Production of Propene from n-Butanol: A Three-Step Cascade Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTJE

Article abstract of DOI:10.1002/cbic.202000378

Propene is one of the most important starting materials in the chemical industry. Herein, we report an enzymatic cascade reaction for the biocatalytic production of propene starting from n-butanol, thus offering a biobased production from glucose. In order to create an efficient system, we faced the issue of an optimal cofactor supply for the fatty acid decarboxylase OleT_JE, which is said to be driven by either NAD(P)H or H₂O₂. In the first system, we used an alcohol and aldehyde dehydrogenase coupled to OleT_JE by the electron-transfer complex putidaredoxin reductase/putidaredoxin, allowing regeneration of the NAD⁺ cofactor. With the second system, we intended full oxidation of n-butanol to butyric acid, generating one equivalent of H₂O₂ that can be used for the oxidative decarboxylation. As the optimal substrate is a long-chain fatty acid, we also tried to create an improved variant for the decarboxylation of butyric acid by using rational protein design. Within a mutational study with 57 designed mutants, we generated the mutant OleT_V292I, which showed a 2.4-fold improvement in propene production in our H₂O₂-driven cascade system and reached total turnover numbers >1000.

Full text of DOI:10.1002/cbic.202000378

Combining Chemistry and Biology
Accepted Article
Title: Production of Propene from n-Butanol: A Three-Step Cascade
Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTJE
Authors: Daniel Bauer, Ioannis Zachos, and Volker Sieber
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1/2020

ChemBioChem
10.1002/cbic.202000378
FULL PAPER
Production of Propene from n-Butanol: A Three-Step Cascade
Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTJE
Daniel Bauer,+[a] Ioannis Zachos,+[a] and Volker Sieber*[a][b][c][d]
[
a]
D. Bauer, I. Zachos, Prof. Dr. V. Sieber
Chair of Chemistry of Biogenic Resources
Campus Straubing for Biotechnology and Sustainability, Technical University of Munich
Schulgasse 16, 94315 Straubing, Germany
E-mail: sieber@tum.de
[
[
[
[
b]
c]
d]
+]
Prof. Dr. V. Sieber
TUM Catalysis Research Center
Technical University of Munich
Ernst-Otto-Fischer-Straße 1, 85748 Garching, Germany
Prof. Dr. V. Sieber
Bio, Electro and Chemocatalysis BioCat, Straubing Branch
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
Schulgasse 11a, 94315 Straubing, Germany
Prof. Dr. V. Sieber
School of Chemistry and Molecular Biosciences, Chemistry Building 68
The University of Queensland
Cooper Road, St Lucia 4072, Queensland, Australia
These authors contributed equally.
Supporting information for this article is given via a link at the end of the document.
Abstract: Propene is one of the most important starting materials in
the chemical industry. Herein, we report an enzymatic cascade
reaction for the biocatalytic production of propene starting from
n-butanol, thus offering a biobased production from glucose. In order
to create an efficient system, we faced the issue of an optimal cofactor
supply for the fatty acid decarboxylase OleTJE, which is said to be
needs to meet the demand for a sustainable future. Therefore,
“green” and innovative solutions for production of bulk chemicals
and “drop-in” compatible fuels as well as niche products need to
be developed. Promising starting points for achieving this goal are
biomass-based materials (e.g. sugars and fatty acids), which are
sustainably available, e.g. from lignocellulose or phototrophic
organisms. In the case of sugars, recent studies have
demonstrated successful in vitro bioconversion of glucose to form
different alcohols like ethanol,[2] isopropanol[3] and n-butanol,[4]
which all are excellent molecular platforms to produce bulk
commodities.
2 2
driven by either NAD(P)H or H O . In the first system, we used an
alcohol and aldehyde dehydrogenase coupled to OleTJE via the
electron transfer complex putidaredoxin reductase/putidaredoxin,
+
allowing regeneration of the NAD cofactor. With the second system,
we intended full oxidation of n-butanol to butyric acid, generating one
equivalent of H
2
O
2
that can be used for the oxidative decarboxylation.
Additionally, fatty acids derived from biological resources like
plants or microorganisms, are important raw materials with high
potential,[ e.g. in the transformation to alkenes, which are ideal
platform molecules. As of recently, this class of molecules can be
produced enzymatically starting from fatty acids, such as the FAD
containing fatty acid photodecarboxylase from Chlorella variabilis
Since the optimal substrate is a long-chain fatty acid, we also tried to
create an improved variant for the decarboxylation of butyric acid
using rational protein design. Within a mutational study with 57
designed mutants, we generated the mutant OleTV292I, which showed
5]
2 2
a 2.4-fold improvement in propene production in our H O driven
NC64A,[ or the non-heme iron oxidase UndA
6]
[7]
and the
cascade system and reached total turnover numbers >1000.
desaturase-like enzyme UndB[8] from Pseudomonas. In 2011,
Rude et al. reported the fatty acid decarboxylase OleTJE
(
CYP152L1) from Jeotgalicoccus sp. ATCC 8456 as being the
Introduction
first
cytochrome
P450
peroxygenase
predominantly
as cosubstrate.[
9]
decarboxylating fatty acids using just H
2
O
2
Material utilization of renewable resources represents one of the
most promising ways of overcoming general global challenges,
such as the growing demand on oil and gas. The field has
gradually broadened as we face a difficult future with irreversibly
decreasing fossil fuel reserves and increasing environmental
concerns like global warming.[1] The use of alternative and
sustainable processes is often desirable for nearly all industrial
sectors. The established petroleum-based industry in particular
Interestingly, later results showed much higher total turnover
numbers (TTN) when using O as an oxidant and NAD(P)H as an
electron donor in combination with several redox partners,
indicating that OleTJE also can act as a monooxygenase.[10–16]
Recent studies refute this theory again and showed a sole activity
based on decoupling processes.[17] Although initial studies with
this enzyme showed only success with long- to medium-chain
fatty acids,[9,10,15,18,19,20,21,22] the substrate scope could be widened
2
1
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ChemBioChem
10.1002/cbic.202000378
FULL PAPER
to a variety of non-native substrates,[23] as well as carboxy
compounds[12,14,16,24,25] down to C4 fatty acids,[11] leading to
decarboxylated, hydroxylated and desaturated products. Besides
OleTJE, further olefin‑producing CYP152 peroxygenases have
been found in recent years (i.e. CYP-Sm46Δ29 from
shown). For that reason, we tried to optimize the reaction to obtain
higher yields by varying each enzyme concentration (Figure 1).
PpAOx and OleTJE had the greatest influence on the cascade,
showing a concentration dependent relation. However, excessive
PpAOx concentrations resulted in a slightly decreased propene
yield (Figure S4 in the Supporting Information), possibly indicating
inactivation of the cytochrome P450 active site or other enzymes
[
26]
Staphylococcus massiliensis S46,
OleTMC from Macrococcus
caseolyticus,[ OleTJH from Jeotgalicoccus halophilus, OleTSQ
from Salinicoccus qingdaonensis,[28] OleTSA from Staphylococcus
aureus[28]), whereby the early CYP152 family members and other
homologues predominantly catalyze the hydroxylation instead of
the decarboxylation of medium- to long-chain fatty acids (i.e.
P450SPα from Sphingomonas paucimobilis,[29] P450BSβ from
Bacillus subtilis,[ P450CLA from Clostridium acetobutylicum,[31]
CYP-Aa162 from Alicyclobacillus acidocaldarius LAA1,[26] CYP-
MP from Methylobacterium populi[32]).
27]
[28]
due to higher amounts of H
Matthews et al. showed a higher H
2
O
2
. It is worth mentioning here that
tolerance of OleTJE
2
O
2
compared to other bacterial P450 monooxygenases like the fatty
acid hydroxylase P450 BM3 heme domain, the M. tuberculosisꢀ
P450s CYP51B1 (sterol demethylase) and CYP121A1
(cyclodipeptide oxidase), which emphasizes its function as a
peroxygenase.[19] We further observed that increasing the
proportion of OleTJE resulted in an enhanced product formation
(Figure S4 and Table S5 in the Supporting Information). Moreover,
we figured out that the cascade reaches equilibrium after 5 h until
the reaction stops completely after 15 h to 18 h (Figure 2).
30]
In this study, we focus on the production of propene, which,
with an annual production of 80 million tons, is the most important
olefin other than ethylene in the chemical industry.[33] It is
assumed that the global demand for propene is expected to
increase to 165 million tons per year by 2030, which is about
1
2 % - 14 % more than the amount of propene that can be
produced by the conventional methods.[34] Currently, propene is
still predominantly produced through steam cracking of
hydrocarbons, notwithstanding the decrease of fossil resources
and global warming.[33] However, bio-based propene is already
being produced from sugar cane via chemical conversion of
bioethanol and ethylene.[35] Propene can also be produced by
enzymatic conversion starting from n-butanol using the oxidative
decarboxylase OleTJE for the last step. The possibility of
producing butanol from syngas opens a path to the fully
biotechnological production of propene from carbon dioxide and
renewable power, thus offering actually sustainable propene
production independent of the food vs. fuel debate. Nevertheless,
the optimal setup of such a cascade, which does not require
external reducing power, remains to be determined. This work is
a case study of novel cascade systems for the production of
propene from n-butanol, which could find an industrial application
in the future.
2 2
Scheme 1. H O dependent enzyme cascade for the production of propene
from n-butanol, using PpAOx, GstAlDH and OleTJE. LpNOx is required for the
cofactor regeneration.
30
AOx
AlDH
NOx
OleT
25
20
15
10
5
0
Results and Discussion
In initial tests, we used a H
system consisting of an alcohol oxidase from Pichia pastoris
PpAOx) and an aldehyde dehydrogenase from Geobacillus
2 2
O generating enzyme cascade
(
stearothermophilus (GstAlDH)[36] to achieve oxidation of
n-butanol to butyric acid in two steps. The last step, the oxidative
decarboxylation of the acid to the final product propene, was
performed by the cytochrome P450 peroxygenase OleTJE, which
simultaneously utilized the H
2
O
2
that was produced in situ by
+
PpAOx in the first step. For the regeneration of the NAD cofactor,
we decided to use NADH oxidase from Lactobacillus pentosus
———————— U L−1 ———————| |—— nM ——|
|
(
LpNOx) (Scheme 1: this cascade is indicated by an orange color
code in the following figures).[37] By using a water-forming instead
of a H -forming NOx, we wanted to avoid the accumulation of
excessive H concentrations, which often lead to rapid
inactivation of enzymes. The whole cascade was performed in
2
O
2
Figure 1. Production of propene at varying enzyme concentrations in H
2 2
O
driven cascade. Reaction conditions: 1 mL total reaction volume in a sealed
2
O
2
−
1
−1
[
38]
10 mL headspace glass vial contained 10 U L PpAOx, 575 U L GstAlDH,
1
77 U L−1 LpNOx, 154 nM OleTJE, 200 µM NAD , 10 mM n-butanol and buffer
+
a closed headspace glass vial system, so the necessary O
not added separately, but diffused from the ambient air.
2
was
(
50 mM Tris-HCl, pH 7.5). Reactions were performed at 30 °C and 170 rpm for
18 h. Varying PpAOx, GstAlDH, LpNOx or OleTJE concentrations while other
conditions remained constant.
Initial experiments with the described system were promising
but showed only modest amounts of propene (< 5 µM, data not
2
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ChemBioChem
10.1002/cbic.202000378
FULL PAPER
To proof whether the last oxidation step is actually driven by H
we added bovine liver catalase to the system. This experiment
was thus initially thought to work as negative control. To our own
2
O
2
,
following figures). Similar to the H
reactions were performed in closed headspace glass vials with no
external addition of O
Like the H dependent system, initial tests for the NAD(H)
2 2
O driven cascade, the
2
.
surprise, we achieved almost
a
doubling of propene
2 2
O
concentrations with catalase present (Figure S5 in the Supporting
Information), although we did not expect an increase due to the
absence of other redox partners for OleTJE. Remarkably, it
appears that 200 U mL−1 catalase is insufficient for completely
removing the peroxide from the system. Similar effects have been
reported before.[39,40] A possible explanation is the low affinity
dependent system were successful, so we followed up on the
optimization by varying the concentrations of the reaction partners
(Figure 3).
towards H
activity at above 50 mM H
high affinity for H (K
d
2
O
2
of bovine liver catalase, which reaches its full
.[41] On the other side a relatively
= 10.4 µM)[22] was determined for
2
O
2
2
O
2
OleTJE.[25] In this case, the catalase solely consumes excess of
peroxides, which leads to a reduced enzyme inactivation and,
therefore, yields in higher propene amounts. Adding 1000 U mL−1
catalase however resulted in decreased yields (Figure S6 in the
Supporting Information). The intermediate concentrations showed
an accumulation of butyric acid. Due to the high concentration of
Scheme 2. Potential monoxygenase-like setup of NAD(H) dependent enzyme
cascade to produce propene from n-butanol using GstADH, GstAlDH and
OleTJE. PdR and Pdx are required for the cofactor regeneration and electron
2 2
the present catalase, there is less amount of H O left for the
oxidative decarboxylation of the acid. It should also be mentioned
that LpNOx has a slight catalase activity.[37]
2 2
transfer to OleTJE. Additionally, H O can be a result of the autoxidation of the
redox donor or the formation of superoxide by OleT-oxy complex. OleTJE uses
only two of four electrons per reaction.
[
17]
2
1
1
1
1
1
000
800
600
400
200
000
900
ADH
AlDH
PdR/Pdx
NAD+
800
700
600
500
800
600
400
200
0
400
300
200
100
0
0
6
12
18
24
30
t / h →
Figure 2. Propene production over time in H
2 2
O driven system. Reaction
conditions: 1 mL total reaction volume in a sealed 10 mL headspace glass vial
−
1
−1
−1
contained 127 U L
5
PpAOx, 575 U L
GstAlDH, 177 U L
LpNOx,
−1
+
.6 mg mL OleTJE, 200 µM NAD , 10 mM n-butanol and buffer (50 mM KPi,
|———— U L−1 ————| |————— µM —————|
pH 7.5). Reactions were performed at 30 °C and 170 rpm for 26 h.
Figure 3. Production of propene at varying enzyme concentrations in NAD(H)
driven cascade. Reaction conditions: 1 mL total reaction volume in a sealed
Furthermore, we wanted to find out whether the cascade can be
run in a H
2
O
2
free system to overcome this issue. Assuming the
−
1
−1
1
0
0 mL headspace glass vial contained 82 U L GstADH, 575 U L GstAlDH,
validity of the theory of OleTJE being a monooxygenase,[
10–16]
we,
−1
−1
.5 µM PdR, 2.5 µM Pdx, 0.44 mg mL OleTJE, 200 U mL catalase, 200 µM
+
therefore, replaced H
2
O
2
producing PpAOx with an alcohol
NAD , 10 mM n-butanol and buffer (50 mM Tris-HCl, pH 7.5). Reactions were
performed at 30 °C and 170 rpm for 18 h. Varying GstADH, GstAlDH, PdR/Pdx
or NAD+ (with 200 U mL (■) or without (■) catalase) while other conditions
remained constant.
dehydrogenase (ADH). We decided to use ADH from Geobacillus
stearothermophilus (GstADH), due to its beneficial activity
towards n-butanol at 30 °C (data not shown). As already shown
by other groups, OleTJE is also able to work in a monooxygenase-
−1
like setup, utilizing O
donor. A wide range of potential redox partner systems have been
investigated for OleTJE
known redox partners putidaredoxin reductase (PdR) and
putidaredoxin (Pdx) from Pseudomonas putida, which acted
simultaneously as a cofactor regeneration system for GstADH
and GstAlDH as well as an electron transfer system for OleTJE
2
as an oxidant and NAD(P)H as an electron
While the first reaction step by GstADH had no significant
influence according to the enzyme concentration, the second
reaction step depended more on the GstAlDH concentration. We
also varied the ratio and concentration of the OleTJE redox
partners PdR and Pdx, showing that a 1 µM : 10 µM (PdR : Pdx)
ratio worked fine for the system. PdR and Pdx are not the natural
redox partners for OleTJE, so it is more likely that the electron
[
10,11,15,16]
.
In our case, we used the well-
(
Scheme 2: this cascade is indicated by a blue color code in the
3
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ChemBioChem
10.1002/cbic.202000378
FULL PAPER
transfer from the NADH cofactor to the cytochrome P450 enzyme
is not ideal and goes via rate-limiting decoupling processes.
Therefore, higher concentrations of the redox partners can
promote a better electron supply. Nevertheless, even higher Pdx
concentrations do not seem applicable in our point of view. The
likely, the cascade can circumvent shortages in electron supply
either by H O or by the NADH cofactor, depending on the most
2 2
favored path for the enzymes. This raises the possibility that
OleTJE actually only works as a peroxygenase and, if any,
additionally together with putidaredoxin as a NADH oxidase.
Recent studies support these facts by showing the production of
+
cofactor NAD had no significant influence above a concentration
of 100 µM. As expected, using the system without catalase raised
2 2
H O as a result of autoxidation, either of the redox cofactor, the
oxy-ferrous complex or from pyridine nucleotide itself.
[
17]
the yield of propene by 80 % - 100 %. This is due to H
2
O
2
, which
was generated by the uncoupling of the cytochrome P450
catalytic cycle[39,40] and which is supposed to be used by the
Given this knowledge and the optimization achieved thus far,
we wanted to improve the cascade by focusing on the last step,
the oxidative decarboxylation. Since OleTJE predominantly
decarboxylates medium- to long-chain fatty acids (C12-C20),
butyric acid (C4) might not bind optimally to the enzyme. We
therefore tried to optimize the active site as well as the substrate
tunnel to make it more suitable for short-chain fatty acids like
butyric acid.
decarboxylase. The production of H
flavoprotein PdR) in the presence of O
However, by comparing the H driven and NAD(H) driven
cascade, both systems seemed to be affected by H . While the
former favors the presence of catalase to get rid of excesses of
2
O
2
by flavin (e.g. from the
is also possible.[17,42]
2
2
O
2
2
O
2
H
H
2
O
O
2
, the latter is best when supported by a certain amount of
, thus making it a H and NAD(H) driven system. Most
2
2
2
O
2
d
200
180
160
140
120
100
80
60
40
20
0
Figure 4. a Active site and substrate tunnel (yellow, arachidic acid) of wild type OleTJE (PDB: 4L40)[22], highlighting all amino acid residues of our mutation studies
teal). Active site with binding pocket of b wild type OleTJE and c mutant OleTVal292Ile: The mutation of Val292 (b, gray residue) to Ile292 (c, pink residue) leads to a
shrinking of the substrate pocket volume, making it more favorable for the binding of short-chain fatty acids like butyric acid. d Mutation landscape. Reaction
(
−1
−1
conditions: 1 mL total reaction volume in a sealed 10 mL headspace glass vial contained 3 U L PpAOx, 2.25 mg mL OleTJE crude cell extract (every approach
for each OleTJE variant was adapted to the same final crude cell extract concentration of 2.25 mg mL−1), 5 mM methanol, 10 mM butyric acid and buffer (50 mM KPi,
pH 7.5). Reactions were performed at 30 °C and 170 rpm for 15 h. Methanol and butyric acid are the standard substances for the enzyme activity assay. The assay
2 2 2 2
consists of the oxidative decarboxylation of butyric acid by OleTJE with H O as substrate. H O is formed in situ by PpAOx, using methanol as substrate. The focus
of this experiment was not the cascade but solely activity screening for OleTJE. Figures were rendered with PyMol after minimization in Yasara using the Amber99
force field.
4
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In a first step, we were looking for key amino acid positions, which
could have a positive influence on the substrate pocket. According
to the work of Du et al. the most influential residues are Leu70,
Phe173, Val292, Phe296 and Ile402. Changing these residues to
those with larger side chains will shrink the volume of the binding
pocket and make a conversion of shorter fatty acids much more
favorable,[ which might have an influence on the decoupling
activity. We widened this spectrum using other residues which
might also have an influence on the binding pocket, the substrate
channel, the substrate positioning, the decarboxylation activity or
polarization (e.g. the proposed key residues Phe79, His85 and
Ile170).[15,18,26,27,44] To do so, we focused on amino acids within a
radius of 5 Å from the ligand. We neglected the residues near the
entrance of the substrate channel, expecting their impact on the
occlusion of the substrate close to the area of the binding pocket
to be only marginal. The remaining residues were selected
according to their structural position and, therefore, their resulting
potential influences on the substrate (Figure 4a). Based on the
model, Leu78 lies near the chain end of bound butyric acid and
might have a stabilization effect by narrowing the tunnel in
addition to a catalytic influence when changing the amino acid
produced nearly the same amount of propene. The same
tendency was shown when using those variants for the H O
2 2
driven cascade (Figure 5b). At this point, OleTV292I reached TTNs
of 1099 and produced 140 % more propene than the wild type
enzyme or OleTH85Q mutant, which both again yielded nearly the
same amount of propene (1.5 mM - 1.8 mM) and only reached
TTNs lower than 460. Using the NAD(H) driven system resulted
in an overall reduced product yield for all variants since we added
catalase to prove again the sole peroxygenase activity (Figure
5c). Interestingly, within this system OleTV292I only showed
marginal improvements in comparison to the wild type, whereas
OleTH85Q showed a strong decrease in propene yield. The clear
decrease in product yield for OleTH85Q using the NAD(H)
dependent system led us to believe that both mutants show higher
43]
2 2
activity in the presence of H O , while the interaction with the
redox partners is not as effective as for the wild type enzyme. This
could be due to conformational changes near the active site as a
result of the mutations. However, it remains unclear why the
NAD(H) dependent system did not work as well as the H
driven cascade for both OleT variants.
2 2
O
(
e.g. to His). Ile74 is located near the tunnel, indicating its
mutation capability for narrowing the substrate channel, which
could lead to a higher dwell time of the substrate in the binding
pocket. Phe291 could be a potential key residue for an alternative
channel for short-chained substrates, providing opportunities to
narrow or widen the channel by mutation.[14] Phe79 mutants have
already been tested by Matthews et al., but only for C10-C20 fatty
acids.[18] However, we wanted to investigate this residue for
butyric acid.
5000
a
b
c
4500
4000
3500
3000
In our screening, four mutants showed marginal to significant
improvements concerning the production of propene (Figure 4d).
In particular, the mutants with small improving effects were
His85Gln, Ile84Val and the double mutant Val292Ile/Ile170Val.
Val292Ile, the most promising mutant, showed a doubling of the
yield in comparison with the wild type. Five mutants (Phe79Leu,
Ile402Met, Ile170Val, Ala83Gly, Phe173Trp) showed up to 50 %
lower turnovers than the wild type enzyme. The rest of the
mutants showed only negligible activities. Earlier studies of other
groups predicted[22,45] and also showed[15,26] worse conversion
towards terminal alkenes for OleTJE His85 mutants due to the
2500
000
1500
000
2
1
500
0
histidine’s postulated role as
a
proton donor in the
decarboxylation reaction. In any case, Matthews et al. showed in
their catalytic studies that His85Gln still favored decarboxylation
reaction similar to the wild type,[18] which is confirmed by our
results. Also, other CYP152 enzymes like P450BSβ from
Bacillus subtilis as well as CYPMP from Methylobacterium populi
lacking the His85 (Gln85 in P450BSβ, Met96 in CYPMP) showed
decarboxylation activity besides the predominantly catalyzed
hydroxylation of fatty acids.[9,32] We had achieved similar results
in our own previous studies, but with lower alkene production
compared to the wild type enzyme and increased α-hydroxylation
Figure 5. Production of propene with OleTJE wild type and mutants Val292Ile
and His85Gln. a Comparison under screening conditions. Reaction conditions:
1
mL total reaction volume in a sealed 10 mL headspace glass vial contained
3 U L−1 PpAOx, 1.16 µM OleTJE variant (every approach for each OleTJE variant
was adapted to the same final concentration), 15 mM methanol, 10 mM butyric
acid and buffer (50 mM Tris-HCl, pH 7.5). Reactions were performed at 30 °C
and 170 rpm for 18 h. b Comparison of
2 2
H O driven cascade. Reaction
conditions: 1 mL total reaction volume in a sealed 10 mL headspace glass vial
−1
−1
−1
contained 127 U L PpAOx, 575 U mL GstAlDH, 177 U mL LpNOx, 4.01 µM
OleTJE variant (every approach for each OleTJE variant was adapted to the same
+
final concentration), 200 µM NAD , 10 mM n-butanol and buffer (50 mM
Tris-HCl, pH 7.5). Reactions were performed at 30 °C and 170 rpm for 18 h.
c Comparison of NAD(H) driven cascade. Reaction conditions: 1 mL total
(
data not shown), indicating that His85 is important but not
essential for the decarboxylation reaction of fatty acids.
reaction volume in
8
variant (every approach for each OleTJE variant was adapted to the same final
concentration), 1000 U mL−1 catalase, 200 µM NAD , 10 mM n-butanol and
buffer (50 mM Tris-HCl, pH 7.5). Reactions were performed at 30 °C and
170 rpm for 18 h.
a
sealed 10 mL headspace glass vial contained
2 U L−1ꢀGstADH, 575 U L ꢀGstAlDH, 1 µM PdR, 10 µM Pdx, 4.01 µM OleTJE
−1
To validate and investigate our results further, we decided to
compare the best mutant OleTV292I and OleTH85Q in their purified
form with the wild type under screening and cascade conditions.
We were able to reproduce our results from the screening when
using both mutants OleTV292I and OleTH85Q as well as wild type
OleTJE in purified form (Figure 5a). OleTV292I produced almost
twice the amount of propene as OleTH85Q and wild type, which
+
5
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ChemBioChem
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FULL PAPER
Examination of the active site of our mutant OleTV292I reveals a
hypothetical reason for the increased product yield in comparison
to the wild type enzyme. As Du et al. postulated in their
calculations,[43] the exchange of valine to the larger isoleucine
caused a narrowing of the substrate tunnel and the binding pocket
The cloning of the expression plasmid pET-28_gstAlDH (pET-28a(+)
vector carrying the GstAlDH gene from Geobacillus stearothermophilus
with C-terminal His-tag) was described previously.[36] The cloning of the
expression plasmid pET-28a_lpNOx (pET-28a(+) vector carrying the
LpNOx gene from Lactobacillus pentosus with N-terminal His-tag) was
previously.[37]
described
The expression plasmid pASK_oleT
(
Figure 4b and Figure 4c). This probably led to a more effective
(
pASK-IBA37plus vector carrying the OleTJE gene from Jeotgalicoccus sp.
binding and stabilization of the short-chained butyric acid,
resulting in higher yields of propene. Interestingly, the recently
characterized peroxygenases CYP152K6 from Bacillus
ATCC 8456) was provided by Robert Kourist (Ruhr-University Bochum,
Germany).[21] The cloning of the expression plasmid pET-22b_PdR (pET-
22b(+) vector carrying the PdR gene from Pseudomonas putida with C-
terminal His-tag) was described previously.[49,50] The cloning and site
directed mutagenesis of the expression plasmid pET-22b_Pdx (pET-
[
46]
methanolicus MGA3 and CYP152N1 from Exiguobacterium sp.
ATIb[
47]
have a phenylalanine at the equivalent V292 position in
2
2b(+) vector carrying the PdxC73S/C85S gene from Pseudomonas putida
OleTJE, most likely inducing a structural kink in the alkyl chain of
the tetradecanoic acid substrate relevant to the hydroxylation
reaction. Similar to the Phe292 residue in CYP152K6 and
CYP152N1, the Ile292 residue in OleTV292I occupies and narrows
the space between the heme and the substrate channel. In our
case, the narrowing seems to be more important for enclosing the
short-chain substrate. We did not detect any side products like
hydroxybutyric acids, which supports the finding of Dennig et al.,
who were also unable to detect hydroxy acids.[11] Furthermore, we
only detected none or marginal amounts of the intermediate
butanal of up to a maximum of 880 µM (Figure S6 and Figure S7
in the Supporting Information), depending on the OleTJE
concentration.
with C-terminal His-tag) was described previously.[
49,50]
OleTJE mutants
were produced by QuikChange mutagenesis. The primers were ordered
from Eurofins Genomics (Table S1 in the Supporting Information).
Reactions (50 µL) contained 10 µL Phusion® HF buffer (5×, New England
BioLabs, Ipswich, MA, USA), 10 µL dNTP Mix (each 10 mM), 1 µL forward
primer (10 µM), 1 µL reverse primer (10 µM), 20 ng template (pASK_OleT
for single mutants, OleTJE single mutant plasmids for double mutants),
5
µL DMSO, 1 µL Phusion® High-Fidelity DNA Polymerase (2000 U mL−1
New England BioLabs, Ipswich, MA, USA) and ddH O. Reactions were
thermocycled as followed: 98 °C for 2 min (step 1); 98 °C for 30 s,
7 °C - 64 °C for 1 min, 72 °C for 3 min (step 2, 16×); 72 °C for 10 min
,
2
5
(
step 3) and 4 °C for storage. Afterwards each sample was digested
overnight with 1 µL DpnI (20000 U mL−1, New England BioLabs, Ipswich,
®
MA, USA) at 37 °C. The DNA was purified using the NucleoSpin Gel and
PCR Clean-up Kit (Merchery-Nagel, Düren, Germany) as described in the
manual. E. coli BL21 (DE3) cells were chemically transformed with 10 µL
of each purified OleTJE mutant DNA (50 ng µL−1 - 80 ng µL−1), before they
were grown overnight on lysogeny broth (LB) agar medium supplemented
with 100 µg mL−1 ampicillin. Three clones were randomly selected and
sequenced from plasmid DNA using the forward or reverse primer
(Eurofins Genomics, Ebersberg, Germany).
Conclusion
In summary, both in vitro cascades provide a first promising basis
as a practical means for producing the relevant bulk chemical
propene from n-butanol, which can be derived from renewable
resources. However, we found strong indications that OleTJE
mainly works as a peroxygenase. With the help of enzyme
engineering, we produced the mutant OleTV292I, which showed an
almost 250 % production rate compared to the wild type. To the
best of our knowledge the highest conversion so far for propene
production with OleTJE was reported to be 5 %.[ Using our
improved system and our new variant, we were able to increase
the alkene production almost tenfold. Focusing on further enzyme
mutations will be one key element for future studies aiming in
enhancing the yield of the cascades. Therefore, suitable high-
throughput screening systems, like a recently established one for
detecting the conversion of medium-chain fatty acids by OleTJE,[48]
could play an essential role in reducing the screening effort.
Enzyme expression: A detailed list of the expression conditions (cells,
plasmids, media, supplements, antibiotics, incubation temperatures) for
the individual enzymes can be found in the Supporting Information
(Table S2 in the Supporting Information). Cells were chemically
transformed with plasmids, before they were grown overnight on LB agar
plates supplemented with antibiotic. 20 mL liquid media with antibiotic in
an 100 mL Erlenmeyer shaking flask were inoculated with one colony and
incubated overnight at 150 rpm on a rotary shaker. Part of this preculture
was used to inoculate the main culture in a 5 L Erlenmeyer shaking flask.
The main culture was incubated at 110 rpm on a rotary shaker for
16 h - 24 h. The cells were harvested by centrifugation (5000 × g, 15 min,
11]
4
°C) and stored at −20 °C.
Enzyme purification: All protein purification steps were carried out at
room temperature using an ÄKTA UPC-900 FPLC system (GE Healthcare,
Munich, Germany). All buffers (Table S3 in the Supporting Information)
were filtered with 0.2 μm regenerated cellulose membranes (VWR
International, Radnor USA).
Experimental Section
Materials: All chemicals, if not stated otherwise, were purchased from
Sigma-Aldrich (Steinheim, Germany) or Carl Roth (Karlsruhe, Germany).
Alcohol oxidase from Pichia pastoris (PpAOx, 21 U mg−1) was obtained
from Sigma-Aldrich (Steinheim, Germany); one unit will oxidize 1.0 μmole
of methanol to formaldehyde per min at pH 7.5 and 25 °C. Lysozyme from
egg white (≥35000 FIP U mg−1) was obtained from Carl Roth (Karlsruhe,
Germany); one FIP unit equates the protein concentration that induces a
reduction of the extinction of 0.001 per min at 450 nm, pH 7.0 and 25 °C,
Purification of GstADH, GstAlDH, LpNOx, PdR and Pdx: The cells were
thawed and resuspended to a 10 % (w/v) (20 % (w/v) for PdR and Pdx)
solution in binding buffer. The cell disruption was carried out once (twice
for PdR and Pdx) using a French press (Basic-Z Cell Disrupter, IUL
Constant Systems, Northants, UK) at 1.37 kbar. DNase I (1 µg mL−1) and
MgCl (2.5 mM) were added to the cell lysate (additionally FAD (50 µM)
2
for LpNOx and lysozyme (5 mg mL−1) for PdR and Pdx), and the solution
was incubated for 25 min at room temperature on a rocking shaker. The
cell debris was removed afterwards by centrifugation (35000 × g, 45 min,
using a solution of Micrococcus luteus as substrate. DNase I (4 U mg−1
)
was obtained from AppliChem GmbH (Darmstadt, Germany).
4
°C). The remaining crude extract was filtered through a syringe filter
(
0.45 µm, VWR International, Radnor, USA), before it was loaded on a
TM
Cloning: The cloning of the expression plasmid pET-28a_gstADH
5 mL HisTrap FF column (GE Healthcare, Munich, Germany), which was
preliminary equilibrated with binding buffer. After removing E. coli proteins
by washing the column with five column volumes binding buffer, protein
(
pET-28a(+) vector carrying the GstADH gene from Geobacillus
stearothermophilus with N-terminal His-tag) was described previously.[
3]
6
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.202000378
FULL PAPER
was eluted by using a gradient from 0 % to 100 % elution buffer. Eluted
fractions were subjected to 12 % SDS-PAGE.[51] The molecular weight of
10 mM n-butanol and buffer (50 mM Tris-HCl, pH 7.5) in a sealed 10 mL
headspace glass vial (Macherey-Nagel, Germany). No O
separately.
2
was added
the desired protein was calculated using the ExPASy ProtParam tool
52]
(
Table S4 in the Supporting Information).[ Fractions with the protein were
pooled and desalted using a HiPrepTM 26/10 Desalting column (GE
Healthcare, Munich, Germany), which was preliminary equilibrated with
desalting buffer. Pooled fractions of the protein (mixed with 1 mM DTT for
PdR and Pdx) were frozen in liquid nitrogen and stored at −80 °C.
NAD(H) driven catalysis: A typical reaction approach consists of 1 mL total
reaction volume with 82 U L−1 GstADH, 575 U L−1 GstAlDH, 0.5 µM PdR,
.5 µM Pdx, a maximum of 50 % (v/v) OleTJE (see captions), 200 U mL−1
2
+
catalase, 200 µM NAD , 10 mM n-butanol and buffer (50 mM Tris-HCl,
pH 7.5) in a sealed 10 mL headspace glass vial (Macherey-Nagel,
Purification of OleTJE and mutants: The cells were thawed and
2
Germany). No O was added separately.
resuspended to a 10 % (w/v) solution in binding buffer supplemented with
small amounts of lysozyme, 1 µg mL−1 DNase I and 2.5 mM MgCl
2
. The
Screening for suitable OleTJE mutants: E. coli BL21 (DE3) cells were
chemically transformed with 5 µL of each purified OleTJE mutant DNA (50-
incubation for 2 h at room temperature was followed by cell disruption with
a French press (Basic-Z Cell Disrupter, IUL Constant Systems, Northants,
UK) at 1.37 kbar. The cell debris was removed afterwards by centrifugation
0 ng µL−1), before they were grown overnight on LB agar medium
8
−1
supplemented with 100 µg mL ampicillin. Two clones of each mutant
(
35000 × g, 45 min, 4 °C). The remaining crude extract was filtered
were randomly selected to inoculate 2 × 1 mL LB medium with 50 µg mL−1
through a syringe filter (0.45 µm, VWR International, Radnor USA) before
it was loaded on a 5 mL HisTrapTM FF column (GE Healthcare, Munich,
Germany), which was preliminary equilibrated with binding buffer. After
removing E. coli proteins by washing the column with five column volumes
binding buffer, protein was eluted by using a gradient of 50 % binding
buffer and 50 % elution buffer. Eluted fractions were subjected to 12 %
SDS-PAGE (Figure S1 in the Supporting Information).[51] The molecular
weight of the desired protein was calculated using the ExPASy ProtParam
tool.[52] Fractions with the protein were pooled and dialyzed (3 × 12 h)
against 50 times excess of dialysis buffer at 4 °C to remove imidazole. The
purified protein was stored at 4 °C (Figure S8 in the Supporting
Information).
ampicillin in a 96 deep well plate (DWP) and incubated overnight at 37 °C
and 800 rpm on a plate shaker. These precultures were combined for each
mutant and used to inoculate 8 × 5 mL expression medium (900 mL TB
medium, 100 mL 10× KPi solution, 250 µL trace element solution,
0 µg mL−1 ampicillin) for each mutant in a 24 DWP, using 210 µL
preculture per 5 mL well. The main cultures were incubated at 37 °C and
5
8
00 rpm on a rotary shaker until an OD600nm of 0.7-0.8, before they were
−1
supplemented with 500 µM δ-aminolevulinic acid, induced with 0.2 mg L
anhydrotetracycline hydrochloride and incubated at 18 °C and 800 rpm for
additional 24 h. The cells were harvested by centrifugation (4500 × g,
15 min, 4 °C) and resuspended in 750 µL resuspension buffer (100 mM
KPi, 300 mM NaCl, pH 7.5). The cells of same mutants were combined
and centrifuged again (4500 × g, 15 min, 4 °C), before they were stored at
−20 °C.
Determination of protein concentration: The protein concentrations
were determined using the Bradford assay Roti®-Quant (Carl Roth,
Karlsruhe, Germany) with BSA as standard, following the instructions for
the microassay. In addition, some concentrations were measured at
The cells were thawed and resuspended in 3 mL desalting buffer (100 mM
KPi, 750 mM NaCl, 20 % (v/v) glycerol, pH 8.0) before being treated with
ultrasound (Sonoplus, BANDELIN and Hielscher LS24d10, 3 × 45 s with
280 nm using NanoPhotometer P 330 (Implen, Munich, Germany) with
parameters under reduced conditions (Table S4 in the Supporting
Information). Reduction was achieved by diluting the enzyme 1:1 with 12 M
guanidine hydrochloride.
4
5 s breaks, 70 % amplitude, 0.5 cycle). The cell debris was removed
afterwards by centrifugation (35000 × g, 45 min, 4 °C). The protein
concentration of the supernatant was determined using the Bradford assay.
For the subsequent assay, the protein concentrations were adjusted to the
lowest concentration of all mutants in order to obtain uniform protein
amounts for each sample. 1 mL total reaction volume in a sealed 10 mL
headspace glass vial (Macherey-Nagel, Germany) contained 3 U L−1
PpAOx, a maximum of 50 % (v/v) OleTJE crude cell extract, 5 mM
CO difference spectroscopy: The amount of active OleTJE was
[
53]
determined using CO difference spectroscopy. 90 µL of the protein in a
microtiter plate were diluted with 70 µL KPi (100 mM, pH 7.5) and 40 µL
2 2 4
CO buffer (1.3 mM KPi, 400 mM Na S O , pH 8.0), either treated with CO
gas for 5 min or not. The samples were measured with the FLUOstar
Omega (BMG Labtech, Ortenberg, Germany) at 400 nm - 500 nm before
the concentration of active enzyme was calculated by the differences of
the absorption A from the CO treated sample and the untreated sample
2
methanol, 10 mM butyric acid and buffer (50 mM KPi, pH 7.5). No O was
added separately. Reactions were performed at 30 °C and 170 rpm for
15 h before being stopped by heating the samples to 80 °C for 10 min. The
samples were then analyzed by GC-FID.
(for standardization, the absorption value at 490 nm was subtracted from
the absorption value at 450 nm). Protein concentrations measured with
CO difference spectroscopy are given in nM or µM. When no CO difference
spectroscopy was possible (due to facility reasons), the OleTJE
concentration was determined by Bradford assay or with the
NanoPhotometer P 330 (Implen, Munich, Germany), taking into account
that not all OleTJE protein was fully loaded. These values are given in
mg mL−1. Results across experiments with different mg mL−1 values are
not comparable.
GC-FID: Qualitatively and quantitatively analysis of propene formation was
carried out by gas chromatography using a Rt-QS-BOND column (15 m by
0
1
.53 mm, 20 µm film, Restek GmbH, Bad Homburg, Germany) on a Trace
310 gas chromatograph (Thermo Scientific, Waltham, MA, USA) with a
FID detector. The samples were incubated at 40 °C for 1 min before 1 mL
of the gaseous phase was injected (syringe temperature: 50 °C) and
separated on the column (inlet and detector 200 °C, 40 °C / hold 1 min,
0 °C min−1 to 50 °C / hold 0 min, 40 °C min−1 to 150 °C / hold 4 min).
1
Airgas standards (C2-C6 olefins, Restek GmbH, Bad Homburg, Germany)
were used for identification of propene (retention time RT = 3.357 min) and
preparation of calibration curves (Figure S2 in the Supporting Information).
Catalytic approach: Biocatalysis was done in two types of three-step
cascades, either depending on
2 2
H O or NAD(H). Reactions were
performed at 30 °C and 170 rpm for 18 h before being stopped by heating
the samples to 80 °C for 10 min. The samples were then analyzed by
GC-FID and GC-MS. In preparation for the HPLC measurements, the
aqueous phase was spin filtrated at 14000 × g for 15 min (centrifugal filter,
modified PES, 10K, VWR International, Radnor, USA) after finishing the
GC measurements.
GC-MS: Analysis of n-butanol, butyraldehyde and propene (Figure S3 in
the Supporting Information) was carried out using Rxi-5Sil-MS column
(
60 m by 0.53 mm, 20 µm film, Restek GmbH, Bad Homburg, Germany)
on a Trace GC Ultra gas chromatograph (Thermo Scientific, Waltham, MA,
USA) with a Quadrupol detector. The samples were incubated at 40 °C for
1
min before 1 mL of the gaseous phase was injected (syringe
H
2
O
2
driven catalysis: A typical reaction approach consists of 1 mL total
temperature: 50 °C) and separated on the column (inlet and detector
200 °C, 40 °C / hold 1 min, 10 °C min−1 to 50 °C / hold 0 min, 40 °C min−1
to 150 °C / hold 4 min).
reaction volume with 127 U L PpAOx, 575 U L−1 GstAlDH, 177 U L
−
1
−1
LpNOx, a maximum of 50 % (v/v) OleTJE (see captions), 200 µM NAD+,
7
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.202000378
FULL PAPER
HPLC: HPLC analytics were done according to previous methods.[54] The
concentrations of n-butanol, butyraldehyde and butyric acid in the aqueous
solutions were quantified using a HPLC system (Dionex, Sunnyvale, CA,
USA) equipped with a Rezex ROA-H+ column (Phenomenex, Torrance,
CA, USA), a refractive index detector (RI 101, Shodex, Tokyo, Japan) and
a PDA detector (210 nm, Dionex, Sunnyvale, CA, USA). The mobile phase
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Acknowledgements
The authors acknowledge financial support by the German
Federal Ministry of Education and Research (BMBF) for Daniel
Bauer (grant No. 03SF0446A) and Ioannis Zachos (grant No.
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Entry for the Table of Contents
Propene is produced in a three-step enzymatic cascade from n-butanol via butanal and butyric acid, with the last step being the oxidative
decarboxylation catalyzed by OleTJE. Variation of the supply of redox donors demonstrates the peroxygenase nature of OleTJE. A
mutational study of OleTJE optimizes the last step as the new variant OleTV292I shows significantly higher activity towards the short-
chain substrate than the wild type.
1
0
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