Consolidated production of coniferol and other high-value aromatic alcohols directly from lignocellulosic biomass

Article abstract of DOI:10.1039/c9gc02359c

Sustainable production of fine chemicals and biofuels from renewable biomass offers a potential alternative to the continued use of finite geological oil reserves. However, in order to compete with current petrochemical refinery processes, alternative biorefinery processes must overcome significant costs and productivity barriers. Herein, we demonstrate the biocatalytic production of the versatile chemical building block, coniferol, for the first time, directly from lignocellulosic biomass. Following the biocatalytic treatment of lignocellulose to release and convert ferulic acid with feruloyl esterase (XynZ), carboxylic acid reductase (CAR) and aldo-keto reductase (AKR), this whole cell catalytic cascade not only achieved equivalent release of ferulic acid from lignocellulose compared to alkaline hydrolysis, but also displayed efficient conversion of ferulic acid to coniferol. This system represents a consolidated biodegradation-biotransformation strategy for the production of high value fine chemicals from waste plant biomass, offering the potential to minimize environmental waste and add value to agro-industrial residues.

Full text of DOI:10.1039/c9gc02359c

Green Chemistry
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Consolidated production of coniferol and
other high-value aromatic alcohols directly
from lignocellulosic biomass†
Cite this: Green Chem., 2020, 22,
44
1
a,b
a
a
Robson Tramontina, James L. Galman, c Fabio Parmeggiani,
a
a
d
Sasha R. Derrington, Timothy D. H. Bugg, Nicholas J. Turner,
Fabio M. Squina
a
and Neil Dixon
*
Sustainable production of fine chemicals and biofuels from renewable biomass offers a potential alterna-
tive to the continued use of finite geological oil reserves. However, in order to compete with current pet-
rochemical refinery processes, alternative biorefinery processes must overcome significant costs and pro-
ductivity barriers. Herein, we demonstrate the biocatalytic production of the versatile chemical building
block, coniferol, for the first time, directly from lignocellulosic biomass. Following the biocatalytic treat-
ment of lignocellulose to release and convert ferulic acid with feruloyl esterase (XynZ), carboxylic acid
reductase (CAR) and aldo-keto reductase (AKR), this whole cell catalytic cascade not only achieved equi-
valent release of ferulic acid from lignocellulose compared to alkaline hydrolysis, but also displayed
efficient conversion of ferulic acid to coniferol. This system represents a consolidated biodegradation–
biotransformation strategy for the production of high value fine chemicals from waste plant biomass,
offering the potential to minimize environmental waste and add value to agro-industrial residues.
Received 10th July 2019,
Accepted 14th October 2019
DOI: 10.1039/c9gc02359c
rsc.li/greenchem
one of the most abundant aromatic units of lignocellulose is
ferulic acid, composing more than 2% yield (wt/wt) of some
Introduction
5
–8
Alternative chemical production processes are of increasing plant biomass. Ferulic acid is an important structural com-
global importance, driven by the need to balance sustainable ponent of the plant cell wall, responsible for cross-linking
6
and efficient resource utilization. Several strategies are being between hemicellulose and lignin, providing physical and
explored to reduce the long-term environmental impacts often mechanical protection to the cell wall. Ferulic acid, along with
incurred in the production of materials, additives and pharma- other aromatic carboxylic acids, their reduced products and
1
ceuticals. Among them, the use of lignocellulose from sugar- derivatives were identified as “Top Value-Added Chemicals
cane bagasse and wheat straw (WS) as a starting material is a from Biomass”. For instance, aromatic aldehydes and alcohols
field of intense study, since plant biomass is a renewable (monolignols), such as cinnamaldehyde, caffeyl alcohol and
source of polysaccharides and phenolic compounds that can coniferol, are essential building blocks for high value-
2
,3
9–12
generate a wide range of molecules with industrial interest.
chemicals, including pharmaceutical compounds.
However,
Lignocellulosic biomass is a suitable source of aromatic these high value monolignols derivatives are generally found in
4
compounds comprising a range 10–30% of its content, and low concentrations in plants, and their total chemical synthesis
1
1,13
involves complex or non-ecofriendly routes.
Coniferyl alcohol (coniferol), a compound with high market
value, is synthesized via the phenylpropanoid biochemical
pathway in plants. Coniferol is widely used as a metabolic
intermediate to elucidate chemical structures of and bio-syn-
thetic pathways, including compounds derived from lignins
a
Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of
Manchester, 131 Princess Street, M1 7DN Manchester, UK.
E-mail: neil.dixon@manchester.ac.uk
b
Postgraduate program in Biosciences and Technology of Bioactive Products (BTPB).
Institute of Biology, State University of Campinas – UNICAMP, 500 Albert Einstein
1
4,15
Av, Zip Code 13083-852 Campinas, SP, Brazil
and lignans.
In addition to direct applications, coniferol is
c
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
also used to synthesize various valuable chemicals, such as
pinoresinol (hypoglycemic agent), sesamin (several bioactive
properties such as anticholesterolemic and antihypertensive
d
Program of Technological and Environmental Processes, Universidade de Sorocaba,
Raposo Tavares Road, km 92.5 – Vila Artura, Zip code 18023-000 Sorocaba, SP,
Brazil
1
6
properties ) and silibinin (used as a hepatoprotective and
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9gc02359c
recently shown to have therapeutic effect for both cancer and
144 | Green Chem., 2020, 22, 144–152
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Fig. 1 Activity and use of compounds derived from plant biomass via the building block intermediate coniferol.
1
7,18
21
arthritis treatment
) (Fig. 1). Moreover, coniferol has also ATP hydrolysis. Further chemical reduction of aromatic alde-
been reported as a precursor of the floral scents, dihydroconi- hydes to alcohols often requires fine control of reaction con-
feryl alcohol, coniferyl acetate and iso-eugenol from Petunia ditions, especially when the compound also contains alkene
1
9
axillaris, and therefore is a potential starting material for the functional groups, such as ferulic acid, due to the possible
cosmetic fragrance industry. non-selective reduction of both the carbonyl and alkenyl func-
The biotechnological production of coniferol, from in- tionalities. Thus, enzymatic reduction with alcohol dehydro-
expensive precursors such as agro-industrial residues presents genases (ADH) and aldo-keto reductases (AKR) are preferable
a promising alternative to supply this compound. The phenyl- since they act on aldehydes in more mild conditions and with
2
4–26
propanoid-dependent synthesis pathway of coniferol involves greater chemoselectivity.
For example, the recently reported
eight enzymes, including two cytochrome P450 enzymes, termite Coptotermes gestroi aldo-keto reductase (CgAKR-1), was
which are difficult to actively express in prokaryotic microor- shown to be active on several fermentation inhibitor aldehydes
2
0
−1
ganisms. According to previous studies, 124.9 mg L coni- commonly found in lignocellulose and therefore presents a
ferol can be obtained by reconstructing the de novo pathway great potential for the synthesis of monolignols and related
1
0
24
from tyrosine in microorganisms using rich growth media.
alcohols. Previously an enzymatic cascade has been reported
Chemical methods for the reduction of carboxylic acids for the de novo production of cinnamyl alcohol from phenyl-
require hazardous reagents such as sodium borohydride, alanine via cinnamic acid using a CAR from Mycobacterium
2
1
expensive metals or complex catalyst formulations.
marinum (mCAR) and an ADH from Saccharomyces cerevisiae
2
5
Accordingly a green alternative for the reduction of aromatic (ScADH). However, the reduction of ferulic acid to coniferol
acids is the use of enzymes. In particular a class of enzymes has not been explored using this proposed catalytic pathway,
called carboxylic acid reductases (CARs) are noted as impor- and indeed to our knowledge there are no reports of such a
tant catalysts in the toolbox for sustainable chemistry for the cascade using heterologous AKR enzymes.
selective reduction of acids to aldehydes. The substrate scope
2
3,27–30
2
2
The generation of these chemicals from abundant biomass
of CARs is exceptionally broad, which offers potential for their sources has many advantages including use of mild reaction
application in diverse synthetic processes, i.e. for the prepa- conditions, compatibility with aqueous media, sourcing of cat-
ration of aldehydes as end products for the flavor and alysts from renewable feedstocks and chemo-, regio- and
fragrance sector and the integration of CARs in cascade reac- enantioselectivity. In this study we explored the use of both an
2
3
tions. CARs have a clear advantage over other enzymes that in vitro enzymatic and whole-cell biocatalytic route to release
are able to perform carboxylic acid reduction, since this reac- ferulic acid from biomass sources and convert it directly into
tion is thermodynamically favoured because it is coupled to coniferol (Fig. 2).
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Fig. 2 Proposed biocatalytic route from lignocellulosic biomass to coniferol (3) via ferulic acid (1) and coniferyl aldehyde (2). Biocatalytic steps per-
formed with (i) chimeric xylanase (XynZ), that contains a glycoside-hydrolase domain (GH10) and a feruloyl esterase domain (CE1), (ii) carboxylic acid
reductase (CAR), and (iii) aldo-keto reductase (CgAKR-1). Both reductions require NADPH, and CAR requires ATP for enzyme activity.
Next, the substrate specificity of CgAKR-1 was assessed by
monitoring NADPH co-factor depletion, revealing CgAKR-1 to
be active against a wide-range of aromatic aldehyde substrates
Results and discussion
Enzymatic reduction of biomass derived aromatic acids
In order to test the feasibility of the enzyme cascade proposed including 2 (Fig. S1†). Next, we developed a two-step enzyme
in Fig. 2, the enzymes were recombinantly produced in E. coli, cascade using different CARs and CgAKR-1 (Table 1). From
purified, and tested for activity in vitro (Methods). Initially, the this, the most efficient double reduction was observed with
carboxylic acid reductase (CAR) step was assessed by monitor- NiCAR-CgAKR-1 and SrCAR-CgAKR-1, producing coniferol 3
ing reduction of ferulic acid 1 to coniferyl aldehyde 2 using from 1 with excellent conversions of 100% and 94% respect-
five different CARs, monitored by LC-MS. Although these CARs ively. A detailed time course experiment was performed to
had their activities evaluated against diverse acids previously, examine the relative activities of NiCAR and SrCAR in the
none of these studies provided a detailed comparison of absence and presence of CgAKR-1 for the generation of 2 and
2
3,27–29
diverse CARs using ferulic acid as a substrate.
The rela- 3 (Fig. 3). NiCAR presented faster conversion of 1 to 2 with
tive activity of the enzyme candidates for the single reduction 88% conversion in 5 h, whereas with SrCAR only 20% conver-
2
7
of 1 to 2 were, Nocardia Iowensis (NiCAR), Segniliparus sion was observed after
5
hours. In comparison, the
NiCAR-CgAKR-1 cascade pushed forward the initial reduction
Mycobacterium marinum (mCAR) and Tsukamurella paurome- reaction relative to NiCAR alone with >62% depletion (vs. 7%
2
5,28
25
rugosus (SrCAR),
Segniliparus rotuduns (SroCAR),
2
5
2
1
tabola (TpCAR) with 100%, 100%, 91%, 80% and 42% for NiCAR-alone) of 1 within 30 min. Enhanced depletion of 1
respectively (Table 1). Both, NiCAR and SroCAR have been pre- was also observed for the SrCAR-CgAKR-1 cascade relative to
viously demonstrated to reduce 1, and NiCAR was reported to SrCAR alone with 50% depletion of 1 within 5 h. Both cascades
2
5,28
have greater activity against this substrate.
Previously produced 3 in excellent yields 98 and 81% with greater pro-
SrCAR, mCAR activity against cinnamic acid and benzoic acid ductivity observed for the NiCAR-CgAKR-1 cascade. Minimal
respectively has been reported, however neither enzyme has intermediate aldehyde 2 was observed for the SrCAR-CgAKR-1
2
2,25
previously been assessed against ferulic acid.
cascade (<10%), compared to the NiCAR + CgAKR-1 cascade (up
0%), this relative productivity was consistent with relative pro-
4
duction levels of 2 for the CAR-alone single reduction results.
Table 1 The effect of CAR and CAR-CgAKR-1 enzymes on the conver-
sion of 1 to 2 and 3. Reaction performed in 100 mM potassium phos-
Whole cell biotransformation of biomass derived aromatics
phate buffer (pH 7.5), 1 mM 1, 10 mM MgCl
1
3
2
, 250 µg CAR enzymes, The application of whole cell biocatalysis for reductive bio-
00 µg CgAKR-1, 10 mM ATP, 4 mM NADPH, final volume 0.5 ml at
0 °C, 250 rpm, 18 h. The compounds were determined via LC-MS and
the concentration calculated according to peak area on a LC device. For
transformations can be advantageous over in vitro multi-
enzyme cascades because the addition of the expensive cofac-
tors ATP and NADPH are unnecessary, rendering the whole cell
biocatalytic process more practical at large scales, as well as,
reducing costs and negative environmental implications
raw values see Table S6†
2
3,31
associated with enzyme isolation.
Thus, in order to
convert 1 to 3 the genes encoding the appropriate biocatalysts
were transferred to a whole cell system. E. coli strains were
transformed with DNA vectors containing the recombinant
genes encoding the enzymes required to convert 1 into 3
CAR + CgAKR-1
CAR
Enzyme
2 (%)
2 (%)
3 (%)
(
Methods). Following induction of gene expression and sub-
MCAR
TpCAR
NiCAR
SroCAR
SrCAR
80
42
100
91
100
13
3
0
7
6
87
75
100
85
94
strate incubation, quantification of the resulting compounds
from the culture medium was performed using LC-MS. To
evaluate the role of recombinant and endogenous aldehyde
reductase enzymes present in the E. coli host, NiCAR and
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Fig. 3 Time course of CAR and CAR-CgAKR-1 production of 2 and 3 from substrate 1. Reaction performed in 100 mM potassium phosphate buffer
pH 7.5), 1 mM 1, 10 mM MgCl , 250 µg NiCAR (a and b) or SrCAR (c and d), CgAKR-1 100 µg (b–d), 10 mM ATP, 6 mM NADPH, final volume 0.5 ml at
0 °C, 250 rpm. Error bars represent standard deviation (SD) of two biological replicates. For raw values see Table S6.†
(
2
3
SrCAR alone or in combination with CgAKR-1 were evaluated the alcohol 3. Unexpectedly NiCAR in the RARE strain also led
using E. coli strains BL21 and RARE, the latter is a strain that to a small production of 3 (12%).
3
2
lacks the endogenous aldehyde reductase enzymes. In BL21
Next we evaluated the whole cell biocatalytic reduction of 2
cells harboring CAR-only 67% and 62% of 1 was reduced by to 3 using the CgAKR-1 in comparison with the alcohol dehy-
SrCAR and NiCAR respectively (equivalent to 582 and drogenase (KRED1) from Paraburkholderia phytofirmans, pre-
1
6
05 mg L− ), affording predominately 3 (47 and 45%), with viously reported to act on cinnamyl aldehyde in a whole cell
2
5
small amounts of 2 (19 and 5% respectively) (Fig. 4). The biocatalytic reaction (Fig. S2†). CgAKR-1 and KRED1 were
observed yields demonstrate enhanced productivity in com- assessed in RARE strains, and both presented efficient
parison to a previous study that reported just 11% conversion reduction of 2. Slightly enhanced activity was observed for
of 1 to 3 using a whole cell SrCAR-dependent biotransform- CgAKR-1, and so this enzyme was selected for subsequent
ation. In the RARE E. coli strain, 1 was reduced to 2 using whole cell cascade reactions with CARs (Fig. S2†).
either NiCAR or SrCAR and gave 68% and 89% product conver- In the presence of the CAR-CgAKR-1 cascades in BL21 cells,
2
8
sion respectively after 24 hours. As such, if the desired product the double reduction was enhanced, relative to the respective
is an aldehyde compound, SrCAR in the RARE host should be CAR-alone, to afford 3 with conversion yields of 75% and 53%
used, as this combination minimizes the double reduction to using NiCAR-CgAKR-1 and SrCAR-CgAKR-1 respectively (equi-
Fig. 4 Quantification of whole cell biotransformation producing 2 and 3 from 1 using CAR and CAR-CgAKR-1, enzymes expressed in BL21 or RARE
−1
−1
(
non-reducing) E. coli strains. The results were given in mg L , medium supplemented with 5 mM (970 mg L ) 1, and quantified after 24 h. Error
bars represent standard deviation (SD) of two biological replicates. *The p-value was <0.05 between SRCAR-CgAKR-1 BL21 and NiCAR-CgAKR-1
BL21 according to the student test. For raw values see Table S6.†
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valent to 690 and 483 mg L−
1
)
(Fig. 4). Collectively, mize side reactions and improve enantioselectivity.
31
NiCAR-CgAKR-1 exhibited higher activities for the production Accordingly, we tested the impact of different media compo-
of 3 compared to SrCAR-CgAKR-1 for both in vitro (Table 1) sition on the production of using the whole cell
3
and the whole cell biocatalytic cascades (Fig. 4) 5.1-fold (p = NiCAR-CgAKR-1 biocatalytic route (Fig. 5A). TB medium was
.8 × 10− ) greater depletion of 1 and 1.4-fold (p = 2.0 × 10 ) shown to be the best media, leading to 94% reduction of 1
6
–2
6
with a 76% conversion rate into 3 (645 ± 19 mg L− ) in com-
1
for the production of 3.
Comparison of the heterologous recombinant enzymes and parison with LB medium that converted 65% of 1 to 3. TB
endogenous aldehyde reductase activity indicates that as medium is a complex media rich in available carbon and nitro-
3
3
expected, little or no aldehyde reduction occurs in the E. coli gen sources, these properties can lead to better protein
RARE strain with CAR (Fig. 4). In the RARE E. coli strain, with expression and increased cell density per liter of medium,
CAR-CgAKR-1 (i.e. heterologous aldehyde reductase only) pro- which may explain the enhanced production of 3. The pro-
duction of 3 between 26–35% was observed, and in BL21 strain duction of 3 in minimal (M9) media was similar with either a
with CARs present (i.e. endogenous aldehyde reductases only) glucose or glycerol carbon source (564 ± 67 and 512 ± 35
gave 47–57% of 3, indicating that endogenous E. coli aldehyde mg L− ). In addition to the reduced cost, one other major
reductase activity is better than CgAKR-1 or that RARE cells advantage of using minimal media is the ease of product
have less NADPH/ATP available. However, NiCAR-CgAKR-1 in identification and isolation from the less complex media
BL21 cells (i.e. with both heterologous and endogenous alde- composition.
1
hyde reductases) greater formation of the alcohol product 3
75%) was observed. Indicating that both endogenous and het- cell growth phase and a substrate conversion phase, two major
erologous recombinant aldehyde reducing enzymes acted in issues affecting biotransformation efficiency can be considered
As efficient whole-cell biocatalytic processes involve both a
(
cooperation improving the conversions of 3 from 1.
as the total amount of cellular biomass and the concentration
of recombinant enzyme per cell. Thus, these factors were
evaluated by setting three variables in the system; the incu-
3
1
Whole cell biotransformation process optimization
The use of whole cell biocatalysts can often require optimiz- bation time pre-induction (2–4 hours); inducer concentrations
ation of reaction conditions to increase product yield, mini- (arabinose and rhammanose from 5 to 10 mM); and induction
Fig. 5 Production of 2 and 3 from 1 using a whole cell biotransformation using an E. coli strain BL21 expressing the NiCAR-CgAKR-1 enzyme
cascade. (a) Activity compared in different growth media and carbon source TB, LB, M9-glucose and M9 glycerol. (b) Time course quantification of
−1
whole cell biotransformation of 1 into 2 and 3 in TB media. Medium supplemented with 5 mM 1 (970 mg L ), samples were taken at 16 h in (a) and
at times indicated in (b). The results were given in mg L− . All the assays were performed in duplicate. For raw values see Table S6.†
1
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time before the addition of substrate 1 (2–4 hours) on the for- into their respective alcohols (Fig. 6). Whereas, the protocate-
−
1
−1
mation of 3 following incubation/biotransformation using chuic 4 (77%–275 mg L ), syringic 5 (68%–185 mg L ),
−1
design of experiments approach (Table S1 and Fig. S3†). caffeic 9 (9%–27 mg L ), and 3,4,5-trimethoxycinnamic 11
Considering maximal production of 3 the optimal conditions (52%–116 mg L− ) acids were only converted to their respective
were: 4 hours pre-induction incubation, 10 mM of both indu- aldehydes without any further reduction to alcohols observed
cers, and 2 hours of recombinant gene expression prior to sub- (Fig. 6). In general, the previously identified substrate scope of
strate 1 addition (ESI Table 1†). These optimized cultivation CARs is exceptionally broad ranging from aliphatic com-
parameters led to near complete depletion of 1, and 97% pro- pounds (from C2 to C18) to (hetero)aromatic compounds and
duction of 3. A full time course experiment was performed a wide range of substituents is tolerated, where the major
with 1 at 5 mM concentration, 81% and 97% conversion (as limitation is the presence of polar groups adjacent to the car-
1
3
6
assessed by LC-MS) to 3 was achieved after a 2 and 10 hours boxylate moiety. Although 4, 5, 9 and 11 were not reduced to
reaction respectively (Fig. 5B). A further scale-up reaction was their respective alcohols it is difficult to assess any clear struc-
performed in order to isolate and chemically characterize 3 ture–activity relationship regarding CgAKR-1 substrate
(Fig. S4 and S5†), here a slightly reduced 74% conversion rate tolerance or indeed the endogenous aldehyde reductases.
was reported. Overall, these results indicate that the whole cell biocatalyst
Other routes to produce 3, and related phenylpropenoic reported here is useful for the production of biomass-derived
acids, have been reported using different enzymatic routes to aromatic alcohols and related molecules, and presents both
1
0,25,30,34
those reported here.
For example, a de novo biosyn- more rapid and more complete conversion than other enzy-
2
3,25,30,34,35
thetic route to 3 from tyrosine was developed using a 4-step matic strategies.
enzyme cascade, consisting of a tyrosine ammonia lyase,
Consolidated biodegradation–biotransformation process
4
-coumaroyl-CoA ligase, cinnamoyl-CoA reductase, and a cin-
namyl alcohol dehydrogenase (TAL-4CL-CCR-CAD) affording In plants, 1 is rarely found in free form and occurs widely
25 mg L−
1 10
.
Two further studies used a similar 3-step linked to various carbohydrates as glycosidic conjugate ester.
37
1
enzyme cascade to produce 3 from 1 using a 4-coumaroyl-CoA Thus we sought to develop a consolidated biodegradation–bio-
ligase, a cinnamoyl-CoA reductase, and a cinnamyl alcohol transformation process to first release ferulic acid 1 from in-
−
1 30
dehydrogenase (4CL-CCR-CAD) yielding 327 mg L
1
and soluble lignocellulosic substrates, followed the conversion of 1
08 mg L−
1 34
of 3 from 1. Finally, a de novo biosynthetic into 3 using the whole cell biocatalytic strains developed
route to cinnamyl alcohol from phenylalanine was developed (Fig. 7). This was performed using wheat straw (WS) and wheat
using a 3-step enzyme cascade, consisting of a phenylalanine arabinoxylan (WAX). WAX is a partially purified polysaccharide
ammonia lyase, carboxylic acid reductase and alcohol dehydro- and contains approximately 1.52 mg g− of 1 and similar
1
−
1
genase (PAL-CAR-ADH) affording 300 mg L
of cinnamyl amounts of coumaric acid (12), whereas WS contains less 1
2
5
7,38
alcohol. In order to determine the relative cost and efficiency and 12.
WS or WAX biomass was incubated with E. coli
for these different enzymatic routes, a value added calculation BL21 expressing NiCAR-CgAKR-1 as described above in the
was performed taking into consideration the cost and volume presence of multi-domain β-xylanase (XynZ) from Clostridium
of the substrate and media used (including carbon-source, thermocellum for 24 hours (Fig. 7). XynZ contains the feruloyl
buffers, inducers and antibiotics), and comparing this to the esterase domain (CE1), and has been reported to release phe-
3
8
product titre and value (Table S4†). From this analysis the nolic compounds from biomass. Alkaline hydrolysis of 2%
CAR-AKR route, developed here, displayed the greatest value (w/v) WAX and WS was performed in order to compare the
added (47-fold), the 4CL-CCR-CAD routes displayed lower enzymatic activity of XynZ for the release of 1. The results
value added (33 and 23-fold), and the TAL-4CL-CCR-CAD route demonstrate that XynZ efficiently releases both ferulic acid 1
displayed the least added value (7-fold). This simple analysis and coumaric acid 12 from recalcitrant substrates after
indicates that the CAR-AKR route is a favorable route for the 24 hours incubation time (Fig. 7). Release of the aromatic
production of 3, however full process analysis would be acids from WAX was ∼3-fold higher than WS (WAX: 1 60
required in the future to accurately determine and compare mg L− and 12 54 mg L
costs at scale.
1
−1
– WS: 1 26 mg L
−1
and 12
17 mg L− ). Moreover, our results were consistent with pre-
1
The developed whole cell biocatalytic system was also vious findings that reported a feruloyl esterase from Aspergillus
further evaluated for the reduction of other aromatic acids, clavatus released phenolic compounds from WAX and sugar-
3
9
and the reaction was performed using M9 media sup- cane bagasse in similar yields.
plemented with glycerol in order to decrease GC-MS analysis The 3-step cascade (XynZ-CAR-AKR) for both biomass sub-
complexity (Fig. S6†). Although different yields were observed strates showed complete conversion of the released acid inter-
for each tested substrate, the reactions were demonstrated to mediates 1 and 12 into their respective alcohols 3 and 14.
be effective across phenylpropenoic acid derivatives (Fig. 6). Consistent with the content of the aromatic acids from the two
The chromatograms of the reactions showed conversion of different biomass substrates, the production of the alcohols
−
1
−1
vanillic 6 (64%–210 mg L ), cinnamic 7 (76%–250 mg L ), was also 3-fold higher from WAX than WS at producing 3 or
−
1
−1
−1
sinapinic 8 (18%–43 mg L ), 3,4 dimethoxycinnamic 10 coumaryl alcohol (14) (WAX: 3 71 mg L and 14 58 mg L
–
1
−1
−1
−1
(
47%–96 mg L− ) and coumaric acid 12 (49%–165 mg L ), WS: 3 26 mg L and 14 17 mg L ) (Fig. 7). No detection of 2
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Fig. 6 Production of other aromatic aldehydes and alcohols for the corresponding acid, via whole cell biotransformation using the BL21 expressing
NiCAR-CgAKR-1 cascade. Activity was assessed 16 h after incubation/induction in M9 media supplemented with glycerol as a carbon source. Results
reported in percentage of conversion of the 5 mM aromatic acid. All the assays were performed in duplicate. Ferulic acid 1, protocatechuic acid 4,
syringic acid 5, vanillic acid 6, cinnamic acid 7, sinapic acid 8, caffeic acid 9, 3,4-dimethoxycinnamic acid 10, 3,4,5-trimethoxycinnamic acid 11, cou-
maric acid 12. For raw values see Table S6.†
and coumaryl aldehyde (13) was reported, possibly because and the product 3 (€275 g− ). As earlier the value added
the lower amounts 1 available compared to a soluble feed- calculation was performed taking into consideration the
stock, which permits more rapid and complete conversion to substrate and media costs (in addition to enzymes
1
3
. Utilization of crude biomass by this method demonstrated production costs), and comparing this to the product titer
high amounts of release of 1 and 12 and conversion to 3 and and value (Table S4†). From this earlier analysis the best
achieving the maximum yield from the WS and route to 3 from 1 was the CAR-AKR route, which displayed
1
4
WAX (Fig. 7). Beyond the applications of 3, the biomass deri- 47-fold value added, whereas here the XynZ-CAR-AKR route,
vate 14 also demonstrates potential as a building block of using WS biomass directly, displayed 74-fold value added
anti-oxidative and anti-inflammatory molecules such as (Table S4†). Further scale up and techno-economic
p-coumaryl fatty acid esters and p-coumaryl alcohol γ-O- analysis would enable full evaluation of this process in a
1
0
methyl ether. The results obtained with crude biomass are biorefinery context, and the viability of this route to out
very promising in terms of value added, considering the rela- compete existing biotechnological production methods to
tive cost of substrates 1 (€5.05 g− ) or wheat straw (€0.07 kg
1
−1
)
monolignols.
150 | Green Chem., 2020, 22, 144–152
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Fig. 7 Consolidated biodegradation and biotransformation of wheat straw (WS) and wheat arabinoxylan (WAX) biomass to release 1 and 12 and
produce coniferol 3 and coumaryl alcohol 14. The samples were taken 24 h after incubation with 2% (w/v) biomass and E. coli BL21 strain expressing
NiCAR-CgAKR-1, and 1 mg g− XynZ/biomass (where indicated). The results were given in mg L . Neither aldehyde intermediate 2 nor 13 were
1
−1
detected. All the assays were performed in duplicate. For raw values see Table S6.†
sents several attractive features including use of plant
Conclusions
biomass-derived feedstocks, use of biocatalytic route (XynZ,
We have demonstrated here that a whole cell biocatalytic NiCAR, and CgAKR-1), ambient, low energy reaction conditions
cascade in E. coli can be applied to produce 3 directly from and significant production rates. Finally, this method opens
biomass via a consolidated biomass degradation–valorisation up new routes for the conversion of biomass to useful products
process. To the best of our knowledge, there are no reports in via synthetic biology approaches to create further designer
literature focusing on the production of 3 either from 1 using organisms and biocatalytic pathways, and provides a toolbox
CARs or directly from biomass following biocatalytic degra- platform for the generation of high-value aromatic molecules
dation of lignocellulose. Therefore this novel method opens up from renewable sources.
a possibility to sustainably produce 3 and other compounds
directly from biomass. The direct use of 1 and other aromatic
acids with the whole cell biocatalytic cascade showed to be Author contributions
very efficient, simple, and ready to be explored at larger scales.
RT designed and carried out all the experiments. JG and FP
Besides being present in untreated lignocellulosic biomass, 1
supported the biochemical characterizations. SRD discovered
and related compounds are also a major component of lignin-
and produced CAR enzymes. RT, FM and ND drafted the
hemicellulose fractions from pre-treated biomass. Process con-
manuscript. TDH, NT, ND conceived the study and partici-
solidation has the potential to increase production efficiency
pated in data interpretation. All the authors read and approved
and also in this biocatalytic scenario reduces/removes the
the final manuscript.
need for chemical pre-treatment of the biomass. The pro-
duction of fine chemicals via this consolidated process
approach has some potential major advantages, both in terms
of sustainability but also the potential higher value of the pro-
Conflicts of interest
ducts, due their naturally-derived sources and use of non-syn-
There are no conflicts to declare.
4
0,41
thetic chemistry processes.
Very few works in literature
have demonstrated the direct release and biotransformation of
aromatic acids from insoluble lignocellulose, previously only
the production of vanillin from biomass has been reported.
Acknowledgements
4
2
In summary, the monolignol coniferol 3 possesses several R. T. was supported a by grant from the São Paulo Research
applications, in lignin research, as a precursor to fragrance Foundation (FAPESP, proc number: 2017/23754-1). ND is sup-
compounds, and as active pharmaceutical ingredients. ported by the BBSRC David Phillips Fellowship (BB/K014773/1)
Through the use of a three-enzyme cascade, we have demon- and BBSRC-FAPESP (FAPPA) grant (BB/L026244/1). ND, NT, JG,
strated the simple production of this compound in high yield and TDHB are supported by BBSRC-FAPESP grant BB/P01738X/
and purity with up to 97% conversion yield. The method pre- 1. FMS is supported by the FAPESP grant (2015/50590-4).
This journal is © The Royal Society of Chemistry 2020
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2
2
3 M. Winkler, Curr. Opin. Chem. Biol., 2018, 43, 23–29.
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