Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

Article abstract of DOI:10.1002/cbic.201800594

Biocatalytic dealkylation of aryl methyl ethers is an attractive reaction for valorization of lignin components, as well as for deprotection of hydroxy functionalities in synthetic chemistry. We explored the demethylation of various aryl methyl ethers by using an oxidative demethylase from Pseudomonas sp. HR199. The Rieske monooxygenase VanA and its partner electron transfer protein VanB were recombinantly coexpressed in Escherichia coli and they constituted at least 25 % of the total protein content. Enzymatic transformations showed that VanB accepts NADH and NADPH as electron donors. The VanA–VanB system demethylates a number of aromatic substrates, the presence of a carboxylic acid moiety is essential, and the catalysis occurs selectively at the meta position to this carboxylic acid in the aromatic ring. The reaction is inhibited by the by-product formaldehyde. Therefore, we tested three different cascade/tandem reactions for cofactor regeneration and formaldehyde elimination; in particular, conversion was improved by addition of formaldehyde dehydrogenase and formate dehydrogenase. Finally, the biocatalyst was applied for the preparation of protocatechuic acid from vanillic acid, giving a 77 % yield of the desired product. The described reaction may find application in the conversion of lignin components into diverse hydroxyaromatic building blocks and generally offers potential for new, mild methods for efficient unmasking of phenols.

Full text of DOI:10.1002/cbic.201800594

Accepted Article
Title: Exploring the selective demethylation of aryl-methyl ethers by a
Pseudomonas Rieske monooxygenase
Authors: Elisa Lanfranchi, Miloš Trajković, Katalin Barta, Johannes G
de Vries, and Dick B Janssen
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10.1002/cbic.201800594
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Exploring the selective demethylation of aryl-methyl ethers by a
Pseudomonas Rieske monooxygenase
Elisa Lanfranchi,*[a]§ Milos Trajkovic,[a] Katalin Barta,[b] Johannes G de Vries,[b,c] and Dick B Janssen[a]
Abstract: Biocatalytic dealkylation of aryl-methyl ethers is an
attractive reaction for valorization of lignin components and for
deprotection of hydroxy functionalities in synthetic chemistry. We
explored the demethylation of various aryl-methyl ethers by an
oxidative demethylase from Pseudomonas sp. HR199. The Rieske
monooxygenase (VanA) and its partner electron transfer protein
(VanB) were recombinantly coexpressed in E. coli and they
constituted at least 25% of the total protein content. Enzymatic
conversions showed that VanB accepts NADH and NADPH as
electron donor. The VanA-VanB system demethylates a number of
aromatic substrates, the catalysis occurs selectively at meta position
of the aromatic ring and the presence of a carboxylic moiety is
essential. The reaction is inhibited by the byproduct formaldehyde.
Therefore, we tested three different cascade/tandem reactions for
cofactor regeneration and formaldehyde elimination; conversion was
especially improved by adding formate dehydrogenase and
formaldehyde dehydrogenase. Finally, the biocatalyst was applied for
the preparation of protocatechuic acid from vanillic acid giving 77%
yield of desired product. The described reaction may find application
in the conversion of lignin components to diverse hydroxyaromatic
building blocks and generally opens prospective to new mild methods
for efficient deprotection of phenols.
that allow more facile demethylation in a mild environment are
therefore desired. Besides in synthetic chemistry, novel
demethylation protocols could also be exploited in the valorization
of compounds derived from lignin. This complex natural polymer
is rich in aryl-methyl ethers groups derived from the precursors
coniferyl and sinapyl alcohol.[4] In the past decade, great progress
was made regarding the development of catalytic strategies for
lignin depolymerization to produce fine chemicals.[5–7]
Independent of the method used, the majority of these processes
result in aromatic compounds that comprise the guaiacyl or
syringyl moiety.[8,9] O-demethylation reactions would generate
hydroxylated aromatics that may serve as building blocks for the
production of value-added chemicals using diverse synthetic
strategies. For example, the demethylation of guaiacyl derivatives
leads to catechols that are present in numerous pharmaceuticals,
pesticides and flavors. Alternatively, catechols are converted by
oxidation to muconic acid which can be hydrogenated to adipic
acid derivatives to be used in polymer synthesis.[10,11]
Green and environmentally friendly O-demethylating solutions
can in principle be addressed by biocatalysis. However, whereas
demethylases are widely present in nature, they have barely been
explored for biocatalytic applications.[12] Reasons might be the
complex architecture of some of these multi-component systems
as well as the requirement for expensive and/or delicate cofactors
(e.g. cobalamin- and/or tetrahydrofolate-dependent enzymes). An
example of such a demethylation system is cytochrome P450
Introduction
CYP199A4 from Rhodopseudomonas palustris,
a
heme-
Phenolic groups occur in numerous natural bioactive compounds
and are important functionalities during the synthesis of specialty
chemicals. However, due to their high reactivity, protection of
phenolic groups is often required prior to further syntheses, for
example by preparing stable and unreactive methyl ethers.[1]
While such methylation strategies are highly effective for the
protection of phenols, the subsequent deprotection of the inert
methyl ethers generally requires harsh reaction conditions such
as the use of strong acids or bases.[2,3] Alternative approaches
dependent monooxygenase which catalyzes the oxidative
demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic
acid.[13,14] The enzyme was recombinantly expressed in E. coli
together with electron transfer proteins and whole cells were
employed for the transformation of veratric acid to vanillic acid.[13]
CYP199A4 only demethylates methoxy groups at the para
position of the phenyl ring. It tolerates other substituents at the
ortho and meta positions, whereas the carboxylate moiety is
critical for substrate recognition.[15] Interestingly, the single
Ser244Asp mutation in the active site of the enzyme unlocked the
carboxylate dependence and extended the substrate scope to
benzene derivatives.[16]
Lignin harbors most of the O-methyl groups at the 3 or 3-5
(meta) positions on the aromatic ring and accordingly
demethylases with different regiospecificity have raised interest.
The tetrahydrofolate (THF)- dependent O-demethylase LigM from
Sphingobium sp. SYK-6 was recently characterized and the
crystal structure was solved.[17–20] The expensive THF cofactor
was required in 10-fold molar excess over substrate in order to
achieve good conversion.[18] Rosini and coworkers proposed a
tandem reaction exploiting a methionine synthase for cofactor
recycling.[18] This enzyme (MetE) converts 5-methyl-THF back to
THF by the transfer of the methyl group to L-homocysteine,
forming L-methionine. With the so developed biotransformation,
the authors were able to obtain 5 mM protocatechuic acid (PCA,
[a]
[b]
Dr. E. Lanfranchi, Dr. M. Trajkovic, Prof. D. B. Janssen
Groningen Biomolecular Sciences and Biotechnology Institute
(GBB)
University of Groningen
Nijenborgh 4, 9726 AG Groningen, The Netherlands
E-mail: elisa.lanfranchi@ucc.ie
Prof. K. Barta, Prof. J. G. de Vries
Synthetic Organic Chemistry - Stratingh Institute for Chemistry
University of Groningen
Nijenborgh 4, 9726 AG Groningen, The Netherlands
Leibniz-Institut für Katalyse e.V.
Albert-Einstein-Strasse 29a, 18059 Rostock, Germany
Current address: School of Food and Nutritional Science Sciences
University College Cork
[c]
§
College Road, Cork T12 YN60, Ireland
Supporting information for this article is given via a link at the end of
the document.
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1b) with only 0.1 mM THF added. Nevertheless, in view of the
high cost of the cofactor, the availability of THF-independent
meta-demethylation systems could offer an attractive alternative.
One example is the new discovered cytochrome P450 from
Amycolatopsis sp. ATCC 39116, which catalyzes the O-
demethylation of guaiacol and its analogs at the meta position of
the aromatic ring.[21]
Early genetic studies indicated that bacteria degrading lignin-
derived components and dimeric model compounds express an
additional type of demethylases to LigM, where demethylation of
vanillic acid (1a) to PCA is catalyzed by a two-component system
consisting of VanA and VanB.[22–24] Sequence similarities suggest
that VanA belongs to the Rieske non-heme iron-dependent
monooxygenases and that it is a homolog of a demethylating
dicamba (3,6-dichloro-2-methoxybenzoic acid) monooxygenase
(DdmC) of which the structure and catalytic mechanism are
known.[25–27] Rieske proteins are distinguished by the presence of
an iron-sulfur cluster [2Fe-2S] that functions as an electron
transfer and electron storage organometallic complex. The active
site contains a single free iron ion involved in the binding and
Scheme 1. Proposed cascade for O-demethylation of aryl-methyl ethers
catalyzed by Rieske non-heme monooxygenase coupled with
regeneration system.
a cofactor
activation
of
molecular
oxygen,
allowing
substrate
hydroxylation.[28,29]
Results and Discussion
One example of such a two-component system is the O-
demethylase from Streptomyces (StVanA) which has been
partially characterized by Nishimura and coworkers.[30] They
explored the substrate scope of StVanA after recombinant protein
expression using whole-cell bioconversions with E. coli. This work
showed the potential value of StVanA-VanB as a whole-cell
biocatalyst, but also underlined the need to improve expression of
soluble protein. Difficulties with heterologous production were
also reported for other iron-sulfur proteins, which make it, together
with protein instability, a major limitation for the exploitation of
Rieske oxygenases (ROs).[31]
A demethylase system that is potentially attractive for applied
biocatalysis is present in Pseudomonas sp. HR199 (DSM 7063),
a strain able to degrade lignin-derived compounds to products of
lower molecular weight.[32] It transforms eugenol to vanillin, which
is further oxidized to vanillic acid and then converted to PCA. The
latter is further metabolized by dioxygenase-mediated cleavage
of the aromatic ring.[33] The sequence of the monooxygenase
component of the vanillate demethylase system from
Pseudomonas HR199 (Ps(HR199)VanA) was identified in the late
nineties and the enzymatic activity was confirmed after
recombinant expression in E. coli.[34] However, the properties of
the protein and selectivity of the enzyme have never been
thoroughly investigated.
Amino acid sequence analysis
A multiple sequence alignment of the monooxygenase VanA
from Pseudomonas HR199 (UniprotKB O05616) with StVanA and
DdmC was performed (Figure S5, Supporting Information).
Ps(HR199)VanA is 36% identical to StVanA and 34% to DdmC.
The latter was the only appreciable hit obtained after a Blast
search of Ps(HR199)VanA against the PDB database. Key
residues and motifs were pinpointed by comparison to the
reference sequence DdmC. Rieske domains are characterized by
two conserved His and two Cys residues which coordinate the
2Fe-2S cluster. Cys47-His49 and Cys66-His69 are the first and
second ligand pairs distinguishable by the typical CXH and CXXH
motifs. Regarding the active site, the putative residues involved in
non-heme Fe ion coordination are His156, His161 and Asp298.
The so called bridging Asp is also conserved (Asp153).[25,35] This
observation suggests a multimeric structure of Ps(HR199)VanA
and, similarly to other ROs enzymes, that the electron transport
chains occurs inter-molecular from the 2Fe-2S cluster of
monomer A to the active site of monomer B. Based on the results
of D’Ordine and coworkers regarding DdmC, the proposed
electron-transfer chain in Ps(HR199)VanA involves αHis49,
αHis69, βAsp153, βHis156 and βHis161.[25] The multiple
sequence alignment suggests a different architecture of the active
site and substrate coordination between DdmC and vanillate
oxygenase. For example, the key residues His251, Asn230 and
Trp285 for the coordination of the carboxylic moiety in DdmC are
not conserved in Ps(HR199)VanA, in congruence with what has
been reported by Dumitru and coworkers. [26] This is explainable
by the different substrates and dealkylation position of the two
enzymes (DdmC catalyzes a demethylation at ortho position of
the aromatic ring).
Here we describe the optimization of recombinant
coexpression of Pseudomonas HR199 VanA and VanB in E. coli.
Subsequently, the catalytic properties of the two-component
system were determined using lyophilized cells and we explored
its suitability for biocatalytic applications. Finally, VanA-VanB
were employed in multi-enzyme cascade reactions for effective
cofactor regeneration and byproduct removal.
VanB (UniprotKB O05617) is the partner reductase enzyme,
indispensable for the supply of electrons to the Rieske domain of
VanA. A protein Blast search of its amino acid sequence against
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the PDB database retrieved only one hit with a high score (37%
identity and 100% query coverage): the phthalate reductase from
Burkholderia cepacia.[36] A pairwise alignment of the two amino
acid sequences was performed (Figure S6, Supporting
Information). A conserved domain search (CDD database)
identified a flavin and a nicotinamide cofactor binding domain, in
agreement with a role in hydride transfer from NAD(P)H to the
flavin cofactor during catalysis. Additionally, the sequence
alignment showed a conserved ferredoxin domain recognizable
by the four conserved Cys residues responsible of the
coordination of the 2Fe-2S cluster (Cys266, Cys271, Cys274 and
Cys304). These results suggested that, similar to other non-heme
oxygenase enzymes, the partner reductase Ps(HR199)VanB
reduces the flavin cofactor by oxidation of NAD(P)H and then
transfers the electrons to the ferredoxin domain, which likely
interacts with the Rieske domain of VanA and provides electrons
necessary for the catalysis.
2
3
4
0.1 mM NADPH, 0.1 mM FeSO4, 10%
DMSO, Regeneration system
44.4 ± 0.6
0.1 mM FeSO4, 10% DMSO, Regeneration
system
20.5 ± 0.2
30.7 ± 0.2
0.5 mM NADH, 0.1 mM FeSO4, 10% DMSO,
Regeneration system
5
6
0.1 mM NADH, 0.1 mM FeSO4, 10% DMSO
7.1 ± 0.8
0.1 mM NADH, 10% DMSO, Regeneration
system
46.7 ± 2.0
7
8
0.1 mM NADH, 1 mM FeSO4, 10% DMSO,
Regeneration system
60.2 ± 0.3
53.3 ± 1.9
≤2
0.1 mM NADH, 0.1 mM FeSO4,
Regeneration system
9
Control 1: No cells
(Conditions entry 1)
10
11
12
13
Control 2:[c] E. coli BL21 (DE3) Star
(Conditions entry 1)
≤2
Coexpression of VanA and VanB in E. coli
To obtain appreciable amounts of the enzymatic
demethylation system, the VanA and VanB proteins were
coexpressed in E. coli BL21 Star (DE3) using a pET-derived
vector named pE2T_Ps(HR199)VanA_VanB. In order to boost
transcription, each coding DNA sequence was inserted
downstream of an independent T7 promoter. Preliminary protein
purification experiments by affinity chromatography failed due to
the partial loss of the [2Fe-2S] complexes and low yields.
Additionally, a detrimental effect of an N-terminally fused 6xHis-
tag was observed on the activity of the purified enzymes (data not
shown). Therefore, the two proteins were coexpressed in their
native form, without any affinity tag. Various cultivation conditions
for IPTG-induced protein production were examined, which
revealed that high-level soluble overexpression of both proteins
was best achieved when additional iron and sulfur sources were
added, suggesting that maturation of the iron-sulfur clusters is a
critical step in protein synthesis (Experimental Section). After
optimization, the two enzymes were well expressed and
constituted approximately 25% of the total soluble protein in cell
lysates, as indicated by two prominent bands at 39 kDa (VanA)
and 35 kDa (VanB) that were observed upon analysis by SDS-
PAGE (Figure S1, Supporting Information).
Control 3:[d] PCA instead of vanillic acid
(Conditions entry 1)
80.5
Control 4:[d] No cells, PCA instead of vanillic
acid (Conditions entry 1)
80.6
Control 5:[d] E. coli BL21 (DE3) Star, PCA
instead of vanillic acid (Conditions entry 1)
92
[a] Determined by HPLC. [b] Regeneration system: 10 µM PTDH and 10 mM
phosphite. [c] Measured in triplicate.[d] Single experiment.
Biocatalytic reaction characterization
Since information on the biochemical properties of vanillate
O-demethylases is scarce, we examined the biocatalytic activity
of the Pseudomonas HR199 VanA-VanB system under various
reaction conditions. In these experiments, 5 mM vanillic acid (1a)
was chosen as the model substrate and the formation of
protocatechuic acid (PCA, 1b) was studied. The conversion was
carried out for 24 h and both substrate and product concentrations
were determined by HPLC (Table 1 and Table S1, Supporting
Information).
Initial experiments were performed by adding rehydrated
freeze-dried cells containing VanA-VanB, NAD(P)H and the
phosphite dehydrogenase (PTDH) cofactor regeneration system
to the reaction mixture (Scheme 1, reaction conditions: Table 1,
entries 1, 2 and 4).[37] The oxidoreductase VanB accepted both
NADPH and NADH as reducing electron donor, with a preference
for the latter. This dual cofactor acceptance is in accordance to
what was previously reported for other Pseudomonas species.[38]
Since omission of NADH and NADPH from reaction mixtures
drastically reduced conversion of vanillic acid (Table 1, entry 3),
we conclude that addition of the reduced nicotinamide-cofactor is
essential to obtain high yields of the demethylated product PCA
and that both NADH and NADPH are incorporated by rehydrated
freeze-dried cells. In situ cofactor regeneration is commonly
adopted for biocatalytic applications of nicotinamide-cofactor
dependent oxidoreductases and avoids the need of stoichiometric
addition of costly NAD(P)H (Scheme 1). As expected, O-
demethylation coupled with PTDH for cofactor regeneration
The high-level recombinant coproduction made it possible to
avoid protein isolation and prepare a stable biocatalyst by
lyophilization of whole-cells. After centrifugation and washing,
14.2 g of wet cell weight (WCW) was obtained per l of culture,
which corresponded to 3.0 g of dry cell weight (DCW) per l of
culture after freeze-drying. The obtained powder containing the
biocatalyst VanA-VanB was used for all following experiments.
Table 1. Initial screening of demethylation reaction conditions with 7.5
mg/mL of freeze-dried cells after 4 h of incubation.
Entry
1
Reaction Conditions
PCA Analytical
Yield (%)[a]
0.1 mM NADH, 0.1 mM FeSO4, 10% DMSO,
Regeneration system[b]
58.8 ± 2.6
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allowed the reaction to proceed beyond stoichiometric
consumption of the reduced cofactor, while the formation of PCA
was much lower when cofactor regeneration was omitted (Table
1, entry 5).
The addition of an excess of iron to the reaction mixture
showed a beneficial effect on the overall conversion, which is in
accordance with the involvement of the metal in catalysis through
oxygen activation. Finally, VanA-VanB tolerated 10% DMSO,
which is advantageous in case of poorly soluble substrates.
Based on the above, we used 0.1 mM NADH, 0.1 mM FeSO4,
10% DMSO and the PTDH-based cofactor regeneration system
for further characterization experiments (Table 1, entry 1).
The described conversions showed
a molar imbalance
between the consumption of the substrate and the production of
PCA (Table S1, Supporting Information), suggesting a partial
conversion of vanillic acid by unknown E. coli enzyme(s) or partial
loss of the catechol due to its instability or further enzymatic
degradation. To examine these possibilities, a series of controls
were designed. Initially, the reaction mixture containing 5 mM
vanillic acid was incubated without the biocatalyst by omission of
cells or by using the E. coli strain lacking VanA-VanB (Table 1,
entries 9 and 10). The latter reaction showed 37% substrate
depletion but no specific product was identified (Table S1,
Supporting Information). Additionally, a partial consumption of
the product was observed when 5 mM PCA was added to the
mixture instead of vanillic acid (Table 1, entries 11, 12 and 13;
Table S2, Supporting Information), confirming the occurrence
of some product loss due to a non-biocatalytic process by
endogenous E. coli enzymes and compound instability.
Figure 1. Effect of antioxidants on the Pseudomonas HR199 VanA-VanB
dealkylation system. A: Effect on enzyme stability. Freeze-dried cells (7.5
mg/mL) were pre-incubated in 100 mM Tris-HCl, pH 7.5, with varying
concentrations of DTT at 30°C and 100 rpm. At the end of the pre-incubation
time, the biotransformation was initiated by addition all remaining components
and product formation was determined after 2 h. PCA formation at 0.25 h pre-
incubation (time required for cells rehydration) was set to 100% for each
condition. B: Effect on bioconversion. Biotransformation was carried out as
described under Experimental Section with addition of different reducing agents
at various concentrations. After 4 h of incubation, the concentrations of product
and substrate were determined by HPLC. Negative control (Neg Ctr): Table 1,
entry 10. Positive control (Pos Ctrl): Table 1, entry 1. BME, b-mercaptoethanol;
GSH, glutathione; DTT dithiothreitol. Error bars indicate the standard deviation
obtained from two independent experiments.
Oxygen sensitivity
Whilst anaerobic conditions might be an optimal environment
for the stability of iron-sulfur proteins, the demethylation reaction
uses molecular oxygen as reactant that obviously cannot be left
out. To examine the possibility that the enzymatic demethylation
was restricted by detrimental effects of molecular oxygen on the
activity and stability of the VanA-VanB system, we examined the
biocatalytic formation of PCA in the presence of antioxidants at
various concentrations (Figure 1).
In the absence of an antioxidant, a dramatic loss of enzyme
activity was observed after 2 h of pre-incubation at 30°C, with only
29% of the initial activity retained and less than 1 mM PCA was
detected. The biocatalyst was essentially inactive after 4 h of pre-
incubation (Figure 1A). The addition of DTT substantially
improved the stability for the first 2 h and more than 80% of the
initial activity was retained (Figure 1A). The beneficial effect of
the antioxidant was also visible when examining the effective
conversion (Figure 1B). Higher product formation was achieved
when glutathione (GSH) or DTT were added. The best result was
recorded in the presence of 5 mM DTT, where 80% analytical
yield was obtained (Figure 1B). These results suggested that
oxygen has a large effect on protein stability.
Substrate scope
To investigate the substrate specificity of VanA, a panel of 19
compounds was selected (Scheme S1, Supporting
Information). Each biocatalytic reaction was set up with cofactor
regeneration as outlined in Scheme 1 and incubations were
carried out for 7 h.
At first, the biocatalyst was tested for the conversion of vanillin,
vanillyl alcohol, acetovanillone and methyl vanillate to their
corresponding catechols. No activity was detected, which
suggests a requirement for a carboxylic moiety for substrate
recognition. The result is in accordance with previous in vivo
studies on Acinetobacter, Streptomyces and P. fluorescens
vanillate demethylases.[30,39,40] The presence of longer aliphatic
chains in the more bulky compounds was unfavorable as well, and
homovanillic acid, ferulic acid, dihydroferulic acid, sinapyl acid
and vanilpyruvic acid were not accepted and no product formation
or substrate consumption was observed.
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Table 2 reports the obtained analytical yield of a series of
methoxy aromatic acids and related products observed by HPLC.
VanA showed high activity for 4-methyl-m-anisic acid (2a),
veratric acid (3a) and m-anisic acid (4a), and ≥85% of substrate
was converted to the corresponding phenol. Depletion of veratric
acid is comparable to vanillic acid, but the amount of product
increased by ca. 20% (Table S3, Supporting Information). This
improvement can be explained by the higher stability of isovanillic
acid (3b) vs the catechol functionality, resulting in a better product
recovery. Additionally, a control reaction showed that 3a is not
significantly subjected to breakdown by endogenous E. coli
enzymes, whereas up to 32% consumption of vanillic acid was
observed in the absence of the biocatalyst VanA-VanB (Table S1
and S4, Supporting Information).
88.6 ± 06
86.8 ± 2.6
n.d.[b]
In spite of the presence of two distinct methoxy functionalities, the
conversion of 3a led to only one product, implying that the
oxidative demethylation occurs regiospecifically at position 3 of
the aromatic ring (meta). To confirm the high regio-selectivity of
VanA, meta-, ortho- and para-anisic acid (4a, 5a and 6a) were
assayed. No product formation was observed after incubation
with 5a and 6a, while 4a showed 86.8% analytical yield.
Interesting, when both positions 3 and 5 bear a methoxy
substituent (7a), 3,4-dihydroxy-5-methoxy benzoic acid (8a) was
obtained in 60% yield, but the reaction did not go any further and
8a was not accepted as substrate. The latter result distinguishes
Ps(HR199)VanA from both AcVanA and StVanA. The first cannot
convert 7a, while StVanA is less specific, and generates a mixture
of 8a and gallate (StVanA) respectively.[30,40]
n.d.
60.0 ± 4.1
n.d.
Biocatalytic production of PCA
To investigate the applicability of the biocatalytic reactions
described here, a semi-preparative bioconversion was set up as
depicted in Scheme 1. An amount of 10 mg of vanillic acid was
added to 12 mL reaction mixture with 360 mg of freeze-dried cells
(Experimental Section). After vanillic acid was completely
consumed the product was isolated and purified and 7.1 mg of
PCA (77% yield) was obtained as white crystals; the product
identity was confirmed by NMR (Figure S13 and S14,
Supporting Information).
n.d
[c]
[a] Determined by HPLC. [b] n.d.: no product was observed. [c] 15 mg/mL
freeze-dried cells.
Table 2. Substrate scope of VanA-VanB. Reaction mixtures contained 7.5
mg/mL of freeze-dried cells, 0.1 mM NADH, cofactor regeneration system,
0.1 mM FeSO4, 10 mM DTT and 5 mM substrate. Product formation was
determined after 7 h of incubation.
Multi enzymatic cascades
The byproduct of oxidative demethylation is formaldehyde,
which is formed in stoichiometric amounts. Hibi and coworkers
showed by deletion studies in E. coli that formaldehyde can be
toxic to the cells, suggesting the necessity of a rapid detoxification
machinery for efficient biotransformation.[38] This raises the
question if formaldehyde is toxic to the overall cell machinery or
specifically affects VanA-VanB activity. To investigate its possible
impact on enzymatic activity, 2 mM formaldehyde was added to
the reaction mixture together with 5 mM vanillic acid. As a result,
the multi enzymatic system was inhibited and conversion dropped
by ~ 50% (Figure 2A).
Product
Observed
Product
Substrate
Analytical
Yield (%)[a]
69
Product inhibition or product toxicity can be overcome by
continuous removal of the undesired compound along the course
of the reaction. One method is to include an additional enzyme
which accepts the product as substrate. Formaldehyde
87.5 ± 0.7
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dehydrogenase (FADH) catalyzes the oxidation of formaldehyde
to formic acid together with the reduction of NAD+ to NADH. We
hypothesized that the combination of the three, VanA, VanB and
FADH, will result in a self-sufficient tandem reaction, where
cofactor regeneration was concomitant to elimination of
formaldehyde. The cascade could be further extended by
introduction of formate dehydrogenase (FDH), driving the reaction
to completion by converting formaldehyde to CO2. This system
would also favor the reaction workup and purification of the
phenolic product.
These proposed enzymatic cascades in which PTDH-driven
cofactor regeneration was omitted were tested for the synthesis
of PCA from 5 mM vanillic acid and compared to the standard
VanA-VanB system with NADH regeneration by PTDH (Figure 2).
Good conversion was achieved by both of the designed cascades
without the need of extensive optimization (one-step cascade with
FADH 56.5±1.9% analytical yield, two-step cascade with
FADH+FDH 84.4±3.3% analytical yield after 4 h reaction time).
However, a 5-fold higher concentration of NADH was required
compared to the 0.1 mM previously adopted. This can be
explained by the different nature of cofactor recycling machinery:
FADH and FDH activity are dependent on formaldehyde
formation by VanA-VanB, while PTDH reduces the cofactor by
phosphite that is present in excess from the beginning.
Conversions stopped after 2 h, probably due to VanA-VanB
instability. Whereas VanA-VanB-FADH seems comparable to
VanA-VanB-PTDH, a significant improvement was observed for
the 2-step aldehyde oxidation (VanA-VanB-FADH-FDH) and 4.1
mM PCA was obtained (Figure 2C).
Figure 2. Formaldehyde inhibition and cascade reaction. A: time course of
enzymatic demethylation of 5 mM vanillic acid under standard conditions
(green) and with addition of 2 mM formaldehyde (white). Dashed lines indicate
vanillic acid depletion and solid lines product formation. B: Cascade reaction for
removal of formaldehyde together with cofactor regeneration. FADH,
formaldehyde dehydrogenase; FDH, formate dehydrogenase. C: Bioconversion
of 5 mM vanillic acid with three different NADH regeneration systems and 15
mg/mL of freeze dried cells. Standard conditions with PTDH (green); one-step
cascade with 6 U/mL FADH (blue); and two-steps cascade with 6 U/mL FADH
and 10 U/mL FDH (purple). 0.5 mM NADH was employed for the conversion
with FADH and FDH. Dashed lines indicate vanillic acid depletion and solid lines
product formation.
Conclusions
The two-component enzymatic system VanA-VanB from
Pseudomonas sp. HR199 was recombinantly coexpressed in E.
coli in good amounts. The resulting biocatalyst governed oxidative
demethylation of methoxy ethers shows high conversion and high
selectivity for small aromatic acids with methoxy groups at the
meta position on the aromatic ring. VanA-VanB conveniently
accepts both NADH and NADPH as cofactor. After a semi-
preparative scale reaction, it was possible to isolate PCA in good
yields. Finally, as proof of concept, multi-enzymatic cascades
were established, demonstrating in situ cofactor regeneration
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Biocatalytic reaction
coupled to simultaneous byproduct removal from the reaction
mixture.
Stock solutions of 50 mM substrate were prepared in DMSO and stored at
4°C. Reaction mixtures (2 mL) were set up as follows unless otherwise
indicated. Samples containing 15, 30 or 60 mg of freeze-dried cells
expressing VanA-VanB (corresponding to 30, 60 or 120 mg of powder)
were weighted into a 15 mL glass vial and allowed to rehydrate in 100 mM
Tris-HCl pH 7.5 containing 10 mM DTT for 15-20 min at 30°C and 100 rpm
(shaker: Innova 40, New Brunswick). Afterwards, 0.1 mM FeSO4, 10 mM
phosphite, 0.01 mM PTDH and 5 mM of methoxy substrate were added to
the mixture. The reaction was initiated by the addition of 0.1 mM NADH
and it was incubated at 30°C at 100 rpm. Each experiment was performed
in duplicate.
The results suggest that VanA-VanB has good potential as
biocatalyst for the removal of methoxy groups from lignin
degradation products and other compounds, offering a valuable
green tool for lignin valorization and synthetic chemistry. For
future practical applications, structural studies would help enzyme
engineering for the improvement of stability and substrate
specificity of the biocatalyst.
Experimental Section
HPLC analysis
General
At different time points 200 µL of the reaction mixture was quenched with
200 µL of 100% acetonitrile (Boom B.V.). The sample was centrifuged for
10 mins at 16,000 g at 4°C and 200 µL of supernatant were transferred to
an HPLC vial. Analysis was performed with Jasco 980-31 HPLC apparatus,
Jasco 2075 Plus UV detector and Jasco MD-2010 Plus PDA detector. An
Alltima HP C18 5u length 250 mm ID 4.6 mm column was used for sample
separation. A solution of 2 mM sodium acetate containing 0.05% formic
acid (Eluent A) and 100% acetonitrile (Eluent B) were chosen as mobile
phase with the following method: 0.5 mL/min flow; 5-10 µL injection; 5 min
isocratic 95% A – 5% B; 15 min gradient to 5% A – 95% B; 5 min isocratic
5% A – 95% B; 5 min gradient to 95% A – 5% B; 10 min isocratic 95% A –
5% B. Substrate and products were detected at 254 nm and 280 nm.
Solvents and organic substrates were purchased from Sigma Aldrich, TCI
Europe and Abcr GmbH unless otherwise specified. NAD+ and NADH were
acquired from Carl Roth. Molecular Biology reagents were purchased from
New England Biolabs or ThermoFischer Scientific. DNA isolation and
purification kits were obtained from Qiagen.
Cloning
The synthetic genes of VanA (UniprotKB O05616) and VanB (UniprotKB
O05617) optimized for E. coli were purchased from GenScript. BsaI sites
suitable for Golden Gate cloning were inserted up and downstream of the
CDS by PCR. pET28(b)_GG was developed as universal and versatile
vector for an easy and highly efficient cloning of multiple fragments using
the Golden Gate method. The vector was prepared by Gibson cloning
using pET28(b)+ as template and a stuffer fragment was inserted. The two
CDS flanked by BsaI sites and a designed synthetic fragment containing
an additional T7 promoter were cloned into the pET28_GG vector in a one
pot reaction by Golden Gate cloning. The final product
pE2T_Ps(HR199)VanA_VanB was transformed in E. coli NEB 10 Beta and
positive clones were selected on LB kanamycin (50 mg/L). Primers, were
obtained from Sigma. Gene fragments were purchased from Integrated
DNA Technology (gBlock) and ThermoFisher Scientific (GeneArt strings).
Details are reported in Supporting Information. Phosphite
dehydrogenase was recombinantly expressed and purified as previously
described.[41]
Preparative scale and product isolation
A 12 mL reaction mixture was set up in a 50 mL bottle. An amount of 720
mg of biocatalyst powder (30 mg of freeze-dried cells expressing VanA-
VanB per mL of reaction mixture) was rehydrated in 100 mM Tris-HCl pH
7.5 (7.31 mL) and 10 mM DTT (0.120 mL of 1 M stock) at 30°C and 100
rpm. Next, the mixture was supplemented with 0.1 mM FeSO4, 10 mM
phosphite and 0.025 mM PTDH. Finally, the reaction was initiated with 0.2
mM NADH and 10 mg vanillic acid. The mixture was incubated overnight
at 30°C at 100 rpm. The complete substrate consumption was confirmed
by HPLC. Then, the reaction mixture was quenched by addition of a 10%
aqueous solution of citric acid, adjusted to pH~3 and extracted with ethyl
acetate (3 times). The combined organic extracts were washed with brine,
dried with anh. MgSO4, filtered and concentrated under reduced pressure.
The crude product was purified by flash chromatography on silica gel
Coexpression of VanA and VanB and biocatalyst preparation
SiliaFlash
P60
40–63
µm
(230-400
mesh)
(SiO2;
A
previously reported protocol was optimized for the heterologous
dichloromethane/methanol = 95:5). NMR spectra were recorded on Agilent
Technologies 400 NMR (1H NMR at 400 MHz, 13C NMR at 100 MHz).
Chemical shifts are expressed in ppm (δ).
coexpression of VanA and VanB in E. coli.[31] Competent cells of E. coli
BL21 Star (DE3) were transformed with the pE2T_Ps(HR199)VanA_VanB
vector. A sample of 200 µL of regenerated culture was immediately added
to 50 mL LB media supplemented with kanamycin (50 mg/L of culture)
followed by overnight growth at 37°C. Then, 5 mL of pre-culture were
transferred to 0.5 L TB medium with a double amount of glycerol (8% v/v)
and cultivation was done with kanamycin selection at 37°C under strong
agitation. At OD600 1, cells were cooled at 20°C for 20-30 min and then
protein expression was induced by addition of 0.5 mM IPTG, FeSO4 (0.1
mg/mL), Fe(III) ammonium citrate (0.1 mg/mL) and 1 mM cysteine. Protein
production was performed at 20°C for 20 h under moderate shaking. At the
end of the cultivation, cells were harvested by centrifugation (4000 rpm,
4°C, 10 min) and washed once with 50 mM Tris-HCl, pH 7.5. Next, the cell
pellet was resuspended in 100 mM Tris-HCl, pH 7.5, (250 mL) and shock
frozen drop by drop in liquid nitrogen. Finally, frozen cell-drops were dried
for 48 h (ice condenser CHRIST Alpha 2-4 LDplus). The obtained fluffy
powder (0.5 g of dried cells/g of powder) was kept at -20°C or at -80°C for
longer storage.
Acknowledgements
This work was supported by the European Commission (Marie
Skłodowska Curie Initial Research Training Network SUBICAT,
Call FP7-PEOPLE-2013-ITN, grant no. 607044) and the BE-Basic
R&D Program (http://www.be-basic.org/), which is financially
supported by an EOS Long Term grant from the Dutch Ministry of
Economic Affairs, Agriculture and Innovation (EL&I). The authors
thank Dr. Ulrich Commandeur and Selin Ece (RWTH Aachen
University) for providing E. coli BL21 Star (DE3) strain, and Dr.
Peter Deuss (University of Groningen) for his inputs.
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Enzymes for free phenols: The
enzymatic demethylation system
VanA-VanB from Pseudomonas was
characterized for the oxidative
demethylation of methoxy aromatic
acids into catechols and phenols.
Three different cascade options for
cofactor regeneration paired to
byproduct removal are proposed,
offering perspectives for innovative
biocatalytic methods for the
E Lanfranchi,* M Trajkovic, K Barta, JG
de Vries, DB Janssen
Page No. – Page No.
Title
dealkylation of aryl-methyl ethers.
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