CN115247156A - Recombinant apple flavonol synthase and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a recombinant apple flavonol synthase and a preparation method and application thereof, wherein the amino acid sequence of the recombinant apple flavonol synthase is shown as SEQ ID No.2, and the invention can efficiently and specifically synthesize quercetin and kaempferol by using a flavonol synthase MdFLS1 catalysis method. The method is the first demonstration at home and abroad that the malflavone alcohol synthase can catalyze dihydroquercetin and dihydrokaempferol to respectively generate quercetin and kaempferol. The invention provides a feasible method for synthesizing the flavonoid substance by an enzyme catalysis method in vitro, and avoids the low efficiency and specificity of obtaining the flavonoid substance by an extraction or chemical synthesis method.

Description

Recombinant apple flavonol synthase and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant apple flavonol synthase and a preparation method and application thereof.

Background

Apple (Malus domestica) is one of four cultivated fruits in the world, and China is the country with the largest planting area and fruit yield in the world, and can meet the wide market consumption demands of a large number of people. With the improvement of living standard of people, the expectation on the fruit quality is more and more increased, and the functional fruit is more and more attractive to consumers. The functional fruit is rich in some special nutrient elements and has strong antioxidant or anticancer activity. Therefore, the screening of high-quality apples is beneficial to the health of people and can increase the income of growers.

The biosynthesis of the flavonoid compound comprises the steps of firstly converting phenylalanine into coumaroyl-CoA through a phenylpropane pathway, enabling the coumaroyl-CoA to enter a flavonoid synthesis pathway to be combined with 3 molecules of malonyl CoA to generate chalcone, and then carrying out intramolecular cyclization reaction to generate the flavanone compound. Flavanone is the main precursor of other flavonoids, and is produced into flavone, isoflavone, flavonol, flavanol, anthocyanidin, etc. through different branched synthetic pathways. Plant flavonoid anabolism has seven major branches: the Anthocyanins (anthocyanines) pathway, procyanidins (PAs) also known as Condensed Tannins (Condensed Tannins), flavonoids (flavanones), flavonols (Flavonols), isoflavones (isoflavanone) and Isoflavonoids (isoflavanoids), tannins (also known as tanning, phoubaphenes) pathway, aurones (Aurones) pathway.

In the flavonol pathway, flavanones can generate dihydroflavonols such as dihydroquercetin and dihydrokaempferol under the action of flavanol-3-hydroxylase (F3H), and then are subjected to desaturation under the action of flavonol synthase (FLS) to form the flavonols. Flavonol synthase (FLS) is a bridge between a flavonoid synthesis pathway and a catechin synthesis pathway, and dihydroflavonol can be subjected to desaturation under the action of the FLS to form flavonol compounds such as quercetin and kaempferol. Xu and the like clone FLS genes from ginkgo leaves, the enzyme can convert dihydrokaempferol into kaempferol in escherichia coli, and meanwhile, the enzyme can also convert naringenin into kaempferol, and the research shows that FLS is a bifunctional enzyme in a flavonoid compound synthesis path.

In recent years, with the intensive research on flavonoids, researchers found that flavonoids in plants have strong antioxidant activity, and that these substances can eliminate free radical-induced oxidative damage to tissues after being ingested. In addition, the substances also have various biological activities of resisting bacteria, viruses, osteoporosis, tumors and the like. Therefore, the method has important application value in the fields of medicine and chemical industry. Besides, the flavonoids have important application value in agriculture. For example, forage grass contains too much flavonoids which can seriously affect the digestion of ruminants and cause abdominal distension, and the appropriate amount of flavonoids can bind to proteins in the forage and prevent the proteins from being fermented by the ruminants, thereby improving the efficiency of converting plant proteins into animal proteins. Therefore, the research significance on flavonoid metabolic pathways and the regulation and control thereof is very important.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a recombinant apple flavonol synthase, a preparation method and a use thereof, which are used for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a recombinant apple flavonol synthase, the amino acid sequence of which is shown in SEQ ID No. 2.

The invention also provides an isolated polynucleotide encoding the recombinant apple flavonol synthase.

The invention also provides a nucleic acid construct comprising the polynucleotide.

The invention also provides an enzyme protein expression system, which comprises the nucleic acid construct or a host cell integrated with the polynucleotide in the genome.

The invention also provides a preparation method of the recombinant apple flavonol synthase, which comprises the following steps: transforming the nucleic acid construct into a host cell, and culturing the host cell to induce expression of the recombinant apple flavonol synthase.

The invention also provides application of the recombinant apple flavonol synthase in preparing flavonol compounds.

The invention also provides a preparation method of the flavonol compound, which comprises the following steps: taking flavanonol compounds as substrates, and adding recombinant apple flavonol synthase into an enzymatic reaction system.

As described above, the recombinant apple flavonol synthase, the preparation method and the application thereof have the following beneficial effects: the invention is proved for the first time at home and abroad that the flavonol synthase can catalyze dihydroquercetin and dihydrokaempferol to respectively generate quercetin and kaempferol. The invention provides a feasible method for synthesizing the flavonoid substance by an enzyme catalysis method in vitro, and avoids the low efficiency and specificity of obtaining the flavonoid substance by an extraction or chemical synthesis method. The recombinant flavonol synthase catalytic method can synthesize quercetin and kaempferol (see figures 1-5) with high efficiency and specificity. The method of the invention brings great economic benefit to the synthesis industry of quercetin and kaempferol.

Drawings

FIG. 1 is a graph showing the results of SDS-PAGE detection of the purified protein. Wherein M is a protein molecular weight marker; lane number 1 is GST tag protein, as control; lane No.2 shows MdFLS1-GST fusion protein.

FIG. 2 is a diagram showing the result of High Performance Liquid Chromatography (HPLC) analysis of the catalytic product formed by catalyzing the production of dihydroquercetin from the purified MdFLS1-GST fusion protein. Wherein, dihydroquercetin is a control group, namely a reaction system of purified GST tag protein and Dihydroquercetin, and no product is synthesized; mdFLS1-GST + Quercetin is an experimental group, namely, a reaction system of the purified MdFLS1-GST fusion protein and dihydroquercetin has 1 more obvious product peaks than a control reaction system only containing GST tag protein, and the product is presumed to be Quercetin.

FIG. 3 shows that the catalytic product of dihydroquercetin was identified by mass spectrometry (LC-MS). Wherein the upper graph shows a standard mass spectrogram of the dihydroquercetin, and the lower graph shows a mass spectrogram of a catalytic product of the dihydroquercetin. The characteristic peaks of the two images are the same, and the fact that the dihydroquercetin forms the quercetin under the catalysis of the enzyme is proved.

FIG. 4 shows that the purified MdFLS1-GST fusion protein catalyzes the formation of kaempferol from dihydrokaempferol, and the catalytic product is analyzed by High Performance Liquid Chromatography (HPLC). Wherein Dihydrokaempferol is used as a control group, and a reaction system of purified GST tag protein and Dihydrokaempferol is adopted, and no product is synthesized; mdFLS1-GST + Kaempferol is an experimental group, and a reaction system of the purified MdFLS1-GST fusion protein and dihydrokaempferol has 1 obvious product peak compared with a control reaction system only containing GST tag protein, and the product is Kaempferol.

FIG. 5 shows that the catalytic product of dihydrokaempferol is identified by mass spectrometry (LC-MS). Wherein the upper graph shows a standard mass spectrum of the dihydrokaempferol, and the lower graph shows a mass spectrum of a catalytic product of the dihydrokaempferol. The characteristic peaks of the two figures are the same, and the fact that the dihydrokaempferol forms the kaempferol under the catalysis of the enzyme is proved.

Detailed Description

The invention provides a recombinant apple flavonol synthase, and the amino acid sequence of the recombinant apple flavonol synthase is shown in SEQ ID No. 2.

In one embodiment, the amino acid sequence of the recombinant apple flavonol synthase has at least 80%, 85%, 90%, 95%, or 99% or more similarity to SEQ ID No. 2. In one embodiment, the recombinant apple flavonol synthase further comprises functional fragments thereof. The functional fragment is, for example, a tag protein.

The gene for coding the recombinant apple flavonol synthase in an organism is an apple flavonol synthase gene MdFLS1. The molecular weight of the recombinant apple flavonol synthase is 36.85KD. The recombinant apple flavonol synthase contained the typical 2-ODD conserved motif from position 196 to position 296 of the N-terminal amino acid sequence as analyzed by BlastP and ScanProsite.

The invention also provides an isolated polynucleotide encoding the recombinant apple flavonol synthase.

The isolated polynucleotide is a nucleotide which is separated from young and tender leaves of apples and can form apple flavonol synthase gene MdFLS1.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

In one embodiment, the isolated polynucleotide has the nucleotide sequence shown as SEQ ID No. 1.

The invention also relates to variants of the above polynucleotides which encode fragments, analogues and derivatives of the enzymes or proteins having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the protein encoded thereby.

The invention also provides a nucleic acid construct comprising the isolated polynucleotide.

In certain embodiments of the invention, the nucleic acid construct is constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vectors.

The invention also provides a composition, which comprises an effective amount of the recombinant apple flavonol synthase, the polynucleotide and one of the nucleic acid constructs and a pharmaceutically acceptable carrier or excipient. In one embodiment, the carrier may be a buffer, water, or the like.

The invention also provides an enzyme protein expression system comprising the nucleic acid construct or a host cell having the exogenous polynucleotide integrated into its genome.

Any cell suitable for expression of the nucleic acid construct may be used as a host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Coli is preferred in the present invention, and XL1-Blue is more preferred.

The invention also provides a preparation method of the recombinant apple flavonol synthase, which comprises the following steps: and transforming the nucleic acid construct into a host cell, culturing the host cell to induce the expression of the recombinant apple flavonol synthase, and separating to obtain the recombinant apple flavonol synthase.

The preparation method also comprises the step of purifying the separated recombinant apple flavonol synthase. The separation and purification may be carried out by a method commonly used by those skilled in the art, and the present invention is not particularly limited. Examples of purification methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

In one embodiment, the host cell is E.coli.

In one embodiment, recombinant apple flavonol synthase expression may be induced by adding IPTG (i.e., isopropyl thiogalactoside) to the culture system of the host cell.

The invention also provides application of the recombinant apple flavonol synthase in preparing flavonol compounds.

The flavonol compounds refer to compounds containing 2-phenyl-3-hydroxy (or oxygen-containing substituted) benzo gamma-pyrone (2-phenyl-3-hydroxy-chromone). Such as quercetin, kaempferol.

In one embodiment, the use is in the preparation of flavonols from dihydroflavonols. The flavanonol compound is a derivative obtained by hydrogenating double bonds at C2-3 position of flavonol. Such as dihydroquercetin, dihydrokaempferol.

In one embodiment, the use is the use of one or more of the recombinant apple flavonol synthase, the isolated polynucleotide, and the nucleic acid construct as a catalyst in the preparation of flavonol compounds.

Further, the application is the application of catalyzing the flavanonol compound to prepare the flavonol compound.

The invention also provides a preparation method of the flavonol compound, which comprises the following steps: taking flavanonol compounds as substrates, and adding recombinant apple flavonol synthase into an enzymatic reaction system.

The enzymatic reaction system also comprises one or more of the following substances: HEPES, mgSO ₄ 、KCl、H ₂ And (O). The addition of HEPES into an enzymatic reaction system can ensure that the pH value of the reaction system is kept constant in a certain range for a long time, and MgSO4 and KCl act to stabilize the enzyme activity.

In one embodiment, the concentration of the substrate in the reaction system at the time of initiation of the reaction is 0.1 to 5mM. For example, the concentration may be 0.1 to 1mM, 1 to 2mM, 2 to 3mM, 3 to 4mM, or 4 to 5mM.

In one embodiment, the recombinant maltulol synthase is added in an amount of 1-2 μ g in a 200 μ l reaction system.

The temperature of the enzymatic reaction is a temperature at which the enzyme is suitable to function. In one embodiment, the reaction temperature is from 30 to 37 ℃.

When it is desired to terminate the reaction, trichloroacetic acid may be added to the enzymatic reaction system to terminate the reaction.

In one embodiment, quercetin may be prepared using dihydroquercetin as a substrate. In another embodiment, kaempferol may be prepared using dihydrokaempferol as a substrate.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention. Example 1 cloning, prokaryotic expression and recombinase protein purification of apple flavonol synthase Gene MdFLS1

1. Cloning of apple flavonol synthase gene MdFLS1

The apple flavonol synthase gene MdFLS1 is cloned from apples by RT-PCR amplification technology. Firstly, RNA is extracted from young and tender leaves of apples by a TRIzol method, and then cDNA of a coding region of the gene is obtained by amplification by an RT-PCR method. A pair of primers used in amplification is: mdFLS1-a: 5-; aaFLS1-b: 5-. The RT-PCR amplification program is as follows: 94 deg.C (pre-denaturation), 5min;94 ℃ (denaturation), 10s;58 ℃ (annealed), 15s;72 deg.C (extension), 2min;36 cycles; 72 deg.C (final extension), 10min. Recovering and purifying the amplification product. And connecting the target gene MdFLS1 obtained by amplification with an EcoRV single-enzyme-digested intermediate vector pBluescript SK to obtain a vector pB MdFLS1, and sequencing the cloned gene.

MdFLS1 sequence information and Properties

After sequencing the cloned flavonol synthase gene MdFLS1, the length of the cDNA coding region of the gene is 1008bp, 335 amino acids are coded, the molecular weight of the coded protein is 36.85KD, and the amino acid sequence is shown as SEQ ID No. 2. By BlastP and ScanProsite analysis, mdFLS1 contains a typical 2-ODD conserved motif from position 196 to position 296 of the N-terminal amino acid sequence, and the motif is the same as the previously cloned FLS of model plants such as Arabidopsis, tobacco, maize and the like, and is a conserved structural domain of the FLS.

3. Construction of prokaryotic expression vector of apple flavonol synthase gene MdFLS1

The prokaryotic expression vector PGEX-3H (preserved in professor HopkiKay university of Shandong) is cut by BamHI and KpnI enzyme, and the vector fragment is recovered for later use. And simultaneously carrying out double enzyme digestion on the vector pB MdFLS1 by using BamHI and KpnI, recovering a target gene fragment, and connecting the target gene fragment into the prokaryotic expression vector to obtain the prokaryotic expression vector pGEX-MdFLS1 of the apple flavonol synthase gene MdFLS1.

4. Transformation of Escherichia coli, induction of protein expression and purification of enzyme protein

And transforming the prokaryotic expression vector pGEX-MdFLS1 into an escherichia coli strain XL1-Blue, storing a strain after PCR and enzyme digestion verification are correct, and using the strain for expression and purification of fusion protein (GST-MdFLS 1 or MdFLS1-GST, namely recombinant apple flavonol synthase fused with GST tag protein).

The above-mentioned bacterial strain was cultured overnight in 2 XYT liquid medium supplemented with ampicillin (100 mg/L) to activate the bacterial strain. The overnight cultures were then expanded to 75ml culture in the ratio 1 ₆₀₀ =0.8. Then IPTG was added to 0.2mM, and the mixture was transferred to 20 ℃ and cultured with shaking for 24 hours to induce the expression of the target protein. After the time, the cells were collected by centrifugation.

2ml Blast Buffer (0.5 mM EDTA,750mM sucrose, 200mM Tris-HCl, pH = 8.0) was added to the cells for resuspension, 14ml diluted 1/2Blast Buffer (2 mg lysozyme was added in advance) was immediately added thereto, the mixture was left on ice for 30min after mixing, the cells were collected by centrifugation, resuspended in PBS (containing 0.2mM PMSF) and left on ice for 30min, centrifuged at 10000rpm for 20min, and the supernatant was collected. Mu.l of glutathione sepharose beads were added to the supernatant, the procedure was followed according to the pGEX protein purification manual, and finally the target protein was eluted with an Elution Buffer (50 mM Tris-HCl,10mM reduced glutathione, pH = 8.0). The purity of the target protein was checked by SDS-PAGE and the protein concentration was determined according to the methods described in the Bradford kit instructions (BSA was used as protein standard).

Example 2 recombinant Malonol synthase catalyzes the synthesis of Quercetin and Kaempferol

1. Enzymatic reaction of dihydroquercetin and dihydrokaempferol

The purified fusion protein (GST-MdFLS 1, which can reflect the catalytic activity of MdFLS1 and belongs to the common method) of the recombinant apple flavonol synthase MdFLS1 is used for carrying out in-vitro enzyme catalytic reaction, and dihydroquercetin and dihydrokaempferol are used as substrates. The enzymatic reaction system used in the catalytic reaction is as follows:

and (3) placing the mixed reaction system in a constant-temperature water bath kettle at 37 ℃ for reaction for 3 hours. The reaction was then stopped by the addition of 20. Mu.l of trichloroacetic acid at a concentration of 240 mg/ml. The reaction tubes were snap frozen in liquid nitrogen and stored at-20 ℃ until HPLC analysis.

2. HPLC identification of enzymatic products

The above reaction mixture was analyzed by High Performance Liquid Chromatography (HPLC) using an apparatus of Shimadzu LC-20AT, a column of C18 (Ultimate, 4.6X 150mm,5 μm), a workstation of Ver 1.21, a diode array detector. The mobile phase is methanol and water (both containing 0.1% of phosphoric acid), and the binary high-pressure gradient elution is carried out, wherein the proportion of the organic phase is 35% -90% respectively. The elution time was 22min. The loading amount was 20. Mu.l.

HPLC analysis shows that, compared with the control (only purified GST tag protein is contained in the reaction system), the GST-MdFLS1 fusion protein can catalyze the synthesis of corresponding quercetin and kaempferol from dihydroquercetin and dihydrokaempferol compounds (see figure 2, 4).

3. Mass spectrometric identification of enzymatic products

The enzyme-catalyzed product was identified by LC-MS using a Surveyor MSQ, mobile phases used were methanol and water (both containing 0.1% phosphoric acid), and organic phase gradient and elution time were the same as those described above for HPLC.

As a result, it was found that the products produced by the enzymatic reaction were each synthesized (see FIGS. 3, 5). It follows that: mallotus philippinensis MdFLS1 can be used for catalyzing dihydroquercetin and dihydrokaempferol to synthesize quercetin and kaempferol.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention is not limited to those specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

The invention belongs to the national key research and development plan, and relates to the technical optimization and quality improvement of stabilizer pharmaceutic adjuvants for novel biological medicines, wherein the project number is SQ2020YFF0422322.

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Claims (10)

1. A recombinant apple flavonol synthase is characterized in that the amino acid sequence of the recombinant apple flavonol synthase is shown in SEQ ID No. 2.

2. An isolated polynucleotide encoding the recombinant apple flavonol synthase of claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide has the nucleotide sequence shown in SEQ ID No. 1.

4. A nucleic acid construct comprising the polynucleotide of claim 2 or 3.

5. An enzyme protein expression system comprising the nucleic acid construct of claim 4 or a host cell having the polynucleotide of claim 2 integrated into its genome.

6. A preparation method of recombinant apple flavonol synthase is characterized by comprising the following steps: transforming the nucleic acid construct of claim 4 into a host cell, culturing the host cell to induce expression of the recombinant apple flavonol synthase, and isolating the recombinant apple flavonol synthase.

7. Use of the recombinant apple flavonol synthase of claim 1 for the preparation of flavonol compounds.

8. Use according to claim 7, characterized in that the use is as a catalyst in the preparation of flavonols, preferably in the preparation of flavonols from flavanonols.

9. A method for preparing flavonol compounds, which is characterized by comprising the following steps: adding the recombinant apple flavonol synthase of claim 1 into an enzymatic reaction system by using a flavanonol compound as a substrate.

10. The method according to claim 9, wherein the enzymatic reaction system further comprises one or more of the following substances: HEPES, mgSO ₄ 、KCl、H ₂ O。

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