Planta (2004) 218: 492–496
DOI 10.1007/s00425-003-1144-y
R AP ID CO MM U N IC A T IO N
Benye Liu Æ Till Beuerle Æ Tim Klundt Æ Ludger Beerhues
Biphenyl synthase from yeast-extract-treated cell cultures
of Sorbus aucuparia
Received: 7 August 2003 / Accepted: 8 October 2003 / Published online: 1 November 2003

Springer-Verlag 2003
Abstract Biphenyls and dibenzofurans are the phytoal-
exins of the Maloideae, a subfamily of the economically
important Rosaceae. The biphenyl aucuparin accumu-
lated in Sorbus aucuparia L. cell cultures in response to
yeast extract treatment. Incubation of cell-free extracts
from challenged cell cultures with benzoyl-CoA and
malonyl-CoA led to the formation of 3,5-dihydroxybi-
phenyl. This reaction was catalysed by a novel polyke-
tide synthase, which will be named biphenyl synthase.
The most efficient starter substrate for the enzyme was
benzoyl-CoA. Relatively high activity was also observed
with 2-hydroxybenzoyl-CoA but, instead of the corre-
sponding biphenyl, the derailment product 2-hydrox-
ybenzoyltriacetic acid lactone was formed.
Keywords Aucuparin Æ Benzoic acid Æ Biphenyl
synthase Æ Phytoalexin Æ Polyketide synthase Æ Sorbus
Abbreviations BIS: biphenyl synthase Æ BPS:
benzophenone synthase Æ DTT: dithiothreitol
Introduction
Plants produce a remarkably diverse array of over
200,000 low-molecular-mass secondary metabolites.
Many of these natural products are phytoalexins, which
have evolved to confer selective advantage against
microbial attack (Dixon 2001). The phytoalexins of the
Maloideae, a subfamily of the Rosaceae, are biphenyls
B. Liu Æ T. Beuerle Æ T. Klundt Æ L. Beerhues (&)
Institut für Pharmazeutische Biologie,
Technische Universität Braunschweig,
Mendelssohnstraße 1, 38106 Braunschweig, Germany
E-mail: l.beerhues@tu-bs.de
Fax: +49-531-3918104
B. Liu
Institute of Botany, Chinese Academy of Sciences,
Nanxincun 20, 100093 Beijing,
Haidian District, China
and dibenzofurans (Kokubun and Harborne 1995). The
Maloideae include many fruit trees such as Malus, Pyrus
and Prunus species. Considering this economic impor-
tance, relatively little is known of the disease resistance
mechanisms present in this subfamily. So far as is
known, the pathogen-induced accumulation of biphe-
nyls and dibenzofurans is unique to the Maloideae.
The best-known biphenyl phytoalexin is aucuparin
(see Fig. 1), which was first isolated from Sorbus aucu-
paria (Erdtman et al. 1963). Since then, biphenyls have
been detected as phytoalexins following fungal infection
in the sapwood of a number of species of the Maloideae
(Kokubun and Harborne 1995 and literature cited
therein). In addition, their accumulation was observed in
isolated leaves of various species upon inoculation with
fungi or treatment with heavy-metal ions (Widyastuti
et al. 1992; Kokubun and Harborne 1994). Recently,
biphenyls have also been found in yeast-extract-treated
cell cultures of Malus domestica (Borejsza-Wysocki et al.
1999).
The biosynthesis of biphenyls is poorly understood. It
has been proposed that their C12 skeleton is formed in
analogy to the stilbene synthase reaction (Sultanbawa
1980). Here we report elicitation of aucuparin formation
and detection of biphenyl synthase activity in S. aucu-
paria cell cultures.
Materials and methods
Chemicals
The sources of CoA esters are described elsewhere (Liu et al. 2003).
[2-14C]Malonyl-CoA and 5-phenylcyclohexane-1,3-dione were
purchased from Amersham Biosciences (Freiburg, Germany) and
ABCR (Karlsruhe, Germany), respectively. Yeast extract was from
Invitrogen (Karlsruhe, Germany).
Synthesis of 3,5-dihydroxybiphenyl
The reaction was conducted according to Nilsson and Norin (1963)
starting from 5-phenylcyclohexane-1,3-dione. The crude reaction

Fig. 1a, b GC–MS analysis of constituents from yeast-extract-
treated (a) and untreated (b) Sorbus aucuparia cell cultures. IS,
internal standard (eicosan); I-1, I-2, solvent impurities; C18:2,
C18:3, C18:0, fatty acids
product was chromatographed twice on silica gel (Merck, Darms-
tadt, Germany) using the solvents diethyl ether:petroleum ether
(7:3, v/v) and pentane:ethyl acetate (6:4, v/v). The NMR spectrum
of the purified product agreed with recently published data (Dol
et al. 1998). For acetylation, 3,5-dihydroxybiphenyl (1 mg) was
dissolved in 10 ll dry pyridine and 10 ll acetic acid anhydride.
After 1 h at room temperature, the reaction was quenched by
addition of 500 ll methanol and an aliquot was analysed by GC–
MS:
– 3,5-Dihydroxybiphenyl: Rf (silica gel, diethyl ether:petroleum
ether 7:3, v/v) 0.69; RI (ZB1) 1,958; 1H NMR (400 MHz,
CDCl3) d 7.53 (d, 2 H, J=7 Hz), 7.42 (t, 2 H, J=7 Hz), 7.34 (t,
1H, J=7 Hz), 6.64 (d, 2H, J=2 Hz), 6.34 (t, 1H, J=2 Hz), 5.06
(s, 2 H, H-O); GC–EIMS, 70 eV, m/z (rel. int.): 186 (100, [M]+),
187 (12), 185 (7), 128 (7), 157 (6), 129 (6), 115 (6), 139 (4), 127
(3), 69 (3).
– 3,5-Di-O-acetylbiphenyl: RI (ZB1) 2,104; GC–EIMS, 70 eV, m/z
(rel. int.): 270 (11, [M]+), 186 (100, [M-2(H2C=C=O)]+), 228
(18, [M-H2C=C=O]+), 43 (13, [C2H3O]+), 187 (12), 157 (5),
229 (3), 128 (3), 115 (2), 129 (2).
Cell cultures and elicitor treatment
Callus of Sorbus aucuparia L. was derived from young shoots.
After surface sterilization, stem segments were put on solid LS
medium (Linsmaier and Skoog 1965). Friable callus was used to
initiate cell suspension cultures, which were grown in liquid LS
medium at 25 C in the dark on an orbital shaker at 100 rpm.
493
Cultured cells (3 g) were transferred into 50 ml fresh medium every
14 days. Five-day-old cell cultures from the linear growth phase
were treated with yeast extract (3 g l)1). Eighteen and 48 h after the
onset of treatment, cultured cells were used for the preparation of
cell-free extract and the analysis of biphenyl derivatives, respec-
tively.
Extraction and analysis of biphenyls
Freshly harvested cells (10 g) were homogenised in 15 ml methanol.
After filtration the residue was extracted twice with 10 ml metha-
nol. The extracts were combined and evaporated under vacuum.
The residue was dissolved in 0.5 ml ethyl acetate, filtered and
analysed by TLC and GC–MS. The cell culture medium was ex-
tracted twice with the same volume of ethyl acetate. The organic
phases were combined, dried over solid sodium sulphate, filtered
and evaporated to dryness. The residue was dissolved in 0.5 ml
ethyl acetate and analysed by TLC and GC–MS.
Enzyme extraction
All steps were carried out at 0–4 C. Cultured cells (30 g) were
mixed with 3 g Polyclar AT (Serva, Heidelberg, Germany) and
homogenised in 20 ml of 0.1 M potassium phosphate buffer
(pH 7.5) containing 1 mM dithiothreitol (DTT). After centrifuga-
tion at 12,000 g for 10 min, the supernatant was subjected to
ammonium sulphate precipitation. The protein fraction obtained
between 50 and 80% saturation was resuspended in 0.1 M potas-
sium phosphate buffer (pH 7.0) and passed through a PD10 column
(Pharmacia, Freiburg, Germany) equilibrated with the same buffer.
Enzyme assays
Incubations were performed with and without radioactively la-
belled malonyl-CoA. The radioactive assay (100 ll) contained
0.1 M potassium phosphate (pH 7.0), 15 lM starter CoA ester,
56 lM malonyl-CoA, 0.93 kBq [2-14C]malonyl-CoA and approx.
10 lg protein. After incubation at 30 C for 30 min, the reaction
was quenched with 11 ll acetic acid (50%). The assay was ex-
tracted twice with 100 ll ethyl acetate and the organic phases were
combined and evaporated to dryness. The residue was taken up in
50 ll ethyl acetate and analysed by TLC and subsequent autora-
diography. Two independent experiments were performed and the
average values calculated.
For HPLC and GC–MS, the assay volume was increased to
500 ll and radioactive malonyl-CoA was omitted. Enzymatic
products were dissolved in either 50 ll methanol (50%) for HPLC
or 50 ll ethyl acetate for GC–MS. Their acetylation was performed
as described above for 3,5-dihydroxybiphenyl.
Analytical procedures
HPLC was carried out on a 1525 Binary HPLC Pump coupled with
a 2487 Dual Absorbance Detector and equipped with the Breeze
software 3.20 (Waters, Eschborn, Germany). Water (A) and
methanol (B) served as the solvents on a Symmetry C18 (5 lm)
column (150 mm long, 4.6 mm i.d.; Waters). The gradient was 40%
B for 3 min, 40 to 70% B in 15 min, 70 to 80% B in 5 min at a flow
rate of 1.0 ml min-1. Detection wavelengths were 225 nm for bi-
phenyls, 288 nm for benzoyltriacetic acid lactone and 327 nm for
benzoyldiacetic acid lactone (Morita et al. 2001).
TLC was carried out on aluminium sheets coated with silica gel
60 F254 (Merck). The solvent was cyclohexane:dichlorome-
thane:ethyl formate:formic acid (35:30:30:1, by vol.). Detection of
biphenyls was under UV light. Radioactively labelled enzymatic
products were analysed on glass plates coated with silica gel 60 F254
494

(Merck). The solvent was chloroform:methanol:water (65:25:4, by Partial enzyme characterisation

vol.). Detection and quantification were carried out using the TLC
radioscanner Rita (Raytest, Straubenhardt, Germany).
GC–MS data were obtained with a Hewlett Packard 5890A gas Enzyme activity was stimulated by DTT, with maximum
chromatograph equipped with a 2 m fused silica guard column (i.d. biphenyl formation occurring at 160 lM (Table 1).
0.32 mm) and a ZB1 or ZB5 (Phenomenex, Aschaffenburg, Ger- DTT, however, also induced the formation of ben-
many) analytical column (30 m long, 0.32 mm i.d.). Injector and zoyldiacetic acid lactone, a derailment product at the
transfer line were set at 280 C. The temperature program was
100 C (3 min) to 300 C (5 min) at 10 C min)1. The split ratio
stage of the triketide intermediate, and was therefore
was 1:20, the injection volume 1 ll and the carrier gas flow 1.6 ml omitted from the assays for enzyme characterization.
min-1 He. The capillary column was directly coupled to a triple Stimulation of product but also side-product formation
quadrupole mass spectrometer Finnigan TSQ 700. The retention by sulfhydryl reagents has long been known with plant
index (RI) was calculated by a set of hydrocarbons (even numbered polyketide synthases (Kreuzaler and Hahlbrock 1975).
C12 to C28) by linear interpolation.
The pH and temperature optima were at 6.5 and 35 C,
respectively. The most efficient substrate for the enzyme
was benzoyl-CoA (Table 2). This starter molecule
Results specificity agreed with the observed pattern of biphenyls
in challenged cell cultures, which contained mainly
Detection of aucuparin aucuparin that has an unsubstituted aromatic ring.
Relatively high activity was also observed with 2-hy-
Yeast-extract-treated cell cultures of S. aucuparia con- droxybenzoyl-CoA but the product formed was, by
tained a prominent constituent that was not detectable comparison with the literature data (Morita et al. 2001),
in control cells, as shown by TLC and GC–MS (Fig. 1). 2-hydroxybenzoyltriacetic acid lactone. As reported
The UV and mass spectra of the elicited compound
agreed with those of aucuparin (Erdtman et al. 1963).
Based on mass-spectroscopical homologies and litera- Table 1 Effect of DTT on biphenyl synthase activity in cell-free
ture data (Malterud and Sandanger Dugstad 1985), low extracts of yeast-extract-treated Sorbus aucuparia cell cultures
amounts of noraucuparin, the putative precursor of
aucuparin, and a hydroxyaucuparin were also detected. DTT concentration Biphenyl formation Side-product formation
(lM) (% of max.) (% of respective
Previously, fungus-challenged sapwood of S. aucuparia biphenyl amount)
has been found to accumulate, besides aucuparin,
2¢-hydroxyaucuparin and two methoxyaucuparins 0 29 0
(Kokubun et al. 1995). The aucuparin content in 10 34 21
cultured cells following yeast extract treatment was 20 39 28
40 69 31
approx. 1.2 mg l)1. The low biphenyl concentration in 80 91 43
the cell culture medium (0.17 mg l)1) was probably due 160 100a 70
to cell lysis. 320 84 98
a
Specific enzyme activity: 9.5 lkat (kg protein))1

Detection of biphenyl synthase

Incubation of cell-free extracts from yeast-extract- Table 2 Starter-substrate specificity of biphenyl synthase in cell-
treated S. aucuparia cell cultures with benzoyl-CoA and free extracts of yeast-extract-treatedS. aucuparia cell cultures
malonyl-CoA led to the formation of an enzymatic
product, as shown by HPLC analysis of the ethyl Substrate Relative enzyme activity (%)
acetate extracts from the enzyme assays. This finding Without With
was confirmed by TLC and subsequent autoradiogra- acidification acidificationa
phy of incubations containing [2-14C]malonyl-CoA. No
product formation occurred when heat-denatured Benzoyl-CoA 100b 100b
enzyme was used and either CoA ester was omitted. To 2-Hydroxybenzoyl-CoA 11 86
3-Hydroxybenzoyl-CoA 8 22
increase the product yield, the enzyme was enriched by Isobutyryl-CoA 5 15
ammonium sulphate precipitation. The 50–80% protein 4-Hydroxybenzoyl-CoA 0 0
fraction exhibited nearly five times the specific activity Cinnamoyl-CoA 0 0
of the crude extract (2.9 vs. 0.6 lkat kg)1) and was 2-Coumaroyl-CoA 0 0
3-Coumaroyl-CoA 0 0
used for further studies. The enzymatic product was 4-Coumaroyl-CoA 0 0
identified as 3,5-dihydroxybiphenyl by comparison with Caffeoyl-CoA 0 0
a sample of chemically synthesised reference com- Feruloyl-CoA 0 0
pound. Besides co-chromatography in HPLC and GC, Sinapoyl-CoA 0 0
the mass spectra of the enzymatic product and its a
Prior to extraction of products, the pH value of the enzyme assays
acetylated derivative matched those obtained with the was adjusted to 3.5
synthetic product. b
Specific enzyme activity: 2.9 lkat (kg protein))1

previously, the quantitative extraction of this derailment
product from the enzyme assays required acidification
(Akiyama et al. 1999). Similarly, incubations with the
starter molecules isobutyryl-CoA and 3-hydrox-
ybenzoyl-CoA led to the formation of the respective
diacetic acid lactones. All the side products observed
result from spontaneous lactonization of tri- and tet-
raketide intermediates that are released prematurely
from the active site (Suh et al. 2000). 4-Coumaroyl-CoA
and related cinnamic acid CoA esters, which are the
preferred starter molecules for chalcone synthase and
stilbene synthase (Schröder 1999), did not serve as sub-
strates.
Discussion
The formation of 3,5-dihydroxybiphenyl was detected
for the first time in yeast-extract-treated cell cultures of
Fig. 2 Reactions of biphenyl synthase (BIS) and benzophenone
synthase (BPS). Aucuparin results from subsequent peripheral
reactions
495
S. aucuparia. The enzyme involved will be termed
biphenyl synthase (BIS). The reaction mechanism
probably resembles that underlying stilbene formation
(Rupprich and Kindl 1978). After sequential condensa-
tion of benzoyl-CoA with three molecules of malonyl-
CoA, the resulting linear tetraketide intermediate
undergoes an intramolecular aldol condensation and
elimination of the terminal carboxyl group (Fig. 2).
Like benzophenone synthase (BPS), which has been
cloned and functionally expressed recently (Liu et al.
2003), BIS uses the same starter substrate for chain
extension. BPS, however, catalyses an intramolecular
Claisen condensation to cyclise the common tetraketide
intermediate (Fig. 2). A pair of enzymes that also cata-
lyse these two different ring closures are chalcone syn-
thase and stilbene synthase which, however, use 4-
coumaroyl-CoA as starter molecule, leading to the for-
mation of stilbenes and flavonoids, respectively (Schrö-
der 1999). Neither BIS nor BPS accepted CoA esters of
cinnamic acids but they were highly active with starter
substrates derived from benzoic acid metabolism.
Acknowledgements We thank the Deutsche Forschungsgemeins-
chaft for financial support (SPP 1152). Benye Liu is grateful to the
Deutscher Akademischer Austauschdienst for a postdoctoral fel-
lowship from the Biosciences Special Program.
References
Akiyama T, Shibuya M, Liu HM, Ebizuka Y (1999) p-Couma-
royltriacetic acid synthase, a new homologue of chalcone
synthase, from Hydrangea macrophylla var. thunbergii. Eur
J Biochem 263:834–839
Borejsza-Wysocki W, Lester C, Attygalle AB, Hrazdina G (1999)
Elicited cell suspension cultures of apple (Malus · domestica)
cv. Liberty produce biphenyl phytoalexins. Phytochemistry
50:231–235
Dixon RA (2001) Natural products and plant disease resistance.
Nature 411:843–847
Dol GC, Kamer PCJ, van Leeuwen PWNM (1998) Synthesis of 5-
substituted resorcinol derivatives via cross-coupling reactions.
Eur J Org Chem 2:359–364
Erdtman H, Eriksson G, Norin T (1963) Aucuparin and meth-
oxyaucuparin, two phenolic biphenyl derivatives from the
heartwood of Sorbus aucuparia L. Acta Chem Scand 17:1151–
1156
Kokubun T, Harborne JB (1994) A survey of phytoalexin induc-
tion in leaves of the Rosaceae by copper ions. Z Naturforsch
Teil C 49: 628–634
Kokubun T, Harborne JB (1995) Phytoalexin induction in the
sapwood of plants of the Maloideae (Rosaceae): biphenyls or
dibenzofurans. Phytochemistry 40:1649–1654
Kokubun T, Harborne JB, Eagles J, Waterman PG (1995) Anti-
fungal biphenyl compounds are the phytoalexins of the sap-
wood of Sorbus aucuparia. Phytochemistry 40:57–59
Kreuzaler F, Hahlbrock K (1975) Enzymatic synthesis of aromatic
compounds in higher plants. Formation of bis-noryangonin (4-
hydroxy-6[4-hydroxystyryl]2-pyrone) from p-coumaroyl-CoA
and malonyl-CoA. Arch Biochem Biophys 169:84–90
Linsmaier EM, Skoog F (1965) Organic growth factor require-
ments of tobacco tissue cultures. Physiol Plant 18:100–127
Liu B, Falkenstein-Paul H, Schmidt W, Beerhues L (2003) Ben-
zophenone synthase and chalcone synthase from Hypericum
androsaemum cell cultures: cDNA cloning, functional expres-
sion, and site-directed mutagenesis of two polyketide synthases.
Plant J 34:847–855
496
Malterud KE, Sandanger Dugstad EK (1985) 4,2¢-Dihydroxy-3,5-
dimethoxybiphenyl, a new phenol from the wood of Salix
caprea L. Z Naturforsch Teil B 40: 853–854
Morita H, Noguchi H, Schröder J, Abe I (2001) Novel polyketides
synthesized with a higher plant stilbene synthase. Eur J Bio-
chem 268:3759–3766
Nilsson B, Norin T (1963) Syntheses of aucuparin and meth-
oxyaucuparin. Acta Chem Scand 17:1157–1159
Rupprich N, Kindl H (1978) Stilbene synthases and
stilbenecarboxylate synthases I: enzymatic synthesis of 3,5,4¢-
trihydroxyxstilbene from p-coumaroyl coenzyme A and
malonyl coenzyme A. Hoppe-Seyler
s Z Physiol Chem
359:165–172
Schröder J (1999) The chalcone/stilbene synthase-type family of
condensing enzymes. In: Sankawa U (ed) Comprehensive natu-
ral products chemistry, vol 1. Elsevier, Amsterdam, pp 749–771
Suh DY, Fukuma K, Kagami J, Yamazaki Y, Shibuya M, Ebizuka
Y, Sankawa U (2000) Identification of amino acid residues
important in the cyclization reactions of chalcone and stilbene
synthases. Biochem J 350:229–235
Sultanbawa MUS (1980) Xanthonoids of tropical plants. Tetra-
hedron 36:1465–1506
Widyastuti SM, Nonaka F, Watanabe K, Sako N, Tanaka K
(1992) Isolation and characterization of two aucuparin-related
phytoalexins from Photinia glabra. Ann Phytopathol Soc Japan
58:228–233