An Engineered Self-Sufficient Biocatalyst Enables Scalable Production of Linear α-Olefins from Carboxylic Acids

Article abstract of DOI:10.1021/acscatal.8b01313

Fusing the decarboxylase OleT_JE and the reductase domain of P450BM3 creates a self-sufficient protein, OleT-BM3R, which is able to efficiently catalyze oxidative decarboxylation of carboxylic acids into linear α-olefins (LAOs) under mild aqueous conditions using O₂ as the oxidant and NADPH as the electron donor. The compatible electron transfer system installed in the fusion protein not only eliminates the need for auxiliary redox partners, but also results in boosted decarboxylation reactivity and broad substrate scope. Coupled with the phosphite dehydrogenase-based NADPH regeneration system, this enzymatic reaction proceeds with improved product titers of up to 2.51 g L^-1 and volumetric productivities of up to 209.2 mg L^-1 h^-1 at low catalyst loadings (～0.02 mol%). With its stability and scalability, this self-sufficient biocatalyst offers a nature-friendly approach to deliver LAOs.

Full text of DOI:10.1021/acscatal.8b01313

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Letter
An Engineered Self-sufficient Biocatalyst Enables Scalable
Production of Linear Alpha Olefins from Carboxylic Acids
Chen Lu, Fenglin Shen, Shuaibo Wang, Yuyang Wang, Juan Liu, Wen-Ju Bai, and Xiqing Wang
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01313 • Publication Date (Web): 23 May 2018
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ACS Catalysis
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An Engineered Self-sufficient Biocatalyst Enables Scalable Produc-
tion of Linear Alpha Olefins from Carboxylic Acids
Chen Lu,†§ Fenglin Shen,†§ Shuaibo Wang,§ Yuyang Wang,‡ Juan Liu,‡ Wen-Ju Bai,*ξ Xiqing
Wang*§
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§ College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu 225009, China
‡ Testing Center, Yangzhou University, Yangzhou, Jiangsu 225009, China
ξ Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA
ABSTRACT: Fusing the decarboxylase OleTJE and the reductase domain of P450BM3 creates a self-sufficient protein,
OleT-BM3R, which is able to efficiently catalyze oxidative decarboxylation of carboxylic acids into linear α-olefins (LAOs)
under mild aqueous conditions using O2 as the oxidant and NADPH as the electron donor. The compatible electron trans-
fer system installed in the fusion protein not only eliminates the need for auxiliary redox partners, but also results in
boosted decarboxylation reactivity and broad substrate scope. Coupled with the phosphite dehydrogenase based NADPH
regeneration system, this enzymatic reaction proceeds with improved product titers of up to 2.51 g L-1 and volumetric
productivities of up to 209.2 mg L−1 h−1 at low catalyst loadings (~0.02 mol%). With its stability and scalability, this self-
sufficient biocatalyst offers a nature-friendly approach to deliver LAOs.
KEYWORDS: Green chemistry, Biocatalysis, Protein engineering, Oxidative decarboxylation, α-Olefins
Linear α-olefins (LAOs) are next-generation fuels and
key feedstock chemicals for the production of surfactants,
lubricants, detergents, and polymers.1-5 Currently, these
olefins are predominantly produced from petroleum via
ethylene oligomerization that only yields even-numbered
terminal olefins.6-9 Considering dwindling fossil fuel re-
serves, pursuing a renewable route to access LAOs is of
urgency.10,11 Pleasingly, abundant and bioavailable fatty
acids (FAs) may provide an alternative source for the sus-
tainable production of LAOs through straightforward
decarbonylative dehydration or oxidative decarboxyla-
tion,12 which can lead to both even- and odd-numbered
LAOs. To achieve this goal, transition-metal catalysts in-
cluding palladium, rhodium, iridium, and iron have been
developed.13-20 While impressive, these approaches usually
suffer from harsh reaction conditions (T ≥110 °C) and
require in situ distillation of the product to maintain ac-
ceptable α-selectivity. Furthermore, activation of the FAs
with stoichiometric anhydrides is often needed, resulting
in extra waste. Nowadays, intensifying environmental
concerns urge scientists to investigate chemical transfor-
mations using green solvents, such as water, or eco-
friendly methods, including electrochemistry21-24 and bio-
catalysis.25-28 Accordingly, we aimed for a green strategy to
supply LAOs from FAs.
Recently, a cytochrome P450 enzyme, OleTJE, was
found to catalyze the oxidative decarboxylation of satu-
rated FAs into terminal olefins using H2O2 as the oxi-
dant.29 H2O2 deactivated this biocatalyst at millimolar
concentration and these H2O2-dependent OleTJE systems
had relatively low catalytic efficiency and narrow sub-
strate scope (FAs ≥ C8).30-35 When coupled with redox
partners, OleTJE could catalyze the same reaction with O2
as the oxidant and NAD(P)H as the electron donor.36-39
Addition of putidaredoxin reductase and putidaredoxin
(CamAB) to the reaction could increase the reactivity and
turnover number of OleTJE.37-39 However, preparation of
the CamAB-containing lysate and calibration of the lysate
versus OleTJE complicated the reaction system and would
limit its application. Previous effort to create a self-
sufficient enzyme by fusing OleTJE to P450RhF reductase
domain achieved H2O2-independent catalysis, but this
engineered enzyme unfortunately displayed low activity,
only being able to decarboxylate FAs C12-C18.36 In this
study, a more compatible electron transfer system is in-
stalled in an OleTJE-containing fusion protein, resulting in
a self-sufficient biocatalyst with increased decarboxyla-
tion reactivity. Our approach, employing O2 as the
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Table 1. The oxidative decarboxylation of various
saturated FAs by OleT-BM3R with NADPH recyclinga
1
2
3
4
Yield
[%]b
Entry
Substrate
Product
5
6
7
8
9
1
CH3(CH2)18CO2H (1a)
CH3(CH2)16CO2H (1b)
CH3(CH2)14CO2H (1c)
CH3(CH2)12CO2H (1d)
CH3(CH2)10CO2H (1e)
CH3(CH2)9CO2H (1f)
CH3(CH2)8CO2H (1g)
CH3(CH2)7CO2H (1h)
CH3(CH2)6CO2H (1i)
CH3(CH2)5CO2H (1j)
CH3(CH2)4CO2H (1k)
CH3(CH2)3CO2H (1l)
CH3(CH2)2CO2H (1m)
CH3(CH2)16CH=CH2 (2a)
CH3(CH2)14CH=CH2 (2b)
CH3(CH2)12CH=CH2 (2c)
CH3(CH2)10CH=CH2 (2d)
CH3(CH2)8CH=CH2 (2e)
CH3(CH2)7CH=CH2 (2f)
CH3(CH2)6CH=CH2 (2g)
CH3(CH2)5CH=CH2 (2h)
CH3(CH2)4CH=CH2 (2i)
CH3(CH2)3CH=CH2 (2j)
CH3(CH2)3CH=CH2 (2k)
CH3CH2CH=CH2 (2l)
70
73
60
52
46
58
70
47
40
70
68
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3
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Figure 1. (A) Structural schematic of OleT-BM3R shown
as the cartoon representation. OleT-BM3R is composed of
OleTJE (residues 1-418, PDB code 4L54, pink), FMN bind-
ing domain (residues 479-630 of P450BM3, PDB code
1BVY, cyan) and FAD/NADPH-binding domain (residues
660-1048 of P450BM3, PDB code 4DQL, blue) of
P450BM3. The cofactors, heme, FMN, FAD and NADP+
are displayed as sticks. (B) Oxidative decarboxylation of
carboxylic acids to olefins by OleT-BM3R (pictured in
lyophilized form) that uses O2 with NADPH recycling.
PTDH = phosphite dehydrogenase.
CH3CH=CH2 (2m)
14
aReaction conditions: purified OleT-BM3R (3 μM), substrate (1
mM), catalase (100 U mL-1), NADPH (200 μM), PTDH (2 μM),
sodium phosphite (0.01 M), 1 mL scale, rt, 12 h. determined by
GC or headspace GC (see the supporting information).
b
oxidant and NADPH as the electron donor, converts
structurally diversified carboxylic acids to their corre-
sponding olefins under mild aqueous conditions. This
stable fusion protein reduces the effort required for pro-
tein production and purification and simplifies the reac-
tion system, making it more suitable for synthetic and
industrial application.
acid (FA C18:0) in the presence of oxygen and NADPH at
ambient temperature [turnover number (TON) = 2520,
turnover frequency (TOF) = 472 h-1, NADPH coupling
efficiency = 61%]. In contrast, when the same reaction was
performed with OleTJE and free BM3R, the TOF and
NADPH coupling efficiency were only 65 h-1 and 29%,
respectively. These results prove an enhanced electron
transfer from BM3R to OleT in our fusion protein. Com-
pared with other redox systems for the decarboxylation of
FAs with OleTJE, this self-sufficient enzyme showed ad-
vantages in turnover number, rate of reaction, and
NADPH coupling efficiency (Table S1). When this reac-
tion was coupled to a phosphite dehydrogenase (PTDH)
based NADPH regeneration system, 44-46 the overall de-
carboxylation activity (TON = 2167) was maintained. Iso-
lated BM3R was susceptible to temperature-induced ac-
tivity loss (inactivation rate = 0.22 min-1 at 30 °C),47 but
the OleT-BM3R fusion protein was stable at 37 °C for 24 h
(Figure S2). Moreover, purified OleT-BM3R can be lyophi-
lized (Figure 1B) and conveniently stored at -20 °C for
months without significant activity loss. These results
demonstrate that OleTJE is able to complement BM3R in
To start, we chose CYP102A1 (P450BM3) from Bacillus
megaterium as the template for the construction of the
self-sufficient OleTJE fusion protein. The heme domain of
P450BM3 is fused with the reductase domain (BM3R) as a
single polypeptide, establishing an efficient electron
transfer to the heme iron that is not contingent on en-
countering a discrete redox partner.40,41 Powered by this
efficient electron transfer chain, P450BM3’s hydroxylase
activity is >1000 fold higher than those of other P450 fatty
acid hydroxylases.42 Superimposition of their crystal
structures revealed a similar structural fold between
30
OleTJE and the heme domain of P450BM343 (RMSD=3.6
Å) (Figure S1). The replacement of the heme domain of
P450BM3 with OleTJE (residue 1-418) resulted in a soluble
protein, named OleT-BM3R (Figure 1A). Preliminary ex-
periments showed that the fusion protein OleT-BM3R
exhibited high decarboxylation activity towards stearic
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ACS Catalysis
Table 2. Further substrate scope study.a
P450BM3, together forming a structurally stable and func-
1
tionally active entity.
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Yield
[%]b
With the engineered OleT-BM3R in hand, we proceed-
ed to explore the reactivity and scope of the oxidative
decarboxylation of FAs (Table 1). Gratifyingly, both long-
and medium-chain saturated FAs 1a-k successfully partic-
ipated in this transformation, delivering the desired LAOs
2a-k up to 73% yield (entries 1-11). Short-chain fatty acids
1l-m also worked albeit with meager yields (entries 12-13).
These results indicate good compatibility between OleT
and BM3R in the fusion protein. Notably, utilizing natu-
rally occurring even-numbered saturated FAs afforded
prohibitively expensive odd-numbered terminal olefins,
such as 2a and 2b, which are inaccessible through the
oligomerization of ethylene. Meanwhile, α- and β-
hydroxylations occurred alongside the desired decarboxy-
lation process. Formation of the nonvolatile α/β-
hydroxylated products was also dependent on chain
length (Table S2). The catalytic cycle of OleTJE involves
abstraction of a Cα/Cβ hydrogen atom of the carboxylic
acid by the iron (IV)-oxo heme π-cation radical interme-
diate (Compound I), followed by a competition between
the ·OH rebound and carbon-carbon scission that delivers
the corresponding hydroxylation and decarboxylation
products.30,48-50 This mechanism guarantees the exclusive
formation of α-alkenes from the decarboxylation pathway.
As a result, this mild enzymatic approach displays ad-
vantages over synthetic methods, which employ transi-
tion-metal catalysts under harsh conditions and thus par-
tially form the thermodynamically stable internal alkenes.
Entry
Substrate
Product
1
30
30
78
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3
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44
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48
42
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40
To extend the synthetic application of this enzymatic
transformation, we further explored its substrate scope
and functional group tolerance (Table 2). To our delight,
both cyclic and acyclic α-branched carboxylic acids 3a-b
underwent the expected reaction, providing the corre-
sponding olefins in reasonable yields (entries 1-2). Simi-
larly, phenylpropanoic acids 3c-d smoothly delivered the
respective vinylbenzenes 4c-d with improved yields (en-
tries 3-4) and only traces of hydroxylation products were
formed in these reactions (Table S2). Functional groups
like olefins and ketones were tolerated and a free-
hydroxyl moiety was also untouched during the reaction
(entries 5-7). Remarkably, sensitive groups such as bromo
and aldehyde, which are vulnerable under conditions us-
ing transition-metal catalysts, survived due to our mild
enzymatic conditions (entries 8-9). When diacid 3j was
examined, the anticipated oxidative decarboxylation oc-
curred on both acids (entry 10). Interestingly, only termi-
nal diene was formed. Although no ω-alkenoic acid in-
termediate was found, its α/β-hydroxy derivatives were
detected (Table S2), indicating that the dicarboxylic acid
underwent tandem decarboxylation via ω-alkenoic acid
intermediate.
9c
11d
10
42
aReaction conditions: see conditions in Table 1. bdeter-
mined by headspace GC. csubstrate (10 mM), sodium
phosphite (0.05 M), 30 mL scale, 20 h. disolated yield.
(entry
1
vs 2). Compared to the previous
OleT/CamAB/FDH cascade system,37 our newly devel-
oped OleT-BM3R/PTDH coupled system offers higher
product titer (1.40 g L-1 vs. 0.93 g L-1) and volumetric
productivity (116.7 mg L-1 h-1 vs. 42.5 mg L-1 h-1), presuma-
bly because of the greater TOF (472 h-1 vs. 53 h-1) and
NADPH coupling efficiency (61% vs. 25%). Meanwhile,
simultaneously elevating the concentrations of both cata-
lyst and substrate further benefited the product titer and
volumetic productivity at the cost of the conversion (en-
tries 3-4). The product titer of our OleT-BM3R/PTDH
system could reach as high as 2.51 g L-1 while the volumet-
ric productivity climbed up to 209.2 mg L−1 h−1 at a low
catalyst loading (~0.02 mol%) (entry 4). Notably, cell-free
lysate allowed the production of 1.26 g L-1 (5.28 mM, 53%
yield) 1-heptadecene from 10 mM stearic acid without the
To testify its potential for preparative application, this
enzymatic transformation was performed in a concentrat-
ed manner with stearic acid as a substrate (Table 3).
Simply increasing the substrate concentration from 1 mM
to 10 mM led to a slightly diminished yield but dramati-
cally improved product titer and volumetric productivity
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Table 3. Oxidative decarboxylation of stearic acid at
Page 4 of 7
ibility of OleTJE and BM3R, and the structural elements in
P450BM3 that facilitate domain-domain interactions and
electron transfer processes altogether contribute to the
boosted decarboxylation reactivity and broad substrate
scope of the fusion protein. The stability and scalability of
this enzyme make it a useful biocatalytic platform for the
sustainable synthesis of LAOs. In addition, high ferricya-
nide and cytochrome c reduction rates (9850 min-1 and
3330 min-1, respectively) were determined for this fusion
protein, indicating that it was powered by the rapid elec-
tron transfer in BM3R.51 But a comparable reaction rate of
P450BM3-catalyzed fatty acid hydroxylation (usually
TOF>400 min-1) was not observed.42 Thus, further engi-
neering this fusion protein for enhanced interaction and
electron transfer between the FMN binding domain and
OleT, which might additionally benefit its catalytic per-
formance, is worth future exploration. Meanwhile, pro-
duction of LAOs in microbial platforms with this biocom-
patible catalyst is also underway in our laboratories.
1
2
3
4
5
6
7
8
various substrate concentrations by OleT-BM3R with
NADPH recycling.a
Entry
OleT-
BM3R
[μM]
Substrate Yield
Titer
[g L-1]
Volumetic
productivity
[mg L-1 h-1]
[mM]
[%]
1
3
1
73
59
40
26
53
54
0.17
1.40
1.91
2.51
1.26
1.29
14.5
9
2
3
4
5
6
3
10
20
40
10
10
116.7
10
11
12
13
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15
16
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40
41
42
43
44
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46
47
48
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52
53
54
55
56
57
58
59
60
9
159.2
9
209.2
105.0
5b
3c
107.5
aReaction conditions: OleT-BM3R (indicated), catalase (100 U
mL-1), NADPH (200 μM), PTDH (2 μM), sodium phosphite
(0.05 M), 1 mL scale, rt, 12 h; bCell-free lysate without
NADPH supplement. cThe activity of lyophilized OleT-BM3R
was tested after 2-month storage at -20 °C.
AUTHOR INFORMATION
Corresponding Author
*E-mail: xiqingwang@yzu.edu.cn, bai1@stanford.edu
Author Contributions
†These authors contributed equally.
ASSOCIATED CONTENT
Supporting Information includes Experimental Procedures
1
GC/Headspace GC results and H Spectra data. This material
is available free of charge via the Internet at
http://pubs.acs.org.
Figure 2. Gram scale enzymatic oxidative decarboxylation
of stearic acid.
ABBREVIATIONS
LAOs, linear α-olefins; FAs, fatty acids ; TON, turnover num-
ber; TOF, turnover frequency; PTDH, phosphite dehydro-
genase; NADP, nicotinamide adenine dinucleotide phos-
phate.
need for protein purification (entry 5). This cell-free ly-
sate system is economically desirable as no additional
NADPH is required. Additionally, lyophilized OleT-BM3R
exhibited comparable reactivity after two-month storage
at -20 °C (entry 6). Moreover, our enzymatic system
proved scalable, and 1g of stearic acid was successfully
converted to 1-heptadecene in 60% yield (Figure 2). As
the obtained product is particularly expensive, our enzy-
matic reaction provides this LAO not only through a sus-
tainable approach, but also in a cost-effective fashion.
Overall, the outstanding stability and scalability of this
self-sufficient biocatalyst demonstrates encouraging po-
tential for preparation of LAOs.
ACKNOWLEDGMENT
We thank Christopher A. Kalnmals (Stanford University) for
critical reading of the manuscript. X. W. acknowledges the
financial support from National Natural Science Foundation
of China (Grants 31400649 and 31670792), Natural Science
Foundation of Jiangsu Province (Grant BK20140477), Natural
Science Research Grant from Department of Education of
Jiangsu Province (Grant 14KJB180026), and Talent Support
Program of Yangzhou University.
In summary, we engineered a catalytically self-
sufficient fusion protein, OleT-BM3R, for oxidative decar-
boxylation of FAs without the need for auxiliary redox
partners. The simplified reaction could be set up with less
effort in protein production and purification and per-
formed under mild conditions in aqueous solution using
O2 as the oxidant and NADPH as the electron donor. It
could also use inexpensive sodium phosphite as the final
electron source when coupled with a PTDH based
NADPH regeneration system. A similar structure between
OleTJE and the heme domain of P450BM3, a good compat-
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