CYP505E3: A Novel Self-Sufficient ω-7 In-Chain Hydroxylase

Article abstract of DOI:10.1002/anie.202001055

The self-sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in-chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω-7 position. It is the first reported P450 to give regioselective in-chain ω-7 hydroxylation of C10–C16 n-alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5-dodecanol and 7-tetradecanol. It shows more than 70 percent regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high-value flavour compound δ-dodecalactone via two routes: 1) conversion of dodecanoic acid into 5-hydroxydodecanoic acid (24 percent regioselectivity), which at low pH lactonises to δ-dodecalactone, and 2) conversion of 1-dodecanol into 1,5-dodecanediol (55 percent regioselectivity), which can be converted into δ-dodecalactone by horse liver alcohol dehydrogenase.

Full text of DOI:10.1002/anie.202001055

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Accepted Article
Title: CYP505E3 – a novel self-sufficient ω-7 in-chain hydroxylase
Authors: Martha Sophia Smit, Mpeyake Jacob Maseme, Alizé Pennec,
Jacqueline van Marwijk, and Diederik Johannes Opperman
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202001055
Angew. Chem. 10.1002/ange.202001055
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10.1002/anie.202001055
Angewandte Chemie International Edition
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CYP505E3 – a novel self-sufficient ω-7 in-chain hydroxylase
M.J. Maseme,a, b A. Pennec,a, b J. van Marwijk,a, b D.J. Opperman,a, b and M.S. Smit,a, b*
Abstract:
A
self-sufficient cytochrome P450 monooxygenase,
chrysosporium preferably hydroxylated 1-dodecanol and
dodecanoic acid at the sub-terminal positions (ω-1 to ω-3), but
also yielded the in-chain hydroxylated products (ω-4 to ω-7) as
minor products[8]. However, to date, no catalyst have been
described for the regioselective in-chain oxyfunctionalization of n-
alkanes, where no directing functional group is available.
CYP505E3, identified in Aspergillus terreus catalyzes regioselective
in-chain hydroxylation of alkanes, fatty alcohols and fatty acids at the
ω-7 position. It thus hydroxylates C10 to C16 alkanes with > 70 %
regioselectivity at the eighth carbon from one methyl terminus, also
displaying remarkably high activity towards decane (TTN ≈ 8 000) and
dodecane (TTN ≈ 2 000). CYP505E3 converts dodecanoic acid to 5-
hydroxydodecanoic acid (24 % regioselectivity), which at low pH
lactonises to δ-dodecalactone. With 1-dodecanol as substrate it
displays 55% regioselectivity towards the ω-7 position to yield 1,5-
dodecanediol, which was converted to δ-dodecalactone by horse liver
alcohol dehydrogenase (HLADH).
Here we report the novel ω-7 in-chain hydroxylase activity of
a self-sufficient cytochrome P450 monooxygenase, CYP505E3,
which we have identified in Aspergillus terreus[9][10]. CYP505E3 is
the first P450 that gives regioselective in-chain ω-7 hydroxylation
of n-alkanes with chain lengths ranging from C10 to C16, enabling
the one step biocatalytic synthesis of rare alcohols such as 5-
dodecanol and 7-tetradecanol. Additionally, CYP505E3’s
regioselectivity for ω-7 hydroxylation also extends to fatty acids
and fatty alcohols (Scheme 1). This ω-7 hydroxylase activity
towards tetradecanoic acid, dodecanoic acid and 1-dodecanol
also makes it an attractive starting point for the development of
biocatalytic processes for production of δ-dodecalactone. δ-
Dodecalactone is a high value flavour compound responsible for
fruity and buttery notes[11] as well as anti-fungal properties[12]. It
occurs naturally in fruits and dairy products in low
Regioselective activation of C-H bonds remains a problem for
which neither chemists nor biochemists have found the perfect
solution. Cytochrome P450 monooxygenases are the enzymes
most extensively studied for this purpose. P450s are known to
hydroxylate linear alkanes, alcohols and fatty acids of different
chain lengths. The most extensively studied alkane and fatty acid
hydroxylating P450s such as the CYP153s and CYP102s mainly
hydroxylate at the terminal or sub-terminal carbons[1–3]. However,
there are several P450s which show in-chain hydroxylation of
medium chain fatty acids such as decanoic acid and dodecanoic
acid, although hydroxylation is rarely more in-chain than the ω-6
position[3]. One exception in this regard is CYP116B46 from
Tepidiphilus thermophiles[4] which gives C5 hydroxylation of
decanoic and dodecanoic acid. It displays in the case of decanoic
acid exquisite regioselectivity (99 %) and enantioselectivity to
produce the “non-natural” (S)-δ-decalactone with > 90 % ee. In
the case of dodecanoic acid C5 regioselectivity was 76 %.
However, these substrates are most likely not the natural
substrates of CYP116B46, since uncoupling with decanoic acid
was 11.5 % and TTNs were 242 with decanoic acid and 27 with
dodecanoic acid. Terminal hydroxylation is the main focus of
reports on n-alkane and fatty alcohol hydroxylation, with very few
reports on the latter[5,6], despite the potential that not only
diterminal diols, but also internal alkane diols hold for the
synthesis of polymers[5] and even lactones and lactams[7].
Recently, Sakai et al. (2018) reported that CYP505D6, a self-
sufficient P450 from the white-rot fungus Phanerochaete
concentrations[13][14]
.
Scheme 1. Regioselective in-chain hydroxylation of alkanes, fatty alcohols and
fatty acids by CYP505E3.
[a]
M.J. Maseme, Dr. A. Pennec, Dr. J. van Marwijk, Prof. D.J.
Opperman, and M.S. Smit
Department of Microbial, Biochemical and Food Biotechnology,
University of the Free State,
P.O. Box 339, Bloemfontein 9300 (South Africa)
e-mail: smitms@ufs.ac.za
South African DST-NRF Centre of Excellence in Catalysis,
c*change, University of Cape Town, South Africa
CYP505E3 and the glucose dehydrogenase (GDH) from Bacillus
megaterium were expressed in Escherichia coli BL-21 Gold (DE3)
and the enzymes were recovered from the cells as cell-free
extracts (CFEs). The P450 concentration in the CFEs derived
from 0.2 g wet cell weight per ml was typically ca. 6 µM (Figure
S1) and the final measured CYP505E3 concentration in the
aqueous biotransformation reaction mixtures (BRMs) ≈ 0.7 – 1.5
µM.
b
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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CYP505E3 containing CFEs showed activity towards the C10 to
C16 alkanes (Figures S2 – S9). The reactions with decane,
yielded 3-decanol as the major product (71 %). Dominant alcohol
products were also observed in the extracts from the dodecane,
tetradecane and hexadecane reactions. When the mass spectra
of these main products were analysed (Figure 1) and the derived
structures closely examined,
a unique regioselectivity of
CYP505E3 for C10 to C16 alkanes became apparent. It appeared
that CYP505E3 preferentially hydroxylated these alkanes on the
8th carbon i.e. at what could be described as a ω-7 position (Figure
2A and 2B, Table S2). Preliminary docking experiments with
decane and dodecane using a homology model of CYP505E3,
placed these n-alkanes in positions which agree with the
observed ω-7 hydroxylase activity.
It shows the “ω-end”
penetrating deep into a hydrophobic pocket parallel to helix I,
placing the ω-7 carbon in close proximity to the oxygen of
compound I (Figure 3).
Figure 1. Mass spectra of major products from GC-MS analysis of
trimethylsilylated samples from reactions of CYP505E3 with decane (A),
Figure 2. Regioselectivity (A and B) and substrate preference (C) of CYP505E3
dodecane (B), tetradecane (C) and hexadecane (D). BRM (1 mL) contained:
towards alkanes, fatty alcohols and fatty acids. BRM (1 mL) contained:
-1
CYP505E3 in cfe (225 μL from 200 g
L
cells, final [CYP] 0.9 μM), GDH in
-1
wcw
CYP505E3 in cfe (225 μL from 200 g
L
cells, final [CYP] 0.9 μM), GDH in
wcw
-1
cfe (25 μL from 200 g
L
cells), 0.1 mM NAD+, 748 μL MOPS buffer (200 mM,
-1
cfe (25 μL from 200 g
L
cells), 0.1 mM NAD+, 748 μL MOPS buffer (200 mM,
wcw
wcw
pH 8, 100 mM glucose, 100 mM glycerol) with 250 µl alkane added.
pH 8, 100 mM glucose, 100 mM glycerol). Regioselectivity calculations were
based on assumption that initial hydroxylation of double hydroxylated products
was at the in-chain position (ω-6, ω-7 or ω-8).
The alkane hydroxylase activity observed with CYP505E3 is not
only remarkable for its regioselectivity, but also for the high activity
observed with decane (TTN ≈ 8 000) and dodecane (TTN ≈ 2 000)
(Table S2) even without any attempt at optimization. In a study
where CYP153s and CYP102A1 mutants with high reported
activities towards alkanes were compared on C6 to C10 alkanes
under similar unoptimized conditions in CFEs, the highest TTNs
obtained with decane were around 2 500[15] (Table S3). The
preferred substrates of these CYPs were n-hexane and n-octane
with TTNs of up to 10 000 obtained with the CYP102A1 R47L-
Y51F mutant on hexane and around 4 000 with CYP153A6 and
some CYP102A1 mutants on n-octane.
CYP505E3’s unique ω-7 regioselectivity was also observed with
the C12 to C16 fatty acids and the C10 to C14 alcohols, when
these were tested as substrates (Figure 2B, Figures S10 – S23,
Table S2), although the products of ω-7 hydroxylation were not in
all cases the major products. No activity was observed with the
C10 fatty acid and the C16 alcohol. In the case of dodecanoic
acid the products of ω-7 hydroxylation, 5-hydroxydodecanoic acid
and δ-dodecalactone (formed by lactonization of the 5-hydroxy
acid at low pH), comprised 24 % of the total products. Activity
towards the fatty acids was similar to the activity towards alkanes
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10.1002/anie.202001055
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-1
-1
with 5.5 mmol LBRM total products formed from 10 mmol LBRM
(Figure S24). In this case no triols were observed and δ-
dodecalactone comprised 55 % of the total products, confirming
earlier estimates of the regioselectivity.
dodecanoic acid added (TTN ≈ 6 000) (Figure 1C, Table S2). The
C14 and C16 fatty acids were double hydroxylated to yield
dihydroxy acids hydroxylated at the ω-7 position as well as a sub-
terminal position (ω-1, ω-2 or ω-3).
In order to test 1-dodecanol conversion on a larger scale, a time
course experiment was run in three 100 mL shake flasks using 10
-1
mL BRM. Two of the flasks contained 180 mmol LBRM
-1
1-dodecanol and the third 60 mmol LBRM
. In flasks with 180
-1
mmol LBRM 1-dodecanol the final total diols reached
a
concentration of ca. 20 mmol LBRM-1, while the triol concentrations
fortuitously remained at less than 10 % (Figure S25.). With 60
-1
mmol LBRM 1-dodecanol the production of diols fell to 12 mmol
LBRM-1, but the percentage conversion improved from 11% to 20%
(Table S4). Addition of extra enzymes and co-factor to this latter
-1
reaction brought the final total diol concentration to 15 mmol LBRM
.
After completion of the reactions the remaining BRMs were
combined, extracted and the 1,5-diol separated by flash
chromatography to yield 50 mg of > 90 % pure 1,5-diol (Figure
S26). 1H- and 13C-NMR spectra were congruent with 1,5-
dodecane diol (Figure S27).
Chiral analysis of the δ-
dodecalactone produced by HLADH oxidation of the purified 1,5-
diol showed that the R-enantiomer, the major enantiomer in
natural δ-dodecalactone, was produced in an enantiomeric
excess of 29% (Figure S28).
Given the relatively low
Figure 3. The lowest energy poses of decane (purple) and dodecane (green)
obtained from docking experiments carried out with a homology model of
CYP505E3. Residues lining the binding pocket are shown as sticks and the
oxygen of compound I as a dotted sphere. Substrate docking in the CYP505E3
homology model was performed with YASARA version 14.12.2 using VINA
AutoDockLGA and the AMBER03 force field. Image was created using PyMOL
software.
enantioselectivity observed with 1-dodecanol, a substrate with a
directing hydroxyl group, chiral separation of other products was
not pursued.
We have thus demonstrated two routes by which CYP505E3 can
be used for the synthesis of δ-dodecalactone (i) via hydroxylation
of dodecanoic acid and lactonisation of the 5-hydroxy fatty acid
and (ii) via hydroxylation of 1-dodecanol followed by oxidation and
lactonisation by an alcohol dehydrogenase (Scheme 2). Both
these routes require extensive optimisation most likely involving
both protein and process engineering. The latter could include
using whole cells of E. coli or perhaps a yeast, screening for other
alcohol dehydrogenases[20] or oxidases[21] for oxidation of the 1,5-
diol as well as in situ product removal to avoid over hydroxylation.
Hydroxylation of tetradecanoic acid followed by one round of β-
oxidation and lactonisation could in principle also yield δ-
dodecalactone, but would require expression of CYP505E3 in an
1-Decanol was the preferred substrate of CYP505E3 with 27
mmol LBRM-1 total products formed (TTN ≈ 30 000) (Figure 2, Table
S2). Although 1,3-decanediol, the product of ω-7 hydroxylation
was a major product (28 % of diol products), regioselectivity was
not as strong as with 1-dodecanol, since equal amounts of 1,7-
decanediol (ω-3 hydroxylation) was also produced. In the case of
1-dodecanol, the substrate with the second highest activity (20
-1
mmol LBRM total products, TTN ≈ 22 000) the product from ω-7
hydroxylation, 1,5-dodecanediol, was the major product (ca. 39 %
of total diol products) with double hydroxylated triol products
forming 25 % of total products (Figure 2, Figure S13, Table S2).
When both diol and triol products were considered regioselectivity
for ω-7 hydroxylation was 55 %. With tetradecanol as substrate
hydroxylation at the ω-7 position was still preferred, but the major
products were double hydroxylated triols.
organism, probably a yeast, with partially disrupted β-oxidation[22]
.
The possibility that 1,5-dodecane diol might also be converted to
δ-dodecalactone did not escape our attention. Oxidation of
alkanediols by horse liver alcohol dehydrogenase (HLADH) for
synthesis of lactones is an established enzymatic reaction[16–19]
.
More specifically, HLADH has been shown to oxidize 1,5-
pentanediol, 1,4-decanediol and 1,5-decanediol with preference
for 1,5-diols[17,18]. Conversion of 1-dodecanol to δ-dodecalactone
was tested by replacing GDH with purified HLADH. GC-MS
analysis of extracts from this reaction showed that the 1,5-
dodecanediol and
a small amount of 1,4-diol (from ω-8
Scheme 2. Routes demonstrated for the production of δ-dodecalactone from
dodecanoic acid (Route i) and 1-dodecanol (Route ii) using CYP505E3.
hydroxylation) were completely converted to δ- and γ-
dodecalactones, respectively, while the other diols remained
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10.1002/anie.202001055
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Here we have reported the first catalyst for the regioselective in-
chain hydroxylation of n-alkanes. Given the high TTNs obtained
and already high regioselectivity, CYP505E3 is a promising
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improved or adjusted regioselectivity and substrate scope to
enable synthesis of unique “natural” alcohols from “natural”
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directed evolution through iterative saturation mutagenesis of
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10.1002/anie.202001055
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COMMUNICATION
COMMUNICATION
Regioselective in-chain C-H
activation: CYP505E3, a self-
sufficient cytochrome P450
M.J. Maseme, A. Pennec, J. van
Marwijk, D.J. Opperman, M.S. Smit*
Page No. – Page No.
monooxygenase from the fungus
Aspergillus terreus, catalyzes
regioselective in-chain hydroxylation
of alkanes, fatty alcohols and fatty
acids at the ω-7 position. It can thus
be used for the biocatalytic production
of δ-dodecalactone from dodecanoic
acid and 1-dodecanol.
CYP505E3 – a novel self-sufficient
ω-7 in-chain hydroxylase
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