Form 2-2（様式２‐２）

Characterization of new type III polyketide synthase from Dendrobium orchid
Name of dispatched researcher（派遣研究者名）
Instructor(指 導 教 官 ， 研 究 指 導 者 )Affiliation of Instructor(所 属 )
Host Collaborator(受 入 研 究 者 )Thaniya Wunnakup

Graduate Student

Wanchai De-Eknamkul

Associate Professor of
Chulalongkorn University

Chulalongkorn University
Hiroyuki Morita

教授

Abstract
Type III polyketide synthases (PKS) are a group of the enzymes that catalyze sequential condensationsof malonyl-CoA and cyclization reaction to produce polyketide scaffold. Bibenzyl synthase (BBS), a type III PKS,
is reportedly involved in the biosynthesis of dihydrostilbenes. In the previous study, we cloned a putative bbs gene(DoBBS) from Dendrobium orchid. To clariefy the DoBBS function, the DoBBS gene was expressed in Pichia
pastoris and the DoBBS in vitro enzyme reaction products derived from dihydro-p-coumaroyl-CoA andp-coumaroyl-CoA with malonyl-CoA were analyzed by LC-MS. The constructed Phichia expression system gave
DoBBS as soluble protein with high efficacy. However, the recombinant DoBBS did not produce any products
from the substrates used in this study, suggesting that further investigation using the other substrate are required.

Keywords: Dendrobium orchid, dihydrostilbene, polyketide, protein expression.

1. Introduction
Polyketides are structurally diverse and pharmaceutically important natural products. They show a wide
range of bioactivities such as antioxidant, anti-inflammatory, anticancer, antibacterial activities, and many
polyketides reportedly recognize as bioactive constituents in the traditional medicines. Previous studies of thebiosynthesis of the plant polyketides revealed that the remarkable structural diversity of the plant polyketides are
principally derived from differences of the starter substrate specificity, the number of malonyl-CoA condensations,and the type of cyclization reaction of structurally simple type III polyketide synthases (PKS) (Austin & Noel,
2003; Abe & Morita, 2010). For example, chaclcone synthase (CHS), which is the pivotal enzyme in thebiosynthesis of Flavonoid, produces naringenin chalcone from condensation of three molecules of malonyl-CoA
with p-coumaroyl-CoA via the formation of the linear tetraketide intermediate and its C6-C1 Claisen type
cyclization reaction (Figure 1). On the other hand, stilbene synthase (STS) generates resveratrol from the common
linear tetraketide intermediate with CHS via an aldol type cyclization reaction. Nowadays, functionally distinct
more than 40 type III PKSs have been identified from plants. Phylogentic analysis has demonstrated that STS
evolves from CHS (Tropf et al., 1994). In addition, the site directed mutagensis studies of CHS converted its
function to STS (Austin et al., 2004).
1Form 2-2（様式２‐２）

Among the reported type III PKSs, bibenzyl synthase (BBS), which is specifically involved in thebiosynthesis of the m-hydrophenylpropionate-derived dihydrostilbene derivatives, has been obtained from theOrchidaceae plants such as Epipactis palustris (Gehlert & Kindle, 1991), Bletilla striata (Reinecke & Kindl,1993), Phalaenopsis sp (Preisigmuller et al., 1995), and Cymbidium hybrida (Wang et al., 2014). The enzyme
catalyzes the STS-like C2-C7 aldol type cyclization reaction of the linear tetraketide intermediate derived fromthree molecules of malonyl-CoAs and dihydro-m-coumaroyl-CoA to generate dihydrostilbene scaffold (Figure 1).

CHS
OH+CoAS

3xCoAS
O

1O

OH
O

OSEnz

OH

O

OHOO

narigenin chalcone

malonyl-CoA

STSOH

OH
OH+CoAS

3xCoASO

7O

OH

OO- CO2O

p-coumaroyl-CoA

HOSEnz

2O

OHO

resveratrolmalonyl-CoA

OH

OH

BBS

OH+CoAS

3xCoAS
O

7O

OH
O

O

dihydro-m-coumaroyl-CoA

O- CO2

HOSEnz2O

Fig. 1OH

6O

p-coumaroyl-CoA

HOOH

OH

O

dihydro-m-hydroxyresveratrolmalonyl-CoA

The Biosynthesis of chalcone, stileben, and dihydrostilebe by type III PKSs

Bibenzyl derivatives such as Phenanthrenes and dihydrostilbenes have also been isolated fromDendrobium genus (Figure 2). However, the BBS enzymes in this genus have not be yet fully elucidated. We
currently isolated putative bbs gene (DoBBS) from D. orchid. In this study, the putative DoBBS gene wasexpressed in Pichia pastoris and its function was evaluated by investigating in vitro reaction products of therecombinant DoBBS.

OHOOOH

Fig. 2HOO
OHO
OH

OHOOHOHOOHO

HOO

HOOOH

Example of polyketides isolated from Dendrobium plants

2. Materials and Methods2.1. Strains and culture conditions
The E. coli strain DH5α was used in subcloning. The cells were cultured in LB medium containing 25µg/ml of Zeocin and incubated at 37 °C. The P. pastoris stain X33 and KM71H was used for expression of2

Form 2-2（様式２‐２）

recombinant DoBBS. Yeast strains were cultured in YPD medium (1% yeast extract, 2% peptone and 2%glucose) and incubated at 30 °C.
2.2. Recombinant enzyme expression
pPICZA with DoBBS cDNA was transformed into the P. pastoris stain KM71H and X33 by using LiClmethod, and cultured on YPD plate containing 100 µg/ml and 500 µg/ml Zeocin, respectively. The transformants
were confirmed with PCR. The KM71H and X33 cells harboring DoBBS cDNA were cultured in BMGHmedium (100 mM potassium phosphate, pH 6.8, 1.34% yeast nitrogen base, 4 x 10-5% biotin, 1% glycerol and0.004% histidine) at 28 °C (200 rpm). After OD was reached to 2–6, the cells were harvested by centrifugation at
3,000 g for 10 min, and then the pellet were resuspended with BMMH medium (100 mM potassium phosphate,pH 6.8, 1.34% yeast nitrogen base, 4 x 10-5% biotin, 1% methanol and 0.004% histidine) to OD600 = 1, and waa
further incubated at 28 °C (200 rpm) for 4 days. During the culture of the cell in BNMH medium, the 1%
methanol was added for everyday to maintain the protein induction.

2.3. Extraction and purification of recombinant protein
The induced cells were harvested by centrifugation at 3,000 g for 10 min (4 °C), and the pellet wasresuspended with 3 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol and 1 mM PMSF) per
1 g of cell. The cell suspension was mixed with 2 g acid-wash glass beads (sigma), and then vortex with highspeed for 30 sec with interval for 30 sec on ice (> 7 times). The lyset was centrifuged at 2500 g for 3 min and thesupernatant was centrifuged at 6,000 g for 10 min (4 °C) to prepare the crude enzyme solution. The crude enzyme
solution was concentrated by 10K cutoff Macrosap Advance Centrifugal Device (Pall Life Sciences), and thensubjected to Ni-NTA affinity column (GE healthcare). The recombinant DoBBS was eluted with 0.5 M imidazole.
Fractions containing the DoBBS protein were determined on the basis of SDS-PAGE analysis, dialyzed, andconcentrated. Protein concentration was measured by the Bradford method.
2.4. Enzyme assay
Crude enzyme (100 µg) or the purified DoBBS (20 µg) were incubated with 0.3 mM malonyl CoA, 0.2
mM p-coumaroyl CoA or 0.2 mM dihydro-p-coumaroyl CoA in 100 µl of 20 mM Tris-HCl buffer, pH 7.0, at
20°C and 30 °C for 12 h and 24 h, respectively. The reaction was stopped by adding 10 µl 20% HCl and theenzyme reaction products were extracted with 200 µl ethyl acetate (triplicate). After the extract was dried under
reduced pressure, the residue was resuspended with 50 µl methanol and subjected to LC/MS using water (A) and
acetonitrile (B) containing 0.1% (v/v) TFA as mobile phase to analyze the enzyme reaction products. The
gradient solvent system was follows: 0-5 min, 10% B; 5-20 min, 20% B; 20-30 min, 60% B; 30-50 min, 100%B; 50-60 min, 10%B. The flow rate was 0.5 ml/min and chromatograms were acquired at 290 nm.

Results and DiscussionThe recombinant DoBBS was heterologously expressed in P. pastoris strain KM71H and X33 as a3

Form 2-2（様式２‐２）

fusion protein with His6-tag at the C-terminus under controlling AOX1 promoter and purified with Ni-NTA
affinity column, respectively. The recombinant DoBBSs expressed in KM71H and X33 migrated as a band with amolecular weight of 44.7 kDa on SDS-PAGE (Figure 3), which agree well with the calculated value of 44.5 kDa,
respectively. Furthermore, the SDS-PAGE analysis indicated that DoBBS was expressed in KM71H with 19times higher efficacy over X33 (Table 1). Thus, the functional analysis of DoBBS was performed with therecombinant DoBBS expressed in KM71H.
Table 1. Summary of protein purification
StrainX33KM71H

Total protein (mg)

34.90

10.00

Ni-NTA column (mg/ml)

0.15

0.83

Target soluble protein (%)

0.22

4.16

Fig. 3

Analysis of the DoBBS expression in P. pastoris strains KM71H and X33 by SDS-PAGE

The expression of DoBBS was evaluated after the recombinant DoBBS was purified by using Ni-NTA affinity
column. M: Molecular weight marker, 1: DoBBS expressed by KM71H, 2: DoBBS expressed by X33. The
recombinant BBS was indicated with arrows.
BBS has been reported to efficiently catalyze the iterative condensation of three molecules ofmalonyl-CoAs with dihydroxy-m-coumaroyl-CoA to generate dihydroresveratrol. Due to the lack ofdihydroxy-m-coumaroyl-CoA, we utilized dihydro-p-coumaroyl-CoA as a substrate with malonyl-CoA in order
to investigate the DoBBS reaction products. Since, functionally related STS has been known to utilize
p-coumaroyl-CoA as a substrate with malonyl-CoA to produce resveratrol, we also carried out the DoBBS
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Form 2-2（様式２‐２）

enzyme reaction using p-coumaroyl-CoA and malonyl-CoA. Boiled DoBBS was used as the negative control.
However, the recombinant DoBBS did not accept dihydro-p-coumaroyl-CoA and p-coumaroyl-CoA withmalonyl-CoA to produce products. These observations suggested that either the recombinant DoBBS wasexpressed in E. coli as inactive enzyme or DoBBS accepts other CoA thioester substrate to produce compound.

Conclusion
DoBBS was expressed in P. pastoris strain KM71H and X33 as a fusion protein with His6-tag at theC-terminus and investigated the enzyme function. The KM71H strain efficiently expressed DoBBS as a soluble
protein. However, the recombinant DoBBS did not produce any products. Thus, further functional analysis ofDoBBS is needed.

References
1.

Abe, I. and Morita, H. (2010) Structure and function of the chalcone synthase superfamily of plant type III
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2.

Austin, M.B., Bowman, M.E., Ferrer, J.-L., et al. (2004) An aldol switch discovered in stilbene synthases
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3.

Austin, M.B. and Noel, J.P. (2003) The chalcone synthase superfamily of type III polyketide synthases,
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4.

Gehlert, R. and Kindl, H. (1991) Induced formation of dihydrophenanthrenes and bibenzyl synthase upon
destruction of orchid mycorrhiza, Phytochemistry, 30, 457−460.

5.

Preisigmuller, R., Gnau, P. and Kindl, H. (1995) The inducible 9,10-dihydrophenanthrene pathway:Characterization and expression of bibenzyl synthase and S-adenosylhomocysteine hydrolase, Archiv
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6.

Reinecke, T. and Kindl, H. (1993) Characterization of bibenzyl synthase catalysing the biosynthesis ofphytoalexins of orchids, Phytochemistry, 35, 63−66.

7.

Tropf, S., Lanz, T., Rensing, S., et al. (1994) Evidence that stilbene synthases have developed from chalcone
synthases several times in the course of evolution, J Mol Evol, 38, 610−618.

8.

Wang, L., Albert, N.W., Zhang, H., et al. (2014). Temporal and spatial regulation of anthocyanin
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983−1002.

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