Development and Optimization of an in Vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin

Article abstract of DOI:10.1021/acs.jafc.8b01299

An in vitro multienzyme synthetic system was developed and optimized to efficiently produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The recombinant proteins were purified through affinity chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase, respectively, followed by development of an in vitro synthetic system for producing kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 0.4% sodium ascorbate, 25 μg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2). When the reaction was carried out at 40 °C for 40-50 min, the yield of kaempferol was 37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%. Overall, this system provides a promising and efficient approach to produce kaempferol economically.

Full text of DOI:10.1021/acs.jafc.8b01299

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Bioactive Constituents, Metabolites, and Functions
Development and Optimization of an in vitro Multienzyme
Synthetic System for Production of Kaempferol from Naringenin
Zhiping Zhang, Yanzhi He, Yue Huang, Li Ding, Lei Chen, Yaxian Liu, Yesen Nie, and Xinyue Zhang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01299 • Publication Date (Web): 18 Jul 2018
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Development and Optimization of an in vitro Multienzyme
Synthetic System for Production of Kaempferol from Naringenin
Zhiping Zhang1, #, Yanzhi He1, #, Yue Huang1, Li Ding1, Lei Chen1, Yaxian Liu1, Yesen Nie1,
Xinyue Zhang1, 2, 3, 4, *
1 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
2 Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for
Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University (26116120),
Yangzhou, Jiangsu 225009, China.
3 Joint International Research Laboratory of Agriculture & AgriꢀProduct Safety, Yangzhou University,
Yangzhou, Jiangsu 225009, China.
4
Jiangsu Coꢀinnovation Center for Prevention and Control of Important Animal Infectious Diseases
and Zoonoses, Yangzhou, Jiangsu 225009, China.
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ABSTRACT
An in vitro multienzyme synthetic system was developed and optimized to efficiently
produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the
biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and
overexpressed in Escherichia coli. The recombinant proteins were purified through affinity
chromatography and showed activities of flavanone 3ꢀhydroxylase and flavonol synthase,
respectively, followed by development of an in vitro synthetic system for producing
kaempferol. The system contains 8.2 mM αꢀketoglutaric acid, 0.01 mM ferrous ion, 25
ꢁg/mL of each recombinant enzyme, and 10% glycerol in 100 mM TrisꢀHCl (pH 7.2).
When the reaction was carried out at 40 °C for 40ꢀ50 min, the yield of kaempferol was
37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%.
Overall, this system provides a promising and efficient approach to produce kaempferol
economically.
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Key Words: kaempferol, in vitro synthesis, optimization, flavanone 3ꢀhydroxylase,
flavonol synthase.
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ꢀ
INTRODUCTION
Flavonoids are a large group of plantꢀderived natural products and can be further classified
into six major subgroups depending on the variations in the heterocyclic Cꢀring: the
1-3
catechins or flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones
.
These compounds show a wide range of biological activities 3-6, leading to quite a number
of studies on these secondary metabolites 7-12
.
Kaempferol (KMF), also known as robigenin or 3,4’,5,7ꢀtetrahydroxyflavone, is a natural
flavonol possessing numerous pharmacological and biological properties including
antioxidant, antiꢀinflammatory, anticancer, antimicrobial, cardioprotective, neuroprotective,
antidiabetic, antiꢀosteoporotic, estrogenic/antiꢀestrogenic, anxiolytic, analgesic, and
antiꢀallergic activities 13, 14. KMF is mainly produced from traditional plant extraction.
However, the cost of KMF production is high due to the very low content of KMF in plants
15, 16. Therefore, it is crucial to develop alternative strategies to reduce the cost for
producing KMF.
Many methods have been developed to synthesize flavonoids 17, 18. However, chemical
synthesis of flavonoids often requires multiple steps, toxic reagents, and extreme reaction
conditions 1, 17. In addition, the chiral synthesis to produce active flavonoid compounds is
another crucial challenge in chemical synthesis. Hence, chemical synthesis is not
economically feasible for largeꢀscale production of flavonoids 1.
Since the biosynthetic pathway of flavonoids has been successfully elucidated in plants 2, it
is a promising alternative strategy to synthesize these natural compounds using a microbial
cell factory. Recently, some flavonoids have been successfully biosynthesized by
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engineered microbes, mainly including Escherichia coli and Saccharomyces cerevisiae 19-23
For example, Duan, et al have constructed a microbial cell factory for KMF production by
introducing a biosynthetic pathway of KMF into the budding yeast S. cerevisiae and
achieved the highest production of KMF at 66.29 mg/L 24. However, not all engineered
microbes produce desired products due to the wellꢀknown complexity of a cellular system,
incompatibility of artificially synthesized genetic elements and hosts, inhibition of host
cells by target products, and instability of an engineered biosystem 25.
.
Another promising alternative strategy is to produce flavonoids using an in vitro
multienzyme synthetic system. Recently, Cheng, et al have reported the successful
multienzyme synthesis of enterocin polyketides by assembling a complete type II
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polyketide synthase enzymatic pathway to natural products in a single reaction vessel
.
This cellꢀfree synthetic biotechnology overcomes the disadvantages of a microbial cell
factory and is economically feasible for commercialꢀscale production of flavonoids 25.
As we know, there are two key enzyme genes, f3h and fls1, involved in the biosynthetic
pathway of KMF 1, 2. These two genes encode flavanone 3ꢀhydroxylase (F3H) and flavonol
synthase (FLS), respectively. In this article, we report a detailed study of the in vitro
multienzyme synthesis of KMF in a single reaction tube from cheap chemicals including
the substrate naringenin (NRN). Firstly, we cloned Atf3h and Atfls1 genes from Arabidopsis
thaliana and expressed them in E. coli BL21(DE3), followed by the purification of
recombinant proteins. Then, we determined the enzyme activities of the purified
recombinant proteins and their kinetic parameters. Finally, we developed a single reaction
system to produce KMF and optimized a series of reaction parameters including pH value,
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reaction temperature and time, and total amount and ratio of two recombinant enzymes to
find the most efficient and economic conditions for KMF production. To our best
knowledge, there is no report on the development of an in vitro multienzyme synthetic
system to produce KMF.
ꢀ
MATERIALS AND METHODS
Plants, Bacterial Strains, Plasmids, and Chemicals. Fourꢀweekꢀold seedlings of
Arabidopsis thaliana were a gift from Dr. Lin Wang of Yangzhou University. Escherichia
coli DH5α and BL21(DE3) were purchased from Beijing CoWin Biotech Co., Ltd. (Beijing,
China) and used for plasmid maintenance and gene expression, respectively. The bacteria
were cultured in Luria–Bertani (LB) broth or on agar supplemented with 100 ꢁg/mL
ampicillin when necessary. Prokaryotic expression vector pETꢀ32a(+) (Novagen) was used
for cloning purposes. Authentic naringenin (NRN), dihydrokaempferol (DHK), kaempferol
(KMF), and other chemicals are products of SigmaꢀAldrich (St. Louis, MO). Restriction
enzymes were bought from New England Biolabs (Hertfordshire, UK). T4 DNA ligase was
a product of Vazyme Biotech Co., Ltd (Nanjing, China). TRIzol® Reagent and Isopropyl
βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) were purchased from Thermo Fisher Scientific Inc.
(MA, USA).
Buffers used. The buffers used for enzyme assays in this study were prepared based on
the published recipes with slight modifications 27, 28. F3H buffer contains 100 mM TrisꢀHCl
(pH 7.5), 0.4% sodium ascorbate, 10% glycerol, 0.174 mM αꢀketoglutaric acid (αꢀKG), and
0.1 mM FeSO4. FLS buffer contains 100 mM TrisꢀHCl (pH 7.2), 0.4% sodium ascorbate,
10% glycerol, 8.2 mM αꢀKG, and 0.01 mM FeSO4. Based on the F3H and FLS buffers, we
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designed a panel of KMF synthetic buffers consisting of 100 mM TrisꢀHCl (pH 7.2 or 7.5),
10% glycerol, 0.4% sodium ascorbate, 0.174 mM or 8.2 mM αꢀKG, and 0.01 mM or 0.1
mM FeSO4, as shown in Table 1.
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Isolation and reverse transcription of total RNA and construction of plasmids. Total
RNA was isolated from seedlings of A. thaliana using TRIzol® Reagent and reversely
transcribed into complementary DNA (cDNA) using the SuperRT cDNA Synthesis Kit
(Beijing CoWin Biotech Co., Ltd., Beijing, China) according to the manufacturer’s
protocol. The cDNAs were used for cloning Atf3h (Genbank accession no.
NM_001203121.1) and Atfls1 (Genbank accession no. NM_120951.3) genes. All primers
used in a specific polymerase chain reaction (PCR) are listed in Table 2 and were
synthesized by GENEWIZ, Suzhou, China. The Atf3h and Atfls1 genes were
PCRꢀamplified and cloned into pETꢀ32a(+) following a standard protocol as described
elsewhere 29. The DNA sequences of cloned Atf3h and Atfls1 genes were determined by
GENEWIZ, Suzhou, China.
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Expression and purification of recombinant proteins. The recombinant plasmids were
transformed into BL21(DE3) and colonies were inoculated into LB broth containing 100
ꢁg/mL ampicillin. Three milliliters of overnight culture were reinoculated into 150 mL LB
broth containing 100 ꢁg/mL ampicillin and cultured at 220 rpm and 37 °C until the optical
density at 600 nm was between 0.4 – 0.6. Then, 0.2 mM IPTG was added into the culture to
induce gene expression at 20 – 22 °C for 3 h. The bacteria were harvested and resuspended
in 1/20 volume of bacterial lysis buffer (50 mM TrisꢀCl, pH8.0, 0.1% Triton Xꢀ100, 1 mM
EDTA, 10% Glycerol, 150 mM NaCl, 0.5 mM DTT, and 0.1 mM PMSF, 1 ꢁg/mL aprotinin,
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1 ꢁg/mL leupeptin, and 1 ꢁg/mL pepstatin). The suspension was sonicated and
centrifugated at 13000 g for 10 min at 4 °C. TrxAꢀHisꢀtagged recombinant proteins were
purified from the supernatant using NiꢀIDA Agarose Resins (Beijing CoWin Biotech Co.,
Ltd., Beijing, China). Purification was performed according to the manufacturer’s protocol.
The purity of the purified proteins was analyzed on a 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDSꢀPAGE) gel and the bands were visualized by
coomassie blue staining.
Enzyme assays. The enzyme assays were analyzed according to the reported protocols
with slight modifications 27, 28, and the final volumes for all assays were 100 ꢁL. In brief,
the F3H assay contained 0.5 mM NRN and 3.3 ꢁg TrxAꢀHisꢀAtF3H in F3H Buffer, and the
FLS assay included 0.5 mM DHK and 1.9 ꢁg TrxAꢀHisꢀAtFLS1 in FLS Buffer. The assays
were incubated at 30 °C in open 2ꢀmL tubes with continuous shaking for 45 min or 3 h,
respectively. The assays were terminated with 100 ꢁL of ethyl acetate and 10 ꢁL of acetic
acid. Two hours later, the organic phases were transferred to 1.5 mL tubes for vacuum
freeze drying in a FreeZone 1 Liter Benchtop Freeze Dry System (Labconco, MO, USA).
The isolated flavonoids were then redissolved in methanol and subjected to polyamide thin
layer chromatography (TLC), high performance liquid chromatography (HPLC), and
electrospray ionization mass spectrometry (ESIꢀMS) analyses. The dots on the TLC plates
were visualized under a Tanonꢀ2500 Gel Imaging System (Tanon Science & Technology
Co. Ltd., Shanghai, China).
To determine the enzyme activity of the TrxAꢀHisꢀAtF3H, the concentration of NRN
was changed into 0.1 mM and the assay was incubated at 30 °C for 45 min. To determine
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that of the TrxAꢀHisꢀAtFLS1, the concentration of DHK was changed into 0.08 mM and
the assay was incubated at 30 °C for 30 min. The calibration curves of authentic DHK and
KMF were made based on the HPLC data and the product amount was thus calculated. The
enzyme activity was evaluated from three individual experiments and expressed as the
amount of the recombinant protein needed to convert 1 ꢁmol substrate into the product at
30 °C within one minute. The values were expressed as mean ± standard deviation.
Determination of the Michaelis-Menten constant Km of the recombinant enzymes.
To determine the MichaelisꢀMenten constant of the TrxAꢀHisꢀAtF3H, a series of NRN
concentrations were prepared in the F3H assay, including 0.04 mM, 0.06 mM, 0.08 mM,
0.10 mM, and 0.12 mM. To determine that of the TrxAꢀHisꢀAtFLS1, the concentrations of
DHK included 0.01 mM, 0.02 mM, 0.04 mM, 0.08 mM, and 0.16 mM in the FLS assay,
respectively. Then, the MichaelisꢀMenten constants were calculated using the data analysis
software OriginPro (version 9.0.0). The measurements were repeated three times and the
Km value of each recombinant enzyme was expressed as mean ± standard deviation.
Development and optimization of an in vitro multienzyme synthetic system for KMF
production. Based on the components in F3H and FLS buffers, a KMF multienzyme
synthetic system was developed to produce KMF from NRN in a single reaction tube. The
system contained 0.5 mM NRN, 2.0 ꢁg TrxAꢀHisꢀAtF3H, and 2.0 ꢁg TrxAꢀHisꢀAtFLS1 in
100 ꢁL of various KMF synthetic buffers as shown in Table 1. The reactions were
incubated at 30 °C with continuous shaking for 3 h and terminated by addition of 100 ꢁL of
ethyl acetate and 10 ꢁL of acetic acid. Two hours later, the organic phase was subjected to
vacuum freeze drying. The powder was dissolved in methanol and analyzed by polyamide
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TLC and HPLCꢀMS.
After initial setup of the synthetic system, the reaction parameters were further
investigated to maximize the efficiency of KMF production. Briefly, the pH value and the
concentrations of αꢀKG and FeSO4 in the reaction system were optimized according to
Table 1 by incubating the reaction tubes at 30 °C for 3 h. After the pH value and the
concentrations of αꢀKG and FeSO4 were fixed at an optimum value, the reaction time was
optimized by incubating the reaction mixture at 30 °C with stirring for 0, 10, 20, 30, 40, 50,
and 60 min. Next, the optimum reaction temperature was explored by incubating the
reaction mixture at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C for an optimum reaction time.
Then, the optimum ratio of the recombinant enzymes was analyzed by setting the ratio of
TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1 at 1:3, 1:2, 1:1, 2:1, and 3:1. Finally, the total
amount of TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1 was optimized by adding 1 ꢁg, 2 ꢁg, 3
ꢁg, 4 ꢁg, 5 ꢁg, and 6 ꢁg of the enzymes at an optimum ratio into a 100 ꢁL of reaction
mixture. All measurements were repeated three times and the results were expressed as
mean ± standard deviation.
TLC analysis. Analysis of flavonoid compounds by TLC was widely reported elsewhere
30. In brief, the reaction products were primarily analyzed by TLC in polyamide 6 plates
(Sinopharm Chemica Reagent Co., Ltd, Shanghai, China) in comparison to authentic
samples. The sampleꢀloaded TLC plates were run in a solvent system consisting of
chloroform/methanol/ethyl acetate/formic acid at a ratio of 5.0:1.5:1.0:0.5. Then, the plates
were sprayed with 1% ethanolic solution of aluminum chloride (AlCl3) and subjected to
UV illumination at 254 nm to visualize spots. The gray value of each spot was analyzed by
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the image processing software ImageJ 1.51j8.
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HPLC-MS analysis. The HPLCꢀMS was performed to analyze the synthesized
flavonoid compounds according to the published work 31 with slight modifications. In brief,
the samples were processed sequentially through 0.45 ꢁm and 0.22 ꢁm filters and analyzed
using an Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole
LC/MS system. Data were collected and analyzed using Agilent MassHunter Workstation
(version B.02.00) and MassHunter Quantitative analysis software (version B.01.04),
respectively. The samples were separated at 30 °C using an Agilent C18 column (150×4.6
mm, 5 ꢁm, Thermo Fisher Scientific. Inc., CA, USA). The separation was achieved using a
combination of two liquids, distilled water and acetonitrile. The column was eluted at 0.2
mL/min by a 10% – 85% (v/v) gradient of acetonitrile in water under the following
conditions: 0 – 10 min, a linear gradient of 10% – 25% (v/v) acetonitrile concomitant with
90ꢀ75% water; 10 – 35 min, a linear gradient of 25% – 50% acetonitrile; 35 – 45 min, a
linear gradient of 50% – 85% acetonitrile; 45 – 50 min, a linear gradient of 85% – 10%
acetonitrile; 50 – 60 min, 10% acetonitrile in 95% water. Absorbance of the eluate was
monitored from 200 nm to 800 nm. Single wavelength chromatograms at 280 nm, 320 nm,
and 370 nm were extracted using the software Xcalibur v2.0.7. The reaction products were
quantified by comparing the peak area of a compound in the reaction sample with that of a
known amount of an authentic compound.
Isolation of KMF. The isolation of KMF was a modification of the method developed
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by Leite, et al. . In brief, the reaction was terminated by adding an equal volume of ethyl
acetate. The organic phase was transferred to 1.5 mL tubes for vacuum freeze drying as
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described above. The dried powder was reꢀdissolved in methanol and processed through a
0.45 ꢁm filter. Next, the solution was subjected to chromatography over a Sephadex LHꢀ20
column eluted with methanol at 0.5 mL/min. The fractions were collected and combined
together according to the results of TLC analysis. The isolated KMF was further confirmed
by HPLCꢀMS analysis as described above and used for calculating the yield.
ꢀ
RESULTS AND DISCUSSION
Cloning, expression and purification of the key enzymes for KMF biosynthesis. As
shown in Figure 1, AtF3H and AtFLS1 are two key enzymes for the biosynthesis of KMF
in plants. To develop an in vitro system for multienzyme synthesis of KMF, we first cloned
Atf3h and Atfls1 genes from A. thaliana into prokaryotic expression vectors.
Generally speaking, soluble expression of recombinant proteins in bacteria maintains its
enzymatic activity. To increase the relative amount of soluble recombinant proteins
expressed in E. coli, we first compared the difference between pETꢀ32a(+) and pGEXꢀ5Xꢀ1,
two common prokaryotic expression vectors, in expressing recombinant proteins AtF3H
and AtFLS1. We found that pETꢀ32a(+) provided a higher level of soluble AtF3H and
AtFLS1 proteins, which possibly resulted from the role of TrxA fragment in pETꢀ32a(+) in
promoting soluble expression. Therefore, we chose pETꢀ32a(+) as a vector for constructing
recombinant plasmids. Sequence analysis verified that there was no mutation occurring in
the PCRꢀgenerated DNA fragments.
Then, we transformed the recombinant plasmids into E. coli strain BL21(DE3) and
optimized the expression conditions mainly by changing two important induction
parameters – the induction temperature and the concentration of IPTG. When we set the
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temperature at 20 – 22 °C and the IPTG concentration at 0.1 – 0.2 mM, we found that the
bacteria expressed a high amount of soluble recombinant proteins. Then, we induced the
expression for 3 h and purified the recombinant proteins by affinity chromatography
through a nickel iminodiacetic acid (NiꢀIDA) agarose resin. All the procedures for protein
purification were carried out at 4 °C to maintain the enzymatic activities of the recombinant
proteins to the highest extent. As shown in Figure 2, the purity of the purified recombinant
proteins, TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1, was >95% on a 10% SDSꢀPAGE gel,
which were pure enough for subsequent enzyme assays.
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Determination of the enzyme activities and the Michaelis-Menten constants of the
purified recombinant proteins. To determine the flavanone 3ꢀhydroxylase activity of the
TrxAꢀHisꢀAtF3H protein, we set up a reaction system containing 0.5 mM NRN and 3.3 ꢁg
purified recombinant protein in F3H buffer. The reaction products were analyzed by
polyamide TLC and HPLCꢀMS. As shown in Figure 3A, there was a new spot on a
polyamide TLC plate with a migration distance similar to that of DHK. The HPLCꢀMS
analysis demonstrated that the new chemical showed a retention time of 12.50 min (Figure
3D) and yielded a quasiꢀmolecular ion peak [MꢀH]ꢀ at m/z 287.1000 (Figure 4B), identical
to those of DHK. These data indicated that the new chemical was DHK and thus the
purified TrxAꢀHisꢀAtF3H protein possessed a flavanone 3ꢀhydroxylase activity. Enzyme
activity analysis indicated that 14.99 ± 0.34 ꢁg of the TrxAꢀHisꢀAtF3H was needed to
convert 1 ꢁmol NRN into DHK at 30 °C in the F3H buffer within one minute. Further
dynamic analysis demonstrated that this recombinant enzyme possessed
a
MichaelisꢀMenten constant of 0.081 ± 0.025 mM.
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To measure the flavonol synthase activity of the TrxAꢀHisꢀAtFLS1 protein, we
established a 100ꢀꢁL reaction system containing 0.75 mM DHK as a substrate and 1.9 ꢁg
purified protein in FLS buffer. Polyamide TLC analysis indicated that the reaction
produced a new spot with a similar migration distance to that of KMF (Figure 3B). HPLC
analysis demonstrated that the new chemical possessed a retention time of 20.46 min
(Figure 3D) and further confirmation by ESIꢀMS generated a quasiꢀmolecular ion peak
[MꢀH]ꢀ at m/z 285.1000 (Figure 4C), identical to those of KMF. These data demonstrated
that the new chemical molecule was KMF and the purified TrxAꢀHisꢀAtFLS1 protein had a
flavonol synthase activity. However, due to an excessive amount of the substrate (0.5 mM)
and less amount of the TrxAꢀHisꢀAtFLS1 in the FLS assay, the peak for KMF was
comparatively low in the HPLC chromatograms (Figure 3D), seemingly indicating a low
catalytic efficiency of the TrxAꢀHisꢀAtFLS1. Therefore, we further analyzed the enzyme
activity of the TrxAꢀHisꢀAtFLS1. The data demonstrated that 10.78 ± 0.61 ꢁg of the
purified recombinant protein was needed to convert 1 ꢁmol DHK into KMF at 30 °C in the
FLS buffer within one minute. Further dynamic analysis showed that the TrxAꢀHisꢀAtFLS1
possessed a MichaelisꢀMenten constant of 0.072±0.052 mM. These data indicated that the
catalytic efficiency of the TrxAꢀHisꢀAtFLS1 was relatively high enough for subsequent
experiments.
Development of the in vitro multienzyme synthetic system. To produce KMF in a
27, 28
single reaction tube, we analyzed the published buffer components for F3H and FLS
and found that both of them are very similar except for the pH value and the concentrations
of αꢀKG and FeSO4. In the beginning, we arbitrarily fixed the pH value at 7.2 and the
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concentrations of αꢀKG and FeSO4 at 8.2 mM and 0.01 mM, respectively, to explore the
possibility that KMF might be synthesized from NRN by AtF3H and AtFLS1 in a single
reaction tube. The reaction was incubated at 30 °C for 3 h. Polyamide TLC analysis
indicated that the reaction produced two new spots with a migration distance similar to that
of DHK and KMF, respectively (Figure 3C). HPLC analysis demonstrated that there were
two new peaks at 12.50 min and 20.46 min, identical to that of DHK and KMF,
respectively (Figure 3D). Further ESIꢀMS analysis confirmed that these two new molecules
yielded quasiꢀmolecular ion peaks [MꢀH]ꢀ at m/z 287.1000 and 285.1000, respectively
(Figure 4B and 4C). These data indicated that we set up an in vitro multienzyme synthetic
system to produce KMF in a single reaction tube.
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Optimization of the in vitro multienzyme synthetic system. To improve the yield of
KMF, we optimized the system by exploring a series of parameters. First, we prepared a
panel of KMF synthetic buffers as listed in Table 1 and compared their efficiency in
producing KMF to find the optimum pH value and concentrations of αꢀKG and FeSO4. The
reaction was incubated at 30 °C with stirring for 3 h. Polyamide TLC analysis showed that
the KMF synthetic buffer 7 demonstrated the highest yield when compared to others (P <
0.01) (Figure 5A and 5E). These data indicated that a relatively lower pH value and a
higher concentration of αꢀKG are beneficial to the synthesis of KMF, whereas the
concentration of ferrous ion is unimportant even though its presence is critical 27, 28
.
The next parameter to be examined was the reaction temperature. First, we examined a
series of temperatures from 25 °C to 45 °C for both recombinant enzymes,
TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1. Our data demonstrated that the enzymatic activity
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of TrxAꢀHisꢀAtF3H increased significantly (P < 0.01) with a rise in temperature and
reached a plateau at 35 °C (Figure 5B and 5F), whereas that of TrxAꢀHisꢀAtFLS1 increased
continuously even up to 45 °C (Figure 5C and 5G). However, when we examined the
temperature parameter for the single reaction system containing both TrxAꢀHisꢀAtF3H and
TrxAꢀHisꢀAtFLS1, we found that when the reaction temperature increased, the yield of
KMF demonstrated a significant increase initially (P < 0.01), reached a peak at 40 °C, and
then started to decrease, as shown in Figure 5D and 5H. The data indicated that 40 °C is the
optimum reaction temperature for this system.
Then, we investigated the dependence of the KMF yield on the reaction time. As shown
in Figure 5I and 5L, the yield of KMF increased significantly (P < 0.01) within the first 30
min and reached a plateau at 40 min. The data demonstrated that the optimum reaction time
is 40 min and further extension of the reaction time is unnecessary for the yield of KMF.
The subsequent parameter to be explored was the ratio of these two recombinant
enzymes. The total protein amount was fixed at 4 ꢁg in a reaction volume of 100 ꢁL. As
shown in Figure 5J and 5M, when the ratio of TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1 was
ranged from 1:3 to 3:1, the yield demonstrated a slight increase and then a slight decrease
and the optimum ratio was 1:1.
The last parameter to be investigated was the total amount of recombinant enzymes.
When we explored this parameter, we set the ratio of TrxAꢀHisꢀAtF3H and
TrxAꢀHisꢀAtFLS1 at 1:1 and other parameters at their optimum values. As expected, the
yield of KMF increased significantly (P < 0.01, P < 0.05) with a rise in the total amount of
recombinant enzymes and reached a plateau at 5 ꢁg in a reaction volume of 100 ꢁL (Figure
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5K and 5N). After optimization, the yield of KMF increased significantly (P < 0.01) by
1.88 folds and was 37.55 ± 1.62 mg/L (Figure 6). The conversion rate from NRN to KMF
was 55.89% ± 2.74%.
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Taken together, KMF was produced most efficiently when the system contains 8.2 mM
αꢀketoglutaric acid, 0.01 mM ferrous ion, 25 ꢁg/mL TrxAꢀHisꢀAtF3H, 25 ꢁg/mL
TrxAꢀHisꢀAtFLS1, and 10% glycerol in 100 mM TrisꢀHCl (pH 7.2), and the reaction was
carried out at 40 °C for 40ꢀ50 min.
In the current study, we cloned two key genes involved in the synthetic pathway of KMF
and purified the expression products. By analysis of the published work, we designed and
optimized an in vitro multienzyme synthetic system to produce KMF in a single reaction
tube. The yield of KMF was as high as 37.55 ± 1.62 mg/L. Compared with the yield of
KMF reported by Duan, et al 24, our system produced less KMF. The relatively low yield of
our synthetic system might result from the comparatively low efficient enzymes, indicating
that our system remains to be further optimized. Future optimization mainly includes
screening for high efficient F3H and FLS enzymes from other plants, mutation of f3h and
fls1 genes, and immobilization of the recombinant enzymes onto inert carriers.
There are obvious advantages for our in vitro multienzyme synthetic system. Firstly, it is
timeꢀ and laborꢀsaving with a reaction time of only 40 min. Secondly, the reaction
conditions are easy to control accurately and make further optimization because all
components of the system is clear and there is no complex physiological regulation as
occurred in the microbial cell factory. Thirdly, it is much easier to purify desired products
from the cellꢀfree multienzyme synthetic system than from cell factory because of the
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simplicity of the components in the system. Fourthly, the reaction system is highly
costꢀeffective because the components of the system are cheap chemicals or prokaryotically
expressed recombinant proteins. Fifthly, the system is easy to scale up and therefore
possesses huge industrialization potential. In addition, this system provides a guide for
other secondary metabolites to produce economically.
ꢀ
AUTHOR INFORMATION
Corresponding Author
*Tel: +86ꢀ514ꢀ87993623. Fax: +86ꢀ514ꢀ87991747. Eꢀmail: zhangxinyue@yzu.edu.cn,
xyzhang_1@163.com.
ORCID
Xinyue Zhang: 0000ꢀ0003ꢀ4290ꢀ4289
Author Contributions
# Z. Z. and Y. H. contributed equally to this work and performed biological and synthetic
experiments, Y. H. and L. C. carried out TLC detection. L. D. and L. C. performed HPLC
and MASS detection, Y. L. and Y. N. performed molecular cloning. Z. Z. and X. Z.
analyzed the data, X. Z. conceived and designed this work and wrote the paper.
Funding
This work was financially supported by Yangzhou University SpeciallyꢀAppointed
Professor Startꢀup Funds, Jiangsu SpeciallyꢀAppointed Professor Startꢀup Funds, Six Talent
Peaks Project in Jiangsu Province (Grant No. 2014ꢀSWYYꢀ016), Topꢀnotch Academic
Programs Project of Jiangsu Higher Education Institutions (TAPP), A Project Funded by
the Priority Academic Program Development of Jiangsu Higher Education Institutions
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(Veterinary Medicine).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank the Testing Center of Yangzhou University for technical support.
ꢀ
ꢀ
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Figure Captions
Figure 1. Schematic representation for the in vitro biosynthesis of kaempferol from
naringenin. F3H, flavanone 3ꢀhydroxylase; FLS, flavonol synthase.
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Figure 2. SDSꢀPAGE Analysis of the purified recombinant proteins. The Atf3h and Atfls1
genes were cloned into pETꢀ32a(+) and expressed in Escherichia coli BL21(DE3). The
recombinant proteins TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1 were purified using a
NiꢀIDA agarose resin. After purification, the proteins showed a high purity on a 10%
SDSꢀPAGE gel. M, protein markers; lane 1, TrxAꢀHisꢀAtF3H; lane 2, TrxAꢀHisꢀAtFLS1.
Figure 3. Determination of the enzyme activities of the purified recombinant proteins by
polyamide TLC and HPLC. (A) Analysis of TrxAꢀHisꢀAtF3Hꢀcatalyzed reaction by
polyamide TLC. 1, reaction mixture. (B) Analysis of TrxAꢀHisꢀAtFLS1ꢀcatalyzed reaction
by polyamide TLC. 1, reaction mixture. (C) Analysis of the in vitro multienzyme synthetic
reaction. 1, reaction mixture. (D) HPLC analysis of reaction mixtures in (A-C).
Figure 4. ESIꢀMS analysis profile of the compounds in various reaction mixtures. (A)
Exact mass of NRN [MꢀH]ꢀ [m/z]ꢀ (270.9000). (B) Exact mass of DHK [MꢀH]ꢀ [m/z]ꢀ
(287.1000). (C) Exact mass of KMF [MꢀH]ꢀ [m/z]ꢀ (285.1000).
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Figure 5. Optimization of the in vitro multienzyme synthetic system to produce KMF from
NRN. (A, E) Polyamide TLC (A) and gray density (E) analyses for optimizing the pH
value and the concentrations of αꢀKG and FeSO4 as shown in Table 1. Digits 1 – 8 indicate
the buffer numbers in the main text. (B, F) Polyamide TLC (B) and gray density (F)
analyses for determining the optimum reaction temperature of TrxAꢀHisꢀAtF3H. Digits 1 –
5 indicate 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C, respectively. (C, G) Polyamide TLC (C)
and gray density (G) analyses for determining the optimum reaction temperature of
TrxAꢀHisꢀAtFLS1. Digits 1 – 5 indicate 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C,
respectively. (D, H) Polyamide TLC (D) and gray density (H) analyses for optimizing the
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the reaction temperature of the in vitro multienzyme synthetic system. Digits 1 – 5 indicate
25 °C, 30 °C, 35 °C, 40 °C, and 45 °C, respectively. (I, L) Polyamide TLC (I) and gray
density (L) analyses for optimizing the reaction time of the in vitro multienzyme synthetic
system. Digits 1 – 7 indicate 0, 10, 20, 30, 40, 50, and 60 min, respectively. (J, M)
Polyamide TLC (J) and gray density (M) analyses for exploring the optimum ratio of
TrxAꢀHisꢀAtF3H and TrxAꢀHisꢀAtFLS1 in the synthetic system. (K, N) Polyamide TLC
(K) and gray density (N) analyses for optimizing the total amount of recombinant enzymes
in the synthetic system. *P < 0.05; **P < 0.01.
Figure 6. Comparison of the yield before and after optimization. (A, C) Polyamide TLC (A)
and gray density (C) analyses for comparison of the yield before and after optimization. (B)
HPLC chromatograms of the reaction before and after optimization. (D) Analysis of the
yield before and after optimization using HPLC data. 1, before optimization; 2, after
optimization. **, P < 0.01.
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Tables
Table 1. KMF synthetic buffers
Buffer #
1
2
3
4
5
6
7
8
pH
7.2
0.174
0.1
100
20
7.5
0.174
0.1
100
20
7.2
8.2
0.1
100
20
7.5
8.2
0.1
100
20
7.2
7.5
7.2
8.2
0.01
100
20
7.5
8.2
0.01
100
20
αꢀKG (mM)
FeSO4 (mM)
TrisꢀHCl (mM)
Sodium ascorbate (mM)
Glycerol (%)
0.174
0.01
100
20
0.174
0.01
100
20
10
10
10
10
10
10
10
10
Table 2. Oligonucleotide primers used in the current study
Sequence (5'ꢀ3')
Purpose
AAGGATCCATGACTCGTCTCGCTCGTGA
Forward primer for cloning Atf3h gene from Arabidopsis
thaliana. BamHI site is attached for cloning into pET32a.
Reverse primer for cloning Atf3h gene from A. thaliana.
EcoRI site is attached for cloning into pET32a.
AAGAATTCCTAAGCGAAGATTTGGTCGA
AAGGATCCATGGAGGTCGAAAGAGTCCA Forward primer for cloning Atfls1 gene from A. thaliana.
BamHI site is attached for cloning into pET32a.
AAGAATTCTCAATCCAGAGGAAGTTTAT
Reverse primer for cloning Atfls1 gene from A. thaliana.
EcoRI site is attached for cloning into pET32a.
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Notes: The restriction site attached for cloning is italicized within the primer sequence.
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Figure 1. Schematic representation for the in vitro biosynthesis of kaempferol from naringenin. F3H,
flavanone 3-hydroxylase; FLS, flavonol synthase.
40x9mm (600 x 600 DPI)
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Figure 2. SDS-PAGE Analysis of purified fusion proteins. The f3h and fls1 genes were cloned into pET-32a(+)
and expressed in Escherichia coli BL21(DE3). The fusion proteins TrxA-His-F3H and TrxA-His-FLS1 were
purified using a Ni-IDA agarose resin. After purification, the proteins showed a high purity on a 10% SDS-
PAGE gel. M, protein markers; lane 1, TrxA-His-F3H; lane 2, TrxA-His- FLS1.
49x83mm (300 x 300 DPI)
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Figure 3. Determination of enzyme activities of the purified fusion proteins by polyamide TLC and HPLC. (A)
Analysis of TrxA-His-F3H-catalyzed reaction by polyamide TLC. 1, reaction mixture. (B) Analysis of TrxA-His-
FLS1-catalyzed reaction by polyamide TLC. 1, reaction mixture. (C) Analysis of the in vitro multienzyme
synthetic reaction. 1, reaction mixture. (D) HPLC analysis of reaction mixtures in (A-C).
183x420mm (300 x 300 DPI)
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