CYP450 gene participating in synthesis of podophyllotoxin compound as well as encoding product and application of CYP450 gene

Abstract

The invention relates to the field of medicinal plant genetic engineering, and particularly discloses a CYP450 gene participating in lignan synthesis in a plant and a coding product thereof. The invention aims to analyze the oxidation modification step of the lignan biosynthetic pathway in the plant, and provides a gene element for the subsequent synthetic biological application. The invention discloses a C-5 hydroxylase gene sequence and an amino acid sequence thereof required by biosynthesis of aryl tetrahydronaphthalene lignans in podophyllum hexandrum for the first time. Reference is provided for subsequent rational design and directional transformation of lignan compound structure modification enzymes, development and utilization of biosynthetic pathway elements and synthetic biology heterologous efficient production of podophyllotoxin compounds.

Description

Technical Field

[0001] The present invention relates to screening CYP450 genes related to the biosynthesis of lignans, a main active ingredient, from a Podophyllum subfamily plant Podophyllum quinata transcriptome library, and performing functional activity analysis on the gene-encoded proteins using a method of feeding with a saccharomyces cerevisiae substrate. The invention relates to CYP450 genes involved in lignan synthesis in Podophyllum subfamily plants, their encoded products, and applications, and belongs to the field of medicinal plant genetic engineering. Background Art

[0002] The subfamily Podophylloideae of the Berberidaceae family includes the genera Sinopodophyllum, Sinopodophyllum, Sinopodophyllum (Royle) Ying, and Sinopodophyllum chinensis. Their active ingredients include lignans, flavonoids, and amino acids. From a phylogenetic perspective, Sinopodophyllum is the most basal group of the subfamily Podophylloideae, and only the species Sinopodophyllum within this genus possesses multiple medicinal properties. Sinopodophyllum was first mentioned in the oldest extant Tibetan medical text, "Yuewang Yaozhen," and was later included in the "Compendium of Materia Medica" for its miraculous anti-toxic properties. Sinopodophyllum is the root and rhizome of Sinopodophyllum hexandrum (Royle) Ying, a plant of the Berberidaceae family. It has the properties of dispelling wind and dampness, promoting blood circulation and relieving pain, and removing phlegm and relieving cough. As one of the "Seven Herbs of Taibai," Sinopodophyllum is rich in lignans, amino acids, vitamins, and trace elements, and possesses extremely high nutritional value. Aryltetralin lignans are the representative active compounds, and related chemical semi-synthetic derivatives such as etoposide and teniposide are widely used anti-cancer drugs in clinical practice. They exert highly effective anti-tumor effects by inhibiting microtubules and DNA topoisomerase II.

[0003] The biosynthetic pathway of aryl tetralin lignans can be divided into four parts: (1) the highly conserved upstream phenylpropanoid pathway to produce coniferyl alcohol (CA); (2) coniferyl alcohol to deoxypodophyllotoxin (DPT); (3) hydroxylation and methylation modification of deoxypodophyllotoxin; and (4) glycosylation modification to produce more water-soluble glycoside compounds. Among them, cytochrome P450 (CYP450) enzymes play an important role in the modification process. Therefore, the ShCYP736A468 gene of the present invention is a key enzyme gene for the formation of hydroxyl groups at the C-5 position and is also a key step in the post-modification of podophyllotoxin compounds. It will provide a reference for the rational design and targeted modification of subsequent podophyllotoxin compound structure modification enzymes, the development and utilization of biosynthetic pathway components, and the efficient production of synthetic biology heterologous production. Before the disclosure of the present invention, there has been no disclosure or report on the C-5 hydroxylase gene sequence and amino acid sequence required for the biosynthesis of aryltetralin lignans in Psoralea corylifolia mentioned in the present invention. Summary of the Invention

[0004] The purpose of the present invention is to provide a CYP450 gene ShCYP736A468 involved in the lignan biosynthesis pathway in the Podophyllotoxin subfamily plant Podophyllum truncatum. The protein encoded by the gene can catalyze the C-5 hydroxylation of (-)-deoxypodophyllotoxin in the cooperation of a suitable CYP450 reductase (cytochrome P450 reductase, CPR) to produce β-peltain A (β-peltain A).

[0005] The present invention provides a key gene in the biosynthesis pathway of aryl tetralin lignans: ShCYP736A468, whose nucleotide sequence is shown in SEQ ID No. 1 in the sequence listing. It also provides a protein encoded by the 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 that has the same activity as the amino acid residue sequence of SEQ ID No. 2.

[0006] The DNA sequence of SEQ ID No. 1 of the present invention consists of 1452 bases and encodes the protein sequence SEQ ID No. 2 in the sequence table consisting of 483 amino acid residues.

[0007] Second-generation transcriptome data showed that the cloned gene ShCYP736A468 was highly expressed in the stems of the Podophyllum subfamily plant, but was hardly expressed in the roots. Furthermore, the expression level in the stems was upregulated by jasmonic acid.

[0008] The present invention also includes the screening method, expression vector, cell line and host bacteria containing the gene ShCYP736A468 of the present invention, the product enrichment preparation method, and the use of this gene in the subsequent rational design and directed modification of aryl tetralin lignan structure modification enzymes, the development and utilization of biosynthetic pathway elements, and the synthetic biology heterologous efficient production of aryl tetralin lignans. pESC-LEU was used as the eukaryotic expression vector for the ShCYP736A468 gene in Saccharomyces cerevisiae BY4741. The CYP450 reductase TwCPR3 from Tripterygium wilfordii Hook.f. was inserted between the restriction endonuclease BamHI and SalI sites. The gene from SEQ ID No. 1 was then cloned into the eukaryotic expression vector pESC-LEU between the restriction endonuclease NotI and SpeI sites to construct the recombinant expression plasmid pESC-LEU::(ShCYP736A468+TwCPR3) carrying the ShCYP736A468 gene. The recombinant expression plasmid was then transformed into the yeast BY4741 expression host and expressed using galactose induction. Functionality was verified by feeding (-)-deoxypodophyllotoxin as a substrate. The reaction product was extracted with ethyl acetate, and the product was analyzed by LC-MS, comparing its retention time and mass spectrometric fragmentation pattern with that of a standard. LC-MS analysis results showed that (-)-deoxypodophyllotoxin was catalyzed by ShCYP736A468 enzyme to generate [M+H] + =415.13794. By analyzing the molecular ion peak of the product and conducting large-scale fermentation, separating and preparing the product and performing nuclear magnetic resonance identification, it is believed that the product is (-)-podophyllotoxin. The research results show that the gene related to the synthesis of aryl tetralin lignan podophyllotoxin of the present invention has a characteristic structural domain of cytochrome P450 gene. The results of substrate feeding experiments show that the gene can catalyze the C-5 hydroxylation of (-)-deoxypodophyllotoxin to form β-peltophyllin A, which has important theoretical and practical significance for the rational design and targeted modification of subsequent aryl tetralin lignan structure modification enzymes and the development and utilization of biosynthetic pathway components. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1 Figure 3 shows the bioinformatics analysis of the protein structure encoded by the ShCYP736A468 gene. Figure A shows the transmembrane structure and analysis of the protein encoded by the ShCYP736A468 gene, and Figure B shows the AlphaFold3 tertiary structure prediction of the protein encoded by the ShCYP736A468 gene. Instructions attached Figure 2 The expression of ShCYP736A468 gene in different tissues of Psoralea corylifolia and different jasmonic acid induction times in Example 3 is shown in the figure; Figure 3Figures 4 and 5 show LC-MS analysis of the ShCYP736A468 gene-encoded protein substrate feeding reaction products. Figure A shows the ion range extraction (mass range = 390-420) of the ShCYP736A468 catalytic reaction: a. Mass range of the standard solution; b. Mass range of the extract product from the substrate feeding reaction of the recombinant expression vector pESC-LEU::(TwCPR3) yeast; c. Mass range of the extract product from the substrate feeding reaction of the recombinant expression plasmid pESC-LEU::(ShCYP736A468+TwCPR3) yeast. Figure B shows the corresponding mass spectrum of the extract product from the substrate feeding experiment of the recombinant expression plasmid pESC-LEU::(ShCYP736A468+TwCPR3) yeast by LC-MS. Instructions attached Figure 4 For the (-)-deoxypodophyllotoxin and the preparation product β-podophyllin A in Example 4 1 H-NMR (800M) and 13 C-NMR (200M) nuclear magnetic identification spectrum: a. Substrate (-)-deoxypodophyllotoxin 1 H-NMR spectrum (800M, CDCl3); b. Preparation of products 1 H-NMR spectrum (800M, CDCl3); c. Substrate (-)-deoxypodophyllotoxin 13 C-NMR spectrum (200M, CDCl3); d. Preparation of products 13 C-NMR spectrum (200M, CDCl3). DETAILED DESCRIPTION

[0009] Example 1: Screening of CYP450 genes in the metabolic pathway of podophyllotoxin compounds

[0010] This experiment used a transcriptome database constructed at different times and tissue locations after jasmonic acid induction in P. truncatula plants to screen for CYP450 genes whose expression levels in roots were higher than in stems and whose expression was upregulated following jasmonic acid induction. A similarity comparison was performed among annotated CYP450 genes in the transcriptome, based on the amino acid sequence of CYP82D61, a gene known to generate the β-hydroxyl group at the C-4 position of (-)-deoxypodophyllotoxin. Furthermore, colinearity analysis was performed between candidate CYP450 genes and known downstream genes in the podophyllotoxin pathway to identify candidate genes. Through these screening methods, 58 candidate genes were identified from a total of 271 CYP450 genes in the second-generation transcriptome data.

[0011] Example 2: Cloning of CYP450 genes in Psoralea corylifolia

[0012] 1. Reversal of the first-strand cDNA of Taoerqi

[0013] The total RNA of Prunus mume was extracted from Prunus mume roots induced with jasmonic acid using the Eastep super total RNA extraction kit from Shanghai Promega Company. The 5' and 3' ends of the synthesized first-strand cDNA were amplified using the SMARTer RACE 5' / 3'Kit from TAKARA to obtain the first-strand cDNA of Prunus mume.

[0014] 2. Cloning and Sequencing of Full-length cDNA

[0015] Based on transcriptome data from the second and third generations of Taoerqi, 58 candidate CYP450 genes were identified. The ORF region was searched, and primers for the full-length genes were designed for amplification using cDNA as a template. Agarose gel electrophoresis revealed a specific fragment at approximately 1500 bp. The target fragment was recovered using an agarose gel extraction kit (Thermo Scientific) and cloned into the pEASY-Blunt Zero vector (Transgen). Positive clones were identified and verified by sequencing for use in the construction of expression vectors.

[0016] Example 3: Bioinformatics analysis, tissue expression and induced expression analysis of ShCYP736A468 gene

[0017] 1. Bioinformatics Analysis of ShCYP736A468 Gene Sequence

[0018] The full-length open reading frame (ORF) of the podophyllotoxin biosynthesis pathway CYP450 gene ShCYP736A468 of the present invention is 1452 bp in length, encoding 483 amino acids. The detailed sequence is shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence table. The results of the primary structure of the protein showed that the amino acid sequence of ShCYP736A468 has a highly conserved proline-rich region in the eukaryotic CYP450, located between amino acids 28-31, and the downstream is the catalytic functional region of ShCYP736A468; the results of the protein transmembrane domain structure showed that residues 3-20 of the ShCYP736A468 protein are anchored on the endoplasmic reticulum membrane, and the rest is a soluble protein; the results of the AlphaFold3 tertiary structure prediction showed that there is a rigid helix anchoring the endoplasmic reticulum membrane at the N-terminus of the protein, and the rest of the region is similar to the known CYP450 protein. The protein with the highest similarity to the ShCYP736A468 protein in the PDB database is the CYP76AH1 protein from Danshen, with a similarity of 30.69% (see the instructions for the attached data). Figure 1 ).

[0019] 2. Analysis of tissue expression and induced expression of ShCYP736A468 gene

[0020] Analysis of the relative expression FPKM value of the ShCYP736A468 gene in the second-generation transcriptome data showed that the expression level of the ShCYP736A468 gene in the stem of P. truncatum was significantly higher than that in the root; the expression level of the ShCYP736A468 gene in the stem induced by jasmonic acid showed a slow upward trend, indicating that jasmonic acid can upregulate the expression level of genes in the β-shield podophyllin A biosynthesis pathway and induce the promotion of β-shield podophyllin A biosynthesis (see the instructions). Figure 2 ).

[0021] Example 4: Eukaryotic expression and functional analysis of ShCYP736A468 gene

[0022] 1. Construction of Yeast Expression Vector

[0023] pESC-LEU, in which the Tripterygium wilfordii CYP450 reductase TwCPR3 was inserted between the restriction endonuclease Bam HI and Sal I sites, was selected as the yeast eukaryotic expression vector. By designing primers with Not I and Spe I restriction sites and performing PCR amplification, the open reading frame of the cloned Prunus armeniaca CYP450 gene was inserted between the Not I and Spe I restriction sites of the yeast expression vector to obtain the recombinant plasmid pESC-LEU::(ShCYP736A468+TwCPR3), which was then identified by PCR and enzyme digestion.

[0024] 2. Inducible expression of ShCYP736A468 in Psoralea corylifolia

[0025] The recombinant plasmid pESC-LEU::(ShCYP736A468+TwCPR3) was transformed into the Saccharomyces cerevisiae expression strain BY4741 using chemical transformation, and expression was induced by galactose. The resulting expression system was used to detect activity in substrate feeding experiments.

[0026] 3. Activity Analysis of ShCYP736A468 in Psoralea corylifolia

[0027] After one day of induction, the substrate (-)-deoxypodophyllotoxin was added to the bacterial suspension at a final concentration of 20 μM, and induction was continued for another two days. The product was extracted with an equal volume of ethyl acetate and analyzed by LC-MS. The LC-MS instrument was a Thermo QExactive HF, with a mobile phase of 0.2% formic acid in acetonitrile at a flow rate of 0.4 ml / min and a column temperature of 30°C. The gradient elution (B%) was as follows: 0 min to 25%, 18 min to 50%, 19 min to 100%, 20 min to 100%, and 21 min to 25%, with an equilibration time of 2 min. The results showed that, with (-)-deoxypodophyllotoxin as substrate, the catalytic group containing the ShCYP736A468 gene recombinant plasmid pESC-LEU::(ShCYP736A468+TwCPR3) produced a new substance at the total ion 415.13794 (m / z) compared with the empty vector control and other gene expression vector reaction products (see the instructions). Figure 3 ).

[0028] 4. Identification of (-)-deoxypodophyllotoxin Products Catalyzed by ShCYP736A468

[0029] To clarify the function of the ShCYP736A468 gene, the hydroxylation product was identified by enriching the product through large-scale fermentation. Using the same strain as the above functional verification, the fermentation system was expanded to 6L, a total of 20mg of substrate (-)-deoxypodophyllotoxin was fed, and the product was extracted for preparation, separation and purification to obtain 3mg of purified product (purity>95%). 2mg of deoxypodophyllotoxin and the purified product were dissolved in deuterated chloroform (CDCl3) and 800M 1 H-NMR and 200M 13 C-NMR analysis, the results are as attached in the manual Figure 4 The NMR data attribution information is shown in Tables 1 and 2. The above results indicate that ShCYP736A468 can catalyze the C-5 hydroxylation of deoxypodophyllotoxin to generate β-podophyllotoxin A. Table 1 (-)-deoxypodophyllotoxin and β-podophyllin A 1 H-NMR spectrum (800M) data assignment Table 2 (-)-deoxypodophyllotoxin and β-podophyllin A 13 C-NMR spectrum (200M) data assignment

Claims

An isolated enzyme having the amino acid sequence shown in SEQ ID NO:

2.

2. A polynucleotide encoding the enzyme according to claim 1. 3 . The polynucleotide according to 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 bacterium according to claim 5 or 6 in regulating and / or synthesizing podophyllotoxin-like compounds.

8. The use according to claim 7, wherein the aryltetralin lignan compound is β-podophyllin A and its 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 thereby.

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