Bio-derived production of cinnamyl alcohol: Via a three step biocatalytic cascade and metabolic engineering

Article abstract of DOI:10.1039/c7gc03325g

The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use of low solvent systems and renewably resourced catalysts in reported examples, many cascades are still dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable fashion. Herein, we report the production of the versatile chemical building block cinnamyl alcohol from the primary metabolite and the fermentation product l-phenylalanine. Through the combination of three biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase) the target compound could be obtained in high purity, demonstrable at the 100 mg scale and achieving 53% yield using ambient temperature and pressure in an aqueous solution. This system represents a synthetic strategy in which all components present at time zero are biogenic and thus minimises damage to the environment. Furthermore we extend this biocatalytic cascade by its inclusion in an l-phenylalanine overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol in mineral media using glycerol and glucose as the carbon sources. This study demonstrates the potential to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol derived, for example, from lipase treated biodiesel.

Full text of DOI:10.1039/c7gc03325g

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Bio-derived production of cinnamyl alcohol via a
three step biocatalytic cascade and metabolic
engineering†
Cite this: DOI: 10.1039/c7gc03325g
Evaldas Klumbys, Ziga Zebec,
Nigel S. Scrutton
Nicholas J. Weise, Nicholas J. Turner
and
*
The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one
of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use
of low solvent systems and renewably resourced catalysts in reported examples, many cascades are still
dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable
fashion. Herein, we report the production of the versatile chemical building block cinnamyl alcohol from
the primary metabolite and the fermentation product L-phenylalanine. Through the combination of three
biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase)
the target compound could be obtained in high purity, demonstrable at the 100 mg scale and achieving
53% yield using ambient temperature and pressure in an aqueous solution. This system represents a syn-
thetic strategy in which all components present at time zero are biogenic and thus minimises damage to
the environment. Furthermore we extend this biocatalytic cascade by its inclusion in an L-phenylalanine
overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol
in mineral media using glycerol and glucose as the carbon sources. This study demonstrates the potential
to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol
derived, for example, from lipase treated biodiesel.
Received 3rd November 2017,
Accepted 12th December 2017
DOI: 10.1039/c7gc03325g
rsc.li/greenchem
In an effort to widen the remit of green chemistry in the pro- from traditional sources, such as petroleum. As such, there is a
duction of materials, additives and pharmaceuticals, several drive to find methods employing biocatalysts for the conversion
avenues are being explored to reduce the long-term environ- of sustainably resourced substrates to products associated with
mental impact often incurred in such processes. One strategy multiple and/or industrially important syntheses.
that has gained sustained attention in recent years is biocataly-
Cinnamyl alcohol is a simple versatile chemical implicated
sis: the use of enzymes to complement and replace chemical in the production of various compounds of applied and com-
routes to various target molecules.1,2 The advantages of using mercial interest. Simple esterification of this alcohol can
enzymes as opposed to organic synthetic methods include afford a variety of cinnamyl esters, which find use in the
mild reaction conditions, compatibility with aqueous media, flavour and fragrance industries,7,8 as well as being used as
sourcing of catalysts from renewable feedstocks and chemo-, precursors for the production of smart polymer materials.9
regio- and enantioselectivity. These features often enable the Examples of these include cinnamyl acetate, which is used to
efficient combination of multiple enzymes within a single reac- confer spicy and floral aromas, and cinnamyl methacrylate – a
tion vessel under common conditions without unwanted cross- monomer which can be polymerised by both radical and
reactivity or the need for complex and wasteful purification of photochemical means to create a dual crosslinked product.
intermediate compounds.3–6 Despite this, a large number of The amination of cinnamyl alcohol has also been reported,
biocatalytic routes make use of starting materials obtained allowing the facile production of various cinnamyl
amines.10–12 Notable examples of these include the clinically
approved drugs flunarizine and naftifine (used for the treat-
Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of
ment of fungal infections and peripheral vascular conditions,
respectively). Besides one-step conversions yielding com-
pounds of interest, cinnamyl alcohol is also reported as a start-
Manchester, 131 Princess Street, M1 7DN Manchester, UK.
E-mail: nigel.scrutton@manchester.ac.uk
†Electronic supplementary information (ESI) available: Complete experimental
ing material for the multistep synthesis of the drug dapoxetine
section, molecular biology protocols, analytical methods and product character-
and a widely used cancer treatment drug Taxol (Fig. 1).13,14
isation. See DOI: 10.1039/c7gc03325g
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Fig. 2 A biocatalytic route to cinnamyl alcohol (4) from bio-derived
L-phenylalanine (1) using a combination of phenylalanine ammonia lyase
(PAL), carboxylic acid reductase (CAR) and alcohol dehydrogenase (AHD)
enzymes. The deamination reaction does not require any additional
cofactors; however both the following reductions need NADPH and ATP
for CAR enzyme activity and either NADPH or NADH for ADH.
represent the first two steps. To this end PAL from the cyano-
bacterium Anabaena variabilis was chosen due to its extensive
use in the biocatalytic literature.24–26 Previous studies report
the use of this enzyme within lyophilised whole cells. As such
a pET-16b plasmid containing the codon-optimised avpal gene
was used to transform E. coli BL21(DE3) cells and sub-
sequently produce the dry whole cell formulation as previously
reported.26 Reactions with varying quantities of AvPAL incu-
bated for 24 h at 30 °C with 10 mM L-phenylalanine showed
increasing conversions up to 90% as seen by reverse-phase
HPLC (Table 1).
Next a CAR from Mycobacterium marinum (MCAR) was pro-
duced in its active form via co-transformation of the encoding
gene from the pET-16a vector with that for a phosphopan-
tetheinyl transferase from Bacillus subtilis in pCDF-1b. This
system was deemed suitable for cascade construction, due to
its successful incorporation into a reported method for the
multi-step enzymatic production of chiral piperidines.27 Initial
studies of the whole lyophilised cells with trans-cinnamic 2
acid gave only 10% conversion to the corresponding aldehyde.
Indeed the major products formed gave a mass spectrum con-
Fig. 1 Examples of industrially relevant chemicals which can be syn-
thesized from cinnamyl alcohol. Cinnamyl amines including naftifine
and flunarizine,10–12 cinnamyl esters including various fragrances7,8 and
photocrosslinking monomers, multi-step synthesis of compounds such
as dapoxetine and Taxol.13,14
The current provision for the bio-derived production of cin-
namyl alcohol is poor, due to the low levels in which it occurs
naturally as a metabolite. As such, the synthesis of this com-
pound usually involves the chemoselective reduction of the
corresponding aldehyde using chemical reducing agents,
finite and expensive metals or complex catalyst formulations,
such as nanotubes or nanoparticles.15–18 The chemical
reduction of allyl alcohols often requires fine control of reac-
tion conditions, due to the possible non-selective reduction of
both the hydroxyl and ene functionalities.19 Whilst there are
examples of alcohol dehydrogenase (ADH) enzymes used to
perform this same reaction in a more mild and chemoselective
fashion,20,21 access to cinnamaldehyde is not as easy as to
various primary metabolites, widely available as fermentation
products. To this end, we envisaged the addition of well-docu-
mented carboxylic acid reductase (CAR)22,23 and phenylalanine
ammonia lyase (PAL) biocatalysts to allow production directly
from the proteinogenic amino acid L-phenylalanine (L-Phe)
(Fig. 2).
Table 1 Effect of catalyst loading on the conversion of L-phenylalanine
(1) to trans-cinnamic acid (2) by PALa
% composition in
aqueous phaseb
Catalyst loading mg−1 mL−1
1
2
0
1
2
3
4
5
>99
44
36
18
13
10
<1
56
74
82
87
90
Results and discussion
a 1–5 mg of lyophilised E. coli cells producing AvPAL, 100 mM potass-
ium phosphate buffer (pH 7.5), 10 mM L-Phe, final volume 1 ml,
30 °C, 250 rpm vertical shaking, 24 h. b Determined via reverse-phase
HPLC analysis on a non-chiral phase.
As an initial test of the feasibility of the three enzyme cascades
proposed in Fig. 2, separate biotransformations were set up to
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sistent with cinnamyl alcohol 4 and 3-phenylpropanol 6. It AvPAL and MCAR in whole cell formulations was indeed found
could be envisaged that these products were formed via the to yield both the unsaturated alcohol 4 and saturated by-
action of CAR and a host cell ene-reductase in either order, fol- product 6 in a ratio of ∼1 : 2.4 within the extracted organic
lowed by host cell aldehyde reduction. It has been demon- layer, with the rest predominantly staying as the acid 2. A more
strated that E. coli ADHs have a broad specificity28 and reduce favourable ratio of ∼4 : 1 (4 : 6) could be achieved through the
cinnamaldehyde as a defence mechanism.29 In addition use of purified MCAR in the presence of an additional GDH-
various ene-reductases are known for their high affinity based cofactor recycling system (Table 2). However, the conver-
towards cinnamaldehyde.30 This situation was found to be sion was found to be lower when using the PAL enzyme in lyo-
remedied (in part) by the use of purified MCAR, with 84% con- philised and MCAR in purified enzyme form compared to both
version of 2 seen, giving a product ratio of ∼20 : 1 (3 : 4) with enzymes in lyophilised cells. The apparent reduction of ene-
steady state kinetic parameters exhibited as seen in Table 2. reductase activity within the system was expected due to the
This was possibly due to the presence of co-purified E. coli lower cell mass used for this biotransformation. Further inves-
ADH enzymes and/or the use of a glucose dehydrogenase tigation revealed the low impact of cinnamaldehyde reduction
(GDH) with low activity for the aldehyde. Evidence of the by the GDH recycling system (∼1% conversion of the
action of endogenous ADH enzymes in both the whole cell substrate).
systems prompted the investigation of PAL-CAR combinations
In order to ensure selective recovery of cinnamyl alcohol
to ascertain whether cinnamyl alcohol could be produced from the reaction mixture, the conversion of trans-cinnamic
without the addition of a third biocatalyst. The use of both acid to the alcohol product was studied further with a combi-
nation of MCAR and a commercially available ADH from
Saccharomyces cerevisiae. It was reasoned that the addition of
this enzyme to biotransformations would act to remove cinna-
Table 2 Conversion of 1, 2 and 3 by the combinations of PAL, CAR and
maldehyde produced by CAR at a higher rate, pushing the
overall conversion higher and minimising the effect of ene-
reductase enzymes, where whole cells were used. The use of
0.25 mg mL−1 ScADH in conjunction with 1 mg mL−1 whole
cells harbouring MCAR was found to give increasingly high
consumption of cinnamate (>90% after 2 hours) to the desired
alcohol, with a small amount of cinnamaldehyde also being
detected after 8 and 24 hour time points. Increasing the MCAR
whole cell biocatalyst component to 2.5 mg mL−1 was found to
give almost complete conversion after 2 hours with only traces
of cinnamate or cinnamaldehyde. Longer reaction times (4, 8
and 24 hours) resulted in accumulation, first of cinnamalde-
hyde and then 3-phenylpropanol contaminants (Fig. 3). These
results indicate the importance of time course assays in the
GDH biocatalystsa
% composition in extracted phaseb
Biocatalyst(s)
Substrate
2
3
4
5
6
CARc
2
2
1
1
3
<1
17
18
45
—
11
79
1
<1
99
33
4
57
44
1
<1
<1
<1
<1
<1
56
<1
24
11
<1
CARd
PAL/CARe
PAL/CARf
GDHg
Steady state kinetic parameters for conversion of 2 to 3 using MCARh
kcat (min−1
184.54 8.9
)
KM (mM)
KI (mM)
kcat/KM (min−1 mM−1
435.24
)
0.424 0.059
24.2 5.3
a General reaction conditions containing PAL: 100 mM potassium
phosphate buffer (pH 7.5), 5 mM substrate, 30 °C, 250 rpm vertical
shaking. For any other reactions containing CAR enzymes additionally
GDH 10 mM MgCl2, 15 mM D-glucose, 10 mM ATP, 10 U GDH, and
500 µM NADP+ were added. b Determined via GC-MS on a non-chiral
phase. c CAR biocatalyst used as a whole lyophilised cell formulation
(1 mg mL−1). d CAR biocatalyst used as an isolated enzyme formulation
(2 µM). e CAR and PAL biocatalysts used as a whole lyophilised cell for-
mulation (1 and 3 mg mL−1, respectively). f CAR biocatalyst used as an
isolated enzyme formulation and PAL as a whole lyophilised cell for-
mulation (2 µM and 3 mg mL−1). g GDH biocatalyst used in recycling
reaction quantities. The conversions were calculated according to peak
area on a GC device. The conversions of PAL are not accounted in this
table and supposed to be around ∼70%, known from Table 1. h MCAR
kinetic parameters were recorded by monitoring the rate of NADPH
oxidation at 340 nm. Reaction conditions: 100 mM KH2PO4/K2HPO4,
pH 7.5, 10 mM MgCl2, 1 mM ATP, 200 µM NADPH, 0.3 µM MCAR
enzyme, 30 °C.
Fig. 3 The effect of catalyst loading on the composition of CAR-ADH
cascade reactions over time. Reaction performed in 100 mM potassium
phosphate buffer (pH 7.5), 5 mM trans-cinnamic acid, 10 mM MgCl2,
MCAR dried cells, 30 mM D-glucose, 10 mM ATP, 10 U GDH, 0.05
mg ml−1 ADH, 500 µM NADP+, 500 µM NAD+, final volume 1 ml at
30 °C, 250 rpm.
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development of an optimised cascade procedure and possibly phase HPLC (to monitor the initial PAL reaction) and GC (to
highlight issues with the reversibility of ScADH in the mitiga- follow product formation with the CAR-ADH cascade). The
tion of side product formation.
reaction was performed with 107 mg (in 65 ml) as the starting
Having demonstrated the possibility of high conversion material, around 85% being converted to 2 after a 22 hour PAL
with minimal side reactions, the system was extended to reaction before complete double reduction (volume increased
include AvPAL starting from L-phenylalanine. The effect of to 130 ml after PAL separation) after a further 5.5 hours
varying the AvPAL whole cell loading on the overall compo- (Fig. 4). The reaction was performed in a sequential manner
sition of the volatile reaction intermediates/products could be separating PAL and CAR with ADH reactions. 6 hours after the
easily tested by the extraction of these from any remaining addition of the second and third biocatalysts, the product
phenylalanine in organic solvents. The full three enzyme reac- could be easily extracted and purified using flash chromato-
tion was performed both as a one-pot cascade and by the graphy from the remaining L-phenylalanine, with an isolated
implementation of the PAL and CAR-ADH portions under tem- yield of 43.5 mg (molar yield 53%). The presence of cinnamyl
poral separation (Fig. S1†). Overall the partition reaction gave alcohol was confirmed by 1H and 13C NMR spectroscopy as
better results with higher percentage compositions of 4, no by- well as GC-MS chromatogram and ionisation spectrum. The
product 6 and only small amounts of 2 or 3 detected. The pres- high resolution MS however did not show any ionisation pat-
ence of the unwanted alcohol 6 and a higher percentage of 2 terns (Fig. S4†).
in the cascade reaction was presumably due to the increased
In order to convert L-phenylalanine to cinnamyl alcohol in
overall cell loading (with associated ene-reductase activity) and an economical and environmentally friendly way, the enzy-
lower stability of CAR and/or ADH enzymes compared to matic reactions were transferred from an in vitro setup to an
AvPAL. In an attempt to increase flux through the second part in vivo system. The substrate, L-phenylalanine, was produced
of the partitioned reaction, loading of ScADH was varied from by E. coli NST strain31 using a glycerol/glucose mixture as a
0.025 to 0.25 mg mL−1 with either a single or double batch carbon source (1 g L−1).32 Additionally, E. coli NST was trans-
addition at t = 0 min and t = 300 min (Fig. S2†). These experi- formed with the vector pZZ-Eva2 (Fig. S6†) enabling the new
ments revealed a siphoning effect resulting in increased purity strain to produce cinnamyl alcohol in TB media. The pro-
of 6 for higher ADH concentrations, with batch addition also duction of cinnamyl alcohol was measured by the extraction of
giving lower percentage compositions of 2 and 3. Additionally, cinnamyl alcohol from the culture medium using ethyl
the effect of altering the ATP concentration was investigated, acetate, further derivatization and final quantification per-
demonstrating that a molar ratio of 1 : 2 (substrate to ATP) was formed by GC, following 24 h, 48 h and 72 h of cell growth
sufficient for conversion of the trans cinnamic acid to cinna- (Fig. 5a). A maximum of 300 mg cinnamyl alcohol per litre of
myl alcohol. Lower molar ratios of substrate to ATP result in culture was produced after 24 h of incubation, with an esti-
insufficient conversion (Fig. S3†), which might be related to mated 30% conversion of L-phenylalanine32 to cinnamyl
the substrate stability or to a lower catalytic activity of alcohol. Cinnamyl aldehyde production was constant at 75
enzymes.
mg L−1 over the entire 72 h, while the production of trans-cin-
Furthermore a full time course experiment was performed namic acid increased over the course of the experiment to a
for the two biocatalytic systems using a combination of reverse maximum of 65 mg L−1. Alternatively we tested the production
Fig. 4 Composition profile for the conversion of 1 to 4 via the addition of PAL (t = 0) performed in 100 mM potassium phosphate buffer (pH 7.5),
3 mg ml−1 of AvPAL dried cells and 10 mM L-Phe (107 mg) at 30 °C, 250 rpm, 65 ml final volume. (t = 1320) The reaction mixture was spun down and
the supernatant supplemented with 10 mM MgCl2, 1 mg mL−1 MCAR dried cells, 30 mM D-glucose, 10 mM ATP, 10 U GDH, 0.05 mg ml−1 ADH,
500 µM NADP+, 500 µM NAD+, final volume 130 ml at 30 °C, 250 rpm.
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Fig. 5 In vivo production of cinnamyl alcohol in E. coli NST using the plasmid pZZ-Eva2, measured by GC after 24 h, 48 h and 72 h. (A) Production
in TB media, and (B) production of cinnamyl alcohol comparing different carbon sources in M9 media. Error bars represent standard deviation (SD)
of three biological replicates.
in mineral M9 media, supplemented with the same glycerol/ with fermentation. However, lipase catalysed biodiesel pro-
glucose mixture and with either glycerol or glucose as the duction38 using different plant oil sources has also reached
carbon source (Fig. 5b). The production of cinnamyl alcohol in industrial levels.39 Glycerol as a by-product of enzymatic
M9 media was the highest with glucose as the carbon source (lipase) biodiesel production is easily removable,40 contains
(80 mg L−1), followed by production with the glycerol/glucose reduced levels of contaminants,41 and should therefore be
mixture (40 mg L−1), and glycerol as the sole carbon source directly suitable as a green carbon source for the production
(20 mg L−1). Regardless of the relatively low cinnamyl alcohol of cinnamyl alcohol.
titres, the major advantage of using mineral M9 media is that
no identifiable side product formation was observed in any of
the biological replicas over the whole course of the
experiment.
Conclusions
Here we demonstrate cinnamyl alcohol production via a
three step biocatalytic cascade and by metabolic engineering
in E. coli NST. The production of cinnamyl alcohol using the
biocatalytic cascade might be advantageous as a result of the
high loading capacity of enzymes, which is consistent with
their use with higher concentrations of substrate/product.
However, such an approach is potentially disadvantageous
regarding the additional cost and reduced simplicity in an
industrial setting (e.g. attributed to biocatalyst preparation
and cofactor supplementation). Nevertheless recent improve-
ments in the field of biocatalysis,34 particularly regarding
coimmobilization35 of enzymes and cofactors and/or using
coimmobilization to enable reuse of different reaction com-
ponents,36 might present a future green and economically
viable solution.
We have also demonstrated that metabolic engineering
in E. coli NST can be applied to produce cinnamyl alcohol
from analytical grade glucose and/or glycerol. Crude gly-
cerol33 is a by-product of biodiesel production and is there-
fore a potential attractive carbon source for E. coli37 in
metabolic engineering programmes that aim to establish
green routes to the production of valuable chemicals. Most
crude glycerol is derived from alkali catalysed biodiesel pro-
duction and contains contaminants that might interfere
The chemical building block cinnamyl alcohol has many uses,
as shown in the literature, as a precursor to various fragrance
compounds, smart materials and commercially available
pharmaceuticals. Through the use of a three enzyme cascade,
we have demonstrated the simple production of this com-
pound in good yield and high purity using biocatalytic func-
tional group interconversion. The optimised method has
several attractive features including a bio-derived starting
material (L-phenylalanine), renewably resourced catalysts
(phenylalanine ammonia lyase, carboxylic acid reductase and
alcohol dehydrogenase enzymes), ambient, low energy reaction
conditions and facile, inexpensive extraction of the final
product. This method opens up routes to the conversion of
biomass to useful products in an industrial setting as well as
synthetic biology approaches to create designer organisms for
the direct fermentative production of cinnamyl alcohol from
primary metabolic processes.
Conflicts of interest
There are no conflicts to declare.
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21 P. Zucca, M. Littarru, A. Rescigno and E. Sanjust, Biosci.,
Biotechnol., Biochem., 2009, 73, 1224–1226.
Acknowledgements
This work was funded by the Biotechnology and Biological 22 M. K. Akhtar, N. J. Turner and P. R. Jones, Proc. Natl. Acad.
Sciences Research Council (BBSRC) and Glaxo-SmithKline (GSK) Sci. U. S. A., 2013, 110, 87–92.
under the Strategic Longer and Larger (sLoLa) grant initiative 23 D. Gahloth, M. S. Dunstan, D. Quaglia, E. Klumbys,
ref. BB/K00199X/1. NSS is an Engineering and Physical Sciences
Research Council (EPSRC) Established Career Fellow. NJT
acknowledges the ERC for an Advanced Grant.
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