A Versatile Microbial System for Biosynthesis of Novel Polyphenols with Altered Estrogen Receptor Binding Activity

Article abstract of DOI:10.1016/j.chembiol.2010.03.010

Isoflavonoids possess enormous potential for human health with potential impact on heart disease and cancer, and some display striking affinities for steroid receptors. Synthesized primarily by legumes, isoflavonoids are present in low and variable abundance within complex mixtures, complicating efforts to assess their clinical potential. To satisfy the need for controlled, efficient, and flexible biosynthesis of isoflavonoids, a three-enzyme system has been constructed in yeast that can convert natural and synthetic flavanones into their corresponding isoflavones in practical quantities. Based on the determination of the substrate requirements of isoflavone synthase, a series of natural and nonnatural isoflavones were prepared and their binding affinities for the human estrogen receptors (ERα and ERβ) were determined. Structure activity relationships are suggested based on changes to binding affinities related to small variations on the isoflavone structure.

Full text of DOI:10.1016/j.chembiol.2010.03.010

Chemistry & Biology
Article
A Versatile Microbial System for Biosynthesis
of Novel Polyphenols with Altered Estrogen
Receptor Binding Activity
Joseph A. Chemler,1,2 Chin Giaw Lim,1 John L. Daiss,2 and Mattheos A.G. Koffas1,
*
1Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA
2First Wave Technologies, Inc., Center of Excellence, Buffalo, NY 14203, USA
*Correspondence: mkoffas@eng.buffalo.edu
DOI 10.1016/j.chembiol.2010.03.010
SUMMARY
numerous developmental and biological interactions and the
precise response is determined by the interplay between
Isoflavonoids possess enormous potential for human estrogen, the estrogen receptors, and an array of modifying
factors (Harris et al., 2005; Leung et al., 2006). Among the
health with potential impact on heart disease and
goals of investigators looking for estrogen analogs has been
the discovery of molecules, referred to as selective estrogen
response modifiers or SERMs (Setchell, 2001), that elicit some
cancer, and some display striking affinities for steroid
receptors. Synthesized primarily by legumes, isofla-
vonoids are present in low and variable abundance
within complex mixtures, complicating efforts to
assess their clinical potential. To satisfy the need
for controlled, efficient, and flexible biosynthesis
of isoflavonoids, a three-enzyme system has been
of estrogen’s effects but not others (Hollmer, 2006). Based on
their striking affinities for the estrogen and other steroid recep-
tors, the isoflavonoids are a potential class of drugs for modifying
estrogen action and metabolism.
The abundance of isoflavonoids from plants is generally low
constructed in yeast that can convert natural and
synthetic flavanones into their corresponding isofla-
and is determined by a host of environmental factors that vary
from year to year and locale to locale, to say nothing of the addi-
vones in practical quantities. Based on the determi- tional variability introduced in the supply chain from field to
processor to distributor to consumer (Mortensen et al., 2009).
In addition, the ‘‘active’’ isoflavonoids are present in undefined
nation of the substrate requirements of isoflavone
synthase, a series of natural and nonnatural isofla-
vones were prepared and their binding affinities for
the human estrogen receptors (ERa and ERb) were
mixtures and, in some natural extracts, components have mutu-
ally off-setting effects (Liew et al., 2003; Liu et al., 2007; Meezan
et al., 2005).
determined. Structure activity relationships are sug-
Chemical synthesis is certainly the preferred method for
gested based on changes to binding affinities related
making most isoflavonoids. However, for compounds with
to small variations on the isoflavone structure.
multiple similar substitution sites like polyphenols or saccha-
rides, partial biosynthesis with natural or mutated enzymes
provides a means for the production of more defined com-
INTRODUCTION
pounds in improved yield. The incorporation of natural and
hybrid biosynthetic pathways into microorganisms has created
Among the classes of plant compounds that exhibit potentially the potential for not only the defined biosynthesis of a multitude
important biological activities are the isoflavonoids, three-ringed of natural products but also the opportunity to generate nonnat-
compounds originating from the condensation of malonyl-CoA ural derivatives with potentially novel or refined pharmaceutical
and p-coumaric acid by a type III polyketide synthase (Chemler activities (Minami et al., 2008; Willits et al., 2004). The ability to
et al., 2008). Isoflavones have been demonstrated to have an do what amounts to combinatorial chemistry in microorganisms
impressive array of pharmacological activities and potential by using a variety of natural or nonnatural precursors and dif-
benefits for human health in multiple areas including cardiovas- ferent combinations of modifying enzymes has opened the
cular disease (Siow et al., 2007; Squadrito et al., 2003), cancer possibility of creating new compounds and at the same time
(Yamamoto et al., 2003), and other potential health-promoting inventing the method for their synthesis (Challis and Hopwood,
activities (Liu et al., 2006; Rasbach and Schnellmann, 2008; 2007; Chemler et al., 2007; Katsuyama et al., 2007a; Straathof
Zhao and Brinton, 2007) although some occur at concentrations et al., 2002).
unlikely to be attained from diet.
Presented here is an approach that combines organic syn-
One particularly striking group of interactions is that of certain thesis and metabolic engineering for the synthesis of natural
isoflavones with steroid receptors (Ji et al., 2006; McCarty, and nonnatural isoflavones in practical quantities in Saccharo-
2006), most famously, the 100 nM interaction of genistein with myces cerevisiae. Based on the essential determination of the
human estrogen receptor a (hERa) and the even more striking substrate requirements of the key enzyme, isoflavone synthase
10 nM affinity of genistein for human estrogen receptor b (hERb) (IFS), this approach is the initial step in the construction of
(Zhao and Brinton, 2005). Estrogen receptors are involved in a system for the potential synthesis of thousands of natural
392 Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Figure 1. Genistein Production Rates from
Naringenin in S. cerevisiae Shake Flask
Cultures
Rates are from the second day of fermentation.
Gm, G. max (soy bean); Tp, T. pratense (red
clover); Ge, G. enchinata (licorice); Mt, M. trunca-
tula (alfalfa); Ps, P. sativum (pea); Sc, S. cerevisiae
(yeast); Cr, C. roseus (Madagascar periwinkle).
Standard deviations are from triplicates.
substrate flexibility and interaction with
coexpressed CPR (Kim et al., 2005), Gly-
cyrrhiza echinata (licorice) because of its
reported productivity in yeast (Kat-
suyama et al., 2007b), and Pisum sativum
(pea) and Medicago truncatula (alfalfa)
because of modest sequence differences
near the putative catalytic site.
The five IFS genes were cloned sepa-
rately into pYES2.1 yeast expression
vector under the control of the GAL1
promoter and transformed in yeast strain
INVSc1, where the necessary NADPH-
derived reducing equivalents would be
provided by the endogenous CPR. Each
strain was then grown in selective mini-
mal medium with galactose as an inducer
and nonnatural isoflavonoids. The pathway for the biosynthesis and carbon source and supplemented with naringenin (Fig-
of the isoflavonoids begins with flavanones, which are converted ure 1A). Conversion of naringenin to genistein was monitored
to isoflavones by the combined action of three enzymes: IFS over 7 days with peak conversion rate observed on the second
(Jung et al., 2000; Kim et al., 2003; Veitch, 2007), 2-hydroxyiso- day. Presented in Figure 1B, maximum observed production of
flavanone dehydratase (HID) (Akashi et al., 2005), and the cyto- genistein by the cloned and expressed IFS genes was ranked
chrome P450 reductase (CPR) (Kim et al., 2005; Seeger et al., as follows: T. pratense > P. sativum > G. max R G. echinata >
2003). The expression and interaction of these three plant M. truncatula. The IFS from T. pratense was selected as the
enzymes in S. cerevisiae enabled the biosynthesis of natural best candidate to continue work toward improving isoflavone
and nonnatural isoflavones that displayed a spectrum of interac- yields.
tions with ERa and ERb.
Conversion of Naringenin to Genistein by IFS
RESULTS AND DISCUSSION
Is Dependent on the Source of the Supporting CPR
NADPH-derived reducing equivalents are transferred from CPR
to a recipient P450 enzyme like IFS by transient, direct contact
between the two membrane-bound enzymes. Interestingly, mul-
tiple eukaryotic microsomal P450 enzymes are supported by
Among Five IFS Genes Tested, Trifolium pratense IFS
Had the Highest Observed Activity When Expressed
in S. cerevisiae
A primary objective was the construction of a yeast strain that a single endogenous CPR, which suggests a universal electron
would efficiently convert a diverse array of flavanone precursors transfer mechanism conserved among higher order species
into natural and nonnatural isoflavones. The central enzyme in allowing the possibility of a CPR to support heterogeneous P450
this process is IFS and only limited data is available on the two enzymes. However, the source of the CPR can influence the effi-
key selection criteria: relative conversion rates and substrate ciency of the coupled redox reaction as seen with P450s of plant
flexibility. In order to find the best natural enzyme, sequences origin (Jennewein et al., 2005; Pompon et al., 1996; Ponnamper-
from the approximately 25 known legume-derived IFS proteins uma andCroteau, 1996) and even the stereochemistry ofthe reac-
were compared. The IFS proteins were found to be highly cons- tion (Kraus and Kutchan, 1995). Using microsomes prepared from
erved; overall sequence identity among these proteins exceeded yeast coexpressing T. pratense IFS and rice CPR, genistein
80%. Consequently, the search for the best IFS was directed production from naringenin increased 4.3-fold (Kim et al., 2005).
toward those that had in some way been distinguished in the Based on these examples, it was of interest to clone NADPH-
literature: Glycine max (soy bean) because of the high levels of dependent CPRsfrom differentsourcesandcomparetheproduc-
isoflavones in soy beans and because of prior experience with tion rates of genistein when overexpressed with the different IFSs.
chimeric soy IFS (Leonard and Koffas, 2007; Tian and Dixon,
To examine the ability of particular CPRs to enhance IFS
2006), Trifolium pratense (red clover) because of reported activity in vivo, the CPR genes from Catharanthus roseus
Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved 393

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
(Madagascar periwinkle), G. max, and S. cerevisiae were coex- coexpressed with either G. max or G. echinata HID in yeast
pressed in S. cerevisiae cells with either IFS from G. max or cultures and the concentration of genistein was measured for
T. pratense (the sequence for the CPR from T. pratense was up to 7 days. For yeast cultures expressing T. pratense IFS,
not available). The CPRs from G. max and C. roseus have the coexpression of G. max HID led to a 75% increase in the max-
66.2% protein identity with each other but only about 30% imum genistein production rate; a 25% increase was observed
protein identity with the CPR from yeast. CPR activity, the trans- when G. echinata HID was expressed. For yeast cultures coex-
fer of electrons from NADPH to the P450 monooxygenase, was pressing G. max IFS and G. max HID, a 2-fold increase in the
determined indirectly by measuring the concentration of genis- maximum production rate was seen while coexpression of
tein produced by the recombinant yeast fed with naringenin G. max IFS with G. echinata HID had no effect on genistein
and cultured over 7 days.
production rate (Figure 1D). These results demonstrate that the
The maximum production rates were then compared to G. max HID augments IFS activity and hence was selected for
yeast strains expressing only IFS supported by the endogenous construction of the isoflavone production pathway.
yeast CPR (Figure 1C). Coexpression of IFS and plant CPR
in yeast cells led to a significant increase in the production rate. Reconstructing the Entire Isoflavone Biosynthesis
For example, the rate of genistein production increased by over System Is the Key for Maximum Production
50% to 15 mg/liter/day, in yeast coexpressing T. pratense IFS In order to reconstitute the optimal functional isoflavone syn-
and G. max CPR. Coexpression of T. pratense IFS with the CPR thesis system within yeast cell cultures, the G. max and T. pra-
from C. roseus or S. cerevisiae had no significant effect on tense IFS genes were coexpressed with the available CPR
production rates compared to control. When G. max IFS was and HID genes. The hypothesis was that the complete system
coexpressed with the CPR from G. max or C. roseus, maximum would have a synergistic effect on the production of genistein
product formation rates increased to 6.5 mg/liter/day, a 4-fold in yeast. HID and CPR genes under separate GAL1 promoters
rate increase over expression of G. max IFS alone. In contrast, were cloned onto yEPlac112; IFS was present on a separate
overexpression of yeast CPR with G. max IFS did not improve pYES2.1 vector. The yeast strains were constructed by trans-
the genistein production rate. These results suggest optimal pair- forming both vectors into INVSc1 yeast cells sequentially and
ing of proteins is crucial for an efficient redox reaction.
the vectors were maintained on selective minimal SC medium.
Coexpression of the best single and double enzyme combina-
tions led to the construction of the yeast strain coexpressing
HID from G. max Enhances Genistein Production Rate
The conversion of 2-hydroxyisoflavanones to isoflavones can T. pratense IFS, G. max CPR, and G. max HID. This construct
proceed spontaneously (Hashim et al., 1990) but in plants it is improved genistein production rate by 15% compared to the
facilitated by the enzyme HID. Only two HID enzymes have coexpression of G. max IFS with G. max HID, by 25% compared
been characterized and share 60.1% protein identity. The HID to T. pratense IFS with G. max CPR, and by 200% compared to
from G. max displays a kinetic bias toward 2-hydroxyisoflava- expression of T. pratense IFS alone (Figure 1E). The combination
nones with a hydroxyl group at the C40 position while HID from of T. pratense IFS, G. max HID, and C. roseus CPR provided no
G. echinata has a higher activity toward substrates that bear improvement and was equivalent to the yeast strain coexpress-
a C40 methoxy substituent (Akashi et al., 1999, 2005).
ing only T. pratense IFS and C. roseus CPR.
The cognate combination of G. max IFS, CPR, and HID yielded
Demonstrations of the requirement for HID have been equiv-
ocal. In one attempt to reconstruct the isoflavone pathway in significant improvements in genistein production rate over any
hairy root cultures of the legume Lotus japonicus, the heterolo- other combinations with the G. max IFS. The three-enzyme
gous expression of G. echinata HID was determined to be a crit- combination was 50% more active compared to just IFS and
ical determinant of the accumulation of intracellular isoflavones CPR, three times higher than IFS with only HID, and over ten
even though the concentration was modest (less than 0.5 mg/ times faster than IFS alone. Despite different initial rates of the
liter) (Shimamura et al., 2007). In contrast, the expression of an strains, the final concentrations of genistein all maximized to
artificial chimera of IFS and CPR in E. coli yielded 4 mg/liter of approximately the same level of about 35 mg/liter (Figure 2).
genistein without an HID (Leonard and Koffas, 2007). In con-
struction of an isoflavone-producing system, it is essential to IFS Activity Is Substantially Inhibited by Genistein
know if the coexpression of HID will increase productivity or and Biochanin A
yield.
One phenomenon observed during fermentations was the con-
To determine whether HID could improve genistein production sistent decrease in genistein production rate over time and
activity in vitro, the soluble protein from yeast cells solely an apparent concentration ‘‘ceiling’’ noted above. The fall in
expressing G. max HID was incubated with microsomes pre- productivity cannot be accounted for by genistein’s low solubility
pared from yeast expressing only T. pratense IFS. The rate of or by consumption of the precursor naringenin. To determine
consumption of naringenin was monitored by HPLC and whether isoflavones like genistein act as product inhibitors of
compared to a control with soluble protein prepared from yeast IFS, yeast microsomes containing T. pratense IFS were incu-
harboring an empty vector. The reaction containing HID bated with a fixed concentration of naringenin and different
increased IFS rate of conversion of naringenin by 45% over concentrations of either genistein, biochanin A, or genistin.
that of microsomes with IFS coincubated with the yeast soluble Similar studies were performed on microsomes prepared con-
protein devoid of HID. taining the other IFSs but the consumption rates were too slow
The ability of HID to improve genistein production rate by IFS to measure accurately. The consumption rate of naringenin
was then examined in vivo. IFSs from T. pratense or G. max were was monitored over 30 min and the rates were calculated as
394 Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Figure 2. Genistein Production Curves from S. cerevisiae Coex-
pressing IFS, CPR, and HID
d: T. pratense IFS, G. max HID, G. max CPR. -: T. pratense IFS, G. max HID,
C. roseus CPR. >: G. max IFS, G. max HID, G. max CPR. 6: G. max IFS,
G. max HID, C. roseus CPR. Standard deviations are from triplicates.
a percentage of the maximum consumption rate when no inhib-
itor was present. When genistein was initially added, significant
inhibition was observed across three orders of magnitude
including a 34% decrease in IFS activity in the presence of
2.5 mM of genistein (Figure 3). An almost identical inhibition
profile was observed when the inhibitor was biochanin A (40-me-
thoxygenistein). In contrast, 0.25 mM genistin (genistein 7-O-
glucoside) exhibited no inhibition of the conversion of naringenin.
Based on these results it appears that binding to IFS is depen-
dent on the presence of an unmodified hydroxyl group at the
C7 position. The majority of isoflavonoids are accumulated in
the form of their glyco- and malonylconjugates in soybean
seeds. Acylation and glycosylation increases isoflavone solu-
bility and stability while enabling compartmentalization in
soy seeds (Heller and Forkmann, 1994). Isoflavone-modifying
enzymes such as glycosyltransferases and methyltransferases
(Dhaubhadel et al., 2008) may also alleviate product inhibition
of IFS by genistein.
Figure 3. Isoflavone Inhibition of the Conversion of 0.25 mM Narin-
genin by IFS
0.0025, 0.025, and 0.25 mM genistein are equivalent to 0.675, 6.75, and
67.5 mg/liter, respectively. Standard deviations are from duplicates.
isoflavones are further differentiated by single or multiple substi-
tutions on the B ring so the library was designed to consist of
flavanones with a variety of hydroxyl, fluoro, chloro, bromo,
methyl, methoxy, and ethoxy substituents at various positions
on the B ring. In total, 7 natural and 19 nonnatural flavanones
were available for assessment of IFS substrate flexibility.
Microsomes bearing the membrane-bound IFS along with the
endogenous S. cerevisiae CPR were prepared and incubated
with each flavanone for 24 hr and the reactions were analyzed
by reverse-phase HPLC. New products were identified in the
elution profiles by comparison with reactions performed with
yeast microsomes that lacked any IFS. Each IFS displayed the
same convertible substrate profile and no diversity in substrate
flexibility was observed. While this might be expected from the
high degree of sequence conservation, the practical conse-
quence is that the range of convertible substrates cannot be
expanded by exploiting natural variations among this ensemble
of IFSs. However, distinct criteria were observed that dictated
whether a flavanone was accepted as a substrate.
All Five IFS Enzymes Have the Same Substrate Flexibility
The second key selection criterion for IFS enzymes was
substrate flexibility. In principle, system substrate flexibility can
be maximized by combining IFS enzymes that accept different
subsets of flavanones as precursors. To determine whether
IFSs from different species had different substrate flexibility,
the five IFSs cloned into yeast were challenged against the
library of natural and nonnatural flavanones (Table 1 and Substrate Requirements for IFS Enzymes
Figure 4).
While the panel of potential flavanone substrates is not compre-
Flavanones were purchased when possible but additional hensive, a few suggestions about IFS substrate requirements
flavanones were synthesized by reacting an appropriate aceta- can be made from a survey of those flavanones that were
phenone and benzaldehyde to form a chalcone that was subse- acceptable substrates. First, a hydroxyl group at the C7 position
quently cyclized to yield the corresponding flavanone (see is required to bind to the IFS as suggested by the conversion of
Supplemental Information available online) (Huang et al., 1999; 7-hydroxyflavanone but not flavanone itself. Second, hydroxyl-
Urgaonkar et al., 2005). Flavanones were designed to mimic ation at the C5 position is not necessary as the synthesis of
plant-derived flavanones and isoflavones. For example, all both genistein and daidzein proceeds without the need for an
natural flavonoids are hydroxylated at the C7 position of the intervening genistein dehydratase. Third, substitutions at the
A ring while the C5 position may or may not be hydroxylated. C20 or C60 position (OH and Cl) were not tolerated, possibly
The resulting library of flavanones had many 7-monohydroxy- due to interference of the hydroxylation reaction at the C2 posi-
lated and 5,7-dihydroxylated members. Natural flavanones and tion. Fourth, substitutions at the C30 and/or C50 position (OH, F,
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Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Table 1. Biotransformations of Flavanones by the Isoflavone Pathway
R1
5
R2
20
H
H
H
H
H
H
H
Flavanone
7
30
40
50
H
H
H
H
H
H
H
H
H
H
H
Primary biotransformation product
Flavanone (1)
H
H
H
H
H
NR
7-Hydroxyflavanone (2)
Pinocembrin (3)
OH
H
H
3,7-Dihydroxydihydroflavonol (D1)
3,5,7-Trihydroxydihydroflavonol (D2)
Genistein (28)
OH OH
OH OH
H
H
Naringenin (4)
H
OH
OH
OH
OH
OH
OCH3
OH
OH
OH
OH
Liquiritigenin (5)
H
OH
OH OH
OH
H
Diadzein (29)
Eriodictyol (6)
Butina (7)
20,40,5,7-Tetrahydroxyflavanonea (8)
OH
OH
H
Orobol (30)
30,40,7-Trihydroxyisoflavone (31)
H
OH OH OH
NR
Hesperitin (9)
OH OH
OH OH
H
H
H
H
H
H
H
H
H
H
H
H
H
F
OH
OCH3
OCH3
OCH3
OCH3
NR
30-Methoxy-40,5,7-trihydroxyisoflavone (32)
40,7-Dihydroxy-30-methoxyisoflavone (33)
Homoeriodictyol (10)
40,7-Dihydroxy-30-methoxyflavanonea (11)
H
OH
30,50-Dimethoxy-40,5,7-trihydroxyflavanonea (12) OH OH
OCH3 30,50-Dimethoxy-40,5,7-trihydroxyisoflavone (34)
OCH3 40,7-Dihydroxy-30,50-dimethoxyisoflavone (35)
40,7-Dihydroxy-30,50-dimethoxyflavanonea (13)
30-Ethoxy-40,5,7-trihydroxyflavanonea (14)
40,7-Dihydroxy-30-ethoxyflavanonea (15)
30-Methyl-40,5,7-trihydroxyflavanonea (16)
40,7-Dihydroxy-30-methylflavanonea (17)
30,50-Dimethyl-40,5,7-trihydroxyflavanonea (18)
40,7-Dihydroxy-30,50-dimethylflavanonea (19)
5,7-Dihydroxy-40-fluoroflavanonea (20)
5,7-Dihydroxy-30-fluoroflavanonea (21)
5,7-Dihydroxy-20-fluoroflavanonea (22)
40-Chloro-5,7-dihydroxyflavanonea (23)
30-Chloro-40,5,7-trihydroxyflavanonea (24)
30-Chloro-40,7-dihydroxyflavanonea (25)
30-Bromo-40,5,7-trihydroxyflavanonea (26)
30-Bromo-40,7-dihydroxyflavanonea (27)
H
OH
OH OH
OH
OH OH
OH
OH OH
OH
OCH2CH3 OH
OCH2CH3 OH
H
30-Ethoxy-40,5,7-trihydroxyflavanone (36)
40,7-Dihydroxy-30-ethoxyflavanone (37)
30-Methyl-40,5,7-trihydroxyisoflavone (38)
40,7-Dihydroxy-30-methylisoflavone (39)
30,50-Dimethyl-40,5,7-trihydroxyisoflavone (40)
40,7-Dihydroxy-30,50-dimethylisoflavone (41)
NR
H
H
CH3
CH3
CH3
CH3
H
OH
OH
OH
OH
F
H
H
H
CH3
CH3
H
H
OH OH
OH OH
OH OH
OH OH
OH OH
F
H
H
NR
H
H
H
NR
H
H
H
H
H
H
Cl
H
NR
Cl
OH
OH
OH
OH
H
30-Chloro-40,5,7-trihydroxyisoflavone (42)
30-Chloro-40,7-dihydroxyisoflavone (43)
30-Bromo-40,5,7-trihydroxyisoflavone (44)
30-Bromo-40,7-dihydroxyisoflavone (45)
H
OH
Cl
H
OH OH
OH OH
Br
H
Br
H
NR, no reaction.
a Chemically synthesized for this study.
Cl, CH3, OCH3, and OCH2CH3) were tolerated if the group 7-hydroxyflavanone and pinocembrin (5,7-dihydroxyflavanone),
was relatively small in size. Fifth, the presence of a C40 hydroxyl were converted by IFS but gave rise to corresponding dihydrox-
group was an absolute requirement for 2-hydroxyisoflavanone yflavonols (Table 1 and Figure 4B). Identical HPLC profiles
synthesis provided that all other criteria were met. Compounds were obtained when the same two substrates were incubated
lacking any substitution on the B ring, as is the case with with flavanone 3b-hydroxylase from Malus domestica, which
Figure 4. Biotransformations of Flavanones
by the Isoflavone Pathway
(A) Conversion of 40-hydroxyflavanones into isofla-
vones.
(B) Conversion of 7-hydroxyflavanone and pino-
cembrin into dihydroflavonols.
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Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
converts flavanones to dihydroxyflavonols (Chemler et al., 2007)
Along with six commercially available natural isoflavones and
(data not shown). The identification of dihydroflavonols corrects one flavanone, 12 compounds of the series produced as
a prior misinterpretation of substrate flexibility (Kim et al., 2003). described above were evaluated for their direct interactions for
Therefore, the C40 hydroxyl group appears to be a strict require- the human estrogen receptors ERa and ERb using commercially
ment for ring migration. All of these observations are consistent available in vitro competitive binding assays and a time-resolved
with a previous model of the mechanism of IFS (Hashim et al., fluorescence resonance energy transfer-capable microplate
1990).
reader (Figure 5). Only 13 out of 20 flavonoids showed binding
The screening of a substrate library against IFS opened up the toward ERa and only 30-bromo-40,5,7-trihydroxyisoflavone was
ability to reexamine the role of key flavanone features required equivalent to genistein for affinity for ERa (35 nM) (Table 2).
for catalytic activity and how they might interact with residues Only 16 out of 20 flavonoids showed binding toward ERb and
within the active site of the enzyme. No crystal structure of none had superior affinity than genistein for ERb (5 nM) (Table
IFS exists but previous docking experiments using homol- 2). All of the tested isoflavonoids had a higher selectivity (ERa
ogy modeling of CYP93C2 (G. enchinata IFS) derived from IC50/ERb IC50) for ERb, with the single exception of 30-chloro-
P450BM3 were performed to help identify key residues in the 40,7-dihydroxyisoflavone with an a/b value of 0.4. The selectivity
catalytic site (Sawada et al., 2002). Using site-directed mutagen- for ERb was greatest for daidzein, genistein, and orobol with a/b
esis, residues Ser 310 and Lys 375 were identified to play roles in values greater than 5. Compounds such as 30-chloro-40,5,7-tri-
the unique aryl migration from the C2 to the C3 position (Sawada hydroxyisoflavone, 30-methyl-40,5,7-trihydroxyisoflavone, 40,7-
et al., 2002). Modification of Lys 375 led to complete abolition of dihydroxy-30-methylisoflavone, 30,40,7-trihydroxyisoflavone, and
2-hydroxyisoflavanone synthesis and resulted in the exclusive biochanin A also exhibited slight preference for ERb with a/b
formation of a dihydroflavonol and/or flavone depending on values just over 2.
additional mutations. A follow up study identified Leu 371 to
The presence of a hydroxyl group at the C5 position signifi-
play an important role in not only catalytic activity but protein cantly enhanced binding affinity for both ERa and ERb. Eight
stability (Sawada and Ayabe, 2005). Both these studies pro- isoflavone pairs differing by only the presence or lack of the
posed that Lys 375 is an absolute requirement for aryl ring C5 hydroxyl group were compared. The genistein analogs
migration and put forth a model that this residue interacts with (with a C5 hydroxyl group) had at least a 4-fold lower IC50 value
the C7 hydroxyl group on the flavanone substrate (Sawada and for ERb than their daidzein counterparts, which lacked a C5
Ayabe, 2005). The screening library used in this study identified hydroxyl group. The same pattern was observed with ERa but
the necessity of a C40 hydroxyl group in order to obtain an isofla- to an even greater degree. On average, the C5 hydroxyl group
vone, and with substrates lacking B ring substituents (7-hydroxy- reduced IC50 values by at least one order of magnitude.
flavanone and pinocembrin) only the respective dihydroflavo- Another trend observed was that substituents in the C30
nols were produced. With these two observations coupled with position generally reduced affinity for both ERa and ERb as
the previous site mutagenesis results, we propose an alternative an inverse to their relative bulk. For genistein analogs, the
role of Lys 375 as the residue directly involved in aryl ring migra- substitution groups with decreasing affinity toward ERa went
tion. That is, Lys 375 binds to a flavanone’s C40 hydroxyl group as follows: Br > H > OH = CH3 > Cl > OCH3 > OCH2CH3. For
and possibly contributes to the shift reaction of the B ring from daidzein analogs, it went as follows: Cl > H > CH3 > OH >
the C2 to the C3 position. In addition, the hydroxyl group at the Br > OCH3 > OCH2CH3. For genistein analogs, the substitution
C7 position appears to be necessary for binding to the catalytic groups with decreasing affinity toward ERb went as follows:
site since no product was observed when flavanone was the H > OH = Br > CH3 = Cl > OCH3 > OCH2CH3. For daidzein
substrate. A more likely residue for the C7 hydroxyl group to analogs, it went as follows: H > CH3 > OH = Cl > OCH3
=
bind to would then be the Ser 310 as the S310T CYP93C2 mutant Br > OCH2CH3. The next generation of isoflavonoids designed
lowered but did not abolish 2-hydroxyisoflavanone production to interact with the estrogen receptors should have small sub-
(Sawada et al., 2002). All of these observations, taken together, stituents at the C30 position.
may guide helpful modifications to a computer-based model of
IFS: substrate interactions (Sawada and Ayabe, 2005).
Modifications of hydroxyl groups give rise to the numerous
variations of isoflavones found in plant extracts. For instance,
the bulk of soy isoflavones are glycosylated (e.g., genistin) and
red clover isoflavones are primarily methylated (e.g., biochanin
A, prunetin, and formononetin). In either case, modifications of
New Guidelines Identified for the Design of Isoflavone
as Potent Estrogen Receptor Agonists
Among the attributes of the soy isoflavones and their derivatives, the hydroxyl group at either the C7 or C40 position greatly
such as equol, is their remarkably high affinity for the human reduced their affinity to either estrogen receptor and increased
estrogen receptors (Choi et al., 2008; Davis et al., 2008; Pinto the IC50 values by at least a factor of ten compared to genistein.
et al., 2008; Zhao and Brinton, 2005). Many of the most prom-
Our understanding of the structure relationship between
ising benefits of isoflavones in general and genistein in particular isoflavones and estrogen receptors has benefited from the
appear to be mediated through interaction with ERb while extensive research on genistein and daidzein. Most notable is
certain of the less encouraging effects take place through ERa that genistein is more estrogenic than daidzein, indicating the
(McCarty, 2006). Further, the observations that estrogen- importance of the C5 hydroxyl group to enhance binding (Fang
replacement therapy promotes both cancer and chronic heart et al., 2001; Miksicek, 1994). After screening a number of natural
fatigue have led to an intensive search for compounds that flavonoids, two QSAR models were proposed to describe the
promote the benefits of estrogen replacement therapy while ERa and ERb individually (Choi et al., 2008). The expanded
reducing some of its worrisome side effects (Hollmer, 2006).
substrate library tested here included novel isoflavones, thereby
Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved 397

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Figure 5. Binding Affinities for ERa and ERb of Isoflavones
(A and B) d: estradiol, 6: 28, ,: 29, B: 30, >: 31.
(C and D) 6: 32, ,: 33, B: 42, >: 44. Standard deviations are from duplicates.
broadening the base of knowledge of the interactions between IFS mechanism was gained after screening the enzyme
phytoestrogens and estrogen receptors. against a library of synthetic flavanone precursors. The IFS
This study confirms the phytoestrogenic character of isofla- system was able to produce 4 natural isoflavones and 14
vones and their remarkable interaction with estrogen receptors unnatural isoflavone analogs. Several isoflavones produced
at nanomolar levels. In particular, the rare natural compound by this system showed significant binding affinities to
orobol and the unnatural 30-bromo-40,5,7-trihydroxyflavone human estrogen receptors, particularly the b isoform. Struc-
exhibited binding capabilities equivalent to that of genistein. ture-activity relationships between the isoflavone structure
The results obtained from the screening library demonstrate and the estrogen receptors were determined. The present
that estrogen receptor binding is strongly increased by hydroxyl study furthers the development of mutasynthesis of natural
groups at positions C40, C5, and C7 and that C30 substituents product analogs, particularly for systems involving cyto-
generally reduce the affinity. With these guidelines, a new series chrome P450 enzymes and their tailoring enzymes. Various
of natural and nonnatural isoflavones can be prepared with the enzymes of plant and microbial origin can be readily incor-
aim of making compounds more specific than genistein for porated together but identification of the most active
ERa and ERb.
enzymes can lead to improved production rates and yields.
The biosynthesis of libraries of natural products and ana-
logs then allows the ability to screen for relevant biological
activities.
SIGNIFICANCE
Isoflavones have been demonstrated to have an impressive
array of pharmacological activities and potential benefits for
human health in multiple areas including cardiovascular
disease and cancer. Therefore, the establishment of an effi-
cient production platform is desired. This paper describes
the construction of an optimized three-enzyme system in
S. cerevisiae for the production of isoflavones. A number
of genes from various plant sources were screened for the
most active enzyme and enzyme combinations resulting in
a final construct with synergistic properties capable of
high isoflavone yields. Additionally, further insight into the
EXPERIMENTAL PROCEDURES
Materials
Estradiol, glucose-6-phosphate, NADPH, and all materials for the chemical
synthesis of flavanones were purchased from Sigma-Aldrich (see Supple-
mental Experimental Procedures for flavanone reaction details). Genistein,
daidzein, biochanin A, prunetin, 30,40,7-trihydroxyisoflavone, genistin, flava-
none, 7-hydroxyflavanone, pinocembrin, liquiritigenin, naringenin, eriodictyol,
butin, homoeriodictyol, and hesperitin were purchased from Indofine. Mate-
rials for the LanthaScreen estrogen receptor assays were obtained directly
from Invitrogen.
398 Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Table 2. Estrogen Receptor IC50 Values of Natural and Unnatural Isoflavones
Ligand
ERa IC50 (nM)
0.1 0.0
ERb IC50 (nM)
0.2 0.1
a/b
0.4
6.4
18.5
4.3
NA
2.0
NA
NA
NA
2.2
7.6
NA
NA
2.6
0.5
1.7
NA
NA
1.4
3.0
1.8
Estradiol
Genistein (28)
34.8 12.0
485.4 145.4
70.8 10.5
>1000
5.4 0.6
Daidzein (29)
26.3 6.7
16.5 5.6
187 96.4
449.8 74.0
706.4 153.9
>1000
Orobol (30)
30,40,7-Trihydroxyisoflavone (31)
30-Methoxy-40,5,7-trihydroxyisoflavone (32)
40,7-Dihydroxy-30-methoxyisoflavone (33)
30-Ethoxy-40,5,7-trihydroxyflavanone (36)
40,7-Dihydroxy-30-ethoxyflavanone (37)
30-Methyl-40,5,7-trihydroxyisoflavone (38)
40,7-Dihydroxy-30-methylisoflavone (39)
30,50-Dimethyl-40,5,7-trihydroxyisoflavone (40)
40,7-Dihydroxy-30,50-dimethylisoflavone (41)
30-Chloro-40,5,7-trihydroxyisoflavone (42)
30-Chloro-40,7-dihydroxyisoflavone (43)
30-Bromo-40,5,7-trihydroxyisoflavone (44)
30-Bromo-40,7-dihydroxyisoflavone (45)
Genistin (genistein-7-O-glucoside)
Prunetin (40,5-dihydroxy-7-methoxyisoflavone)
Biochantin A (5,7-dihydroxy-40-methoxyisoflavone)
Naringenin (4)
880.3 287.0
>1000
>1000
>1000
>1000
69.3 12.4
540.9 159.5
>1000
30.8 9.5
70.8 20.9
155.4 55.5
>1000
>1000
101.4 32.3
104.4 41.7
28.4 10.5
>1000
39.5 6.0
208.7 49.8
17.1 4.2
841.5 183.7
>1000
>1000
413.3 91.5
465.4 115.2
328.8 127.2
299.4 51.7
157.1 24.1
186.4 32.6
Standard deviation from duplicates. NA, not applicable.
Plasmids and Strains
and/or tryptophan. Yeast cells were then quantified by spectrophotometry
at 600 nm. Yeast cultures at final OD600 of 0.8 were used to inoculate 5 ml
of SC dropout media containing 2% galactose and supplemented with
1.0 mM of the flavanone substrate naringenin [equivalent to 0.5 mM (2S)-nar-
ingenin]. Cultures were incubated at 30ꢀC to produce genistein. For isofla-
vone production analysis, 150 ml of culture was harvested and frozen for later
analysis. The thawed cultures were centrifuged to remove cell debris and the
supernatant was analyzed by HPLC. Naringenin and genistein concentrations
were quantified from 50 ml of sample directly injected into an HPLC system
IFS from Glycine max (soy), Glycyrrhiza echinata (licorice), Medicago truncatula
(alfalfa), Pisum sativum (pea), and Trifolium pratense (red clover), HID from
Glycine max and Glycyrrhiza echinata, and CPR from Catharanthus roseus
(Madagascar periwinkle) and Glycine max were isolated from their respective
plants. First, the seeds were germinated in the dark at 30ꢀC in a growth
chamber to obtain etiolated seedlings. Seedlings were wounded either by
cutting small slits (ꢁ1 mm sections) or exposed to UV for 5 min. Incubation
in the dark continued for 6 hr before harvesting. Harvested plant tissues
were flash frozen using liquid nitrogen and stored at ꢂ70ꢀC until further use.
RNAs from plant tissue were isolated using the RNeasy Plant Mini Kit
(QIAGEN). cDNAs were obtained from the RNAs first by reverse transcription
and then by PCR using the SuperScript One-Step RT-PCR with Platinum
Taq System (Invitrogen). Primers included in the reaction were designed
based on their respective sequence in GenBank. CPR from S. cerevisiae
was obtained directly using PCR of DNA isolated from yeast strain INVSc1
using the DNeasy Blood and Tissue Kit (QIAGEN) accordingly. Once each
gene was obtained, they were cloned into the pYES2.1-TOPO vector (Invitro-
gen), downstream of the GAL1 promoter. E. coli strain TOP10F0 (Invitrogen)
was used for plasmid maintenance and manipulation. All genes were
sequenced and errors found were corrected using the QuickChange Site-
Directed Mutagenesis Kit (Stratagene). Each HID and each CPR under a
GAL1 promoter was then cloned separately into YEplac112 (ATCC). The
YEplac112 plasmid harboring HID from G. max was then used to clone sepa-
rately CPR from G. max and C. roseus with their own GAL1 promoter. pYES2.1
plasmids harboring an IFS transformed into S. cerevisiae strain INVSc1 (Invi-
trogen) or in combination with a YEplac112 plasmid harboring HID and/or
CPR. Positive transformants were selected by growth on uracil- and/or trypto-
phan-deficient SC yeast medium agar plates. A complete list of plasmids can
be found in Table S1.
(1100 series; Agilent) equipped with
a ZORBAX SB-18 column (5 mm,
4.6 3 150 mm) held at 25ꢀC and a diode array detector. The isocratic mobile
phase at a flow rate of 1 ml/min consisted of 50% water, 35% methanol, and
15% acetonitrile. The elution time of naringenin was 7.08 min and of genistein
was 8.20 min. Concentrations were determined using authentic standards;
naringenin was quantified at 290 nm while genistein was quantified at
260 nm.
Measurement of Inhibition of IFS by Isoflavones
Yeast microsomes from yeast harboring pYES2.1-TpIFS were prepared
according to the procedure previously described (Pompon et al., 1996), and
microsomal proteins were quantified colorimetrically (Protein Assay Dye
Reagent; Bio-Rad). Enzyme assays for determining the inhibition of the rate
at which IFS consumed naringenin by various isoflavones were performed at
25ꢀC in 2 ml vials at a final volume of 250 ml. Yeast microsomes (250 mg micro-
somal protein/reaction) were incubated with 0.0025, 0.025, and 0.25 mM
isoflavone (genistein, genistin, or biochanin A), 1 mM EDTA, and 0.25 mM nar-
ingenin in 50 mM Tris-HCl (pH 7.4). Nicotinamide cofactor recycle was per-
formed using 0.25 U/reaction of glucose-6-phophate dehydrogenase from
S. cerevisiae (Sigma-Aldrich) and 2.5 mM glucose-6-phosphate. Reactions
were initiated by adding 1 mM NADPH. Samples of 25 ml were taken every
10 min for 1 hr and injected directly for HPLC analysis. Percent inhibition of
the rate of consumption of naringenin was determined for three different isofla-
vone concentrations and compared to when no isoflavone was present
initially.
Production Curves of Genistein
Recombinant yeast cells were grown to saturation in 3 ml cultures for 2 days
at 30ꢀC on SC dropout media containing 2% glucose and lacking uracil
Chemistry & Biology 17, 392–401, April 23, 2010 ª2010 Elsevier Ltd All rights reserved 399

Chemistry & Biology
Nonnatural Isoflavonoids for Estrogen Receptors
Measurement of In Vitro Rate of Naringenin Consumption
of IFS with HID
Received: October 15, 2009
Revised: March 20, 2010
Accepted: March 24, 2010
Published: April 22, 2010
Soluble yeast protein from yeast harboring pYES2.1-GmHID was prepared
according to the procedure previously described (Pompon et al., 1996) but
the supernatant obtained following cell lysis was saved and the insoluble
proteins and cell debris discarded. The total soluble protein was quantified
colorimetrically (Protein Assay Dye Reagent). In vitro assays using yeast
microsomes containing IFS from T. pratense were performed as previously
described but 125 mg of soluble yeast protein containing HID from G. max
was added. Percent increase of the rate of consumption of naringenin was
determined and compared to when no HID was added.
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SUPPLEMENTAL INFORMATION
Supplemental Information includes two tables and Supplemental Experi-
mental Procedures and can be found with this article online at doi:10.1016/
j.chembiol.2010.03.010.
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