP is
also similar to MnP, horseradish peroxidase (HRP) and cyto-
chrome C peroxidase (CCP) in catalyzing the oxidation of
substrate by H2O2. However, there are some important features
distinguishing this enzyme from other oxidoreductases, such as
their very low pH optima and much higher redox potentials. The
optimum pH for steady-state turnover of LiP is near 2.0, which is
lower than those of all other peroxidases25
For LiP, veratryl alcohol, produced by ligninolytic fungi, has
been proposed to act as a redox mediator: it is oxidized to the
corresponding cation radical that acts as a diffusible oxidant;26,27
however, the veratryl alcohol cation radical formed after one
electron transfer has a very short lifetime.28 The active site heme
of LiP is only accessible to small LiP substrates, which can
undergo direct electron transfer to heme. However, long-range
electron transfer is also possible, via a mobile Trp-171 residue,
which can form a stable radical via reaction with heme
This journal is ª The Royal Society of Chemistry 2011
 compound I, and can then flip to the protein surface.20,26 The
location of Trp-171 at the LiP surface is in a very acidic envi-
ronment that could stabilize cation radicals formed after one
electron transfer.27 Multiple sequence alignments show that Trp-
171 is conserved in all LiP sequences, and site-directed muta-
genesis experiments suggest that Trp-171 is a redox-active
residue in LiP.26 The reaction of P. chrysosporium LiP with b-aryl
ether lignin model compounds has been studied in detail, and
found to proceed via Ca–Cb bond cleavage.29
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
Although the reactions of LiP and MnP with lignin model
compounds are well characterised, these enzymes have not been
shown to oxidise native polymeric lignin, implying that other
reactions are also involved in the initial stage of lignin depoly-
merisation.30 Fungal LiP and MnP have been shown to oxidise
synthetic polymeric lignin substrates in vitro, but a mixture of
depolymerisation and repolymerisation was observed.31,32
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2.3 Involvement of extracellular laccases in lignin degradation
Laccases catalyse the oxidation of various phenolic substrates via
the reduction of oxygen to water.33 The substrate range can be
extended to non-phenolic compounds in the presence of small
mediator molecules, such as 2,20 -azinobis(3-ethylbenzothiazo-
line-6-sulfonic acid) (ABTS) and 1-hydroxybenzotriazole
(HBT).34 Laccases have been found in a variety of organisms from
bacteria and fungi, to plants and insects. In plants they are
involved in the biosynthesis of lignin, whilst in fungi it is thought
that they play a role in the degradation of lignin, although this role
is unclear and controversial.35,36 Laccases contain four copper
atoms that have different spectral and paramagnetic properties.
The mononuclear Type 1 (T1) copper is responsible for the
characteristic blue colour of these enzymes, and shows a strong
absorption band around 600 nm. The remaining copper atoms are
arranged in a trinuclear cluster consisting of a Type 2 (T2) copper
and two Type 3 (T3) coppers. The T2 copper has a characteristic
EPR spectrum and the T3 coppers show absorption at around 320
nm. Substrate oxidation is thought to occur at the mononuclear
T1 copper and electrons are transferred to the trinuclear T2/T3
centre where oxygen is reduced to water.37–39
Several lignin-degrading white rot fungi, such as Trametes
versicolor and Phlebia radiata, produce laccases in addition to
peroxidases,40,41 and it has been reported that the lignin
degrading ability of laccase-deficient mutants is impeded,42,43
implying a role in lignin degradation. Laccases have also been
shown to cleave phenolic lignin model compounds44 and non-
phenolic compounds via the mediator system.45 However, it has
also been shown that laccase is not essential for lignin degrada-
tion. Studies inhibiting laccase activity with antibodies or thio-
glycollic acid, a copper-chelating reagent, showed that strains
were still able to decompose lignin.46 Furthermore, the genome of
Phanerochaete chrysosporium, one of the most efficient and well-
studied fungal lignin degraders, contains no classical laccases.47
A multicopper oxidase with a secretory signal was found, but this
is thought to have activity as a ferroxidase.48
2.4 Use of Fenton chemistry in brown-rot fungi
In contrast to white-rot fungi, which produce an arsenal of
extracellular oxidative enzymes for lignin degradation, brown-
This journal is ª The Royal Society of Chemistry 2011
rot fungi preferentially hydrolyse the cellulose component of
lignocellulose, but also partially oxidise lignin. Analysis of lignin
oxidation fragments has led to the proposal that a hydroxyl
radical is produced via Fenton oxidation chemistry.49–51 The
involvement of Fenton chemistry in biology has been reviewed.52
A redox cycling process has been proposed in Gloeophyllum
trabeum, using two quinones that are produced extracellularly,
2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and 4,5-dime-
thoxy-1,2-benzoquinone (4,5-DMBQ),53 as shown in Fig. 4. Each
hydroquinone is able to reduce Fe3+ to Fe2+, in each case chelated
to oxalate, which is the predominant form of iron found extra-
cellularly. The iron(II) oxalate complex then reacts with hydrogen
peroxide to generate hydroxyl radicals, which attack lignin. The
oxidised quinone is then recycled to the hydroquinone form by
the fungus.53 Transcriptomic analysis of brown-rot fungus Postia
placenta indicates up-regulation of genes involved in iron
acquisition, consistent with this redox-cycling hypothesis.54 The
production of hydroxyl radicals via a redox cycling mechanism
has also been proposed for white-rot fungi.55
2.5 Bacterial lignin degraders and emerging enzymology
Although the microbial degradation of lignin has been most
intensively studied in white-rot and brown-rot fungi, there are
a number of reports of bacteria that can break down lignin.56,57
Several streptomycetes have been reported to break down
lignin,56 of which the best-studied strain is Streptomyces vir-
idosporus T7A.58 This strain produces several extracellular
peroxidases, which have been shown to catalyse oxidative
cleavage of b-aryl ether lignin model compounds.58 A radio-
chemical assay for breakdown of 14C-labelled lignin has been
used to identify strains of Nocardia and Rhodococcus that can
break down lignin.57 Catabolic pathways for breakdown of lignin
components have been studied extensively in Sphingomonas
paucimobilis SYK-6, and will be discussed in Section 4.59 More
recently, Ahmad et al. have developed two spectrophotometric
assays for lignin breakdown: one involving fluorescently-labelled
lignin; and a UV-vis assay involving chemically nitrated lignin,
which releases nitrated phenols upon breakdown.60 Several
Fig. 4 A scheme of the Fenton redox cycle found in brown-rot fungi.
Nat. Prod. Rep., 2011, 28, 1883–1896 | 1887
 known aromatic degraders were found also to be lignin degraders
using these assays, including Pseudomonas putida mt-2 and
Rhodococcus jostii RHA1.60 The activity of these bacterial strains
in the assay was much less than that of white-rot fungus Pha-
nerochaete chrysosporium using the same assays, but was
comparable to other lignin-degrading fungi. Pseudomonas putida
and Rhodococcus RHA1 were found to break down lignocellu-
lose in small-scale incubations, releasing low molecular weight
phenolic products.60 The majority of bacterial lignin degraders
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
identified to date fall into three classes, the actinomycetes, a-
proteobacteria, and g-proteobacteria, representatives of which
have also been found in the digestive systems of wood-infesting
termites and insects.61
The enzymology for bacterial lignin degradation is much less
well-studied than that of white-rot fungi, yet there are indications
from the work on Streptomyces viridosporus T7A that bacteria
use extracellular peroxidases for lignin degradation.58 Using
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a colorimetric assay for lignin breakdown, the activity for
Streptomyces viridosporus was found to be highly dependent
upon hydrogen peroxide, consistent with the use of extracellular
lignin peroxidase enzymes, whereas P. putida and Rhodococcus
RHA1 both show activity in the absence of hydrogen peroxide,
suggesting either the use of oxygen-utilising laccase enzymes, or
extracellular enzymes for hydrogen peroxide generation.60 A
dyp-type peroxidase has recently been identified from bio-
informatic analysis of the genome of Rhodococcus jostii RHA1,
and peroxidase DypB has been shown to be active for lignin
degradation by spectrophotometric assay, and active against
Kraft lignin and lignin model compounds.62 This enzyme is
activated by Mn2+ ions, and a potential site for Mn2+ binding has
been identified in the crystal structure of R. jostii DypB.63 A dyp-
type peroxidase has also been recently reported in the secretome
Table 1 Aromatic breakdown products detected from lignin breakdown
Compound
Benzoic acid 4-hydroxy
4-hydroxy-3-methoxy
4-hydroxy-3-methoxy-6-carboxy
4-hydroxy-3-methoxy-5-carboxy
3,4-dimethoxy
3,4-dimethoxy-2-carboxy
2-hydroxy-3-methoxy
2,3-dihydroxy
2,3,4-trihydroxy
Benzaldehyde 4-hydroxy-3-methoxy
3,4,5-trimethoxy
Cinnamic acid 4-hydroxy
4-hydroxy-3-methoxy
Biphenyl-5,50 -dicarboxylic acid,
2,20 -dihydroxy, 3,30 -dimethoxy
Diphenyl ether
Propiophenone-30 -hydroxy 4-hydroxy-3-methoxy
Acetophenone 4-hydroxy-3-methoxy
Phenol 2-methoxy
2-methoxy-4-vinyl
1888 | Nat. Prod. Rep., 2011, 28, 1883–1896
of cellulose-degrading Thermobifida fusca.64 Another extracel-
lular heme-dependent enzyme with activity against lignin model
compounds has been recently reported in Amycolatopsis sp.
72vi2.65
3 Small molecule products from lignin metabolism
Since lignin is composed of phenylpropanoid units, then the
oxidative breakdown of lignin could potentially release low
molecular weight products, which might be useful renewable
chemicals, provided that the breakdown process could be
controlled.
Studies of spruce wood lignin breakdown by Phanerochaete
chrysosporium has identified 28 low molecular weight products, 10
of which were aromatic carboxylic acids,66 and a further 13 of which
were acyclic 2,4-hexadiene-1,6-dioic acids resulting from oxidative
ring cleavage.67 The aromatic acids were all benzoic acid derivatives,
indicating that they had arisen from Ca–Cb oxidative cleavage of
lignin components, and the compounds included a biphenyl
dicarboxylic acid, and a diphenyl ether dicarboxylic acid, derived
from the biphenyl and diphenyl ether components of lignin,
respectively. The breakdown of Kraft lignin by Trametes versicolor,
which produces extracellular laccase and Mn peroxidase enzymes,
has been studied using 14C-labelled lignin material.68 After an initial
polymerisation of the lignin, depolymerisation then took place,
releasing alkali-extractable material, and then soluble products,
which ultimately formed most of the degradation products, though
in this case the products were found to be oligomeric fragments,
rather than monomeric ones.68
Similar phenolic products have been observed from treatments
of lignin or lignocellulose by bacterial lignin degraders
(See Table 1). Small molecule phenolic metabolites, such as
Fungal Bacterial
lignin degrader lignin degrader
P. chrysosporium A. aneurinilyticus
P. chrysosporium A. aneurinilyticus
P. chrysosporium
P. putida, R. jostii RHA1
P. chrysosporium
P. chrysosporium
A. aneurinilyticus, P. putida
Bacillus sp.
S. paucimobilis
Bacillus sp.
Bacillus sp.
Bacillus sp., P. putida, R. jostii RHA1
P. chrysosporium
P. chrysosporium
S. paucimobilis. P. putida, R. jostii RHA1
Soil metabolite
Soil metabolite
Soil metabolite
This journal is ª The Royal Society of Chemistry 2011
 4-hydroxy-3-methoxy-30 -hydroxy-propiophenone and 4-
hydroxy-5-methoxy-3-carboxybenzoic acid, have been observed
in biotransformations of lignocellulose using Pseudomonas
putida and Rhodococcus RHA1,60 and have been observed using
Sphingomonas paucimobilis.59 Degradation of Kraft lignin by
Bacillus sp. and Aneurinibacillus aneurinilyticus has been studied
by GC-MS, and a number of products were identified: trans-4-
hydroxycinnamic acid, 3,4,5-trimethoxy benzaldehyde, gallic
acid and ferulic acid.69 In a similar study, breakdown products
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
from waste paper effluent by Aeromonas formicans were identi-
fied by GC: 4-hydroxybenzoic acid, vanillic acid, protocatechuic
acid, syringic acid, trans-4-hydroxycinnamic acid and ferulic
acid.70 Phenol and guaiacol, resulting from decarboxylation of
the benzoic acids noted above, are known lignin metabolites
detected in soil, together with acetovanillone, and 4-vinyl-
guaiacol.71 The observed metabolites are summarised in Table 1.
Oxidised polymeric intermediates have also been isolated in
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several cases. Streptomyces viridosporus metabolises lignin to
form a water-soluble intermediate, acid-precipitable poly-
phenolic polymeric lignin (APPL),72 which is also produced by
other bacteria, such as Thermobifida fusca.73
4 Catabolic pathways for breakdown of lignin
components
Although lignin-degrading enzymes have been identified and
characterised using model substrates, information about the
catabolic pathways by which oxidised lignin fragments are
broken down in vivo is incomplete, and is based upon pathways
elucidated in bacteria for degradation of certain lignin compo-
nents. Knowledge of such pathways is potentially important for
biotechnological applications of lignin degradation.
4.1 b-Aryl ether degradation pathways
Bacterial catabolic pathways for several lignin components have
been studied extensively in Sphingomonas paucimobilis SYK-6.59
A b-aryl ether lignin dimer compound is metabolised via the
pathway shown in Fig. 5. Initial oxidation of the a-hydroxyl
group to the corresponding ketone is catalysed by the NAD-
dependent dehydrogenase LigD.74 Then, an unusual reductive
ether cleavage reaction takes place, via a novel glutathione-
dependent b-etherase enzyme,75 that is a member of the
Fig. 5 Pathways for the metabolism of b-aryl ether components of
lignin. Gene designations are those identified in S. paucimobilis.
This journal is ª The Royal Society of Chemistry 2011
glutathione-S-transferase superfamily.76 The LigEFG gene
products are responsible for this transformation: LigE and LigF
were found to catalyse the reaction of reduced glutathione with
the two different enantiomers of the aryl ether substrate,77 to give
a b-thioether intermediate, and LigG catalysed the reaction with
a second molecule of reduced glutathione,76 as shown in Fig. 6.
Degradation of this b-aryl ether model compound has also been
reported in Pseudomonas acidovorans.78 The ketone product has
been detected as a by-product of lignocellulose breakdown using
Pseudomonas putida and Rhodococcus jostii RHA1, where it
might arise via the b-etherase cleavage reaction, or via a b-elim-
ination reaction.60
The ketone product is then metabolised to vanillic acid,
probably via oxidation of the g-hydroxyl group to the carboxylic
acid, and then a C–C cleavage reaction analogous to fatty acid
b-oxidation, as shown in Fig. 5. Decarboxylation of the
carboxylic acid intermediate might explain the formation of the
methyl ketone acetovanillone, which is a known lignin break-
down product, and is a mediator for fungal laccases.79 A
b-etherase enzyme has also been identified in the fungal asco-
mycete strain 2BW-1.80
Demethylation of vanillic acid to protocatechuic acid in S.
paucimobilis SYK-6 is catalysed by a tetrahydrofolate-dependent
demethylase LigM, which generates 5-methyl tetrahydrofolate as
a by-product.81 In contrast, the same demethylation reaction in
Pseudomonas and Acinetobacter is catalysed by a non-heme iron-
dependent demethylase enzyme.82,83 The product, protocatechuic
acid, is then a substrate for oxidative ring cleavage, as will be
described below.
In white-rot fungi, metabolism of b-aryl ether model
compounds is known to take place in Phanerochaete chrys-
osporium predominantly via Ca-Cb oxidative cleavage, to give
vanillin as a product.84 It has been confirmed in vitro that P.
chrysosporium lignin peroxidase catalyses this oxidative cleavage
reaction.29 Other oxidative products are also observed, including
the benzylic ketone product.11 Vanillin is then oxidised to vanillic
acid by vanillate dehydrogenase, which is encoded by the ligV
gene in S. paucimobilis SYK-6,85 and by the vdh gene in Pseu-
domonas sp. HR199.86 The by-product of oxidative C–C cleavage
is 2-hydroxyacetaldehyde. It seems likely that this metabolite is
oxidised to oxalic acid, which is a known metabolite in fungal
lignin degradation that is used to complex Mn2+ for lignin attack
by manganese peroxidase.20 Oxalic acid has also been detected as
a metabolite in bacterial breakdown of lignocellulose.60
4.2 Biphenyl degradation pathways
Bacterial degradation of biphenyls has been extensively studied,
since polychlorinated biphenyls are an important class of envi-
ronmental pollutants that occur in electrical fluids such as
Fig. 6 A scheme for reductive b-etherase cleavage by LigEFG.
Nat. Prod. Rep., 2011, 28, 1883–1896 | 1889
 transformer oils.87 A number of soil bacteria are able to degrade
biphenyl and lightly chlorinated biphenyls via oxidation to
2,3-dihydroxybiphenyl, followed by oxidative meta-cleavage.87
The biphenyl linkage is one of the main components of lignin,
commonly occurring between two guaiacyl units. Oxidative
cleavage of the C3 sidechain attached to the biphenyl would
generate 2,20 -dihydroxy-3,30 -dimethoxy,-5,50 -dicarboxy-
biphenyl, which can be utilised for growth by S. paucimobilis
SYK-6, via the pathway shown in Fig. 7. Demethylation of one
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
methoxy group is catalysed by a non-heme iron-dependent
demethylase enzyme LigX.88 The catecholic product of LigX is
then a substrate for oxidative meta-cleavage by the extradiol
dioxygenase LigZ,89 and the ring fission product is then cleaved
by C–C hydrolase LigY, to form 5-carboxyvanillic acid and 4-
carboxy-2-hydroxypentadienoic acid.90 Two decarboxylase
enzymes LigW and LigW2 have been identified in S. paucimobilis
SYK-6 that convert 5-carboxyvanillic acid into the central
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intermediate vanillic acid.91,92 LigW has been shown to incor-
porate deuterium from D2O into the product, and its amino acid
sequence shows 20% sequence similarity with C–C hydrolase
LigY, even though they catalyse quite different reactions.91 By
analogy with other aromatic degradation pathways, where the
product of hydrolytic C–C cleavage is often the substrate for
a hydratase enzyme,93 the by-product of LigY-catalysed
cleavage, 4-carboxy-2-hydroxypentadienoic acid, is probably
hydrated to form 4-hydroxy-4-methyl-2-oxoglutarate, for which
an aldolase enzyme has been identified in S. paucimobilis SYK-
6,94 and in Pseudomonas ochraceae NGJ1.95 Aldolase-catalysed
cleavage would then generate two molecules of pyruvic acid.
Since a number of soil bacteria are biphenyl degraders, and
since much of the biphenyl-containing compounds in soil will
come from lignin degradation, it is likely that there is some
connection between biphenyl degradation and lignin degrada-
tion. It has been observed recently that PCB-degrading Rhodo-
coccus jostii RHA1 has activity for lignin degradation.60
Furthermore, a survey of bacteria identified as lignin degraders
has found that many such organisms are aromatic degraders.61
4.3 Diarylpropane degradation pathways
The fungal breakdown of diarylpropane lignin model
compounds has been studied in detail.11 Oxidative cleavage of the
Ca–Cb bond occurs in P. chysosporium, to give aromatic alde-
hyde products (see Fig. 8).96 This reaction is catalysed by lignin
Fig. 7 The pathway for the degradation of the biphenyl component of
lignin. Gene designations are those identified in S. paucimobilis.
1890 | Nat. Prod. Rep., 2011, 28, 1883–1896
peroxidase,97 and the mechanism of this reaction has been
studied in detail.98
In bacteria, an enzyme activity which catalyses a novel frag-
mentation reaction, namely elimination of formaldehyde and
water (see Fig. 8), from a diarylpropane model compound, has
been identified and purified from Pseudomonas paucimobilis
TMY1009.99 The gene encoding this enzyme has not been iden-
tified to date, however, the metabolism of the product of this
reaction, lignostilbene, has been well studied. Lignostilbene
dioxygenase is a non-heme iron-dependent dioxygenase enzyme
that catalyses the oxidative cleavage of lignostilbene to give two
molecules of vanillin. Isozymes of lignostilbene dioxygenase have
been purified and characterised from Pseudomonas paucimobilis
TMY1009,100 and the corresponding genes identified.101 This
enzyme has sequence similarity with the wider family of carot-
enoid cleavage dioxygenases, responsible for the biosynthesis of
apocarotenoid signalling molecules in plants.102 Related genes
have been identified in Novosphingobium aromaticivorans DSM
12444 and Bradyrhizobium sp., and the gene products were found
in the former case to cleave lignostilbene, and in the latter case to
cleave carotenoid substrates.103
4.4 Degradation of phenylcoumarane and pinoresinol lignin
components
Breakdown of the phenylcoumarane component of lignin has
been studied in Phanerochaete chrysposporium and Fusarium
solani.104,105 Degradation of the alkylated phenylcoumarane by
white-rot fungus P. chrysosporium was found to proceed initially
by oxidation of the sidechain, then oxidation of the heterocyclic
ring to a furan, and finally oxidative Ca–Cb bond cleavage,104 as
shown in Fig. 9. In contrast, breakdown of the phenolic pheny-
coumarane by filamentous fungus Fusarium solani M-13-1 pro-
ceeded via direct Ca–Cb bond to give 5-acetylvanillone.105 Loss of
the b-hydroxyl group could be rationalised by the formation of
an epoxide intermediate, followed by hydrolytic cleavage of the
Ca–Cb bond, as shown in Fig. 9. In bacteria, S. paucimobilis
SYK-6 is reported to degrade phenylcoumarane lignin model
compounds,59 but the genes responsible have not yet been iden-
tified. It seems quite possible that there could be a role for
lignostilbene dioxygenase in bacterial phenylcoumarane break-
down, since the product of a-hydroxylation would ring open to
form a benzylic ketone. Reduction of this ketone would generate
Fig. 8 Fungal and bacterial pathways for the degradation of the diaryl
propane component of lignin.
This journal is ª The Royal Society of Chemistry 2011
 the diaryl propane skeleton, which could then be broken down
via the pathway shown in Fig. 8.
Breakdown of the pinoresinol component of lignin has also
been studied in Fusarium solani M-13-1.106 Oxidation of the
benzylic position was observed, as shown in Fig. 10, leading to
C–O bond cleavage, to form a monocyclic ketone intermediate.
Further aryl–alkyl oxidation yielded a carboxylic acid product,
and the corresponding lactone.106 In bacteria, S. paucimobilis
SYK-6 is reported to degrade pinoresinol lignin model
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
compounds,59 but the genes responsible have not yet been iden-
tified. The catabolic pathways for both heterocyclic lignin
components appear to involve a-hydroxylation as an initial step.
It is therefore interesting to note that there is chemical precedent
for a-hydroxylation of phenylpropane compounds using iron
porphyrin biomimetic models.107
4.5 Bacterial degradation of ferulic acid
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Another phenolic component of lignocellulose is ferulic acid,
which is attached via ester linkages to hemicellulose, and is
released by esterase enzymes that are found in fungi and
bacteria.108 Ferulic acid esterase has been purified and charac-
terised from Aspergillus niger,109 and esterase enzymes have also
been identified in Pseudomonas fluorescens110 and Streptomyces
avermitilis111 that can release ferulic acid from hemicelluloses.
Ferulic acid is degraded in bacteria by two catabolic pathways,
shown in Fig. 11. In S. paucimobilis SYK-6, a feruloyl CoA
synthetase enzyme FerA converts ferulic acid into feruloyl CoA,
which is then converted to vanillin and acetyl CoA by a feruloyl
CoA hydratase/lyase FerB.112 Related genes have been identified
in Pseudomonas fluorescens,113 Pseudomonas sp. HR199,114
Pseudomonas putida,115 and Amycolatopsis sp. HR167.116
Other bacteria contain a decarboxylase enzyme that converts
hydroxycinnamic acids into the corresponding 4-vinyl aromatic
compounds, which was first identified in Bacillus sp. BP-7.117
Similar decarboxylases have been identified in Lactobacillus118
and in Enterobacter sp. Px6-4, and an X-ray crystal structure of
the Enterobacter decarboxylase has been solved.119 The corre-
sponding 4-ethyl phenol is a phenolic aroma compound found in
wine, caused by the presence of Brettanomyces bruxellensis, and
an NADPH-dependent reductase enzyme has been identified in
this organism, which can reduce the 4-vinyl phenol to the 4-ethyl
Fig. 9 Pathways for the fungal breakdown of phenylcoumarane
components of lignin.
This journal is ª The Royal Society of Chemistry 2011
Fig. 10 The pathway for the degradation of the pinoresinol component
of lignin by Fusarium solani.
phenol.120 4-Ethylphenol is also a malodourant in meat waste,
generated from plant material by lactobacilli, and similar
transformations have been observed in a Lactobacillus sp.
isolate.121
4.6 Oxidative cleavage of protocatechuic acid
Many of the catabolic pathways described above lead to the
production of vanillin, or its oxidation product vanillic acid,
which is converted via demethylation (see Section 4.1) to proto-
catechuic acid. Protocatechuic acid can be degraded by many soil
bacteria via oxidative ring cleavage, using non-heme iron-
dependent catechol dioxygenase enzymes, which have been
reviewed previously.122,123 Two types of oxidative cleavage reac-
tions are observed: intradiol cleavage between the two phenolic
hydroxyl groups, catalysed by iron(III)-dependent dioxygenases,
and extradiol cleavage adjacent to the phenolic hydroxy groups,
catalysed by iron(II)-dependent dioxygenases.122,123
In Sphingomonas paucimobilis SYK-6, a protocatechuate
4,5-dioxygenase enzyme LigAB has been identified,124 and an
X-ray crystal structure of this enzyme has been solved.125 The
product of this extradiol cleavage is 4-carboxy-2-hydroxy-
muconate semialdehyde (CHMS), which exists mainly in a cyclic
form.59 Oxidation of CHMS is catalysed by CHMS dehydroge-
nase (LigC), to give 2-pyrone-4,6-dicarboxylate (PDC), which is
hydrolysed by LigI.59 Hydratase LigJ and aldolase LigK then
produce pyruvate and oxaloacetate.59 S. paucimobilis SYK-6 can
also degrade syringyl lignin model compounds via the trisubsti-
tuted gallic acid and 3-methyl-gallic acid, which are cleaved by
catechol dioxygenases DesB and DesZ, respectively, both iron
Fig. 11 Bacterial catabolic pathways for the degradation of ferulic acid.
Nat. Prod. Rep., 2011, 28, 1883–1896 | 1891
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
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Fig. 12 Ortho- and meta-cleavage pathways for catabolism of protocatechuic acid and gallic acid. Gene designations are those found in S. paucimobilis.

(II)-dependent dioxygenases.126 The pathways for breakdown of
guaiacyl and syringyl lignin components therefore converge after
ring cleavage, as shown in Fig. 12.
In other bacteria, intradiol cleavage of protocatechuic acid
occurs, via protocatechuate 3,4-dioxygenase, to give cis,cis-muconic
acid, which is then metabolised via the b-keto-adipate pathway, or
ortho-cleavage pathway, as shown in Fig. 12. This pathway has been
well characterised in Pseudomonas putida and Acinetobacter cal-
coaceticus, and is believed to operate in a range of other bacteria.127
There is therefore a question of how widely the ortho- vs. meta-
cleavage pathways occur in bacteria that can metabolise lignin and
lignin fragments, which will be discussed in Section 5.

Table 2 Occurrence of homologues for lignin degradation pathway enzymes across the bacterial kingdom. The table lists the number of hits in each
bacterial class, giving >30% sequence identity to the named sequence probe, using the BLAST algorithm on the NCBI server

Gram-positive bacteria Gram-negative bacteria

Lignin Protein Actino- a-proteo- b-proteo- g-proteo- d-proteo- cyano-

component sequence probe Source organism bacteria bacilli clostridia bacteria bacteria bacteria bacteria bacteroides bacteria

b-aryl ether Peroxidase R. jostii RHA1 70 28 0 20 31 168 0 27 0

DypB
Dehydrogenase Sphingomonas 46 3 1 9 24 21 3 0 0
LigD paucimobilis SYK-6
b-etherase LigE S. paucimobilis 0 0 0 67 6 24 1 0 25
b-etherase LigF S. paucimobilis 0 0 0 44 9 12 2 0 1
b-etherase LigG S. paucimobilis 0 0 0 6 2 5 1 0 0
Vanillic acid Vanillin Rhodococcus 39 27 0 56 26 58 1 0 4
dehydrogenasea erythropolis
S. paucimobilis 46 3 1 9 24 21 3 0 0
Biphenyl Demethylase S. paucimobilis 4 0 0 10 23 5 0 0 2
LigX
Dioxygenase S. paucimobilis 3 0 0 2 2 0 0 0 0
LigZ
Diaryl-propane Lignostilbene Pseudomonas 18 0 0 14 4 7 3 1 24
dioxygenase fluorescens WH6
Proto- 4,5-PCD S. paucimobilis 3 0 0 21 22 37 0 0 0
catechuate a subunit
metabolism (LigA)
3,4-PCD Rhodococcus jostii 41 0 0 90 78 45 0 0 1
a subunita RHA1
P. putida GB-1 42 0 0 93 76 69 1 0 0
2,3-PCD Paenibacillus sp. 2 4 0 0 1 0 0 0 0
(formerly Bacillus
macerans)
a
In the cases of vanillin dehydrogenase and protocatechuate 3,4-dioxygenase, both Gram-positive and Gram-negative sequence probes were used, since
there is some sequence diversity between Gram-negative and Gram-positive sequences.

1892 | Nat. Prod. Rep., 2011, 28, 1883–1896 This journal is ª The Royal Society of Chemistry 2011
 5 Occurrence of lignin degradation pathways in
Gram-positive and Gram-negative bacteria
Much of the information about bacterial pathways has come
from the extensive studies on Sphingomonas paucimobilis SYK-
6,59 however, other lignin-degrading bacteria are being discov-
ered, especially in the actinobacteria and the a- and g-proteo-
bacteria.60,61 Hence, one would like to know whether the
pathways described in Section 4 are found widely across the
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
bacterial kingdom, or not. Similarly, for the DypB peroxidase
recently discovered in Rhodococcus jostii RHA1:62 is this enzyme
found widely in the bacterial kingdom, or not? We therefore
include a bioinformatic analysis of several of the pathways
mentioned in Section 4. We have used protein sequences for
a number of the enzymes mentioned in Section 4 to probe the
current Genbank sequence database for matches above 30%
identity, across the bacterial kingdom. The results are shown in
Table 2.

Table 3 A survey of bacterial strains containing 3,4-protocatechuate dioxygenase (3,4-PCD, ortho-cleavage) vs. 4,5-protocatechuate dioxygenase
(4,5-PCD, meta-cleavage). The strains listed are those that gave >30% identity to the 3,4-PCD and 4,5-PCD protein sequences used as probes in Table 2.
In cases where all strains grouped into one category, only the bacterial genus is given; in cases where strains from one bacterial genus appeared in more
than one category, then individual strains are listed, in which case numbers in brackets indicate the number of strains from the same species in the same
category

Contain 3,4-PCD
Class Order Contain 3,4-PCD and 4,5-PCD Contain 4,5-PCD
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a-proteobacteria Rhizobiales Agrobacterium, Brucella, Bradyrhizobium sp. (2)

Bradyrhizobium japonicum,
Chelativorans, Hoflea,
Mesorhizobium,
Ochrobactrum, Rhizobium,
Sinorhizobium
Rhodobacterales Citreicella, Jannaschia,
Labrenzia, Oceanicola,
Pelagibaca, Pseudovibrio,
Rhodobacter, Roseibium,
Roseobacter, Roseovarius,
Ruegeria, Sagittula,
Sulfitobacter
Rhodospirillales Azospirillum Magnetospirillum
Sphingomonadales Sphingomonas
Caulobacterales Caulobacter
b-proteobacteria Burkholderiales Burkholderia pseudomallei Burkholderia multivorans, Burkholderia sp. (2),
(19), B. mallei (12), B. cepacia B. graminis, B. phymatum B. cepacia (1)
(1), B. cenocepacia (5),
B. xenovorans, 15 other
Burkholderia.
Ralstonia pickettii, Ralstonia solanacerum (2) Comamonas
R. eutropha, R. solanacearum
(4)
Rhodocyclales Cupriavidus Azoarcus
g-proteobacteria Pseudomonadales Pseudomonas aeruginosa, Pseudomonas putida (4) Pseudomonas putida (1),
P. fluorescens, P. stutzeri, P. straminea, P. resinovorans
P. syringae, P. savastanoi
Acinetobacter, Azotobacter,
Xanthomonadales Xanthomonas campestris (1), Xanthomonas campestris (4), Xanthomonas campestris (1)
X. albilineans X. axonopodis, X. oryzae
Enterobacteriales Klebsiella sp. (1), Serratia Klebsiella pneumoniae, Klebsiella sp. (1)
K. variicola
Vibrionales Vibrio
Actinobacteria Micrococcineae Arthrobacter arilaitensis, Arthrobacter sp.(1)
A. aurescens,
A. chlorophenolicus
Brevibacterium, Kocuria,
Terrabacter
Corynebacterineae Corynebacterium, Rhodococcus equi (1)
Mycobacterium, Rhodococcus
erythropolis, R. equi (2),
R. jostii, R. opacus
Pseudonocardineae Saccharomonospora
Actinomycetales Catenulispora,
Micromonospora,
Thermobispora
Streptomycineae Streptomyces coelicolor, Streptomyces hygroscopicus, Kitasatospora
S. lividans, S. scabiei, 6 other S. violaceusniger
Streptomyces
Frankineae Geodermatophilus

This journal is ª The Royal Society of Chemistry 2011 Nat. Prod. Rep., 2011, 28, 1883–1896 | 1893
 For b-aryl ether breakdown (section 4.1), the b-etherase genes
encoded by ligEFG in S. paucimobilis SYK-6 are not found at all
in actinobacteria, and are clustered mainly in a- and g-proteo-
bacteria. Homologues of LigE and LigF gene products are found
much more widely than LigG homologues, and all three genes
are found in only a small number of a-proteobacteria (6) and
g-proteobacteria (5), implying that this is not a universal
strategy. However, homologues of the first enzyme on the
pathway, dehydrogenase LigD, are found more commonly in
Published on 15 September 2011 on http://pubs.rsc.org | doi:10.1039/C1NP00042J
actinobacteria, suggesting that this enzyme might have
a different function in these organisms. The DypB peroxidase
identified in Rhodococcus jostii RHA162 has homologues in
a wide range of bacteria, especially in the actinobacteria and
g-proteobacteria, suggesting that lignin degraders in these
families might use peroxidase cleavage for b-aryl ether break-
down. Homologues of the vanillate dehydrogenase genes of S.
paucimobilis and Rhodococcus erythropolis occur widely across
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the actinobacteria, bacilli, and proteobacteria, implying that
utilisation of vanillin and vanillic acid is relatively common in
bacteria.
Homologues of the demethylase LigX and dioxygenase LigZ
involved in biphenyl degradation in S. paucimobilis (Section 4.2)
occur in a smaller proportion of bacteria, and both are found in
only three actinobacteria, two a-proteobacteria, and two b-
proteobacteria, though a higher proportion contain LigX
homologues. These observations suggest that this biphenyl
degradation pathway occurs relatively infrequently (though
other bacteria might perhaps use a different dioxygenase enzyme
to effect ring cleavage).
The occurrence of lignostilbene dioxygenase homologues
(Section 4.3) is mainly in the actinobacteria (18), a-proteobac-
teria (14), g-proteobacteria (7), and, surprisingly, in cyanobac-
teria (24). The occurrence of this enzyme appears to correlate
quite well with lignin-degrading bacterial strains,61 so some role
in lignin breakdown seems plausible. There are reports of cya-
nobacteria that are able to break down lignin,128–130 and the
recent report that lignin is present in seaweed131 suggests that
cyanobacteria might utilise lignin in seaweed as an additional
carbon source.
Finally, for the downstream metabolism of lignin degradation
products (Section 4.6), there is a clear choice between the ortho-
and meta-cleavage pathways for oxidative cleavage of proto-
catechuic acid. The data in Table 2 indicates that homologues of
protocatechuate 3,4-dioxygenase (ortho-cleavage pathway) are
found widely across the actinobacteria and proteobacteria,
whereas homologues of protocatechuate 4,5-dioxygenase (meta-
cleavage pathway) are found less widely, and almost exclusively
in the proteobacteria. Homologues of protocatechuate
2,3-dioxygenase are found only in bacilli (4), and selected acti-
nobacteria (2) and b-proteobacteria (1). A more detailed survey
of bacterial strains containing 3,4-PCD and 4,5-PCD genes is
shown in Table 3. In the a-proteobacteria, many nitrogen-fixing
rhizobiales that are symbiotic with plant roots contain 3,4-PCD,
and many phototrophic rhodobacterales also contain 3,4-PCD.
The well-studied Sphingomonas paucimobilis contains only
4,5-PCD, and is one of relatively few a-proteobacteria to do so.
The b- and g-proteobacteria show mixed behaviour, with strains
of Burkholderia, Ralstonia, Pseudomonas, Xanthomonas and
Klebsiella containing either 3,4-PCD, 4,5-PCD, or in some cases
1894 | Nat. Prod. Rep., 2011, 28, 1883–1896
both enzymes. The Gram-positive actinobacteria, in which
a number of lignin-degrading bacteria are found,57 contain
predominantly 3,4-PCD, and therefore will use the ortho-
cleavage pathway for aromatic degradation, with a small number
of exceptions.
The high proportion of lignin-degrading genes in the proteo-
bacteria and actinobacteria provide a rationalisation for why
lignin-degrading strains identified thus far cluster into these
classes,57 with the proviso that there are likely to be more lignin-
degrading enzymes and pathways as yet unidentified.
6 Conclusions
The microbial breakdown of lignin is a topic of considerable
relevance to the commercial utilisation of plant lignocellulose for
production of biofuels and renewable chemicals. Despite exten-
sive work on the fungal degradation of lignin,11 the catabolic
pathways for degradation of lignin are still incomplete, and in
practice are hard to elucidate, given the complex mixture of
components found in lignin, and its recalcitrance to breakdown.
The detailed studies on catabolism of lignin components in
Sphingomonas paucimobilis SYK-6 59 have given considerable
insight into the downstream pathways, but a wider range of
lignin-degrading bacteria are now coming to light.60,61 The bio-
informatic analysis in Section 5 suggests that there is diversity
across the bacterial kingdom in the catabolic pathways for
degradation of lignin components. It seems likely that there are
additional enzymes and pathways yet to be identified, and that
the entire lignin catabolism system might have some complexity,
but an understanding of this system will be important in the
development of lignocellulosic biorefineries for the 21st century.
7 Acknowledgements
Research in the authors’ laboratory was supported by the
BBSRC IBTI Biorefinery Club (grant BB/H004270/1), and the
IMRC ‘‘Wealth out of Waste’’ project.
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