Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast

May 30, 2016 - Masaki Kamakura,. † and Toshiyuki Sakaki. †,‡. †. Department of Biotechnology, Faculty of Engineering, Toyama Prefectural Unive...
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Biosynthesis of Drug Glucuronide Metabolites in Budding Yeast Saccharomyces cerevisiae Shinichi Ikushiro, Miyu Nishikawa, Yuuka Masuyama, Tadashi Shouji, Miharu Fujii, Masahiro Hamada, Noriyuki Nakajima, Moshe Finel, Kaori Yasuda, Masaki Kamakura, and Toshiyuki Sakaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00954 • Publication Date (Web): 30 May 2016 Downloaded from http://pubs.acs.org on June 1, 2016

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Molecular Pharmaceutics

-TITLE PAGE

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae

Shinichi Ikushiroa*, Miyu Nishikawaa,b, Yuuka Masuyamaa, Tadashi Shoujia, Miharu Fujii b, Masahiro Hamadaa, Noriyuki Nakajimaa, Moshe Finelc, Kaori Yasudaa, Masaki Kamakuraa, and Toshiyuki Sakakia,b

a

Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University,

5180 Kurokawa, Imizu, Toyama, 939-0398, Japan b

Imizu Institute, TOPU BIO RESEARCH Co., Ltd, 5180 Kurokawa, Imizu, Toyama, 939-0398,

Japan c

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of

Helsinki, Finland

*To whom correspondence should be addressed: Shinichi Ikushiro, Ph. D.

Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University,

5180 Kurokawa, Imizu,, Toyama

939-0398, Japan

Phone: +81-766-56-7500, FAX: +81-766-56-2498

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e-mail: [email protected] ABBREVIATIONS: P450, cytochrome P450; 7HC,7-hydroxycoumarin; UGDH, UDP-glucose-6-dehydrogenase; UDPGA, UDP-glucuronic acid; UGT,

UDP-glucuronosyltransferase

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KEYWORDS: acyl glucuronide; UDP-glucuronosyltransferase; budding yeast;

UDP-glucose-6-dehydrogenase; UDP-glucuronic acid

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ABSTRACT Glucuronidation is one of the most common pathways in mammals for detoxification and

elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however,

can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often

needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites

frequently limits such toxicological analyses. To overcome this hurdle we have developed a new

approach, the essence of which is a co-expression system containing a human, or another

mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase

(UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using

resting yeast cells co-expressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate,

in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid.

Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting

suitable mammalian UGT enzyme for the co-expression system we could obtain the desired

glucuronides of various compounds, including molecules with multiple conjugation sites and acyl

glucuronides of several carboxylic acid containing drugs, namely mefenamic acid, flufenamic acid

and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been

developed that exhibits a capacity for efficient production of various glucuronides, including acyl

glucuronides.

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ABSTRACT GRAPHIC

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INTRODUCTION The glucuronidation of many endogenous and exogenous compounds including bilirubin,

steroids,

different

drugs

and

environmental

pollutants,

is

catalyzed

by

microsomal

UDP-glucuronosyltransferases (UGTs) and plays important roles in the biotransformation and pharmacokinetics of hydrophobic xenobiotics1. UGTs catalyze the transfer of the glucuronic acid

moiety from the cofactor, UDP-glucuronic acid (UDPGA) to a nucleophilic group, mostly a

hydroxyl or amino group, on the acceptor substrate molecule. There are 19 human UGTs in

subfamilies 1A, 2A and 2B, which mainly contribute to xenobiotic glucuronidation. The 9 members of subfamily 1A are all encoded by a single gene complex, ugt1a2 that undergoes exon sharing and

alternative splicing. The resulting mature mRNA for each UGT of subfamily 1A contains a different

exon 1 that encodes the N-terminal domain, along with identical exons 2-5 that encode the rest of the protein, its C-terminal domain3. Hence, the construction of different UGT1As to enable

detoxification of a diverse range of xenobiotic substrates using the same cofactor occurs by combining an N-terminal variable region with the C-terminal commonly shared region1.

In most cases, glucuronidation of xenobiotics, including drugs, is associated with their

inactivation and excretion. Nevertheless, productions of biologically active or even reactive metabolites were also reported, such as in the case of acyl glucuronides4-6. Furthermore, polymorphisms within the UGT genes may sometimes lead to adverse effects of drug therapy7,8.

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Consequently, an effective method is needed for the production and purification of glucuronides for

toxicity studies, as well as for reference standards. Traditionally, such glucuronides were obtained

either by conventional, often cumbersome organic chemical synthesis, by time-consuming

purification of metabolites from biological samples such as bile or urine, or by enzyme-dependent biosynthesis using hepatic microsomes9-12. An alternative approach of glucuronides formation using

glycosynthase has also been recently developed. Ma et al. reported that a library of steroid

glucuronides was prepared using the glucuronysynthase derived from Escherichia coli

β-glucuronidase13. Budding yeast cells were previously used for heterologous expression of UGTs14-17. Nevertheless,

due to their inability to generate UDPGA, resting budding yeast cells are unable to convert drugs

into glucuronides using the expressed UGTs. The latter was changed, however, when heterologous

expression of plant UGDH enabling budding yeast to produce UDP-glucuronic acid by de novo synthesis from intrinsic UDP-glucose18. Following that breakthrough report, Dragan et al.

19

developed whole-cell biotransformation system for glucuronides production, using co-expression of

human UGDH and UGTs in the fission yeast, Schizosaccharomyces pombe, achieving bioconversion

of pharmaceutical compounds to glucuronides without requiring the expensive co-substrate, UDPGA.

That was a significant progress, but some disadvantages, or limitations, were also observed. For

example, UGT1A6, did not show full glucuronidation activity toward 4-methylumbelliferone in

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comparison to another enzyme, UGT1A919. Another one was that in addition to the glucuronide

metabolite, a glucosylated metabolite of ibuprofen (acyl glucoside) was produced by fission yeast

cells co-expressing expressing UGT2B7 and UGDH, probably due to excessive production of UDP-glucose20.

Budding yeast is widely used in biotechnological platforms for the production of various compounds, including P450-dependent drug metabolites21,22. We have now developed a biosynthesis

system for glucuronides, including acyl glucuronides, that is based on the budding yeast,

Saccharomyces cerevisiae, rather than the fission yeast, that co-expresses the rat UGDH and a

mammalian UGT enzyme of choice. The new and flexible yeast system is a useful and versatile

platform for glucuronides synthesis at low cost and high efficiency.

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MATERIALS AND METHODS

Chemicals. 7-Hydroxycoumarin (7HC), 11α-hydroxyprogesterone, mycophenolic acid, and 7-hydroxycoumarin-β-D-glucuronide (7GC) were purchased from Sigma-Aldrich (St. Louis, MO).

Diclofenac acyl glucuronide, phenolic β-D-glucuronide and acyl-β-D-glucuronide derivatives of

mycophenolic acid were purchased from Toronto Research Chemicals Inc. (Toronto, Canada).

Acetonitrile [high-performance liquid chromatography (HPLC) grade] was purchased from KANTO

CHEMICAL CO., Inc. (Tokyo, Japan). All other chemicals used in this study were purchased from

Nacalai Tesque (Kyoto, Japan). The chemical structures of the compounds that were used for

glucuronidation in this study (UGTs’ substrates) are shown in Figure S1 of the Supporting

Information.

Isolation and subcloning of genes, and construction of yeast vectors for co-expression of rat UGDH with various UGT enzymes. All the UGT genes used in this study are listed in Table S1

(Supporting Information). The cDNAs of the rat UGT members of subfamilies 1A and 2B, as well as the human UGTs 1A7, 1A8 and 1A10, were obtained as previously described23,24. The cDNAs of

human UGT1A5, UGT2A1, UGT2B17, porcine UGT2B18, UGT2B31, UGT2C1 and bovine

UGT1A6 were obtained as a synthetic gene constructs optimized for S. cerevisiae expression by

Eurofins MWG Operon (Tokyo, Japan). The cDNAs of other UGTs of human, mouse and porcine,

as well as the rat UDP-glucose-6-dehydrogenase (UGDH; GenBank accession no. NM_031325)

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were amplified by PCR from the corresponding liver cDNA libraries (OriGene Technologies Inc.,

Rockville, MD) using primers containing flanking HindIII sites.

The construction scheme for the yeast co-expression vectors containing UGDH and selected UGT

enzymes is shown in Figure S2 (Supporting information). Some UGT enzymes (human UGTs 1A1,

1A4, and 1A9) exhibited relatively low expression level in the yeast cell expression system (Upper panel in Figure 1). Their expression level was enhanced by replacing their original N-terminal sequences (putative signal sequence) with the corresponding signal sequence encoding segment of

UGT1A7 (see Table 2, Supporting Information, for the N-terminal signal peptide sequences of UGTs 1A1, 1A4, 1A7 and 1A9). To enable replacement of the original N-terminal signal peptide of UGT1A1, 1A4 or 1A9 with the segment from UGT1A7, PCR amplifications were performed using

pUC119 with UGT cDNA and specific primers containing the modified sequences. The resultant

modifications, following confirmation by DNA sequencing, were ligated into the HindIII site of

pGYR. To insert the UGDH gene, along with the GAPDH promoter and terminator, into the NotI

site of the genome-integrating yeast expression vector pAUR101 (Takara, Otsu, Japan), a NotI site was generated in the multiple cloning site of the vector using QuickChangeTM mutagenesis (Agilent

Technologies). The UGDH gene, along with the GAPDH promoter and terminator, was obtained from pGYR/UGDH by digestion with NotI and cloned into pAUR-N using InFusionTM Advantage

PCR cloning kit (Takara, Otsu, Japan), resulting in pAUR-UGDH. The genome-integrating yeast

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expression vectors, containing rat UGDH and a selected UGT, were constructed using pAUR-UGDH

with a single NotI site. The UGT genes along with the GAPDH promoter and terminator was

digested from pGYR/UGT with NotI and ligated into pAUR/UGDH at the NotI site.

Transformation of yeast cells with UGDH/UGT co-expression plasmids. The yeast (Saccharomyces cerevisiae) strain AH22 was used for protein expression, as previously reported14-17.

Since the genome-integrating vector pAUR has an aureobasidin A-resistant gene, AH22

transformants with pAUR are able to grow in YPD medium (1% (w/v) yeast extract, 2% (w/v)

polypeptone, 2% (w/v) glucose), supplemented with 0.5 µg/mL aureobasidin A (Takara, Otsu,

Japan). The expression plasmids were digested by BsiWI prior to transformation (0.5 – 1 µg DNA/10 µL, 10 µL), which was done using the lithium chloride method26. Colony-PCR was used to

confirm the introduction of the pAUR expression vector into the genome of aureobasidin A-resistant

yeast cells, i.e. the transformed cells.

Assay of cell dependent glucuronidation using resting yeast cells. Transformed yeast strains

were used to inoculate 5 mL of YPD supplemented with 0.5 µg/mL aureobasidin A and the culture

was incubated at 30°C for 2 days. The culture was then transferred to 500 mL of synthetic minimal

(SD) medium (2% (w/v) D-glucose and 0.67% (w/v) yeast nitrogen base without amino acids),

supplemented with 20 µg/mL L-histidine and 100 µg/mL L-leucine ( SD+His+Leu), and incubated

for 40 h. The yeast cells were recovered by centrifugation (3,000×g, 5 min, room temperature) and

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resuspended in 0.2 mL of 100 mM potassium phosphate buffer (pH 7.4) containing 8% (w/v)

glucose. Glucuronidation substrate was then added to a final concentration of 1 mM by the addition

of 2 µL of 100 mM stock solution in DMSO. The assay was carried out in a 96-deep well microplate for 24 h at 30°C and shaking at 180 rpm, using a constant temperature incubator shaker (micro tube

Maximizer MBR-022UP; TAITEC Co., Saitama, Japan). Following incubation, a 2.5–fold volume of

chloroform:methanol (3:1, v/v) was added to the reaction medium containing the yeast cells. The

extracts were clarified into an upper aqueous and lower organic phase by centrifugation (12,000×g,

10 min, room temperature). The lower organic phase was collected, evaporated and redissolved in

100 µL acetonitrile and each phase was analyzed using the UPLC system. For analysis of the amount

of glucuronide on the outside and inside of the yeast cells, the reaction medium was separated by

centrifugation before extraction of the glucuronide in the yeast cell, followed by extractions of the

two fractions, separately.

The detection of all the glucuronides and parent compounds in this study was carried out using a

UPLC system equipped with a Cosmosil 2.5C18-MS-II column (2.0 mm x 100 mm, Nacalai Tesque).

For analysis of 7-hydroxycoumarin and its glucuronide, the column temperature was 45°C, the flow

rate was 0.5 mL/min and detection at 320 nm. The gradient elution conditions were:

water-acetonitrile with 1 % (v/v) trifluoroacetic acid, 10% (v/v) acetonitrile (4 min), 10-70%

acetonitrile (6 min), 70-10% acetonitrile (2 min), 10% acetonitrile (4 min). For analysis of

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mycophenolic acid and its glucuronides the flow rate was 0.5 mL/min, detection at 250 nm and

column temperature of 40°C. The gradient elution conditions were: water-acetonitrile with 1% (v/v)

trifluoroacetic acid, 20-40% (v/v) acetonitrile (7 min), 40% acetonitrile (2 min), 40-20% acetonitrile

(2 min), 20% acetonitrile (2 min). For analysis of acyl glucuronides from non-steroidal

anti-inflammatory drugs, such as diclofenac, mefenamic acid, flufenamic acid and zomepirac, the

flow rates were 0.5mL/min, detection at 250 nm and column temperature of temperature 40°C. The

gradient elution conditions were: water-acetonitrile with 1% (v/v) trifluoroacetic acid, 5-40% (v/v)

acetonitrile (5 min), 40-100% acetonitrile (3 min) 100% acetonitrile (1 min), 100-5% acetonitrile (5

min), 5% acetonitrile (3 min). In each case, confirmation of glucuronide production was performed

by β-glucuronidase-dependent hydrolysis. Standard curves for quantification of glucuronides in the analyses were prepared using authentic glucuronides, when available. In other cases, the parent

compound (aglycone substrate) absorbance was used as a close approximation for the glucuronide absorbance when detection was performed by UV absorbance27.

Production of UDP-glucuronic acid in resting yeast cells expressing the rat UGDH gene. Production of UDPGA in budding yeast cells was confirmed by detection of intracellular UDPGA.

Following cultivation of yeast transformants expressing the UGDH gene in selective medium at

30°C for 48 h, 2.5–fold volume of chloroform:methanol (3:1, v/v) was added to the yeast cells

pellets. The upper aqueous phase was then separated by centrifugation (12,000×g, 10 min, room

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temperature). Intracellular UDPGA in the yeast cells was isolated and detected using a WakoPack

Navi C30-5 (Wako Pure Chemical Industries, Tokyo, Japan) -column- HPLC system and an

isocratic elution with 20 mM triethylamine-acetate (pH 7.0) as the mobile phase, at flow rate of 0.35

mL/min and detection at 260 nm.

Analysis of expressed UGDH and UGT enzymes in yeast cells by immunobloting. To confirm

the expression of rat UGDH and different human UGT1As, whole yeast proteins were analyzed by

immunoblot analysis. Yeast pellets were treated with 0.5 mg/mL zymolyase 100T (Nacalai Tesque)

for 30 min at 30°C and then subjected to SDS-PAGE, 10% acrylamide gel. The resulting

polypeptide bands were transferred to nitrocellulose membranes and polyclonal antibodies against

the UGT enzymes or rat UGDH were used for detecting the immunoreactive bands. Anti-human

UGT1A or anti-rat UGDH antibodies recognize the corresponding specific peptide region, i.e., 516-GKGRVKKSHKSKTH-529 and 420-FKELDYERIHKRML-433 as described previously28. The

target proteins were visualized by chemical luminescence (ECL detection kit; Amersham

Biosciences Inc., Piscataway, NJ) and the level of each protein was determined densitometrically,

using the ImageJ software.

Preparative biosynthesis and purification of diclofenac acyl glucuronide. Transformed yeast

strains were inoculated into 5 mL of YPD supplemented with 0.5 µg/mL aureobasidin A and

incubated for 2 days at 30°C. The cultures were then transferred to 200 mL YPD without

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aureobasidin A for 12 h at 30°C. These cultures were used to inoculate 10 L of SD medium,

supplemented with 20 µg/mL L-histidine and 100 µg/mL L-leucine, using 10 L of single use Culture Bag (Fujimori Kogyo Co., Ltd., Tokyo, Japan), and incubated for 40 h. Following this incubation,

the yeast cells were centrifuged (3,000g, 5 min, room temperature) and resuspended in 400 mL of

100 mM potassium phosphate buffer (pH 7.4) containing 8 % (w/v) glucose. Substrate was added to

a final concentration of 1 mM by adding 4 mL of 100 mM diclofenac from stock solutions in DMSO.

After bioconversion in the resting cell for 48 h, the reaction medium containing diclofenac acyl

glucuronide was separated from the yeast cells by centrifugation (3,000×g, 5 min, room temperature).

Purification of diclofenac acyl glucuronide was performed using preparative RP chromatography

with a Cosmosil 140C18-OPN column (140 µm, 2.7 x 20 cm) by stepwise elution of water-methanol. Each fraction was evaluated for purity by UPLC and the pure fractions were pooled and evaporated

to reduce solvent volume. Purification was also performed using a shorter Cosmosil 140C18-OPN column (140 µm, 2.7 x 10 cm). Fractions containing glucuronide were pooled, evaporated and lyophilized. The purity of intact acyl glucuronide of diclofenac was confirmed by comparison of its

UPLC elution pattern with the authentic glucuronide and examining the effect of β-glucuronidase treatment. Further structural confirmations of acyl glucuronide were performed by measurements

of MS and NMR spectra (See Figure S5 and S6., in the Supporting Information).

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RESULTS

Enhancing the expression levels of UGTs 1A1, 1A4 and 1A9 in yeast The initial immunoblot analyses of the expressed human UGTs 1A1, 1A4, and 1A9 in the

budding yeast system revealed relatively low expression levels in comparison to the other human

UGTs (Figure 1, upper panel). In order to enhance the expression level of these 3 UGTs, we have

replaced their signal peptides with the corresponding segment from UGT1A7 since the expression

level of UGT1A7 was the highest among the human UGTs (Figure 1, upper panel). The positive

effect of this replacement was particularly large for UGT1A4, but it was also clear for UGT1A1 and

UGT1A9 (Figure 1, lower panel. The amino acid sequences of the N-terminal signal peptides of

human the UGTs 1A1, 1A4, 1A7 and 1A9 are shown in Table S2 of the supporting information). The

results show that the expression levels of the modified UGTs, with the signal peptide of UGT1A7,

increased 2 to 4-fold, resulting in a similar expression level for all the UGT1A enzymes (Figure 1,

lower panel). A comparable incremental increase in glucuronidation activity of 7HC was observed in

the microsomal fractions (results not shown).

Production of UDP-glucuronic acid in yeast cells expressing rat UGDH For production of UDPGA in yeast, the rat UGDH gene was introduced into the yeast genome

using the genome-integrating expression vector pAUR-UGDH (Figure S2, Supporting Information).

Zymolyase-treated yeast cells that incorporated pAUR-UGDH were analyzed by immunodetection

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to confirm the expression of this protein with an apparent molecular mass of 53 kDa (Figure S3,

Supporting Information). Production of UDPGA in yeast cells was verified by examination of the

endogenously produced sugar nucleotides (Figure S4, Supporting Information). As could be seen

from the figure, while only UDP-glucose was detected as the sugar-nucleotide in control yeast cells

(line B), a significant peak of UDPGA, in addition to UDP-glucose, was detected in line A (Figure

S4, Supporting Information). Based on an estimation of the amount of UDPGA, the intracellular

concentration of this sugar-nucleotide in those rat UGDH-expressing yeast cells was about 1.2 - 1.6

mM.

Biosynthesis of glucuronide in yeast cells transformed with pAUR-UGDH/UGT For production of glucuronides in yeast cells, we have constructed co-expression vectors

encoding a human, or another mammal, UGT enzyme and the rat UGDH, generally termed

pAUR-UGDH/UGT (Figure S2, Supporting Information). Time course of 7HC conversion to its

glucuronide in these yeast transformants with the highest glucuronidation activity, the yeast strain

AH22 transformed with the co-exspression plasmid pAUR-ratUGDH/humanUGT1A6, revealed a

near complete conversion to 7HC-glucuronide within 24h (Figure 2). Treatment of the product with

β-glucuronidase in the reaction medium confirmed the formation of β-linked glucuronide of 7HC rather than glycoside (results not shown). Examination of the dependence of the conversion on the

glucose concentration indicated that complete conversion of 7HC to its glucuronide was highest at

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8% (w/v) of glucose (Figure 3). Furthermore, based on analyses of the amount of glucuronide in the

medium and inside the cells, it was found that about 90% of the formed glucuronide was secreted to

the medium and could be collected from it following removal of the cells by centrifugation.

Comparison of glucuronide formation between budding and fission yeasts A previous study reported glucuronides production by whole-cell biotransformation using genetically engineered fission yeast, Schizosaccharomyces pombe19. To compare the glucuronides

production ability of the budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces

pombe) yeasts, both containing UGDH and UGT, several transformants from our new system were

selected, namely those expressing the human UGTs 1A1 and 1A6. Table 1 presents a comparison of

4-methylumbelliferone glucuronide production between budding and fission yeast. The production

rates of this glucuronide in the budding yeast with UGT1A1 and UGT1A6 were 10- and 50-fold

higher than in fission yeast, respectively. The amounts of glucuronide per dry weight of budding

yeast indicated a 20 to 100-fold increase in comparison to fission yeast.

Biosynthesis of glucuronides using human and other mammalian UGTs The UGT genes in mammalians, including human, belong to a gene family containing the UGT1

and UGT2 subfamilies. Each UGT enzyme exhibits a different, even if often partly overlapping,

substrate- and region-specificity toward xenobiotic compounds. In order to produce glucuronides of

structurally diverse compounds in yeast at high efficiency, cDNA sequences encoding either one of

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the 15 human UGTs, 10 rat UGTs, 8 mouse UGTs, 5 porcine UGTs or bovine UGT1A6 were

inserted into the pAUR-UGDH vector (the full list is presented in Table S1, Supporting information).

Yeast transformation with each one of these co-exspressing pAUR vectors resulted in a set of 39

different yeast strains that co-express a given mammalian UGT with the rat UGDH. The

glucuronidation activity in all these strains was evaluated using, separately, 3 substrates, 7HC,

diclofenac and 11α-hydroxyprogesterone (Figure S1, Supporting information). The reasons for this substrates selection were that 7HC is a small phenolic substrate that is glucuronidated, at different

rates, by most UGTs; diclofenac has a carboxylic acid and is glucuronidated to form an acyl

glucuronide, whereas 11α-Hydroxyprogesterone is a steroid substrate. The results of this comparison are presented in Table S3 (Supporting information) and it shows that the majority of these 39

co-expressing yeast strains exhibited glucuronides production from at least one of the 3 selected test

substrates. The transformats carrying human UGT1A6, human UGT2A1, rat UGT2B1 and mouse

Ugt1a6 exhibited relatively high efficiency in the production of 7HC-glucuronide. Significant

specific production rates of diclofenac acyl-glucuronide were observed in cells expressing rat

UGT2B1 and mouse Ugt2b1. Among the UGT2 subfamily member enzymes, rat UGT2B6 exhibited

the highest specific production rates of 11α-hydroxyprogesterone glucuronide. The range of specific

production rates was from 0.001 to 10.26 (µmol/d/g wet weight). Hence, the results demonstrate that a combination of human or mammalian UGT enzymes with rat UGDH allows genetically modified

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strains of the budding yeast to produce a variety of glucuronides.

Biosynthesis of regiospecific glucuronides with multiple conjugation sites One of the major advantages of enzymatic biosynthesis of glucuronides, in comparison to

chemical synthesis, is regiospecific glucuronidation in the case of substrates with multiple potential

conjugation sites. Mycophenolic acid, which is used as an immunosuppressant drug, has two such

potential glucuronidation sites that yield either phenolic- or carboxyl-linked conjugates. Figure 4

shows mycophenolic acid glucuronides production using resting yeast cells with the UGT-UGDH

genes system. Human UGT1A9 in yeast cells can specifically catalyze phenolic glucuronide

formation of mycophenolic acid, as the recombinant human UGT1A9 that was previously expressed in HEK293 cells29. On the other hand, rat UGT2B1 produces the acyl glucuronide of mycophenolic

acid (Figure 4). Thus, by selecting suitable mammalian UGT enzyme, derived not only from humans

but also other species, we could obtain the desired glucuronides of various compounds containing

multiple conjugation sites.

Biosynthesis and purification of acyl glucuronide in yeast

1β-O-acyl glucuronide of some drugs with carboxylic acid groups were reported to be reactive metabolites that may bind covalently to proteins, causing potential toxicity4-6. Consequently, acyl

glucuronides are among the most important metabolites for safety testing during drug development

and the stability and reactivity of 1β-O-acyl glucuronide were previously examined using chemically

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synthesized acyl glucuronide30,31. Among the transformed yeast strains with human or other

mammalian UGTs, the most suitable enzymes for biosynthesis of acyl glucuronide were selected by

assessing diclofenac glucuronidation activity. As with mycophenolic acid acyl glucuronide, the rat

UGT2B1 was found to be highly effective in mediating diclofenac glucuronidation and the effects of

reaction medium pH on this activity were also examined (Figure 5). Table 2 presents the

biosynthesis of acyl glucuronides of several drugs with carboxylic acid groups using resting yeast

cells harboring the rat UGT2B1-UGDH gene system. The chemical structures of these drugs are

shown in Figure S1 (Supporting information). The range of specific production rate of acyl

glucuronide was between 0.39 and 4.19 (µmol/day/g wet weight). Based on the results above, production of diclofenac acyl glucuronide was performed using rat

UGT2B1-expressing budding yeast strain under optimized conditions. The diclofenac acyl

glucuronide was purified and the HPLC profiles of the sample (Figure 6) revealed a highly pure

diclofenac acyl glucuronide without acyl migration. The identity of the product, intact acyl

glucuronide of diclofenac, was confirmed by co-elution in HPLC with a commercially available

standard of acyl glucuronide (data not shown). LC-MS analysis of purified diclofenac acyl

glucuronide shows a protonated molecular ion of glucuronide (m/z 472) and an aglycone ion (m/z

296) produced from the loss of the glucuronic acid due to in-source fragmentation, thereby

confirming the formation of mono-glucuronide of diclofenac (Figure S5, supporting information).

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Furthermore, 1H- and

13

H-NMR spectrum of the purified diclofenac acyl glucuronide that was

biosynthezysed in this strain, co-expressing the rat UGT2B1 and UGDH (Figures S6A and B,

supporting information) revealed the characteristic NMR signals for β configuration at the C1 of the

glucuronic acid to be δΗ 5.66 (1Η, d, J=8.0 Hz, 1`-H) for the anomeric proton and δC 95.8 for the anomeric carbon, as previously reported32. Using 50 g wet weight of yeast cells, about 40 mg of

diclofenac acyl glucuronide was obtained in the final purification step. The yield and purity of

diclofenac acyl glucuronide was 21% and 96%, respectively. Hence, the methodology described in

this paper is suitable for producing tens of milligrams of pure acyl glucuronides at reasonable cost.

Taken together, our results suggest that the genetically modified budding yeast S. cerevisiae,

harboring a UGDH gene and a suitable UGT, that could be selected and optimized for different

needs, is a useful system for the production of UGT metabolites via whole-cell dependent

biosynthesis.

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DISCUSSION Genetically modified budding yeast is widely used in biotechnological platforms for the production of various compounds, including P450-dependent drug metabolites21,22. In this study

budding yeast strains co-expressing a selected mammalian UGT enzyme, one of the 39 that we have

developed thus far, together with the rat UGDH, were constructed for biosynthesis of glucuronides

as phase II drug metabolites. Several lines of evidence indicated that budding yeast, as host cells,

could be used for the expression of UGT enzymes by employing the pGYR vector with a yeast glyceraldehyde 3-dehydrogenase promoter and terminator14-17,25. When expressing human UGTs in

budding yeast, some of them, particularly UGT1A4, exhibited lower expression level than others

(Figure 1). We found that replacing the signal peptide of the low-level expressed UGTs with the

signal peptide of the human UGT1A7 stimulated their expression and led to a nearly similar

expression levels for all the human UGT1As (Figure 1). Likewise, in the heterologous expression of

cytochrome P450 in membranes, mutation of the N-terminal region favors the expression of protein in host cells33.

Bureik and co-workers developed a glucuronide production system using whole-cell biotransformation with the fission yeast, Schizosaccharomyces pombe (S. pombe) 19,20,34,35. Both S.

cerevisiae and S. pombe are classified as yeasts, which are thought to have branched off during their evolution some 300-400 million years ago36. Although the two yeasts have similar genome sizes and

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numbers of genes, there are marked differences between them in chromosome number and growth

characteristics. We have identified some advantages of the budding yeast for whole-cell glucuronide production and demonstrate it here using 4-methylumbelliferone as a substrate (Table 1). The production ability of glucuronides in budding yeast containing rat UGDH and the human UGT1A1

or UGT1A6 is between 10- and 50-fold higher than in fission yeast carrying the corresponding genes,

respectively. There are some possibilities for these differences in production ability between budding

and fission yeasts, such as promoter-dependent expression level or vector-dependent copy number of

the genes. Another possibility is more efficient excretion of the formed glucuronides in the budding

yeast, preventing their accumulation that could have slowed down further synthesis. Further

investigation is needed for elucidation these and, probably, other reasons for the production

differences between budding yeast and fission yeast, but this is outside the scope of this study. In

addition to high efficiency of glucuronides production, the budding yeast strains yielded biomass

concentrations between 4 and 5 g/L after 2 days growth, in contrast to biomass yields of between 1 and 2 g/L for fission yeast19.

An additional important outcome of this work is that most of the produced glucuronides were

exported to the reaction medium after complete conversion of the substrate (Figure 3). Thus, a

tedious processing step for glucuronide purification, such as disruption of yeast cells, was not

required. The presence of ABC transporters in budding yeast probably facilitates the export of

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glucuronides from into the growth medium37. Thus, it appears that in spite of the high degree of

similarity with the fission yeast system, there are several significant benefits in using budding yeast

for glucuronides production.

The major advantage of enzymatic synthesis is the regiospecific glucuronidation of drugs

containing

several potential conjugation

sites. Indeed, synthesis of such compounds,

mono-glucuronides conjugated on each single and desired site at a time, could be very challenging in

conventional chemical synthesis. Kittelmann and co-workers reported the preparative enzymatic

synthesis of acyl glucuronide of mycophenolic acid using horse liver homogenates and excessive UDPGA with several efforts for screening for optimal condition38 The different UGT enzymes possess variable N-terminal halves and highly conserved C-terminal halves1. The substrate- and

regio-specificity of glucuronidation mainly depends on the variable region of the UGTs, the

N-terminal domain. Desirable glucuronides could be obtained using the budding yeast whole cell

system by selecting suitable UGT enzymes according to the target drug. For example, in this study

phenolic and acyl glucuronide conjugates of mycophenolic acid were produced by human UGT1A9

and rat UGT2B1-expressing yeast strains, respectively (Figure 4).

Most mammalian UGTs (e.g., human, rat, mouse, pig and cow) are able of catalyzing

glucuronidation of suitable substrates in budding yeast cells (Table S4, Supporting information).

Introduction of cDNA of different UGTs from genome analyses into the pAUR-rUGDH expression

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vector allowed us to characterize their UGT function, including substrate specificity and

regiospecificity, without the need to prepare membrane fractions for in vitro assays. In addition to

human UGTs, yeast strains having a set of UGTs from preclinical animal, such as rat and mouse,

could be useful tools in the development of high throughput screening systems for identification of

the UGT(s) that are responsible for the conjugation of test drug candidates.

The FDA guidance in 2008 paid attention for safety assessment of the metabolites identified

only in human plasma, or metabolites present at disproportionately higher levels in humans than in any of the animal test species39. The formation of acyl glucuronides of drug candidates is assessed

during the safety testing procedures. Based on screening experiments for the most suitable isoform for diclofenac acyl glucuronide production, rat UGT2B1, an orthologue of the human UGT2B740,

was selected for reactions requiring acyl glucuronide biosynthesis. Table 2 shows the biosynthesis of

acyl glucuronides of various drugs containing carboxylic acid groups using resting yeast cells

harboring the rat UGT2B1-UGDH gene system. Among the drugs tested, zomepirac is classified into

the safety category of “withdrawn” due to its idiosyncratic drug toxicity risk because the

corresponding acyl glucuronide displays chemical instability and reactivity under physiological conditions41. Interestingly and importantly, despite that instability, it is possible to biosynthesize the

acyl glucuronide of zomepirac in the yeast glucuronide production system.

In conclusion, the genetically modified budding yeast S. cerevisiae harboring the UGDH gene is

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a useful host organism to characterize the function of UGTs from various mammalian species and for

the production of UGT metabolites via whole-cell dependent biosynthesis at rather low cost and high

efficiency.

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(27) Court, M.H. Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods Enzymol. 2005, 400, 104-116. (28) Ikushiro, S.; Emi. Y.; Kato, Y.; Yamada, S.; Sakaki, T. Monospecific antipeptide antibodies against human hepatic UDP- glucuronosyltransferase 1A subfamily ( UGT1A) isoforms.

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(29) Miles, K.K.; Stern, S.T.; Smith, P.C.; Kessle,r F.K.; Ali, S.; Ritter, J.K. An investigation of human and rat liver microsomal mycophenolic acid glucuronidation: evidence for a principal role of UGT1A enzymes and species differences in UGT1A specificity. Drug Metab Dispos. 2005, 33, 1513-1520. (30) Stachulski, A.V.; Harding, J.R.; Lindon, J.C.; Maggs, J.L.; Park, B.K.; Wilson, I.D. Acyl glucuronides: biological activity, chemical reactivity, and chemical synthesis. J Med Chem. 2006, 49, 6931-6945.

(31) Berry, N.G.; Iddon, L.; Iqbal, M.; Meng, X.; Jayapal, P.; Johnson, C.H.; Nicholson, J.K.; Lindon, J.C.; Harding, J.R.; Wilson, I.D.; Stachulski, A.V. Synthesis, transacylation kinetics and computational chemistry of a set of arylacetic acid 1β-O-acyl glucuronides. Org Biomol Chem. 2009,7, 2525-2533. (32) Bowkett, E.R.; Harding, J.R.; Maggs, J.L.; Park, B.K.; Perrie, J.A.; Stachulski, A.V. Efficient synthesis of 1β-O-acyl glucuronides via selective acylation of allyl or benzyl D-glucuronate.

Tetrahedron 2007, 63, 7596-7605. (33) Barnes, H. J.; Arlotto, M. P.; Waterman, M. R. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.Proc Natl Acad Sci U S A. 1991, 88, 5597-5601. (34) Zöllner, A.; Buchheit, D.; Meyer, M. R.; Maure,r H. H.; Peters, F.T.; Bureik, M. Production of human phase 1 and 2 metabolites by whole-cell biotransformation with recombinant microbes.

Bioanalysis.2010, 2, 1277-1290. (35) Buchheit, D.; Schmitt, E. I.; Bischoff, D.; Ebner, T.; Bureik, M. S-Glucuronidation of 7-mercapto-4-methylcoumarin by human UDP glycosyltransferases in genetically engineered fission yeast cells. Biol Chem. 2011, 392, 1089-1095. (36) Wixon, J. Featured organism: Schizosaccharomyces pombe, the fission yeast.

Comp Funct Genomics 2002, 3,194-204.

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(37) Paumi, C.M.; Chuk, M.; Snider, J.; Stagljar, I.; Michaelis, S. ABC transporters in Saccharomyces cerevisiae and their interactors: new technology advances the biology of the ABCC (MRP) subfamily.Microbiol. Mol Biol Rev. 2009,73, 577-593. (38) Kittelmannn, M.; Rheinegge,r U.; Espiga,t A.; Oberer, L.; Aichholz, R.; Francotte, E.; Ghisalba, O. Preparative enzymatic synthesis of the acylglucuronide of mycophenolic acid. Adv.Synth.Catal. 2003, 345, 825-829. (39) Anderson, S.; Knadler, M. P.; Luffer-Atlas, D. Overview of metabolite safety testing from an industry perspective. Bioanalysis 2010, 2, 1249-1261. (40) King, C.; Tang, W.; Ngu,i J.; Tephly, T.; Braun, M. Characterization of rat and human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac. Toxicol Sci. 2001, 61, 49-53. (41) Sawamura, R.; Okudaira, N.; Watanabe, K.; Murai, T.; Kobayashi, Y.; Tachibana, M.; Ohnuki, T.; Masuda, K.; Honma, H.; Kurihara, A.; Okazaki, O. Predictability of idiosyncratic drug toxicity risk for carboxylic acid-containing drugs based on the chemical stability of acyl glucuronide.

Drug Metab Dispos. 2010, 38, 1857-1864.

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ACKNOWLEDGEMENTS We would like to thank Prof. Matthias Bureik, School of Pharmaceutical Science and Technology

(SPST), Tianjin University, Tianjin, China, for helpful discussion about the use of

11α-hydroxyprogesterone as universal glucuronidating substrate. We would also like to thank Mrs. Shizuka Sakaki, Nanami Nishiguchi, Ririi Koike and Mr. Takashi Hasegawa, Masashi Kuroda for

skilled technical assistance.

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FOOTNOTES This work was supported in part by a Grant-in-Aid for Scientific Research of the Ministry of

Education, Culture, Sports, Science and Technology of Japan. This work is part of the following

patent application: Ikushiro, S., Sakaki, T., and Yasuda, K. (2010) Japanese Patent Application No.

JP 2010-040150 and (2011) International Application No. PCT/JP2011/053016

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FIGURE LEGENDS Figure 1. Immunoblot analysis of expressed human UGTs in budding yeast. Immunodetection

was performed using anti-UGT1A antibody. The same amount of microsomal protein, 10 µg, from either control untransformed cells (C), or human UGT1A gene-transformed yeast strains, was loaded

onto each lane. The upper panel shows the expression of wild type human UGTs, whereas the lower

panel presents the expression of both wild type UGTs and N-terminal modified UGTs 1A1, 1A4, and

1A9.

Figure 2. Time dependence of the production of 7-hydroxycoumarin glucuronide in genetically modified budding yeast strain expressing rat UGDH and human UGT1A6. The production of

7HC glucuronide in the resting cell was measured at several time points during incubation. Circles

and triangles represent 7HC and 7HC-glucuronide, respectively. The HPLC elution profiles of

metabolites at the start (A) and following 24 h incubation (B) are shown in the inset. The retention

times of 7HC-glucuronide and the substrate 7HC were 2.4 min and 6.1 min, respectively.

Figure 3. Glucose concentration dependence of 7HC-glucuronide production in genetically

modified budding yeast strain expressing human UGT1A6 and rat UGDH. The production of 7HC-glucuronide in resting cells was measured at several glucose concentrations.

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Open and shaded areas in each bar represent the amount of glucuronide outside and inside the yeast

cells, respectively.

Figure 4. Regiospecific formation of mycophenolic acid glucuronides in genetically modified budding yeast strain expressing rat UGDH with either human UGT1A9 or rat UGT2B1. (A)

Elution profile of authentic glucuronide and substrate. 1: phenolic β-D-glucuronide of mycophenolic

acid. 2: acyl-β-D-glucuronide of mycophenolic acid. 3: mycophenolic acid. (B) Elution profile of the

metabolite produced by yeast cells expressing the human UGT1A9. (C) Elution profile of the

metabolites from yeast cells expressing rat UGT2B1.

Figure 5. The effects of reaction medium pH on diclofenac acyl glucuronide formation and stability. HPLC elution profiles of diclofenac metabolite that was biosynthezysed in yeast strain

expressing rat UGDH and rat UGT2B1 under medium pH condition of either 8.5 (A), 7.5 (B), or 6.5

(C). In the HPLC system employed here, the retention times of intact diclofenac acyl glucuronide

and the substrate diclofenac are 6.25 min and 7.20 min, respectively.

Figure 6. Biosynthesis and purification of diclofenac acyl glucuronide in genetically modified budding yeast strain expressing rat UGDH and rat UGT2B1. (A) Elution profile of the

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biosynthesized and purified diclofenac acyl glucuronide. (B) Elution profile of diclofenac

metabolism extract (before purification) from the yeast strain expressing rat UGDH and rat UGT2B1.

(C) Elution profile of diclofenac metabolism extract from wild type yeast strain.

the retention times of diclofenac and its acyl glucuronide.

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See Figure 5 for

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Table 1. Comparison of glucuronide production between UGT-UGDH expressing S. cerevisiae and

S. pombe

4-Methylumbelliferone glucuronidation Human UGT

(a

µM / day

µmol / day / g dry weight

S.cerevisiae

S.pombe(a

S.cerevisiae

S.pombe(a

UGT1A1

20.2

1.8

2.6

0.14

UGT1A6

195.7

3.5

24.8

0.22

Drăgan CA, Buchheit D, Bischoff D, Ebner T, Bureik M (2010) Drug Metab Dispos. 38,

509-515

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Table 2. Production of acyl glucuronides by rat UGT2B1 expressing yeast cells

specific production rate

Substrates

( µmol / day / g wet weight )

Mycophenolic acid

0.54

Mefenamic acid

0.56

Flufenamic acid

1.12

Diclofenac

4.19

Naproxen

0.60

Loxoprofen

0.43

Zomepirac

0.39

Each value of specific production rate was an average of duplicate experiments

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254x190mm (96 x 96 DPI)

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Figure 1. Immunoblot analysis of expressed human UGTs in budding yeast. 254x190mm (96 x 96 DPI)

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Figure 2. Time dependence of the production of 7-hydroxycoumarin glucuronide in genetically modified budding yeast strain expressing rat UGDH and human UGT1A6. 254x190mm (96 x 96 DPI)

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Figure 3. Glucose concentration dependence of 7HC-glucuronide production in genetically modified budding yeast strain expressing human UGT1A6 and rat UGDH. 254x190mm (96 x 96 DPI)

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Figure 4. Regiospecific formation of mycophenolic acid glucuronides in genetically modified budding yeast strain expressing rat UGDH with either human UGT1A9 or rat UGT2B1. 254x190mm (96 x 96 DPI)

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Figure 5. The effects of reaction medium pH on diclofenac acyl glucuronide formation and stability. 254x190mm (96 x 96 DPI)

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Figure 6. Biosynthesis and purification of diclofenac acyl glucuronide in genetically modified budding yeast strain expressing rat UGDH and rat UGT2B1. 254x190mm (96 x 96 DPI)

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