CYP716 gene participating in triterpene synthesis in corydalis tuber as well as encoding product and application of CYP716 gene

Abstract

The invention aims to analyze CYP450 modified by oxidizing a pentacyclic triterpenoid in rhizoma corydalis, and discloses a C-28-site hydroxylation gene sequence and an amino acid sequence thereof required for forming homoroot diol in rhizoma corydalis for the first time, which is a triterpenoid component 3beta-hydroxy-oleanane-11, 11 ', 12'-triterpenoid separated from subsequent rhizoma corydalis. A basis is provided for full analysis of a biosynthetic pathway of the 1, 13-diene-28-acid, and a reference is provided for heterologous efficient generation of high root diol as a gene element.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and relates to a CYP450 gene involved in triterpenoid synthesis in Papaveraceae plants, its encoded product and applications. By analyzing the genome library of Corydalis yanhusuo, a plant of the genus Corydalis in Papaveraceae, a CYP450 gene related to the biosynthesis of triterpenoid components in Corydalis yanhusuo was screened, and co-expressed with upstream pathway genes in a Saccharomyces cerevisiae heterologous system to analyze the functional activity of the encoded protein of the screened gene. Background Art

[0002] The Papaveroideae includes the genera Corydalis, Papaver, Eschscholtzia, Macleaya, etc., and the active ingredients are alkaloids, triterpenoids, organic acids, etc. Corydalis yanhusuo was first recorded as "Yanhusuo" in Lei Gong Pao Zhi Lun during the Southern and Northern Dynasties, and was called Yuanhusuo in the ancient herbal classics of subsequent dynasties. It was renamed the well-known Corydalis yanhusuo in Kai Bao Ben Cao of the Song Dynasty, specifically referring to the dried tuber of Corydalis yanhusuo W.T.Wang, a plant of the Papaveraceae family. As one of the famous "Eight Flavors of Zhejiang", it has effects such as relieving pain, promoting blood circulation, and regulating qi. Corydalis yanhusuo is rich in benzylisoquinoline alkaloid components. With the separation and research of the chemical components of Corydalis yanhusuo, its triterpenoid components have also attracted attention. 3β-Hydroxy-olean-11,13-diene-28-oic acid can be isolated and identified from Corydalis yanhusuo. At present, there are few related studies on this compound, but it can be clearly known that as a pentacyclic triterpenoid compound widely existing in nature, there is also a corresponding biosynthetic pathway and specific catalytic enzymes in Corydalis yanhusuo.

[0003] According to the skeleton structure of the chemical component 3β-hydroxy-olean-11,13-diene-28-oic acid, it can be classified into β-amyrin-type pentacyclic triterpenoid compounds. The biosynthetic pathway of this type of compound has been clarified in various medicinal plants and model plants, and can be simply divided into three parts: (1) The mevalonic acid (MVA) pathway and the 2-methyl-D-erythritol-4-phosphate (MEP) pathway synthesize the common precursor 2,3-oxidosqualene; (2) 2,3-Oxidosqualene forms pentacyclic triterpenoid components with different skeletal conformations under the action of 2,3-oxidosqualene cyclases (OSCs); (3) The pentacyclic triterpenoid skeleton forms various pentacyclic triterpenoid components under the post-modification of Cytochrome P450 (CYP450) and glycosyltransferase (GT). Summary of the Invention

[0004] The object of the present invention is to provide a CYP450 gene CyCYP716A467 involved in the synthetic pathway of pentacyclic triterpenoids in Corydalis of Papaveroideae. The protein encoded by it can catalyze the hydroxylation of C-28 of β-amyrin in cooperation with a suitable cytochrome P450 reductase (CPR) to generate erythrodiol.

[0005] The present invention provides a key gene in the biosynthetic pathway of oleanane-type pentacyclic triterpenoids: CyCYP716A467, whose nucleotide sequence is shown as SEQ ID No.1 in the sequence listing. And a protein encoded by the above gene, a protein encoded by the amino acid sequence of SEQ ID No.2 in the sequence listing, a protein having the amino acid residue sequence of SEQ ID No.2 in the sequence listing and a protein derived from SEQ ID No.2 having the same activity as the amino acid residue sequence of SEQ ID No.2.

[0006] Another object of the present invention is to provide a polynucleotide sequence encoding the Tripterygium wilfordii triterpene synthase protein, as shown in SEQ ID No.1 in the sequence listing. The full-length sequence of the polynucleotide or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention for the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequence. Corydalis is a tetraploid plant with a relatively high heterozygosity and complex genes. Multiple homologous genes can be cloned using a pair of primers.

[0007] The DNA sequence of SEQ ID No.1 of the present invention consists of 1455 bases and encodes the protein sequence SEQ ID No.2 composed of 484 amino acid residues in the sequence listing.

[0008] Another object of the present invention is to provide an expression vector, which contains a polynucleotide encoding the Corydalis triterpene synthase protein or its variant of the present invention. The expression vector contains a promoter and a terminator, wherein the promoter is operably linked to the polynucleotide, and the polynucleotide is operably linked to the transcription terminator.

[0009] Another object of the present invention is to provide a host cell, which comprises the polynucleotide molecule encoding the protein CyCYP716A467 or its variant according to the present invention, or comprises the expression vector described above in the present invention. The host cell is selected from: bacteria, prokaryotic cells (such as Escherichia coli), fungal cells, yeast cells, insect cells, mammalian cells or plant cells. Preferably, it is a yeast cell or a plant cell.

[0010] The screening method, expression vector, cell line and host bacterium containing the gene CyCYP716A467 of the present invention, as well as the subsequent verification of various catalytic activity functions using this gene are also within the protection scope of the present invention. The protein encoded by CyCYP716A467 in Corydalis yanhusuo can be developed and utilized as a post-modification element for pentacyclic triterpenoids, and at the same time promote the complete analysis of the biosynthetic pathway of 3β-hydroxy-olean-11,13-diene-28-oic acid. The pESC-LEU was selected as the eukaryotic expression vector of the CyCYP716A46 gene in Saccharomyces cerevisiae GQ1. The CYP450 reductase TwCPR3 derived from Tripterygium wilfordii Hook.f. was inserted between the restriction enzyme sites of BamHI and Sal I, and the gene of SEQ ID No.1 was cloned between the restriction enzyme sites of Not I and Spe I of the eukaryotic expression vector pESC-LEU to construct a recombinant expression plasmid pESC-LEU::(CyCYP716A46 + TwCPR3); it was transferred into the yeast strain GQ1, and the expression host bacterium was induced to express with galactose. GQ1, as the modified Saccharomyces cerevisiae strain, can endogenously produce high-yield 2,3-oxidosqualene, and co-express heterologously to construct TwOSC1 with high-yield β-amyrin FBR The pYES2 of FBR and the recombinant plasmid pESC-LEU::(CyCYP716A46 + TwCPR3) were used to extract the reaction product with ethyl acetate, derivatize it with bis(trimethylsilyl)trifluoroacetamide (BSTFA), and analyze the retention time and mass spectrometry fragmentation pattern of the product by comparing it with the standard. The GC-MS analysis results showed that under the catalysis of the CyCYP716A467 enzyme, β-amyrin generated a characteristic ion peak M / Z = 216. By analyzing the molecular ion peak of the product and comparing it with the peak of the standard compound of gaulberdiol derivatized under the same gas phase conditions, it was considered that the product was gaulberdiol. The research results showed that the gene related to the synthesis of pentacyclic triterpenoids in the present invention has the characteristic domain of cytochrome P450 gene. The results of yeast heterologous co-expression experiments proved that this gene can catalyze β-amyrin to form gaulberdiol, which has theoretical and practical significance for the subsequent study of the functional promiscuity of CyCYP716A467 and the development and utilization of the biosynthetic pathway components of oleanane-type pentacyclic triterpenoids. Description of the Drawings

[0011] Instructions attached Figure 1 For Example 3, in Figure A, the CyCYP716A467 gene is located on the chromosome of Corydalis yanhusuo, and in Figure B, the gene family analysis is shown.

[0012] Instructions attached Figure 2 For Example 3, it is a bioinformatics analysis diagram of the protein structure encoded by the CyCYP716A467 gene. In Figure A, it is the transmembrane structure and analysis of the protein encoded by the CyCYP716A467 gene, and in Figure B, it is the prediction of the AlphaFold3 tertiary structure of the protein encoded by the CyCYP716A467 gene.

[0013] Instructions attached Figure 3 For Example 4, it is a GC-MS analysis diagram of the heterologous expression product extraction of the protein encoded by the CyCYP716A467 gene. In Figure A, it is the gas phase diagram of the ion extraction (M / Z = 216) of the catalytic reaction of CyCYP716A467: a. The gas phase diagram of the standard product of homogentisic alcohol; b. The gas phase diagram of the sample co-expressed by CyCYP716A467 and TwOSC1 FBR co-expressed sample gas phase diagram; c. The gas phase diagram of the sample co-expressed by the empty pESC-LEU and TwOSC1 FBR co-expressed sample gas phase diagram; In Figure B, it is the corresponding mass spectrum diagram of the GC-MS product extraction of the recombinant plasmid pESC-LEU::(CyCYP716A46+TwCPR3): a. The mass spectrum diagram of the standard product of homogentisic alcohol; b. The mass spectrum diagram of the sample co-expressed by CyCYP716A467 and TwOSC1 FBR co-expressed sample mass spectrum diagram. Detailed implementation manners

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0016] Example 1: Screening of the pentacyclic triterpene post-modification enzyme CYP716 gene in Corydalis yanhusuo

[0017] According to the relevant annotation information in the genome, a total of 24 genes annotated as β-amyrin 28-monooxygenase / oxidase were screened. The open reading frame (ORF) lengths of the candidate genes were checked using the ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) website, and the candidate genes with ORF lengths greater than 1300bp were further selected. Finally, 9 full-length candidate genes of β-amyrin 28-monooxygenase / oxidase were obtained.

[0018] Example 2: Cloning of the cDNA sequence of Corydalis CYP716

[0019] 1. Total RNA extraction from Corydalis and acquisition of the first strand of cDNA

[0020] Take 4 portions of frozen Corydalis tissues, grind them into powder for RNA extraction, and use the RNAprep Pure PlantPlus Kit (Tiangen Biotech Co., Ltd., Beijing, China) to extract total RNA according to the kit instructions. Identify the integrity of RNA by 1.2% agarose gel electrophoresis, measure the purity of RNA by OD260 / OD280, and measure the purity of RNA by NanoDrop One (ThermoFisher Scientific, Massachusetts, USA). The experimental results prove that the quality of the extracted RNA samples is good. Use the FastKing cDNA First Strand Synthesis Kit (Tiangen, Beijing, China) to extract cDNA according to the instructions.

[0021] 2. Cloning and sequencing of full-length cDNA

[0022] Based on the 8 candidate CYP716 gene information screened from the Corydalis genome data, design full-length gene primers according to the ORF region, and use cDNA as a template for amplification. Agarose gel electrophoresis shows that a specific fragment appears at about 1500 bp. Recover the target fragment with an agarose gel recovery kit (Thermo), clone it into the pEASY-BluntZero vector (Transgen), identify positive clones and perform sequencing verification (Beijing Ruibo Xingke), which is used for the construction of the expression vector. Corydalis is a tetraploid with 32 chromosomes and a relatively high heterozygosity of about 1.69%. Multiple genes with extremely high homology can be cloned with a pair of primers, and a total of 16 genes are cloned.

[0023] Example 3: Bioinformatics analysis and structural sequence analysis of the CyCYP716A467 gene

[0024] 1. Bioinformatics analysis of the CyCYP716A467 gene

[0025] The full-length open reading frame of the CyCYP716A467 gene involved in the formation of pentacyclic triterpenoids in Corydalis yanhusuo is 1455 bp in length and encodes 484 amino acids. The detailed sequence can be seen in SEQ ID No.1 and SEQ ID No.2 in the sequence listing. Using the chromosome assembly information to view the location of the CyCYP716 gene on the chromosome, it was found that 8 candidate genes were respectively located on 5 chromosomes. The full-length open reading frame of CyCYP716A467 was subjected to a homology search in the NCBI database using the BLAST program. The alignment analysis at the amino acid level showed that this gene had a relatively high homology with the β-amyrin 28-monooxygenase gene (NP_001268115.1) in the Vitaceae family of the Vitales order, with a similarity of 70.60%, and phylogenetic tree analysis was performed using the published CYP716 genes of different species. (See the appendix of the specification Figure 1 )

[0026] 2. Sequence analysis of CyCYP716A467 gene

[0027] The results of the primary protein structure showed that the eukaryotic CYP450 of the CyCYP716A467 amino acid sequence had a highly conserved proline-rich region located between amino acids 40-42, and the downstream amino acids 292-300 were the catalytic oxidation active functional region of CyCYP716A467; the results of the protein transmembrane domain showed that the residues 4-26 of the CyCYP716A467 protein were anchored on the endoplasmic reticulum membrane, and the rest were soluble proteins. To further understand the protein structure information, the tertiary structure of the protein was predicted using Alphafold3. (See the appendix of the specification Figure 2 )

[0028] Example 4: Eukaryotic expression and functional analysis of CyCYP716A467 gene

[0029] 1. Construction of yeast expression vector

[0030] The pESC-LEU containing Tripterygium wilfordii CYP450 reductase TwCPR3 inserted between the restriction enzyme sites of Bam HI and Sal I was selected as the yeast eukaryotic expression vector. By designing primers with Not I and Spe I restriction enzyme sites and PCR amplification, the open reading frame of the cloned Corydalis yanhusuo CYP450 gene was inserted between the Not I and Spe I restriction enzyme sites of the yeast expression vector to obtain the recombinant plasmid pESC-LEU::(CyCYP716A467+TwCPR3), which was transformed into the competent cells of Escherichia coli Trans-T1 (Transgen). After inverting the plate and culturing the cells for 24 h, colony PCR identification and sequencing verification were performed (Beijing Ruibo Xingke).

[0031] 2. Yeast heterologous expression

[0032] Extract the plasmid from the verified positive bacterial solution, and use the lithium acetate method to co-transform it into the modified Saccharomyces cerevisiae strain (MATahis3Δ1leu2Δ0met15Δ0ura3Δ0tHMG1 ERG1 ERG20 ERG9 ERG1rox1Δbts1Δyjl064wΔypl062wΔ) together with TwOSC FBR Co-transform it into the modified Saccharomyces cerevisiae strain (MATahis3Δ1leu2Δ0met15Δ0ura3Δ0tHMG1 ERG1 ERG20 ERG9 ERG1rox1Δbts1Δyjl064wΔypl062wΔ). Incubate it upside down on an SD-Leu-Ura (2% Glu) double-deficient plate at 37 °C for 3 days to verify plasmid transformation. Pick well-growing bacterial cells and culture them overnight with 5 mL of SD-Leu-Ura (2% Glu). Inoculate them into a 100 mL shake flask containing 20 mL of SD-Leu-Ura (2% Glu) at a final OD600 = 0.05, and culture them on a shaker (30 °C, 200 rpm) for 24 h of expansion culture. Replace the medium with an equal volume of SD-Leu-Ura (2% Gala) and ferment for 48 h.

[0033] 3. Product extraction and detection

[0034] Extract three times by ultrasonic treatment with an equal volume of ethyl acetate, collect all the extracts and concentrate them. Add 100 μL of pyridine and 100 μL of BSTFA to the yeast extract, derivatize at 70 °C for 2 h, then dry it under nitrogen, re-dissolve it with 60 μL of ethyl acetate, and perform detection and analysis by GC / MS. Use an Agilent 7000 gas chromatograph and a DB-5MS capillary column (15 m × 250 μm, 0.1 μm). Then inject 1 μL of the concentrated organic phase with a He flow rate of 2.25 mL / min. The temperature program is 50 °C, with gradient heating, from 50 °C to 280 °C at 60 °C / min, then heat to 305 °C at 1 °C / min and hold for 1 min. The temperature of the ion trap is 230 °C and the electron energy is 70 eV. Record the spectrum in the range of 10 - 550 m / z. Identify all prominent peaks in the GC chromatogram by comparing with the database and standards.

[0035] It was found that in the extracted ion chromatogram at M / Z: 216 for the recombinant plasmid containing pESC-LEU::(CyCYP716A467 + TwCPR3), there were new peaks compared with the blank vector pESC-LEU, and the peak emergence time was consistent with that of the hecogenol derivatization standard. By comparing the mass spectrometry diagrams of the peak emergence times, it was found that the fragment ion peaks could match, indicating that it has the function of catalyzing the formation of hecogenol from β-amyrin. (See attached Figure 3 ) Sequence listing <110>Capital Medical University <120>A CYP716 gene involved in triterpenoid synthesis in Corydalis yanhusuo, its encoded product and applications <160>2 <170>WIPO Sequence 2.3.0 <210>1 <211>1455 <212>DNA <213>Corydalis yanhusuo (Y.H.Chou & C.C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu <400>1 <210>2 <211>484 <212>PRT <213>Corydalis yanhusuo (Y.H.Chou & C.C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu <400>2

Claims

1. An isolated enzyme having an amino acid sequence as shown in SEQ ID NO:

2.

2. A polynucleotide encoding the enzyme according to claim 1. 3 . The polynucleotide of claim 2 , wherein the polynucleotide has a nucleic acid sequence as shown in SEQ ID NO:

1.

4. An expression vector comprising the polynucleotide of claim 2 or 3.

5. A recombinant host bacteria comprising the polynucleotide according to claim 2 or 3, or the expression vector according to claim 4. The recombinant host bacteria according to claim 5 , wherein the host bacteria is Saccharomyces cerevisiae.

7. Use of the enzyme according to claim 1, or the polynucleotide according to claim 2 or 3, or the expression vector according to claim 4, or the recombinant host bacteria according to claim 5 or 6 in regulating and / or synthesizing pentacyclic triterpenoid compounds.

8. The use according to claim 7, wherein the pentacyclic triterpenoid compounds are erythrodiol, oleanolic acid, uracil, uracil acid, betulin, betulic acid and their derivatives.

9. A composition comprising the enzyme of claim 1.

10. A polyamino acid sequence comprising the amino acid sequence of claim 1 with a similarity of more than 90% and an enzyme encoded by the polyamino acid sequence.

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