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>
	<li>Falcone Ferreyra, M. L., Rius, S. P., Casati, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoids:+biosynthesis,+biological+functions,+and+biotechnological+applications.">Flavonoids: biosynthesis, biological functions, and biotechnological applications.</a> Frontiers in Plant Science. 3, 222 (2012).</li><li>Fang, F., Tang, K., Huang, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+flavonol+synthase+and+flavonol+contents+during+grape+berry+development.">Changes of flavonol synthase and flavonol contents during grape berry development.</a> European Food Research and Technology. 237 (4), 529-540 (2013).</li><li>Cui, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthocyanins+and+flavonols+are+responsible+for+purple+color+of+Lablab+purpureus+(L.)+sweet+pods.">Anthocyanins and flavonols are responsible for purple color of Lablab purpureus (L.) sweet pods.</a> Plant Physiology and Biochemistry. 103, 183-190 (2016).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+class+of+flavonol-based+anti-prostate+cancer+agents:+Design,+synthesis,+and+evaluation+in+cell+models.">A new class of flavonol-based anti-prostate cancer agents: Design, synthesis, and evaluation in cell models.</a> Bioorganic &amp; Medicinal Chemistry Letters. 26 (17), 4241-4245 (2016).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Wnt+signaling+activity+for+growth+suppression+induced+by+quercetin+in+4T1+murine+mammary+cancer+cells.">Regulation of Wnt signaling activity for growth suppression induced by quercetin in 4T1 murine mammary cancer cells.</a> International Journal of Oncology. 43 (4), 1319-1325 (2013).</li><li>Kimura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activities+and+structural+characterization+of+flavonol+O-glycosides+from+seeds+of+Japanese+horse+chestnut+(Aesculus+turbinata+BLUME).">Antioxidant activities and structural characterization of flavonol O-glycosides from seeds of Japanese horse chestnut (Aesculus turbinata BLUME).</a> Food Chemistry. 228, 348-355 (2017).</li><li>Cassidy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+dietary+anthocyanin+and+flavonol+intakes+are+associated+with+anti-inflammatory+effects+in+a+population+of+US+adults.">Higher dietary anthocyanin and flavonol intakes are associated with anti-inflammatory effects in a population of US adults.</a> The American Journal of Clinical Nutrition. 102 (1), 172-181 (2015).</li><li>Chao, H. C., Tsai, P. F., Lee, S. C., Lin, Y. S., Wu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Myricetin-Containing+Ethanol+Solution+on+High-Fat+Diet+Induced+Obese+Rats.">Effects of Myricetin-Containing Ethanol Solution on High-Fat Diet Induced Obese Rats.</a> Journal of Food Science. 82 (8), 1947-1952 (2017).</li><li>Serban, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Quercetin+on+Blood+Pressure:+A+Systematic+Review+and+Meta-Analysis+of+Randomized+Controlled+Trials.">Effects of Quercetin on Blood Pressure: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.</a> Journal of the American Heart Association. 5 (7), (2016).</li><li>Nakagawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+memory+recall+by+quercetin+in+rodent+contextual+fear+conditioning+and+human+early-stage+Alzheimer's+disease+patients.">Improvement of memory recall by quercetin in rodent contextual fear conditioning and human early-stage Alzheimer's disease patients.</a> Neuroreport. 27 (9), 671-676 (2016).</li><li>Muthukrishnan, S. D., Kaliyaperumal, A., Subramaniyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+determination+of+flavonoids,+carotenoids+and+chlorophyll+concentration+in+Cynodon+dactylon+(L.)+by+HPLC+analysis.">Identification and determination of flavonoids, carotenoids and chlorophyll concentration in Cynodon dactylon (L.) by HPLC analysis.</a> Natural Product Research. 29 (8), 785-790 (2015).</li><li>Agar, O. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Studies+on+Phenolic+Composition,+Antioxidant,+Wound+Healing+and+Cytotoxic+Activities+of+Selected+Achillea+L.+Species+Growing+in+Turkey.">Comparative Studies on Phenolic Composition, Antioxidant, Wound Healing and Cytotoxic Activities of Selected Achillea L. Species Growing in Turkey.</a> Molecules. 20 (10), 17976-18000 (2015).</li><li>Yang, R. Y., Lin, S., Kuo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Content+and+distribution+of+flavonoids+among+91+edible+plant+species.">Content and distribution of flavonoids among 91 edible plant species.</a> Asia Pacific Journal of Clinical Nutrition. 17, 275-279 (2008).</li><li>Tang, L. J., Zhang, S. F., Yang, J. Z., Gao, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Synthetic+Methods+of+Flavones.">New Synthetic Methods of Flavones.</a> Chinese Journal of Organic Chemistry. 24 (8), 882-889 (2004).</li><li>Lu, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+luteolin+and+kaempferol+(author's+transl).">Synthesis of luteolin and kaempferol (author's transl).</a> Yao Xue Xue Bao. 15 (8), 477-481 (1980).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Optimization+of+an+In+vitro+Multienzyme+Synthetic+System+for+Production+of+Kaempferol+from+Naringenin.">Development and Optimization of an In vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin.</a> Journal of Agricultural and Food Chemistry. 66 (31), 8272-8279 (2018).</li><li>Malla, S., Pandey, R. P., Kim, B. G., Sohng, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regiospecific+modifications+of+naringenin+for+astragalin+production+in+Escherichia+coli.">Regiospecific modifications of naringenin for astragalin production in Escherichia coli.</a> Biotechnology and Bioengineering. 110 (9), 2525-2535 (2013).</li><li>Zhu, S., Wu, J., Du, G., Zhou, J., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+synthesis+of+eriodictyol+from+L-tyrosine+in+Escherichia+coli.">Efficient synthesis of eriodictyol from L-tyrosine in Escherichia coli.</a> Applied and Environmental Microbiology. 80 (10), 3072-3080 (2014).</li><li>Trantas, E., Panopoulos, N., Ververidis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+the+complete+pathway+leading+to+heterologous+biosynthesis+of+various+flavonoids+and+stilbenoids+in+Saccharomyces+cerevisiae.">Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae.</a> Metabolic Engineering. 11 (6), 355-366 (2009).</li><li>Miyahisa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+biosynthesis+of+flavones+and+flavonols+in+Escherichia+coli.">Combinatorial biosynthesis of flavones and flavonols in Escherichia coli.</a> Applied Microbiology and Biotechnology. 71 (1), 53-58 (2006).</li><li>Leonard, E., Yan, Y., Koffas, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+a+P450+flavonoid+hydroxylase+for+the+biosynthesis+of+plant-specific+hydroxylated+flavonols+in+Escherichia+coli.">Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli.</a> Metabolic Engineering. 8 (2), 172-181 (2006).</li><li>Koopman, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+production+of+the+flavonoid+naringenin+in+engineered+Saccharomyces+cerevisiae.">De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae.</a> Microbial Cell Factories. 11, 155 (2012).</li><li>Winkel-Shirley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoid+biosynthesis.+A+colorful+model+for+genetics,+biochemistry,+cell+biology,+and+biotechnology.">Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology.</a> Plant Physiology. 126 (2), 485-493 (2001).</li><li>Duan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+and+engineering+of+kaempferol+in+Saccharomyces+cerevisiae.">Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae.</a> Microbial Cell Factories. 16 (1), 165 (2017).</li><li>Cheng, Q., Xiang, L., Izumikawa, M., Meluzzi, D., Moore, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+total+synthesis+of+enterocin+polyketides.">Enzymatic total synthesis of enterocin polyketides.</a> Nature Chemical Biology. 3 (9), 557-558 (2007).</li><li>Connolly, M. A., Clausen, P. A., Lazar, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+RNA+from+plant+tissue+using+trizol.">Preparation of RNA from plant tissue using trizol.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+RNA+from+cells+and+tissues+by+Acid+phenol-guanidinium+thiocyanate-chloroform+extraction.">Purification of RNA from cells and tissues by Acid phenol-guanidinium thiocyanate-chloroform extraction.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+cDNA+Libraries+Stage+1:+Synthesis+of+First-strand+cDNA+Catalyzed+by+Reverse+Transcriptase.">Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+cloning+into+plasmid+vectors.">Directional cloning into plasmid vectors.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cloned+Genes+in+E.+coli+Using+IPTG-inducible+Promoters.">Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Histidine-tagged+Proteins+by+Immobilized+Ni2++Absorption+Chromatography.">Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography.</a> Cold Spring Harbor Protocols. (1), (2006).</li><li>Halbwirth, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+flavonoid+enzyme+activities+in+tissues+of+fruit+species.">Measuring flavonoid enzyme activities in tissues of fruit species.</a> Journal of Agricultural and Food Chemistry. 57 (11), 4983-4987 (2009).</li><li>Prescott, A. G., Stamford, N. P., Wheeler, G., Firmin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+properties+of+a+recombinant+flavonol+synthase+from+Arabidopsis+thaliana.">In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana.</a> Phytochemistry. 60 (6), 589-593 (2002).</li></ol>

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 ...

Research

JoVE Journal

Biochemistry

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...