Engineered alkane-hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties

Article abstract of DOI:10.1002/anie.200702616

(Figure Presented) Divide, evolve, and conquer: A domain-based strategy (see scheme) was used to engineer high catalytic and coupling efficiency for propane hydroxylation in a multidomain cytochrome P450 enzyme. The engineered enzymes exhibit high total activities in whole-cell bioconversions of propane to propanol under mild conditions, using air as oxidant.

Full text of DOI:10.1002/anie.200702616

Communications
DOI: 10.1002/anie.200702616
Directed Evolution
Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting
Nativelike Catalytic Properties**
Rudi Fasan, Mike M. Chen, Nathan C. Crook, and Frances H. Arnold*
Cytochrome P450 enzymes (P450s) are exceptional oxygen-
Selective hydroxylation of short alkanes is a long-standing
problem, for which no practical catalysts are available. In
[
1]
[13]
ating catalysts with enormous potential in drug discovery,
[
2,3]
chemical synthesis, bioremediation, and biotechnology.
an effort to produce P450BM3-based biocatalysts for selective
hydroxylation of small alkanes, we previously engineered this
Compared to their natural counterparts, however, engineered
P450s often exhibit poor catalytic and cofactor coupling
[
14]
enzyme to accept propane and ethane (35E11 variant).
[
3]
efficiencies. Obtaining native-like catalytic proficiencies is a
mandatory first step towards utilizing the power of these
versatile oxygenases in chemical synthesis.
Despite greater than 5000 total turnover (TTN) supported
in vitro, the utility of this catalyst remained limited because of
its poor in vivo performance (see below), which was mostly
due to the low efficiencies for coupling the product formation
to cofactor consumption (17.4% for propane and 0.01% for
ethane oxidation).
Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes
the subterminal hydroxylation of long-chain (C –C ) fatty
1
2
20
[
4]
acids. Its high activity and catalytic self-sufficiency (heme
and diflavin reductase domains are fused in a single polypep-
Our goal was to engineer a P450BM3 variant with native-
like activity and coupling efficiency towards a structurally
challenging, nonnative substrate (propane) and evaluate the
impact of these features on performance in preparative-scale
biotransformations. To this end, we used a domain-based
protein-engineering strategy, in which the heme, flavin
mononucleotide (FMN), and flavin adenine dinucleotide
(FAD) domains of the 35E11 variant were evolved separately
in the context of the holoenzyme, and beneficial mutations
were recombined in a final step (Figure 1). Previous work
suggested that mutations in the reductase and linker regions
[
2,4,5]
tide chain)
make P450BM3 an excellent platform for
biocatalysis. However, despite numerous reports of the
heme domain being engineered to accept nonnative sub-
strates, including short-chain fatty acids, aromatic compounds,
[
6–8]
alkanes, and alkenes,
tions of P450BM3 remain scarce.
reports of preparative-scale applica-
[
9]
P450BM3 function is finely regulated through conforma-
tional rearrangements in the heme and reductase domains
[
4,10]
and possibly also through hinged domain motions.
Hy-
droxylation of fatty acids occurs almost fully coupled to
cofactor (NADPH) utilization (93–96% depending on the
[
14,15]
can affect catalytic properties.
However, no systematic
[
11]
substrate). In the presence of nonnative substrates or when
amino acid substitutions are introduced, the mechanisms
engineering efforts had been undertaken to engineer the
complete 1048 amino acid holoenzyme.
Holoenzyme libraries outlined in Figure 1 were created
using random, saturation, and site-directed mutagenesis and
[
12]
controlling efficient catalysis in P450s are disrupted,
leading to the formation of reactive oxygen species and
[
4]
rapid enzyme inactivation. High coupling efficiencies on
substrates whose physicochemical properties are substantially
different from the native substrates have not been achieved,
and coupling efficiencies ranging from less than 1% to 30–
[
7,8]
4
0% are typical.
Strategies for addressing this “coupling
problem” are needed in order to take engineered P450s to
larger-scale applications.
[
*] Dr. R. Fasan, M. M. Chen, N. C. Crook, Prof. Dr. F. H. Arnold
Department of Chemistry and Chemical Engineering
California Institute of Technology
1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
Fax: (+1)626-568-8743
E-mail: frances@cheme.caltech.edu
[
**] This work was supported by a Swiss National Science Foundation
fellowship to R.F., a U.S. NSF Fellowship to M.M.C. and by the U.S.
Army Research Office, ARO Contract DAAD19-03-D-0004. We thank
Dr. Christopher Snow for providing the homology model of P450BM3
reductase and Dr. Matthew W. Peters, Dr. Peter Meihnold, and Dr.
Marco Landwehr for helpful discussions regarding the biotransfor-
mations and for providing access to DasGip fermenter.
Figure 1. Outline of the domain engineering strategy used to improve
cytochrome P450BM3 heme and reductase domains. HL=heme domain
libraries, RL=reductase domain libraries.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Angew. Chem. Int. Ed. 2007, 46, 8414 –8418

Angewandte
Chemie
[
8]
screened for activity on a propane surrogate, dimethyl ether.
Positives were confirmed in a re-screen, purified, and
challenged with propane in sealed vials in the presence of a
cofactor regeneration system. As a cumulative measure of
both catalytic and coupling efficiency, improvement in total
turnover (moles of propanol produced per mole of enzyme)
was used as the sole selection criterion.
Measurement of the half-denaturation temperature of
3
5E11 heme domain demonstrated a considerable reduction
in its stability as a consequence of the 15 accumulated
mutations (T = 43.48C vs. 55.08C for wild type). We there-
5
0
fore subjected 35E11 to an initial thermostabilization step
HL1), in which stabilizing mutations from a thermostable
(
[
16]
P450BM3 peroxygenase were tested singly and in combina-
tion in the 35E11 background (see the Supporting
Information). Variant ETS8 (DT = + 5.18C, DTTN =
propane
5
0
À1250) showed the best combination of increased stability
with little decrease in TTN and was selected for further
directed evolution. Using ETS8 as parent, heme-domain
random mutagenesis libraries were generated by error-prone
PCR (HL2). Variant 19A12, with about twofold increase in
TTN (Table 1), was then used to create a pool of active-site
libraries (HL3) in which 17 positions along the substrate
channel (Figure 2a) and near the active site (Figure 2b) were
subjected individually to saturation mutagenesis. Further
improvements in propane-hydroxylating activity were ach-
ieved in multiple variants, including 11-3. Recombination of
the beneficial mutations from the active-site libraries (HL4)
led to variant 1-3. Further fine-tuning of the active site was
pursued with a series of recombination/site-saturation libra-
ries (HL5, see Supporting Information). From these libraries,
Figure 2. a) Substrate channel and b) active-site residues targeted for
saturation mutagenesis mapped on the palmitate-bound structure of
[
26]
P450BM3 heme domain (PDB 1FAG ). Heme (white) and fatty acid
orange) are shown in space-filling mode.
(
heme domain. Improved 11-3-derived variants were found to
contain G443A, V445R, P654K, T664G, D698G, and E1037G
mutations and showed TTN between 16000 and 20000. In the
final step, a library containing the beneficial reductase
domain mutations was fused to the heme domain of variant
7-7 (L9). The most active variant isolated from this library,
P450PMOR2, supported more than 45000 turnovers and
produced 2- and 1-propanol in a 9:1 ratio. As we expected,
the increase in productivity strongly correlates with the
increase in coupling efficiency, which in the best variant
(P450PMOR2, 98.2%) reaches levels comparable to those
measured for wild-type in the hydroxylation of myristate
7
-7 emerged as the most active variant, supporting 20500
turnovers with propane.
Meanwhile, two libraries were constructed in which
random mutations were targeted to the FMN- and FAD-
binding domains of 35E11 (RL1 and RL2, respectively).
Screening of more than 5000 members from each library led
to the identification of eight beneficial mutations (G443D,
V445M, T480M, T515M, P654Q, T664M, D698G, and
[
11]
(88%), palmitate (93%), or laurate (96%).
The sequence of mutational events leading to P450PMO
generation reveals a continuous rearrangement of substrate-
channel and active-site residues (Table 1), presumably in
search of an optimal configuration for accommodating
propane. Additional beneficial mutations in the hydroxylase
domain include L188P and G443A. Leucine 188 is located
along helix F, which together with helix G forms a lid covering
[
17]
E1037G).
These positions were further optimized by
saturation mutagenesis in a holoenzyme construct having
the 11-3 heme domain (RL3, RL4). Swapping the heme
domains this way serves to remove mutations whose benefi-
cial effect is solely dependent on the presence of the 35E11
[
a]
Table 1: In vitro propane oxidation activities of most representative P450BM3 variants.
[
b]
[c]
À1
[d]
Variant
Library
Mutations versus 35E11
Rate [equivmin
]
Coupling [%] Total turnovers
heme domain
reductase domain
3
1
1
6
1
7
5E11
9A12
1-3
98E5
-3
–
–
–
–
–
210
420
390
295
320
150
455
370
17.45650
HL2
HL3
RL3
HL4
HL5
L52I, L188P, I366V
L52I, A74S, L188P, I366V
L52I, A74S, L188P, I366V
L52I, A74S, V184A, L188P, I366V
L52I, A74E, S82G, A184V, L188P, I366V
44.2
55.3
65.3
72.1
90.9
94.4
98.2
10550
13200
17300
19200
20500
35600
45800
D698G
–
–
-7
P450PMOR1 L9
P450PMOR2 L9
L52I, A74E, S82G, A184V, L188P, I366V, G443A P654K, E1037G
L52I, A74E, S82G, A184V, L188P, I366V, G443A D698G
[
a] Mean values from at least three replicates Æ10% error. [b] Mutations in 35E11 are R47C, V78F, A82S, K94I, P142S, T175I, A184V, F205C, S226R,
H236Q, E252G, R255S, A290V, A328F, L353V, E464G, I710T. [c] Over the first 20 s. [d] Ratio between propanol formation rate and NADPH oxidation
rate in propane-saturated buffer.
Angew. Chem. Int. Ed. 2007, 46, 8414 –8418
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Communications
[
18]
À1
the active site. Glycine 443 lies on a loop at the C-terminal
0.9 gcdwL ; cdw = cell dry weight) were used to avoid
[
18]
À1
end of hairpin b4, which inserts into the active site.
oxygen-transfer limitations. Activities of 80–120 Ug cdw
(where 1 U = 1 mmolpropanolmin ) were measured for
À1
Interestingly, the activity-enhancing substitutions in the
reductase domain are clustered in the same region in the FAD
domain (T664G, D698G, E1037G) and nearby linker to the
FMN domain (P654K; see map in the Supporting
Information). Perturbation of electrostatic charge distribu-
tion appears to be a prevailing trend, suggesting a more
P450PMOR1 and P450PMOR2 in various E. coli strains (Fig-
ure 3a, Table 2). The experiment was repeated in a larger
fermenter (0.3 L, pH and dissolved oxygen control). A
suspension of P450-expressing DH5a cells was fed with a
1:1 mixture of pure oxygen and propane, and propanol
important role of these forces in P450
function than
BM3
[
19]
previously proposed. A smaller contribution was obtained
by mutating the FMN domain. This effect may reflect its
higher sensitivity to mutagenesis, as judged by the signifi-
cantly lower fraction of functional variants in the FMN
libraries compared to the FAD libraries (data not shown).
Chemical and thermal denaturation studies have shown that,
among the three cofactors, FMN is most weakly bound to the
[a]
Table 2: In vivo propane oxidation activities of P450BM3 variants.
[
b]
[b,c]
Variant
Oxidant
propane/oxidant
ratio)
Activity
Productivity
À1
(
[Ug cdw] [mmol propanol
0.5 h 3 h
À1
À1
g P450h ]
3
1
7
5E11
9A12
-7
air (1:1)
air (1:1)
air (1:1)
9
41
2
9
12
44
74n.d. 88
[
20]
enzyme.
P450PMOR1 air (1:1)
P450PMOR2 air (1:1)
118 73 119
10468 106
A common strategy to reduce the prohibitive costs of
NADPH-driven biotransformations is the use of cofactor
P450PMOR1
P450PMOR2 O (1:1)
O
(1:1)
176 63 96
119 39 94
2
[
9c,21]
regeneration systems.
such as alkane hydroxylation, these in vitro approaches are
For bulk chemical transformations
2
[a] Mean values from two biological replicates Æ15% error. n.d.=not
[
22]
À1
not viable. The propane-hydroxylating P450 variants were
therefore evaluated in whole-cell biotransformations using
resting E. coli cells (Figure 3).
determined. [b] At cell density=0.5–0.9 gcdwL . [c] Calculated from
the first hour of biotransformation.
The expression levels of these variants in minimal medium
(
initially less than 0.5% of total cell mass) were first
formation was monitored for up to 9 h (Figure 3b). Under
these conditions, very high activities (up to 180 Ug cdw)
À1
optimized to achieve 6–11% of total cell mass as soluble
P450 enzyme. Experiments were carried out in a 100-mL
fermenter using cell suspensions in nitrogen-free minimal
medium (supplemented with glucose) and a propane/air
mixture as substrate and oxidant feed. Under these condi-
were obtained. In comparison, maximal activities of
À1
30 Ug cdw on n-nonene were reported for the natural
AlkB alkane hydroxylase system in both homologous (P.
[
23a]
oleovorans) and heterologous strains (E. coli).
À1
À1
tions, cell densities less than 1 gcdwL (typically 0.5–
At moderately higher cell densities (ca. 4 gcdwL ),
propanol accumulated to a concentration of more than
1
5 mm over 4 h (Figure 4, upper panel). The improved
coupling efficiencies result in considerably extended periods
of whole-cell activity (6 vs. 0.5 h, Figures 3b and 4). To
investigate the possible causes of the decrease in productivity
over time, we monitored the biocatalyst concentration over
the course of the biotransformation (Figure 4, lower panel).
At the end of the experiment, approximately 52% of the
Figure 3. Whole-cell biotransformation of propane. a) Initial activities
of selected P450BM3 variants in different E. coli strains using air/
propane (1:1) feed (pH 7.2, 258C). b) Time course of propane
biotransformation using recombinant DH5a cells using oxygen/pro-
pane (1:1) feed (pH 7.2, 258C). Product amount is given per gram cell
dry weight to facilitate comparison among variants. Control: no
propane in the gas feed.
Figure 4. Concentration of propanol during biotransformation of pro-
pane with DH5a cells expressing P450PMOR1 (&) and P450PMOR2 (~) at
medium cell density (4gcdwL ). In the lower panel, relative P450
concentration as determined from CO-binding difference spectra on
cell lysate; OD=optical density.
À1
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Angewandte
Chemie
initial P450PMOR2 was still correctly folded in the cells.
Control experiments using P450PMOR2-expressing cells and
propanol concentrations up to 30 mm showed no product
inhibition nor overoxidation to acetone, suggesting that host-
related rather than biocatalyst-dependent factors are limiting.
Indeed, 40–60% of the initially measured activity could be
restored by resuspending cells from the plateau phase (i.e.
after 4–6 h reaction) in fresh medium. In addition, the rate of
biocatalyst inactivation could be reduced by varying the
relative concentration of oxygen in the gas feed, with more
extended whole-cell activity periods obtained at a propane/
oxygen ratio of 4:1 compared to 1:1 (Table 2). Optimization
of this parameter as well as the availability of more robust
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[
[
22]
host strains is expected to further enhance the whole-cell
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.
Overall, a domain-based directed evolution strategy has
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Received: June 15, 2007
Published online: September 20, 2007
Keywords: alkane oxidation · biotransformations ·
cytochrome P450 · directed evolution · protein engineering
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[
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www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 8414 –8418