Name: biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade
Uploaded: 2019-08-14T12:30:00
Description: Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

This work was financially supported by Yangzhou University Specially-Appointed Professor Start-up Funds, Jiangsu Specially-Appointed Professor Start-up Funds, Six Talent Peaks Project in Jiangsu Province (Grant No. 2014-SWYY-016), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Veterinary Medicine). We thank the Testing Center of Yangzhou University for HPLC and MS analyses of flavonoids.

1. Isolate total RNA from plant tissues26,27
<ol>
	<li>Homogenize the plant tissues.
	<ol>
		<li>Collect 100 mg of a fresh plant tissue (e.g., 4-week-old seedlings from Arabidopsis thaliana). Freeze the tissue and a pestle and mortar with liquid nitrogen, followed by grinding the tissue into powder.</li>
		<li>Add 1 mL of RNA isolation reagent (see Table of Materials) into the mortar. The reagent will be frozen immediately. Homogenize ...

<ol>
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Quite a number of studies are focused on the derivation of flavonols due to their potential application in health care and food industry. However, traditional plant extraction using organic solvents and chemical synthesis possess intrinsic disadvantages, which restrict their use in the production of flavonols. Here, we report a detailed method for producing a flavonol from a flavanone in one pot by establishing an in vitro bienzymatic cascade. The critical steps in this protocol are: 1) obtaining pure recombinant enzymes...

Flavonols are a major subclass of plant flavonoids and are involved in plant development and pigmentation1,2,3. More importantly, these compounds possess a wide range of health-beneficial activities, such as anti-cancer4,5, anti-oxidative6, anti-inflammatory7, antiobesity8, anti-hypertensive9, and memory recall properties10, leading to a large number of studies on these plant-derived secondary metabolites. Traditionally, these compounds are mainly derived from plant extraction using organic solvents. However, due to their very low contents in plants11,12,13, the production cost for most flavonols remains high, which imposes great restrictions on their application in healthcare and the food industry.
During the past decades, scientists have developed quite a number of methods to derive flavonoids14,15. However, chemical synthesis of these complicated molecules possesses a variety of intrinsic disadvantages16. It requires not only toxic reagents and extreme reaction conditions, but also many steps to produce a target flavonoid compound14,17. Moreover, another important challenge in this strategy is the chiral synthesis of active flavonoid molecules. Therefore, it is not an ideal strategy to produce flavonoids at a commercial scale via chemical synthesis16,17.
Recently, scientists have developed a promising alternative strategy to produce these complicated natural compounds by engineering microbes with a pathway for flavonoid biosynthesis18,19,20,21,22, which has been successfully deciphered in plants23. For example, Duan et al. introduced a biosynthetic pathway into the budding yeast Saccharomyces cerevisiae to produce kaempferol (KMF)24. Malla et al. produced astragalin, a glycosylated flavonol, by introducingflavanone 3-hydroxylase (f3h), flavonol synthase (fls1), and UDP-glucose:flavonoid 3-O-glucosyltransferase UGT78K1 genes into Escherichia coliBL21(DE3)17. Even though there are quite a few paradigms, not all genetically engineered microbes produce the products of interest due to the complexity of a cellular platform, the incompatibility between artificially synthesized genetic elements and hosts, the inhibitory effect of target products against host cells, and the instability of an engineered cellular system itself16.
Another promising alternative strategy for flavonoid production is to establish a multienzymatic cascade in vitro. Cheng et al. have reported that enterocin polyketides can be successfully synthesized by assembling a complete enzymatic pathway in one pot25. This cell-free synthetic strategy circumvents the restrictions of a microbial production factory and thus is feasible for producing some flavonoids in large quantity16.
Recently, we have successfully developed a bienzyme synthetic system to convert naringenin (NRN) into KMF in one pot16. Here, we describe this system in great details and the methods involved in analyzing the products. We also present two examples that use this system to produce KMF from NRN and quercetin (QRC) from eriodictyol (ERD). In addition, we discuss crucial steps of this method and future research directions in the biosynthesis of flavonoids.

<table>
	<tbody>
		<tr>
			<td>2&#215; Pfu MasterMix</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0717A</td>
			<td>PCR amplification of genes with high fidelity</td>
		</tr>
		<tr>
			<td>Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>an equipment for analysis of flavonoids by HPLC/MS</td>
		</tr>
		<tr>
			<td>Agilent MassHunter Workstation (version B.03.01)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for collection of the data from the Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
		</tr>
		<tr>
			<td>dihydrokaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>91216</td>
			<td>intermediate product for producing kaempferol from naringenin</td>
		</tr>
		<tr>
			<td>dihydroquercetin</td>
			<td>Sichuan Provincial Standard Substance Center for Chinese Herbal Medicine</td>
			<td>PCS0371</td>
			<td>intermediate product for producing quercetin from eriodictyol</td>
		</tr>
		<tr>
			<td>DNA Clean-up Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2301</td>
			<td>purification of PCR-amplified or gel-purified DNA</td>
		</tr>
		<tr>
			<td>eriodictyol</td>
			<td>Shanghai Yuan Ye Biotechnology Co., Ltd.</td>
			<td>B21160</td>
			<td>substrate for producing quercetin</td>
		</tr>
		<tr>
			<td>Escherichia coli BL21(DE3)</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0809</td>
			<td>bacteria strain for expressing target genes</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0808</td>
			<td>bacteria strain for plasmid proliferation</td>
		</tr>
		<tr>
			<td>FreeZone 1 Liter Benchtop Freeze-Dry System</td>
			<td>Labconco Corporation</td>
			<td>7740020</td>
			<td>an equipment for freeze-drying of flavonoids dissolved in organic solvent</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2302</td>
			<td>purification of a DNA band from an agarose gel</td>
		</tr>
		<tr>
			<td>Gel Imaging System</td>
			<td>Shanghai Tanon Science &#38; Technology Co. Ltd.</td>
			<td>Tanon- 
			2500</td>
			<td>an equipment for visualization of DNA band on an agarose gel or flavonoid spot on a polyamide TLC plate</td>
		</tr>
		<tr>
			<td>GenElute Plasmid Miniprep Kit</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>PLN350-1KT</td>
			<td>minipreparation of plasmids</td>
		</tr>
		<tr>
			<td>kaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>60010</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>MassHunter Quanlitative Analysis (version B.01.04)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC/LC/MS data</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-8000-GL</td>
			<td>an equipment for determination of DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>naringenin</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>N5893</td>
			<td>substrate for producing kaempferol</td>
		</tr>
		<tr>
			<td>Ni-IDA Agarose Resin</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0010</td>
			<td>purification of His-tagged fusion proteins</td>
		</tr>
		<tr>
			<td>pET-32a(+)</td>
			<td>Novagen</td>
			<td>69015-3</td>
			<td>plasmid for cloning and expressing target genes</td>
		</tr>
		<tr>
			<td>plasmid sequencing</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>sequencing of recombinant plasmids</td>
		</tr>
		<tr>
			<td>primer synthesis</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>synthesis of PCR primers</td>
		</tr>
		<tr>
			<td>quercetin</td>
			<td>Shanghai Aladdin Biochemical Technology Co.,Ltd.</td>
			<td>Q111273</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>SuperRT cDNA Synthesis Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0741</td>
			<td>synthesis of the first strand of cDNA from total RNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0016</td>
			<td>ligation of an insert into a linearized vector DNA</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td>isolation of total RNA</td>
		</tr>
		<tr>
			<td>Vector NTI Advance</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605099</td>
			<td>a software for PCR primer design and DNA sequence analysis</td>
		</tr>
		<tr>
			<td>Xcalibur v2.0.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC data</td>
		</tr>
	</tbody>
</table>

biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

F3H and FLS1 are two important key enzymes in the conversion of a flavanone into a flavonol in plants as shown in Figure 1. To develop an in vitro biosynthetic system for producing a flavonol from a flavanone, Atf3h (GenBank accession no.NM_114983.3) and Atfls1 (GenBank accession no. NM_120951.3) genes were cloned from the seedlings of 4-week-old A. thaliana into a prokaryotic expression vector pET-32a(+). The recombinant plasmids w...

Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Using this protocol, we can easily produce quite a number of flavonols from flavanones in one pot. This technique is labor-and time-saving, highly cost-effective, and easy to control, so it has a huge industrialization potential. Yes, this method can provide insight into economic production of other secondary metabolites, but it needs some modifications when applied to it.He will usually get nothing or very low yield. My advice is to handle recombinant enzymes on ice and add 10%glycerin to the stock solution of enzymes. That's because only through visual demonstration can a new hand understand the important details and truth affecting the successful execution of the experiment.First prepare buffers. Make two times synthetic buffer by dissolving appropriate amount of TRIS-base, sodium ascorbate, alpha ketoglutaric acid into de-ionized water, and pipette appropriate amount of glycerol to the solution. Use a pipette to add hydrochloric acid to adjust the pH to 7.2, and adjust the volume by adding de-ionized water.Store the two times synthetic buffer at four degrees Celsius for future use. Then make a 100 times stock solution by dissolving ferrous sulfate heptahydrate in deionized water. Make a stock solution of 25 millimolar flavonoid by dissolving flavonoid in methanol, and store the solution at minus 20 degrees Celsius.The most critical step is to set up a synthetic system. To set up a synthetic system to produce a flavanol from a flavanone, first prepare the synthetic system in a two milliliter tube according to the reagents listed in this table. Place the open tube in a shaking heat block at 40 degrees Celsius, at 600 rpm for forty minutes.After that, terminate the reaction by adding 10 microliters of acetic acid, and 100 microliters of ethyl acetate. Two hours later, use a pipette to tranfer the top organic phase to a 1.5 milliliter tube, and place the tube in a hood for air drying at room temperature. To perform TLC analysis, first retrieve the tubes from the hood, and redissolve the flavanoid powder in 160 microliters of methanol in a tube.Prepare authentic flavonoid samples with serial concentrations, by diluting the 25 millimolar flavonoid stock solution in methanol. Load one microliter of the redissolved reaction sample, and one microliter of the authentic flavonoid sample, onto a polyamide plate. Do the same for the other concentrations of authentic flavonoid on the same plate.Then in a fume hood, put the sample loaded plate in a chromatography cylinder filled with a solvent system. Attach the plate to the bottom of the cylinder, and run the plate in the solvent system for 20 minutes. Air dry the plates at room temperature.With a sprayer bottle, spray the plates with 1%ethanolic solution of aluminum chloride, followed by air drying again at room temperature for 30 minutes. Visualize the spots on the plates under a UV light at 254 nanometers, and take images, then on the computer, open the software ImageJ. Click file, open, to open the image to be analyzed.Click the left most rectangular selection tool in the ImageJ user interface. Outline the ROI in the image with the mouse, and press one to label the first ROI. Move the rectangular selection with the mouse right to the next ROI, and press two to label the second ROI.Repeat to label all other ROIs by pressing two. Press three to generate profile plots for all ROIs, in a pop-up window. Then, use the straight line selection tool to drop baselines so as to define a closed area for each peak of interest.Activate the wand tool, by clicking the corresponding icon in the ImageJ user interface. Click inside the peak to display results for all peaks in a pop-up window. Next, in Excel, plot the gray values from the pop-up window against the corresponding flavonoid concentrations, to make a TLC based standard curve of the authentic flavonoid.Then calculate the yield of the flavonoid of interest produced in this protocol, according to the resulting formula. Use a syringe to process each sample sequentially through a 0.45 micrometer and 0.22 micrometer filters. Load the samples into an HPLC-MS system through suction nozzle, and separate the samples at 30 degrees Celsius using a C18 column.Elute the column at one milliliter per minute by a gradient of 10 to 85 volume per cent acetyl nitrile in water, and monitor the absorbance of the eluate from 200 to 800 nanometers. Perform the LC-MS analysis in a negative ion mode, with a drying nitrogen flow of 10 liters per minute at 300 degrees Celsius, in a sheath gas flow of seven liters per minute at 250 degrees Celsius, and collect data using a built in software. To extract single wavelength chromatographs, open the qualitative analysis program, and click file, open datafile.Select the file to be analyzed in the open data file window, and click open to open the file. Right click the mouse in the chromatogram results window and then extract chromatograms in a pop-up menu. In the type list, click on other chromatograms.In the detector combo box, select DAD1, then click okay to display the HPLC results in the chromatogram results window. Click the manual integration icon docked at the top of the chromatogram results window. Draw baseline for the peak required for manual integration analysis with the mouse.Click view, integration peak list, to display the results. Copy the results of the peak areas to Excel, and plot an HPLC based standard curve of the authentic flavonoid against the corresponding flavonoid concentrations, then calculate the yield of the flavonoid of interest produced in this protocol according the resulting formula. Then repeat the same procedures to extract single wavelength chromatograms to analyze the MS data for the exact mass of flavonoid compounds.Click the range select icon on the chromatogram results toolbar. Select the peak of interest. Right click the mouse in the selected range, and click the extract MS spectrum in the pop-up menu to display the results in the MS spectrum results window.To determine whether this in-vitro system can be used for the conversion of a flavanone into a flavonol, NRN was added into the system. There were two new spots emerged on a polyamide TLC plate. One spot showed a migration distance similar to that of DHK, and the others similar to that of KMF.Further analysis by HPLC and LC-MS demonstrated that the new chemical showed a retention time of 12 minutes and 20 minutes respectively, in a quasi-molecular ion peak M over Z at 287 and 285 which were identical to those of DHK and KMF. To determine whether the system can be used for the conversion of other flavanones into their corresponding flavonols, ERD was added into the system. Two new spots on a polyamide TLC plate displayed a migration distance similar to that of DHQ and QRC respectively.HPLC and LC-MS analyses demonstrated that these new chemicals revealed a retention time of 10 minutes and 16 minutes respectively, and a quasi-molecular ion peak M over Z at 303 and 301, which exactly corresponded to those of DHQ and QRC. When preparing the synthetic system, you should add the enzyme lastly, and add the ferrous sulfate second to last. Yes, it provides a guide for the economical production of other secondary metabolites.

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