Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s

Article abstract of DOI:10.1039/c6cc01749e

A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

Full text of DOI:10.1039/c6cc01749e

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DOI: 10.1039/C6CC01749E
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Whole-cell microtiter plate screening assay for terminal
hydroxylation of fatty acids by P450s†
Received 00th January 20xx,
Accepted 00th January 20xx
Martin J. Weissenborn,§a Sandra Notonier,§a Sarah-Luise Lang,a Konrad B. Otte,a Susanne Herter,b
Nicholas J. Turner,b Sabine L. Flitschb and Bernhard Hauer*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
A readily available galactose oxidase (GOase) variant was used to more rapid solid-phase assays.11 Also detection methods relying
develop a whole cell screening assay. This endpoint detection upon the use of alcohol dehydrogenases, which oxidise the
system was applied in a proof-of-concept approach by screening a hydroxylated compound of interest into the corresponding carbonyl
focussed mutant library. This led to the discovery of the thus far product thereby producing NAD(P)H, are limited due to the
most active P450 Marinobacter aquaeolei mutant catalysing the aforementioned reasons.12 The background activity with other
terminal hydroxylation of fatty acids.
alcohol dehydrogenases is another factor which has been
addressed by the design of artificial cofactors for other enzyme
systems recently.13,14
Directed evolution techniques enable the development of tailor-
made biocatalysts exhibiting enhanced catalytic activities, stabilities
and substrate selectivities.1 Enzyme engineering requires sensitive,
simple to implement and reliable systems of detection.2 Employing
evolution methods as a tool to generate large libraries of variants
necessitates a smart screening strategy and selection methodology,
respectively.3 Accordingly, the system applied for the detection of
the desired enzyme activities should be highly sensitive but
concomitantly relatively unresponsive to side-reactions and, in
particular, applicable in the range of micromolar substrate
concentrations.4
Cytochrome P450 monooxygenases (CYPs or P450s) are
remarkable enzymes catalysing a broad variety of reactions under
mild conditions.5 Previous and successful efforts to engineer P450s
focussed on the improvement and variation of activity, chemo-,
regio-, and stereoselectivity as well as the catalysis of unnatural
carbene reactions.6–9
Alternative and rather indirect methods for the detection of P450
activities are based on the use of unnatural substrates to generate
colorimetric or fluorescent signals. For instance, indole which
spontaneously forms the insoluble dye indigo after P450-catalysed
hydroxylation has been successfully applied to the screening for
new variants of P450cam from Pseudomonas putida and P450BM3
from Bacillus megaterium (Scheme 1a).15,16 In order to use a
substrate which is more similar to the actual substrate of interest,
Schwaneberg et al. developed a very elegant strategy to screen and
to directly monitor the hydroxylation of aliphatic compounds by
employing various fatty acids with terminal p-nitrophenol (PNP)
ether moieties (p-nitrophenoxycarboxylic acids).3,17,18 The
chromophore PNP gets released upon P450 catalysed hydroxylation
of the PNP-binding carbon (Scheme 1b, top). A similar principle was
applied in the so-called Purpald® assay.19 Here a methyl ether
derivative is employed as a substrate-analogue. The P450 catalysed
The reducing equivalents of P450s are mostly provided by the
cofactor NAD(P)H. Therefore, monitoring the NAD(P)H consumption
as a measure of P450 activity in presence of a substrate seems to be
a valid and convenient detection method.10 However, previous
work has shown that this technique can yield misleading results
(‘false positives’) due to uncoupling events – the consumption of
NAD(P)H without the formation of hydroxylated product.2 Even
with high coupling efficiencies do NAD(P)H depletion assays suffer
from high background signals and hence are not applicable to the
a. Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569
Stuttgart, Germany. E-mail: bernhard.hauer@itb.uni-stuttgart.de
b. School of Chemistry, University of Manchester, Manchester Institute of
Biotechnology, 131 Princess Street, M1 7DN Manchester, United Kingdom
§ These authors contributed equally to this work
†
Electronic Supplementary Information (ESI) available: Experimental details,
Scheme 1 Comparison of different P450 assays with the herein established
additional tables and figures. See DOI: 10.1039/x0xx00000x
detection methodology.
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hydroxylation of the methoxide group results in the formation of accurate and sensitive and shortens the otherwise laborious and
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formaldehyde (Scheme 1b, bottom). The formaldehyde then reacts time intensive fatty acid analysis by a factor DoOf sI:e1v0e.1n0.39/C6CC01749E
with the Purpald® reagent and forms a dark purple colour.
We started our investigations into a direct P450 assay by
All of these assays, however, rely on substrate-analogues. These studying different oxidases for the cofactor independent oxidation
analogues are different to the actual substrate of interest. of alcohols on our example substrate 12-hydroxydodecanoic acid
Following the paradigm of Arnold et al. – you get what you screen (ω-OHC12). In order to design an assay which is broadly applicable in
for – it would be of great use to avoid variations of the substrate in any laboratory, we focussed on commercially available oxidases: (i)
an activity assay intended for the identification of new enzyme glucose oxidase, (ii) alcohol oxidase from Pichia pastoris and (iii)
variants and activities.20 Comparing a P450 substrate dodecanoic GOase from Dactylium dendroides. However these oxidases showed
acid (C12) with the substrate-analogue for the purpald assay – no activity towards ω-OHC12 and other aliphatic alcohols (data not
decanoic-acid-methylether – reveals two major differences with shown). We therefore employed the evolved galactose oxidase
respect to the enzymatic hydroxylation: i) the additional ability of variant GOaseM3-5 which has been previously shown to possess a
the methoxide to accept hydrogen bonds and ii) the lowered wide substrate scope including primary and secondary alcohols.29
stability of the C-H bond of the methoxide for homolytic cleavage With this variant in hand, we tested a range of different alcohols
by approximately 6 kcal/mol (correlated from ethane for C12 and and hydroxylated fatty acids (ω-OHFA) with medium chain length
dimethylether for the decanoic acid methylether).21
via the commonly used horseradish peroxidase (HRP) ABTS assay
Another obstacle in the screening process of P450s is the and were pleased to find GOaseM3-5 to be active towards all of the
variation in enzyme expression rates which can lead to ‘false compounds tested (Table S2). The enzyme displayed higher activity
negatives'. The variation in the concentration of active protein towards primary alcohols when compared to ω-OHFA, whereas the
between different expression trials is especially significant for P450s best activity was determined for 1-hexanol while ω-OHC6 was only
and presumably reasoned by their cell toxic activities.22 In this poorly oxidised.
context, an evaluation of the expression level of each P450 variant
prior to the activity measurement would be highly useful to find focussed on ω-OHC12 as an example substrate intended to develop
promiscuous and more active mutants.23,24
a P450 activity assay. The terminal hydroxylation of fatty acids is of
Encouraged by the results from the activity screen, we next
In the present study, we were interested in developing an assay industrial relevance and since the analysis of fatty acids by gas
for the terminal fatty acid hydroxylase CYP153A from Marinobacter chromatography (GC-FID) requires an extraction and additional
aquaeolei in whole cells (use of CYP153AM.aq-CPRBM3).25–28 The assay derivatisation step there is a high demand for a quick and
relies on the substrate conversion by a readily available galactose quantitative assay (Fig. 2). The assay was performed using resting
oxidase (GOase) mutant (Scheme 1c). It is applicable in whole cell cells expressing CYP153AM.aq-CPRBM3 and incubated with 2 mM C12
P450 transformations for the screening of terminal fatty acid at 25 °C for 2 h. The biotransformation reaction was terminated by
hydroxylases and has accuracy in the micromolar range thereby centrifugation yielding a cell-free supernatant followed by the
using the actual substrate of interest (Fig. 1). Implementing a whole addition of the GOaseM3-5 enzyme in combination with HRP and
cell P450-CO assay – which detects the P450 concentration in whole ABTS, which resulted in a typical colour formation (ABTSox) and an
cells – allowed correlating “cell-activity” to the concentration of increase in absorbance at λ = 420 nm. However, the change in
active P450 proteins. This enabled the identification of active absorbance could only be observed after several hours, depending
variants based on the protein concentration and not only based on on the amount of product formed. This GOase-related lag phase is
the efficiency of substrate conversion. The assay reported herein is known, but so far not fully elucidated.30 We hypothesised that the
Fig. 1 The developed P450 assay: ω-OHC12 formation by CYP153AM.aq
-
CPRBM3 in whole cells followed by the addition of GOase to the reaction
supernatant. GOase is able to oxidize the primary alcohol to the aldehyde
which results in the formation of H2O2 as a by-product. The H2O2 can be
detected in situ by the common horseradish peroxidase ABTS assay.
Fig. 2 The analysis of fatty acids by gas chromatography and by the GOase
based assay.
2 | J. Name., 2012, 00, 1-3
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GOase can be inhibited by either an unstable inhibitor or that concentration in whole cells was then validated in a proof of
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residual metabolic activity consumed the oxygen required for the concept approach. A focussed P450 mutanDt OlibI:r1a0r.y10w39a/sC6gCeCn0e1r7a4te9dE
GOase reaction. We therefore implemented a heat deactivation after the creation of a homology model and docking studies and
step to the procedure after the whole cell reaction and prior to the tested for improved activities towards C12 (Table 1). The variants
GOase addition. The supernatant containing the fatty acid product were compared to the so far most active CYP153A mutant
was heated for 30 min at 90 °C. We were able to circumvent the lag G307A.26,32 Three positions within the active site, previously shown
phase this way and gained an instant increase of absorption upon to be substrate-interacting, and three positions at the substrate
GOase addition (Fig. S1).
entrance tunnel were selected for mutations (Table S3). The
The change in absorption over time correlated well with substituted residues were chosen based on amino acid frequencies
product formation of the whole cell P450 reaction as validated by after sequence alignments of the P450 families as described
parallel analysis of the product formation by GC-FID (Fig. 3). The elsewhere.33,34 The library mutants were expressed in a 2 mL final
detection limit was found to be in the range of 10 - 20 µM of ω- volume in 24 deep-well plates. The cell material from each well was
OHC12. Similar experiments with higher concentrations of ω-OHC12 split in two 1-mL-parts: one was screened via the GOase assay for
added to the resting cells confirmed the correlation between the terminal hydroxylation activity and the other was treated with CO
intensity of the signal in the GOase assay and the amount of to determine the P450 concentration. In order to be able to
product being formed (Fig. S2). Further controls included the compare the product concentrations formed and validate the MTP-
absence of GOase, HRP and ABTS, respectively (Fig. S3). The assay, the reactions were additionally analysed by GC-FID (Table 1
selectivity of the assay for ω-OHC12 was shown by performing the and Table S4). To be able to compare the results within different
reaction with P450BM3 in place of CYP153AM.aq-CPRBM3. P450BM3 is systems and mutants, we set the P450 G307A mutant results to
known to hydroxylate fatty acids in ω-1, ω-2 and ω-3 position.31 By 100 % for the MTP-assay and the GC-FID analysis. The results
performing the GOase assay with the supernatant of the P450BM3 obtained with mutants were calculated relative to the G307A
whole cell reaction, no increase in absorption above the conversion (relative conversion). Judging from the obtained relative
background level could be observed even though product formation conversions, no improved CYP153A variant was found. However, by
was evident by GC-FID analysis (Fig. S4). This confirms the selectivity parallel analysis of the P450 concentration, it was noticed that
of the assay for terminally hydroxylated substrates.
Apart from the determination of P450 activity, we also designed G307A variant showed a concentration of 1.3 µM. Calculating the
the assay for the evaluation of the expression of active P450 relative specific conversion
which includes the enzyme
variant S453A had only a concentration of 0.8 µM whereas the
–
protein. The expression of P450s in E. coli very often suffers from concentration – resulted in a 19 % more active mutant S453. These
large variations and irreproducibility, partially evoked by inclusion results with the novel most active CYP153A could be confirmed by
bodies.22 Previous work has shown that the P450 concentration can GC-FID.
be assessed via a CO binding assay in whole cells.24 By incubating
In conclusion, a new microtiter plate-based P450 assay has
the whole cells with sodium dithionite for 30 min followed by the been developed which utilises the exact substrate of interest and a
addition of CO and incubation for 1 h, we were able to apply this previously reported GOase variant. The assay has been validated for
method in microtiter plates (MTPs). A combination of the CO a range of different substrates and was applied to a focussed
binding assay with the GOase assay enabled us to determine the mutant library. By implementing an additional CO assay to the work
activity of the P450s and thus to correlate these to the enzyme flow the inherent expression problem of P450s could be taken into
concentration. Both steps are applicable to MTPs and do not account to avoid false negatives. The presented assay is
require cell lysis.
quantitative and applicable for small, medium and large mutant
Determining not only the enzyme activity, but also its libraries. By comparing the current extraction and GC-FID protocol
applied to the analysis of fatty acids and related derivatives with
the herein newly developed microtiter plate assay, an economy of
time is evident as 96 samples can be screened in 2.5 rather than
28 h (Fig. 2).
SN received funding from the People Programme (Marie Curie
Actions) of the European Union's 7th Framework Programme
(FP7/2007-2013) ITN P4FIFTY under REA Grant Agreement 289217.
We also wish to thank Łukasz Gricman (University of Stuttgart) for
his help in the generation of the small focussed mutant library and
Jens Schmid for the initial experiments. NJT and SLF thank the Royal
Society for a Wolfson Research Merit Award.
Fig.
3 Plot representing the production of ω-OHC12 after whole-cell
biotransformation and via GOase assay assessment beside GC-FID analysis.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Table 1 Assessment of a small focussed mutant library for terminal hydroxylation activity towards C12 evaluated by the herein developed GOase assay and
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compared to GC-FID analysis. The relative conversion of each variant was calculated based on the conversion of mutant G307A which was set to 100 %. The
DOI: 10.1039/C6CC01749E
relative specific conversion was calculated by dividing the obtained values by the P450 concentration. The specific activities were based on the amount of product
formed (from GC analysis) per minute and per µM of enzyme. (AS: active site; SE: substrate entrance, MTP: microtiter plate)
Mutants
Mutation
locations
AS
Rel. conversion
MTP-Assay [%]
100
P450 conc.
[µM]
Rel. specific conversion
MTP-Assay [%]
100
Rel. specific
conversion GC-FID [%]
100
Specific activity
[µM min-1 µM-1]
2.62 ± 0.57
G307A
V306I
1.3 ± 0.03
1.5 ± 0.22
AS
87 ± 0.03
69 ± 0.04
62 ± 0.13
1.65 ± 1.12
G307R
F455V
D134V
I145L
AS
AS
SE
SE
0
0
0
0
0
0
0
0.9 ± 0.21
1.4 ± 0.02
1.3 ± 0.10
0
0
85 ± 0.01
82 ± 0.02
67 ± 0.01
45 ± 0.04
51 ± 0.08
1.17 ± 0.32
65 ± 0.01
1.34 ± 0.66
S453A
SE
-
86 ± 0.02
0.8 ± 0.14
119 ± 0.03
116 ± 0.03
3.06 ± 0.28
Empty
-
0
0
0
0
pET28a(+)
18
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