Optimization of the biosynthesis of b-ring ortho-hydroxy lated flavonoids using the 4-hydroxyphenylacetate 3-hydroxylase complex (Hpabc) of escherichia coli

Article abstract of DOI:10.3390/molecules26102919

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L^?1, an induction temperature of 28^?C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caf-feic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.

Full text of DOI:10.3390/molecules26102919

molecules
Article
Optimization of the Biosynthesis of B-Ring Ortho-Hydroxy
Lated Flavonoids Using the 4-Hydroxyphenylacetate
3
-Hydroxylase Complex (HpaBC) of Escherichia coli
Longji Wang 1 , Xiubing Ma
,2,†
1,2,†
, Haixiang Ruan , Yang Chen , Liping Gao , Ting Lei , Yan Li , Lin Gui ,
1,2
1
1
1
1
1
Lina Guo , Tao Xia and Yunsheng Wang 1,2,
1
2
*
1
School of Life Science, Anhui Agricultural University, Hefei 230036, China; wlj18326662118@163.com (L.W.);
8855112725@163.com (X.M.); 13645516418@163.com (H.R.); chenyangshouji2hao@126.com (Y.C.);
1
gaolp62@126.com (L.G.); 18755195565@163.com (T.L.); liyantea@126.com (Y.L.); lin.gui@anlongen.com (L.G.);
gln2570874851@163.com (L.G.)
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University,
Hefei 230036, China; xiatao62@126.com
2
*
Correspondence: wangyunsheng@ahau.edu.cn
†
These authors contributed equally to this work.
Abstract: Flavonoids are important plant metabolites that exhibit a wide range of physiological and
pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory,
antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and phar-
maceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo
production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed,
and the corresponding products were successfully detected by feeding naringenin to vector-carrying
strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity
was significantly affected. The optimal culture conditions were determined, including a substrate
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Wang, L.; Ma, X.; Ruan, H.;
Chen, Y.; Gao, L.; Lei, T.; Li, Y.; Gui, L.;
Guo, L.; Xia, T.; et al. Optimization of
the Biosynthesis of B-Ring
−1 ◦
Ortho-Hydroxy Lated Flavonoids
Using the 4-Hydroxyphenylacetate
concentration of 80 mg
time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying
strains fed naringin was up to 57.67 3.36%. The same strategy was used to produce catechin and caf-
feic acid, and the highest conversion efficiencies were 35.2 3.14 and 32.93 2.01%, respectively. In
·
L
, an induction temperature of 28 C, an M9 medium, and a substrate delay
3
-Hydroxylase Complex (HpaBC) of
Escherichia coli. Molecules 2021, 26,
919. https://doi.org/10.3390/
±
±
±
2
this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated
for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo
B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a
bacterial expression system.
molecules26102919
Academic Editor: Armando Zarrelli
Received: 29 March 2021
Accepted: 8 May 2021
Published: 14 May 2021
Keywords: B-ring ortho-hydroxylation; Escherichia coli; flavonoids; 4-hydroxyphenylacetate
3
-hydroxylase; biosynthesis
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
Flavonoids are naturally occurring important secondary metabolites predominantly
originating from plants and fungi, and they display diverse bioactivities and distinguished
application potential [ ]. Several studies have shown that these metabolites possess
1,2
significant pharmacological activities, such as antioxidant [
cinogenic [5] and antibacterial [6] properties.
Chemically, the skeletal structure of flavonoids has 15 carbons, which consists of two
phenyl rings (A and B) and a heterocyclic ring (C). Naturally, flavonoids are plant-derived
products and they are found in different parts of the plants, which comprise of six ma-
jor subgroups, including chalcones, flavones, flavonols, flavan-3-ols, anthocyanins and
3], antimutagenic [4], anticar-
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
proanthocyanins [
7]. A number of phenolic hydroxy groups of flavonoids are shown to
have antioxidant activity, the capacity of free radical scavenging and many other special
4
.0/).
Molecules 2021, 26, 2919. https://doi.org/10.3390/molecules26102919
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2919
2 of 13
biological activities. Several studies have indicated that hydroxyl radical scavenging ac-
tivity is positively correlated with the number of hydroxy groups on ring B; for instance,
0
0
quercetin (Q, ortho-catechol 3 ,4 -OH on ring B) has a stronger antioxidant capacity than
0
kaempferol (K, 4 -OH on ring B) [
P450s) in plants, flavonoid 3 -hydroxylase (F3 H) and flavonoid 3 , 5 -hydroxylase, deter-
mined (F3 5 H) the presence and the number of hydroxy groups on the B-ring of flavonoids
8,
9
]. Two cytochrome P450-dependent monooxygenases
0
0
0
0
(
0
0
0
0
0
0 0
(F3 5 H) [10
,
11]. Previous research has demonstrated that F3 H and F3 5 H can be success-
fully expressed in a yeast system [12
–
14], whereas these genes are difficult to express in a
0
0
bacterial expression system. One successful example of the co-expression of a plant F3 5 H
and other flavonoid genes in Escherichia coli showed that flavonols could be synthesized
from phenylpropanoid acids, while the catalytic activity was relatively low [15]. To address
this hurdle, much effort has been focused on finding suitable enzyme replacements for the
P450-catalyzed hydroxylation of flavonoids.
The 4-hydroxyphenylacetate 3-monooxygenase (HpaB) and NAD (P)H-flavin oxidore-
ductase (HpaC) genes from E. coli encode the 4-hydroxyphenylacetate 3-hydroxylase com-
plex [16]. It has been suggested that HpaC uses NADH to generate reduced flavin mononu-
−
−
cleotides (FMNH ), and HpaB utilizes FMNH to catalyze the hydroxylation of phenolic
compounds. Previous research has shown that this complex demonstrates gram-scale
conversion of a variety of substrates, including p-coumaric acid, tyrosol, coniferaldehyde
and umbelliferone, to their corresponding ortho-hydroxylated counterparts [17]. Further
research confirmed the ability of the HpaBC hydroxylase complex to convert naringenin (N)
and afzelechin (Af) to the corresponding ortho-hydroxylated flavonoids [18]. However, the
comparison of their catalytic efficiency for different para-hydroxylated flavonoid substrates
needs further systematic analysis.
In this paper, we have constructed a variety of HpaBC expression vectors, and the
corresponding products were successfully detected by feeding of N. To increase the con-
version efficiency of fermentation products further, we optimized fermentation conditions
including medium, induction temperature, substrate concentration and substrate delay
time. Ultimately, using optimum conditions, we demonstrated the ability of the HpaBC
hydroxylase complex to act on p-coumaric acid (p-CA), N, dihydrokaempferol (DHK),
kaempferol (K) and Af to form the corresponding ortho-hydroxylated products. This study
demonstrated that, using a bacterial expression system, it is feasible to efficiently synthesize
ortho-hydroxylated flavonoids in vivo, such as catechins, flavanols and flavonols.
2. Materials and Methods
2.1. Chemicals
Cyanidin (CYA), pelargonidin (PEL), N, eriodictyol (E), K, quercetin (Q), DHK, di-
hydroquercetin (DHQ), catechin (C), p-CA and caffeic acid (CA) were purchased from
Shanghai TOT Chemicals Firm (Shanghai, China). Af was purchased from Yuan ye Biotech-
nology Co., Ltd. All other chemicals were analytical grade chemicals.
2.2. Media, Bacterial Strains and Vectors
The media, bacterial strains and vectors used in this study are given in Table 1. The P1
and P2 is pRSFDuet vector and the two genes were inserted with different sites. In the P1
pRSFDuet vector HpaB gene is inserted into the first multiple cloning site of the pRSFDuet
vector, and the HpaC gene is inserted into the second multiple cloning site. Similarly, in
the P2 pRSFDuet vector the HpaC gene was inserted into the first multiple cloning site,
and the HpaB gene is inserted into the second multiple cloning site. P3 and P4 is pETDuet
vector with different cloning sites. In P3 PETDuet vector, HpaB gene is inserted into the
first multiple cloning site and the other gene HpaC gene is inserted into the second multiple
cloning site; in the P4 PETDuet vector the HpaC gene is inserted into the first multiple
cloning site of the PETdut vector, and the HpaP gene is inserted into the second multiple
cloning site. The P1 and p2 were transformed into E. coli BL21 for co-expression.

Molecules 2021, 26, 2919
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Table 1. Strains and plasmids used in this study.
Strains and Plasmids
Relevant Characteristics
Source or Reference
Plasmids
pRSFDuet
pETDuet
P1
Double T7 promoter, ColE1 ori. KanR
Double T7 promoter, ColE1 ori. Amp
pRSFDuet carrying (MCS-1)-HpaB and HpaC (MCS-2)
pRSFDuet carrying (MCS-1)-HpaC and HpaB (MCS-2)
pETDuet carrying (MCS-1)-HpaB and HpaC (MCS-2)
pETDuet carrying (MCS-1)-HpaC and HpaB (MCS-2)
Novagen
Novagen
This study
This study
This study
This study
R
P2
P3
P4
Strains
DH5α
General cloning host
Invitrogen
Novagen
BL21 * (DE3)
BL21-P1
BL21-P2
BL21-P3
BL21-P4
BL21-P2&P3
BL21-P1&P4
Host for flavonoid production and gene clones
General expression strain of pRSFDuet P1
General expression strain of pRSFDuet P2
General expression strain of pETDuet P3
General expression strain of pETDuet P4
General co-expression strain of P2 and P3
General co-expression strain of P1 and P4
This study
This study
This study
This study
This study
This study
LB medium was used for inoculum preparation and protein expression. Modified
M9 (M9) medium and Terrific Broth (TB) were used for feeding experiments and de novo
production of target compounds. LB medium contained NaCl (1%, w/v), tryptone (1.0%,
w/v) and yeast extract (0.5%, w/v) per liter. M9 medium contained glucose (0.4%, w/v),
Na HPO (40 mM), NaCl (0.25%, w/v), KH PO (17 mM), NH Cl (19 mM), MgSO (2 mM),
2
4
2
4
4
4
MCaCl (1 mM) then set volume to 1 L. The one-liter TB liquid medium contained tryptone
2
(
1.2%, w/v), yeast extract (2.4%, w/v), glycerol (0.4%, v/v), KH PO (17 mM), and K HPO
2 4 2 4
(
72 mM). The bacterial strains and plasmids that were used or constructed in this study
was used to propagate all plasmids, while strain BL21 *
DE3) was used as the host for flavonoid production. The vectors pRSFDuet and pETDuet
Novagen) were used as the basis for all plasmid construction and pathway expression.
are listed in Table 1. E. coli DH5
(
(
α
2.3. Construction of the HpaB and HpaC Expression Plasmids
The amplified DNA fragments of HpaB and HpaC were digested with Nde I and Xho
I and then inserted into multiple cloning site 2 (MCS-2) of the pETDuet or pRSFDuet
plasmid. On the basis of these plasmids, we transferred the genes into multiple cloning
site 1 (MCS-1) of the pETDuet or pRSFDuet plasmid using a one-step cloning method.
The constructed recombinant expression plasmids are shown in Table 1, and the primers
used are shown in Table S1. The resulting plasmids were amplified in DH5α cells. The
recombinant plasmids were confirmed by sequencing, and these plasmids were introduced
into BL21 * (DE3) cells by transformation.
Single colonies were inoculated into 4 mL of LB media containing ampicillin or
◦
kanamycin and were cultured overnight at 37 C. The overnight cultures were inoculated
◦
into 10 mL of M9 or TB medium. The cultures were allowed to grow at 37 C until the
optical density at 600 nm (OD600) reached 0.6 and then were induced with 1 mM IPTG at
◦ ◦ ◦
2
0 C, 28 C or 37 C for 4 h, 6 h or 8 h. The bacterial strains were fed with substrates for
ortho-hydroxylated flavonoid production. The samples collection was performed at regular
time intervals. The OD600 was measured for cell growth, and the concentrations of the
products and intermediates were analyzed by high-performance liquid chromatography
(
HPLC) and LC-MS. The products were extracted with ethyl acetate, and all experiments
were performed in duplicate.
2
.4. HPLC and LC-MS Analysis
The HPLC analysis was performed using a C18 column (150
µm C18), Phenomenex, Torrance, CA, USA) with an LC-10Avp system (Shimadzu, Kyoto,
×
4.6 mm i.d.: Luna®
5
Japan). The mobile phase comprises of acetonitrile (solvent A) and water (solvent B) (both

Molecules 2021, 26, 2919
4 of 13
·
min 1. The HPLC program was as
−
contained 1% formic acid) at a flow rate of 0.4 mL
follows: 10% to 15% B (v/v) for 5 min, 15% to 40% B from 5 to 15 min, 40% to 60% B from 20
to 22 min, and 10% B for 22 to 25 min. N, E, K, Q, DHK, DHQ, C and Af were monitored at
2
5
80 nm; p-CA and CA were monitored at 340 nm; and the anthocyanins were monitored at
30 nm. For further identification of the products, a liquid chromatography mass spectrum
(LC-MS) system was used as previously described [19]. The quantitative products of Caffeic
acid, Eriodictyol, Catechin, Quercetin and Dihydroquercetin were respectively used and
their standard curves were plotted at 280 nm.
2.5. Statistical Analysis
Statistical differences were analyzed with SPSS 19.0 using one-way analysis of variance.
The results were expressed as the means
represent the standard deviation for at least three replicates.
±
the standard errors of the mean. The error bars
3. Results
3.1. Expression of HpaB and HpaC in E. coli
The open reading frames (ORFs) of HpaB, which encodes for the monooxygenase com-
ponent (GenBank CAD6019151.1), and HpaC which encodes for oxidoreductase component
(GenBank CAD6019161.1), were cloned into the expression vectors pETDuet and pRSFDuet
(Novagen, Carlsbad, CA, USA), respectively. The following results indicate that HpaB and
HpaC were successfully and recombinantly expressed in E. coli cells. SDS-PAGE analysis
revealed that there were the presence of major bands corresponding to HpaB (58.5 kDa) and
HpaC (18.6 kDa) in samples prepared from the soluble fractions of E. coli cells (Figure 1).
Figure 1. SDS-PAGE of the proteins HpaB and HpaC. The protein expression of different plasmids in
BL21 cells. P1: pRSFDuet-HpaBC; P2: pRSFDuet-HpaCB; P3: pETDuet-HpaBC; P4: pETDuet-HpaCB;
P2&3: co-expression of P2 and P3; and P1&4: co-expression of P1 and P4. The locations of the HpaB
and HpaC proteins are indicated by the arrows on the right. The molecular weights of the marker
proteins (180 kDa, 100 kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are also shown.
Several HpaBC expression vectors were constructed, and a two-step fermentation
−
1
was performed using N as a substrate (final concentration of 200 mg
·
L
); N and E were
detected by HPLC [18]. The HPLC results showed that the strains had remarkably different
ortho-hydroxylation activities (Figure 2). The enzyme activities of the strains carrying the
P1 and P3 (containing HpaB in MCS-1 and HpaC in MCS-2) plasmids were significantly
lower than those of strains carrying the P2 and P4 (containing HpaC in MCS-1 and HpaB
in MCS-2) plasmids. For example, the conversion efficiency of the P2-carrying strain
−
1
(
7.47
and that of the P4-carrying strain (6.3
than that of the P3-carrying strain. In addition, we also co-expressed pRSFDuet-HpaC
MCS-2) and pETDuet-HpaB (MCS-2), and the results showed very low ortho-hydroxylation
±
0.41%,15.81
±
0.86 mg
·
L
) was 3.81-fold higher than that of the P1-carrying strain,
−
1
±
0.30%,13.36
±
0.63 mg
·
L
) was 2.86-fold higher
(
activity (results not shown). These findings suggested that when the HpaC gene was
attached to the MCS-2 locus with an S-Tag (15 amino acids) at the carboxyl-terminus, the
enzyme activity could be affected.

Molecules 2021, 26, 2919
5 of 13
Figure 2. Construction strategy for all engineered Duet vectors (grey shadowed area) with HpaB and
HpaC genes (colored boxes). The ortho-hydroxylation activities of different strains; (
HpaBC (P1), ( ): pRSFDuet-HpaCB (P2), ( ): pETDuet-HpaBC (P3), ( ): pETDuet-HpaCB (P4), (
co-expression of P2 and P3, and ( ): co-expression of P1 and P4. ‘His His His’ represents the three
a
): pRSFDuet-
b
c
d
e):
f
amino acid composition of His-Tag, and’ Lys Phe Ser ‘represents the label composition of S-Tag. Final
substrate concentration of 200 mg·L−1, n = 3.
We expressed P1&4 and P2&3 to obtain a strain with higher activity than those
mentioned above. The data showed that the ortho-hydroxylation activity was significantly
improved, and the conversion efficiency of the P2&3-carrying strain (10.40
±
0.72%, product
) was 1.39-fold higher than that of the P2-carrying
strain, and that of the P1&4-carrying strain (6.7 0.43%, product concentration was
) was 1.07-fold higher than that of the P1-carrying strain, while the
−
1.54 mg L
1
concentration was 22.02
±
·
±
−
0.91 mg L
1
1
4.37
±
·
conversion efficiency of the P1&4-carrying strain was still lower than that of the P2-carrying
strain. Therefore, on the basis of these observations, we have selected these two strains,
including the P2&3 and P2 plasmids, and further optimized the fermentation conditions.
3.2. Optimization of the Induction Temperature and Substrate Delay Time
To investigate the effect of culture temperature on enzyme activity, three tempera-
◦
◦
◦
tures (20 C, 28 C and 37 C) were selected for fermentation (Figure 3a). The conversion
◦
efficiency of P2&3-carrying strain for E production at 37 C was 8.46
centration was 17.92
P2-carrying strain at the same temperature. At the culture temperature of 28 C, the conver-
±
0.43% (product con-
−
1
±
0.92 mg
·
L
), which was 1.3 times higher than that produced by the
◦
sion efficiency of E produced by the P2&3-carrying strain was up to 12.92
±
0.59% (product
−
L
1
concentration was 27.36
ing P2 to produce E was 10.08
±
1.26 mg
·
), and the conversion efficiency of the strain carry-
−
1
±
0.69% (product concentration was 21.35
±
1.49 mg
·
L
).
However, the amount of product decreased, as the temperature was further reduced to
◦
2
0 C. At this temperature, the conversion efficiency of P2&3- and P2-carrying strains
for E production was only 5.87
±
1.24% and 3.45
±
0.74% (product concentration was
−
1
−1
1
2.43
±
2.63 mg
·
L
and 7.31
±
1.57 mg
·
L
), respectively. This result indicates that in
certain temperature range, the production of bioactive protein increases with the increase
◦
of temperature and reached the peak at the culture temperature of 28 C.

Molecules 2021, 26, 2919
6 of 13
Figure 3. Production of E from the corresponding substrate, N. The substrate (final concentration of
−
1
2
00 mg
·
L
) was added to the cell culture in LB medium. (a): Conversion efficiency of E at different
◦
◦
◦
induction temperatures. The strains were induced for 8 h at 20 C, 28 C or 37 C. (
b
): Conversion
efficiency of E at different substrate delay times after IPTG induction. Bacterial culture medium was
◦
induced for 4 h, 6 h or 8 h at 28 C. Data are shown as the means ± s.d.s (n = 3).
To determine the optimal substrate delay time, bacterial growth medium was sup-
◦
plemented at 28 C for 4 h, 6 h and 8 h after IPTG induction. The conversion efficiency
of E increased gradually with increasing induction time and then reached the production
peak at 6 h after IPTG induction (Figure 3b). The conversion efficiencies of the P2&3- and
P2-carrying strains reached 16.47
±
1.01% and12.50
±
1.00% (product concentration was
−
1
−1
3
3.98
±
2.12 mg
·
L
and 26.48
±
2.12 mg
·
L
), respectively. After 8 h of induction, the
conversion efficiencies of the P2&3- and P2-carrying strains decreased to 13.47
±
00.63 and
−
1
−1
1
0.29
±
0.71% (product concentration was 28.53
±
1.33 mg
·
L
and 21.81
±
1.57 mg
·
L
),
respectively. These results show that it is possible to achieve high-density culture of re-
◦
combinant bacteria and high expression of products at the optimal temperature (28 C)
and IPTG induction time (6 h). Therefore, we chose these fermentation conditions for the
following study.
3.3. Optimization of the Substrate Concentration and Medium to Improve Catalytic Efficiency
Previous studies have shown that when the medium contains high concentrations of
phenylpropanoic acids or flavonoids, the growth of bacteria was significantly inhibited [20,21]
.
This experiment was conducted to study the effect of the initial concentration of N on the
catalytic efficiency of the P2&3- and P2-carrying strains. As shown in Figure 4a,b it can be
−
1
seen that at 200 mg
2 h after substrate addition. Figure 4a shows that the cell concentration of the P2-carrying
strain was the lowest, and the final OD600 (cell concentration) was only 1.201 0.09, while
). Figure 4b also shows
that the growth curve of the P2&3-carrying strain showed the most obvious downtrend,
and the OD600 of the P2&3-carrying strain was 1.597 0.06. When the initial concentration
(Figure 4c,d), the growth of the P2 and P2&3-carrying strains did
not decrease with the addition of the substrate. The final OD600 of the P2-carrying strain
·
L
concentration of N, the cell growth rate was significantly reduced
1
±
−
2.01 mg L
1
the conversion efficiency of E was 5.81
±
0.95% (12.32
±
·
±
−
of N was 80 mg L
1
·
−
1
reached 1.73
±
0.08, and the concentration of E was 27.63
The final OD6 of the P2&3-carrying strain was 2.46
±
2.31% (23.40
±
1.96 mg
·
L
).
±
0.18, and the highest conversion
00

Molecules 2021, 26, 2919
7 of 13
−
1); these results indicate that a
2.35 mg L
high initial concentration of N produces a certain degree of inhibition on the growth of the
bacterial strains.
efficiency of E reached 38.49
±
2.77% (32.60
±
·
Figure 4. Growth curve of bacterial culture at different substrate concentrations and the conversion
efficiency of E at different incubation times. The hollow boxes show the growth curve of bacterial
cells, and the solid circles represent the titer of E at different incubation times. The IPTG induction
time is shown by a red arrow, and the red squares indicate the substrate addition time. (
a
,
b
): Substrate
−1
L ) in LB medium. Data are shown as the
−
1
(
200 mg
·
L
) in LB medium; (
c,d
): Substrate (80 mg
·
means ± s.d.s (n = 3).
In addition, Figure 4 shows that as the fermentation time increased, the conversion
efficiency of the product increased continuously with the cultivation time. The conversion
efficiency of E could be maximized 10 h after substrate addition (this result was applicable
−
1
−1
to various concentrations of 80 mg
·
L
or 200 mg
·
L ). The co-transformed strain had the
highest catalytic ability, which corresponded to the results in Figure 2.
We further studied the effects of substrate concentration and culture medium on
the catalytic ability with N as a substrate based on the above results. Three types of
media (including LB, TB and M9) and five substrate concentrations were selected for
this study (Figure 5). The results showed that the ideal substrate concentration was
−
1
8
0 mg
·
L
, and the optimal medium for E production was M9. The highest conversion
−
1
efficiency of E of the P2-carrying strain was 39.58
final substrate concentration of 80 mg
±
3.6% (31.67
±
2.89 mg
·
L
), with a
−
1
·
L
in M9 medium, followed by that in TB medium
−
1
−1
(27.87
±
2.52 mg
·
L
), while that in LB medium was the lowest (22.72
±
1.14 mg
·
L
). The
most exciting result was that the conversion efficiency of E produced by the P2&3-carrying
−
1
strain in the conversion efficiency was up to (46.84
±
2.85 mg
N were chosen as the optimal medium and substrate concentration, respectively,
for the subsequent study.
·
L
). Hence, M9 medium and
−
0 mg L
1
8
·

Molecules 2021, 26, 2919
8 of 13
Figure 5. Conversion efficiency of E in different media (LB, TB and M9) and substrate concentrations
−
1
−1
(
substrate concentrations from 40 mg
P2-carrying strain in LB, TB and M9 media. (
strain in LB, TB and M9 media. Data are shown as the means ± s.d.s (n = 3).
·
L
to 120 mg
b
·
L
). (
a): the conversion efficiency of E of the
): the conversion efficiency of E of the P2&3-carrying
3.4. Substrate Diversity Analysis of the HpaBC Complex
To further investigate the diversity of substrates, in addition to flavanone (N), a
monohydroxylated phenolic acid (p-coumaric acid, p-CA), dihydroflavonol (DHK), flavonol
K), flavan-3-ol (afzelechin, Af) and anthocyanin (pelargonidin, PEL) were fed under the
(
optimal conditions, and the fermentation products were detected by HPLC and LC-MS
methods (Figure 6). Previous studies have suggested that the HpaBC complex has in vivo
activity towards p-CA, N, and Af, with high catalytic efficiency. Our results showed
that those substrates (except anthocyanin) could be converted to the corresponding ortho-
products under the optimal conditions. Similarly, the conversion of these substrates
by P2&3-carrying strains was also significantly higher than that of P2-carrying strains.
The conversion rate from N to E was 58%, followed by that for Af (35.2
±
3.14), p-CA
(
32.93 2.01), DHK (23.74 1.75) and K (23.84 0.88), while activity on the substrate PEL
±
±
±
was not detected (Table 2). These results provided evidence that the HpaBC complex had
in vivo activity towards monohydroxylated flavanol and flavonol.

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Figure 6. HPLC analysis of the enzymatic products of the HpaBC complex, when feeding with different substrates. HPLC
chromatogram (left) and standard compound (right) analyses of the enzymatic reaction. N, E, K, Q, DHK, DHQ, C and Af
were monitored at 280 nm, and p-CA and CA were monitored at 340 nm. The substrates and corresponding products were
detected by HPLC and LC-MS. The ortho-hydroxylation activities of (a): p-CA to CA; (b): N to E; (c): Af to C; (d): K to Q;
and (e): DHK to DHQ. Final substrate concentration of 80 mg·L−1, n = 3.
Table 2. The yield and conversion rate of ortho-hydroxylated flavonoids for the HpaBC complex
when feeding with different substrates. The horizontal lines in the table indicate that no activities has
been detected. Data are shown as the means ± s.d.s (n = 3).
Substrates
Products
Yield (mg·L−1)
Conversion Rate (%)
28.91 ± 1.77
32.93 ± 2.01
p-Coumaric acid
Caffeic acid

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Table 2. Cont.
Substrates
Products
Yield (mg·L−1)
Conversion Rate (%)
4
6.84 ± 2.85
57.67 ± 3.36
35.2 ± 3.14
Naringenin
Afzelechin
Eriodictyol
2
9.81 ± 2.66
Catechin
2
2
0.14 ± 0.75
0.05 ± 1.48
23.84 ± 0.88
Kaempferol
Quercetin
Dihydroquercetin
Cyanidin
23.74 ± 1.75
Dihydrokaempferol
_ns__
_ns__
Pelargonidin
4. Discussion
Flavonoids are essential health-promoting secondary compounds produced at very
low levels in plants and fungi. Metabolic engineering of flavonoids has become an ex-
tensively researched area. However, the hydroxylation on the B-ring is the bottleneck of
metabolic engineering of flavonoids in Escherichia coli expression systems. In this paper,
we used the 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) complex to replace P450-
catalyzed hydroxylation and constructed a variety of HpaBC expression vectors. We found
that conversion activity was significantly reduced when the HpaC gene was attached to the
MCS-2 locus with an S-Tag (15 amino acids) at the carboxyl-terminus (C-terminal). HpaC is
−
a flavin reductase that catalyzes reduction of FMN to generate FMNH , which is used by
HpaB in the hydroxylation of flavonoids. Several studies have indicated that the C-terminus
of HpaC is a self-inhibiting protein domain and that, upon binding, the effector undergoes
conformational changes to allow faster flavin reduction and release [20]. We hypothesized
that the C-terminus of HpaC connected with an expressional tag might inhibit the release of
flavin, thereby inhibiting the catalytic activity of the HpaBC complex in vivo. In addition,
by comparing the catalytic activities of different strains, we obtained two strains containing
the P2 or P2&3 plasmids with increased conversion efficiency. The results showed that the
co-expression of the multiple cloning site sites of HpaB and HpaC genes connected to the
N-terminal of pRSFDuet vector could have a higher catalytic efficiency, but the individual
plasmids will be lost in the process of culture, which inhibits the catalytic substrate process.

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Therefore, further studies are required to optimize the conditions for the expression of the
two genes separately to improve the catalytic efficiency.
The optimum conditions, including substrate concentration, induction temperature,
substrate delay time and medium, were studied in this paper using the P2- and P2&3-
carrying strains. Our research found that high concentrations of the substrate (N) sig-
nificantly inhibited the growth rate and conversion activity of these cells. The growth
rate of the cells (the P2- and P2&3-carrying strains) was significantly reduced 12 h after
−
1
adding 200 mg
significant antibacterial effects in vitro
might be more conducive to an increase in the growth and activity of the recombinant
·
L
N as the substrate. Previous reports have shown that flavonoids have
[
20 21]. A low concentration of flavonoid substrates
,
−
L
1
strains. Through optimizing the concentration of substrate, we determined that 80 mg
was the optimum substrate concentration. On this basis, the conversion efficiency of the
ortho-hydroxylated flavonoid product was the highest with 80 mg
after 6 h of induction at 28 C in M9 medium. It is worth noting that, in the process of
optimization of the optimal medium, the M9 medium was the most nutrient rich, and the
catalytic efficiency of strains was the highest in M9 medium. The results of the optimal
medium indicated that sufficient nutrients were needed for the culturing of the of strains,
so in the industrial production process the composition of nutrients could be gradually
increased to achieve the maximum efficiency. Therefore, our corresponding experiments
focus on the optimization of nutrient composition in the culture medium.
·
−
1
·
L
N as the substrate
◦
Substrate diversities of the HpaBC complex have been studied in previous studies,
which have verified the catalytic activity of these complexes towards p-coumaric acids [21].
In addition, Jones et al. confirmed that HpaBC also has a catalytic effect on Af and N. In
this study, we adopted optimal fermentation conditions to produce ortho-hydroxylated
flavonoids in the E. coli expression system (Figure 7). Recent studies have reported that the
N to E conversion rate of the recombinant HpaBC complex reached 22.12
±
0.95% (substrate
−
1
−1
concentration was 300 mg
·
L
, product concentration was 62.7
±
2.7 mg
·
L
) in vivo [18],
and the maximum conversion rate was increased to 57.67
±
3.36 (substrate concentration
−
1
−1
was 80 mg
·
L
, product concentration was 46.84
±
2.85 mg
·
L
) in this study. We also
obtained the corresponding products C and CA by using the corresponding substrates
(
Af and p-CA, respectively). Unfortunately, the catalytic efficiency of the recombinant
−
1
HpaBC complex for p-CA to CA in this study has not reached 3.82 g
·
L
as mentioned in
the literature [17], so we will further explore this catalytic condition in future studies. In
addition, the activity of HpaBC with K and DHK as substrates was demonstrated in this
paper. To the best of our knowledge, this is the first report regarding HpaBC production of
quercetin and dihydroquercetin.
Figure 7. The catalytic process of HpaBC with different substrates. The red color is the first discovered
catalytic activity in this study.

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In the future, our research will focus on screening strains with tolerances to high concen-
trations of substrate. It is speculated that the production of ortho-hydroxylated flavonoids
might be further increased if the optimal concentration of substrates was enhanced.
5. Conclusions
The hydroxylase complex, HpaBC, was selected to efficiently produce ortho-hydroxylated
flavonoids in vivo. A variety of HpaBC expression vectors were constructed, and the corre-
sponding products were successfully detected when feeding naringenin to the recombinant
−
1
strains. The optimal culture conditions were a substrate concentration of 80 mg
·
L
, in-
◦
duction temperature of 28 C, medium of M9 medium, and substrate delay time of 6 h
after IPTG induction. With the optimal conditions, the conversion efficiency of eriodictyol
from P2&3-carrying strains fed naringin was up to 57.67
±
3.36 (substrate concentration
−
1
−1
was 80 mg
·
L
, product concentration was 46.84
±
2.85 mg
·
L
). The same strategy
was used for catechin and caffeic acid production, and the highest conversion efficiency
were 35.2 3.14% and 32.93 2.01%, respectively. The HpaBC activities towards di-
±
±
hydrokaempferol and kaempferol were evidenced in this paper. This study provides a
feasible way to efficiently synthesize in vivo the B-ring of dihydroxy flavonoids and lays a
foundation for the de novo synthesis of flavonoids by E. coli.
Supplementary Materials: The following are available online. Figure S1. HPLC and HPLC-MS/MS
analysis of the enzymatic products of the HpaBC complex, when feeding with different substrates.
Table S1. Primers of HpaB and HpaC for construction of recombinant expression plasmids.
Author Contributions: L.W. and X.M. validation; L.G. (Liping Gao) and T.X. formal analysis; H.R.,
T.L., Y.C. and L.G. (Lina Guo) methodology; L.G. (Lin Gui) and Y.L. data curation; Y.W. writing; All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (no.
3
1770729), the National Key R&D Program of China (no. 2018YFD1000601) and the Natural Science
Foundation of China (no.31870677, 31870676).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank Yan Chang from the School of Life Sciences
for assisting with HPLC detection and analysis. This work was supported by the National Nat-
ural Science Foundation of China (no. 31770729), the National Key R&D Program of China (no.
2
018YFD1000601) and the Natural Science Foundation of China (no.31870677, 31870676).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples for all compounds in this article are available from the authors.
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