CYP267A1 and CYP267B1 from sorangium cellulosum so ce56 are highly versatile drug metabolizers

Article abstract of DOI:10.1124/dmd.115.068486

The guidelines of the Food and Drug Administration and International Conference on Harmonization have highlighted the importance of drug metabolites in clinical trials. As a result, an authentic source for their production is of great interest, both for t

Full text of DOI:10.1124/dmd.115.068486

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2016/02/03/dmd.115.068486.DC1.html
1521-009X/44/4/495–504$25.00
DRUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.115.068486
Drug Metab Dispos 44:495–504, April 2016
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are
Highly Versatile Drug Metabolizers s
Fredy Kern, Yogan Khatri, Martin Litzenburger, and Rita Bernhardt
Department of Biochemistry, Saarland University, Saarbruecken, Germany
Received November 19, 2015; accepted February 2, 2016
ABSTRACT
The guidelines of the Food and Drug Administration and International the in vitro experiments and Escherichia coli–based whole-cell bio-
Conference on Harmonization have highlighted the importance of conversions. We were able to detect activity of CYP267A1 toward
drug metabolites in clinical trials. As a result, an authentic source for seven out of 22 drugs and the ability of CYP267B1 to convert 14 out of
their production is of great interest, both for their potential application 22 drugs. Moderate to high conversions (up to 85% yield) were
as analytical standards and for required toxicological testing. Since observed in our established whole-cell system using CYP267B1 and
we have previously shown promising biotechnological potential of expressing the autologous redox partners, ferredoxin 8 and ferredoxin-
cytochromes P450 from the soil bacterium Sorangium cellulosum So
NADP+ reductase B. With our existing setup, we present a system
ce56, herein we investigated the CYP267 family and its application for capable of producing reasonable quantities of the human drug me-
the conversion of commercially available drugs including nonsteroidal tabolites 49-hydroxydiclofenac, 2-hydroxyibuprofen, and omeprazole
anti-inflammatory, antitumor, and antihypotensive drugs. The CYP267 sulfone. Due to the great potential of converting a broad range of
family, especially CYP267B1, revealed the interesting ability to convert substrates, wild-type CYP267B1 offers a wide scope for the screening
a broad range of substrates. We established substrate-dependent of further substrates, which will draw further attention to future
extraction protocols and also optimized the reaction conditions for biotechnological usage of CYP267B1 from S. cellulosum So ce56.
Introduction
Although the purification of major metabolites from urine is relative-
ly easy and cheap (Gao et al., 2012), a constant and subject-independent
large-scale production of drug metabolites, for instance, by using
human liver, is hindered by very limited availability. To circumvent
such limitations, alternative approaches of using microorganisms have
been practiced, in which several microorganisms such as the fungus
Cunninghamella sp. or bacterial variants of Streptomyces strains were
shown to transform drugs and xenobiotics to mammalian metabolites
(Zhang et al., 1996; Asha and Vidyavathi, 2009; Bright et al., 2011;
Murphy and Sandford, 2012; Diao et al., 2013). However, because of
the release of side products and the difficulty on handling the microbes
during such biotransformation process, there is now a great interest
in cytochrome P450 (P450) enzymes for the production of drug metab-
olites. In general, P450 enzymes are versatile, heme-containing enzymes,
which catalyze a variety of reactions highlighting them as essential
candidates for biotechnological and pharmaceutical research (Bernhardt
and Urlacher, 2014). It has been shown that the utilization of human P450
enzymes enables the sufficient production of human drug metabolites
employing baculovirus-infected insect cells expressing CYP3A4 or
CYP2D9 (Rushmore et al., 2000), fission yeast expressing CYP2D6
(Peters et al., 2007) or CYP2C9 (Drǎgan et al., 2011; Neunzig et al.,
2012), and Escherichia coli cells expressing CYP3A4, CYP2C9, and
CYP1A2 (Vail et al., 2005). Since it is not mandatory to employ
associated human P450 enzymes to synthesize human drug metabo-
lites (Schroer et al., 2010; Geier et al., 2015), microbial, especially
bacterial, P450 enzymes serve as a good alternative because they are
convenient to handle and usually hold higher expression levels and
activities, recommending the possibility to employ them as useful
The emergence of new diseases, rising concerns about drug re-
sistance, and the decreasing efficacy of the existing drugs are of great
pharmaceutical concern. As a result, drug research during the past
century, driven by chemistry, pharmacology, and clinical science, has
shown an increasing contribution to the development of new therapeu-
tic agents (Drews, 2000). Despite the success in combating the majority
of genetic, infectious, and bacterial diseases, novel drugs and drug
derivatives are consistently demanded. However, the efficacy of such
drugs and their related metabolites need to be tested and approved. The
guidelines published by the Food and Drug Administration and the
International Conference on Harmonization highlight the importance of
qualifying metabolite exposure in clinical trials, in which a metabolite
formed greater than 10% needs to be specifically tested for toxicity
(Food and Drug Administration, 2008; International Conference on
Harmonization, 2009, 2012). Due to the frequent introduction of novel
drugs and drug candidates with new or modified chemical structure,
implementation of costly multistep chemical syntheses may not be
sufficient enough to overcome the demand of the respective metabolites
(Rushmore et al., 2000).
This work was supported by Deutsche Forschungsgemeinschaft [Grant
Be1343/23].
This manuscript describes original work and is not under consideration by any
other journal. All authors approved the manuscript and this submission. The
authors declare no competing financial interests.
dx.doi.org/10.1124/dmd.115.068486.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: AdR, adrenodoxin NADP+ reductase; Adx4-108, truncated adrenodoxin; BM3, cytochrome P450 102A1; CO, carbon monoxide;
FdR_B, ferredoxin-NADP+ reductase B; Fdx8, ferredoxin 8; Fpr, ferredoxin NADP+ reductase; HPLC, high-performance liquid chromatography;
MS/MS, tandem mass spectrometry; P450, cytochrome P450.
495

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Kern et al.
enzymes. The resulting plasmid pCDF_F8B contains the genes for expressing
the autologous Fdx8 and FdR_B from S. cellulosum So ce56 (see also
Supplemental Fig. 1).
Expression and Purification. CYP267A1 and CYP267B1 have been
expressed and purified as previously described (Khatri et al., 2015) using
the T7 promoter–based expression construct of CYP267A1 and CYP267B1
(pET22b_CYP267A1 and pET22b_CYP267B1). The electron transfer part-
ners Fdx8 and FdR_B were expressed and purified as noted elsewhere (Ewen
et al., 2009).
Media and Buffers. For the heterologous expression of CYP267A1 and
CYP267B1, terrific broth medium (24 g yeast extract, 12 g peptone, 4 ml glycerol,
2.31 g K2HPO4, and 12.54 g KH2PO4 per liter H2O) was used. The whole-cell
conversions were performed in M9CA medium (6 g Na2HPO4, 3 g KH2PO4, 0.5 g
NaCl, 1 g NH4Cl, 4 g Bacto Casamino Acids (BD Diagnostics, Sparks, NV), 4 g
glucose, 50 ml 1M CaCl2, 2 ml 1 M MgSO4, 2 ml of trace elements solution per
liter H2O; trace elements solution contained 2.5 g EDTA, 250 mg FeSO4, 25 mg
ZnCl2, and 5 mg CuSO4 per 50 ml H2O).
Spectral Characterization of the CYP267 Family. UV-visible spectra for
the purified P450 enzymes were recorded at room temperature on a double-beam
spectrophotometer (UV-2101PC, Shimadzu, Kyoto, Japan). The enzyme solu-
tion (2 mM) in 10 mM potassium phosphate buffer, pH 7.4, was dosed with a few
grains of sodium dithionite to reduce the heme-iron and the sample was split into
biocatalysts (Bernhardt, 2006). The genetic manipulation of bacte-
rial P450 enzymes toward a drug metabolizing activity has been
successfully demonstrated for several P450 enzymes including the
most studied P450 102A1 (BM3), CYP102A1 (Whitehouse et al.,
2012; Ren et al., 2015). However, the engineering of enzymes against
their native, narrow substrate range, or in general the screening for
enzymes to produce certain drug metabolites, is time consuming and
complex, which can be overcome by employing versatile wild-type
P450 enzymes showing an untypically broad substrate range (Yin
et al., 2014).
During our recent investigations on P450 enzymes from the
myxobacterium Sorangium cellulosum So ce56, several interesting
enzymes displaying new properties and substrate specificities have been
discovered, which lead us to investigate the potential of these P450 en-
zymes for an application as drug metabolizing biocatalysts (Khatri et al.,
2010, 2013; Schifrin et al., 2015; Litzenburger and Bernhardt, 2016).
Therefore, we used bioinformatics analysis to identify myxobacterial
P450 enzymes that are closely related to drug metabolizing P450
enzymes. Among others, the two members of the CYP267 family,
CYP267A1 and CYP267B1, were found to be potential candidates.
Although purified CYP267A1 and CYP267B1 have been previously two cuvettes. The baseline was recorded between 400 and 700 nm. The sample
cuvette was bubbled gently with carbon monoxide (CO) for 1 minute and a
spectrum was recorded. The concentration of the P450 enzymes was estimated
by CO-difference spectroscopy assuming molar extinction coefficient « (450–
490 nm) = 91 mM21cm21 according to the method of Omura and Sato (1964).
In Vitro Conversions. A reconstituted in vitro system containing the
corresponding P450 (0.5 mM), FdR_B (1.5 mM), and Fdx8 (10 mM), and a
cofactor regenerating system with glucose-6-phosphate (5 mM) and glucose-6-
phosphate dehydrogenase (2 U/ml) in potassium phosphate buffer (20 mM, pH 7.4,
1% glycerin) was used. The potential substrates, except olanzapine and omeprazole
(both dissolved in dimethylsulfoxide), were dissolved in ethanol (10 mM) and added
to an end concentration of 200 mM. The total volume of the reaction was 250 ml.
The reaction was started by the addition of NADPH (800 mM) and carried out for
3 hours at 30ꢀC. For substrates 1, 9, 11–15, and 18–21, 1 M glycine buffer (pH 11)
or acetate buffer (pH 4) was added after reaction to enable improved recovery of the
analytes. Therefore, the reaction was stopped by adding buffer (300 ml) or
organic solvent (500 ml). The extraction was performed twice with 500 ml of the
appropriate solvent (see (Supplemental Table 0). A negative control without
P450 was implemented for each substrate to verify the P450-dependent
reaction.
Whole-Cell Conversions. The experiments were performed with C43(DE3)
cells, which were transformed with two plasmids, one encoding CYP267A1
(pET22b_CYP267A1) or CYP267B1 (pET22b_CYP267B1) and the second one
encoding the redox partners Fdx8 and FdR_B (pCDF_F8B). For the main culture,
50 ml M9CA medium containing ampicillin (100 mg/ml) and streptomycin
(50 mg/ml), inoculated with the corresponding overnight culture in lysogeny broth
medium (dilution 1:100), was used. At an optical density of 0.9–1, the induction
was initiated by adding 1 mM isopropyl b-D-1-thiogalactopyranoside and 0.5 mM
5-aminolevulinic acid and the temperature was set to 28ꢀC. After 21 hours of
expression, the temperature was set to 30ꢀC and the substrate was added to a final
concentration of 200 mM. To enable higher substrate conversion, EDTA (20 mM)
or polymyxin B (32 mg/l) was added to increase permeability and substrate uptake
of the E. coli cells (Janocha and Bernhardt, 2013). After 48 hours at 30ꢀC, a 500 ml
sample was removed, quenched by adding buffer or organic solvent, and extracted
two times with 500 ml of organic solvent (see Supplemental Table 0). The organic
phases were collected and evaporated to dryness. The extracts were stored at
220ꢀC until analysis. All experiments were done twice, including a negative
control (cells only transformed with pCDF_F8B).
shown to convert certain drugs (Kern et al., 2015; Litzenburger et al.,
2015) and CYP267A1 was found to catalyze the hydroxylation of
fatty acids (Khatri et al., 2015), a broader analysis of their substrate
spectrum has never been tested. Therefore, in this study, we have
tested the in vitro and whole-cell conversion of 22 widely used drugs
(Fig. 1) by CYP267A1 and CYP267B1 for the first time. Compounds
showing significant in vitro conversion were further chosen for a
whole-cell biotransformation to upscale the metabolite production for
the structural elucidation of the product(s) via NMR spectroscopy. We
demonstrate that seven out of 22 drugs can be converted by CYP267A1
and that CYP267B1 shows activity toward 14 out of 22 drugs.
Materials and Methods
Chemicals. Amitriptyline, amodiaquine, haloperidol, losartan, olanzapine,
quinine, repaglinide, ritonavir, tamoxifen, and thioridazine were kindly pro-
vided by Dr. Stephan Lütz (Novartis, Basel, Switzerland). Isopropyl-b-D-1-
thiogalactopyranoside and d-aminolevulinic acid were purchased from Carbolution
Chemicals (Saarbruecken, Germany). Bacterial media were purchased from
Becton Dickinson (Heidelberg, Germany). All other chemicals were obtained
from standard sources at the highest purity available.
Strains. The E. coli strains Top 10 and NovaBlue Singles Competent Cells for
cloning purpose were obtained from Invitrogen (San Diego, CA) and Merck
(Duesseldorf, Germany). The E. coli strains BL21-Gold(DE3) for the heterol-
ogous expression of CYP267A1 and C43(DE3) for the heterologous expression
of CYP267B1 were purchased from Agilent Technologies (Santa Clara, CA),
whereby C43(DE3) was also used for whole-cell conversions.
Plasmids. The genes of CYP267A1 and CYP267B1 were cloned into a
pET22b plasmid (Novagen, Darmstadt, Germany) as described elsewhere
(Litzenburger et al., 2015). The pKKHC plasmids for the heterologous
expression of the autologous redox partners ferredoxin 8 (Fdx8) and ferredoxin-
NADP+ reductase B (FdR_B) originate from previous work done in our laboratory
(Ewen et al., 2009).
For the expression of the redox partners in the whole-cell system, the pCDF_dFA
plasmid from Litzenburger et al. (2015) was used and changed as follows. The gene
encoding ferredoxin NADP+ reductase (Fpr) was excised using the restriction
enzymes NdeI and KpnI and substituted with the FdR_B gene obtained from
Analysis of the In Vitro and Whole-Cell Conversions via High-
pKKHC_FdR_B using the same restriction enzymes. The ligation reactions were Performance Liquid Chromatography (HPLC). The HPLC analysis was
performed with the Fast-Link DNA Ligase Kit (Biozym Scientific GmbH, Hessisch
Oldendorf, Germany). From the resulting plasmid pCDF_AFB containing the
performed on a Jasco (Gross-Umstadt, Germany) HPLC 2000 system consisting
of a PU-2080 Plus Pump, a AS-2050 Plus Sampler and a UV-2075 Plus UV/
truncated adrenodoxin (Adx4-108) from Bos taurus and ferredoxin-NADP+ Vis-Detector. For the HPLC analysis, samples of substrates 3, 10, 13, and 22 were
reductase FdR_B from S. cellulosum So ce56, the gene of Adx4-108 was excised with
NcoI and HindIII. The resulting open plasmid was ligated with the gene of
Fdx8 previously obtained from pKKHC_Fdx8 using the mentioned restriction
dissolved in 75 ml acetonitrile and 75 ml Milli-Q water. The remaining substrates
were dissolved in the same volumes of solvents containing 0.1% trifluoroacetic
acid. Analyses were performed on a reversed-phase column (125/4 Nucleodur

CYP267A1 and CYP267B1 Are Highly Versatile Drug Metabolizers
497
Fig. 1. Structural illustration of the tested drugs. In the case of drugs 13, 19, 20, and 21, racemic mixtures were used in all experiments.
100-5 C18ec; Macherey Nagel, Düren, Germany) at a flow rate of 1 ml/min and a
Upscaling of the Whole-Cell Biotransformation System and Purification
temperature of 40ꢀC. The injection volume was set to 30 ml. Due to the amount of of the Products. To obtain sufficient amounts of products for structure elucidation
different substrates the HPLC methods (including detection wavelengths) were via NMR analysis, the previously described whole-cell conversions were up-scaled
substrate dependent and are presented in the Supplemental Material (Supplemental to a total volume of 2.5 l. After 48 hours, the reaction was stopped with the same
Table 1).
volume of the appropriate solvent (see Supplemental Table 0). Before extraction of

498
Kern et al.
the product of substrate 13, the culture was set to a pH of 11 using 2 M KOH and
subsequently purified as described previously (Litzenburger et al., 2015). The
purification of product 13a was done in the absence of trifluoroacetic acid.
NMR Analysis. The structures of the products were analyzed by NMR
spectroscopy (Institute for Pharmaceutical Biology, Saarland University). The
1H- and 13C-NMR were recorded on a Bruker (Rheinstetten, Germany) 500 MHz
NMR spectrometer. Two-dimensional NMR spectra were recorded as gs-H,
H-COSY, gs-HSQC, and gs-HMBC. All chemical shifts are relative to CDCl3
(d = 77.00 for 13C-NMR; d = 7.24 for 1H-NMR) or CD3OD (d = 49.00 for
13C-NMR; d = 3.31 for 1H-NMR) using the standard d notion in parts per million.
Tandem Mass Spectrometry (MS/MS) Analysis. Product 13a was addi-
tionally characterized by MS/MS analysis (Institute of Bioanalytical Chemistry,
Saarland University) with a API 2000 Qtrap (ABSciex, Darmstadt, Germany).
Detailed settings of the MS/MS experiments can be found in section 8 (MS/MS
settings) of the Supplemental Material.
P450 enzymes such as CYP107E4 from Actinoplanes sp. ATCC 53771
and CYP105 and CYP256 from Rhodococcus jostii RHA1, showing
amino acid sequence identities of 38.9%, 34.2%, and 33.4%, respectively
(Fig. 2; Supplemental Table 2). These bacterial P450 enzymes are
considered to be active drug metabolizers for several drugs including
amitriptyline, chlorpromazine (2), and diclofenac (5) (Prior et al., 2010;
Kulig et al., 2015). In addition, we have previously shown that CYP264A1
was able to convert tricyclic drug molecules (Litzenburger et al., 2015),
and CYP265A1 and CYP266A1 were able to hydroxylate the antitumor
drug epothilone D (Kern et al., 2015). As a result, our homology study
with the drug metabolizing bacterial P450 enzymes suggested that
the two members of the CYP267 family (in addition to CYP265A1
and CYP266A1) are potential candidates for metabolizing certain drugs.
The amino acid sequence alignment of CYP267A1 and CYP267B1
showed an identity of 38%. CYP267B1 possesses a conserved heme-
binding domain (347FGGGIHFCLG356), the conserved threonine in the
I-helix (243AGHETT248), and glutamic acid and arginine in the K-helix
Results
Bioinformatics Studies and Comparison of CYP267A1 with
CYP267B1
(
281EEALR285), whereas in CYP267A1 the conserved phenylalanine in
the heme-domain is replaced by leucine (L366) (Khatri et al., 2015).
To identify homologs of potentially drug metabolizing P450 Amino acid sequence alignment of the CYP267 family with human
enzymes from S. cellulosum So ce56, all of the 21 P450 enzymes P450 enzymes demonstrated that CYP267B1 showed the highest
(Khatri et al., 2010) from this bacterium were aligned with the known identities of 20.7%, 20.2%, 19.6%, 19.3%, and 19.0% with CYP2W1,
bacterial drug metabolizing P450 enzymes (Supplemental Table 2). We CYP2C8, CYP2A6, CYP2D6, and CYP3A4, respectively (Supplemental
observed that the CYP265A1, CYP266A1, and the CYP267 family of Table 2), which are considered to be efficient drug metabolizers (Wrighton
this bacterium clustered within the same clan of the drug metabolizing and Stevens, 1992; Guengerich, 1999).
Fig. 2. The radial view of an unrooted phylogenetic tree obtained by MEGA4 (version 4.0, (Tamura 2007)) analysis for the determination of relatedness of the 21 P450
enzymes from S. cellulosum So ce56 (in black) with respect to drug metabolizing bacterial P450 enzymes CYP105D1 (P26911.1) from Streptomyces griseus; CYP51_RHA1
(Q0S7M9), CYP105_RHA1 (Q0SDH7), CYP116_RHA1 (Q0RUR9), CYP125_RHA1 (Q0S7N3), CYP256_RHA1 (Q0RXF8), and CYP257_RHA1 (Q0RVH0) from
Rhodococcus jostii RHA1; CYP107E4 (ACN71221.1) from Actinoplanes sp. ATCC 53771; and CYP116B4 (EAV41564.1) from Labrenzia aggregate (in gray). The cluster
of drug metabolizing P450 enzymes is shown in the gray clan. The bar in the tree indicates 0.5 amino acid substitutions per amino acid for the branch length.

CYP267A1 and CYP267B1 Are Highly Versatile Drug Metabolizers
499
Expression, Purification, and Characterization of the CYP267 Optimization of the E. coli–Based Whole-Cell Bioconversion
Family Members
System
Since we observed a significant increase in the product formation
First expression studies for CYP267B1 were realized using the
vector pCWori+. However, the yield of CYP267B1 using the pCWori+- when using the autologous redox partners, we investigated the coex-
based expression construct was very low (,20 nmol/l E. coli culture pression of the autologous redox partners Fdx8 and FdR_B for our
after purification). Therefore, the protein has been expressed and whole-cell bioconversion experiments. For comparison, a whole-cell
purified using a T7-based expression construct (pET22b_CYP267B1) system coexpressing Adx4-108 and Fpr was used, since it has been shown
(Litzenburger et al., 2015), in which the protein yield was increased previously that the whole-cell conversion of 4-methyl-3-phenyl-coumarin
5-fold (100 nmol/l).
by CYP264A1 from S. cellulosum So ce56 increased when Fpr instead
As shown in Fig. 3, the UV-visible absorption spectrum of the of AdR was used (Ringle et al., 2013). However, when substituting
oxidized CYP267B1 showed the presence of the Soret band (g) at Adx4-108/Fpr with the autologous redox partners Fdx8/FdR_B, the yield
416 nm, and the Q-bands at 567 nm (b) and 533 nm (a). The reduction of the product was even further increased. In the case of CYP267A1, a
of CYP267B1 with sodium dithionite showed a slightly diminished nine times higher conversion of substrate 20 was observed (from 5% to
absorption peak for the Soret band at 410 nm and a single peak in the 45% thioridazine-5-sulfoxide 20a) (Table 1). Likewise, the formation of
Q-region (538 nm). The reduced CO-complex of CYP267B1 showed a chlorpromazine sulfoxide (2a) was nearly doubled by the CYP267B1-
typical peak maximum at 449 nm. The purified CYP267A1 showed the Fdx8-FdR_B whole-cell bioconversion system compared with CYP267B1-
same spectroscopic features as previously described (Khatri et al., Adx4-108-Fpr (from 18% to 30% 2a) (Table 1). In addition, we also
2015), with the characteristic peak maximum at 448 nm in the CO- observed a higher yield (from 7.5% to 26%) of the product
difference spectroscopy experiment and a peak maximum at 418 nm in 10-hydroxyamitriptyline from amitriptyline (Table 1). On the basis
the oxidized form of CYP267A1.
of these results, all further drug conversions were performed using
Fdx8/FdR_B as redox partners in all in vitro and E. coli–based whole-
cell bioconversion experiments.
Optimization of the In Vitro Conversion
When establishing a whole-cell system for P450 enzymes, indole-
dependent inhibition should also be considered. The metabolism of
tryptophan by the tryptophanase TnaA of E. coli results in the formation
of indole (Li and Young, 2013). Since tryptophan is present in terrific
broth medium, a concentration of over 600 mM indole was detected after
72 hours (Ringle et al., 2013). It was observed that indole acts as an
inhibitor of CYP264A1 from S. cellulosum So ce56 (Ringle et al., 2013)
and CYP109B1 from Bacillus subtilis (Girhard et al., 2010). In addition,
some P450 enzymes are known to convert indole, and in this way
competing with the normal substrate (Gillam et al., 2000). In case of the
two members of the CYP267 family, the presence of 600 mM indole
decreases the product formation in vitro by up to 40% for CYP267B1 and
by up to 85% in the case of CYP267A1 (Supplemental Fig. 3). Therefore,
we performed the whole-cell bioconversions in a defined M9CA minimal
medium, which was previously shown to exhibit a very low amount of
indole when using E. coli as a host (,5 mM) (Ringle et al., 2013).
Furthermore, the effect of the additives EDTA and polymyxin B was
also investigated during the whole-cell conversions, since it has been
shown previously that the presence of EDTA or polymyxin B enhances
substrate uptake of E. coli cells for resin acid diterpenoid conversion by
a CYP105A1-based whole-cell biocatalyst (Janocha and Bernhardt,
2013). We observed that the highest product formation was obtained
when 20 mM EDTA for substrates 1, 2, 5, 7, 9, 15, and 16, and 32 mg ml21
polymyxin B for substrates 11, 13, 18, 19, and 20 were applied in the
whole-cell system. Higher concentrations of both additives did not
alter the product pattern for the tested drugs.
Since P450 enzymes are monooxygenases, they require electrons
from NADPH, which are transferred by autologous or heterologous
redox partners (Hannemann et al., 2007). The substrate turnover also
depends on the coupling and the efficiency of the redox partner
proteins. In the case of the CYP267 family we observed limited in
vitro conversions (Kern et al., 2015; Litzenburger et al., 2015) when
using the bovine redox partners Adx4-108/adrenodoxin NADP+
reductase (AdR). Therefore, we substituted the heterologous Adx4-108
and AdR with the autologous redox partners Fdx8 and FdR_B from
S. cellulosum So ce56, which were previously shown to increase the
CYP267B1-dependent epothilone D conversion (Kern et al., 2015). In
our previous studies on the conversion of tricyclic antipsychotics and
antidepressants (Litzenburger et al., 2015), thioridazine (20) could be
identified as a substrate for CYP267A1 and amitriptyline as well as
chlorpromazine (2) for CYP267B1, respectively; however, with low
in vitro and whole-cell conversion. The substitution of Adx4-108/AdR
with the autologous redox partners Fdx8/FdR_B showed increased
thioridazine-5-sulfoxide (20a) formation (from 43% to 50%) during the
in vitro conversion of substrate 20. Likewise, the in vitro conversion of
amitriptyline by CYP267B1 showed a significant enhancement of
10-hydroxyamitriptyline formation (from 15% to 60%), whereas the in
vitro conversion of chlorpromazine (2) showed no difference.
Optimization of Product Extraction and HPLC Conditions for the
Investigation of Drug Conversions
Due to the diverse chemical structures and functional groups of the
tested drug molecules, we established a substrate-dependent extraction
protocol to improve our experimental conditions for efficient analyses
and product purification (Supplemental Table 0). We also optimized the
HPLC conditions as listed in the Supplemental Table 1.
CYP267-Dependent Substrate Conversion
In Vitro and Whole-Cell Conversions by CYP267A1. The in
vitro conversions revealed that seven out of 22 drugs were converted by
CYP267A1. In comparison with CYP267B1, the product pattern of
Fig. 3. Spectroscopic characterization of CYP267B1. The UV-visible spectra of
oxidized (black line), dithionite reduced (dashed line), and CO-bound (gray line)
CYP267B1 were recorded in 10 mM potassium phosphate buffer, pH 7.5.

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Kern et al.
TABLE 1
Comparison of the whole-cell conversions with CYP267A1 and CYP267B1 using different redox partners
Conversion with
Conversion with
Model Substrate
CYP267
Product
heterologous Adx4-108/Fpra autologous Fdx8/FdR_B
%
%
Amitriptyline
Chlorpromazine (2)
Thioridazine (20)
B1
B1
A1
7.5
18
5
26
30
45
10-Hydroxyamitriptylinea
Chlorpromazine sulfoxidea (2a)
Thioridazine-5-sulfoxidea (20a)
aTaken from Litzenburger et al. (2015).
CYP267A1 differs only for chlorpromazine (2; 2.4%) for which one addition, amodiaquine (1, 10.2%), losartan (9; 8.9%), noscapine (11;
single product was observed. For ibuprofen (7), tamoxifen (18), and 12.1%), olanzapine (12; 16.5%), omeprazole (13; 13.7%), papaverine
terfenadine (19) the yields of the product formation were significantly (15; 12.6%), and tamoxifen (18; 15%) were converted by CYP267B1.
lower compared with the respective CYP267B1-dependent conversions Very low conversion was observed for ritonavir (17; 1.4%) and
(7: 1.5%, 18: 3.7%, and 19: 2.1%). Despite having lower conversions terfenadine (19; 4.1%). However, during the in vitro experiments, also
with CYP267A1, five new substrates for CYP267A1 were identified one minor side product was observed for substrates 7 (2.2% 6 0.1%),
(4, 6, 7, 18, and 19) (Fig. 4A). However, during in vitro conversion of 9 (1.9% 6 0.4%), 11 (1.85% 6 0.45%), 12 (2% 6 0.1%), 13 (7% 6
substrates 18 and 20, one side product has been observed for substrate 0.4%), 15 (2.2% 6 0.1%), 16 (7.8% 6 1.4%), and 18 (2.55% 6
18 (1.9% 6 1.1%) and in the case of substrate 20 two minor side 0.45%). In the case of substrate 14, two minor side products were
products (3.7% 6 0.3% and 2.6% 6 0.1%) were found.
found (5.1% 6 1.0% and 3% 6 0.5%).
Only dextromethorphan (4), haloperidol (6), and thioridazine (20)
The 14 drugs identified as substrates for CYP267B1 during the in
were further employed for the investigation in the whole-cell system vitro experiments were further investigated in the corresponding whole-
with CYP267A1-Fdx8-FdR_B, in which compounds 4 and 6 showed cell experiments, where the highest yield was observed for omeprazole
no conversion. Due to the higher in vitro conversion of substrates 2, 18, (13; 78.1%). The compounds 2 (30.3%), 5 (38%), and 7 (44.1%)
and 19 with CYP267B1, these substrates were only tested in the showed similar yields compared with the corresponding in vitro
CYP267B1-Fdx8-FdR_B whole-cell system. However, using CYP267A1, experiments. We also observed a high conversion of losartan (9) to
substrate 20 was successfully converted to product 20a yielding a one product; however, without expressing CYP267B1 in the whole-cell
44.7% product formation in our E. coli–based whole-cell bioconver- experiment. This observation leads to the assumption that substrate
sion (Fig. 4A).
9 might have been oxidized by E. coli C43(DE3) to losartan carboxy
In Vitro and Whole-Cell Conversions by CYP267B1. It is very acid. Due to the limited availability of a reference standard, we were not
interesting to note that the in vitro conversions of the 22 tested drugs able to further investigate this assumption. The CYP267B1-Fdx8-FdR_B
showed that CYP267B1 was able to convert 14 out of 22 compounds whole-cell system was also able to convert substrates 1, 11, 15, 16, 18,
(Fig. 4B), seven more than CYP267A1. The highest in vitro yield was and 19, but in lower yields (#10%). In the CYP267B1-dependent whole-
observed for oxymetazoline (14; 77.7%) and moderate in vitro cell experiments, minor side products were only formed in the case of
conversions were detected for chlorpromazine (2; 37%), diclofenac substrates 11 (1.25% 6 0.15%), 13 (5.3% 6 0.2%), 15 (5.4% 6 0.5%),
(5; 37.5%), ibuprofen (7; 31.1%), and repaglinide (16; 41.4%). In and 16 (5.2% 6 0.4%), showing identical retention time to those
Fig. 4. The main metabolite formation in vitro (black bar) and in the whole-cell (gray bar) system by CYP267A1-Fdx8-FdR_B (A) and CYP267B1-Fdx8-FdR_B (B). (A)
The compounds 1, 3, 5, 8–17, 21, and 22 were not converted by CYP267A1 and are therefore not shown. Only the substrates 4, 6, and 20 were further tested in the whole-cell
system, whereby compounds 4 and 6 showed no conversion. The highest whole-cell conversion for substrate 20 was achieved with the supplement of polymyxin B
(32 mg/ml). (B) The highest yields for the whole-cell conversions of compounds 1, 2, 5, 7, 9, 15, and 16 by CYP267B1 were achieved in the presence of 20 mM EDTA. In
the case of compounds 11, 13, 18, and 19, the highest conversions were observed with the addition of polymyxin B (32 mg/ml). Despite the in vitro conversions of
compounds 12 and 14 by CYP267B1, no conversion was observed in the corresponding whole-cell experiments. Due to the absence of conversion in the in vitro
experiments, compounds 3, 4, 6, 8, 10, and 20–22 are not presented in this diagram.

CYP267A1 and CYP267B1 Are Highly Versatile Drug Metabolizers
501
observed in the corresponding in vitro experiments. For compounds compounds of pharmaceutical interest has continuously grown (Julsing
12 and 14, all attempts to utilize them in the whole-cell conversion et al., 2008). This progress can directly be of use for efficient and time-
system were unsuccessful, despite having high in vitro conversions saving production of human drug metabolites. High yields and conver-
(16.5% for compound 12 and 83% for compound 14). For the substrates sion rates can already be achieved by using corresponding human
that were not converted (3, 4, 6, 8, 10, 20, 21, and 22) or showed poor (Rushmore et al., 2000; Vail et al., 2005; Schroer et al., 2010; Geier
conversion (17) during the in vitro assay, attempts of investigating these et al., 2012; Schiffer et al., 2015) or suitable nonhuman (Taylor et al.,
drugs within the whole-cell system were discarded. Due to the high 1999; Otey et al., 2006; Sawayama et al., 2009; Reinen et al., 2011; Di
conversion in the whole-cell experiments, compounds 2, 5, 7, and 13 Nardo and Gilardi, 2012; Kiss et al., 2015; Ren et al., 2015) P450
were further chosen for upscale and product characterization.
enzymes in a whole-cell system to produce respective metabolites. The
majority of published bacterial P450 enzymes used for the conversion
of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.
We investigated the native myxobacterial P450 enzymes from
S. cellulosum So ce56 for their application as drug metabolizers since
soil bacteria should be able to convert and metabolize different
xenobiotics present in their environment. Therefore, special attention
was given to the CYP267 family.
It is interesting to note that CYP267B1 revealed the remarkable
ability to accept substrates with completely different chemical struc-
tures and functions. In addition to the capability of converting tricyclic
compounds (Litzenburger et al., 2015), the large 16-membered macro-
lide epothilone D (Kern et al., 2015), and small structures such as
apocarotenoids (Litzenburger and Bernhardt, 2016), CYP267B1 also
showed activity toward the conversion of 14 out of 22 different drugs.
In contrast, CYP267A1 was only able to convert seven out of 22 drugs.
The drugs converted by CYP267A1 and CYP267B1 feature a variety of
chemical structures such as heterocyclic aromatics, morphinan class
compounds, and alkaloids, thus increasing both the known substrate
spectrum for this P450 family and the conceivable fields of their
application. The metabolism of a drug molecule by a human P450
usually results in the formation of several side products (Table 2) since
the main aspect of drug metabolism is excretion out of the body. Hence,
the application of myxobacterial P450 enzymes is favorable in en-
abling the production of a single human drug metabolite, highlighting
their ability for biotechnological processes to produce a metabolite in
large quantity.
However, an important bottleneck in the application of P450
enzymes in biotechnological processes is often the efficiency of the
redox system (Bernhardt and Urlacher, 2014). Therefore, we first
identified efficient autologous redox partners to transfer electrons to the
CYP267 family. Although the autologous redox system Fdx8/FdR_B
from S. cellulosum So ce56 has already been shown to transfer electrons
to myxobacterial CYP109D1, CYP260A1, and CYP264A1 (Khatri
et al., 2010; Ringle et al., 2013), the heterologous bovine Adx4-108 with
AdR or the E. coli Fpr has been shown to be more efficient (Khatri et al.,
2013; Ringle et al., 2013). However, in this study, the substitution of
Adx4-108/Fpr by Fdx8/FdR_B showed a significant increase of product
yields for drug molecules (Table 1) when using the members of the
CYP267 family. As a result, an E. coli–based whole-cell bioconversion
system has been established containing the autologous redox partners of
the CYP267 family.
Another bottleneck in whole-cell conversions that we faced during
our studies was substrate uptake and indole inhibition. As a result, we
established a substrate-dependent protocol, where EDTA and poly-
myxin B, which are shown to enhance substrate uptake into E. coli cells
(Janocha and Bernhardt, 2013), significantly increased the limited
whole-cell conversions (Fig. 4, A and B). To overcome inhibition of
CYP267A1 and CYP267B1 by indole (Supplemental Fig. 3), we
performed the whole-cell experiments in defined M9CA medium.
However, for substrates 2, 11, 14, 16, 18, and 19 the CYP267B1-
dependent whole-cell conversion showed lower yields compared with
Production of Drug Metabolites Using an E. coli–Based Whole-
Cell Bioconversion System and Purification of Products via
Preparative HPLC
The whole-cell system of CYP267B1-Fdx8-FdR_B was up-scaled to
2.5 l of M9CA medium for substrates 2, 5, and 7. For compound 13, an
upscaling to 500 ml was sufficient enough to produce 5 mg of product
13a. In the case of whole-cell system CYP267A1-Fdx8-FdR_B and
substrate 20, the conversion was also up-scaled to 2.5 l M9CA medium.
For the compounds converted in a larger scale, comparable yields as
previously described (Fig. 4, A and B) were observed, revealing the
great potential of the established bioconversion system for future
biotechnological upscaling. The products were purified via preparative
HPLC and the purity of the isolated products was further verified by an
additional HPLC measurement. The chromatograms of the purified
products (5a, 7a, and 13a) and the pure substrates (5, 7, and 13) are
shown in the Supplemental Material (Supplemental Fig. 2), confirming
the high purity of the corresponding products. For products 2a and 20a,
the chromatograms coincide with previous data and can be found in the
Supplemental Material (Litzenburger et al., 2015).
Drug Metabolites Formed by the CYP267 Family
As previously presented, CYP267A1 is able to convert seven out of
22 drugs and CYP267B1 catalyzes the conversion of 14 out of 22 drugs in
vitro. However, only drug 20 for CYP267A1 and 10 drugs (1, 2, 5, 7, 11,
12, 15, 16, 18, and 19) for CYP267B1 were successfully converted in our
whole-cell system. For drugs 2, 5, 7, 13, and 20 showing high yields after
whole-cell biotransformation, the respective metabolites were additionally
elucidated with an up-scaled production and via NMR measurements. A
comprehensive overview of the analyzed drugs, and the human metab-
olites produced by the two members of the CYP267 family, is presented in
(Supplemental Fig. 6). CYP267B1 is able to catalyze an aromatic
hydroxylation of drug 5 to the human metabolite 49-hydroxydiclofenac
(5a) and an aliphatic hydroxylation of drug 7 to 2-hydroxyibuprofen (2a).
Furthermore, the sulfoxidation of drugs 2 and 13 is catalyzed by
CYP267B1 and the sulfoxidation of drug 20 is catalyzed by CYP267A1.
All products were obtained with high purity and sufficient amounts (5–
10 mg) for the structure elucidation via NMR spectroscopy. The NMR (1H
and 13C) data for products 5a, 7a, and 13a are shown in the (Supplemental
Material). In the case of product 13a, an additional MS/MS measurement
provided an unambiguous assignment to omeprazole sulfone (Supple-
mental Fig. 5). The NMR data of products 2a and 20a were identical to
those previously described (Litzenburger et al., 2015) and match the
corresponding reference standards (Zhang et al., 1996; Morrow et al.,
2005). However, we achieved significantly increased yields in this study
by our new whole-cell constructs (see Supplemental Fig. 1), which also
gave better access to high product amounts for characterization of the
respective products.
Discussion
In recent years, the number of publications about the potential the in vitro assay, which might be a limitation caused by the low per-
applications of P450 enzymes for the production of drug or drug-related meability of the E. coli cells for these substrates during the whole-cell

502
Kern et al.
TABLE 2
Comprehensive overview of human drug metabolites formed by the members of the CYP267 family from S. cellulosum So ce56 and the corresponding human P450 enzymes
#
Metabolites marked with and * are selectively formed by CYP267A1 and CYP267B1, respectively. The order of the listed metabolites is not representative for the product distribution.
Drug
Human P450
Desired Metabolite
Expected Side Products of Human P450 Enzymes
Reference
Cashman et al. (1993)
Chlorpromazine (2)
CYP2D6
CYP3A4
CYP1A2
Chlorpromazine sulfoxide (2a) *
7-Hydroxychlorpromazine
Chlorpromazine-N-oxide
—
Diclofenac (5)
49-Hydroxydiclofenac (5a) *
Bort et al. (1999)
CYP2C8
CYP2C18
CYP2C19
5-Hydroxydiclofenac
4,5-Dihydroxydiclofenac
CYP2C9
3-Hydroxydiclofenac
5-Hydroxydiclofenac
Ibuprofen (7)
CYP2C8
CYP2C9
2-Hydroxyibuprofen (7a) *
Omeprazole sulfone (13a) *
3-Hydroxyibuprofen
Carboxyibuprofen
Hamman et al. (1997); Neunzig et al. (2012)
Omeprazole (13)
Thioridazine (20)
CYP3A4
CYP2D6
5-Hydroxyomeprazole
N-desmethylthioridazine
7-Hydroxythioridazine
Mesoridazine
Yamazaki et al. (1997); Abelö et al. (2000)
Daniel et al. (2000)
#
Thioridazine-5-sulfoxide (20a)
conversion. In contrast, substrates 7 and 13 showed higher yields in the yield (nearly 80%, 68 mg/l of drug 13) and high selectivity (,5%
whole-cell system compared with the in vitro conversion, suggesting that formation of unknown side product). Although CYP3A4 is responsible
the conditions established for these substrates support an efficient for the formation of omeprazole sulfone (13a) in the human body
metabolite production with our whole-cell system.
(Yamazaki et al., 1997), to the best of our knowledge, the bio-
It has been shown recently that several members of a P450 fusion technological production of product 13a with CYP3A4 (Table 2) or
library, constructed by P450 enzymes and their autologous redox another P450 has thus far not been described.
system RhfRED from R. jostii RHA1, are able to convert five out of
The established CYP267-Fdx8-FdR_B whole-cell systems are an
48 selected drugs (Kulig et al., 2015). Compared with our system excellent starting point for further optimizations in view of bio-
consisting of the wild-type CYP267B1 and its autologous redox part- technological upscaling and optimization (Bernhardt and Urlacher,
ners Fdx8 and FdR_B, we observed a larger substrate range (14 out of 2014). Optimizations such as changing expression and reaction
22 drugs) and significantly higher activity toward the conversion of conditions or engineering P450 enzymes could be potential topics of
drugs. This leads to the suggestion that CYP267B1 features a great interest. Several approaches for increasing the performance of the
potential for the biotechnological production of various drug metabo- whole-cell system have been published, describing that an increased
lites when using bacterial P450 enzymes. Our thus far not optimized number of ferredoxin gene copies (Schiffer et al., 2015) or coexpressing
whole-cell system was able to convert 38% of 160 mg of diclofenac (5) a NADPH regenerating system (Zehentgruber et al., 2010) could
within 48 hours, which is a good starting point for the production of enhance product formation. In a recent review, numerous approaches
49-hydroxydiclofenac (5a). In this regard, the production of product 5a and examples were presented to enhance the catalytic activity of P450
has previously been shown using an optimized fermentation process. enzymes toward potential practical purposes (Gillam, 2008). It is
The recombinant expression of CYP2C9 in fission yeast strain CAD68 remarkable that the wild-type CYP267B1 is already able to catalyze
resulted in an efficient formation of product 5a (468 mg/l) after the three different reaction types (hydroxylation, sulfoxidation, and epoxi-
optimization of the pH value, the glucose concentration, and the dation) without any directed or evolutionary modification. In fact, the
establishment of a favorable host organism for the hydroxylation of catalyzed hydroxylation reactions can be diversified to aliphatic (in the
substrate 5 (Drǎgan et al., 2011). The engineering of BM3 toward the case of drug 7), allylic (as described for sesquiterpenes) (Litzenburger
metabolism of drugs resulted in the BM3 mutant Asp251Gly/ and Bernhardt, 2016), and aromatic (as shown for drug 5) hydroxyl-
Gln307His, capable of the metabolism of drug 5 to product 5a in vitro ations, whereby the aromatic hydroxylation has not yet been described
(Tsotsou et al., 2012). This BM3 mutant was also shown to produce for a myxobacterial P450 enzyme.
2-hydroxyibuprofen (7a) from ibuprofen (7) (Tsotsou et al., 2012);
Although in previous studies the bioconversion of drugs and
however, experiments were only done in vitro or in a microtiter plate. xenobiotics was performed using different strains of Streptomyces or
Likewise, the production of product 7a in a preparative in vitro scale Cunninghamella sp. (Zhang et al., 1996; Asha and Vidyavathi, 2009;
was described yielding 74.3 mg of product 7a (96% conversion) Bright et al., 2011; Murphy and Sandford, 2012; Diao et al., 2013), the
(Rentmeister et al., 2011). However, in this study, we were able to systems were not selective and side products were usually observed. In
produce product 7a in a more relevant, biotechnological way using an addition, the nonoptimized media conditions used in these approaches
E. coli–based whole-cell bioconversion system for CYP267B1 con- could also interfere with the product identification. In contrast, in this
sisting of autologous redox partners and necessary cofactors within the study, we established a whole-cell biocatalyst for the conversion of
cells. In this regard, the conversion of 115 mg of drug 7 to product 7a widely used therapeutically important drugs and xenobiotics using two
giving 44% product yield by the wild-type CYP267B1 demonstrated a bacterial P450 enzymes (CYP267A1 and CYP267B1) and we also
promising scope for further optimization since we have not yet focused optimized this whole-cell system to allow convenient and effective
on the optimization or the engineering of the respective P450 toward product isolation for identification by NMR and MS/MS. In this way,
higher space-time yields. In addition, our CYP267B1-Fdx8-FdR_B the substrate spectrum of the CYP267 family—and especially for
whole-cell system also presents the first method to produce omeprazole CYP267B1—was extended to commercially used drugs and their
sulfone (13a) using a biotechnological approach with a high conversion associated diverse chemical structures, demonstrating the potential of

CYP267A1 and CYP267B1 Are Highly Versatile Drug Metabolizers
503
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CYP267B1 is an efficient and promising candidate for further substrate
screening and protein engineering attempts, particularly with regard to
its biotechnological applicability.
Khatri Y, Hannemann F, Ewen KM, Pistorius D, Perlova O, Kagawa N, Brachmann AO, Müller
R, and Bernhardt R (2010) The CYPome of Sorangium cellulosum So ce56 and identification
of CYP109D1 as a new fatty acid hydroxylase. Chem Biol 17:1295–1305.
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Acknowledgments
The authors thank Dr. Stephan Lütz, Novartis (Basel, Switzerland), for
providing some of the substrates and Dr. Josef Zapp, Institute of Pharmaceutical
Biology (Saarland University), for measuring the NMR samples. The authors
also thank Tobias K. F. Dier, Institute of Bioanalytical Chemistry (Saarland
University) for the MS/MS measurement of omeprazole and omeprazole sulfone.
Kiss FM, Lundemo MT, Zapp J, Woodley JM, and Bernhardt R (2015) Process development for
the production of 15b-hydroxycyproterone acetate using Bacillus megaterium expressing
CYP106A2 as whole-cell biocatalyst. Microb Cell Fact 14:1–13.
Authorship Contributions
Participated in research design: Kern, Bernhardt.
Conducted experiments: Kern, Litzenburger, Khatri.
Performed data analysis: Kern, Litzenburger, Khatri.
Wrote or contributed to the writing of the manuscript: Kern, Khatri,
Litzenburger, Bernhardt.
Kulig JK, Spandolf C, Hyde R, Ruzzini AC, Eltis LD, Grönberg G, Hayes MA, and Grogan G
(2015) A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its eval-
uation for the biotransformation of drug molecules. Bioorg Med Chem 23:5603–5609.
Li G and Young KD (2013) Indole production by the tryptophanase TnaA in Escherichia coli is
determined by the amount of exogenous tryptophan. Microbiology 159:402–410.
Litzenburger M and Bernhardt R (2016) Selective oxidation of carotenoid-derived aroma com-
pounds by CYP260B1 and CYP267B1 from Sorangium cellulosum So ce56. Appl Microbiol
Biotechnol DOI:10.1007/s00253-015-7269-7 [published ahead of print].
Litzenburger M, Kern F, Khatri Y, and Bernhardt R (2015) Conversions of tricyclic antide-
pressants and antipsychotics with selected P450s from Sorangium cellulosum So ce56. Drug
Metab Dispos 43:392–399.
Morrow RJ, Millership JS, and Collier PS (2005) Facile syntheses of the three major metabolites
of thioridazine. Helv Chim Acta 88:962–967.
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Address correspondence to: Dr. Rita Bernhardt, Institut für Biochemie,
Universität des Saarlandes, Campus Geb. B2.2, 66123 Saarbrücken, Germany.
E-mail: ritabern@mx.uni-saarland.de