Polygonum multiflorum PmFMO3 protein, its encoding gene and its application in hydroxylation modification of stilbene components

Abstract

The present invention discloses a PmFMO3 protein of Polygonum multiflorum, a coding gene and its application in the hydroxylation modification of stilbene components, and belongs to the field of biomedicine. The amino acid sequence of the PmFMO3 protein of Polygonum multiflorum is shown in SEQ ID No.2, and the nucleotide sequence of the coding gene is shown in SEQ ID No.1. The present invention obtains a specific stilbene 2-hydroxylase, i.e., flavin monooxygenase PmFMO3, from Polygonum multiflorum, which consists of 519 amino acids and the coding gene nucleotide is 1560bp. Experiments have shown that PmFMO3 can catalyze stilbene components such as resveratrol to generate corresponding 2-hydroxylated catalytic products, laying the foundation for the heterologous synthesis of 2,3,5,4'-tetrahydroxystilbene glycosides and other polyhydroxystilbene products. The present invention provides a new material for stilbene 2-hydroxylase.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a Polygonum multiflorum PmFMO3 protein, a coding gene and an application thereof in the hydroxylation modification of stilbene components. Background Art

[0002] Stilbenes are a class of non-flavonoid polyphenol compounds with 1,2-stilbene as the skeleton structure. Figure 1 ). Due to the famous "French Paradox", the health and medicinal value of stilbenes represented by resveratrol has attracted widespread attention, among which the antioxidant, anti-inflammatory, and anti-tumor activities have been widely confirmed. However, due to its low water solubility and low bioavailability, the commercial promotion and application of resveratrol is severely limited, and it is urgently needed to improve its related properties through structural modification. Among them, hydroxylation modification is an important means to increase water solubility and antioxidant activity. At present, it mainly relies on chemical methods to modify the stilbenes structure.

[0003] However, chemical modification has problems such as high harm of synthetic reagents, harsh synthetic conditions, and low yield due to lack of specificity. Therefore, the research direction has begun to turn to biosynthesis, which has the characteristics of mild process, environmental friendliness and strong product specificity. Biosynthesis requires the participation of functionally specific catalytic enzymes, but at present, few enzymes that perform hydroxylation function on diphenylethylene have been reported, and the 2-hydroxylase has not yet been identified. Summary of the invention

[0004] The purpose of the present invention is to provide a Polygonum multiflorum PmFMO3 protein, a coding gene and an application thereof in the hydroxylation modification of stilbene components to solve the problems existing in the above-mentioned prior art. The present invention discovers a hydroxylase (i.e., flavin monooxygenase PmFMO3) that specifically hydroxylates stilbene components at position 2 from Polygonum multiflorum. Experiments have shown that PmFMO3 can catalyze stilbene components such as resveratrol to generate corresponding 2-hydroxylated catalytic products, laying a foundation for the heterologous synthesis of 2,3,5,4'-tetrahydroxystilbene glycosides and other polyhydroxystilbene products.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Polygonum multiflorum PmFMO3 protein, whose amino acid sequence is shown in SEQ ID No.2.

[0007] The present invention also provides a gene encoding the Polygonum multiflorum PmFMO3 protein, and the nucleotide sequence of the gene is shown in SEQ ID No.1.

[0008] The present invention also provides a primer pair for amplifying the gene, and the nucleotide sequence of the primer pair is shown as SEQ ID No. 3-4.

[0009] The present invention also provides an expression cassette or an expression vector containing the gene.

[0010] The present invention also provides a recombinant bacterium containing the expression cassette or expression vector.

[0011] The present invention also provides the use of the Polygonum multiflorum PmFMO3 protein or the gene or the primer pair or the expression cassette or expression vector or the recombinant bacteria in any of the following:

[0012] (1) Application in the preparation of hydroxylase;

[0013] (2) Application in the synthesis of diphenylene compounds;

[0014] (3) Application in catalyzing the hydroxylation modification of stilbene components;

[0015] (4) Application in the preparation of hydroxylase for catalyzing the hydroxylation modification of stilbene components;

[0016] (5) Application in the preparation of hydroxylase for catalyzing the conversion of stilbene components into 2-hydroxylated stilbene components.

[0017] The present invention also provides a hydroxylase for catalyzing the hydroxylation modification of stilbene components, which contains the Polygonum multiflorum PmFMO3 protein.

[0018] Preferably, the Polygonum multiflorum PmFMO3 protein catalyzes the generation of 2-hydroxylated stilbene components from stilbene-like components.

[0019] The present invention also provides a method for catalyzing the hydroxylation modification of stilbene components, comprising the step of using the Polygonum multiflorum PmFMO3 protein as a hydroxylase to carry out hydroxylation modification on the stilbene components.

[0020] Preferably, the hydroxylation modification comprises hydroxylation modification of the 2-position of the stilbene component.

[0021] The present invention discloses the following technical effects:

[0022] The present invention mines and obtains a specific stilbene 2-hydroxylase in Polygonum multiflorum, namely flavin monooxygenase PmFMO3, through genome, metabolome, transcriptome, co-expression analysis, enzyme activity analysis and identification. The flavin monooxygenase PmFMO3 consists of 519 amino acids and has a coding gene nucleotide of 1560bp. Experiments have shown that PmFMO3 can catalyze stilbene components such as resveratrol to generate corresponding 2-hydroxylated catalytic products, laying the foundation for the heterologous synthesis of 2,3,5,4'-tetrahydroxystilbene glycosides and other polyhydroxystilbene products.

[0023] The PmFMO3 disclosed in the present invention can directly and specifically add a hydroxyl group to the 2-position of the A ring of the stilbene component, thereby providing a catalytic element for producing polyhydroxylated stilbene components by means of synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the structure of diphenylethylene;

[0026] Figure 2 It is a schematic diagram of common stilbene components and the inferred synthesis route;

[0027] Figure 3 Identification and phylogenetic analysis of flavin monooxygenase (FMOs) family members based on the genome of Polygonum multiflorum;

[0028] Figure 4 The results of differential expression analysis of 28 FMOs encoding genes in Polygonum multiflorum in 15 different tissues;

[0029] Figure 5 Why is the difference in THSG content in 15 tissue parts of Polygonum multiflorum analyzed?

[0030] Figure 6 The results of correlation analysis between the expression levels of 28 PmFMOs members and THSG content;

[0031] Figure 7 Why is the EIC diagram of the stilbene component catalyzed by PmFMO3 in tobacco?

[0032] Figure 8 Why is the primary mass spectrometry identification result of peak 2 of the stilbene component catalyzed by PmFMO3 in tobacco?

[0033] Fig. 9 Why is the secondary mass spectrometry identification result of peak 2 of the diphenylethylene component catalyzed by PmFMO3 in tobacco? DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Polygonum multiflorum contains a large number of stilbenes. Unlike grapes and Polygonum cuspidatum, which mainly exist in the form of resveratrol (3,5,4′-trihydroxystilbene, resveratrol) and Polydatin (3,5,4′-trihydroxystilbene-3-O-β-D-glucoside, polydatin), Polygonum multiflorum mainly exists in the form of a special 2,3,5,4′-tetrahydroxystilbene glucoside (THSG) ( Figure 2), which provides experimental materials for the present invention to discover stilbene 2-hydroxylase. The present invention discovers the specific stilbene 2-hydroxylase in Polygonum multiflorum through genome, metabolome, transcriptome, co-expression analysis, enzyme activity analysis and identification, and can directly add a hydroxyl group to the 2-position of the A ring of the stilbene component, providing a catalytic element for producing polyhydroxylated stilbene components by synthetic biology. The following is further explained with specific examples.

[0040] Example 1 Screening and phylogenetic analysis of flavin monooxygenase genes based on the genome, second-generation transcriptome and THSG content data of Polygonum multiflorum

[0041] 1. Experimental methods

[0042] The Arabidopsis flavin monooxygenase protein sequence and the conserved domain of flavin monooxygenase protein (Pfam ID: PF00743) were used as seed files to perform BLASTP and HMM searches on the protein sequence files of the Polygonum multiflorum genome to find homologous genes. The obtained sequences were combined and submitted to the NCBI database to verify the conserved domains, thereby determining the FMO family members in Polygonum multiflorum. Multiple sequence alignment was completed using MUSCLE, and the phylogenetic tree was constructed using IQtree and the maximum likelihood method, with bootstrap selection repeated 5000 times. At the same time, the Illumina NovaSeq6000 high-throughput sequencing platform was used to perform second-generation sequencing and THSG content determination in 15 different tissue parts of Polygonum multiflorum (enlarged root ERT, unenlarged root URT, cutting stem CS, thick old vine TcBLC, thin old vine TnBLC, vine bark BC, vine heart XC, young vine YGC, mature leaf ML, mature petiole PML, young leaf YL, bud B, flower F, inflorescence axis R and seed S). The Clean Reads obtained from the second-generation transcriptome were aligned with the genome sequence using HiSAT2 to obtain the transcript location information. RSEM software was used to quantitatively analyze the expression levels of transcripts in different tissue parts and calculate the FPKM value. Then, the FPKM values ​​of FMO family members were extracted and correlated with the THSG content.

[0043] 2. Results and Analysis

[0044] Based on the genome data of Polygonum multiflorum, a total of 28 PmFMOs were screened, and phylogenetic analysis clustered these 28 PmFMOs into three subgroups (see Figure 3 The expression levels of these 28 PmFMOs members and THSG content in 15 tissue parts of Polygonum multiflorum were extracted, and it was found that the THSG content and the expression of these members were tissue-specific (see Figure 4 and Figure 5Based on correlation analysis, it was found that the expression levels of PmFMO3 and PmFMO6 were highly correlated with the THSG content (see Figure 6 ), but the expression level of PmFMO6 in each tissue was low (less than 2), so it was speculated that PmFMO3 may have stilbene hydroxylase activity.

[0045] Example 2 Cloning of candidate PmFMO3 gene and construction of binary expression vector

[0046] 1. Experimental methods

[0047] The pK7WG2D-PmFMO3 expression vector was obtained by Gateway homologous recombination method, and the primer sequences were designed (see Table 1). The cDNA of Polygonum multiflorum was used as a template, and the PmFMO3 gene fragment was cloned using PrimeSTAR® Max DNA Polymerase high-fidelity enzyme (purchased from Bio-Technology (Beijing) Co., Ltd., product number R047A). The total volume of the cloning system program was 50 μL: 25 μL PrimeSTAR® Max DNA Polymerase, 1 μL template, 1.5 μL forward primer, 1.5 μL reverse primer and 21 μL water. The PCR reaction program is shown in Table 2.

[0048] The obtained gene fragment was recombined into the pDONR221 vector (purchased from Thermo Fisher Scientific (China) Co., Ltd., product number 12536017) using Gateway™ BP Clonase™ enzyme mixture (purchased from Thermo Fisher Scientific (China) Co., Ltd., product number 11789013), and the recombinant product was directly transformed into TOP10 Escherichia coli competent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B528412), and positive clones were selected for sequencing. The total volume of the colony PCR system was 20 μL: 10 μL 2×Taq PCR Mix, 1 μL template, 1 μL M13F primer (5'-TGTAAAACGACGGCCAGT-3', SEQ ID No. 5), 1 μL M13R primer (5'-CAGGAAACAGCTATGACC-3', SEQ ID No. 6) and 7 μL water. The colony PCR reaction procedure is shown in Table 3. The correctly sequenced entry vector was recombined into the pK7WG2D binary expression vector using Gateway™ LRClonase™ enzyme mixture (purchased from Thermo Fisher Scientific (China) Co., Ltd., product number 11791100) to recombinant the PmFMO3 gene fragment. The recombinant product was directly transformed into TOP10 competent Escherichia coli (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B528412), and the positive clones were selected and expanded and cultured, and the bacteria were collected and the plasmid was extracted using the SanPrep column plasmid DNA mini-extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B518191).

[0049] Table 1 Primer sequences for constructing pK7WG2D-PmFMO3 expression vector

[0050]

[0051] Table 2 KOD high-fidelity enzyme PCR reaction program

[0052]

[0053] Table 3 Colony PCR reaction procedure

[0054]

[0055] 2. Results and Analysis

[0056] The cloned PmFMO3 gene sequence is shown in SEQ ID No. 1. The gene sequence contains 1560 nucleotides, which encodes the protein shown in SEQ ID No. 2. The protein sequence consists of 519 amino acids. The protein is named PmFMO3 according to the chromosomal location.

[0057] SEQ ID No.1:

[0058]

[0059] SEQ ID No.2:

[0060] MERSIAIVGAGISGLLACKYAVSKGYCPVVFESQCELGGVWAKTLETTKLQTPKMIYQFSDFPWPSTVTDMNPTGDQVLDYVRGYAEHFGLLSHIRFNTKVLSIEFEGVDEREMEAWSLWNCTGEGFGNM GKWSLLLQDGATQSKQVMQFAFVIICVGQFSDLPKLPEFPAGKGPEVFRGKVMHSMDYSAMANKDARSLVEGKRVVVVGFSKSALDVAAECAASNGKNNPCHLLYRTPRWILSNFTAWGLPYPFLYFNRF AELLLHKPGEGLFHSLLATVLSPLRWLINKFAESDVRHKHPIKKLGLLPEYSLLQSLSSCVFAIAPEGFFKHIDQGSIVLKKAQKFSFCKEGVVVQGQDDKPLEADLVILATGFHGDRKLRNIFASSMFR DCIMGSPTSILPLYRQCIHPRIPQLAVIGYSESIWNLYTSEMRSRWVMELLDGTFKLPEINDMEKDISRWDKYMKTYAPNHYRKSCIAKLHIWYNDQLCKDMGWNPKRKKGLFSELFEPYGPLDYVNSS.

[0061] Example 3 Functional verification of candidate PmFMO3 encoding genes

[0062] 1. Experimental methods

[0063] The pK7WG2D-PmFMO3 binary expression vector obtained in Example 2 was transformed into GV3101 Agrobacterium tumefaciens competent cells (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZC141). Positive colonies were picked and cultured overnight, activated and resuspended in infection buffer (10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid and 100 μM acetosyringone), and the OD was adjusted. 600The value reached 0.5, and the tobacco leaves were injected after 4 hours of rest. The leaves were cultured normally for 3 days before the substrate, resveratrol, was injected. After 1 day of reaction, the leaves were collected and freeze-dried under negative pressure, and the catalytic products were detected using Agilent Technologies 1290 Infinity Ⅱ and 6530 Q-TOF LC-MS instruments. Chromatographic column: Waters Acquity UPLC® BEHC18 column (1.7 μm, 150 × 2.1 mm), column temperature: 40 ℃, injection volume 2 μL. Chromatographic conditions: mobile phase A: water (containing 0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.3 mL / min, elution program: 0 min, 10% B; 8 min, 18% B; 14min 22% B; 19 min 33% B; 22 min 90% B; 26 min 90% B; 26.1 min 10% B; 30 min 10%. The Q-TOF mass spectrometry parameters were as follows: ESI source, negative ion mode, 50-1000 m / z; drying gas temperature, 300 °C, flow rate, 9 L / min; sheath gas temperature, 400 °C, flow rate, 11.0 L / min; nebulizer, 35 psi; VCap, 3500 V; metabolites were detected in Auto-MS / MS mode and Target-MS / MS mode; and ion fragments were obtained using a collision energy of 35 V.

[0064] 2. Results and Analysis

[0065] The catalytic results showed that PmFMO3 could convert the substrate resveratrol in tobacco leaves to produce a product peak with the same retention time as the THSG standard (see Figure 7 ), the peak molecular weight of the product is [MH] - :m / z=405.1195(see Figure 8 ), and the secondary mass spectrometry showed that the product peak had a completely consistent fragmentation pattern with the THSG standard (see Fig. 9 ). Therefore, it was determined that PmFMO3 has the ability to hydroxylate the 2-position of stilbene components.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Polygonum multiflorum PmFMO3 protein, characterized in that Its amino acid sequence is shown in SEQ ID No.

2.

2. The gene encoding the Polygonum multiflorum PmFMO3 protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

1.

3. An expression cassette or expression vector containing the gene according to claim 2.

4. A recombinant bacterium containing the expression cassette or expression vector according to claim 3.

5. Use of the Polygonum multiflorum PmFMO3 protein according to claim 1, the gene according to claim 2, the expression cassette or expression vector according to claim 3, or the recombinant bacteria according to claim 4 in any of the following: (1) Application in catalyzing the hydroxylation modification of stilbene components; (2) Application in the preparation of hydroxylase for catalyzing the hydroxylation modification of stilbene components; The stilbene component is resveratrol.

6. Use of the Polygonum multiflorum PmFMO3 protein according to claim 1, or the gene according to claim 2, or the expression cassette or expression vector according to claim 3, or the recombinant bacterium according to claim 4 in the preparation of a hydroxylase that catalyzes stilbene components to generate 2-hydroxylated stilbene components, characterized in that: The stilbene component is resveratrol.

7. A hydroxylase that catalyzes the hydroxylation modification of resveratrol, characterized in that: It contains the Polygonum multiflorum PmFMO3 protein described in claim 1.

8. A method for catalyzing the hydroxylation modification of resveratrol, characterized in that: The method comprises the steps of transferring an expression vector containing the PmFMO3 gene into tobacco, injecting substrate resveratrol into the tobacco after culturing, and collecting the tobacco after reaction.

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