A novel P450-based biocatalyst for the selective production of chiral 2-alkanols

Article abstract of DOI:10.1039/c4cc00647j

A P450 monooxygenase from Nocardia farcinica (CYP154A8) catalyses the stereo- and regioselective hydroxylation of n-alkanes, still a challenging task in chemical catalysis. In a biphasic reaction system, the regioselectivity for the C2-position of C₇-C₉ alkanes was over 90%. The enzyme showed strict S-selectivity for all tested substrates, with enantiomeric excess (ee) of up to 91%. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00647j

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A novel P450-based biocatalyst for the selective
production of chiral 2-alkanols†
Cite this: Chem. Commun., 2014,
50, 4089
Clemens J. von Buhler and Vlada B. Urlacher*
¨
Received 24th January 2014,
Accepted 24th February 2014
DOI: 10.1039/c4cc00647j
www.rsc.org/chemcomm
A P450 monooxygenase from Nocardia farcinica (CYP154A8) catalyses A very large and intensively studied group of enzymes catalysing
the stereo- and regioselective hydroxylation of n-alkanes, still a hydroxylation reactions are the heme containing cytochrome P450
challenging task in chemical catalysis. In a biphasic reaction system, monooxygenases (P450s). Several wild type and engineered P450s
the regioselectivity for the C2-position of C7–C9 alkanes was over are known to accept alkanes as substrates and especially CYP102A
90%. The enzyme showed strict S-selectivity for all tested substrates, enzymes are known to produce 2-alcohols.7
with enantiomeric excess (ee) of up to 91%.
Recently, we characterised two novel P450 enzymes belonging
to the CYP154 family.8 CYP154A8 from Nocardia farcinica IFM
Chiral linear alcohols are widely used as building blocks in the 10152 proved to be quite regioselective. Thus, this enzyme was
synthesis of pharmaceuticals, agrochemicals, pheromones and chosen for a detailed investigation of its suitability as a biocatalyst
liquid crystals and therefore synthetic routes to these products are for n-alkane oxidation.
of high interest.1 Besides the difficult and less selective chemical
CYP154A8 was expressed in E. coli in soluble form and
routes,2 the enzymatic reduction of prochiral ketones catalysed by purified as reported earlier.8 A catalytically active P450 system was
alcohol dehydrogenases is an established enzymatic process for the then reconstituted by the addition of the flavodoxin YkuN from
production of chiral alcohols.3 However, a prerequisite for this Bacillus subtilis and E. coli flavodoxin reductase FdR.8 Reactions were
enzymatic approach is the availability of functionalised substrates, performed in 500 ml reaction volume under NADPH cofactor
namely ketones. Therefore, an enzymatic approach for the produc- regeneration by glucose dehydrogenase. The following alkanes were
tion of chiral secondary alcohols via direct hydroxylation of inert tested as substrates: heptane (C7), octane (C8), nonane (C9), decane
alkanes by a monooxygenase seems to be more beneficial. Alkanes (C10) and dodecane (C12). After extraction and derivatisation, the
are cheap starting compounds that are easily produced from mineral reaction mixtures were analysed via achiral and chiral GC coupled
oil and hence are available in high amounts. Enzyme-catalysed with MS. The stereoselectivity of the enzyme was chain length
selective hydroxylation of alkanes comes along with the typical dependent. In comparison to authentic standards it could be
advantages of biocatalytic processes: no requirement for toxic determined that CYP154A8 showed strict S-selectivity for all tested
metal catalysts or high reaction temperatures and only limited alkanes: 84% ee for both 2-(S)-heptanol and 2-(S)-nonanol, 63% ee
use of potentially toxic additional organic solvents.
for 2-(S)-decanol and a maximum of 91% ee was found for
Several enzyme groups have been described that are capable 2-(S)-octanol (Fig. S1, ESI†). Dodecane was accepted by CYP154A8
of this type of reaction.4 The number of oxygenases producing but only trace amounts of products were formed and hence this
secondary and particularly 2-alcohols is however limited. The soluble alkane was not investigated further. In the case of decane oxida-
methane monooxygenase (sMMO) and the particulate methane tion, 27% 3-decanol was formed with 89% ee.
monooxygenase (pMMO) from Methylococcus capsulatus produce
By monitoring the octane conversion over a period of 24 h it
2-alcohols from short chain alkanes but suffer from low expression.5 could be demonstrated that the ee value of the formed 2-(S)-octanol
Furthermore fungal peroxygenases accept small and medium was not dependent on the degree of conversion, as is expected for
chain alkanes as substrates but display moderate regioselectivity.6 an asymmetric synthesis (Fig. S2, ESI†).
So far the best-characterised P450s that catalyse this
¨
¨
Institute of Biochemistry, Heinrich-Heine University Dusseldorf, Universitatsstraße
reaction with high stereoselectivity are the CYP102A1 mutants
F87V/A328F,9 1-12G10 and 139-3,10,11 which all produce 2-octanol,
but with significantly lower ee-values, i.e. 46% ee for 2-(R)-octanol,
39% ee for 2-(R)-octanol and 58% ee for 2-(S)-octanol, respec-
tively. Thus CYP154A8 is the first P450 enzyme that is
¨
1, 40225 Dusseldorf, Germany. E-mail: Vlada.Urlacher@uni-duesseldorf.de;
Fax: +49 211 8113117; Tel: +49 211 8113866
† Electronic supplementary information (ESI) available: Materials and methods.
GC/MS chromatograms, time dependence of octane conversion, and supplemen-
tation experiments. See DOI: 10.1039/c4cc00647j
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Scheme 1 Hydroxylation of octane catalysed by CYP154A8 in a biphasic reaction system consisting of an organic substrate phase and an aqueous
phase. The product scavenging effect of the organic phase prevents formation of higher oxidation products. YkuN (flavodoxin from B. subtilis), FdR (E. coli
flavodoxin reductase), GDH (glucose dehydrogenase from B. megaterium).
shown to produce highly enantiomerically enriched 2-(S)-octanol
from octane.
However, substrate conversion in aqueous solution resulted
in the formation of higher oxidation products, diols, which are
produced upon exposure of the formed 2-alcohol to the enzyme
as described earlier.8 In order to circumvent the formation of the
undesired products, the reaction setup was modified: by employing
the substrate as a second phase (20 vol% of the aqueous phase) a
double benefit over the homogenous aqueous reaction system was
achieved. First, the concentration of the substrate available to the
enzyme was maximised, and second, the continuous extraction
of formed products into the organic phase minimised or even
prohibited the formation of higher oxidation products (Scheme 1).
Partition coefficients for all 2-alcohols have been determined in the
respective biphasic alkane–buffer-systems. Values ranged between
log PC7 1.0 Æ 0.03 for 2-heptanol and log PC10 1.42 Æ 0.1 (Table S1
and Section S4.4, ESI†). This altered reaction setup yielded
2-alcohols as main reaction products with over 80% selectivity.
Considering that some of the 2-alcohol molecules were obviously
further oxidised by CYP154A8 to the corresponding ketones, an
overall preference towards the C2-position of up to 90% can be
calculated (Fig. 1A–C and Fig. S3, ESI†).
Similar to the stereoselectivity, the regioselectivity of CYP154A8
was chain length dependent and decreases for C10 down to 70%
Fig. 1 Product distribution after hydroxylation of various alkanes: (A) heptane,
(Fig. 1D and Fig. S3, ESI†). For decane a significant amount of
3-decanol as well as its overoxidation product 3-decanone was
detected. GC/MS data confirmed that 3-alkanones were not
present for any other substrate but decane.
(B) octane, (C) nonane, and (D) decane, determined from GC/MS peak
areas and plotted as percentage of a specific product to the total amount
of product (Fig. S3, ESI†). Where error bars cannot be seen, they are smaller
than the linewidth.
The benchmark for our reaction was the data published by
Peters et al. (2003) for the CYP102A1 mutant 1-12G,11 and data for
In the next step challenges inherent to biphasic reaction
the CYP102A1 mutant F87V/A328F designed in our group.9 The systems such as interfacial surface area, low substrate solubility
mutant enzyme 1-12G produces 2-alcohols from alkanes ranging in the aqueous phase, and the choice of protein stabilising
from C7–C10 with a corresponding selectivity of 76, 82, 86, and additives have been addressed. The optimised biphasic system
86%. However, in all cases alcohols with a hydroxy group at the included catalase to decompose H2O2, which might be formed by
positions C1, C3 and C4 were observed as by-products. Compared a shunt reaction in the catalytic cycle of P450s,12 BSA for protein
to 1-12G, CYP154A8 showed a higher or similar regioselectivity stabilisation,13 ethanol as a cosolvent and the use of a rotator for
depending on the chain length of the substrate, but its product mixing (Section S8 and Fig. S4, ESI†).
distribution was limited to the C2- and partially C3-position only.
The following product titres were obtained after 24 h in the
The mutant F87V/A328F is very selective for the C2-position of optimised biphasic reaction system: 3 mM 2-nonanol; 1.9 mM
octane (92%).9 Thus, the total regioselectivity of CYP154A8 for the 2-heptanol; 2.2 mM 2-octanol; and 1.2 mM 2-decanol. Compared
C2-position was comparable to that of F87V/A328F.
to values found for CYP102A1 mediated octane hydroxylation9,11
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Table 1 Parameters for the 2-hydroxylation of heptane, octane, nonane
and decane catalysed by CYP154A8
This in turn may explain the observed higher final concentrations of
2-nonanol as compared to 2-octanol (Table 1).
In conclusion, CYP154A8 from N. farcinica was identified as
a novel regio- and stereoselective biocatalyst for the hydroxylation
of inert n-alkanes. Depending on the substrate chain length,
enzyme preference for oxidation at the C2-position was more than
90%. To the best of our knowledge, CYP154A8 is the only P450
monooxygenase where high regioselectivity for medium chain
alkane hydroxylation coincides with a moderate to high enantio-
meric excess of the formed 2-(S)-alcohols and high total turnover
numbers. Furthermore, by means of reaction optimisation the total
product amounts were significantly increased to levels that can
compete with the best performing and well-established CYP102A1-
based systems. But unlike those evolved CYP102A1 systems,
CYP154A8 wild type shows already high selectivity, which might
be even further increased by protein engineering methods.
Compared to other enzymes like fungal peroxygenases both
regio- and stereoselectivity of 2-alkanol production by CYP154A8
is significantly higher.6 This study demonstrates the potential of
CYP154A8 in alkane oxidation. Simultaneously, the described
biocatalyst implies additional optimisations and the transfer to a
whole cell system which are currently under investigation.
Product
Couplingb
(%)
Conc.c
(mM)
ee-(S)c
(%)
Sub.
formation ratea,b
TTNc,d
C7
C8
C9
C10
1.13 Æ 0.08
4.6 Æ 1.04
3.73 Æ 0.44
0.37 Æ 0.05
7.3 Æ 0.9
21.1 Æ 4.2
15. 9 Æ 1.8
4.0 Æ 0.6
1.9
2.2
3.0
1.2
2800
3200
4400
1700
84
91
84
63
a
b
Given as nmol of 2-alcohol per nmol P450 per minute. Determined
using a 200 ml aqueous reaction system in multiwell plates with
1 mM substrate. After 24 h in 500 ml of a biphasic reaction system.
Calculated as nmol of 2-alcohol per nmol P450.
c
d
the total turnover numbers (TTNs) of CYP154A8 were much higher
than those reported for F87V/A328F and in the same order of
magnitude as those of 1-12G (Table 1). Moreover, these results
demonstrated that even though the catalytic system consisted
of five enzymes, this complexity apparently does not have a
negative effect on product formation.
In P450 systems at various stages of the electron transfer chain,
electrons can be diverted from a productive reaction, leading to
unproductive cofactor consumption, which reduces the catalytic
performance of the system. This can take place between redox
partners as well as through the earlier mentioned shunt reactions
in the P450 catalytic cycle. To evaluate the performance of the
CYP154A8 system we calculated the coupling between NADPH
consumption and product formation (Table 1 and ESI†). The
rather low values (Table 1) are however in accordance with
those reported for other artificial P450 redox chains oxidising
non-natural substrates.14
Notes and references
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2 R. H. Crabtree, J. Chem. Soc., Dalton Trans., 2001, 2437.
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Biol., 2004, 8, 120.
At a closer look these data seem to be contradictory: the total
turnover number (TTN) and the final product concentration
were highest for nonane but its product formation rate and
coupling were lower than for octane.
Since we attributed this discrepancy to the stability of the
involved enzymes we designed experiments to verify our hypothesis:
octane conversion was supplemented after 5.5 h with either addi-
tional glucose, glucose dehydrogenase (both for cofactor regenera-
tion), YkuN/FdR or YkuN/FdR/P450. After a total reaction time of
24 h each case was compared to a standard reaction setup without
this extra addition. Neither the addition of glucose nor that of
glucose regenerating GDH resulted in an elevated final product
4 (a) Y. Ji, G. Mao, Y. Wang and M. Bartlam, Front. Microbiol., 2013,
4, 58; (b) J. B. Van Beilen and E. G. Funhoff, Appl. Microbiol.
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9 E. Weber, A. Seifert, M. Antonovici, C. Geinitz, J. Pleiss and
V. B. Urlacher, Chem. Commun., 2010, 46, 944.
10 A. Glieder, E. T. Farinas and F. H. Arnold, Nat. Biotechnol., 2002, 20, 1135.
11 M. W. Peters, P. Meinhold, A. Glieder and F. H. Arnold, J. Am.
Chem. Soc., 2003, 125, 13442.
concentration. As opposed to this, the supplementation of the 12 P. R. Ortiz de Montellano, Cytochrome P450. Structure, mechanism,
and biochemistry, Kluwer Academic/Plenum Publishers, New York,
3rd edn, 2005.
13 S. C. Maurer, K. Ku¨hnel, L. A. Kaysser, S. Eiben, R. D. Schmid and
reaction with the redox partners or the P450 led to about 15% more
product which could even be further maximised (45%) by the
simultaneous addition of YkuN, FdR and CYP154A8 (Fig. S5, ESI†).
These results indicated that as suggested the stability of the redox
partner proteins and that of CYP154A8 turned out to have a major
influence on the degree of conversion. In the C7 to C12 alkane series
V. B. Urlacher, Adv. Synth. Catal., 2005, 347, 1090.
14 (a) A. Schallmey, G. den Besten, I. G. P. Teune, R. F. Kembaren and
D. B. Janssen, Appl. Microbiol. Biotechnol., 2011, 89, 1475; (b) M. Girhard,
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15 National Library of Medicine (US), Division of Specialized Informa-
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the solubility decreases from 3.4 mg LÀ1 to 5.2 10À2 mg LÀ1 15 It is
.
therefore reasonable to assume that enzyme denaturation is
decreased with a longer alkane chain, due to reduced exposure.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 4089--4091 | 4091