Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of Lithocholic Acid

Article abstract of DOI:10.1002/anie.202012675

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β-position but LCA is exclusively hydroxylated at the 6β-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic “small but smart” library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

Full text of DOI:10.1002/anie.202012675

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Accepted Article
Title: Engineering Regioselectivity of a P450 Monooxygenase Enables
the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of
Lithocholic Acid
Authors: Sascha Grobe, Christoffel Badenhorst, Thomas Bayer, Emil
Hamnevik, Shuke Wu, Christoph Grathwol, Andreas Link,
Sven Koban, Henrike Brundiek, Beatrice Großjohann, and
Uwe Bornscheuer
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202012675
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Engineering Regioselectivity of a P450 Monooxygenase Enables
the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of
Lithocholic Acid
[
a]
[a]
[a]
[a]
[a]
Sascha Grobe, Christoffel P. S. Badenhorst, Thomas Bayer, Emil Hamnevik, Shuke Wu,
[
b]
[b]
[c]
[c]
[d]
Christoph W. Grathwol, Andreas Link, Sven Koban, Henrike Brundiek, Beatrice Großjohann,
[
a]
and Uwe T. Bornscheuer*
[
a]
M. Sc. S. Grobe, Dr. C. P. S. Badenhorst, Dr. T. Bayer, Dr. E. Hamnevik, Dr. S. Wu, Prof. Dr. U. T. Bornscheuer
Department of Biotechnology and Enzyme Catalysis
Institute of Biochemistry, University of Greifswald
Felix Hausdorff-Str. 4, 17487 Greifswald, Germany
E-mail: uwe.bornscheuer@uni-greifswald.de
Dr. C.W. Grathwol, Prof. Dr. A. Link
[
[
[
b]
c]
d]
Institute of Pharmacy, University of Greifswald
Friedrich-Ludwig-Jahn-Str. 17, 17487 Greifswald, Germany
M. Sc. S. Koban, Dr. H. Brundiek
Enzymicals AG
Walther-Rathenau-Str. 49, 17487 Greifswald, Germany
Prof. Dr. B. Großjohann
HERBRAND PharmaChemicals GmbH
Betriebsstätte Anklam, An der Redoute 1, 17390 Murchin, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: We engineered the cytochrome P450 monooxygenase
CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and
regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield
ursodeoxycholic acid (UDCA). OleP was previously shown to
hydroxylate testosterone at the 7β-position but LCA is exclusively
hydroxylated at the 6β-position, forming murideoxycholic acid
Alternatively, semi-synthetic UDCA can be produced from cholic
acid (CA)[6] or CDCA.[7,8] The synthesis route starting from CA
forms CDCA within 5 steps, including a Wolff-Kishner reduction,
and an epimerization at C7 to produce UDCA (Scheme 1a,
Scheme S1).[9] The yields of this pathway do not exceed 30%. To
overcome these limitations, a shorter synthesis route based on
the biocatalytic epimerization of CDCA to UDCA (Scheme 1a) has
been developed.[7,10]
(
MDCA). Structural and 3DM analysis, and molecular docking were
used to identify amino acid residues F84, S240, and V291 as
specificity-determining residues. Alanine scanning identified S240A
as a UDCA-producing variant. A synthetic 'small but smart' library
based on these positions was screened using a colorimetric assay for
UDCA. We identified a nearly perfectly regio- and stereoselective
triple mutant (F84Q/S240A/V291G) that produces 10-fold higher
levels of UDCA than the S240A variant. This biocatalyst opens up new
possibilities for the environmentally friendly synthesis of UDCA from
the biological waste product LCA.
LCA is an abundant and inexpensive waste product of meat
production[ as this bile acid is found in farmed animals like
sheep[12], cattle[12], and pigs.[13] Currently, no biotechnological
process[14] utilizing LCA originating from these sources is known,
making it a desirable starting material for the synthesis of UDCA.
A few microbial organisms have been reported to form UDCA
from LCA.[15] For example, the fungus Fusarium equiseti converts
LCA to a number of products, including UDCA at 35% yield.[16]
However, there is currently no enzyme known to selectively
hydroxylate LCA at the 7β-position to form UDCA and its
synthesis pathway in microbial organisms, starting from LCA,
remains enigmatic. An enzyme for direct 7β-hydroxylation would
11]
Introduction
be a valuable tool for direct conversion of LCA to UDCA without
involving the complex metabolism of fungi that invariably[16]
produce multiple undesired side products,[15] complicating
Ursodeoxycholic acid (UDCA) is a valuable bile acid frequently
prescribed for the treatment of cholecystitis as it can solubilize
cholesterol gallstones with fewer side effects than
chenodeoxycholic acid (CDCA).[1] UDCA also has anti-
inflammatory properties[2] and is applied in the therapy of cystic
fibrosis[3] and liver diseases like primary biliary cholangitis. The
downstream processing.
A major challenge in the enzymatic conversion of LCA to
UDCA is the hydrophobicity and thus, extremely low water
[4]
solubility of LCA compared to the more widely used CA or
CDCA.[17]
[5]
major natural source of UDCA is bear bile , a popular traditional
medicine obtained by biliary catheterization of farmed bears.
1
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RESEARCH ARTICLE
Scheme 1. a) Chemoenzymatic synthesis routes towards UDCA starting from CA or CDCA. b) The desired 7β-hydroxylation of LCA towards UDCA by an engineered
OleP variant and wild-type OleP mediated formation of MDCA, starting from LCA.
P450s are heme-containing enzymes capable of stereo- and
regioselective hydroxylation reactions of wide variety of
substrates, using molecular oxygen as oxidant.[18]
structures[21,22] of OleP with bound inhibitors and a natural
substrate analog, we were able to dock LCA into the OleP active
site in multiple conformations (Figure 1). In this evaluation
process, a docked structure was found matching three desired
criteria. First, LCA had to be positioned at most 5 Å away from the
heme iron. Second, the 6β-hydrogen had to be oriented towards
the heme, mimicking the binding of LCA for MDCA formation. This
resulted in the elimination of perpendicular orientations. Finally,
since the steroid nucleus can flip in the horizontal orientation, we
used the most frequently occurring conformations in our docking
experiment (Figure 1a). Based on this docking pose, residues in
a zone of 5–14 Å around the heme were chosen. Important P450
specificity-determining residues known from literature[21,24],[27]
were also selected. Finally, residues interacting with the substrate
analog[24] were also selected. In total, 24 active site residues were
identified for further investigation.
a
This enzyme class has been intensively investigated due to
its potential for late-stage hydroxylation of industrially relevant
precursor compounds.[19] Protein engineering is often employed
to produce P450 monooxygenases that meet the requirements of
excellent regio- and stereoselectivity for the desired
applications.[20]
The monooxygenase CYP107D1 (OleP), originally described
for an epoxidation[21,22] in the oleandomycin biosynthesis
pathway,[23] was found to accept 12-membered macrolactone
substrates[24] as well. OleP can also hydroxylate testosterone at
the positions 6β, 7β, 12β and 15β.[25] However, bile acids like LCA
and deoxycholic acid (DCA) are hydroxylated exclusively at the
6
β-position, forming MDCA (Scheme 1b) and 3α-, 6β-, 12α-
trihydroxy-5β-cholan-24-oic acid, respectively.[26]
Alanine scanning was used to generate space in the active
site to allow LCA to shift from the 6β- towards 7β-hydroxylation
orientation and to investigate the contributions of residues to
hydroxylation activity.
To open up a novel reaction pathway towards UDCA, as no
enzyme is known for selective 7β-hydroxylation, and to avoid
using whole cell fungi, wherein the action of multiple P450s
frequently results in side-product formation, we decided to
engineer OleP and create an Escherichia coli (E. coli)-based
whole-cell system for the regio- and stereoselective hydroxylation
of LCA at the 7β-position to form UDCA (Scheme 1b).
Of the 24 residues selected (Figure 1b), four were already
alanine or glycine. The remaining 20 were exchanged to alanine
by site-directed mutagenesis. The variants were co-expressed
with the redox partner proteins putidaredoxin (PdX) and
putidaredoxin reductase (PdR), employing a two-plasmid system
in E. coli C43(DE3). Whole-cell biotransformations were
performed with 5 mM LCA. HPLC analysis revealed five UDCA-
producing variants (F84A, V93A, L94A, S240A, and V291A).
L179A and S295A produced no product and the remaining
variants produced exclusively MDCA, like wild-type OleP.
Results and Discussion
Initially, we determined which amino acid residues could influence
the positioning of LCA in the OleP active site. Using crystal
2
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RESEARCH ARTICLE
designed a 'small but smart' 3DM library[29] focused on F84, S240,
and V291 (Table S2). The library, containing 4,480 unique
variants, was ordered from Twist Bioscience thereby eliminating
bias, and cloned into the pET-28a vector.[30] E. coli C43(DE3) was
co-transformed with the library and the redox partner system
pACYCDuet-1-pdR/pdX. Sequencing a random sample revealed
only the desired codons. Cultivation and whole-cell
biotransformations took place in 24 deep-well plates, which
allowed us to standardize the process. Detection of the product
initially depended on HPLC measurements.
Screening of libraries towards desired regio- and
stereoselective hydroxylation of steroids and bile acids has with
some exceptions[31] mostly been done by chromatographic
methods. A colorimetric UDCA assay, employing an NADP+-
dependent 7β-hydroxysteroid dehydrogenase,[32] allowed us to
screen 600 variants per round (Scheme S2).
The S240A variant, which was the most active variant
resulting from the initial alanine scanning, was used as a positive
control during screening (Figure S5). Screening a set of 3,400
clones actually led to the identification of S240A as a hit. In total,
3
4 clones, with up to 18-fold higher absorbance than that of the
S240A variant, were identified (Figure S5). Of these 34 hits, 32
were confirmed to produce UDCA by HPLC in comparison to a
commercial standard.
We observed a clear correlation between the colorimetric
UDCA assay signals and the number of side products formed. An
increased signal in the 7β-HSDH assay correlated with fewer side
products as confirmed by HPLC (Figure 2). The variants showing
the highest colorimetric signal mostly produced MDCA and UDCA
in different ratios ranging from 25.9% UDCA formation
(
(
(
F84Q/S240A),
through
31.9%
UDCA
formation
F84C/S240A/V291A), up to 72.8% UDCA formation
F84Q/S240A/V291D) (Table S3). The specificity of
F84Q/S240A/V291D for UDCA formation was remarkable,
considering that wild-type OleP exclusively produces MDCA and
the S240A variant forms only 8.2% UDCA. None of the UDCA-
producing mutants formed the 7α-hydroxylation product (CDCA),
demonstrating perfect stereoselectivity.
Figure 1. a) Structure of OleP (PDB ID: 4XE3) with docked LCA. b) Active site
residues surrounding the docked LCA. The heme cofactor is colored blue and
the iron atom in red, LCA is orange with its surface shown in white. Residues
selected for alanine scanning are colored white and the selectivity influencing
residues F84, S240, and V291 are green.
We continued by sequencing the HPLC-confirmed UDCA-
producing variants and found very clear amino acid preferences
at each of the randomized positions (Figure S6). In UDCA-
producing variants, F84 was most frequently mutated to Q (44%)
or T (19%). For S240, a clear preference for A (87%) was
observed. For V291, the most common variants were G (34%), A
The variants F84A, S240A, and V291A were most promising as
they produced mostly MDCA and UDCA, compared to V93A and
L94A, which produced several unidentified products.
The residue F84 of the BC loop is associated with substrate
recognition.[22] S240 is part of the I-helix and coordinates parts of
the hydrogen-bond network, which connects OleP with the
(
14%) or E (14%). By the introduction of a polar and more flexible
amino acid like Q or T at position F84, the flexibility of the BC-loop
is enhanced. This loop is involved in substrate recognition and
stabilization of the bile acid. Since S240 is both a key residue
establishing the water network of the active site and associated
with the water-mediated coordination of substrates, we
hypothesize that with the introduction of a nonpolar and small
residue, the water network in the active site is remodeled, favoring
the 7β-hydroxylation. This concept is in line with recent findings
3
substrate. V291 is within a β-hairpin (β ) and part of a hydrophobic
bulge facilitating coordination of the substrate by van der Waals
interactions.[22] This suggested that manipulation of the water
network and taking advantage of the hydrophobic interactions,
which have significant effects in the positioning of the substrate,
could enhance the 7β-hydroxylation of LCA to form UDCA.
A 3DM[28] database of P450 monooxygenases was used to
evaluate the most frequently occurring amino acids at each of the
residues altered in the initial UDCA-forming variants (F84A, V93A,
L94A, S240A, and V291A). Each residue was mutated to the four
most frequently occurring amino acids. The aim was to diversify
these positions without the introduction of amino acids which are
evolutionarily unfavored and likely to result in inactive variants.
With these variants created, UDCA formation and the occurrence
of side products were analyzed by HPLC. Substitutions of F84,
S240, and V291 resulted in increased UDCA production. We then
reported for
a testosterone hydroxylation using the P450
monooxygenase BM3.[34] As V291 is part of a hydrophobic cleft,
its mutation to glycine could be beneficial for the 7β-hydroxylation
by the generation of space to position the more dented bile acid
in the active site better compared to flat steroids such as
testosterone.
3
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RESEARCH ARTICLE
screened employing a high-throughput colorimetric UDCA assay.
Starting from an enzyme which did not even have trace activity in
the formation of the target compound, we were able to create a
variant that produces mainly UDCA, with only traces of MDCA
formed. The near complete inversion of regioselectivity from 6β-
(
MDCA formation) to 7β-hydroxylation (UDCA production)
demonstrates how protein engineering can be used to create
custom biocatalysts, even for reactions where no naturally
occurring counterpart has yet been identified.
Acknowledgements
We thank the Technologie-Beratungs-Institut GmbH for funding of
the P450-OH project (grant number: TBI-V-1-198-VBW-068). We
are also grateful to Dr. Henk-Jan Joosten for useful discussions
related to the 3DM database used. T.B. was supported by the
Austrian Science Fund (FWF) through the Erwin Schrödinger
Fellowship (grant number: J4231-B21). S.W. was supported by a
Humboldt research fellowship.
Figure 2. UDCA-producing variants with increased selectivity. OleP variants
show increasing selectivity towards UDCA (blue) and increased UDCA
formation (red). UDCA was quantified by HPLC of three replicate reactions.
Conflict of interest
S.K. and H.B. are employees of Enzymicals AG and B.G. is an
employee of HERBRAND PharmaChemicals GmbH.
To get further insights into the contribution of each position and to
find the most selective variant, we created a set of 37 single,
double, and triple mutants at residues F84, S240A, and V291.
This selection was based on UDCA:MDCA ratios of the individual
variants and sequencing of UDCA producing variants (Figure S6
and Table S3). All newly created variants were analyzed by the
colorimetric assay as well as HPLC. Several single, double, and
triple mutants (Figure 2) were capable of producing UDCA
Keywords: Ursodeoxycholic acid • 7β-hydroxylation • lithocholic
acid • protein engineering • cytochrome P450 monooxygenase
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RESEARCH ARTICLE
Entry for the Table of Contents
We report engineering of a P450 monooxygenase for the stereo- and regioselective 7β-hydroxylation of lithocholic acid to produce
ursodeoxycholic acid (UDCA). Structural and 3DM analysis, and molecular docking identified selectivity-influencing residues. A ‘small
but smart’ mutant library was then screened with a selective colorimetric assay. The best mutant has nearly perfect regio- and
stereoselectivity, enabling a new route for UDCA synthesis.
Institute and/or researcher Twitter usernames: @Wissen_lockt
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