Microbial Production of Natural and Unnatural Monolignols with Escherichia coli

Article abstract of DOI:10.1002/cbic.201800673

Phenylpropanoids and phenylpropanoid-derived plant polyphenols find numerous applications in the food and pharmaceutical industries. In recent years, several microbial platform organisms have been engineered towards producing such compounds. However, for the most part, microbial (poly)phenol production is inspired by nature, so naturally occurring compounds have predominantly been produced to date. Here we have taken advantage of the promiscuity of the enzymes involved in phenylpropanoid synthesis and exploited the versatility of an engineered Escherichia coli strain harboring a synthetic monolignol pathway to convert supplemented natural and unnatural phenylpropenoic acids into their corresponding monolignols. The performed biotransformations showed that this strain is able to catalyze the stepwise reduction of chemically interesting unnatural phenylpropenoic acids such as 3,4,5-trimethoxycinnamic acid, 5-bromoferulic acid, 2-nitroferulic acid, and a “bicyclic” p-coumaric acid derivative, in addition to six naturally occurring phenylpropenoic acids.

Full text of DOI:10.1002/cbic.201800673

Accepted Article
Title: Microbial production of natural and non-natural monolignols with
Escherichia coli
Authors: Jennifer Aschenbrenner, Patrick Marx, Joerg Pietruszka, and
Jan Marienhagen
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201800673
Link to VoR: http://dx.doi.org/10.1002/cbic.201800673
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10.1002/cbic.201800673
ChemBioChem
Microbial production of natural and non-natural
monolignols with Escherichia coli
Jennifer Aschenbrenner[a], Patrick Marx[a], Jörg Pietruszka[a,b]
and Jan Marienhagen [a] *
,
[a]Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich
GmbH, D-52425 Jülich, Germany
[b]Institute of Bioorganic Chemistry, Heinrich Heine University of Düsseldorf at
Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
# Corresponding author:
Dr. Jan Marienhagen
Phone: +49 2461 61 2843
E-mail: j.marienhagen@fz-juelich.de
ORCID: 0000-0001-5513-3730
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Abstract
Phenylpropanoids and phenylpropanoid-derived plant polyphenols find numerous
applications in food and pharmaceutical industries. In recent years, several microbial
platform organisms were engineered towards producing such compounds. However,
for the most part, microbial (poly)phenol production is inspired by nature, and thus
predominantly naturally occurring compounds have been produced to this date.
Here, we took advantage of the promiscuity of enzymes involved in phenylpropanoid
synthesis and exploited the versatility of an engineered E. coli strain harboring a
synthetic monolignol pathway to convert supplemented natural and non-natural
phenylpropenoic acids to their corresponding monolignols. Performed
biotransformations showed that this strain is able to catalyze the stepwise reduction of
chemically interesting non-natural phenylpropenoic acids such as 3,4,5-
trimethoxycinnamic acid, 5-bromoferulic acid, 2-nitroferulic acid, and a ‘bicyclic’ p-
coumaric acid derivative in addition to six naturally occurring phenylpropenoic acids.
Keywords
biocatalysis, p-coumaric acid, monolignols, natural products, phenylpropanoids
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10.1002/cbic.201800673
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Introduction
Many plant polyphenols such as flavonoids, stilbenes or lignans are important
compounds for the food and pharmaceutical industries.[1] Here they find an application,
e.g., as flavors, colorants, therapeutic agents or antibiotics. General precursor
molecules of these valuable compounds are phenylpropanoids, which in turn are
derived from the aromatic amino acids L-phenylalanine or L-tyrosine (1).
Phenylpropanoid synthesis starts with the non-oxidative deamination of the aromatic
amino acid yielding the typical phenylpropanoid core structure: a phenyl group
attached to a propene tail (Figure 1).[1–3] This decisive reaction is catalyzed by
ammonia lyases, either phenylalanine ammonia lyases (PAL) or tyrosine ammonia
lyases (TAL).
COOH
NH2
COOH
4CL
COSCoA
CCR
CHO
CH2OH
(1)
(2)
(3)
(4)
(5)
TAL
CAD
NADPH NADP+
+ CoA
NADPH NADP+
AMP
+ PPi
NH3
CoASH
OH + ATP
OH
L-tyrosine
OH
OH
OH
p-coumaroyl-CoA
p-coumaryl
aldehyde
p-coumaric acid
p-coumaryl
alcohol
Figure 1. Biosynthetic pathway for p-coumaryl alcohol synthesis from L-tyrosine. TAL, tyrosine
ammonia-lyase; 4CL, 4-coumaroyl-CoA ligase; CCR, cinnamoyl-CoA reductase; CAD,
cinnamyl alcohol dehydrogenase.
In case of L-tyrosine, the resulting phenylpropenoic acid p-coumaric acid (2) is
subsequently activated by 4-coumarate-CoA ligases (4CL) yielding 4-coumaroyl-CoA.
This CoA-activated compound 3 can subsequently serve as precursor molecule for the
synthesis of flavonoids and stilbenes. Alternatively, p-coumaroyl-CoA can be stepwise
reduced to the respective alcohol 5, which is also referred to as monolignol. The
required two reduction steps are catalyzed by cinnamoyl-CoA reductases (CCR) and
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10.1002/cbic.201800673
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cinnamyl alcohol dehydrogenases (CAD), respectively. In plants the resulting
monolignols represent key building blocks for the synthesis of lignin, but are also
necessary for the synthesis of the pharmaceutically interesting group of lignans.[4,5]
In principle, phenylpropanoids and phenylpropanoid-derived polyphenols can be
isolated from plants as their natural producers, but polyphenol concentrations in the
plant usually account for less than one percent of the plant dry weight only.[6]
Furthermore, plant extraction is also limited by slow plant growth as well as
environmental and regional factors affecting overall product yields.[7,8] Total chemical
synthesis represents an interesting alternative, but depending on the complexity of the
target compound the synthesis route comprises a number of individual steps with
intermediate purifications.[9–11] Microbial phenylpropanoid production offers a
promising alternative to the uneconomic isolation from plant material as modern
molecular tools allow for the functional implementation of plant biosynthetic pathways
into the microbial metabolism.[1] Following this strategy, many microbial strains for plant
phenol synthesis were developed in recent years, especially for the production
phenylpropanoid-derived flavonoids and stilbenes.[12,13]
In this context, an Escherichia coli strain has been engineered to accumulate up to
52 mg/L p-coumaryl alcohol (5) without supplementation of any precursor
molecules.[14] The strain harbors a full synthetic phenylpropanoid pathway, which is
plasmid-encoded by a tetracistronic operon. Interestingly, all four enzymes
participating in monolignol biosynthesis have been previously described to be
promiscuous with regard to their substrate specificities.[15] This finding could enable
biosynthesis of other natural, and possibly also non-natural monolignols with
interesting applications from supplemented precursor molecules.[15,16] However,
practicability of this concept has been only demonstrated for the microbial production
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of cinnamyl alcohol (6), caffeoyl alcohol (7) and coniferyl alcohol (8) from
supplemented natural cinnamic acid derivatives.[17–19] In addition, individual enzymes
of the monolignol pathway were successfully used for the microbial synthesis of
different non-natural flavanones and stilbenes from various precursors.[20–25] Here,
more detailed studies exploring the catalytic promiscuity of the enzymes of the
monolignol pathway will not only help to gain a deeper understanding of the enzymes
involved, but might also provide access to new compounds with interesting chemical
or pharmaceutical properties.[26–30]
In this study, we set out to explore the catalytic versatility of a synthetic monolignol
pathway in E. coli by supplementing naturally and non-natural occurring cinnamic acid
derivatives.
Results and Discussion
Microbial synthesis of naturally occurring monolignols with E. coli
Recently, E. coli BL21-Gold (DE3) lacIQ1 pALXtreme-tal-4cl-ccr-cad was designed and
constructed, which can synthesize the monolignol p-coumaryl alcohol (5).[14] This strain
harbors a synthetic monolignol pathway composed of a tyrosine ammonia lyase from
Rhodobacter sphaeroides (TALRs, GenBank: ABA81174.1), a 4-coumarate: CoA ligase
from Petroselinum crispum (4CLPc, GenBank: X13324.1), a cinnamoyl-CoA reductase
from Zea mays (CCRZm, GenBank: Y15069.1) and a cinnamyl-alcohol dehydrogenase
from Z. mays. All four genes, organized as synthetic operon under control of the IPTG-
inducible T7 promoter are plasmid-encoded. Initially, it was tested, if this monolignol
pathway is also capable of reducing cinnamic acid (9), caffeic acid (10), ferulic acid
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(11), hydroxyferulic acid (12) and sinapic acid (13) as the most abundant naturally
occurring cinnamic acid derivatives (Figure 2).
O
OH
O
O
O
O
O
OH
OH
OH
OH
OH
(A)
MeO
HO
OMe
OH
OMe
OMe
OH
OH
OH
OH
OH
Cinnamic acid
p-Coumaric acid
Caffeic acid
Ferulic acid
Hydroxyferulic acid
Sinapic acid
(13)
(9)
(12)
(10)
(11)
(12)
O
O
O
OH
OH
OH
O
OH
(B)
NO2
MeO
Br
OMe
OMe
OMe
OMe
OH
OH
OH
5-Bromoferulic
2-Nitroferulic
3,4,5-Trimethoxy-
cinnamic acid
3-(4-Hydroxynapthalen-1-yl)
(22)
acid
acid
(18)
(20)
(16)
prop-2-enoic acid
(referred to as 'bycyclic'
p-coumaric acid)
Figure 2. (A) Naturally occurring and (B) non-natural cinnamic acid derivatives used in this
study.
For this purpose, all phenylpropenoic acids were individually supplemented to cultures
of growing E. coli cells at a concentration of 2.5 mM right at the start of the cultivation.
After 17 hours of cultivation, the concentrations of supplemented acid precursor
molecules as well as their corresponding monolignols in the supernatant were
determined by HPLC.
As a result of this systematic approach, it could be confirmed that the synthetic
pathway, although comprised of enzymes originating from three different organisms,
is indeed capable to reduce all supplemented natural cinnamic acid derivatives to their
corresponding monolignols in E. coli (Table 1). In case of cinnamic acid (9) as
chemically “most simple” precursor without any additional substituent on the aryl ring,
a product titer of 195 mg/L (1.46 mM) cinnamyl alcohol (6) could be determined (Table
1). In the past, cinnamyl alcohol (6) was produced in E. coli with a different set of
enzymes yielding 300 mg/L (2.24 mM) after 24 h.[17] However, biotransformations in
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this study were performed using TB media containing a glycerol/glucose mixture
(1 g/L), which served as carbon and energy source as this turned out to be the most
suitable medium for monolignol synthesis with E. coli in previous studies.[14]
Table 1. Monolignol titers obtained through biotransformations with E. coli BL21-Gold (DE3)
lacIQ1 pALXtreme-tal-4cl-ccr-cad from supplemented phenylpropenoic acids. For production,
E. coli cells were cultivated in 50 mL LB medium and 2.5 mM of the respective cinnamic acid
derivatives were individually supplemented. All biotransformations were performed at 25 °C
for 17 h. Data represents average values and standard deviations from three biological
replicates.
Monolignol
concentration
[mg/L]
[mM]
Natural monolignols
Cinnamyl alcohol
p-Coumaryl alcohol
Caffeoyl alcohol
195 ± 62
121 ± 5
5 ± 1
1.46
0.81
0.03
1.82
0.52
0.14
Coniferyl alcohol
327 ± 10
102 ± 30
30 ± 3
Hydroxyconiferyl alcohol
Sinapyl alcohol
Non-natural monolignols
3,4,5-Trimethoxycinnamyl alcohol
5-Bromoconiferyl alcohol
2-Nitroconiferyl alcohol
4 ± 1
462 ± 40
74 ± 15
25 ± 44
0.02
1.78
0.33
0.13
‘Bicyclic’ p-coumaryl alcohol
In addition to cinnamyl alcohol (6), the already described capability of this strain to
produce p-coumaryl alcohol (5) could be confirmed as a concentration 121 mg/L
(0.81 mM) of this monolignol could be determined in culture supernatants under the
cultivation conditions described. Caffeic acid (10), characterized by an additional O-
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methyl group on the aryl-ring in comparison to p-coumaric acid, was the least favored
substrate for the synthetic pathway as only 5 mg/L (0.03 mM) caffeoyl alcohol (7)
accumulated in the supernatant. In previous studies, the microbial production of
caffeoyl alcohol (7) with E. coli was achieved by using immobilized cells.[18] The
engineered strain equipped with a different set of enzymes produced up to 39 mg/L
(0.24 mM) caffeoyl alcohol (7) in LB medium within eight hours. In another recent
study, 534 mg/L (3.22 mM) caffeoyl alcohol (7) could be produced with an engineered
E. coli strain, but in total 4 mM caffeic acid (10) were supplemented at several time
points during 22 hours of cultivation using an optimized cultivation protocol and M9-
medium with yeast extract supplementation.[19] Interestingly, ferulic acid (11) turned
out to be the preferred natural substrate in this study since a product titer of 327 mg/L
(1.82 mM) of the corresponding coniferyl alcohol (8) could be determined in our
experiments. Extraction of coniferyl alcohol (8) from one liter culture supernatant
yielded 280 mg (1.55 mmol) of the pure compound.
For the first time the microbial production of hydroxyconiferyl alcohol (14) and sinapyl
alcohol (15) from supplemented hydroxyferulic acid (12) and sinapic acid (13),
respectively, could be demonstrated in vivo. After 17 h of cultivation, monolignol
concentrations of 102 mg/L (0.52 mM) and 30 mg/L (0.14 mM), respectively could be
determined (Table 1).
Noteworthy, not converted phenylpropenoic acids were not degraded and could be
detected in the supernatants of the E. coli cultures (data not shown).
Microbial synthesis of non-natural monolignols with E. coli
Hitherto, only the microbial synthesis of naturally occurring monolignols has been
described. This is somewhat surprising, as access to non-natural monolignols would
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also enable the synthesis pharmaceutically interesting lignans with novel properties.
With the aim to explore the catalytic flexibility of the established synthetic pathway for
the synthesis of such compounds, we attempted the conversion of four structurally very
different non-natural phenylpropenoic acids in order to probe the scope of the
approach. In particular, 5-bromoferulic acid (18) and 2-nitroferulic acid (20) were
chosen based on their potential for further diversification, e.g., through palladium-
catalyzed cross-couplings or after reduction to the corresponding aniline derivative.
Among these, 3,4,5-trimethoxycinnamic acid (16) and 5-bromoferulic acid (18) were
commercially available, but the substrates 2-nitroferulic acid (20) and 3-(4-
hydroxynaphthalen-1yl)prop-2enoic acid (22) needed to be synthesized (see
Supporting Information). In addition, the corresponding monolignols of all four non-
natural substrates tested were chemically synthesized to serve as reference
compounds for qualitative and quantitative analyses (see Supporting Information).
First experiments with 3,4,5-trimethoxycinnamic acid (16), a compound closely related
to sinapic acid (13) revealed that only a small fraction of 0.02 mM (4 mg/L) of this
substrate could be efficiently reduced to 3,4,5-trimethoxycinnamyl alcohol (17) (Table
1). In contrast, 5-bromoferulic acid (18) was rapidly reduced by the synthetic
monolignol pathway and a final product titer of 462 mg/L (1.78 mM) 5-bromoconiferyl
alcohol (19) could be determined in the supernatant (Table 1). Interestingly, under the
conditions tested, 5-bromoferulic acid (18) proved to be a much better substrate
compared to any of the naturally occurring phenylpropenoic acids used in this study.
The engineered E. coli strain also successfully reduced 2-nitroferulic acid (20) to 2-
nitroconiferyl alcohol (21). After 17 hours of biotransformation 74 mg/L (0.33 mM) 2-
nitroconiferyl alcohol (21) accumulated in the supernatant (Table 1). Motivated by
these results, the conversion of ‘bicyclic’ p-coumaric acid (22) as sterically most
challenging substrate was also attempted (Figure 2). This naphthalene derivative also
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10.1002/cbic.201800673
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proved to be a suitable substrate as 25 mg/L (0.13 mM) of the corresponding
monolignol ‘bicyclic’ p-coumaryl alcohol 23 could be detected in culture supernatants
(Figure 1). Noteworthy, qualitative NMR experiments revealed that the pathway
intermediate ‘bicyclic’ p-coumaryl aldehyde accumulated in the supernatants of the
E. coli cultures. This indicates that this aldehyde is not a favored substrate for the CAD,
which catalyzes the last reduction step of the synthetic monolignol pathway.
Optimization of the microbial 5-bromoconiferyl alcohol production
Subsequently, the 5-bromoconiferyl alcohol (19) production with the engineered E. coli
strain was further optimized. Until this point substrate concentrations of 2.5 mM were
used in all biotransformations since natural cinnamic acid derivatives are known to
have an inhibitory effect on microbial growth.[31,32] With the aim to balance microbial
growth and product yield, biotransformations with different 5-bromoferulic acid (18)
concentrations were performed in 48-well microtiter plates in a microbioreactor system.
Unfortunately, 5-bromoferulic acid (18) concentrations exceeding 4 mM led to
substrate precipitation, which rendered determination of the culture backscatter over
time impossible (data not shown). This in turn impeded the evaluation of the impact of
elevated substrate concentrations on microbial growth. However, performed cultivation
experiments with substrate concentrations ranging from 0 mM and 2.5 mM already
revealed, that presence of 5-bromoferulic acid (18) has a growth-inhibiting effect
similar to the naturally phenylpropenoic acids tested here and in other studies (Figure
3A).[31,32] With regard to the maximum achievable product titer when considering the
cytotoxic effects of this compound for the cells, substrate concentrations between 2.5
mM and 3 mM turned out to most suitable as up to 0.9 mM 5-bromoferulic acid (18)
could be efficiently converted 5-bromoconiferyl alcohol (19) (Figure 3B). Higher
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10.1002/cbic.201800673
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substrate concentrations of up to 6 mM 5-bromoferulic acid (18) resulted in a reduced
product formation, most likely due to the (probably) even more pronounced growth-
inhibitory effect of substrate concentrations exceeding 2.5 mM. The observed
substrate toxicity could be circumvented by stepwise addition of 5-bromoferulic acid
(18) during the biotransformation as it was also previously demonstrated for the
microbial production of p-coumaryl alcohol (5) and caffeoyl alcohol (7).[19] For microbial
monolignol production at reactor-scale, fed-batch fermentations are a suitable
option.[33] For future experiments at smaller scale, a slow-release technique could be
used to avoid growth inhibiting effects of elevated phenylpropenoic acid
concentrations. This technique is based on a diffusion-driven substrate release and
requires a feed reservoir filled with a concentrated substrate solution.[34,35] Here, a
dialysis membrane separating the reservoir from the E. coli cells, enables the diffusion
of the substrate into the culture medium.[34,35] In principle, this approach could be also
used for biotransformations at microtiter plate-scale.[36]
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Figure 3. Impact of different 5-bromoferulic acid (18) concentrations on cell growth and 5-
bromoconiferyl alcohol (19) production. A) Growth of the engineered E. coli strain in the
presence of 5-bromoferulic acid (18) concentrations ranging from 0 mM to 2,5 mM B) Obtained
5-bromoconiferyl alcohol (19) concentrations in the presence of varying 5-bromoferulic acid
(18) concentrations. E. coli BL21-Gold (DE3) lacIQ1 pALXtreme-tal-4cl-ccr-cad was cultivated
in 900 µL LB medium with different 5-bromoferulic acid (18) concentrations in 48-well microtiter
plates at 25 °C and 900 rpm. Heterologous gene expression was induced with 1 mM IPTG at
the time point of inoculation. 5-Bromoconiferyl alcohol (19) concentrations were determined by
HPLC. Data represents average values and standard deviations from three biological
replicates.
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Conclusions
In this study, E. coli BL21-Gold (DE3) lacIQ1 pALXtreme-tal-4cl-ccr-cad was
characterized with regard to the biosynthetic versatility of the heterologous monolignol
pathway. In this context, it could be shown that this strain represents a suitable catalyst
for the production of six naturally and four non-natural occurring monolignols. Key to
the success was the relaxed substrate specificity of the enzymes within this synthetic
pathway, which accept a broad range of phenylpropanoid-like compounds as
substrate.
In the context of this study, microbial synthesis of the naturally occurring
hydroxyconiferyl alcohol (14) and sinapyl alcohol (15) could be demonstrated for the
first time. In addition, the chemically interesting monolignols 3,4,5-trimethoxycinnamyl
alcohol (17), 5-bromoconiferyl alcohol (19), 2-nitroconiferyl alcohol (21) and the
‘bicyclic’ p-coumaryl alcohol 23 could be synthesized by this E. coli strain. These
compounds represent interesting starting points for the synthesis of more complex
plant-inspired active agents.
Experimental Section
Bacterial strains, plasmids, media and growth conditions
E. coli BL21-Gold (DE3) lacIQ1 pALXtreme-tal-4cl-ccr-cad was used for monolignol
production.[37] The pALXtreme vector backbone was constructed from a pET-28a(+)
standard vector (Merck KGaA, Darmstadt, Germany) by removing 63 % of its
sequence[38]. The resulting smaller vector was originally designed to improve the
transformation efficiency in the context of screening campaigns, in which the efficient
cloning and transformation of large and genetically diverse libraries is required.
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Redesign of this plasmid required the genomic integration of the lacIQ1 gene from pET-
vector system yielding E. coli BL21-Gold (DE3) lacIQ1. Hence, pALXtreme can be only
used in combination with this strain.[38] E. coli was cultivated aerobically in Luria Bertani
(LB) medium on a rotary shaker (130 rpm) or on LB plates (LB medium with 1.5 %
agar) at 37°C.[39] Where appropriate, kanamycin (50 µg/mL) was added to the medium.
Growth was determined by following the optical density at 600 nm (OD600).
Chemical synthesis of phenylpropenoic acids and monolignols
Cinnamic acid derivatives and cinnamyl alcohol derivatives were either commercially
available or synthesized (see Supporting Information). The compounds were
supplemented during microbial monolignol synthesis and used standards for HPLC-
analyses.
Microbial monolignol production with E. coli
For monolignol production in 500 mL baffled shake flasks, 50 mL LB medium
containing 2.5 mM of the respective phenylpropenoic acid substrate was inoculated
with an E. coli over-night culture to an OD600 of 0.1. The culture was incubated at 37 °C
and 120 rpm until an OD600 of 0.2 was reached. Subsequently, the cultivation
temperature was decreased to 25 °C and heterologous gene expression was induced
with 1 mM IPTG when an OD600 of 0.6 was reached. Samples were taken 17 h after
IPTG addition for substrate/product analyses. All cultivations were performed in
biological triplicates.
For the microbial production of monolignols at microtiter plate-scale, E. coli cells were
cultivated using a BioLector device (m2p-labs GmbH, Germany). For this purpose,
cultivations were performed in 900 µL LB medium using 48-well flower plates. These
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plates were incubated at 900 rpm and 25 °C, a humidity of 85% and a throw of ø 3
mm. When using this cultivation format, heterologous gene expression was induced
with 1 mM IPTG at the time point of inoculation. All cultivations were performed in
biological triplicates.
Quantification of phenylpropenoic acids and monolignols
Concentrations of phenylpropenoic acids and monolignols in cell free culture
supernatants were determined by HPLC using an Agilent 1260 infinity LC device
(Santa Clara, CA, USA) coupled with a DAD detector. For analyses, a mixture of water
with 2 % (v/v) acetic acid (buffer A) and acetonitrile with 2 % (v/v) acetic acid (buffer B)
as the mobile phases was used. LC separation was carried out using a ZORBAX
Eclipse AAA (3.5 μm, 4.6 × 75 mm) column with a guard cartridge (4.6 × 12.5 mm) at
50°C. For an efficient separation, 85 % buffer A and 15 % buffer B were used for a
maximum of 35 min with one additional minute as post time. Substrates and products
were detected by monitoring the absorbance at a defined single wavelength (Table 2).
Benzoic acid (final concentration 100 mg/L, 0.82 mM) was used as internal standard.
Authentic metabolite standards were either purchased from Sigma-Aldrich
(Schnelldorf, Germany) or chemically synthesized in-house. Six different
concentrations of each standard dissolved in acetonitrile were measured for each
calibration curve. Calibration curves were calculated based on analyte/internal
standard ratios for the obtained area values.
Coniferyl alcohol extraction from culture supernatant
Culture supernatants were carefully acidified to pH 6.0 using 1 M hydrochloric acid.
Subsequently, the coniferyl alcohol was extracted three times with 450 mL ethyl
acetate. The combined organic layers were dried with MgSO4, filtrated and the solvent
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was removed under reduced pressure. The resulting product was purified via column
chromatography (n-pentane: ethyl acetate 60:40).
Acknowledgements
We would like to thank the German Federal Ministry of Education and Research, BMBF
(“BioLiSy” Bioeconomic Lignan Synthesis; Grant No. 031A554) for financial support.
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10.1002/cbic.201800673
ChemBioChem
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