Available online at www.sciencedirect.
com
ScienceDirect
Biochemical transformation of lignin for deriving valued
commodities from lignocellulose
Daniel L Gall1, John Ralph1,2, Timothy J Donohue1,3 and
Daniel R Noguera1,4
The biochemical properties of lignin present major obstacles to
deriving societally beneficial entities from lignocellulosic
biomass, an abundant and renewable feedstock. Similar to
other biopolymers such as polysaccharides, polypeptides, and
ribonucleic acids, lignin polymers are derived from multiple
types of monomeric units. However, lignin’s renowned
recalcitrance is largely attributable to its racemic nature and the
variety of covalent inter-unit linkages through which its
aromatic monomers are linked. Indeed, unlike other
biopolymers whose monomers are consistently inter-linked by
a single type of covalent bond, the monomeric units in lignin are
linked via non-enzymatic, combinatorial radical coupling
reactions that give rise to a variety of inter-unit covalent bonds
in mildly branched racemic polymers. Yet, despite the chemical
complexity and stability of lignin, significant strides have been
made in recent years to identify routes through which valued
commodities can be derived from it. This paper discusses
emerging biological and biochemical means through which
degradation of lignin to aromatic monomers can lead to the
derivation of commercially valuable products.
Addresses
1
U.S. Department of Energy’s Great Lakes Bioenergy Research Center,
Wisconsin Energy Institute, University of Wisconsin, Madison, WI 53726,
United States
2 useful monomeric products. This paper focuses on recent
Department of Biochemistry, University of Wisconsin, Madison,
WI 53706, United States
3
Department of Bacteriology, University of Wisconsin, Madison,
WI 53706, United States
4
Department of Civil & Environmental Engineering, University of
Wisconsin, Madison, WI 53706, United States
Corresponding author: Noguera, Daniel R (dnoguera@wisc.edu)
Current Opinion in Biotechnology 2017, 45:120–126
This review comes from a themed issue on Energy biotechnology
Edited by Scott Banta and Brian Pfleger
For a complete overview see the Issue and the Editorial
Available online 24th March 2017
http://dx.doi.org/10.1016/j.copbio.2017.02.015
0958-1669/ã 2017 The Authors. Published by Elsevier Ltd. This is an
open access article under the CC BY-NC-ND license (http://creative-
commons.org/licenses/by-nc-nd/4.0/).
Introduction
Lignocellulosic biomass is composed of Earth’s three
most abundant terrestrial biopolymers (cellulose, hemi-
celluloses, and lignin) and accounts for the majority of
Current Opinion in Biotechnology 2017, 45:120–126
organic carbon in the global budget [1]. Both abundant
and renewable, polysaccharides (cellulose and hemicel-
luloses) are attractive feedstocks for the production of
paper, ethanol, and next-generation fuels [2]. Converting
lignocellulose to these and other valued commodities is,
however, made challenging by the physiochemical con-
straints placed on polysaccharides by a third biopolymer,
lignin. Comprising between 15 and 30% (dry weight) of
vascular plant cell walls [3], lignin is a complex and
combinatorial polymer derived from a variety of aromatic
monomers linked together via heterogeneous chemical
bonds, and to which plant polysaccharides may be cova-
lently bound, particularly in grasses [4–6]. Processes
designed to simply remove lignin and convert the result-
ing cellulosic and hemicellulosic components to valued
commodities [7,8] often result in the release of aromatic
compounds that may be inhibitory to microbial conver-
sion processes [9]. Although the utility of lignin polymers
in a variety of industries [10] is attributable to lignin’s
complexity and stability, these same characteristics create
a challenge for its use as a chemical feedstock by bioma-
nufacturers striving to derive low molecular weight
products from it. However, there is emerging evidence
that bioconversion processes may help convert lignin to
advances in biotechnology that may enable the derivation
of desirable aromatic monomers and certain aliphatic
compounds from lignin. It will not address the industrial
applications of lignin polymers that have been described
elsewhere [10,11,12,13–15].
Lignin’s molecular structure
Lignin is crucial to a plant’s structural rigidity and plays
critical roles in water and nutrient transport [16], as well
as conferring resistance to mechanical stress, photode-
gradation, and various infectious agents [17,18]. The
heterogeneous and combinatorial lignin polymers are
produced via oxidative coupling of the ‘monolignols,’
primarily coniferyl alcohol, sinapyl alcohol and, in
grasses, their p-coumarate esters [6,19,20]. Radical con-
densation of the monomers, primarily with the growing
polymer [21], gives rise to lignin containing guaiacyl and
syringyl monoaromatic units that may be sidechain-acyl-
ated with p-coumarate (Figure 1a) [3,6]. The inherent
structural rigidity of lignin is attributable to the non-
selective nature with which these monomeric units
become covalently cross-linked into the polymer during
lignification.
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 Biological valorization of lignin Gall et al. 121

Figure 1

(a) (b)
5 γ OH
HO 6 p-coumarate
4 p-coumarate β
ester ester
3 7 9 O HO
1 8 OH
2 OMe γ
7 9 O 5
HO 8 (v )
RO RO β
γ γ (i ) O MeO
α O 4
γ α O OMe
α β α β MeO γ β 4 ( iv )
β HO
1 1 β α O
γ
6 2 6 2 OMe β–5
5 3 5 3 O α
( ii ) 4 5 ( iii ) phenyl-
4 OMe MeO 4 OMe
O OMe coumaran
OH OH
guaiacyl monomers: syringyl monomers: β–β OMe OH β–O–4
– coniferyl alcohol (R = H) – sinapyl alcohol (R = H) resinol ether
– coniferyl p-coumarate – sinapyl p-coumarate 4–O–5
(R = p-coumarate) (R = p-coumarate) ether

Current Opinion in Biotechnology

The structural components of lignin in monocots. (a) The biosynthetic precursors, coniferyl (blue) and sinapyl (red) alcohols, as well as their
cognate p-coumarate (green) conjugated esters, coniferyl p-coumarate and sinapyl p-coumarate, undergo radical coupling to give rise to (b)
guaiacyl (blue) and syringyl (red) units along with their pendant p-coumarates (green) in lignin polymers. A representative oligomer arising from the
coupling of sinapyl alcohol (monomers i and iv), coniferyl alcohol (ii and v), and coniferyl p-coumarate (iii) shows the most prominent of the various
types of inter-unit covalent linkages in lignin polymers. Ester linkages formed before lignification are labeled (unshaded text). All inter-unit bonds
formed during lignification, as well as the hydroxyl groups resulting from post-coupling rearomatization of quinone methide intermediates, are
shown in black. The linkages specifically formed by radical coupling reactions are bolded and labeled (shaded text) with the type of unit produced
in the polymer.

Lignin biosynthesis is well established to result from the
enzymatic radicalization of monolignols and subsequent
non-enzymatic coupling of those radicals in the plant
apoplast [19,22]. Thus, unlike other biopolymers, lignin’s
guaiacyl and syringyl monomers are not linked together
by a single type of chemical bond. Rather, as monolignol
radicals couple with the growing polymer, the bond
structure manifested between two monomer units will
be characterized by one of several inter-unit linkage types
typically found in lignin (Figure 1b): resinols (b–b), 4–O–
5 di-aryl ethers, phenylcoumarans (b–5) and, most prom-
inently, b–O–4-aryl ether linkages (termed b-ethers here-
after) [6,23]. Further, because these inter-unit linkages
are formed through non-enzymatic coupling of mono-
lignol radicals and the phenolic end-group (radical) of
the growing lignin polymer, and because monolignols
invariably couple at their b-positions, chiral centers are
created at these b-positions with each chain-extending
step, and at their a-positions during the subsequent
rearomatization [6]. These non-stereoselective coupling
reactions therefore result in heterogeneous inter-unit
linkage types with variable stereochemical configurations
and yield lignin polymers that are racemic and, conse-
quently, more biologically recalcitrant [6,24,25].
Conversion of lignin polymers to
monoaromatic products
Finding industrial applications for lignin polymers has
been a decades-long goal, as large amounts of lignin are
produced by the pulp and paper industry. New processes
for lignin purification and fractionation have been imple-
mented at an industrial scale [26,27] and applications of
purified lignin polymers range from their use as concrete
additives to the manufacturing of composite materials in
the production of plastics, resins, and textiles [10].
Although these are industrially important applications,
the direct use of lignin polymers is not the subject of this
paper.
Of emerging interest is the possibility of complete lignin
depolymerization to monomers. This is a fascinating
research frontier that could lead to the production of
valuable aromatic or aliphatic chemicals that are currently
produced from fossil fuel resources. Many guaiacyl and
syringyl [i.e., compounds bearing a p-hydroxy group ( para
to the aliphatic sidechain) and either one or two meta-
methoxy groups] monoaromatic compounds, such as van-
illin and syringaldehyde (Figure 2), are potential lignin
derivatives in demand by the biofuel, food, pharmaceuti-
cal, and cosmetic industries [28]. Furthermore, this route
for lignin valorization is even more promising when
biotechnological advances in transgenic plants are taken
into account. For example, the recently discovered ability
of plants to also utilize monolignol ferulate conjugates as
monomers for lignification holds significant promise for
incorporating ester bonds into the lignin backbone itself,
rendering lignins more amenable to chemical depolymer-
ization [29] and potentially with improved yields of

www.sciencedirect.com Current Opinion in Biotechnology 2017, 45:120–126

122 Energy biotechnology
Figure 2
O OH O H O OH
OMe OMe MeO OMe MeO
OH OH OH
vanillic acid vanillin syringic acid
Current Opinion in Biotechnology
Valued commercial monoaromatic carboxylates and aldehydes that
may be derived from lignins. Guaiacyl and syringyl compounds are
shown in blue and red, respectively.
monoaromatic compounds. Natural lignins that are essen-
tially homopolymers, such as those derived solely from
caffeyl alcohol or 5-hydroxyconiferyl alcohol, have been
unearthed [30,31]. However, most attempts to derive
aromatic monomers from lignin have, to date, targeted
the prominent b-ether inter-unit linkage in the natural
polymer’s backbone (Figure 1b). In fact, cleavage of
b-ether linkages is considered to be so crucial to lignin
degradation that extensive effort has gone into biotech-
nological studies that have successfully increased the
proportion of syringyl units, and consequently of
b-ethers, in Arabidopsis and hybrid poplars without sig-
nificant detriment to plant growth and development
[32,33].
Chemical methods
The goal of lignin depolymerization for biochemical
applications should be to achieve the highest possible
yield of aromatic monomers, but concentrated in the
smallest possible number of different products. Thus,
chemical depolymerization methods that produce com-
plex mixtures of hundreds of aromatic monomers, such as
pyrolysis, are not suitable for biochemical upgrading.
Depolymerization with methods that selectively cleave
b-ether linkages, such as Derivatization Followed by
Reductive Cleavage (DFRC) [34,35] and thioacidolysis
[36], produce simpler mixtures of aromatic monomers.
DFRC involves an initial treatment with acetyl bromide,
producing an a-bromo intermediate (Figure 3b), and
subsequent reductive cleavage with zinc dust, ultimately
yielding monoaromatic products. In the case of a syringyl-
b-guaiacyl lignin dimer model, these products are guaia-
col (Figure 3c) and the acetate of sinapyl alcohol; DFRC
is unique in being able to release the monolignols origi-
nally used in lignification (Figure 3d), and is therefore
ideal for lignin characterization, but the original analytical
method has limited application to industrial scale lignin
depolymerization; other reactions that fall under the
DFRC class, such as benzylic oxidation (chemically or
electrochemically) [37], followed by reductive cleavage
(again chemically or electrochemically), may well prove
to be scalable. Although ranging in selectivity, hydroge-
nolytic methods can also yield guaiacol (Figure 3c) and a
Current Opinion in Biotechnology 2017, 45:120–126
small variety of other monomers in high yields [12,38–
40]. There have also been advances on promising metal-
O H free, oxygen-dependent chemoselective methods for
b-ether cleavage that use relatively inexpensive organo-
catalysts for an initial benzylic oxidation to an a-ketone
OMe intermediate (Figure 3b) and subsequent formic acid-
OH
catalyzed b-ether cleavage, yielding, in the case of the
syringaldehyde
syringyl-b-guaiacyl model, guaiacol (Figure 3c) and a
syringyl a,b-diketone (Figure 3d) as monoaromatic pro-
ducts [37,41]. When oxidative depolymerization was
applied to woody lignin, about 60% of the lignin was
recovered as soluble monomers [37]. More recently,
hydrogenolysis using a method that protects the released
monomers from acid-catalyzed condensation reactions,
has been shown to produce near-theoretical yields of
monomers and, with the high-syringyl poplar mentioned
above, a striking 78% yield of a simplified array of
monomers [42].
Biological b-ether based depolymerization methods
Perhaps not surprisingly, Nature appears to have previ-
ously developed strategies for biochemical cleavage of
b-ether linkages. In the bacterial Sphingobium sp. strain
SYK-6 [43] and related organisms known as the
‘sphingomonads’ [44], the so-called ‘Lig’ enzymes serve
as the biocatalysts of the b-etherase pathway. There are
four known nicotinamide adenine dinucleotide (NAD+)-
dependent dehydrogenases that catalyze oxidation of the
benzylic hydroxyl in the syringyl-b-guaiacyl model sub-
strate (Figure 3a) to yield an a-ketone (Figure 3b) [45,46].
In subsequent reactions, a set of glutathione (GSH)-
dependent enzymes catalyze reductive b-ether cleavage
to yield guaiacol (Figure 3c) and an a-ketone (Figure 3d)
as monoaromatic products [44,47–50,51]. The require-
ment for multiple Lig enzymes for each of the b-etherase
pathway reactions illustrates how bacteria have evolved
complementary stereospecific enzymes to overcome the
racemic nature of lignin polymers. Because chiral centers
exist at both the a- and b-positions, separate a-dehydro-
genases and b-etherases are typically used to catalyze
reactions with substrates of either the R- or S-configura-
tion at each center. There are reports suggesting that
some of these enzymes may exhibit activity toward both
R- and S-configured substrates [52] with preference for
one over the other, and crystal structures of b-etherase
pathway enzymes are starting to emerge [53,54,55],
suggesting that the structural reasons for stereospecificity
will soon be elucidated.
Given the low solubility of lignin oligomers and the
presumed difficulties with transporting large polymers
into bacterial cells, biological depolymerization with
the Lig pathway in vivo may have limited industrial
application. In vitro depolymerization may be more prom-
ising if the challenges of lignin solubility and cofactor
regeneration are overcome. The former awaits the engi-
neering of solvent resistant enzymes, and the latter could
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 Biological valorization of lignin Gall et al. 123

Figure 3

(a) (b) (c) (d)

AcO

Br
O HO AcO
OMe OMe
HO monoaromatic
product,guaiacol
MeO OMe
HO (DFRC, chemical, biological)
O OH
OMe MeO OMe
intermediate α-bromide
(DFRC) OH
MeO OMe monoaromatic product
HO (DFRC)
OH HO
O
syringyl-β-guaiacyl O O O
lignin model compound OMe O

MeO OMe
MeO OMe MeO OMe
OH
OH OH
intermediate α-ketone monoaromatic product monoaromatic product
(chemical, biological) (biological) (chemical)
Current Opinion in Biotechnology

Routes for cleavage of b-ether bonds in dimeric lignin model compounds where guaiacyl and syringyl units are shown in blue and red,
respectively. In parentheses is indicated the conversion method, either the Derivatization Followed by Reductive Cleavage (DFRC), aerobic
chemoselective oxidation, formic acid-catalyzed b-ether cleavage (chemical), or the sphingomonad b-etherase pathway (biological) through which
each intermediate or monoaromatic product arises. Panel (a) shows a syringyl-b-guaiacyl lignin model compound with dashed lines indicating
where the model would be covalently bound within a lignin polymer. Panel (b) shows the a-bromide (obtained via the DFRC method) and a-ketone
(chemical and biological methods) intermediates that undergo b-ether cleavage. Panel (c) shows the compound, guaiacol, a monoaromatic
product that is common to each method. Panel (d) shows the second of two monoaromatic products that are produced by each method.

be tackled with enzymes that recycle the b-etherase
pathway coproducts NADH and glutathione disulfide
(GSSG) to their original oxidation-reduction states
(NAD+ and GSH), as recently described [56]. However,
it remains unclear whether or not the reactions that this
pathway’s enzymes carry out with model compounds can
also be achieved with bona fide lignin polymers [57,58].
Interesting applications of the b-etherase pathway con-
tinue to be explored, with a recent study expressing a
bacterial a-dehydrogenase in transgenic Arabidopsis,
which resulted in a mild increase of a-ketones
(Figure 3b) observed in its isolated lignins [59], thereby
further advancing the potential of other chemical and
biological methods that cleave a-keto-b-ether linkages
[37,41]. It appears that the sphingomonads also possess
enzymes that target some of the other types of inter-unit
linkages between lignin monomers (Figure 1b), but the
products of these reactions and the potential of these
enzymes to be utilized in biotechnological applications
have yet to be investigated [60–63].
Other biological depolymerization methods
Other biological mechanisms for lignin degradation
have been identified that require O2 as a cosubstrate.
Generally, white rot and brown rot fungi secrete lignin
peroxidases, manganese peroxidases and laccases that are
involved in initial degradation of lignin [64,65]. Because
the reactions catalyzed by these enzymes are nonselec-
tive, they yield a wide variety of partially oxidized aro-
matic and ring-opened products [64,66,67]. Fungal deg-
radation methods are therefore not attractive from the
point of view of using the depolymerized products for
further biological upgrading. However, superoxide dis-
mutases have been recently identified in a sphingomonad
bacterium that has been shown to degrade polymeric
lignin and yields a more limited set of monoaromatic
derivatives, including guaiacol (Figure 3c) or catechol (i.
e., demethylated guaiacol), than those derived by the
aforementioned fungal enzymes [68]. Developments
in the understanding of bacterial depolymerization meth-
ods that do not use peroxidases may therefore offer
interesting and complementary alternatives to consider
in the future.
Biochemical modifications of lignin-derived
aromatic monomers to valued commodities
Mixtures of monoaromatic carboxylates and aldehydes
that may be obtained from lignin via chemical or

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124 Energy biotechnology
biological depolymerization (e.g., those shown in Figure 2)
have a wide variety of uses in the biofuel, food, pharma-
ceutical, and cosmetic industries [28]. Given the complex
nature of monomers from depolymerized lignin, a con-
cept gaining acceptance is the use of aromatic-cataboliz-
ing organisms to create “biological funnels” that receive
heterogeneous aromatic substrates and convert them to a
few products or even a single valuable product. This
funneling of depolymerized lignin products has been
recently demonstrated with strains of Pseudomonas putida
producing medium-chain polyhydroxyalkanoates [69]
and adipic acid [70], and engineered strains of Rhodop-
seudomonas palustris producing benzoic and p-hydroxy-
benzoic acids [71]. Beyond these initial proof-of-concept
demonstrations, future advances in the derivation of
valued commodities from lignin will likely rely on genetic
engineering of pathways that increase the repertoire of
aromatic monomers that can be funneled to intermediate
products, and further engineering the organisms to pro-
duce higher value products. Moreover, we envision a
future in which a lignin-to-bioproduct processing chain
will take advantage of chemical depolymerization of
lignin to low molecular weight aromatics, followed by
biochemical funneling to a single bioproduct.
Summary
Developments in chemical and biochemical strategies for
deriving high yields of a small number of low molecular
weight products from lignin have recently begun to
approach the pace of genetic advances for modifying
lignin in plants. As research emerges to bridge the gaps
between these two fields, there is renewed promise in the
development of (a) viable plants that produce lignins with
a high proportion of semi-labile inter-unit linkages, (b)
strategies for obtaining valued monoaromatic derivatives
from isolated lignins, and (c) methodologies for upgrading
monoaromatics to aliphatic compounds and other socie-
tally beneficial commodities. Given the abundance and
renewability of lignocellulose, and the potentially vast
supplies of ‘waste’ lignin, these prospective lignin-to-
bioproduct pipelines may eventually replace existing
infrastructure and relieve strain on manufacturing indus-
tries currently relying on non-renewable, finite, natural
resources.
Acknowledgment
This work was supported by the Department of Energy Office of Science’s
Great Lakes Bioenergy Research Center, Grant DE- FC02-07ER64494.
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significantly exceed anything found in Nature, in plants that aear to be
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