Ascorbate peroxidase and its encoding gene and application

Abstract

The present application relates to ascorbate peroxidase and its coding gene and application. The ascorbate peroxidase of Fritillariae Pallidicaulis Herb is disclosed for the first time, which has the ability of synthesizing protocatechuic aldehyde and can catalyze p-hydroxybenzaldehyde to generate protocatechuic aldehyde. The present application also discloses polynucleotide coding the ascorbate peroxidase, vector and host cell expressing the ascorbate peroxidase, and method for producing protocatechuic aldehyde.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and plant biology; more specifically, this invention relates to an ascorbic acid peroxidase derived from Amaryllidaceae plants, its encoding gene, and its applications. Background Technology

[0002] Ascorbate peroxidase (APS), present in all photosynthetic eukaryotes from algae to higher plants, is a key enzyme in the breakdown of H₂O₂. APS utilizes ascorbic acid as a specific electron donor to reduce H₂O₂ to H₂O, participating in the regulation of H₂O₂ and ascorbic acid levels. At the cellular level, APS is located in all subcellular compartments that produce H₂O₂, including ectoplasts, cytoplasm, plastids, chloroplasts, mitochondria, and peroxisomes, either in soluble form or attached to organelle membranes. Within these organelles, APS participates in the formation of the ascorbate-glutathione cycle. The ascorbate-glutathione cycle plays a central role in the cellular redox system network, supporting energy-sensitive communication within different cellular compartments and integrating plant signaling pathways. APS plays a crucial role in protecting plant cells from various environmental stresses and in plant growth and development. In addition to ascorbic acid, ascorbic acid peroxidase can also oxidize aromatic substrates such as p-cresol, o-dianisidine, guaiacol, and p-coumarate, exhibiting a broad substrate spectrum.

[0003] Protocatechuic aldehyde, also known as 3,4-dihydroxybenzaldehyde, is a naturally occurring biomolecule. A derivative of benzaldehyde, it is an aromatic aldehyde with one formyl group and two phenolic hydroxyl groups, belonging to the phenolic class of compounds. Thanks to these functional groups, protocatechuic aldehyde exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, antimicrobial, anti-apoptotic, and antitumor effects. Furthermore, it can participate in regulating cardiovascular remodeling, protect endothelial cells, and treat cardiovascular damage. In recent years, protocatechuic aldehyde has been shown to effectively prevent and treat ischemic cardiovascular and cerebrovascular diseases. In nature, protocatechuic aldehyde is widely found in vegetables, fruits, and crops. It is also an effective component of some traditional Chinese medicines, such as Danshen (Salvia miltiorrhiza). Protocatechuic aldehyde is also an important intermediate in the biosynthesis of plant natural products, such as galantamine—an alkaloid from the Amaryllidaceae family that has therapeutic effects on Alzheimer's disease.

[0004] *Lycoris aurea* is a perennial herbaceous bulbous medicinal plant belonging to the genus *Lycoris* in the family Amaryllidaceae. It can synthesize numerous plant natural products with important biological activities and applications, such as protocatechuic aldehyde and galantamine. However, the ascorbate peroxidase and its encoding gene from *Lycoris* plants have not yet been isolated and cloned. Therefore, it is necessary to develop ascorbate peroxidase from *Lycoris aurea*. This protein and its encoding gene can be used for the biotransformation and biosynthesis of protocatechuic aldehyde and galantamine through plant transgenic and heterologous expression. Summary of the Invention

[0005] The purpose of this invention is to provide an ascorbic acid peroxidase from Amaryllidaceae plants, wherein the ascorbic acid peroxidase is selected from:

[0006] (a) A protein with the amino acid sequence shown in SEQ ID NO:1; or

[0007] (b) A protein derived from (a) having ascorbate peroxidase activity, formed by substitution and / or deletion and / or addition of one or more (e.g., 1–50) amino acid residues of the amino acid sequence SEQ ID NO:1; or

[0008] (c) A protein derived from (a) that has at least 75% sequence identity with the amino acid sequence of SEQ ID NO:1 and has ascorbic acid peroxidase activity.

[0009] The protein shown in SEQ ID NO:1 of this invention is a novel ascorbate peroxidase isolated from Lycoris aurea. For ease of description, the protein shown in SEQ ID NO:1 is named LaAPX24999.

[0010] In a preferred embodiment, the ascorbic acid peroxidase activity refers to the generation of water (H2O) using hydrogen peroxide (H2O2) as a substrate.

[0011] In another preferred embodiment, the ascorbic acid peroxidase has the activity of synthesizing protocatechuic aldehyde using p-hydroxybenzaldehyde as a substrate.

[0012] In another preferred embodiment, the sequence (c) further includes a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (a) or (b).

[0013] Another object of the present invention is to provide an isolated polynucleotide that encodes the ascorbic acid peroxidase.

[0014] In a preferred embodiment, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:2.

[0015] It should be understood that, considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons appropriate for specific species as needed. Therefore, the polynucleotide of ascorbic acid peroxidase of the present invention also includes a nucleotide sequence encoding ascorbic acid peroxidase activity obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO:2.

[0016] Another object of the present invention is to provide a vector containing the aforementioned polynucleotide. The vector is obtained by operatively linking the polynucleotide encoding the ascorbic acid peroxidase of the present invention to an expression vector, thereby obtaining a recombinant expression vector capable of expressing the ascorbic acid peroxidase of the present invention or a gene silencing vector that inhibits the expression of the polynucleotide encoding the ascorbic acid peroxidase of the present invention.

[0017] In a preferred embodiment, the vector is a recombinant expression vector pET28a-LaAPX24999 containing the sequence encoded by SEQ ID NO:2 of the ascorbic acid peroxidase.

[0018] In another preferred embodiment, the vector is a recombinant expression vector pET28a-LaAPX24999(ΔC45) containing polynucleotides encoding positions 1-738 of the sequence shown in SEQ ID NO:2 of the ascorbic acid peroxidase.

[0019] Another object of the present invention is to provide a host cell containing the aforementioned vector or genome incorporating the aforementioned polynucleotides. The host cell is a prokaryotic or eukaryotic cell. Commonly used prokaryotic host cells include *Escherichia coli*, *Bacillus subtilis*, *Pseudomonas motilityis*, and lactic acid bacteria; commonly used eukaryotic host cells include fungal cells, plant cells, insect cells, and mammalian cells. The fungal cells include yeast cells. By introducing the aforementioned recombinant expression vector or gene silencing vector into the appropriate host cell, genetically engineered strains, transgenic cell lines, transgenic calluses, transgenic tissues, transgenic plants, or genetically engineered plants expressing the ascorbate peroxidase described in this invention are obtained.

[0020] Another object of the present invention is to provide the use of the aforementioned ascorbic acid peroxidase for the synthesis of protocatechuic aldehyde.

[0021] In a preferred embodiment, the substrate is p-hydroxybenzaldehyde.

[0022] Another object of the present invention is to provide an expression construct. The expression construct includes the encoding genes of the following enzymes and / or gene expression cassettes:

[0023] (a) the ascorbate peroxidase described above; and / or

[0024] (b) Biosynthetic enzymes of ascorbic acid peroxidase substrates.

[0025] The gene expression cassette is a biological element required for the expression and regulation of enzymes in host cells, including promoters, enhancers, attenuators, ribosome binding sites, Kozak sequences, introns, and / or transcription terminators; in addition, it may also include tag coding sequences and / or signal (peptide) coding sequences.

[0026] In a preferred embodiment, the expression construct includes the gene encoding the ascorbic acid peroxidase.

[0027] In another preferred embodiment, the expression construct further includes a gene encoding p-hydroxybenzaldehyde synthase (HBS) and a gene encoding tyrosine aminolyase (TAL).

[0028] In another preferred embodiment, when transforming Escherichia coli cells, the expression construct further includes gene expression cassettes such as an Escherichia coli promoter, an Escherichia coli ribosome binding site, and / or an Escherichia coli transcription terminator.

[0029] Another object of the present invention is to provide a host cell. The host cell includes the aforementioned expression construct. The host cell is a prokaryotic or eukaryotic cell. Commonly used prokaryotic host cells include *Escherichia coli*, *Bacillus subtilis*, *Pseudomonas motilityis*, and lactic acid bacteria; commonly used eukaryotic host cells include fungal cells, plant cells, insect cells, and mammalian cells. Preferably, the host cell is a cell that endogenously possesses a substrate of ascorbate peroxidase or a precursor thereof.

[0030] Another object of the present invention is to provide the use of the expressed construct for the synthesis of protocatechuic aldehyde.

[0031] Another object of the present invention is to provide a method for producing protocatechuic aldehyde.

[0032] In a preferred embodiment, the method includes: transforming a host cell with the expression construct, and using the transformed host cell to catalyze the synthesis of protocatechuic aldehyde from p-hydroxybenzaldehyde; wherein the host cell is a prokaryotic or eukaryotic cell. Commonly used prokaryotic host cells include *Escherichia coli*, *Bacillus subtilis*, *Pseudomonas motilityis*, and lactic acid bacteria; commonly used eukaryotic host cells include fungal cells, plant cells, insect cells, and mammalian cells. The fungal cells include yeast cells.

[0033] In another preferred embodiment, the method includes: transforming a host cell with the expression construct, culturing the transformed *E. coli* cells, and synthesizing protocatechuic aldehyde using a simple carbohydrate (such as glucose). The host cell is a cell containing a substrate of ascorbic acid peroxidase; preferably, the host cell is a cell endogenously containing p-hydroxybenzaldehyde or a precursor thereof.

[0034] This invention discloses for the first time an ascorbate peroxidase LaAPX24999 derived from the Amaryllidaceae plant *Lysimachia christinae*, which possesses the ability to catalyze the synthesis of protocatechuic aldehyde from p-hydroxybenzaldehyde. This invention also discloses the polynucleotide encoding the ascorbate peroxidase, the expression vector expressing the ascorbate peroxidase, and the host cell. This invention utilizes ascorbate peroxidase derived from Amaryllidaceae plants to perform the biotransformation and biosynthesis of the plant natural product protocatechuic aldehyde. Attached Figure Description

[0035] Figure 1 This is the result of cloning the gene encoding ascorbic acid peroxidase LaAPX24999.

[0036] Figure 2 These are the biotransformation detection results of recombinant strains Eco-LaAPX24999 and Eco-LaAPX24999(ΔC45).

[0037] Figure 3 These are the mass spectrometry analysis results of the biotransformation products of the recombinant strain Eco-LaAPX24999.

[0038] Figure 4 This is the mass spectrometry analysis result of the protocatechuic aldehyde standard. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0040] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0041] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0042] Example 1: Cloning of the gene encoding ascorbate peroxidase LaAPX24999

[0043] Two primers were synthesized, each with the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing.

[0044] Using cDNA obtained by reverse transcription of RNA extracted from *Lysimachia christinae* as a template, PCR amplification was performed using the two primers SEQ ID NO:3 and SEQ ID NO:4 as described above. The DNA polymerase used was from Nanjing Novizan Biotechnology Co., Ltd. Super-Fidelity DNA polymerase. The PCR amplification program was: 95℃ for 5 min; 94℃ for 45 s, 56℃ for 45 s, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min, then cooled to 10℃. The PCR products were detected by agarose gel electrophoresis, and the results are as follows: Figure 1 .

[0045] Under UV light irradiation, the target DNA band was excised. Then, using a multifunctional DNA purification kit (centrifuge column type) (Beijing Biotech Biotechnology Co., Ltd.), the target DNA was recovered from the agarose gel, which was the amplified DNA fragment encoding the ascorbate peroxidase LaAPX24999 gene. Using the pMD19-T cloning kit from Takara Bio Engineering (Dalian) Co., Ltd. (TaKaRa), the recovered PCR product was cloned into the pMD19-T vector, and the constructed vector was named pMDT-LaAPX24999. Sequencing yielded the encoding gene sequence of ascorbate peroxidase LaAPX24999.

[0046] The gene encoding ascorbic acid peroxidase LaAPX24999 has the nucleotide sequence of SEQ ID NO:2 in the sequence listing. Nucleotides 1-876 from the 5' end of SEQ ID NO:2 constitute the open reading frame (ORF) of ascorbic acid peroxidase LaAPX24999, nucleotides 1-3 from the 5' end of SEQ ID NO:2 constitute the start codon ATG of the gene encoding ascorbic acid peroxidase LaAPX24999, and nucleotides 874-876 from the 5' end of SEQ ID NO:2 constitute the stop codon TGA of the gene encoding ascorbic acid peroxidase LaAPX24999. The gene encoding ascorbic acid peroxidase LaAPX24999 encodes a protein containing 291 amino acids, with the amino acid sequence of SEQ ID NO:1. Software predictions indicate that the theoretical molecular weight of this protein is 32 kDa, and its isoelectric point (pI) is 6.20.

[0047] Example 2: Construction of the recombinant expression vector for ascorbate peroxidase LaAPX24999

[0048] (1) Two primers with the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing were synthesized. Nco I and Xho I restriction enzyme sites and their protective base sequences were set at the 5' ends of the synthesized primers SEQ ID NO:5 and SEQ ID NO:6, respectively. PCR amplification was performed using the pMDT-LaAPX24999 vector as a template. The PCR amplification procedure was the same as in Example 1. The PCR amplification product was detected by agarose gel electrophoresis, separated, and recovered by gel cutting. It was then digested with Nco I and Xho I and ligated into the pET28a vector (Novagen), which had also been digested with Nco I and Xho I, using Takara Bio Engineering (Dalian) Co., Ltd. (TaKaRa) T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and plated on LB agar plates supplemented with 25 μg / mL kanamycin. Positive transformants were obtained by colony PCR verification. Sequencing further confirmed the successful construction of the recombinant plasmid pET28a-NcoI-LaAPX24999-XhoI, which contained the full-length polynucleotide sequence of SEQ ID NO:2 between the Nco I and Xho I restriction sites. The obtained recombinant plasmid was named pET28a-LaAPX24999.

[0049] (2) Two primers with the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:7 in the sequence listing were synthesized. Nco I and Xho I restriction enzyme sites and their protective base sequences were added to the 5' ends of the synthesized primers SEQ ID NO:5 and SEQ ID NO:7, respectively. PCR amplification was performed using the pMDT-LaAPX24999 vector as a template. The PCR amplification procedure was the same as in Example 1. The PCR amplification products were detected by agarose gel electrophoresis, separated, and recovered by gel cutting. They were then digested with Nco I and Xho I and ligated into the pET28a vector (Novagen) which had also been digested with Nco I and Xho I using T4 DNA ligase (purchased from Takara Bio Engineering (Dalian) Co., Ltd. (TaKaRa)). The ligation products were transformed into competent E. coli DH5α cells (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and plated on LB plates supplemented with kanamycin (final concentration 25 μg / mL). Positive transformants were verified by colony PCR. Sequencing further confirmed the successful construction of the recombinant plasmid pET28a-NdeI-LaAPX24999(ΔC45)-XhoI, which contained nucleotides 1-738 of the polynucleotide sequence in SEQ ID NO:2 between the Nco I and Xho I restriction sites. The obtained recombinant plasmid was named pET28a-LaAPX24999(ΔC45).

[0050] Example 3: Synthesis of protocatechuic aldehyde from p-hydroxybenzaldehyde catalyzed by ascorbate peroxidase LaAPX24999

[0051] (1) Preparation of culture media. Induction medium (1L): 30g / L glycerol, 12g / L casein hydrolysate, 24g / L yeast extract, 2.31g / L KH2PO4, 12.54g / L K2HPO4, 5mM MgSO4, 1% trace element stock solution, 1μg / L VB1, 0.5μg / L VH. Biotransformation medium (1L): 100mM MOPS, 28.71mM K2HPO4, 25.72mM KH2PO4, 26.50mM (NH4)2HPO4, 10mM citric acid, 0.02mM phenol red, adjusted to pH 7.2. The trace element stock solution formula (1L) is: 36.00mM FeSO4, 7.82mM ZnSO4, 4.00mM CuSO4, 2.00mM MnSO4, 0.60mM Na2B4O7, 13.60mM CaCl2, 0.08mM (NH4)6MO7O 24 It dissolves in 100mM HCl.

[0052] (2) The recombinant plasmids pET28a-LaAPX24999 and pET28a-LaAPX24999(ΔC45) were transformed into Escherichia coli BL21(DE3) cells to obtain recombinant strains BL21(DE3) / pET28a-LaAPX24999 and BL21(DE3) / pET28a-LaAPX24999(ΔC45), which were named strains Eco-LaAPX24999 and Eco-LaAPX24999(ΔC45), respectively. Single clones of each strain were picked and inoculated into LB culture medium supplemented with kanamycin (final concentration 25 μg / ml) and cultured overnight at 37°C and 200 rpm.

[0053] (3) Dilute the overnight culture 100 times in 50 mL of induction medium containing kanamycin (final concentration 25 μg / mL) and culture. When the bacterial culture grows to an absorbance of 0.6-0.8 at 600 nm, add the inducing agent isopropyl-β-D-thiogalactoside (IPTG) (final concentration 0.1 mmol / L) for induction culture for 16 h at 30 °C.

[0054] (4) Collect the bacterial culture medium after 16 h of induction, centrifuge at 12000 rpm and 4℃ for 5 min, discard the supernatant and wash the cells once with 40 mL of biotransformation medium. Concentrate the cells with biotransformation medium, add 1 mM p-hydroxybenzaldehyde and 5 mM ascorbic acid to a final concentration, and incubate at 30℃ and 220 rpm.

[0055] (5) Take 1 ml of the biotransformation solution and freeze-thaw it at 4℃. Add an equal volume of methanol and shake to mix. Centrifuge at 12000 rpm for 5 min at room temperature. Filter the supernatant through a 0.22 μm pore size filter membrane. Analyze the filtrate using a high-performance liquid chromatograph (HPLC). The analytical conditions were as follows: LC-20A HPLC system (Shimadzu, Japan), InertSustain C18 column (5 μm, 4.6 mm × 250 mm), column temperature 35℃, diode array detector, wavelength 310 nm, injection volume 10 μl, mobile phase A 0.1% (v / v) formic acid aqueous solution, mobile phase B 100% acetonitrile, flow rate 1.0 mL / min, gradient elution. The analytical results are as follows: Figure 2 , Figure 3 and Figure 4 The results showed that both ascorbate peroxidase LaAPX24999 and LaAPX24999(ΔC45) could catalyze the synthesis of protocatechuic aldehyde from the substrate p-hydroxybenzaldehyde.

[0056] Example 4: Construction of recombinant Escherichia coli Eco-LaAPX24999-TAL

[0057] (1) Two primers with nucleotide sequences of SEQ ID NO:8 and SEQ ID NO:9 respectively were synthesized, and the T7-RBS-TAL-T7ter operon fragment was amplified by PCR using the recombinant plasmid p15a-TAL as a template. The PCR amplification program was as follows: 95℃ for 5 min; 94℃ for 45 s, 56℃ for 45 s, 72℃ for 3 min, for a total of 30 cycles; 72℃ for 10 min, then cooled to 10℃. The PCR products were separated and recovered by agarose gel electrophoresis. The recovered DNA fragment had 25 bp sequences at both ends that were homologous to the Sph I restriction site on both sides of the pET28a-LaAPX24999 vector. The pET28a-LaAPX24999 vector was linearized using Sph I restriction endonuclease (purchased from Takara Bio Engineering (Dalian) Co., Ltd. (TaKaRa)). Using a one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), the T7-RBS-TAL-T7ter fragment was introduced into the Sph I restriction site of the pET28a-LaAPX24999 vector according to the product instructions. Colony PCR was performed using synthesized universal sequencing primers T7 and T7ter to verify the recombinant expression plasmid pET28a-LaAPX24999-TAL. Further sequencing confirmed the correct sequence of the T7-RBS-TAL-T7ter fragment in the recombinant plasmid.

[0058] (2) The recombinant plasmid pET28a-LaAPX24999-TAL was transformed into Escherichia coli BL21(DE3) cells to obtain the recombinant strain BL21(DE3) / pET28a-LaAPX24999-TAL, which was named strain Eco-LaAPX24999-TAL.

[0059] Example 5: Fermentation synthesis of protocatechuic aldehyde by recombinant Escherichia coli Eco-LaAPX24999-TAL

[0060] (1) Preparation of fermentation medium (1L): 12g / L casein hydrolysate, 24g / L yeast extract, 31g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5.0g NH4Cl, 0.24g MgSO4, 0.01g CaCl2, 30g Glucose, 1mL trace element stock solution. The formula for the trace element stock solution is the same as in Example 3.

[0061] (2) Select a single clone of strain Eco-LaAPX24999-TAL and inoculate it with LB culture supplemented with kanamycin (final concentration of 25 μg / ml) and culture overnight at 37℃ and 200 rpm.

[0062] (3) Dilute the overnight culture 100-fold in 50 mL of fermentation medium containing kanamycin (final concentration 25 μg / mL) and culture. When the bacterial culture grows to an absorbance of 0.6-0.8 at 600 nm, add isopropyl-β-D-thiogalactoside (IPTG) (final concentration 0.1 mmol / L) to induce fermentation. Change the culture temperature to 30 °C and ferment for 72 h.

[0063] (4) Take 1 ml of fermentation broth and freeze-thaw it at 4℃, add an equal volume of methanol, and shake to mix. Centrifuge at 12000 rpm for 5 min at room temperature. Filter the supernatant through a 0.22 μm pore size filter membrane, and analyze the filtrate using high-performance liquid chromatography (HPLC). The analytical conditions are the same as in Example 3. The analytical results show that the constructed Escherichia coli strain can synthesize protocatechuic aldehyde, with a product concentration of 4.8 mg / L. This can be understood as follows: based on microbial strains capable of synthesizing the substrate of ascorbic acid peroxidase LaAPX24999 and its upstream metabolites, ascorbic acid peroxidase LaAPX24999 can perform de novo biosynthesis of protocatechuic aldehyde.

[0064] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An ascorbic acid peroxidase, characterized in that, The ascorbic acid peroxidase is a protein with the amino acid sequence shown in SEQ ID NO:

1.

2. The polynucleotide encoding the ascorbic acid peroxidase of claim 1, characterized in that, The nucleotide sequence of this polynucleotide is shown in SEQ ID NO:

2.

3. A carrier, characterized in that, It contains the polynucleotide as described in claim 2.

4. The use of the carrier according to claim 3, characterized in that, The ascorbic acid peroxidase of claim 1 is expressed.

5. An expressive construct, characterized in that, The expression construct includes the gene expression cassette of ascorbic acid peroxidase as described in claim 1.

6. A host cell, characterized in that, The host cell contains the vector of claim 3 or the expression construct of claim 5, or the genome integrates the polynucleotide of claim 2.

7. The use of the ascorbate peroxidase according to claim 1, characterized in that, Catalyzing the synthesis of protocatechuic aldehyde from p-hydroxybenzaldehyde.

8. A method for producing protocatechuic aldehyde, characterized in that, Protocatechuic aldehyde is produced using the ascorbic acid peroxidase described in claim 1; the method includes: culturing the host cells described in claim 6 to synthesize protocatechuic aldehyde.

Images