Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing monooxygenases expressing budding yeast

Article abstract of DOI:10.1016/j.dmpk.2020.01.007

Flavin containing monooxygenases (FMOs) represent one of the predominant types of phase I drug metabolizing enzymes (DMEs), and thus play an important role in the metabolism of xeno- and endobiotics for the generation of their corresponding oxides. These oxides often display biological activities, however they are difficult to study since their chemical or biological synthesis is generally challenging even though only small amounts are required to evaluate their efficacy and safety. Previously, we constructed a DME expression system for cytochrome P450, UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell dependent manner. In this study, we developed a heterologous expression system for human FMOs, including FMO1–FMO5, in Saccharomyces cerevisiae and examined its N- and S-oxide productivity. The recombinant yeast cells expressed each of the FMO successfully, and the FMO4 transformant produced N- and S-oxide metabolites at several milligrams per liter within 24 h. This whole-cell dependent biosynthesis enabled the production of N- and S-oxides without the use of the expensive cofactor NADPH. Such novel yeast expression system could be a powerful tool for the production of oxide metabolites.

Full text of DOI:10.1016/j.dmpk.2020.01.007

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Drug Metabolism and Pharmacokinetics
journal homepage: http://www.journals.elsevier.com/drug-metabolism-and-
pharmacokinetics
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Whole-cell dependent biosynthesis of N- and S-oxides using human
flavin containing monooxygenases expressing budding yeast
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Q5 Yuuka Masuyama , Miyu Nishikawa , Kaori Yasuda , Toshiyuki Sakaki ,
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Q1 Shinichi Ikushiro
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Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0351, Japan
Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0351, Japan
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Article history:
Flavin containing monooxygenases (FMOs) represent one of the predominant types of phase I drug
metabolizing enzymes (DMEs), and thus play an important role in the metabolism of xeno- and endo-
biotics for the generation of their corresponding oxides. These oxides often display biological activities,
however they are difficult to study since their chemical or biological synthesis is generally challenging
even though only small amounts are required to evaluate their efficacy and safety. Previously, we con-
structed a DME expression system for cytochrome P450, UDP-glucuronosyltransferase (UGT), and sul-
fotransferase (SULT) using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell
dependent manner. In this study, we developed a heterologous expression system for human FMOs,
including FMO1eFMO5, in Saccharomyces cerevisiae and examined its N- and S-oxide productivity. The
recombinant yeast cells expressed each of the FMO successfully, and the FMO4 transformant produced
N- and S-oxide metabolites at several milligrams per liter within 24 h. This whole-cell dependent
biosynthesis enabled the production of N- and S-oxides without the use of the expensive cofactor
NADPH. Such novel yeast expression system could be a powerful tool for the production of oxide
metabolites.
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Received 24 October 2019
Received in revised form
16 January 2020
Accepted 27 January 2020
Available online xxx
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Keywords:
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Flavin containing monooxygenase (FMO)
N- and S-oxygenation
Biosynthesis
Heterologous expression system
Xenobiotic metabolism
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2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
contribute to the metabolism of about 5% and catalyze about 2% of
the more than 4000 oxidoreduction reactions undergone by these
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Xenobiotics such as small molecular drugs, environmental
pollutants, and dietary polyphenols are preferably metabolized by
drug metabolizing enzymes (DMEs). This results in a decreased
biological activity as well as increased water solubility of these
xenobiotic compounds, which allow for their faster elimination
from the body. Flavin-containing monooxygenases (FMOs; EC
1.14.13.8) are one of the most important families of non-
cytochrome P450 (non-P450) enzymes involved in phase I meta-
bolism. Among 860 drugs surveyed, FMOs were found to
drug [1].
FMOs catalyze the oxidative metabolism of a broad range of
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structurally diverse chemicals containing soft nucleophiles such as
sulfur or nitrogen. For this purpose, they require flavin adenine
dinucleotide (FAD) as prosthetic group, NADPH as cofactor, and
molecular oxygen as cosubstrate. FMOs are present in all kingdoms
of life including bacteria, fungi, and plants [2,3], and are located on
the membranes of the endoplasmic reticulum in eukaryotes.
Humans possess five functional FMO genes, designated as
FMO1eFMO5 [4]. FMO1 is expressed in fetal liver [5] and adult
kidney [6], while FMO2 is expressed in lung tissue [7]. In the case of
human FMO2, two alleles have been described: FMO2*1 is a func-
tional full length version that is found with high frequency in Sub-
Saharan African populations [8], whereas the FMO2*2 gene codes
for a sequence with a premature stop codon that results in a non-
functional protein [7]. FMO3 is considered as the prominent func-
tional FMO form expressed in adult human liver [9,10]. Mutations of
FMO3 produce phenotypes associated with the inherited disorder
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Abbreviations: FMO, flavin containing monooxygenase; DME, drug metabolizing
enzymes; P450 or CYP, cytochrome P450; UGT, UDP-glucuronosyltransferase; SULT,
sulfotransferase; FAD, flavin adenine dinucleotide; UGDH, UDP-glucose dehydro-
genase; PAPS, phosphoadenosine phosphosulfate; HPLC, high performance liquid
chromatography; LC-MS/MS, HPLC-tandem mass spectrometry; ESI, electrospray
ionization; MRM, multiple reaction monitor; MTS, methyl p-tolyl sulfide; BZD,
benzydamine; MTSO and BZDO, oxidized forms of MTS and BZD.
* Corresponding author.
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E-mail address: ikushiro@pu-toyama.ac.jp (S. Ikushiro).
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https://doi.org/10.1016/j.dmpk.2020.01.007
1347-4367/© 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

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trimethylaminuria [11]. The FMO4 gene is expressed in several
tissues, however its expression level is very low. Finally, FMO5 is
expressed in adult human liver [9,10].
Unlike mammals, yeast (Saccharomyces cerevisiae) does not
possess several FMO isoforms, but a single one called yFMO that
does not accept xenobiotic compounds. On the other hand, yFMO
aids the folding of proteins that contain disulfide bonds by cata-
Methyl p-tolyl sulfoxide was purchased from Apollo Scientific Ltd.
(Manchester, UK).
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Rabbit polyclonal anti-FMO1 (ab97720), anti-FMO2 (ab95977),
and anti-FMO3 (ab126711) antibodies were purchased from Abcam
(Cambridge, UK), while anti-FMO5 (GTX114414) was purchased
from GeneTex, Inc. (Irvine, CA). Goat polyclonal anti-FMO4 (T14, sc-
104258) was acquired from Santa Cruz Biotechnology Inc. (Dallas,
USA). Alkaline phosphatase (AP)-conjugated goat polyclonal anti-
body to rabbit IgG (7054) was obtained from Cell Signaling Tech-
nology (Massachusetts, USA). AP-conjugated rabbit polyclonal
antibody against goat IgG (A4187) was purchased from Sigma-
Aldrich Corp. All chemicals and solvents were of the highest
commercially available grade.
lyzing the O
[12,13].
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and NADPH-dependent oxidation of biological thiols
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Throughout the drug discovery and development process, drug
metabolites may serve as analytical references for structure
elucidation. Furthermore, with the introduction of the metabo-
lites in safety testing (MIST) guidelines by the US Food and Drug
Administration in 2008, all metabolites present in the human
metabolism at >10% relative to the parent compound have to be
subjected to toxicity studies [14]. The chemical preparation of
authentic drug metabolites often requires multiple synthetic
steps including several protection and deprotection reactions of
various functional groups. To circumvent this issue, we aimed at
the generation of scalable mimics of single steps of phase 1
metabolism reactions in vitro. This could be accomplished with
heterologously expressed human enzymes in the form of ready-
to-use biocatalysts such as E. coli and yeast. The use of whole-
cell catalysts is beneficial in many aspects, since the enzyme
performing the actual biotransformation does not need to be
isolated and purified, which saves time and costs. FMOs have been
successfully expressed in E. coli, and the whole-cell bioconversion
of drug metabolites was obtained by human FMO3 and generated
human FMO2 [15,16].
Other heterologous expression systems have also been devel-
oped for DMEs such as cytochrome P450 (P450), UDP-
glucuronosyltransferases (UGTs), and sulfotransferases (SULTs).
S. cerevisiae offers several advantages for the expression of DMEs.
More than three decades ago, Sakaki et al. [17] successfully
expressed mammalian P450 in S. cerevisiae AH22 cells, and these
recombinant yeast cells were reported to effectively metabolize
drugs, environmental pollutants, flavonoids, and dietary compo-
nents [17e21]. Following the description of the use of the
S. cerevisiae AH22 strain to express UGT [22], we established a
whole-cell system to obtain glucuronides without the need of
adding UDP-glucuronic acid by simultaneously expressing UGT and
UDP-glucose dehydrogenase (UGDH) [23]. Furthermore, we suc-
cessfully synthesized sulfo-conjugates without adding the expen-
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2.2. Construction of an FMO expression system in yeast
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In order to construct an FMO expression system in yeast, the
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Escherichia coli (E. coli)eyeast shuttle vector pGYR was used. Such
vector contains glyceraldehyde-3-phosphate dehydrogenase
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promoter and terminator derived from Zygosaccharomyces rouxii,
and has been used for P450 expression in previous studies [26].
Synthetic cDNA fragments were obtained from Thermo Fisher
Scientific Inc. (Waltham, MA); they contained 15bp additional se-
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quences at the 5 - and 3 -end, which are the homologous sequences
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to the 3 - and 5 -end of linearized pGYR by Hind III, respectively,
and were optimized for codon usage in Saccharomyces cerevisiae.
These cDNA fragments were ligated with linearized pGYR by Hind
III using the In-Fusion HD cloning kit (TaKaRa Bio Inc., Otsu, Japan).
GenBank information of human FMO genes were described below
(FMO1; NM_001282692, FMO2; NM_001460, FMO3; NM_006894,
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FMO4; NM_002022, FMO5; NM_001461).
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The transformation of S. cerevisiae AH22 yeast cells (ATCC
38626) was performed by using the lithium acetate method as
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previously described [23]. The resultant clones were genotyped by
direct colony PCR using KOD FX Neo (Toyobo Co., Ltd., Osaka,
Japan).
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2.3. Cultivation of the recombinant yeast cells expressing FMOs
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The cultivation of the recombinant yeast cells was performed as
previously described [24]. A glycerol stock of FMO transformant
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was spread on SD þ His agarose plates, which were preincubated at
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sive cofactor 3 -phosphoadenosine-5 -phosphosulfate (PAPS) using
a SULT expression system in yeast [24]. The S. cerevisiae system was
found to have a higher productivity than a similar system using the
fission yeast Schizosaccharomyces pomb [25].
ꢀ
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C for 2e3 days. After picking the colonies, the cells were
ꢀ
cultivated in 1 L SD þ His medium at 30 C overnight upon shaking
at 200 rpm. The cells were harvested at a cell density of 2.0e2.5
OD660 and subsequently resuspended in an equal volume of
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In the present study, we describe the expression of five human
FMO isoforms (FMO1-5) in S. cerevisiae AH22 yeast cells as well as
the potential use of this system in the production of N- and S-
oxidants.
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distilled water. The cells were stocked at ꢁ80 C and used as bio-
catalyst after thawing.
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2.4. Immunoblot analysis
2. Materials and methods
Whole yeast proteins were analyzed by immunoblot analysis as
previously described [23,24]. Yeast pellets were treated with
0.5 mg/mL zymolyase 20T (Nacalai Tesque, Kyoto, Japan) for
2.1. Materials
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5 min at 30 C, and then subjected to SDS-PAGE using 10% acryl-
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Yeast nitrogen base with ammonium sulfate was purchased
from MP Biomedicals (Santa Ana, USA). L-Histidine and midazolam
amide gel. The resulting polypeptide bands were transferred to
nitrocellulose membranes (Bio-Rad Laboratories, Berkeley, CA, US),
equilibrated with methanol, and subsequently blocked with 10%
bovine serum albumin (BSA) in a TBS buffer at room temperature
for 1 h. After incubation with primary and AP-conjugated second-
ary antibodies, respectively, the detection was performed using the
BCIP-NBI solution kit for alkaline phosphatase stain, nuclease
tested (Nacalai Tesque Inc., Kyoto, Japan).
were acquired from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). Zymolyase from Arthrobacter luteus was purchased from
Seikagaku Corporation (Tokyo, Japan). Methyl p-tolyl sulfide and
benzydamine hydrochloride were purchased from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan). Benzydamine N-oxide hydrogen
maleate was obtained from Sigma-Aldrich Corp. (St. Louis, MO).
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Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

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2.5. Whole-cell-dependent biosynthesis of N-oxides and sulfoxides
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The enzymatic activities of the FMO transformants were
evaluated using the universal substrates methyl p-tolyl sulfide
(MTS) and benzydamine (BZD). The optimization of the reaction
conditions was carried out varying the different reaction pa-
rameters, and the optimized conditions were as follows. Wet
yeast cells were suspended at a concentration of 25% (w/v) in
100 mM KPi buffer (pH 8.3) containing 8% (w/v) glucose, and
10 mM substrate in DMSO was added to the cell suspension at a
final concentration of 0.1 mM. The reactions were conducted at
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30 C with shaking. Following incubation, a 2.5-fold volume of
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chloroform:methanol (3:1, v/v) was added to the reaction me-
dium containing the yeast cells. The extracts were clarified into
an upper aqueous and lower organic phase by centrifugation
(12,000ꢂg, 10 min, room temperature). The lower organic phase
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was dried and resolved in 100
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subsequent HPLC and LC-MS/MS analysis.
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2.6. HPLC analysis
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The rate of S-oxygenation of methyl p-tolyl sulfide in the yeast
cells was determined by using a HPLC system consisting of a Waters
695 separations module, Waters 2487 dual- absorbance UV de-
m, 4.6 ꢂ 150 mm;
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tector, and Cosmosil 5C18-Ar-II column (5
Waters, Milford, MA).
m
The gradient elution conditions were watereacetonitrile with
.1% (v/v) formic acid, 10% acetonitrile (2 min), 10e80% acetonitrile
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8 min), 80% acetonitrile (2 min), and 10% acetonitrile (3 min). The
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flow rate was set at 1.0 mL/min, the column temperature was 40 C,
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and the injection volume was 10 ml. The formation of methyl p-tolyl
sulfoxide was monitored at a wavelength of 254 nm, and the
quantitation was carried out with authenticated standards.
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.7. LC-MS/MS analysis
Fig. 1. Protein expression of human FMO transformants. Immunodetection was per-
formed using anti-FMO1, FMO2, FMO3, FMO4, and FMO5 antibodies, respectively. The
same amount of whole yeast protein (10 mg) from either the control pGYR vector alone
(ꢁ) or human FMO transformed yeast strains (þ) was loaded onto each lane.
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The rate of N-oxygenation of benzydamine was analyzed by
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using a HPLC-tandem mass spectrometry (LC-MS/MS) system
consisting of an Agilent 1200 series LC system (Agilent technologies
Inc., Santa Clara, CA) coupled with an API 3200 QTRAP mass spec-
trometer (AB Sciex, Toronto, Canada) equipped with a Capcell Pak
As shown in Fig. 1, the successful expression of the various FMO
isoforms in S. cerevisiae AH22 cells was confirmed.
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C18 UG120 column (5
The isocratic mobile phase conditions were 20% acetonitrile con-
m
m, 4.6 ꢂ 250 mm; Shiseido, Tokyo, Japan).
In order to determine the N- and S-oxygenation activities of
the FMO transformants, methyl p-tolyl sulfide (MTS) and ben-
zydamine (BZD) were used as universal substrates (Fig. 2a and
Fig. 3a). MTS was successfully converted to its S-oxide form
MTSO by FMOs (Fig. 2b). The FMO4 transformant efficiently
catalyzed also the N-oxygenation of benzydamine (Fig. 3b). MTSO
and BZDO were successfully generated in a whole-cell dependent
manner (not only the FMO transformants, but also pGYR alone)
without the need of adding an exogenous supplement of NADPH,
suggesting that the endogenous NADPH present in the yeast cells
was used by the FMOs to catalyze the N- and S- oxygenation
reactions. Time-dependent conversions to MTSO and BZDO were
performed using FMO4 and control pGYR transformants (Fig. 4).
The time course analysis of MTS oxidation revealed that half of
taining 0.1% formic acid, the flow rate was set at 0.8 mL/min, the
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column temperature was 40 C, and the injection volume was 2
ml.
The MS analysis was carried out by electrospray ionization (ESI)
with a positive ion mode scan in a multiple reaction monitor
MRM) mode. MRM transitions were monitored independently
corresponding to the precursor and product ion pairs for benzyd-
amine N-oxide (m/z transition 326 / 102), benzydamine (m/z
transition 310 / 86), and the internal standard midazolam (m/z
transition 326 / 291). For the quantitative determination of ben-
zydamine N-oxide, a calibration curve was prepared using the peak
area ratio of the authentic standard to the internal standard over a
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(
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concentration range of 0.1e30 mM.
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Fig. 4a).
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3. Results
3.1. Establishment of a whole-cell dependent production system of
N- and S-oxide metabolites using recombinant yeast cells expressing
human FMOs
3.2. Optimization of the reaction conditions for whole-cell
dependent N- and S-oxide production
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In order to improve the productivity of N- and S-oxide pro-
duction in the yeast cells, the reaction conditions were optimized.
The effect of the cell amount on the S-oxygenation was examined
The expression level of the FMOs transfected in yeast was
determined by immunoblot analysis using FMO-specific antibodies.
Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

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pH > 8.0 to ensure maximal FMO activity [27]. The benzydamine N-
oxygenation catalyzed by FMO3 significantly increased when the
reaction was conducted in a buffer at pH 8.3 compared with buffers
at pH 6.8 and 7.4 (Fig. 5b). In contrast, the MTS S-oxygenation
catalyzed by FMO4 was not significantly affected by changes in
buffer pH (Fig. 5c).
As shown in Fig. 5, the FMO-expressing yeast showed an optimal
conversion ratio towards MTSO and BZDO under the conditions of
8.3 buffer pH and 25% (w/v) yeast cells. We next compared the
metabolic activity towards MTS and BZD among the five FMO
transformants (Fig. 6). The oxidation activity towards MTS was
confirmed for the FMO1, FMO3, and FMO4 transformants, with the
highest activity observed for FMO4. The productivity per volume (L)
is important from a practical standpoint, since the resultant N- and
S-oxides are the purified by preparative HPLC. The highest pro-
ductivity of MTSO and BZDO in the presence of the FMO4 trans-
formant was 6.9 and 1.6 mg/L, respectively.
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4. Discussion
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The N- and S-oxides of some drugs are often formed by P450s
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and FMOs. The relative contribution of CYP vs FMO-dependent
metabolism generally varies with the xenobiotic. Taniguchi et al.
reported that N-oxygenation of BZD could be mainly catalyzed by
FMO1/3 while N-demethylation could be catalyzed by P450 2D6
27]. They suggested that the determination of the contribution of
FMO isoforms to metabolism of new drug candidates might be paid
careful attention.
Earlier, we developed DME expression systems expressing P450,
UGT, and SULT using yeast cells, and successfully produced various
metabolites of xeno- and endobiotics [17,23,24]. In the present
study, we further constructed FMO expression systems in yeast.
Subsequently, transformants expressing the FMO4 isoform were
used to effectively produce several milligrams per liter of N- and S-
oxide metabolites without the addition of NADPH. In vitro systems
using subcellular fractions or purified FMO enzymes require an
expensive cofactor (NADPH) for oxidation, therefore bypassing the
requirement for exogenous NADPH offers a great advantage for the
[
Fig. 2. S-oxygenation potential of the human FMO4 transformant towards methyl p-
tolyl sulfide. (a) Reaction scheme of MTS S-oxygenation. (b) HPLC profile of 0.1 mM
MTS incubated with the FMO4 transformant for 0 and 6 h.
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using FMO3 and FMO4 transformants. As expected, the conversion
rate of MTS to MTSO increased depending on the cell amount up to
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5% (w/v) (Fig. 5a). Further increases of the cell amount led to a
high viscosity of the reaction solution and low yield of MTSO. In
addition, we examined the effect of the buffer pH on the N- and S-
oxygenation, since FMO reactions are commonly conducted at a
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Fig. 3. N-oxygenation potential of the human FMO4 transformant towards benzydamine. (a) Reaction scheme of benzydamine N-oxygenation. (b) LC-tandem mass spectrometry
quantitation using the MRM mode of 0.1 mM BZD incubated with the FMO4 transformant for 0 and 24 h. TIC chromatograms for the transitions m/z 310 / 86 (BZD), 326 / 102
(BZDO), and 326 / 291 (MDZ, IS). No analytical interference between the amine and N-oxide was detected. TIC, total ion current; MRM, multiple reaction monitoring.
Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

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Fig. 4. Time-dependent conversion of MTS and BZD to MTSO (a) and BZDO (b) by the pGYR alone (open circle) and FMO4 transformant (filled circle). 0.1 mM of MTS and BZD were
incubated with 25% (w/v) biomass. The values are shown as mean ± SEM (n ¼ 3).
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Fig. 5. Optimization of the reaction conditions using the MTS and BZD substrates. (a) Conversion of MTS to MTSO using different amounts of biomass. For each amount of biomass,
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.2 and 0.1 mM of MTS were incubated with the FMO3 and FMO4 transformant, respectively. The values are shown as mean ± SEM (n ¼ 2e3). Effect of the buffer pH on the N-
oxygenation of BZD (b) and S-oxygenation of MTS (c). 0.1 mM MTS and BZD was incubated with 10% (w/v) biomass of pGYR alone (open bar) and FMO3 or FMO4 (filled bar),
respectively. The values are shown as mean ± SEM (n ¼ 3).
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industrial production of oxidants. Recently, Hanlon et al. [15] and
Geier et al. [16] successfully produced N- and S-oxide metabolite
using recombinant FMOs expressed in bacterial systems. Both E. coli
and S. cerevisiae are considered suitable hosts for the whole-cell
dependent production of N- and S-oxide metabolites owing to
their ability to produce NADPH endogenously. In S. cerevisiae, FMO
isoforms were expressed at a sufficient level without a concomitant
reduction of inclusion body formation compared to bacterial
expression systems, and whole-cell biosynthesis using frozen
stocks of the recombinant yeast resulted in a stable expression
along with good reproducibility.
The regio- or enantioselective production of metabolites is one
of the greatest features of biosynthesis. In our previous study, we
successfully produced conjugates of polyphenols in a regioselective
manner. The regioselective oxidation ability of each FMO isoform
towards ranitidine and phosphor-NSAID was reported [28,29];
therefore, it can be expected that our yeast expression system for
FMOs could be used for predicting metabolic pathways of candidate
compounds in humans.
In addition to this, drug metabolites are required as analytical
standards for structure elucidation throughout the drug discovery
and development process. Furthermore, 2008 MIST guidelines by
the FDA established that all metabolites present at >10% relative
to the parent compound in the human metabolism have to be
subjected to toxicity studies [14]. Our yeast expression system for
FMOs could be used in the production of oxide metabolites for
quantitative analysis, however, improvements are necessary to
attain sufficient amounts (~g order) of metabolites for toxicity
studies.
In conclusion, the heterologous expression system for FMOs in
yeast cells developed in this study offers two main advantages: (1)
bypassing of the requirement for exogenous NADPH, (2) high re-
covery of N-and S-oxide metabolites from the yeast culture me-
dium through a relatively simple procedure. Therefore, this FMO
expression system could be useful for revealing the biological ac-
tivity of FMO-dependent metabolites, and by combining it with the
P450, UGT, and SULT expression systems, it can be envisaged to
accelerate the study of the metabolism and involved metabolites.
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Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

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Fig. 6. Conversion of MTS and BZD to MTSO and BZDO, respectively, in each FMO transformant. 0.1 mM MTS (a) and BNZ (b) were incubated with the pGYR alone (open bar) and
FMO transformant (filled bar). The values are shown as mean ± SEM (n ¼ 3).
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Authors contributions
[11] Yamazaki H, Shimizu M. Survey of variants of human flavin-containing
monooxygenase (FMO3) and their drug oxidation activities. Biochem
3
Pharmacol 2013;85:1588e93. https://doi.org/10.1016/j.bcp.2013.03.020.
12] Suh JK, Poulsen LL, Ziegler DM, Robertus JD. Molecular cloning and kinetic
characterization of a flavin-containing monooxygenase from Saccharomyces
cerevisiae. Arch Biochem Biophys 1996;336:268e74. https://doi.org/10.1006/
abbi.1996.0557.
Y.M. T.S., and S.I. designed the research and wrote the paper;
Y.M., M.N., K.Y., and S.I conducted the experiments and analyzed
the data. S.I. had primary responsible for the final contents.
[
[
13] Suh JK, Robertus JD. Yeast flavin-containing monooxygenase is induced by the
unfolded protein response. Proc. Natl. Acad. Sci. U.S.A 2000;97:121e6. https://
doi.org/10.1073/pnas.97.1.121.
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Declaration of competing interest
[
14] Smith DA, Obach RS. Metabolites in safety testing (MIST): considerations of
mechanisms of toxicity with dose, abundance, and duration of treatment.
Chem Res Toxicol 2009;22:267e79. https://doi.org/10.1021/tx800415j.
Q4
There are no conflicts of interest to declare.
[15] Hanlon SP, Camattari A, Abad S, Glieder A, Kittelmann M, Lütz S, et al.
Expression of recombinant human flavin monooxygenase and moclobemide-
N-oxide synthesis on multi-mg scale. Chem Commun 2012;48:6001e3.
https://doi.org/10.1039/c2cc17878h.
References
3
7
3
3
4
8
9
0
[16] Geier M, Bachler T, Hanlon SP, Eggimann FK, Kittelmann M, Weber H, et al.
Human FMO2-based microbial whole-cell catalysts for drug metabolite syn-
thesis. Microb Cell Factories 2015;14:82. https://doi.org/10.1186/s12934-015-
[
1] Rendic S, Guengerich FP. Survey of human oxidoreductases and cytochrome
P450 enzymes involved in the metabolism of xenobiotic and natural chem-
icals. Chem Res Toxicol 2015;28:38e42. https://doi.org/10.1021/tx500444e.
2] Mascotti ML, Lapadula WJ, Juri Ayub M. The origin and evolution of baeyerd
villiger monooxygenases (BVMOs): an ancestral family of flavin mono-
0
262-0.
[17] Sakaki T, Oeda K, Miyoshi M, Ohkawa H. Characterization of rat cytochrome P-
50MC synthesized in Saccharomyces cerevisiae. J Biochem 1985;98:167e75.
[
4
1
4
4
4
4
4
4
2
3
4
5
6
oxygenases.
PloS
One
2015;10:e0132689.
https://doi.org/10.1371/
https://doi.org/10.1093/oxfordjournals.jbchem.a135255.
journal.pone.0132689.
[
18] Kasai N, Ikushiro S, Hirosue S, Arisawa A, Ichinose H, Wariishi H, et al.
[3] Mascotti ML, Ayub MJ, Furnham N, Thornton JM, Laskowski RA. Chopping and
0
Enzymatic properties of cytochrome P450 catalyzing 3 -hydroxylation of
changing: the evolution of the flavin-dependent monooxygenases. J Mol Biol
naringenin from the white-rot fungus Phanerochaete chrysosporium. Bio-
chem Biophys Res Commun 2009;387:103e8. https://doi.org/10.1016/
j.bbrc.2009.06.134.
2016;428:3131e46. https://doi.org/10.1016/j.jmb.2016.07.003.
[4] Hernandez D, Janmohamed A, Chandan P, Phillips IR, Shephard EA. Organi-
zation and evolution of the flavin-containing monooxygenase genes of human
and mouse: identification of novel gene and pseudogene clusters. Pharma-
cogenetics 2004;14:117e30.
[5] Koukouritaki SB, Simpson P, Yeung CK, Rettie AE, Hines RN. Human hepatic
flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental
expression. Pediatr Res 2002;51:236e43. https://doi.org/10.1203/00006450-
4
7
[19] Oeda K, Sakaki T, Ohkawa H. Expression of rat liver cytochrome P-450MC
cDNA in Saccharomyces cerevisiae. DNA 1985;4:203e10. https://doi.org/
4
4
5
8
9
0
10.1089/dna.1985.4.203.
[
20] Sakaki T, Shinkyo R, Takita T, Ohta M, Inouye K. Biodegradation of poly-
chlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A
subfamily. Arch Biochem Biophys 2002;401:91e8. https://doi.org/10.1016/
S0003-9861(02)00036-X.
5
1
200202000-00018.
5
5
5
5
5
2
3
4
5
6
[6] Yeung CK, Lang DH, Thummel KE, et al. Immunoquantitation of FMO1 in
[
21] Sakaki T, Yamamoto K, Ikushiro S. Possibility of application of cytochrome
P450 to bioremediation of dioxins. Biotechnol Appl Biochem 2013;60:65e70.
https://doi.org/10.1002/bab.1067.
human liver, kidney, and intestine. Drug Metab Dispos 2000;28:1107e11.
[
[
[
7] Dolphin CT, Beckett DJ, Janmohamed A, Cullingford TE, Smith RL, Shephard EA.
The flavin-containing monooxygenase 2 gene (FMO2) of humans, but not of
other primates, encodes a truncated, nonfunctional protein. J Biol Chem
1998;273:30599e607. https://doi.org/10.1074/jbc.273.46.30599.
8] Veeramah KR, Thomas MG, Weale ME, Zeitlyn D, Tarekegn A, Bekele E. The
potentially deleterious functional variant flavin-containing monooxygenase
[
22] Ikushiro S, Sahara M, Emi Y, Yabusaki Y, Iyanagi T. Functional co-expression of
xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-
glucuronosyltransferase 1A6, in yeast microsomes. Biochim Biophys Acta
2004;1672:86e92. https://doi.org/10.1016/j.bbagen.2004.02.012.
23] Ikushiro S, Nishikawa M, Masuyama Y, Shouji T, Fujii M, Hamada M, et al.
Biosynthesis of drug glucuronide metabolites in the budding yeast Saccha-
romyces cerevisiae. Mol Pharm 2016;13:2274e82. https://doi.org/10.1021/
acs.molpharmaceut.5b00954.
5
7
[
[
[
5
5
6
8
9
0
2
*1 is at high frequency throughout sub-Saharan Africa. Pharmacogenetics
Genom 2008;18:877e86. https://doi.org/10.1097/FPC.0b013e3283097311.
9] Koukouritaki SB, Poch MT, Cabacungan ET. Discovery of novel flavin-
containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and
functional analysis of upstream haplotype variants. Mol Pharmacol 2005;68:
383e92. https://doi.org/10.1124/mol.105.012062.
24] Nishikawa M, Masuyama Y, Nunome M, Yasuda K, Sakaki T, Ikushiro S. Whole-
cell-dependent biosynthesis of sulfo-conjugate using human sulfotransferase
expressing budding yeast. Appl Microbiol Biotechnol 2018;102:723e32.
https://doi.org/10.1007/s00253-017-8621-x.
6
1
6
6
6
6
2
3
4
5
[
10] Shimizu M, Denton T, Kozono M, Cashman JR, Leeder JS, Yamazaki H. Devel-
opmental variations in metabolic capacity of flavin-containing mono-oxy-
genase 3 in childhood. Br J Clin Pharmacol 2011;71:585e91. https://doi.org/
10.1111/j.1365-2125.2010.03876.x.
25] Dr ꢀa gan CA, Buchheit D, Bischoff D, Ebner T, Bureik M. Glucuronide production
by whole-cell biotransformation using genetically engineered fission yeast
Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007

DMPK313_proof ■ 3 March 2020 ■ 7/7
Y. Masuyama et al. / Drug Metabolism and Pharmacokinetics xxx (xxxx) xxx
7
1
2
3
4
5
6
7
8
Schizosaccharomyces pombe. Drug Metab Dispos 2010;38:509e15. https://
doi.org/10.1124/dmd.109.030965.
26] Yasuda K, Ikushiro S, Kamakura M, Ohta M, Sakaki T. Metabolism of sesamin
by cytochrome P450 in human liver microsomes. Drug Metab Dispos 2010;38:
2117e23. https://doi.org/10.1124/dmd.110.035659.
27] Taniguchi-Takizawa T, Shimizu M, Kume T, Yamazaki H. Benzydamine N-
oxygenation as an index for flavin-containing monooxygenase activity and
benzydamine N-demethylation by cytochrome P450 enzymes in liver mi-
crosomes from rats, dogs, monkeys, and humans. Drug Metabol Pharmaco-
kinet 2015;30:64e9. https://doi.org/10.1016/j.dmpk.2014.09.006.
[28] Chung WG, Park CS, Roh HK, Lee WK, Cha YN. Oxidation of ranitidine by
isozymes of flavin-containing monooxygenase and cytochrome P450. Jpn J
Pharmacol 2000;84:213e20. https://doi.org/10.1254/jjp.84.213.
[29] Xie G, Wong CC, Cheng KW, Huang L, Constantinide PP, Rigas B. Regioselective
oxidation of phospho-NSAIDs by human cytochrome P450 and flavin mono-
oxygenase isoforms: implications for their pharmacokinetic properties and
9
10
11
12
13
14
15
16
[
[
safety. Br
J Pharmacol 2012;167:222e32. https://doi.org/10.1111/j.1476-
5381.2012.01982.x.
Please cite this article as: Masuyama Y et al., Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing
monooxygenases expressing budding yeast, Drug Metabolism and Pharmacokinetics, https://doi.org/10.1016/j.dmpk.2020.01.007