catalysts

Communication
An Enzyme Cascade Synthesis of Vanillin
Tobias Klaus, Alexander Seifert, Tim Häbe, Bettina M. Nestl and Bernhard Hauer *
Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet
Stuttgart, Allmandring 31, 70569 Stuttgart, Germany; tobias.klaus@curevac.com (T.K.);
alexander.seifert@gmx.net (A.S.); tim.t.haebe@ernaehrung.uni-giessen.de (T.H.);
bettina.nestl@itb.uni-stuttgart.de (B.M.N.)
* Correspondence: bernhard.hauer@itb.uni-stuttgart.de; Tel.: +49-711-685-64523




Received: 30 January 2019; Accepted: 21 February 2019; Published: 12 March 2019


Abstract: A novel approach for the synthesis of vanillin employing a three-step two-enzymatic
cascade sequence is reported. Cytochrome P450 monooxygenases are known to catalyse the selective
hydroxylation of aromatic compounds, which is one of the most challenging chemical reactions.
A set of rationally designed variants of CYP102A1 (P450 BM3) from Bacillus megaterium at the amino
acid positions 47, 51, 87, 328 and 437 was screened for conversion of the substrate 3-methylanisole
to vanillyl alcohol via the intermediate product 4-methylguaiacol. Furthermore, a vanillyl alcohol
oxidase (VAO) variant (F454Y) was selected as an alternative enzyme for the transformation of one of
the intermediate compounds via vanillyl alcohol to vanillin. As a proof of concept, the bi-enzymatic
three-step cascade conversion of 3-methylanisole to vanillin was successfully evaluated both in vitro
and in vivo.

Keywords: cytochrome P450; vanillin; enzyme cascade; vanillyl alcohol oxidase; flavor compound

1. Introduction
Vanillin is one of the most widely used flavouring compounds worldwide. Besides being a flavour
ingredient for food and beverages, it is used as a fragrance ingredient in perfumes and cosmetics [1],
as well as an intermediate in the chemical and pharmaceutical industries for the production of
antifoaming agents, herbicides and drugs [2]. However, natural vanillin extracted from vanilla pods is
very expensive, and the chemical synthesis of vanillin has several disadvantages including the lack
of substrate selectivity. Therefore, a variety of biotechnological approaches was developed for the
production of vanillin from eugenol, isoeugenol, ferulic acid, lignin, glucose, phenolic stilbene, vanillic
acid or aromatic amino acid starting materials from renewable feedstocks [3–5].
The biotechnological synthesis of vanillin from ferulic acid has gained increasing attention in
recent years. Artificial cascades combining a decarboxylase with an oxygenase were developed for
the one-pot synthesis of vanillin [6,7]. Aiming at the enzymatic synthesis of vanillin by the selective
hydroxylation of an aromatic substrate, cytochrome P450 monooxygenases (P450s) constitute ideal
candidates [8]. In general, P450s catalyse oxidation reactions introducing a single oxygen-atom from
molecular oxygen into the substrate with the concomitant reduction of the other oxygen atom to water [9].
The electrons necessary for P450-catalysed reactions are usually provided by NAD(P)H and transferred
via flavoprotein and/or iron–sulphur redox partner proteins. Recently, these haem-containing proteins
have been engineered for non-natural chemistries, thus expanding the biocatalytic repertoire [10–12].
One of the most investigated cytochrome P450 monooxygenases is CYP102A1 (P450 BM3) from
Bacillus megaterium. It is a natural self-sufficient fusion protein, consisting of a monooxygenase and
a diflavin reductase domain [13]. Originally, the wild-type enzyme was found to be a medium-
to long-chain fatty-acid hydroxylase, which requires only NADPH and oxygen to function and

Catalysts 2019, 9, 252; doi:10.3390/catal9030252 www.mdpi.com/journal/catalysts

Catalysts 2019, 9, 252 2 of 17

hydroxylates exclusively at sub-terminal positions [14]. In the last years, the substrate profile of
CYP102A1 has been extended by directed evolution and rational protein design approaches [15–22].
In previous work, a minimal and highly enriched mutant library of CYP102A1 has been constructed
by the combination of five hydrophobic amino acids (Ala, Leu, Ile, Phe and Val) at the two positions 87
and 328. These amino acid positions have been identified as hotspots of selectivity and specificity [23],
as they are located directly above the haem group and thus are expected to influence the accessibility
of the activated haem oxygen from opposite sides of the haem access channel [24]. Both positions were
already addressed in numerous previous studies [25]. The focused library, consisting of the CYP102A1
wild-type and 24 variants, was screened for conversion of the terpene substrates (4R)-limonene,
nerylacetone, geranylacetone and (+)-valencene. Eleven variants were found, that converted at least
one of the substrates with improved regio- or stereoselectivity [24]. Furthermore, variants of CYP102A1
were successfully applied to screen for hydroxylation of cyclic and acyclic alkanes as well as aromatic
substrates [21,26,27].
The vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum provides an alternative to
CYP102A1 and its variants. Similar to eugenol oxidase that has been reported for the synthesis of
vanillin from vanillyl alcohol [28], VAO is a flavoprotein with a covalently bound FAD cofactor [29].
It is known to be active with a wide range of 4-hydroxybenzylic compounds [30] and for its catalytic
cycle with 4-alkylphenols [31]. Both 4-methylguaiacol and vanillyl alcohol are known to be substrates
for VAO [32]. In 2004, van den Heuvel and coworkers identified several single variants of VAO
in a random mutagenesis approach, aiming at the generation of an enhanced reactivity concerning
the conversion of 4-methylguaiacol to vanillin, showing up to 40-fold increase in catalytic efficiency
(kcat /Km ) compared with the wild-type enzyme [33].
In the present work, we introduce the concept of enzymatic cascade reactions as an effective
means to selectively oxidise aromatic compounds for the synthesis of vanillin from the simple
aromatic phenylalkane 3-methylanisole (Scheme 1). In recent years, a plethora of reviews covering
multi- and chemo-enzymatic cascades for the production of various natural and non-natural
products have been reported [34–43]. By simultaneously screening the minimal and highly enriched
CYP102A1 mutant library and by generating CYP102A1 and VAO variants by rational protein design,
an alternative system for the synthesis of vanillin was formed as demonstrated by first in vitro and
then in vivo transformations.
Catalysts 2019, 9, 252 3 of 17
Catalysts 2018, 8, x FOR PEER REVIEW 3 of 18

Scheme 1. Proposed reaction pathway for the oxidation of 3-methylanisole (1) to vanillin (5) via the
Scheme 1. Proposed reaction pathway for the oxidation of 3‐methylanisole (1) to vanillin (5) via the
intermediate products 3-methoxybenzyl alcohol (2) or 4-methylguaiacol (3) and vanillyl alcohol (4).
intermediate products 3‐methoxybenzyl alcohol (2) or 4‐methylguaiacol (3) and vanillyl alcohol (4).
The reaction steps are intended to be catalysed by a combination of variants of the cytochrome P450
The reaction steps are intended to be catalysed by a combination of variants of the cytochrome P450
monooxygenase CYP102A1
monooxygenase CYP102A1andand
vanillyl alcohol
vanillyl oxidase.
alcohol oxidase.The
The preferred routeisishighlighted
preferred route highlighted in blue.
in blue.

2. Results and Discussion

In the present work, we introduce the concept of enzymatic cascade reactions as an effective
means to selectively oxidise aromatic compounds for the synthesis of vanillin from the simple
2.1. Screening of the Small Focused CYP102A1 Library
aromatic phenylalkane 3‐methylanisole (Scheme 1). In recent years, a plethora of reviews covering
multi‐
We and chemo‐enzymatic
studied the P450 enzyme cascades
CYP102A1 for the
for production of various
the synthesis natural
of vanillin fromand
annon‐natural products in
aromatic compound
have been reported
an enzyme-catalysed [34–43].
cascade By simultaneously
reaction, screening and
as both wild-type the minimal
variantsand
of highly enriched
CYP102A1 CYP102A1
were previously
mutant library and by generating CYP102A1 and VAO variants by rational protein design,
shown to successfully convert various aromatic substrates [21,25]. Hence, we tested the focused library an
alternative system for the synthesis of vanillin was formed as demonstrated by first in vitro and then
of CYP102A1 consisting of the wild-type and 24 variants (single and double variants constructed
in vivo transformations.
by the combination of five hydrophobic amino acids Ala, Leu, Ile, Phe and Val in two positions 87
and 328). The library
2. Results was screened for conversion of the substrate 3-methylanisole (1) as well as
and Discussion
the intermediate products 3-methoxybenzyl alcohol (2) and 4-methylguaiacol (3) by HPLC. Substrate
2.1. Screening
concentrations of the
of 0.5 mM Small
wereFocused
usedCYP102A1
in the firstLibrary
screening. To investigate a possible overoxidation of
the intended We studied the P450 enzyme CYP102A1 for thewas
product vanillyl alcohol (4), this compound also applied
synthesis for the
of vanillin screening.
from Whilst
an aromatic
3-methoxybenzyl
compound in an enzyme‐catalysed cascade reaction, as both wild‐type and variants of CYP102A1 any
alcohol (2), 4-methylguaiacol (3) and vanillyl alcohol (4) were not converted with
of the CYP102A1 variants,
were previously shownconversion of 3-methylanisole
to successfully convert various (1) was detected
aromatic with
substrates the wild-type
[21,25]. Hence, we as well as
tested
thevariants
22 of the focused (Table
library S1).
of CYP102A1
Only two consisting of the wild‐type
variants displayed and 24
no activity variants
towards (single and double
3-methylanisole (1) and
six variants, including the wild-type, showed only low activity (less than 5% conversion). However,
for six variants, a conversion of 3-methylanisole (1) higher than 25% was detected, with five of them
having a substitution of phenylalanine at position 87 by valine (F87V). Subsequently, a focus was set
Catalysts 2019, 9, 252 4 of 17

on the variants with the highest conversion results for 3-methylanisole (1), namely the single variants
A328L and F87V and the four double variants F87V/A328F/I/L/V. These variants, including the
wild-type for comparison, were investigated in more detail by GC–FID for the exact determination of
the conversion of substrate 3-methylanisole (1) and the respective product yields (Table 1 and Table S2).
Unfortunately, all of the investigated variants with relatively high activity displayed only low selectivity.
In addition to the desired intermediate products 3-methoxybenzyl alcohol (2) and 4-methylguaiacol (3),
generated by benzylic or aromatic hydroxylation, respectively, four other byproducts could be detected:
4-methoxy-2-methylphenol (6), 2-methoxy-6-methylphenol (8), m-cresol (9) and methylhydroquinone
(10) (Figure 1). Little product formation of 2-methoxy-6-methylphenol (8) (less than 1%) was detected
only using CYP102A1 variants F87V and F87V/A328F (data not shown in Table 1).
Whilst the CYP102A1 wild-type enzyme showed only very low conversion (<1%) of 3-methylanisole
(1) with compound 4-methoxy-2-methylphenol (6) as sole product, all single and double variants
were much more active towards the substrate. The main product for all reactions was 4-methoxy-2-
methylphenol (6), except for the variant F87V/A328I, where a mixture of 3-methoxybenzyl alcohol (2),
4-methoxy-2-methylphenol (6), and m-cresol (9) was produced in comparable amounts. The highest activity
of about 59% was achieved with F87V/A328L, whereas the respective single variants F87V and A328L
displayed only about 15% and 31% total conversion, respectively. Moreover, with F87V/A328L the product
yields of 3-methoxybenzyl alcohol (2) and m-cresol (9) raised to a maximum and methylhydroquinone
(10) came up as a new product. Biotransformations with m-cresol (9) and 4-methoxy-2-methylphenol
(6) demonstrated that the formation of methylhydroquinone (10) resulted from the conversion of
m-cresol (9). No product formation of methylhydroquinone (10) was observed in the transformation
of 4-methoxy-2-methylphenol (6). While F87V/A328L showed the highest conversion of 3-methylanisole
(1) to intermediate product 3-methoxybenzyl alcohol (2) (9.1%), the highest production of 4-methylguaiacol
(3) was achieved with the single variant A328L (2.7%). Formation of byproduct 7 was not detected.
Overoxidation was detected only for F87V/A328L and F87V/A328V. Methylhydroquinone (10) was
identified not to be the product of O-demethylation of 4-methoxy-2-methylphenol (6) but to be the
product of aromatic hydroxylation of m-cresol (9) (data not shown). We assume that the rate-enhancing
effect and the site-directing influence by the methyl and methoxy substituents lead to a preference
for the aromatic hydroxylation in ortho and para position of the methyl group (4-methylguaiacol (3),
4-methoxy-2-methylphenol (6), and 2-methoxy-6-methylphenol (8)) and in para position of the methoxy
group (4-methoxy-2-methylphenol (6)). As a result, 4-methoxy-2-methylphenol (6) was the main product
of the conversion of 3-methylanisole (1). Aromatic hydroxylation in meta position giving intermediate
3-methoxy-2-methylphenol (7) was clearly not the favoured reaction (Figure 1).

Table 1. Selected results of the conversion of 3-methylanisole (1) with variants of CYP102A1. Results
for all investigated variants of CYP102A1 are shown in Table S2.

CYP102A1 Variant Product Yield (% of Initial Substrate) Total

2 3 6 9 10 Conversion (%)

wild-type – – <1 – – <1

A328L <1 2.7 ± 0.2 26.6 ± 2.5 <1 – 31.0 ± 2.8
F87V <1 <1 12.5 ± 0.2 <1 – 14.9 ± 0.3
F87V/A328L 9.1 ± 0.2 1.6 ± 0.1 23.2 ± 0.4 12.0 ± 0.8 13.5 ± 0.8 59.3 ± 1.8
F87V/A328L/L437I 20.0 ± 0.9 <1 14.0 ± 0.7 7.4 ± 1.3 19.3 ± 0.5 61.3 ± 2.9
R47L/Y51F <1 – 1.5 ± 0.1 – – 1.6 ± 0.1
R47L/Y51F/F87V/A328V 5.0 ± 0.3 <1 24.0 ± 0.8 3.0 ± 0.1 2.1 ± 0.2 34.9 ± 1.4
R47L/Y51F/F87V/A328L/L437I 13.5 ± 0.4 <1 10.1 ± 0.5 7.7 ± 0.2 17.8 ± 0.8 50.4 ± 1.8
–, not detected or <0.05%; 2, 3-methoxybenzyl alcohol; 3, 4-methylguaiacol; 6, 4-methoxy-2-methylphenol;
9, m-cresol; 10, methylhydroquinone. Negative controls showed no conversion. Reactions were performed
in triplicates using a volume of 1 mL in Tris–HCl buffer pH 7.5 (50 mM) containing a glucose-6-phosphate
(5 mM)/glucose-6-phosphate dehydrogenase (5 U/mL and 1 mM MgCl2 ) cofactor regeneration system. Substrate
was added at a final concentration of 0.5 mM from a 25 mM stock solution in dimethyl sulphoxide (DMSO).
CYP102A1 and variants thereof were used at a final concentration of 1 µM. Reactions were run at 30 ◦ C and 180 rpm
for 2 h. Samples were derivatised and analysed by GC–FID.
substituents lead to a preference for the aromatic hydroxylation in ortho and para position of the
methyl group (4‐methylguaiacol (3), 4‐methoxy‐2‐methylphenol (6), and 2‐methoxy‐6‐methylphenol
(8)) and in para position of the methoxy group (4‐methoxy‐2‐methylphenol (6)). As a result, 4‐
methoxy‐2‐methylphenol (6) was the main product of the conversion of 3‐methylanisole (1).
Aromatic hydroxylation in meta position giving intermediate 3‐methoxy‐2‐methylphenol (7) was
Catalysts 2019, 9, 252 5 of 17
clearly not the favoured reaction (Figure 1).

Figure 1. Products of the conversion of 3-methylanisole (1) with selected CYP102A1 variants. The
Figure 1. Products of the conversion of 3‐methylanisole (1) with selected CYP102A1 variants. The
three potential reaction pathways for vanillin synthesis comprise (indicated with colored arrows;
three potential reaction pathways for vanillin synthesis comprise (indicated with colored arrows;
respective reaction product is framed): benzylic hydroxylation (blue), aromatic hydroxylation (red) and
respective reaction product is framed): benzylic hydroxylation (blue), aromatic hydroxylation (red)
O-demethylation (green). Formation of compound 3-methoxy-2-methylphenol (7) was not detected.
and O‐demethylation (green). Formation of compound 3‐methoxy‐2‐methylphenol (7) was not
Main product 4-methoxy-2-methylphenol (6) is highlighted in bold.
detected. Main product 4‐methoxy‐2‐methylphenol (6) is highlighted in bold.
In this work, we have identified that single and double variants displayed a much higher substrate
In this compared
conversion work, we tohave identified that
the wild-type, singlethose
especially and double
having avariants displayed
valine instead of aaphenylalanine
much higher
substrate conversion compared to the wild‐type, especially those having a valine
at position 87. We assume that this is due to the smaller size of the side-chain of valine comparedinstead of toa
phenylalanine at
phenylalanine, position
thus 87. We
allowing assume
a better that and
access this positioning
is due to theof
smaller size of the
the substrate to side‐chain
the activatedof valine
haem
compared to phenylalanine, thus allowing a better access and positioning of the substrate
oxygen in the active site. This effect of the F87V mutation on the hydroxylation of aromatic compounds, to the
activated haem oxygen in the active site. This effect of the F87V mutation on
resulting in an improved NADPH-consumption rate and coupling efficiency, was described in the hydroxylation of
aaromatic
previouscompounds,
work [44]. resulting in an improved
Unfortunately, the regio-NADPH‐consumption
and chemoselectivityrate andmost
of the coupling
activeefficiency,
variants
was described in a previous work [44]. Unfortunately, the regio‐ and chemoselectivity
was rather low as a variety of oxidation and demethylation products could be detected. Oxidation of the mostof
the aromatic ring occurred in three of the four possible positions with a preference for the para position
to the methoxy group, yielding 4-methoxy-2-methylphenol (6) as main product. Meta hydroxylation
did not occur in this work, which is in agreement with the results of a recent study [45].

2.2. Screening of Additional CYP102A1 Variants

As the activity concerning the conversion of 3-methylanisole (1) could be strongly enhanced
by generation of double variants of CYP102A1, additional CYP102A1 variants (triple variants)
were investigated with the aim to further improve the enzyme´s activity and especially selectivity.
In a previous work, it was shown that in addition to the amino acids at positions 87 and 328, the amino
acid at position 437 (leucine in the CYP102A1 wild-type) affects the orientation of the substrate to the
activated haem oxygen in the active site (Figure 2) and thus, influences both activity and selectivity of
the respective variant [46]. In analogy to this work, the variant F87V/A328L, which revealed the highest
activity to 3-methylanisole (1), was chosen for the combination with an additional mutation at position
L437. Additional mutations in position 437 were previously shown to be beneficial for the synthesis of
perillyl alcohol from (4R)-limonene, presumably due to the strong influence of the respective amino
Catalysts 2019, 9, 252 6 of 17

acid at this position on the orientation of the substrate in the active site [46]. The wild-type leucine was
replaced by alanine, phenylalanine, isoleucine and valine, as substrate-interacting amino acids in P450s
are preferentially hydrophobic [24]. The respective CYP102A1 triple variants were finally investigated
concerning the conversion of the 3-methylanisole (1) (Table 1 and Table S2), as well as the compounds
3-methoxybenzyl alcohol (2), 4-methylguaiacol (3) and vanillyl alcohol (4). While 3-methoxybenzyl
alcohol (2), 4-methylguaiacol (3) and vanillyl alcohol (4) were not converted by any of the triple variants
(data not shown), the mutations of L437 clearly influenced the enzyme activity and selectivity in the
conversion of 3-methylanisole (1). Replacement of leucine at amino acid position 437 by alanine or
phenylalanine dramatically decreased the total conversion of 3-methylanisole (1), whereas isoleucine
and valine at position 437 had no effect on the enzymatic activity. However, in this case, the product
distribution was different. About twice the amount of product 3-methoxybenzyl alcohol (2) was formed;
the concentration of produced compound 4-methylguaiacol (3), 4-methoxy-2-methylphenol (6) and
m-cresol (9) was reduced approximately by half; and the overoxidation product methylhydroquinone
(10), generated via 9, increased slightly. None of the investigated CYP102A1 variants was able to
catalyse the second reaction step of the cascade, accepting either 3-methoxybenzyl alcohol (2) or
Catalysts 2018, 8, x FOR(3)
4-methylguaiacol PEER
as aREVIEW
substrate. 7 of 18

Figure 2.
Figure Haem access
2. Haem access region
region in
in CYP102A1
CYP102A1 (PDB
(PDB ID:
ID: 1BU7,
1BU7, chain
chain A).
A). The
The image
image was
was generated
generated by
by
PyMOL. Active
PyMOL. Activesite
siteresidues,
residues,including
includingthe
the haem
haem group
group (green),
(green), a section
a section of the
of the I-helix,
I‐helix, B/C with
B/C loop loop
with F87, a section of the substrate recognition site 5 (SRS5) with A328, residue L437 (positioned
F87, a section of the substrate recognition site 5 (SRS5) with A328, residue L437 (positioned at the at
the substrate entrance to the active site cavity) and substrate channel residues R47 and Y51 are shown.
substrate entrance to the active site cavity) and substrate channel residues R47 and Y51 are shown.
The entrance route of the substrate to the active site is indicated by a red arrow.
The entrance route of the substrate to the active site is indicated by a red arrow.

So far many of the variants showed conversion of 3‐methylanisole (1) to intermediate product
3‐methoxybenzyl alcohol (2) and 4‐methylguaiacol (3) in various amounts in our study.
Unfortunately, due to the low selectivity of the enzyme variants, intense byproduct formation was
also detected. The aromatic hydroxylation in para position to the methyl group is less favoured
compared to the benzylic hydroxylation of substrate 3‐methylanisole (1), as intermediate product 3‐
Catalysts 2019, 9, 252 7 of 17

So far many of the variants showed conversion of 3-methylanisole (1) to intermediate product
3-methoxybenzyl alcohol (2) and 4-methylguaiacol (3) in various amounts in our study. Unfortunately,
due to the low selectivity of the enzyme variants, intense byproduct formation was also detected.
The aromatic hydroxylation in para position to the methyl group is less favoured compared to the
benzylic hydroxylation of substrate 3-methylanisole (1), as intermediate product 3-methoxybenzyl
alcohol (2) was detected in higher amounts than intermediate product 4-methylguaiacol (3) (product
formation of about 22% compared to less than 3%, respectively).

2.3. Molecular Dynamics Simulation

Based on the enzymatic preference for the benzylic hydroxylation compared to the aromatic
hydroxylation in para position to the methyl group, as mentioned above, the second pathway via
benzylic hydroxylation of the intermediate compound 4-methylguaiacol (3) to the product vanillyl
alcohol (4) was chosen for further investigation. Though many of the applied single, double and triple
variants of CYP102A1 showed methyl group hydroxylation of 3-methylanisole (1) and in spite of the
fact that 3-methylanisole (1) differs from 4-methylguaiacol (3) by only one hydroxyl group, the latter is
not converted by any of the minimal library variants. In order to unveil whether 4-methylguaiacol (3)
is able to bind in productive orientations in the active site cavity of CYP102A1 variant F87V/A328I,
multiple 100 ns molecular dynamics (MD) simulations (30) of the enzyme substrate complex were
carried out. In the simulations, 4-methylguaiacol (3) was frequently observed in three different
regions of the substrate binding cavity (Figure S1). In five out of 15 simulations, 4-methylguaiacol (3)
stayed close to its initial position in a distance of 10 Å from the activated oxygen. In six simulations,
4-methylguaiacol (3) approached the activated oxygen in different orientations to a distance suitable
for oxidation. As an example, in one simulation 4-methylguaiacol (3) moved towards the haem during
the first 10 ns and stayed stable for the remaining 90 ns with its ring methyl group exposed to the
activated haem oxygen with a C-OFE distance < 4 Å (Figure S2). This indicates that 4-methylguaiacol
(3) binds in a productive conformation to the active site of variant F87V/A328I. Interestingly, in the
remaining four simulations, 4-methylguaiacol (3) moved from its initial position to a pocket formed by
the β1 sheet and the A’ helix. The pocket is located in ~20 Å distance from the active site haem in the
substrate access channel. There, 4-methylguaiacol (3) was stabilised by polar interaction/hydrogen
bonds with residues R47 and Y51 (Figure S3). The stabilisation of the molecule in this pocket might
hamper its approach to the haem active site, which could explain the lacking activity of the variant
towards 4-methylguaiacol (3). As a consequence, the substitution of R47 and Y51 to nonpolar residues
should abolish these polar interactions, and therefore facilitate the approach of 4-methylguaiacol (3) to
the active site enabling its conversion.

2.4. Screening of Quadruple CYP102A1 Variants for Substrate Conversion

As a result of the MD simulations, R47L/Y51F-mutations were inserted in the previously
investigated single and double variants and in one of the triple variants, which showed the highest
activity for the conversion of 3-methylanisole (1). Subsequently, these quadruple variants and the one
quintuple variant were screened for the conversion of 4-methylguaiacol (3) as well as 3-methylanisole (1),
3-methoxybenzyl alcohol (2) and vanillyl alcohol (4). 4-Methylguaiacol (3), which was neither accepted
as a substrate by the CYP102A1 wild-type nor by any of the previously applied CYP102A1 single,
double and triple variants, was converted by all of the designed R47L/Y51F-variants, except for
R47L/Y51F and R47L/Y51F/A328L (Table S3). The highest conversion of 4-methylguaiacol (3) with
concomitant production of vanillyl alcohol (4) was measured with the variant R47L/Y51F/F87V/A328V
(4.5%). This indicates that the blocking of the substrate channel by hydrogen bond formation with
4-methylguaiacol (3) could be overcome. Besides, for all quadruple variants and the one quintuple variant,
a very small peak of an unknown byproduct was detected (data not shown). Due to the missing reference
substance, the amount of this compound could not be determined. Whilst compounds 3-methoxybenzyl
alcohol (2) and vanillyl alcohol (4) were not converted at all, conversion of 3-methylanisole (1) was
Catalysts 2019, 9, 252 8 of 17

achieved with all variants (Table 1 and Table S2). The R47L/Y51F-variant of the CYP102A1 wild-type
showed only little conversion of 3-methylanisole (1). Compared to the respective single, double and triple
variants, variants with the additional mutations R47L and Y51F demonstrated varying performance in
enzyme activity. Only variant R47L/Y51F/F87V/A328V displayed no change in activity and product
distribution compared to the respective double variant (F87V/A328V). The highest total conversion
of 50% was detected for the quintuple variant R47L/Y51F/F87V/A328L/L437I. Furthermore, product
distribution of the conversion of 3-methylanisole (1) with the R47L/Y51F-variants compared to the
variants without these mutations did not change dramatically, except that the overoxidation product
methylhydroquinone (10) was detected for some more variants.

2.5. In Vitro One-Pot Cascade Reactions

As CYP102A1 variants for both reaction steps from 3-methylanisole (1) to product vanillyl alcohol
(4) have now been identified, a one-pot two-step biocatalytic cascade sequence of cytochrome P450
monooxygenase-catalysed oxidation reactions was set up with the aim of the production of vanillyl
alcohol (4). For this purpose, the CYP102A1 variants A328L and R47L/Y51F/F87V/A328V were
chosen, as they were shown to be the most efficient variants concerning the conversion of 2 mM
of 3-methylanisole (1) to intermediate product 4-methylguaiacol (3) and further conversion of this
compound to product vanillyl alcohol (4), respectively. 3-Methylanisole (1) was converted to about
25% with 4-methoxy-2-methylphenol (6) as main product (19.3%) and small amounts of intermediate
4-methylguaiacol (3) (1.2%) (Table 2). A successive addition of the second enzyme after half of the
reaction time led to an increase in total conversion up to about 30%. Besides, the amount of produced
compounds 4-methylguaiacol (3) and 4-methoxy-2-methylphenol (6) increased slightly, whereas the
formation of compounds 3-methoxybenzyl alcohol (2) and m-cresol (9) (data not shown) decreased.
However, the intended product vanillyl alcohol (4) could not be detected in any of the two reaction
setups by GC–FID analysis. This was also confirmed by HPLC analysis of the samples (data not
shown). For this reason, we investigated a different cascade reaction setup with a known vanillyl
alcohol oxidase (VAO) from P. simplicissimum as a second enzyme in combination with the CYP102A1
variant A328L. This VAO is not only known to catalyse the conversion of vanillyl alcohol (4) to vanillin
(5), but also to catalyse the conversion of 4-methylguaiacol (3) via vanillyl alcohol (4) to vanillin (5).
As there is some information about improved variants [33], we chose the VAO variant F454Y according
to the published data and created it by site-directed mutagenesis. A comparison of the VAO wild-type
and F454Y variant in an in vitro conversion of 4-methylguaiacol (3) confirmed the much higher activity
of the single variant F454Y (data not shown). Therefore, VAO_F454Y was applied in combination with
A328L (best CYP102A1 variant for conversion of 3-methylanisole (1) to intermediate 4-methylguaiacol
(3) in the in vitro one-pot cascade reactions with VAO and P450 (Table 2). We assume that this results
from the low amount of 4-methylguaiacol (3) that is produced by A328L in the first reaction step and
the competitive conversion of 3-methylanisole (1) and 4-methylguaiacol (3), with a preference for
3-methylanisole (1), by the R47L/Y51F/F87V/A328V variant in the second reaction step. A successive
addition of the second enzyme after half of the reaction time improved the total conversion but was
not sufficient for the synthesis of vanillyl alcohol (4).
Though the total conversion and the formation of 4-methoxy-2-methylphenol (6) did not change
dramatically compared to the setup with the two CYP102A1 variants, in the system with the VAO
variant F454Y as a second enzyme, less 4-methylguaiacol (3) was detected. Moreover, the final
target product vanillin (5) could be detected by GC–FID. The formation of vanillyl alcohol (4) was
not observed. This clearly indicates that A328L catalysed the conversion of 3-methylanisole (1) to
4-methylguaiacol (3), which was then oxidised by the VAO variant F454Y via the intermediate product
vanillyl alcohol (4) to the product vanillin (5) (complete conversion of vanillyl alcohol to vanillin).
Byproducts m-cresol (9) and methylhydroquinone (10) were only detected in very low amounts (data
not shown).
Catalysts 2019, 9, 252 9 of 17

Table 2. Results of the in vitro one-pot conversion of 3-methylanisole (1) with a combination of the
CYP102A1 variants A328L and R47L/Y51F/F87V/A328V and a combination of A328L and the vanillyl
alcohol oxidase (VAO) variant F454Y.

Reaction Product Yield (% of Initial Substrate) Total

Enzymes
Time (h) 2 3 5 6 Conversion (%)

A328L 2 2.2 ± 0.1 1.2 ± 0.1 – 19.3 ± 1.3 24.7 ± 1.6

R47L/Y51F/F87V/A328V 1+1 1.2 ± 0.2 2.4 ± 0.5 – 25.6 ± 4.8 30.3 ± 5.7
A328L 2 <1 <1 <1 19.8 ± 2.7 22.9 ± 3.1
VAO_F454Y 1+1 1.0 ± 0.1 <1 1.2 ± 0.1 25.0 ± 2.2 29.0 ± 2.5
–, not detected or <0.05%; 2, 3-methoxybenzyl alcohol; 3, 4-methylguaiacol; 5, vanillin; 6, 4-methoxy-2-methylphenol;
and conversion time “1+1” indicates addition of the second enzyme after 1 h. Negative controls showed no
conversion. Reactions were performed in triplicates using a volume of 1 mL in Tris–HCl buffer pH 7.5 (50 mM).
Substrate was added at a final concentration of 2 mM from a 100-mM stock solution in dimethyl sulphoxide (DMSO).
CYP102A1 and variants thereof were used as lysate at a final concentration of 1 µM. Reactions were run at 30 ◦ C
and 180 rpm for 2 h. Samples were derivatised and analysed by GC–FID.

2.6. In Vivo Cascade Reactions

Finally, we were interested in the conversion of 3-methylanisole (1) to vanillin (5) in vivo with
E. coli as expression host. The advantages of an in vivo conversion with whole cells compared
to an in vitro system are especially the higher stability of the enzymes in a cellular system and the
employment of the NADPH cofactor and cofactor regeneration system of the host cells. Therefore, E. coli
BL21(DE3) cells were transformed with plasmids harboring genes for the enzymes CYP102A1_A328L
and VAO_F454Y. After protein expression, the cells were harvested and applied in the whole-cell
conversion setup. The results of the in vivo biotransformation are shown in Figure 3 and Table S4.
4-Methoxy-2-methylphenol (6), the main byproduct in all of our earlier investigations, was the only
product detected during the first hour of reaction time and stayed the main product throughout the
whole reaction. Besides, the intermediate products 3-methoxybenzyl alcohol (2) and 4-methylguaiacol
(3), byproduct m-cresol (9) and the target product vanillin (5) were detected after 3 h. Very interestingly,
the product yield of vanillin (5) increased in the course of reaction time, whilst the concentration
of -methoxybenzyl alcohol (2), 4-methylguaiacol (3) and m-cresol (9) stayed at a very low level and
vanillyl alcohol (4) was not detected at all. These findings confirm the functionality of the reaction
pathway from 3-methylanisole (1) to vanillin (5) in vivo. However, after 12 h, the total conversion
(11.7%) and the yield of product 5 (1.1%) reached the maximum, though glycerol and glucose were
supplemented again to the biotransformation medium. The successful formation of vanillin (5) was
confirmed by GC–MS analysis and comparison of the fragmentation pattern of the respective product
peak and a vanillin reference (Figure S4).
The high demand for natural flavor has resulted in the development of novel alternative
production processes for natural vanillin. Natural vanillin is extracted from the orchids Vanilla
planifolia, Vanilla tahitiensis, and Vanilla pompon [3,47]. However, the production of natural vanillin
from vanilla pods is very time consuming and expensive. Moreover, weather conditions as well as
plant diseases directly influence the market volume and process for vanillin extracted from vanilla
pods. Recently, the conversion of certain raw material to synthesize vanillin through biotechnological
means has been presented. Vanillin produced from alternate natural raw materials can be classified as
natural product. Biotechnological approaches for vanillin production are mostly based on conversion
of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid or aromatic amino acids. In view of the
above, it is considered desirable to further explore the raw materials that would lead to a possible
novel vanillin production pathway. In this work, the microbial transformation of the cheap and
readily available substrate 3-methylanisole to synthesise vanillin has been studied. This substrate
has shown promise due to its production by both chemical and biotechnological processes. In the
biotechnology-based approach, m-cresol, biosynthesised from acetyl-CoA and malonyl-CoA, can be
4‐methylguaiacol (3), byproduct m‐cresol (9) and the target product vanillin (5) were detected after 3
h. Very interestingly, the product yield of vanillin (5) increased in the course of reaction time, whilst
the concentration of ‐methoxybenzyl alcohol (2), 4‐methylguaiacol (3) and m‐cresol (9) stayed at a
very low level and vanillyl alcohol (4) was not detected at all. These findings confirm the functionality
of the reaction pathway from 3‐methylanisole (1) to vanillin (5) in vivo. However, after 12 h, the total
Catalysts 2019, 9, 252 10 of 17
conversion (11.7%) and the yield of product 5 (1.1%) reached the maximum, though glycerol and
glucose were supplemented again to the biotransformation medium. The successful formation of
vanillin (5)towas
converted theconfirmed by GC–MS analysis
desired 3-methylanisole andO-methyltransferase,
with an comparison of the fragmentation
which is thenpattern of the
deployed as
respective product
alternative peakmaterial
natural raw and a vanillin
for the reference
microbial(Figure S4). of vanillin.
production

Catalysts 2018, 8, x FOR PEER REVIEW 11 of 18

Figure
Figure 3. Total
3. Total conversionwithin
conversion within24
24hhreaction
reaction time
time (A) and
and product
productformation
formationwithin
within 1212 h reaction
h reaction
time
time (B)(B)
of of
thethe
in in vivo
vivo 3‐methylanisole(1)
3-methylanisole (1)cascade
cascade conversion
conversion with
withaacombination
combination of of
thethe
enzymes
enzymes
CYP102A1_A328L
CYP102A1_A328L andandVAO_F454Y.
VAO_F454Y.Due Dueto tothe
the fact
fact that
that no
no further
furtherconversion
conversionofofsubstrate
substrate after 12 h
after 12 h
reaction
reaction time
time was
was observed,product
observed, productformation
formation within
within this
this time
timeframe
frameisisshown.
shown.2, 2,
3‐methoxybenzyl
3-methoxybenzyl
alcohol;
alcohol; 3, 4‐methylguaiacol;5,5,vanillin;
3, 4-methylguaiacol; vanillin;6,6,4-methoxy-2-methylphenol;
4‐methoxy‐2‐methylphenol; 9,9,m‐cresol.
m-cresol.

The high
3. Materials demand for natural flavor has resulted in the development of novel alternative
and Methods
production processes for natural vanillin. Natural vanillin is extracted from the orchids Vanilla
3.1.planifolia,
MaterialsVanilla tahitiensis, and Vanilla pompon [3,47]. However, the production of natural vanillin
from vanilla pods is very time consuming and expensive. Moreover, weather conditions as well as
All chemicals were purchased in analytical-reagent grade or higher quality. Tryptone from casein
plant diseases directly influence the market volume and process for vanillin extracted from vanilla
was obtained from Fluka (Buchs, Switzerland), yeast extract and vanillin from Roth (Karlsruhe,
pods. Recently, the conversion of certain raw material to synthesize vanillin through biotechnological
Germany) and β-NADPH tetrasodium salt from Acros Organics (Geel, Belgium). Glucose-6-phosphate
means has been presented. Vanillin produced from alternate natural raw materials can be classified
dehydrogenase from Saccharomyces cerevisiae was purchased from Roche Diagnostics (Mannheim, Germany).
as natural product. Biotechnological approaches for vanillin production are mostly based on
conversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid or aromatic amino acids. In
view of the above, it is considered desirable to further explore the raw materials that would lead to a
possible novel vanillin production pathway. In this work, the microbial transformation of the cheap
and readily available substrate 3‐methylanisole to synthesise vanillin has been studied. This substrate
has shown promise due to its production by both chemical and biotechnological processes. In the
Catalysts 2019, 9, 252 11 of 17

3-Methylanisole, 4-methylguaiacol, 3-methoxybenzyl alcohol, vanillyl alcohol, vanillin and 3-methoxy-

5-methylphenol were obtained from Alfa Aesar (Karlsruhe, Germany), 2-methoxy-6- methylphenol from
Frinton Laboratories (Hainesport, NJ, USA), D-Glucose-6-phosphate disodium salt hydrate, m-cresol,
4-methoxy-2-methylphenol, methylhydroquinone and all other chemicals, solvents and buffer components
from Sigma-Aldrich (Schnelldorf, Germany). Oligonucleotides were bought from Metabion (Martinsried,
Germany) in salt-free form.

3.2. Plasmids and Strains

Escherichia coli DH5α from Invitrogen (now Life Technologies, Darmstadt, Germany) was used
as host for cloning. For recombinant gene expression, the plasmids pET-22b(+), pET-28a(+) and the
strain E. coli BL21(DE3) from Novagen (Madison, Wisconsin, USA) were used as expression vectors
and expression host, respectively.

3.3. Molecular Biology Techniques and Enzyme Expression

General molecular biological and microbiological experiments were carried according to standard
procedures [48]. The QuikChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, California,
USA) was used to introduce site-directed mutations, following the manufacturer’s protocol. Heterologous
expression of the CYP102A1 wild-type and variants in E. coli was done as reported previously [49].
Expression and cultivation was performed as described in the PhD thesis of Tobias Klaus (University
of Stuttgart, Germany) [50]. The codon-optimised vaoA gene from P. simplicissimum was obtained as
a synthetic gene (Life Technologies GmbH, Darmstadt, Germany). After cloning the wild-type vaoA
gene into the NdeI and XhoI cloning sites of the T7 promoter-based pET-22b(+) vector, the variant F454Y
was constructed by site-directed mutagenesis following the manufacturer’s protocol, mentioned above.
Successful cloning was verified by automated DNA-sequencing (GATC Biotech, Konstanz, Germany).
For protein expression, the pET-22b(+)_VAO_F454Y vector construct was transformed into competent
E. coli BL21(DE3) cells and transformants were selected on LB agar plates with ampicillin (100 µg/mL).
A volume of 400 mL LB medium supplemented with 100 µg/mL of ampicillin was inoculated with 2 mL
overnight culture—grown from a single colony—and grown at 37 ◦ C and 180 rpm. At an OD600 of 0.7–0.8,
the culture was supplemented with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and grown for
another 18 h at 20 ◦ C and 140 rpm. Harvest of cells was done by centrifugation (10,800× g, 20 min, 4 ◦ C),
followed by resuspension in buffer solution (50 mM Tris-HCl buffer pH 7.5 containing 500 mM NaCl, 5%
glycerol, 0.1 mM phenylmethylsulphonyl fluoride (PMSF)). Disruption of cells was done by sonication
on ice (6 × 1 min, interspaced by 30 s). Subsequently, the cell debris was removed by centrifugation
(37,000× g, 30 min, 4 ◦ C), and the soluble protein fraction was recovered and filtered through a 0.45-µm
sterile filter.

3.4. Determination of Protein Concentration

The concentrations of the CYP102A1 variants were determined by the CO differential spectral
assay as described previously [51,52] using an extinction coefficient of 91 mM−1 cm−1 . The Bradford
protein assay was used for the determination of total protein concentration [53].

3.5. In Vitro Biotransformations

Biotransformations were performed as described in the PhD thesis of Tobias Klaus (University
of Stuttgart, Germany) [50] using a final volume of 1 mL in 50 mM Tris-HCl buffer pH 7.5,
containing 1 mM MgCl2 , 5 mM glucose-6-phosphate (G6P) and 5 U/mL glucose-6-phosphate
dehydrogenase (G6PDH) for cofactor regeneration. For biotransformations, substrates 3-methylanisole,
4-methylguaiacol, 3-methoxy-benzyl alcohol and vanillyl alcohol were added at a final concentration
of 0.5 mM (from a 25 mM stock solution in dimethyl sulphoxide). A concentration of 1 µM or 0.5 µM
of CYP102A1 enzyme (lysate) was added for the conversion of 3-methylanisole or 4-methylguaiacol,
Catalysts 2019, 9, 252 12 of 17

3-methoxybenzyl alcohol and vanillyl alcohol, respectively. The reaction was started by the addition of
0.2 mM NADPH. Samples were incubated at 30 ◦ C and 180 rpm for 2 h.
One-pot cascade reactions using CYP102A1 variants A328L and R47L/Y51F/F87V/A328V were
conducted as described in the PhD thesis of Tobias Klaus (University of Stuttgart, Germany) [50] with
CYP102A1 enzyme variants (lysate) and the substrate 3-methylanisole added at a final concentration of
1 µM and 2 mM, respectively. For further evaluation, in some experiments, the second enzyme variant
was added after 1 h (total reaction time was 2 h). One-pot cascade reactions with a combination of the
CYP102A1 variant A328L and the VAO variant F454Y were performed with VAO_F454Y-lysate added
at a final VAO-lysate protein concentration of 1 mg/mL. Lysate of empty vector expression (pET-22b(+)
or pET-28a(+) in E. coli BL21(DE3)) was used as a negative control for the in vitro biotransformations.
Experiments were performed in triplicate.

3.6. In Vivo Biotransformations

Whole-cell biotransformations were performed as described in the PhD thesis of Tobias Klaus
(University of Stuttgart, Germany) [50]. Competent E. coli BL21(DE3) cells were transformed with
the plasmids pET-28a(+)_CYP102A1_A328L and pET-22b(+)_VAO_F454Y. Protein expression was
performed as described above with minor modifications. LB medium was supplemented with
both kanamycin (30 µg/mL) and ampicillin (100 µg/mL) and protein expression was induced by
adding 1 mM IPTG, 0.1 mM 5-aminolevulinic acid and 0.1 mM FeSO4 . After incubation at 20 ◦ C
and 140 rpm for 18 h, cells were harvested by centrifugation (2700× g, 20 min, 4 ◦ C), washed twice
with 50 mM Tris-HCl buffer pH 7.5, followed by resuspension in biotransformation medium. In vivo
biotransformations were carried out in 100 mL shake flasks with a volume of 30 mL biotransformation
medium. For bioconversions, fresh cell suspension (50 gcww /L) in 50 mM Tris-HCl buffer pH 7.5
with 1% (w/v) glycerol, 0.4% (w/v) D-glucose, 30 µg/mL kanamycin and 100 µg/mL ampicillin
was prepared. Biotransformations were started by the addition of substrate 3-methylanisole (from
a 200 mM stock solution in dimethyl sulfoxide) at a final concentration of 4 mM and incubated at 30 ◦ C
and 180 rpm for 24 h. After 12 h reaction time, cells were fed with a glycerol/glucose mixture (1% (w/v)
glycerol, 0.4% (w/v) D-glucose). For analysis, samples were collected at time points 0, 15 and 30 min
and 1, 2, 3, 4, 6, 8, 12 and 24 h after substrate addition. As negative controls, E. coli BL21(DE3) cells
transformed with empty vectors pET-28a(+) and pET-22b(+) were used. Experiments were performed
in triplicate.

3.7. Sample Treatment

Preparation of samples for GC or HPLC analysis was conducted as described in the PhD thesis of
Tobias Klaus (University of Stuttgart, Germany) [50]. Substrate conversion was stopped with 20 µL
concentrated HCl. For initial library screening by high-performance liquid chromatography (HPLC),
substrate and products were extracted from the aqueous reaction mixture by the addition of 0.5 g of
K2 CO3 and 0.5 mL of a 40/60 (% v/v) mixture of n-hexane/2-propanol. Samples were mixed for 2 min
and centrifuged for 3 min at 20,200× g. For analysis, 100 µL of organic phase was mixed with 100 µL
of n-hexane. In order to determine substrate conversion and product distribution, transformations
were extracted using 0.25 mL ethyl acetate containing the internal standard benzaldehyde (0.1 mM) or
catechol (0.5 mM) for gas chromatography (GC) analysis or HPLC analysis, respectively.

3.8. GC Analysis
GC analyses were performed as described in the PhD thesis of Tobias Klaus (University of
Stuttgart, Germany) [50]. Biotransformations were analysed on a GC–FID (GC-2010 from Shimadzu
(Kyoto, Japan) with flame ionization detector) equipped with a DB-5 capillary column (length, 30 m;
internal diameter, 0.25 mm; film thickness, 0.25 µm) from Agilent Technologies (Waldbronn, Germany).
Hydrogen was used as carrier gas (flow rate, 0.88 mL/min; linear velocity, 30 cm/s). The injector
and detector temperatures were set at 200 ◦ C and 330 ◦ C, respectively. The temperature program of
Catalysts 2019, 9, 252 13 of 17

the column oven was as follows: 100 ◦ C for 3 min, 10 ◦ C/min to 190 ◦ C, 75 ◦ C/min to 320 ◦ C and
hold for 3 min. Reference materials were used for the determination of substrate conversion and
product distribution. In this case, solutions with 0.01–0.5 mM reference substances in 50 mM Tris-HCl
buffer pH 7.5 were extracted with ethyl acetate containing internal standard, followed by analysis with
GC–FID to obtain straight-line calibration plots (determination of the internal response factor).
In addition to GC–FID, GC–MS measurements were performed on a GC-2010 with GCMS-QP2010
from Shimadzu (Kyoto, Japan) equipped with a DB-5MS capillary column (length, 30 m; internal
diameter, 0.25 mm; film thickness, 0.25 µm) from Agilent Technologies (Waldbronn, Germany). Helium
was used as carrier gas (flow rate, 4.3 mL/min; linear velocity, 30 cm/s). The injector temperature was
set at 250 ◦ C. Mass spectra were collected using electrospray ionisation (70 eV). The temperature
program of the column oven was as follows: 80 ◦ C for 1 min, 60 ◦ C/min to 140 ◦ C, hold for
3 min, 20 ◦ C/min to 160 ◦ C, hold for 5 min, 75 ◦ C/min to 320 ◦ C and hold for 2 min. The use
of reference materials and analysis of respective fragmentation patterns supported the identification of
reaction products.

3.9. HPLC Analysis

HPLC analyses were performed as described in the PhD thesis of Tobias Klaus (University of
Stuttgart, Germany) [50]. Biotransformations were analysed by normal phase high-performance
liquid chromatography on a 1200 Series HPLC from Agilent Technologies (Waldbronn, Germany)
equipped with a Luna Silica 5-µm column (particle size, 5 µm; pore size, 100 Å; length, 150 mm;
inner diameter, 4.6 mm) from Phenomenex (Aschaffenburg, Germany). A mixture of 2-propanol
with 0.2 M formic acid (A), 2-propanol (B) and n-hexane (C) was used as mobile phase. Gradient
elution was applied with solution A (constant 5%), solution B (0%–25% in 4 min, 25%–45% in 4 min)
and solution C to complement to 100% at 30 ◦ C and a flow rate of 0.8 mL/min. For initial library
screening, 40 µL of extracted sample was injected and analysed at a wavelength of 272 nm. The analysis
of 4-methylguaiacol conversion and product distribution was performed at 280 nm with 15 µL of
extracted sample. For calibration, standard solutions in the range of 0.01–0.5 mM reference substances
in 50 mM Tris-HCl buffer pH 7.5 were prepared and were extracted with ethyl acetate containing
internal standard.

3.10. Molecular Dynamics Simulations

Molecular dynamics (MD) simulations were performed with the GROMACS 4.5.3 software
(Department of Biophysical Chemistry, University of Groningen, Groningen, Netherlands) [54,55]
at T = 298K and 1 bar, using the leap-frog integrator [56] with a time step of 2 fs. Systems were
equilibrated for 2 ns with position restraints on the protein and the substrate during the first 50 ps,
while applying Nose-Hoover [57,58] temperature coupling and Parrinello-Rahman [59] pressure
coupling. This coupling scheme was also applied to the 100-ns production runs that were the basis
for analysis. Independent coupling for the protein group, the substrate group and the water/ions
group were applied every 5 ps for pressure and every 0.5 ps for temperature. Hydrogen-bond lengths
were constrained with the LINCS algorithm [60]. Long-range electrostatics was calculated with the
particle-mesh Ewald method (PME) [61,62]. Lennard-Jones interactions were treated with a cutoff and
capped at 1.2 nm. The CYP102A1 crystal structure PDB ID: 1BU7A (1.65 Å resolution) was chosen as the
protein coordinate file. The Pymol 0.99 program [63] was used to introduce the amino acid exchanges
at positions 87 and 328. The protein was parameterised with the Amber03 force field [64], with the
exception of the haem group, which was parameterised as previously described [24]. The substrate was
manually placed into the active site in a distance of 10 Å from the haem center. Substrate parameters
were derived from the AMBER03 force field and the general AMBER force field (GAFF) [65] as needed,
while charge parameters were calculated using the RESP ESP charge Derive Server (REDS) [66], using
the GAMESS-US quantum mechanics program [67] in conjunction with the RESP-A1B charge model.
Catalysts 2019, 9, 252 14 of 17

Water was described by the SPC/E model [68]. Counter-ions were added to neutralise the overall
system charge.

4. Conclusions
This research generated a variety of CYP102A1 single, double and triple variants to oxidise the
starting material 3-methylanisole to vanillyl alcohol, an important precursor of vanillin. Variants were
identified catalysing the conversion of 3-methylanisole (1) to both 3-methoxybenzyl alcohol (2) and
4-methylguaiacol (3). MD simulations have identified two additional mutation sites, and variants
thereof were successfully applied for the conversion of 4-methylguaiacol (3) to vanillyl alcohol (4).
Successful synthesis of vanillin (5) was finally achieved both in an in vitro and an in vivo whole-cell
cascade reaction, starting with 3-methylanisole (1) as a substrate and employing a combination of
variants of two different enzymes (CYP102A1_A328L and VAO_F454Y). The in vivo synthesis of
vanillin (5) reached a maximum of 1.1% after 12 h. Thus, as a proof of principle, a new biotechnological
pathway for the synthesis of vanillin (5) is reported in this work. Further optimisations of this cascade
synthesis need to be addressed in the future.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/3/252/s1,

Figure S1: Regions in CYP102A1 variant F87V/A328I active site cavity where 4-methylguaiacol (3) was frequently
observed during molecular dynamics (MD) simulations. The orange molecule shows the starting position,
green—the region close to the haem center and blue—the pocket formed by the β1 sheet and the A’ helix.
The substrate access channel is indicated by a red arrow. (B) is zoomed in on (A). Figure S2: Distance of
4-methylguaiacol (3) carbon atoms from the activated oxygen of the haem in a 100 ns MD run. The molecule
approaches the haem oxygen to a distance <4 Å (C7) where it stays stable for 90 ns. Lines show a moving average
of 20 data points. Figure S3: Representation of four exemplary MD simulations from a pool of 15 MD simulations.
The distance between all possible hydrogen bond donors and acceptors of 4-methylguaiacol (3) and the side chain
of Y51 (green, red and magenta lines) and R47 (all remaining lines) are shown. A hydrogen bond is considered
present at a hydrogen bond donor to acceptor distance ≤3.5 Å. Lines show a moving average of 20 data points.
Figure S4: GC–MS analysis chromatogram (A) and fragmentation patterns of reference compound (B) (0.05 mM
vanillin) and sample (C) (after 12 h conversion time) of the in vivo biotransformations. Table S1: Initial screening
results for the conversion of 3-methylanisole (1) catalysed by all 24 variants plus wild type of the CYP102A1
minimal mutant library. Lysate of empty vector expression (pET-22b(+), pET-28a(+)) was used as a negative
control and showed no conversion. Table S2: Results of the conversion of 3-methylanisole (1) with all investigated
variants of CYP102A1. Table S3: Results of the conversion of 4-methylguaiacol (3) with R47L/Y51F-variants of
CYP102A1. Table S4: Results of the in vivo conversion of 3-methylanisole (1) with a combination of the CYP102A1
variant A328L and the vanillyl alcohol oxidase (VAO) variant F454Y.
Author Contributions: T.K. performed the experiment, T.H. screened the first enzyme library, A.S. performed
molecular dynamics simulations and T.K., B.M.N. and B.H. wrote the paper.
Funding: This work was supported by the German Federal Ministry of Education and Research (BMBF) in the
frame of the “Systems Biology in Pseudomonas for Industrial Biocatalysis” project. We want to thank. Jürgen
Pleiss for bioinformatic support of this study.
Conflicts of Interest: The authors declare no conflict of interest.

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