Development and Optimization of an In Vitro Multienzyme Synthetic

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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 Downloaded from http://pubs.acs.org on July 19, 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 & Agri-Product Safety, Yangzhou University,

Yangzhou, Jiangsu 225009, China. 4

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases

and Zoonoses, Yangzhou, Jiangsu 225009, China.

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ABSTRACT

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An in vitro multienzyme synthetic system was developed and optimized to efficiently

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produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the

4

biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and

5

overexpressed in Escherichia coli. The recombinant proteins were purified through affinity

6

chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase,

7

respectively, followed by development of an in vitro synthetic system for producing

8

kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 25

9

µg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2).

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When the reaction was carried out at 40 °C for 40-50 min, the yield of kaempferol was

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37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%.

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Overall, this system provides a promising and efficient approach to produce kaempferol

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economically.

14

15

16

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Key Words: kaempferol, in vitro synthesis, optimization, flavanone 3-hydroxylase,

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flavonol synthase.

19

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Flavonoids are a large group of plant-derived natural products and can be further classified

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into six major subgroups depending on the variations in the heterocyclic C-ring: the

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catechins or flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones 1-3.

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These compounds show a wide range of biological activities

26

of studies on these secondary metabolites 7-12.

27

Kaempferol (KMF), also known as robigenin or 3,4’,5,7-tetrahydroxyflavone, is a natural

28

flavonol possessing numerous pharmacological and biological properties including

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antioxidant, anti-inflammatory, anticancer, antimicrobial, cardioprotective, neuroprotective,

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antidiabetic, anti-osteoporotic, estrogenic/anti-estrogenic, anxiolytic, analgesic, and

31

anti-allergic activities

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However, the cost of KMF production is high due to the very low content of KMF in plants

33

15, 16

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producing KMF.

35

Many methods have been developed to synthesize flavonoids

36

synthesis of flavonoids often requires multiple steps, toxic reagents, and extreme reaction

37

conditions

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another crucial challenge in chemical synthesis. Hence, chemical synthesis is not

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economically feasible for large-scale production of flavonoids 1.

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Since the biosynthetic pathway of flavonoids has been successfully elucidated in plants 2, it

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is a promising alternative strategy to synthesize these natural compounds using a microbial

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cell factory. Recently, some flavonoids have been successfully biosynthesized by

INTRODUCTION

3-6

, leading to quite a number

13, 14

. KMF is mainly produced from traditional plant extraction.

. Therefore, it is crucial to develop alternative strategies to reduce the cost for

17, 18

. However, chemical

1, 17

. In addition, the chiral synthesis to produce active flavonoid compounds is

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engineered microbes, mainly including Escherichia coli and Saccharomyces cerevisiae 19-23.

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For example, Duan, et al have constructed a microbial cell factory for KMF production by

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introducing a biosynthetic pathway of KMF into the budding yeast S. cerevisiae and

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achieved the highest production of KMF at 66.29 mg/L

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microbes produce desired products due to the well-known complexity of a cellular system,

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incompatibility of artificially synthesized genetic elements and hosts, inhibition of host

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cells by target products, and instability of an engineered biosystem 25.

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Another promising alternative strategy is to produce flavonoids using an in vitro

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multienzyme synthetic system. Recently, Cheng, et al have reported the successful

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multienzyme synthesis of enterocin polyketides by assembling a complete type II

53

polyketide synthase enzymatic pathway to natural products in a single reaction vessel

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This cell-free synthetic biotechnology overcomes the disadvantages of a microbial cell

55

factory and is economically feasible for commercial-scale production of flavonoids 25.

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As we know, there are two key enzyme genes, f3h and fls1, involved in the biosynthetic

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pathway of KMF 1, 2. These two genes encode flavanone 3-hydroxylase (F3H) and flavonol

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synthase (FLS), respectively. In this article, we report a detailed study of the in vitro

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multienzyme synthesis of KMF in a single reaction tube from cheap chemicals including

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the substrate naringenin (NRN). Firstly, we cloned Atf3h and Atfls1 genes from Arabidopsis

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thaliana and expressed them in E. coli BL21(DE3), followed by the purification of

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recombinant proteins. Then, we determined the enzyme activities of the purified

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recombinant proteins and their kinetic parameters. Finally, we developed a single reaction

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system to produce KMF and optimized a series of reaction parameters including pH value,

24

. However, not all engineered

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.

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reaction temperature and time, and total amount and ratio of two recombinant enzymes to

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find the most efficient and economic conditions for KMF production. To our best

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knowledge, there is no report on the development of an in vitro multienzyme synthetic

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system to produce KMF.

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MATERIALS AND METHODS

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Plants, Bacterial Strains, Plasmids, and Chemicals. Four-week-old seedlings of

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Arabidopsis thaliana were a gift from Dr. Lin Wang of Yangzhou University. Escherichia

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coli DH5α and BL21(DE3) were purchased from Beijing CoWin Biotech Co., Ltd. (Beijing,

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China) and used for plasmid maintenance and gene expression, respectively. The bacteria

74

were cultured in Luria–Bertani (LB) broth or on agar supplemented with 100 µg/mL

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ampicillin when necessary. Prokaryotic expression vector pET-32a(+) (Novagen) was used

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for cloning purposes. Authentic naringenin (NRN), dihydrokaempferol (DHK), kaempferol

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(KMF), and other chemicals are products of Sigma-Aldrich (St. Louis, MO). Restriction

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enzymes were bought from New England Biolabs (Hertfordshire, UK). T4 DNA ligase was

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a product of Vazyme Biotech Co., Ltd (Nanjing, China). TRIzol® Reagent and Isopropyl

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β-D-1-thiogalactopyranoside (IPTG) were purchased from Thermo Fisher Scientific Inc.

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(MA, USA).

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Buffers used. The buffers used for enzyme assays in this study were prepared based on

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the published recipes with slight modifications 27, 28. F3H buffer contains 100 mM Tris-HCl

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(pH 7.5), 0.4% sodium ascorbate, 10% glycerol, 0.174 mM α-ketoglutaric acid (α-KG), and

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0.1 mM FeSO4. FLS buffer contains 100 mM Tris-HCl (pH 7.2), 0.4% sodium ascorbate,

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10% glycerol, 8.2 mM α-KG, 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 Tris-HCl (pH 7.2 or 7.5),

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10% glycerol, 0.4% sodium ascorbate, 0.174 mM or 8.2 mM α-KG, and 0.01 mM or 0.1

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mM FeSO4, as shown in Table 1.

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Isolation and reverse transcription of total RNA and construction of plasmids. Total

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RNA was isolated from seedlings of A. thaliana using TRIzol® Reagent and reversely

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transcribed into complementary DNA (cDNA) using the SuperRT cDNA Synthesis Kit

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(Beijing CoWin Biotech Co., Ltd., Beijing, China) according to the manufacturer’s

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protocol. The cDNAs were used for cloning Atf3h (Genbank accession no.

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NM_001203121.1) and Atfls1 (Genbank accession no. NM_120951.3) genes. All primers

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used in a specific polymerase chain reaction (PCR) are listed in Table 2 and were

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synthesized by GENEWIZ, Suzhou, China. The Atf3h and Atfls1 genes were

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PCR-amplified and cloned into pET-32a(+) following a standard protocol as described

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elsewhere

100

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

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transformed into BL21(DE3) and colonies were inoculated into LB broth containing 100

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µg/mL ampicillin. Three milliliters of overnight culture were reinoculated into 150 mL LB

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broth containing 100 µg/mL ampicillin and cultured at 220 rpm and 37 °C until the optical

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density at 600 nm was between 0.4 – 0.6. Then, 0.2 mM IPTG was added into the culture to

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induce gene expression at 20 – 22 °C for 3 h. The bacteria were harvested and resuspended

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in 1/20 volume of bacterial lysis buffer (50 mM Tris-Cl, pH8.0, 0.1% Triton X-100, 1 mM

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EDTA, 10% Glycerol, 150 mM NaCl, 0.5 mM DTT, and 0.1 mM PMSF, 1 µg/mL aprotinin,

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1 µg/mL leupeptin, and 1 µg/mL pepstatin). The suspension was sonicated and

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centrifugated at 13000 g for 10 min at 4 °C. TrxA-His-tagged recombinant proteins were

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purified from the supernatant using Ni-IDA Agarose Resins (Beijing CoWin Biotech Co.,

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Ltd., Beijing, China). Purification was performed according to the manufacturer’s protocol.

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The purity of the purified proteins was analyzed on a 10% sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS-PAGE) gel and the bands were visualized by

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coomassie blue staining.

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Enzyme assays. The enzyme assays were analyzed according to the reported protocols 27, 28

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with slight modifications

, and the final volumes for all assays were 100 µL. In brief,

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the F3H assay contained 0.5 mM NRN and 3.3 µg TrxA-His-AtF3H in F3H Buffer, and the

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FLS assay included 0.5 mM DHK and 1.9 µg TrxA-His-AtFLS1 in FLS Buffer. The assays

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were incubated at 30 °C in open 2-mL tubes with continuous shaking for 45 min or 3 h,

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respectively. The assays were terminated with 100 µL of ethyl acetate and 10 µL of acetic

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acid. Two hours later, the organic phases were transferred to 1.5 mL tubes for vacuum

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freeze drying in a FreeZone 1 Liter Benchtop Freeze Dry System (Labconco, MO, USA).

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The isolated flavonoids were then redissolved in methanol and subjected to polyamide thin

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layer chromatography (TLC), high performance liquid chromatography (HPLC), and

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electrospray ionization mass spectrometry (ESI-MS) analyses. The dots on the TLC plates

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were visualized under a Tanon-2500 Gel Imaging System (Tanon Science & Technology

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Co. Ltd., Shanghai, China).

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To determine the enzyme activity of the TrxA-His-AtF3H, the concentration of NRN

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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 TrxA-His-AtFLS1, the concentration of DHK was changed into 0.08 mM and

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the assay was incubated at 30 °C for 30 min. The calibration curves of authentic DHK and

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KMF were made based on the HPLC data and the product amount was thus calculated. The

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enzyme activity was evaluated from three individual experiments and expressed as the

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amount of the recombinant protein needed to convert 1 µmol substrate into the product at

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30 °C within one minute. The values were expressed as mean ± standard deviation.

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Determination of the Michaelis-Menten constant Km of the recombinant enzymes.

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To determine the Michaelis-Menten constant of the TrxA-His-AtF3H, a series of NRN

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concentrations were prepared in the F3H assay, including 0.04 mM, 0.06 mM, 0.08 mM,

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0.10 mM, and 0.12 mM. To determine that of the TrxA-His-AtFLS1, the concentrations of

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DHK included 0.01 mM, 0.02 mM, 0.04 mM, 0.08 mM, and 0.16 mM in the FLS assay,

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respectively. Then, the Michaelis-Menten constants were calculated using the data analysis

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software OriginPro (version 9.0.0). The measurements were repeated three times and the

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Km value of each recombinant enzyme was expressed as mean ± standard deviation.

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Development and optimization of an in vitro multienzyme synthetic system for KMF

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production. Based on the components in F3H and FLS buffers, a KMF multienzyme

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synthetic system was developed to produce KMF from NRN in a single reaction tube. The

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system contained 0.5 mM NRN, 2.0 µg TrxA-His-AtF3H, and 2.0 µg TrxA-His-AtFLS1 in

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100 µL of various KMF synthetic buffers as shown in Table 1. The reactions were

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incubated at 30 °C with continuous shaking for 3 h and terminated by addition of 100 µL of

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ethyl acetate and 10 µL of acetic acid. Two hours later, the organic phase was subjected to

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vacuum freeze drying. The powder was dissolved in methanol and analyzed by polyamide

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TLC and HPLC-MS.

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After initial setup of the synthetic system, the reaction parameters were further

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investigated to maximize the efficiency of KMF production. Briefly, the pH value and the

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concentrations of α-KG and FeSO4 in the reaction system were optimized according to

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Table 1 by incubating the reaction tubes at 30 °C for 3 h. After the pH value and the

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concentrations of α-KG and FeSO4 were fixed at an optimum value, the reaction time was

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optimized by incubating the reaction mixture at 30 °C with stirring for 0, 10, 20, 30, 40, 50,

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and 60 min. Next, the optimum reaction temperature was explored by incubating the

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reaction mixture at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C for an optimum reaction time.

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Then, the optimum ratio of the recombinant enzymes was analyzed by setting the ratio of

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TrxA-His-AtF3H and TrxA-His-AtFLS1 at 1:3, 1:2, 1:1, 2:1, and 3:1. Finally, the total

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amount of TrxA-His-AtF3H and TrxA-His-AtFLS1 was optimized by adding 1 µg, 2 µg, 3

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µg, 4 µg, 5 µg, and 6 µg of the enzymes at an optimum ratio into a 100 µL of reaction

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mixture. All measurements were repeated three times and the results were expressed as

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mean ± standard deviation. TLC analysis. Analysis of flavonoid compounds by TLC was widely reported elsewhere

168 169

30

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(Sinopharm Chemica Reagent Co., Ltd, Shanghai, China) in comparison to authentic

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samples. The sample-loaded TLC plates were run in a solvent system consisting of

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chloroform/methanol/ethyl acetate/formic acid at a ratio of 5.0:1.5:1.0:0.5. Then, the plates

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were sprayed with 1% ethanolic solution of aluminum chloride (AlCl3) and subjected to

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UV illumination at 254 nm to visualize spots. The gray value of each spot was analyzed by

. In brief, the reaction products were primarily analyzed by TLC in polyamide 6 plates

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the image processing software ImageJ 1.51j8.

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HPLC-MS analysis. The HPLC-MS was performed to analyze the synthesized

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flavonoid compounds according to the published work 31 with slight modifications. In brief,

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the samples were processed sequentially through 0.45 µm and 0.22 µm filters and analyzed

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using an Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole

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LC/MS system. Data were collected and analyzed using Agilent MassHunter Workstation

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(version B.02.00) and MassHunter Quantitative analysis software (version B.01.04),

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respectively. The samples were separated at 30 °C using an Agilent C18 column (150×4.6

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mm, 5 µm, Thermo Fisher Scientific. Inc., CA, USA). The separation was achieved using a

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combination of two liquids, distilled water and acetonitrile. The column was eluted at 0.2

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mL/min by a 10% – 85% (v/v) gradient of acetonitrile in water under the following

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conditions: 0 – 10 min, a linear gradient of 10% – 25% (v/v) acetonitrile concomitant with

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90-75% water; 10 – 35 min, a linear gradient of 25% – 50% acetonitrile; 35 – 45 min, a

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linear gradient of 50% – 85% acetonitrile; 45 – 50 min, a linear gradient of 85% – 10%

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acetonitrile; 50 – 60 min, 10% acetonitrile in 95% water. Absorbance of the eluate was

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monitored from 200 nm to 800 nm. Single wavelength chromatograms at 280 nm, 320 nm,

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and 370 nm were extracted using the software Xcalibur v2.0.7. The reaction products were

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quantified by comparing the peak area of a compound in the reaction sample with that of a

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known amount of an authentic compound.

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Isolation of KMF. The isolation of KMF was a modification of the method developed

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by Leite, et al. 32. In brief, the reaction was terminated by adding an equal volume of ethyl

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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 re-dissolved in methanol and processed through a

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0.45 µm filter. Next, the solution was subjected to chromatography over a Sephadex LH-20

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column eluted with methanol at 0.5 mL/min. The fractions were collected and combined

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together according to the results of TLC analysis. The isolated KMF was further confirmed

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by HPLC-MS analysis as described above and used for calculating the yield.

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RESULTS AND DISCUSSION

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Cloning, expression and purification of the key enzymes for KMF biosynthesis. As

204

shown in Figure 1, AtF3H and AtFLS1 are two key enzymes for the biosynthesis of KMF

205

in plants. To develop an in vitro system for multienzyme synthesis of KMF, we first cloned

206

Atf3h and Atfls1 genes from A. thaliana into prokaryotic expression vectors.

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Generally speaking, soluble expression of recombinant proteins in bacteria maintains its

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enzymatic activity. To increase the relative amount of soluble recombinant proteins

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expressed in E. coli, we first compared the difference between pET-32a(+) and pGEX-5X-1,

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two common prokaryotic expression vectors, in expressing recombinant proteins AtF3H

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and AtFLS1. We found that pET-32a(+) provided a higher level of soluble AtF3H and

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AtFLS1 proteins, which possibly resulted from the role of TrxA fragment in pET-32a(+) in

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promoting soluble expression. Therefore, we chose pET-32a(+) as a vector for constructing

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recombinant plasmids. Sequence analysis verified that there was no mutation occurring in

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the PCR-generated DNA fragments.

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Then, we transformed the recombinant plasmids into E. coli strain BL21(DE3) and

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optimized the expression conditions mainly by changing two important induction

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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

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bacteria expressed a high amount of soluble recombinant proteins. Then, we induced the

221

expression for 3 h and purified the recombinant proteins by affinity chromatography

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through a nickel iminodiacetic acid (Ni-IDA) agarose resin. All the procedures for protein

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purification were carried out at 4 °C to maintain the enzymatic activities of the recombinant

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proteins to the highest extent. As shown in Figure 2, the purity of the purified recombinant

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proteins, TrxA-His-AtF3H and TrxA-His-AtFLS1, was >95% on a 10% SDS-PAGE gel,

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which were pure enough for subsequent enzyme assays.

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Determination of the enzyme activities and the Michaelis-Menten constants of the

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purified recombinant proteins. To determine the flavanone 3-hydroxylase activity of the

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TrxA-His-AtF3H protein, we set up a reaction system containing 0.5 mM NRN and 3.3 µg

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purified recombinant protein in F3H buffer. The reaction products were analyzed by

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polyamide TLC and HPLC-MS. As shown in Figure 3A, there was a new spot on a

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polyamide TLC plate with a migration distance similar to that of DHK. The HPLC-MS

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analysis demonstrated that the new chemical showed a retention time of 12.50 min (Figure

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3D) and yielded a quasi-molecular ion peak [M-H]- at m/z 287.1000 (Figure 4B), identical

235

to those of DHK. These data indicated that the new chemical was DHK and thus the

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purified TrxA-His-AtF3H protein possessed a flavanone 3-hydroxylase activity. Enzyme

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activity analysis indicated that 14.99 ± 0.34 µg of the TrxA-His-AtF3H was needed to

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convert 1 µmol NRN into DHK at 30 °C in the F3H buffer within one minute. Further

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dynamic

240

Michaelis-Menten constant of 0.081 ± 0.025 mM.

analysis

demonstrated

that

this

recombinant

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possessed

a

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To measure the flavonol synthase activity of the TrxA-His-AtFLS1 protein, we

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established a 100-µL reaction system containing 0.75 mM DHK as a substrate and 1.9 µg

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purified protein in FLS buffer. Polyamide TLC analysis indicated that the reaction

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produced a new spot with a similar migration distance to that of KMF (Figure 3B). HPLC

245

analysis demonstrated that the new chemical possessed a retention time of 20.46 min

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(Figure 3D) and further confirmation by ESI-MS generated a quasi-molecular ion peak

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[M-H]- at m/z 285.1000 (Figure 4C), identical to those of KMF. These data demonstrated

248

that the new chemical molecule was KMF and the purified TrxA-His-AtFLS1 protein had a

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flavonol synthase activity. However, due to an excessive amount of the substrate (0.5 mM)

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and less amount of the TrxA-His-AtFLS1 in the FLS assay, the peak for KMF was

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comparatively low in the HPLC chromatograms (Figure 3D), seemingly indicating a low

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catalytic efficiency of the TrxA-His-AtFLS1. Therefore, we further analyzed the enzyme

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activity of the TrxA-His-AtFLS1. The data demonstrated that 10.78 ± 0.61 µg of the

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purified recombinant protein was needed to convert 1 µmol DHK into KMF at 30 °C in the

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FLS buffer within one minute. Further dynamic analysis showed that the TrxA-His-AtFLS1

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possessed a Michaelis-Menten constant of 0.072±0.052 mM. These data indicated that the

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catalytic efficiency of the TrxA-His-AtFLS1 was relatively high enough for subsequent

258

experiments.

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Development of the in vitro multienzyme synthetic system. To produce KMF in a 27, 28

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single reaction tube, we analyzed the published buffer components for F3H and FLS

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and found that both of them are very similar except for the pH value and the concentrations

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of α-KG and FeSO4. In the beginning, we arbitrarily fixed the pH value at 7.2 and the

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concentrations of α-KG and FeSO4 at 8.2 mM and 0.01 mM, respectively, to explore the

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possibility that KMF might be synthesized from NRN by AtF3H and AtFLS1 in a single

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reaction tube. The reaction was incubated at 30 °C for 3 h. Polyamide TLC analysis

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indicated that the reaction produced two new spots with a migration distance similar to that

267

of DHK and KMF, respectively (Figure 3C). HPLC analysis demonstrated that there were

268

two new peaks at 12.50 min and 20.46 min, identical to that of DHK and KMF,

269

respectively (Figure 3D). Further ESI-MS analysis confirmed that these two new molecules

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yielded quasi-molecular ion peaks [M-H]- at m/z 287.1000 and 285.1000, respectively

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(Figure 4B and 4C). These data indicated that we set up an in vitro multienzyme synthetic

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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

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KMF, we optimized the system by exploring a series of parameters. First, we prepared a

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panel of KMF synthetic buffers as listed in Table 1 and compared their efficiency in

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producing KMF to find the optimum pH value and concentrations of α-KG and FeSO4. The

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reaction was incubated at 30 °C with stirring for 3 h. Polyamide TLC analysis showed that

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the KMF synthetic buffer 7 demonstrated the highest yield when compared to others (P