Designer lignins: Harnessing the plasticity of lignification

Abstract

Lignin is a complex polyphenolic constituent of plant secondary cell walls. Inspired largely by the recalcitrance of lignin to biomass processing, plant engineering efforts have routinely sought to alter lignin quantity, composition, and structure by exploiting the inherent plasticity of lignin biosynthesis. More recently, researchers are attempting to strategically design plants for increased degradability by incorporating monomers that lead to a lower degree of polymerisation, reduced hydrophobicity, fewer bonds to other cell wall constituents, or novel chemically labile linkages in the polymer backbone. In addition, the incorporation of value-added structures could help valorise lignin. Designer lignins may satisfy the biological requirement for lignification in plants while improving the overall efficiency of biomass utilisation.

Full text

Designer
lignins:
harnessing
the
plasticity
of
lignification
Yaseen
Mottiar
1
,
Ruben
Vanholme
2,3
,
Wout
Boerjan
2,3
,
John
Ralph
4,5
and
Shawn
D
Mansfield
1,4
Lignin
is
a
complex
polyphenolic
constituent
of
plant
secondary
cell
walls.
Inspired
largely
by
the
recalcitrance
of
lignin
to
biomass
processing,
plant
engineering
efforts
have
routinely
sought
to
alter
lignin
quantity,
composition,
and
structure
by
exploiting
the
inherent
plasticity
of
lignin
biosynthesis.
More
recently,
researchers
are
attempting
to
strategically
design
plants
for
increased
degradability
by
incorporating
monomers
that
lead
to
a
lower
degree
of
polymerisation,
reduced
hydrophobicity,
fewer
bonds
to
other
cell
wall
constituents,
or
novel
chemically
labile
linkages
in
the
polymer
backbone.
In
addition,
the
incorporation
of
value-added
structures
could
help
valorise
lignin.
Designer
lignins
may
satisfy
the
biological
requirement
for
lignification
in
plants
while
improving
the
overall
efficiency
of
biomass
utilisation.
Addresses
1
Department
of
Wood
Science,
University
of
British
Columbia,
Vancouver,
BC
V6T
1Z4,
Canada
2
Department
of
Plant
Biotechnology
and
Bioinformatics,
Ghent
University,
B-9052
Gent,
Belgium
3
Department
of
Plant
Systems
Biology,
VIB,
B-9052
Gent,
Belgium
4
Department
of
Energy
Great
Lakes
Bioenergy
Research
Center,
Madison,
WI
53726-4084,
USA
5
Department
of
Biochemistry,
Wisconsin
Energy
Institute,
University
of
Wisconsin,
Madison,
WI
53706-1544,
USA
Corresponding
author:
Ralph,
John
(jralph@wisc.edu)
Current
Opinion
in
Biotechnology
2016,
37:190–200
This
review
comes
from
a
themed
issue
on
Plant
biotechnology
Edited
by
Anne
Osbourn
and
John
A
Morgan
http://dx.doi.org/10.1016/j.copbio.2015.10.009
0958-1669/#
2015
The
Authors.
Published
by
Elsevier
Ltd.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Lignin
is
a
complex
polyphenolic
constituent
of
the
secondary
cell
walls
of
vascular
plants,
accounting
for
18–35%
of
the
biomass
by
weight
[1].
It
is
a
crucial
element
of
water
conduction
and
plant
defence
systems
in
tracheophytes,
and
it
contributes
significantly
to
the
compressive
strength
of
secondary
xylem
tissues.
Conse-
quently,
lignification
of
the
plant
vascular
system
repre-
sents
an
important
evolutionary
milestone
for
land
plants.
Lignin
is
one
of
the
most
abundant
biopolymers
on
the
planet
and
is
an
immensely
important
global
carbon
sink.
However,
the
chemical
recalcitrance
of
lignin
poses
a
major
challenge
for
industrial
biomass
processing,
most
notably
in
pulp
and
paper
production
and
in
the
emerging
cellulosic
biofuels
industry
[2,3].
In
addition,
the
lignin
content
of
forage
crops
is
an
important
consideration
in
animal
nutrition
and
feed
conversion
rates
in
agriculture
[4].
Polymeric
lignin
is
constructed
primarily
from
three
4-hydroxyphenylpropanoids
known
as
monolignols
—
p-coumaryl
alcohol,
coniferyl
alcohol
and
sinapyl
alcohol
—
that
differ
only
in
the
degree
of
aromatic
ring
methoxylation
(Figure
1)
[5].
Once
incorporated
into
a
lignin
polymer,
they
produce
p-hydroxyphenyl
(H),
guaiacyl
(G)
and
syringyl
(S)
moieties.
These
canonical
monolignols
are
synthesised
in
the
cytoplasm
prior
to
export
to
the
site
of
polymerisation
in
the
cell
wall
where
laccase
and
peroxidase
enzymes
generate
monolignol
radicals
by
dehydrogenation,
either
through
direct
action
on
the
monolignols
or
via
a
redox
shuttle
[6].
The
incor-
poration
of
monolignols
into
a
growing
lignin
polymer
via
the
combinatorial
coupling
of
radicals
results
in
highly
variable
racemic
polymers
with
different
physicochemical
features
[7].
For
example,
gymnosperm
lignin
is
more
resistant
to
degradation
largely
because
it
is
composed
primarily
of
G
subunits
that
yield
abundant
b–5,
5–5,
and
b–b
carbon–carbon
bonds
in
addition
to
the
less
resilient
ether-type
b–O–4
linkages
that
feature
more
prominently
in
the
S-rich
lignins
of
angiosperms.
The
amount,
com-
position,
and
structure
of
lignins
are
highly
diverse
across
plant
taxa,
cell
types,
developmental
stages,
and
across
the
cell
wall
layers.
Lignin
is
embedded
in
the
cell
walls
of
the
plant
vasculature,
notably
in
the
xylem
fibres
and
vessel
elements
that
constitute
the
bulk
of
secondary
xylem
tissues,
but
also
in
the
sclerenchyma
fibres
and
sclereids
in
xylem
and
phloem,
and
in
the
cortex
cells
of
the
periderm.
Lignin
recalcitrance
has
received
considerable
research
attention
and
recent
advances
in
our
understanding
of
lignin
biogenesis
have
provoked
novel
approaches
in
plant
biotechnology
[8–10].
In
this
review,
we
summarise
the
progress
in
lignin
engineering,
highlight
develop-
ments
within
the
past
three
years
in
the
area
of
designer
lignins,
and
chart
a
course
forward
to
producing
less
recalcitrant
or
more
valuable
lignins
thereby
highlighting
the
potential
to
enhance
the
overall
utility
of
this
abun-
dant
natural
polymer.
Lignin
pathway
engineering
Monolignol
biosynthesis
occurs
via
the
shikimate
and
general
phenylpropanoid
pathways
prior
to
export
and
Available
online
at
www.sciencedirect.com
ScienceDirect
Current
Opinion
in
Biotechnology
2016,
37:190–200
www.sciencedirect.com

Designer
lignins
Mottiar
et
al.
191
Figure
1
O
O
MeO
OMe
O
O
OMe
OMe
OHOH
O
HO
HO
O
OMe
MeO
O
HO
OH
OMe
OH
OH
OOH
OH
O
MeO
MeO
OMe
OHOH
MeO
OMe
O
OHOH
MeO
OMe
O
OHOH
HO
MeO
MeO
OMe
O
HO
HO
MeO
OMeO
HO
MeO
OH
O
MeO
OH
O
OH
MeO
OMe
HO
O
HO
OH
OMe
MeO
HO
HO
O
OH
HO
OOMe
HO
O
HO
MeO
OMe
OH
OH
O
OH
OMe
OMe
MeO
OMe
HO
OMe
OH
HO
OH
HO
MeO
OH
HO
MeO
MeO
O
MeO
MeO
O
O
MeO
HO
MeO
O
OH
MeO
OH
OH
O
MeO
OH
O
MeO
OH
O
MeO
OH
O
MeO
OH
O
MeO
OH
O
MeO
O
MeO
O
MeO
O
OH
MeO
H
O:
H
vessel element
xylem fibres
Glucosyl-
transferase
Membrane
transport
syringyl unit (S)
guaiacyl unit (G)
p-coumaryl alcohol p-hydroxyphenyl unit (H)
coniferyl alcohol
sinapyl alcohol
Radical coupling
Nucleophilic H2O addition
Site of biosynthesis
in the plastid and cytosol
Site of polymerisation
in the cell wall
Monolignol
export
Passive
diffusion
Active
transport
Vesicle-
mediated
exocytosis
Laccase
Peroxidase
S3
S2
S1
P
(a)
(d)
(b)
(c)
Radical stabilization
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Current
Opinion
in
Biotechnology
2016,
37:190–200

deposition
into
the
plant
cell
wall
(see
Figure
2
for
a
detailed
overview
of
the
biosynthetic
pathways
including
enzyme
abbreviations)
[5].
Seven
enzyme-catalysed
steps
of
the
plastid-localised
shikimate
pathway
convert
pho-
tosynthate-derived
phosphoenolpyruvate
and
erythrose
4-phospate
into
chorismate,
and
further
transformations
yield
the
aromatic
amino
acids
phenylalanine,
tyrosine
and
tryptophan.
Phenylalanine
is
then
shuttled
to
the
cytosol
where
the
first
dedicated
step
of
the
general
phenylpropanoid
pathway
is
its
deamination
to
produce
cinnamic
acid.
Thereafter,
an
intricate
succession
of
aromatic
ring
hydroxylations
and
catechol
unit
O-methy-
lations
interspersed
with
the
activation
of
cinnamate
as
a
CoA
thioester
and
subsequent
reduction
via
the
aldehyde
eventually
yield
the
monolignols.
There
are
numerous
inter-species
variations
in
the
architecture
of
the
lignin
biosynthetic
pathway,
particularly
in
the
preferred
route
through
the
metabolic
grid.
For
example,
grasses
are
uniquely
capable
of
using
tyrosine
in
addition
to
phenyl-
alanine,
and
gymnosperms
are
generally
unable
to
pro-
duce
sinapyl
alcohol
because
the
requisite
hydroxylase
is
absent
[11].
Lignin
engineering
efforts
were
initially
focused
largely
on
the
enzymes
directly
involved
in
the
general
phenyl-
propanoid
and
monolignol
biosynthetic
pathways.
Mutants
or
transgenics
with
targeted
downregulation
of
key
biosynthetic
genes
in
diverse
plant
species
have
shown
varying
levels
of
reduced
lignin
production
[8].
However,
monolignol
biosynthesis
is
highly
plastic,
allowing
plants
to
substitute
monolignols
when
one
or
more
of
the
genes
is
disrupted
or
misregulated
such
that
lignin
composition
and
quantity
are
often
concomitantly
altered.
For
example,
suppression
of
C30H
in
hybrid
poplar
resulted
in
a
60%
reduction
in
lignin
as
well
as
a
shift
toward
H
units
[12],
whereas
knockdown
of
PAL
in
Brachypodium
led
to
43%
less
lignin
and
a
relative
increase
in
S
and
H
units
[13].
On
the
other
hand,
the
Arabidopsis
fah1
mutant
lacks
a
functional
F5H,
corre-
sponding
to
a
near-complete
loss
of
S
units
but
no
significant
change
in
total
lignin
content
[14].
As
lignin
structure
is
a
reflection
of
the
monomers
available
during
polymerisation
and
the
prevalence
of
different
linkage
types
is
a
major
determinant
of
chemical
resilience,
lignin
composition
represents
an
important
parameter
in
bio-
mass
recalcitrance
that
rivals
finite
lignin
content.
For
instance,
the
S
to
G
lignin
ratio
in
transgenic
hybrid
poplar
lines
has
been
positively
correlated
with
cell-wall
bioconversion
rates
[15].
Non-canonical
monolignols
are
also
amenable
to
lignifi-
cation,
further
emphasising
the
plasticity
of
lignin
bio-
genesis.
In
transgenic
poplar
for
example,
downregulation
of
CCR
has
been
shown
to
lead
to
the
low-level
incorpo-
ration
of
ferulic
acid
[16],
downregulation
of
CAD
results
in
the
incorporation
of
hydroxycinnamaldehydes
[17,18],
and
a
reduction
in
COMT
expression
leads
to
a
lignin
derived,
in
part,
from
5-hydroxyconiferyl
alcohol
[19].
Similarly,
suppression
of
CCoAOMT
in
transgenic
pine
cell
cultures
leads
to
the
incorporation
of
caffeyl
alcohol
[20].
Interestingly,
the
seed
coats
of
Vanilla
planifolia
and
several
Cactaceae
species
contain
a
lignin
derived
almost
entirely
from
this
monomer
or
the
related
5-
hydroxyconiferyl
alcohol
[21

,22].
More
evidence
of
lig-
nin
plasticity
is
offered
by
the
incorporation
of
g-acylated
monolignol
conjugates,
namely
acetylated
monolignols
in
kenaf,
sisal,
palm
and
abaca,
p-hydroxybenzoylated
monolignols
in
poplar,
willow
and
palm,
and
p-coumar-
oylated
monolignols
in
the
commelinid
monocots
[23].
In
effect,
any
compatible
phenolic
compounds
present
at
the
site
of
polymerisation
in
the
cell
wall
are
‘candidates’
for
radical
transfer,
radical–radical
coupling,
and
lignin
polymerisation
[24].
Although
covalent
bonds
have
been
shown
to
link
lignin
and
some
hemicelluloses,
most
nota-
bly
via
the
dehydrodimerisation
of
ferulate
on
arabinox-
ylans
in
grasses
[25],
the
direct
attachment
of
lignin
to
hemicellulose
remains
difficult
to
authenticate
[26].
Simi-
larly,
cell
wall
proteins
could
also
become
covalently
bound
to
lignin,
but
this
too
remains
largely
unexplored
[27].
There
has
been
remarkable
progress
in
altering
the
amount
and
composition
of
lignin
by
targeting
the
general
phenylpropanoid
and
monolignol
biosynthetic
pathways,
but
lignin-modified
plants
often
exhibit
developmental
defects
ranging
from
stem
lodging
to
dwarfism.
It
may
simply
be
that
reduced
lignin
content
alters
structural
integrity
and
impairs
water
transport,
and
such
plants
often
have
collapsed
xylem
cells
[28].
However,
perturbations
to
the
biosynthetic
pathway
may
also
result
in
the
overpro-
duction
of
other
phenylpropanoids
and
glycoside
deriva-
tives
that
provoke
diverse
pleiotropic
effects
[29].
Besides
lignin,
the
shikimate
and
phenylpropanoid
pathways
also
give
rise
to
an
array
of
other
primary
and
secondary
metabolites.
Moving
forward,
lignin
engineering
strategies
192
Plant
biotechnology
(
Figure
1
Legend
)
Overview
of
plant
cell
wall
lignification.
(a)
Lignin
is
produced
primarily
in
the
secondary-thickened
cell
wall
layers
of
xylem
tissues;
shown
here
are
several
xylem
fibres
and
part
of
a
vessel
element,
shown
in
purple.
The
blue-highlighted
cell
depicts
the
primary
(P)
and
three
secondary
cell
wall
layers
(S1,
S2
and
S3).
Biosynthesis
of
the
three
canonical
monolignols
occurs
in
the
cytoplasm,
depicted
in
the
green-
highlighted
cell.
Finally,
three
proposed
models
of
monolignol
export
to
the
cell
wall
are
shown
in
the
brown-highlighted
cell
and
polymerisation
of
monolignols
occurs
within
the
cell
wall,
highlighted
in
yellow.
(b)
Laccase
and
peroxidase
enzymes
present
in
the
cell
wall
generate
monolignol
radicals
that
are
stabilised
by
electron
delocalisation
(shown
for
coniferyl
alcohol)
prior
to
radical
coupling
reactions.
(c)
The
major
polymerisation
reaction
is
the
end-wise
coupling
of
a
monolignol
radical,
invariably
at
its
b-position,
with
the
radical
of
the
phenolic
end-unit
of
the
growing
polymer
(shown
for
the
b–O–4-coupling
of
coniferyl
alcohol
with
a
guaiacyl
radical).
(d)
An
example
of
a
typical
polymer
model,
derived
from
20
monolignols,
for
poplar
lignin.
For
an
explanation
of
colour
coding,
see
the
caption
for
Figure
3.
Current
Opinion
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Biotechnology
2016,
37:190–200
www.sciencedirect.com

Designer
lignins
Mottiar
et
al.
193
Figure
2
CSE
prephenate
chorismate
5-enolpyruvylshikimate-3-phosphate
shikimate-3-phosphate phosphoenolpyruvate
shikimate
+
3-dehydroshikimate
3-dehydroquinate 3-deoxy- -arabinoheptulosonate
7-phosphate
erythrose 4-phosphate
phosphoenolpyruvate
+
p-hydroxyphenylpyruvatephenylpyruvate
caffealdehyde
caffeoyl alcohol
sinapoyl-CoA
caffeate
sinapateferulate
C4H/C3′H
CCR CCR CCR
CAD
COMT COMT
COMT
COMT
COMT COMT
4CL 4CL 4CL 4CL
CAD
GENERAL PHENYLPROPANOID AND MONOLIGNOL BIOSYNTHESIS
ADH
PDH
DHQD-SDH DHQD-SDH DHQS
DAHPS
HPPATPATPPAT
CM
CS
EPSPS
SK
PDT
ADT
SHIKIMATE PATHWAY
EXPORT OF PHENYLALANINE AND
TYROSINE TO THE CYTOSOL
TAL
AROMATIC AMINO ACID BIOSYNTHESIS
SYMPLASTIC OR APOPLASTIC
DELIVERY OF PHOTOSYNTHATE
SUCROSE
METABOLISM
phosphoenolpyruvate
GLYCOLYSIS
IMPORT OF
PHOSPHOENOLPYRUVATE
INTO THE PLASTID
OXIDATIVE PENTOSE
PHOSPHATE PATHWAY
IMPORT OF
GLUCOSE-6-PHOSPHATE
INTO THE PLASTID
EXPORT OF MONOLIGNOLS
TO THE SITE OF POLYMERISATION
IN THE CELL WALL
TRANSIENT STARCH
METABOLISM
PAL
p-coumarate
p-coumaraldehyde
p-coumaryl alcohol
CCR
4CL
CAD
C4H
C3′H
HCT
caffeoyl-CoA
CCoAOMT
caffeoyl
shikimate
HCT
coniferaldehyde
coniferyl alcohol
CCR
CAD
feruloyl-CoA
COMT
sinapyl alcohol
CAD
sinapaldehyde
-glucose-6-phosphate
p-coumaroyl-CoA
cinnamate
ER
5-hydroxyconiferyl alcohol
5-hydroxyferulate
5-hydroxyconiferaldehyde
p-coumaroyl
shikimate
F5H
F5H
F5H
5-hydroxyferuloyl-CoA
PHYSIOLOGICAL AND
DEVELOPMENTAL CUES
MYB58 MYB63 MYB85
MED5B
MED5A
TRANSCRIPTION
OF MONOLIGNOL
BIOSYNTHETIC GENES
MYB46 MYB83
SND1 NST2
VND7
NST1
VND6
MYB4
MYB32
MYB7
KNAT7
plastid cytosol cell wall
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should
contemplate
the
effects
of
altered
metabolic
flux
on
related
pathways
and
metabolites.
Moreover,
rewiring
these
metabolic
networks
will
be
an
important
element
of
innovative
strategies
that
incorporate
alternative
mono-
mers
into
designer
lignins.
Designer
lignins
It
is
now
eminently
feasible
to
produce
genetically
engi-
neered
plants
with
severely
reduced
lignin
levels
deliv-
ering
improved
biomass
processing;
however,
such
plants
are
often
less
vigorous
and
agronomically
inferior
[28,29].
By
engineering
the
chemical
structure
of
lignin
without
drastically
altering
lignin
content
or
functionality,
it
may
be
possible
to
satisfy
the
biological
requirement
for
lignification
while
concomitantly
reducing
recalcitrance.
Novel
physicochemical
properties
could
render
lignin
more
easily
extractable
during
processing
and
could
even
create
new
avenues
in
biomass
utilisation
[30

].
At
least
five
types
of
these
designer
lignins
have
been
proposed:
(A)
lignins
with
a
lower
degree
of
polymerisation,
(B)
lignins
that
are
less
hydrophobic,
(C)
lignins
with
fewer
bonds
to
structural
carbohydrates,
(D)
lignins
containing
chemically
labile
bonds,
and
(E)
lignins
designed
to
harbour
value-added
chemical
moieties
(Figure
3).
Sev-
eral
examples
of
these
designer
lignins
will
be
described
here
to
illustrate
the
breadth
of
possibilities.
A.
Shorter
lignin
chains
Although
conventional
wisdom
maintains
that
lignin
polymers
have
a
high
molecular
weight
and
are
exten-
sively
cross-linked,
recent
evidence
suggests
that
native
lignin
comprises
relatively
short
oligomeric
chains
with
only
minimal
branching
[31].
Nonetheless,
lignins
with
reduced
degrees
of
polymerisation
may
be
more
readily
extracted.
Incorporation
of
monomers
capable
only
of
single
coupling
reactions
that
either
initiate
(‘starters’)
or
terminate
(‘stoppers’)
chain
elongation
could
reduce
average
lignin
polymer
chain
lengths.
For
example,
dihy-
droconiferyl
alcohol
found
in
the
lignin
of
gymnosperms
[17],
as
well
as
hydroxyphenylglycerols
(Figure
3e)
and
hydroxybenzenoids
detected
in
a
variety
of
plants,
can
serve
as
lignin
initiators
[7,32

].
A
greater
prevalence
of
these
groups
during
active
polymerisation
may
lead
to
more
polymer
initiation
events
resulting
in
more
lignin
chains
that
are
shorter.
Consistent
with
this
approach,
expression
of
the
bacterial
hydroxycinnamoyl-CoA
hydra-
tase-lyase
gene
in
Arabidopsis
lignifying
cells
did
not
alter
plant
growth
or
overall
lignin
content
but
did
lead
to
side-
chain
shortening
of
monomers,
the
incorporation
of
p-
hydroxybenzaldehyde
and
p-hydroxybenzoate
groups,
reduced
lignin
chain
length,
and
improved
saccharifica-
tion
[32

].
An
analogous
tactic
is
to
exploit
the
relative
oxidation
potential
of
different
monomers
in
an
effort
to
introduce
more
end-groups.
For
example,
p-coumaryl
alcohol
favours
radical
transfer
reactions
and
frequently
occurs
as
free-phenolic
endgroups
such
that
H-rich
lig-
nins
have
a
lower
degree
of
polymerisation
[33].
The
S-
rich
lignin
from
transgenic
poplar
overexpressing
F5H
also
appears
to
have
a
remarkably
shorter
chain
length
with
an
average
degree
of
polymerisation
of
approximate-
ly
10
(Figure
3b)
[34].
B.
Less
hydrophobic
lignins
Lignin
is
largely
hydrophobic
in
nature
and
designer
lignin
polymers
containing
more
hydrophilic
constituents
could
be
more
soluble
during
delignification
processes.
Additionally,
increased
hydrophilicity
could
reduce
hy-
drophobic
interactions
in
the
cell
wall
and
improve
en-
zyme
accessibility
during
saccharification.
Various
alternative
strategies
are
conceivable
and
candidate
monomers
may
contain
additional
hydroxyl
groups
or
conjugated
hydrophilic
moieties,
such
as
carbohydrates
[35]
(e.g.,
Figure
3e–g).
The
opposite
strategy
could
be
favourable;
molecular
dynamics
simulations
of
lignin
with
increased
hydrophobicity
predicted
a
reduction
of
non-
covalent
associations
between
lignin
and
hemicelluloses
[36]
that
could
make
the
cell
wall
polysaccharides
more
accessible
during
saccharification.
194
Plant
biotechnology
(
Figure
2
Legend
)
Biosynthesis
of
monolignols
via
the
shikimate,
aromatic
amino
acid,
and
phenylpropanoid
pathways.
Photosynthate,
supplied
as
sucrose
and
stored
transiently
as
starch,
is
metabolised
in
sink
tissues
via
glycolysis
and
the
oxidative
pentose
phosphate
pathway
to
produce
phosphoenolpyruvate
and
erythrose
4-phosphate.
These
in
turn
are
converted
via
the
shikimate
pathway
into
chorismate.
The
aromatic
amino
acid
pathway
yields
phenylalanine,
tyrosine
and
tryptophan
(not
shown).
Finally,
the
monolignols,
primarily
p-coumaryl
alcohol,
coniferyl
alcohol
and
sinapyl
alcohol,
are
produced
through
the
general
phenylpropanoid
pathway
and
the
monolignol-specific
biosynthetic
pathway.
Enzymatic
reactions
and
metabolite
shuttling
are
shown
in
orange,
the
plastid
as
well
as
membrane
transporters
and
the
inner
and
outer
plastid
membranes
are
coloured
in
green,
the
ER
and
ER
membranes
are
depicted
in
blue,
and
the
plasma
membrane
and
cell
wall
are
highlighted
in
yellow.
Note
that
not
all
routes
shown
have
been
demonstrated
in
all
plants;
for
example:
TAL
has
been
found
only
in
monocots,
CSE
activity
has
only
been
demonstrated
in
Arabidopsis
so
far,
F5H
is
absent
from
most
gymnosperms,
and
the
route
from
p-coumarate
directly
to
caffeate
has
only
been
demonstrated
in
poplar.
The
cascade
of
transcriptional
regulation
is
shown
in
purple
and
includes
proteins
from
the
MYB,
KNAT
and
NAC
families
of
transcription
factors
as
well
as
two
subunits
of
the
Mediator
transcriptional
co-regulator
complex.
Enzyme
abbreviations:
3-deoxy-D-arabino-
heptulosonate-7-phosphate
synthase
(DAHPS),
3-dehydroquinate
synthase
(DHQS),
3-dehydroquinate
dehydratase–shikimate
dehydrogenase
(DHQD-SDH),
shikimate
kinase
(SK),
5-enolpyruvylshikimate-3-phosphate
synthase
(EPSPS),
chorismate
synthase
(CS),
chorismate
mutase
(CM),
prephenate
aminotransferase
(PAT),
arogenate
dehydrogenase
(ADH),
arogenate
dehydratase
(ADT),
prephenate
dehydrogenase
(PDH),
4-
hydroxyphenylpyruvate
aminotransferase
(HPPAT),
prephenate
dehydratase
(PDT),
phenylprephenate
aminotransferase
(PPAT),
phenylalanine
ammonia
lyase
(PAL),
tyrosine
ammonia
lyase
(TAL),
cinnamate-4-hydroxylase
(C4H),
4-coumarate
CoA
ligase
(4CL),
p-hydroxycinnamoyl-CoA:
shikimate/quinate
p-hydroxycinnamoyltransferase
(HCT),
p-coumaroyl-shikimate/quinate-3-hydroxylase
(C30H),
caffeoyl
shikimate
esterase
(CSE),
caffeoyl-CoA
O-methyltransferase
(CCoAOMT),
ferulate/coniferaldehyde-5-hydroxylase
(F5H),
caffeic
acid/5-hydroxyconiferaldehyde
O-
methyltransferase
(COMT),
cinnamoyl-CoA
reductase
(CCR),
and
cinnamyl
alcohol
dehydrogenase
(CAD).
Other
abbreviations:
inorganic
phosphate
(P
i
),
S-adenosylmethionine
(SAM),
S-adenosylhomocysteine
(SAH).
Current
Opinion
in
Biotechnology
2016,
37:190–200
www.sciencedirect.com

Designer
lignins
Mottiar
et
al.
195
Figure
3
(a)
(b)
(c) (d)
(e)
(f)
(g)
(h)
(i)
Current Opinion in Biotechnology
Lignin
models
and
designer
lignins.
(a)
A
poplar
lignin
model
containing
20
units,
S:G
=
13:7.
4–O–5-Coupling
was
thought
to
produce
branching,
but
this
is
now
being
questioned
[31];
the
unit
(near
the
bottom
of
the
structure)
is
shown
here
as
a
free-phenolic
unit;
p-hydroxybenzoates
acylating
some
g-OH
groups
are
not
shown.
(b)
An
all-S
poplar
lignin
model,
showing
only
the
two
types
of
primary
units,
b–b
and
b–O–4;
such
lignins
are
essentially
linear
and
may
have
a
low
degree
of
polymerisation
(i.e.,
are
chain-shortened)
[34].
(c)
Almost
completely
homogeneous
(in
terms
of
interunit
linkage
type)
and
linear
lignins
are
produced,
in
vivo
and
in
vitro,
from
atypical
monolignols
caffeyl
alcohol
(no
5-OMe)
and
5-
hydroxyconiferyl
alcohol
[21,22];
such
lignins
are
also
devoid
of
lignin–polysaccharide
cross-linking
as
rearomatisation
of
the
quinone
methide
intermediate
following
b–O–4-coupling
is
via
fast
internal
trapping
by
the
o-phenol.
(d)
A
model
of
a
high-zip
lignin
from
16
coniferyl
alcohol
monomers
and
4
coniferyl
ferulate
conjugates
(magenta)
showing
some
of
the
ways
that
both
the
ferulate
and
the
monolignol
moiety
may
couple
into
the
polymer
to
form
a
potentially
more
cross-linked
polymer
that
nevertheless
readily
falls
apart
during
pretreatment
[23,39

];
readily
cleavable
ester
bonds
are
shown
hashed.
Cleaving
this
oligomer
containing
4
zip-conjugates
cleaves
this
model
lignin
into
5
fragments;
in
general,
a
polymer
containing
n
zip-conjugates
will
cleave
into
(n
+
1)
fragments.
(e)
A
fragment
of
a
chain-shortened
polymer
created
by
lignification
using
monomers,
such
as
the
guaiacylglycerol
shown
here,
that
can
only
start
a
lignin
chain;
this
polymer
is
logically
also
more
hydrophilic.
(f)
A
fragment
of
a
particularly
hydrophilic
lignin
polymer
created
in
part
using
monolignol
g–O–1b-glucosides
in
vitro
[52].
(g)
A
fragment
of
lignin
www.sciencedirect.com
Current
Opinion
in
Biotechnology
2016,
37:190–200

C.
Lignins
with
less
structural
cross-linking
to
carbohydrates
In
addition
to
non-covalent
interactions,
lignin
is
con-
jectured
to
be
linked
to
hemicelluloses
through
various
types
of
covalent
bonds.
Whenever
a
monolignol
couples
via
its
b
position
to
the
growing
polymer
(or
to
another
monomer),
a
quinone
methide
intermediate
is
produced
and,
in
the
case
of
the
dominant
b-ether
units,
this
is
overwhelmingly
quenched
and
re-aromatised
via
nucleo-
philic
attack
from
water
(Figure
1c)
[7].
Alcohol
and
carboxylic
acid
groups
inherent
to
hemicelluloses
may
also
theoretically
serve
as
nucleophiles
giving
rise
to
the
benzyl
ether
or
benzyl
ester
bonds
that
contribute
to
recalcitrant
lignin–carbohydrate
complexes,
but
compel-
ling
evidence
for
this
is
still
lacking.
Potentially
useful
novel
units
containing
o-diphenol
groups,
such
as
those
arising
from
caffeyl
alcohol
or
5-hydroxyconiferyl
alcohol,
result
in
quinone
methide
intermediates
that
are
rapidly
internally
trapped,
forming
benzodioxane
structures
(Figure
3c)
before
any
possibility
of
external
nucleophilic
attack
can
occur.
A
number
of
similar
candidate
mono-
mers
that
could
reduce
polysaccharide–lignin
cross-link-
ing
have
recently
been
validated
in
biomimetic
in
vitro
studies,
including
rosmarinic
acid,
epicatechin,
ethyl
gal-
late
and
epigallocatechin
[35,37].
As
noted
above,
mono-
cots
have
a
distinctive
mechanism
for
cross-linking
cell
wall
polymers
to
strengthen
the
wall;
arabinoxylan
is
acylated
with
ferulate
moieties
that
can
radically
cross-
couple
to
adjacent
feruloylated
hemicellulose
chains
or
to
lignin
resulting
in
extensive
polysaccharide–polysaccha-
ride
and
polysaccharide–lignin
cross-linking
(Figure
3i)
[23].
Several
groups
are
currently
working
to
identify
the
acyltransferase
enzyme(s)
responsible
for
acylating
the
arabinosyl
units
on
arabinoxylans
with
ferulate
[38].
D.
Lignins
with
novel
chemically
labile
bonds
As
has
been
shown,
various
perturbations
of
the
mono-
lignol
biosynthetic
pathway
result
in
a
shift
in
monomer
composition
that
may
yield
more
chemically
labile
bonds
[5,8,30

].
For
example,
ferulic
acid
incorporates
into
the
lignin
of
CCR-deficient
plants
producing
acid-labile
acet-
als
[16],
and
overexpression
of
F5H
in
hybrid
poplar
results
in
a
lignin
containing
nearly
98%
S
units
yielding
more
alkali-labile
b-ethers
(Figure
3b)
[34].
One
of
the
most
highly
sought
after
objectives
has
been
the
intro-
duction
of
S
lignin
units
into
conifer
tree
species
that
normally
contain
the
more
recalcitrant
G-rich
lignin.
Recently,
an
important
proof-of-principle
milestone
was
achieved
with
the
simultaneous
introduction
of
COMT
and
F5H
genes
into
transgenic
pine
cell
cultures
[11].
The
‘zip-lignin’
strategy
has
also
been
heralded
as
a
major
breakthrough
in
designer
lignins
[23,39

].
Inspired
by
the
proven
incorporation
of
ferulates
integrally
into
grass
lignins,
an
exotic
feruloyl-CoA:monolignol
transfer-
ase
from
Angelica
sinensis
was
introduced
into
hybrid
poplar
and
resulted
in
a
lignin
in
which
chemically
labile
ester
bonds
had
been
integrated
into
the
polymer
back-
bone
(Figure
3d),
improving
cell
wall
digestibility
after
mild
alkaline
pretreatment
[39

].
Recently,
a
bacterial
Ca-dehydrogenase
was
shown
to
oxidise
the
a-hydroxyl
groups
in
lignin
and,
when
introduced
into
transgenic
Arabidopsis,
yielded
low
levels
of
novel
chemically
labile
a-keto-b-ether
units
in
lignin
[40

].
Finally,
as
amino
acids
are
also
capable
of
cross-linking
to
lignin,
an
alter-
native
approach
is
to
direct
tyrosine-rich
or
cysteine-rich
peptides
to
the
cell
wall
such
that
protease
enzymes
could
digest
these
cross-links
and
accelerate
lignin
digestion
[41].
E.
Value-added
lignins
Lignin
extracted
during
industrial
biomass
processing
is
frequently
used
for
its
calorific
value,
being
burnt
to
provide
process
energy.
But
recently,
aspirations
of
com-
plete
biomass
utilisation
within
the
modern
biorefinery
have
inspired
an
array
of
lignin-derived
high-value
pro-
ducts
[42].
Research
in
this
area
has
primarily
focused
on
optimising
lignin
recovery
and
developing
products
from
lignin
that
can
compete
with
existing
petroleum-derived
materials.
However,
moving
forward,
inherently
valuable
lignin
polymers
could
be
developed
to
facilitate
the
production
of
novel
high-value
products
using
industrial
lignin
waste
streams.
The
abundant
aldehyde
groups
in
the
above-mentioned
hydroxycinnamaldehyde-derived
lignins
from
CAD-deficient
plants
[17,18]
create
enor-
mous
potential
for
functionalisation
in
diverse
applica-
tions.
Lignins
derived
solely
from
caffeyl
alcohol
or
5-hydroxyconiferyl
alcohol
monomers
produce
homoge-
neous
linear
lignin
chains
of
b–O–4-derived
benzodiox-
ane
units
(Figure
3c)
[21

,22].
Such
regularity
would
likely
be
beneficial
in
applications
such
as
the
generation
of
lignin-derived
carbon
fibres,
due
to
its
homogeneous
structure
and
lower
complexity.
However,
as
intriguing
as
the
possibilities
are,
it
is
not
yet
clear
that
large,
healthy
plants
can
be
produced
with
such
lignins.
Nature
herself
196
Plant
biotechnology
(Figure
3
Legend
Continued)
containing
rosmarinic
acid
(purple)
that
has
been
incorporated
into
the
chain
via
radical
coupling
[37].
Such
lignins
would
display
various
features,
including
being
more
hydrophilic
(due
to
the
acid
group),
having
benzodioxane
units
(like
the
polymers
in
c)
that
preclude
lignin-polysaccharide
cross-linking
at
those
sites,
and
zip-lignin
signatures
allowing
the
polymer
to
be
readily
cleaved
by
mild
base
(the
cleavable
ester
linkage
is
shown
hashed).
(h)
An
example
of
a
high-value
component
in
lignin–tricin
end-units
(magenta)
occur
in
grass
lignins
[43

].
Tricin
is
a
flavonoid
and
it
is
synthesised
outside
the
monolignol
biosynthetic
pathway.
(i)
Model
of
the
extensive
polysaccharide–
polysaccharide
(via
arabinoxylan-bound
ferulate
dimerisation)
and
polysaccharide–lignin
(via
incorporation
of
the
ferulates
and
diferulates
(red,
on
arabinoxylan)
into
the
lignin
polymer)
in
all
commelinid
monocots
[23,25];
again,
readily
cleavable
ester
bonds
are
shown
hashed.
a–i.
The
bonds
formed
by
radical
coupling
reactions
are
bolded;
bonds
formed
during
post-coupling
rearomatisation
are
grey,
as
are
the
OH
groups
from
water
addition
(see
Figure
1c).
In
all
of
the
models,
units
derived
from
lignin
monomers
are
in
cyan
(G)
and
green
(S),
whereas
novel
units
are
coloured
uniquely.
Current
Opinion
in
Biotechnology
2016,
37:190–200
www.sciencedirect.com

is
revealing
pathways
by
which
valued
products
could
naturally
be
produced
in
lignins
and
is
even
showing
how
they
can
be
arranged
to
be
on
the
end
of
a
chain
where
they
are,
presumably,
easiest
to
cleave
off.
For
example,
it
was
recently
discovered
that
the
flavonoid
tricin
is
naturally
incorporated
into
monocot
lignins,
as
(starting)
endgroups
(Figure
3h)
[43

].
This
discovery,
further
illustrating
the
inherent
plasticity
of
lignification,
was
particularly
remarkable
because
tricin
is
not
produced
via
the
monolignol
biosynthetic
pathway
so
it
exemplifies
how,
through
thoughtful
metabolic
engineering,
it
may
be
possible
to
incorporate
other
unique
and
valuable
chemi-
cal
constituents
into
lignin.
Given
the
abundance
of
surplus
lignin
available
in
industry,
it
is
plausible
that
even
low-level
production
of
value-added
lignins
could
help
economise
total
biomass
utilisation
in
biorefineries.
Perspectives
on
lignin
engineering
A
number
of
potential
alternative
monomers
has
been
proposed,
many
of
which
have
overlapping
functionalities
in
designer
lignins
[30

].
For
example,
rosmarinic
acid
is
a
hydrophilic
compound
with
a
chemically
labile
ester
linkage
and
two
o-diphenol
groups
(Figure
3g)
[37].
The
resulting
lignin
polymers
would
therefore
be
less
hydro-
phobic,
possess
fewer
links
to
hemicelluloses,
and
have
readily
cleavable
ester
bonds
within
lignin
chains.
Before
embarking
on
plant
metabolic
engineering
with
novel
lignin
monomers,
in
vitro
experiments
and
biomimetic
test
systems
can
be
used
to
validate
design
strategies
[35,37].
For
example,
with
rosmarinic
acid,
these
tests
revealed
no
barriers
to
radical
formation
or
lignin
polymerisation
in
maize
cell
walls
and
pointed
to
significant
improvements
in
saccharification
[37].
A
number
of
additional
alternative
monomers
are
currently
being
evaluated
through
a
similar
pipeline
and
plant
engineering
work
with
the
most
prom-
ising
candidates
will
soon
follow
[30

].
The
‘zip-lignin’
strategy
to
introduce
backbone
esters
exemplifies
the
potential
of
monolignol
acyltransferases
in
lignin
engineering
[39

].
Presently,
genes
encoding
p-
coumaroyl-CoA:monolignol
transferase
and
feruloyl-CoA:
monolignol
transferase
have
been
discovered
[39

,44],
whereas
genes
for
acetyl-CoA
and
p-hydroxybenzoyl-
CoA
analogues
remain
elusive.
Recent
surprising
discov-
eries
in
the
chemical
variability
of
natural
lignins
leave
room
to
ponder
what
additional
monolignol
acylation
pos-
sibilities
may
exist
and
whether
corresponding
acyltrans-
ferases
might
be
found
in
nature.
Coupled
with
innovative
metabolic
pathway
engineering
to
supply
these
alternative
monomers,
transformational
changes
in
lignin
engineering
appear
to
be
within
reach.
As
the
regulation
of
monolignol
biosynthesis
is
largely
enacted
through
transcriptional
control,
considerable
at-
tention
has
been
devoted
to
demystifying
these
processes
and
harnessing
them
to
actively
switch
biosynthesis
on
or
off
at
will.
In
addition
to
the
array
of
MYB
family
transcription
factors
that
activate
monolignol
biosynthetic
genes
and
the
NAC
family
transcription
factors
that
serve
as
master
switches
for
secondary
cell
wall
biosynthesis,
subunits
of
the
transcriptional
co-regulator
Mediator
have
recently
been
identified
as
homeostatic
repressors
of
monolignol
biosynthesis
and
all
of
these
show
promise
in
lignin
engineering
[45,46

]
(Figure
2).
Although
many
designer
monomers
may
originate
from
the
phenylpropa-
noid
pathway
directly,
other
monomers
may
have
different
metabolic
origins
and
regulatory
constraints.
It
also
remains
to
be
seen
how
designer
lignins
might
ensure
adequate
cell
wall
properties
in
the
context
of
recently
proposed
cell
wall
integrity
models
[29,47].
Although
monolignol
transport
mechanisms
remain
obscure,
recent
evidence
points
to
active
transport
through
membrane-bound
transporter
pro-
teins
[48].
The
realisation
that
alternative
monomers
are
also
translocated
to
the
site
of
polymerisation
in
the
cell
wall
casts
some
doubt
on
these
concepts.
It
may
be
that
monolignol
transporters
are
exceptionally
non-specific,
or
perhaps
that
monolignols
are
not
exported
by
active
trans-
port
whatsoever.
Similarly,
several
peroxidase
and
laccase
enzymes
have
been
implicated
in
the
dehydrogenation
of
monolignols
[49];
however,
these
proteins
are
also
apparently
non-selective
as
non-canonical
monomers
are
routinely
incorporated.
Alternatively,
it
may
be
that
these
monomers
are
not
accepted
directly
and
rely
solely
on
radical
transfer
reactions
to
enter
polymerisation.
Although
there
are
no
obvious
barriers
for
export,
radical
formation,
and
incorporation
of
non-canonical
monomers
into
lignin
polymers,
these
mechanisms
represent
underexploited
avenues
in
lignin
engineering.
Designer
lignins
invariably
possess
novel
physicochemi-
cal
properties
that
could
potentially
have
adverse
effects
on
plant
physiology
and
development.
Manipulations
of
the
monolignol
biosynthetic
pathway
have
been
known
to
produce
plants
with
reduced
growth,
developmental
defects,
increased
susceptibility
to
disease
and
water
transport
difficulties
[29,50].
Although
not
all
lignin-mod-
ified
plants
are
compromised
and
such
widespread
pleio-
tropic
phenotypes
may
not
be
purely
a
consequence
of
reduced
lignin
content
or
altered
composition,
it
empha-
sises
the
biological
importance
of
lignin
in
plants.
In
an
effort
to
produce
agronomically
viable
plants,
broader
plant
metabolism
should
be
considered
and
it
may
be
prudent
to
use
tissue-specific
promoters
to
drive
trans-
gene
expression.
Spatiotemporal
control
may
restrict
de-
signer
lignins
to
xylem
tissues,
or
xylem
fibres
more
specifically
[9].
Additionally,
yield
penalties
could
per-
haps
be
overcome
by
vessel-specific
complementation
[51].
Given
the
diversity
of
lignin
structures
tolerated
by
plants
in
nature,
there
is
every
reason
to
believe
that
some
novel
designer
lignins
will
be
compatible
with
normal
plant
growth.
Moreover,
it
is
imperative
that
all
these
novel
transgenic
strategies
are
appropriately
field-tested
to
ensure
that
the
observed
trait
modifications
withstand
normal
growing
conditions.
Designer
lignins
Mottiar
et
al.
197
www.sciencedirect.com
Current
Opinion
in
Biotechnology
2016,
37:190–200

Conclusions
Lignin
engineering
has
evolved
beyond
simple
perturba-
tions
of
the
general
phenylpropanoid
and
monolignol
biosynthetic
pathways,
culminating
in
a
suite
of
designer
lignins
with
novel
physicochemical
properties.
Much
like
the
Trojan
horse
used
to
covertly
circumvent
the
wall
defences
of
the
ancient
city
of
Troy,
designer
lignins
may
satisfy
the
biological
requirement
for
lignin
in
plant
cell
walls
while
providing
improved
biomass
utilisation
effi-
ciency.
Moreover,
high-value
designer
lignins
have
been
conceived
that
could
valorise
lignin
waste
streams
in
biomass
refineries,
and
could
be
targeted
toward
upgrad-
ing
processing.
Lignification
shows
remarkable
plasticity,
but
the
development
of
agronomically
viable
biomass
feedstocks
featuring
designer
lignins
will
require
thoughtful
selection
of
non-canonical
monomers,
valida-
tion
in
biomimetic
systems,
careful
metabolic
engineer-
ing,
thorough
assessment
of
pleiotropic
effects
that
could
potentially
accrue
from
the
incorporation
of
novel
com-
ponents,
field
trials,
and
the
generation
of
sufficient
volumes
of
material
for
industrially
relevant
bioconver-
sion
assessment.
Although
possibilities
abound,
maintain-
ing
plant
health
is
paramount
and,
ultimately,
the
plants
themselves
will
dictate
which
of
these
approaches
can
be
tolerated.
At
the
dawn
of
this
new
era
in
lignin
engineer-
ing,
we
are
limited
only
by
the
biological
constraints
of
lignification
and
by
our
collective
imagination.
Acknowledgements
We
gratefully
acknowledge
the
support
of
the
Natural
Sciences
and
Engineering
Research
Council
of
Canada
through
the
Discovery
Research
Grant
Program
to
SDM
and
a
postgraduate
scholarship
to
YM.
YM,
SDM,
and
JR
were
funded
in
part
by
the
US
Department
of
Energy
Great
Lakes
Bioenergy
Research
Center
(DOE
BER
Office
of
Science
DE-FC02-
07ER64494).
RV
and
WB
acknowledge
funding
from
the
Multidisciplinary
Research
Partnership
‘Biotechnology
for
a
Sustainable
Economy’
(01MRB510W)
of
Ghent
University
and
from
the
Agency
for
Innovation
by
Science
and
Technology
(IWT)
for
the
SBO
project
‘ARBOREF’.
RV
is
indebted
to
the
Research
Foundation-Flanders
(FWO)
for
a
postdoctoral
fellowship.
WB
and
JR
were
funded
in
part
by
Stanford
University’s
Global
Climate
and
Energy
Project
(GCEP).
References
and
recommended
reading
Papers
of
particular
interest,
published
within
the
period
of
review,
have
been
highlighted
as:

of
special
interest

of
outstanding
interest
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a
lignin
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constructed
entirely
from
caffeyl
alcohol,
a
non-canonical
monolignol.
As
this
had
only
previously
been
observed
in
transgenic
pine
systems
with
reduced
levels
of
CCoAOMT
(Ref.
[20]),
this
report
provides
further
proof
that
lignification
is
highly
diverse
in
nature,
that
plants
can
tolerate
variability
in
lignin
polymers,
and
that
linear
homogeneous
lignins
are
possible
in
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RA:
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Current
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in
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37:190–200
www.sciencedirect.com

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lignin.
Phytochemistry
2013,
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28.
Voelker
SL,
Lachenbruch
B,
Meinzer
FC,
Kitin
P,
Strauss
SH:
Transgenic
poplars
with
reduced
lignin
show
impaired
xylem
conductivity,
growth
efficiency
and
survival.
Plant
Cell
Environ
2011,
34:655-668.
29.
Bonawitz
ND,
Chapple
C:
Can
genetic
engineering
of
lignin
deposition
be
accomplished
without
an
unacceptable
yield
penalty?
Curr
Opin
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30.

Vanholme
R,
Morreel
K,
Darrah
C,
Oyarce
P,
Grabber
JH,
Ralph
J,
Boerjan
W:
Metabolic
engineering
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novel
lignin
in
biomass
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New
Phytol
2012,
196:978-1000.
This
review
article
provides
an
overview
of
lignin
biosynthesis
and
examines
potential
alternative
monomers
that
could
be
used
for
future
efforts
in
designer
lignins.
The
authors
identified
over
160
alternative
‘monomers’
that
satisfy
the
minimal
criteria
for
producing
less
recalcitrant
lignins.
31.
Crestini
C,
Melone
F,
Sette
M,
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R:
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32.

Eudes
A,
George
A,
Mukerjee
P,
Kim
JS,
Pollet
B,
Benke
PI,
Yang
F,
Mitra
P,
Sun
L,
Persil-C¸
etinkol
O
¨et
al.:
Biosynthesis
and
incorporation
of
side-chain-truncated
lignin
monomers
to
reduce
lignin
polymerization
degree
and
enhance
saccharification.
Plant
Biotech
J
2012,
10:609-620.
The
authors
report
on
xylem-specific
expression
of
a
bacterial
hydroxycinnamoyl-CoA
hydratase-lyase
in
Arabidopsis.
Side-chain
truncation
reactions
led
to
the
incorporation
of
hydroxybenzaldehyde
and
hydroxybenzoate
endgroups
in
lignin
and,
consequently,
a
lower
degree
of
polymerisation
while
not
altering
total
lignin
content
or
plant
growth.
This
study
demonstrates
the
potential
of
alternative
monomers
and
designer
lignins
and
emphasises
the
importance
of
tissue-specific
expression
in
lignin
engineering.
33.
Lapierre
C,
Rolando
C:
Thioacidolyses
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pre-methylated
lignin
samples
from
pine
compression
and
poplar
woods.
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1988,
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34.
Stewart
JJ,
Akiyama
T,
Chapple
CC,
Ralph
J,
Mansfield
SD:
The
effects
on
lignin
structure
of
overexpression
of
ferulate
5-
hydroxylase
in
hybrid
poplar.
Plant
Physiol
2009,
150:621-635.
35.
Grabber
JH,
Ress
D,
Ralph
J:
Identifying
new
lignin
bioengineering
targets:
impact
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quercetin
glycoside,
and
gallate
derivatives
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the
lignification
and
fermentation
of
maize
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Food
Chem
2012,
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36.
Carmona
C,
Langan
P,
Smith
JC,
Petridis
L:
Why
genetic
modification
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lignin
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37.
Tobimatsu
Y,
Elumalai
S,
Grabber
JH,
Davidson
CL,
Pan
X,
Ralph
J:
Hydroxycinnamate
conjugates
as
potential
monolignol
replacements:
in
vitro
lignification
and
cell
wall
studies
with
rosmarinic
acid.
ChemSusChem
2012,
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38.
Molinari
HB,
Pellny
TK,
Freeman
J,
Shewry
PR,
Mitchell
RA:
Grass
cell
wall
feruloylation:
distribution
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bound
ferulate
and
candidate
gene
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Brachypodium
distachyon.
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Plant
Sci
2013,
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39.

Wilkerson
CG,
Mansfield
SD,
Lu
F,
Withers
S,
Park
J-Y,
Karlen
SD,
Gonzales-Vigil
E,
Padmakshan
D,
Unda
F,
Rencoret
J
et
al.:
Monolignol
ferulate
transferase
introduces
chemically
labile
linkages
into
the
lignin
backbone.
Science
2014,
344:90-93.
This
study
was
the
first
to
demonstrate
the
potential
of
‘zip-lignins’
in
reducing
lignin
recalcitrance.
The
authors
report
on
the
xylem-specific
expression
of
a
feruloyl-CoA:monolignol
transferase
from
Angelica
sinen-
sis
in
hybrid
poplar
resulting
in
novel
chemically
labile
ester
bonds
within
the
lignin
backbone.
Transgenic
poplars
outperformed
control
plants
in
lignin
digestibility
upon
mild
alkaline
pretreatment.
As
lignin
polymer
integrity
was
not
altered
during
normal
growth,
this
approach
exemplifies
the
potential
of
designer
lignins
to
improve
biomass
usability
while
permitting
normal
plant
development
and
physiology.
40.

Tsuji
Y,
Vanholme
R,
Tobimatsu
Y,
Ishikawa
Y,
Foster
CE,
Kamimura
N,
Hishiyama
S,
Hashimoto
S,
Shino
A,
Hara
H
et
al.:
Introduction
of
chemically
labile
substructures
into
Arabidopsis
lignin
through
the
use
of
LigD,
the
Ca-
dehydrogenase
from
Sphingobium
sp.
strain
SYK-6.
Plant
Biotechnol
J
2015,
13:821-832.
The
authors
report
on
the
introduction
into
Arabidopsis
of
a
newly-
discovered
bacterial
dehydrogenase
that
oxidises
a-hydroxyl
groups
of
lignin.
Analysis
of
the
transgenic
plants
revealed
a
modest
increase
in
a-keto-b–O–4-units
in
lignin,
despite
challenges
in
apoplast
targeting.
As
such
linkages
would
be
more
chemically
labile
than
typical
a-hydroxy-
b–O–4-units,
this
approach
may
still
hold
promise
in
designer
lignins.
41.
Liang
HY,
Frost
CJ,
Wei
XP,
Brown
NR,
Carlson
JE,
Tien
M:
Improved
sugar
release
from
lignocellulosic
material
by
introducing
a
tyrosine-rich
cell
wall
peptide
gene
in
poplar.
Clean-Soil
Air
Water
2008,
36:662-668.
42.
Ragauskas
AJ,
Beckham
GT,
Biddy
MJ,
Chandra
R,
Chen
F,
Davis
MF,
Davison
BH,
Dixon
RA,
Gilna
P,
Keller
M
et
al.:
Lignin
valorization:
improving
lignin
processing
in
the
biorefinery.
Science
2014,
344:1246843.
43.

Lan
W,
Lu
F,
Regner
M,
Zhu
Y,
Rencoret
J,
Ralph
SA,
Zakai
UI,
Morreel
K,
Boerjan
W,
Ralph
J:
Tricin,
a
flavonoid
monomer
in
monocot
lignification.
Plant
Physiol
2015,
167:1284-1295.
This
reference
demonstrates
the
first
natural
incorporation
of
a
valuable
phenolic
from
outside
the
monolignol
biosynthetic
pathway
into
the
lignin
polymer.
44.
Petrik
DL,
Karlen
SD,
Cass
CL,
Padmakshan
D,
Lu
F,
Liu
S,
Le
Bris
P,
Antelme
S,
Santoro
N,
Wilkerson
CG
et
al.:
p-Coumaroyl-
CoA:Monolignol
Transferase
(PMT)
acts
specifically
in
the
lignin
biosynthetic
pathway
in
Brachypodium
distachyon.
Plant
J
2014,
77:713-726.
45.
Schuetz
M,
Smith
R,
Ellis
B:
Xylem
tissue
specification,
patterning,
and
differentiation
mechanisms.
J
Exp
Bot
2013,
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46.

Bonawitz
ND,
Kim
JI,
Tobimatsu
Y,
Ciesielski
PN,
Anderson
NA,
Ximenes
E,
Maeda
J,
Ralph
J,
Donohoe
BS,
Ladisch
M
et
al.:
Disruption
of
Mediator
rescues
the
stunted
growth
of
a
lignin-
deficient
Arabidopsis
mutant.
Nature
2014,
509:376-380.
This
study
describes
the
role
of
subunits
of
the
Mediator
transcriptional
coregulatory
complex
as
homeostatic
repressors
of
lignin
biosynthesis.
In
parallel
to
the
large
body
of
work
on
transcription
factors
involved
in
transcriptional
regulation,
these
findings
emphasise
the
complexity
of
regulation
in
lignin
biosynthesis.
And
as
perturbation
of
Mediator
genes
in
a
c30h
mutant
led
to
a
recovery
from
the
dwarfism
phenotype
but
no
change
in
the
near-100%
H-lignin
composition,
this
study
supports
a
view
that
plants
can
tolerate
abnormal
lignin
composition
provided
the
phy-
siological
demand
for
lignin
is
satisfied.
47.
Yang
F,
Mitra
P,
Zhang
L,
Prak
L,
Verhertbruggen
Y,
Kim
JS,
Sun
L,
Zheng
K,
Tang
K,
Auer
M
et
al.:
Engineering
secondary
cell
wall
deposition
in
plants.
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Biotechnol
J
2013,
11:325-335.
48.
Alejandro
S,
Lee
Y,
Tohge
T,
Sudre
D,
Osorio
S,
Park
J,
Bovet
L,
Lee
Y,
Geldner
N,
Fernie
AR
et
al.:
AtABCG29
is
a
monolignol
transporter
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lignin
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Biol
2012,
22:1207-1212.
49.
Lu
S,
Li
Q,
Wie
H,
Chang
M-J,
Tunlaya-Anukit
S,
Kim
H,
Liu
J,
Song
J,
Sun
Y-H,
Yuan
L
et
al.:
Ptr-miR397a
is
a
negative
regulator
of
laccase
genes
affecting
lignin
content
in
Populus
trichocarpa.
Proc
Natl
Acad
Sci
USA
2013,
110:10848-10853.
50.
Zhao
Q,
Dixon
RA:
Altering
the
cell
wall
and
its
impact
on
plant
disease:
from
forage
to
bioenergy.
Ann
Rev
Phytopathol
2014,
52:69-91.
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51.
Petersen
PD,
Lau
J,
Ebert
B,
Yang
F,
Verhertbruggen
Y,
Kim
JS,
Varanasi
P,
Suttangkakul
A,
Auer
M,
Loque
´D
et
al.:
Engineering
of
plants
with
improved
properties
as
biofuels
feedstocks
by
vessel-specific
complementation
of
xylan
biosynthesis
mutants.
Biotechnol
Biofuels
2012,
5:84.
52.
Tobimatsu
Y,
Takano
T,
Kamitakahara
H,
Nakatsubo
F:
Studies
on
the
dehydrogenative
polymerizations
(DHPs)
of
monolignol
b-glycosides:
Part
4.
Horseradish
peroxidase-catalyzed
copolymerization
of
isoconiferin
and
isosyringin.
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2008,
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200
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