Name: Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade (Video) | JoVE Journal
Uploaded: 2019-08-14T12:30:00.000+00:00
Duration: 9 min 50 s
Description: Learn a one-pot bienzymatic cascade method for efficient flavonol biosynthesis from flavanones using recombinant enzymes. This cost-effective protocol offers high yields and significant industrial potential for healthcare and food applications.

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5 minutes

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.

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<li>Tang, L. J., Zhang, S. F., Yang, J. Z., Gao, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+Synthetic+Methods+of+Flavones%5BTitle%5D)+AND+%22Chinese+Journal+of+Organic+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Synthesis+of+luteolin+and+kaempferol+%28author%27s+transl%29%5BTitle%5D)+AND+%22Yao+Xue+Xue+Bao%22%5BJournal%5D)">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/pubmed?term=((Development+and+Optimization+of+an+In+vitro+Multienzyme+Synthetic+System+for+Production+of+Kaempferol+from+Naringenin%5BTitle%5D)+AND+%22Journal+of+Agricultural+and+Food+Chemistry%22%5BJournal%5D)">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>
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<li>Zhu, S., Wu, J., Du, G., Zhou, J., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+synthesis+of+eriodictyol+from+L-tyrosine+in+Escherichia+coli%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">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/pubmed?term=((Metabolic+engineering+of+the+complete+pathway+leading+to+heterologous+biosynthesis+of+various+flavonoids+and+stilbenoids+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Metabolic+Engineering%22%5BJournal%5D)">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/pubmed?term=((Combinatorial+biosynthesis+of+flavones+and+flavonols+in+Escherichia+coli%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Functional+expression+of+a+P450+flavonoid+hydroxylase+for+the+biosynthesis+of+plant-specific+hydroxylated+flavonols+in+Escherichia+coli%5BTitle%5D)+AND+%22Metabolic+Engineering%22%5BJournal%5D)">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/pubmed?term=((De+novo+production+of+the+flavonoid+naringenin+in+engineered+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Microbial+Cell+Factories%22%5BJournal%5D)">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/pubmed?term=((Flavonoid+biosynthesis.+A+colorful+model+for+genetics%2C+biochemistry%2C+cell+biology%2C+and+biotechnology%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((Biosynthesis+and+engineering+of+kaempferol+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Microbial+Cell+Factories%22%5BJournal%5D)">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/pubmed?term=((Enzymatic+total+synthesis+of+enterocin+polyketides%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">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/pubmed?term=((Preparation+of+RNA+from+plant+tissue+using+trizol%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Purification+of+RNA+from+cells+and+tissues+by+Acid+phenol-guanidinium+thiocyanate-chloroform+extraction%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Construction+of+cDNA+Libraries+Stage+1%3A+Synthesis+of+First-strand+cDNA+Catalyzed+by+Reverse+Transcriptase%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Directional+cloning+into+plasmid+vectors%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Expression+of+Cloned+Genes+in+E.+coli+Using+IPTG-inducible+Promoters%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Purification+of+Histidine-tagged+Proteins+by+Immobilized+Ni2%2B+Absorption+Chromatography%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography.</a> Cold Spring Harbor Protocols. (1), (2006).</li>
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</ol>

The authors declare that they have no competing financial interests.

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&amp;times; 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>&lt;em&gt;Escherichia coli&lt;/em&gt; BL21(DE3)</td><td>Beijing CoWin Biotech Co., Ltd</td><td>CW0809</td><td>bacteria strain for expressing target genes</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha;</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 &amp;amp; Technology Co. Ltd.</td><td>Tanon-&lt;br/&gt; 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

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...

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 ...

biosynthesis-flavonol-from-flavanone-establishing-one-pot-bienzymatic

Research

JoVE Journal

Biochemistry

The synthesis of asymmetric species of ferrocene is challenging using solution techniques.  This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis.  Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Ionic liquids (ILs) mediate fast, simple and cheap access to 1,6-ketoesters in high diastereoselectivities and good yields. The reaction protocol is robust and the 1,6-ketoesters can be obtained in gram scale after a simple filtration protocol. Moreover, the 1,6-ketoesters are potent gelators in hydrocarbon solvents.

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Enantiomerically enriched bispiro[γ-butyrolactone-pyrrolidin-4,4'-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling generation.

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.

Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food industry.

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles

In the present work, we elucidate the green synthesis of quinoline-based Ionic Liquid (IL), namely, 1-Hexadecylquinolin-1-ium bromide {[C16quin]Br} by mixing quinoline with an excess of 1-Bromohexadecane, along with its detailed characterization using Nuclear Magnetic Resonance and Infrared spectroscopic measurements.

Green Synthesis of Quinoline-Based Ionic Liquid

Learn a one-pot bienzymatic cascade method for efficient flavonol biosynthesis from flavanones using recombinant enzymes. This cost-effective protocol offers high yields and significant industrial potential for healthcare and food applications.

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

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