CYP450 gene participating in lignan synthesis 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-4-site alpha-hydroxylase gene sequence and an amino acid sequence of the C-4-site alpha-hydroxylase gene sequence required by biosynthesis of aryl tetrahydronaphthalene lignan podophyllotoxin 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.

Description

Technical Field

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

[0002] The subfamily Podophyllotoxin of the Berberidaceae family includes the genera Sinopodophyllum, Royle, Royle and Royle, and its active ingredients are lignans, flavonoids and amino acids. From the perspective of systematic evolution, the genus Sinopodophyllum is the most basal group of the subfamily Podophyllotoxin, and only the species Sinopodophyllum in this genus has multiple medicinal effects. Sinopodophyllum was first recorded in the earliest existing Tibetan medical book "Yuewang Yaozhen" in my country, and was later included in "Ben Cao Gangmu Shiyi" for its miraculous anti-toxic effect. It is the root and rhizome of Sinopodophyllum hexandrum (Royle) Ying, a plant of the genus Sinopodophyllum of the Berberidaceae family, and has the effects of dispelling wind and dampness, promoting blood circulation and relieving pain, and removing phlegm and relieving cough. As one of the "Seven Taibai Medicines", Sinopodophyllum is rich in lignans, amino acids, vitamins and trace elements, and has 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 clinical anti-cancer drugs, which exert highly effective anti-tumor effects by inhibiting microtubules and DNA topoisomerase II.

[0003] The biosynthetic pathway of aryltetralin 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 of deoxypodophyllotoxin; (4) glycosylation to produce more water-soluble glycoside compounds. Among them, cytochrome P450 (CYP450) enzymes play an important role in the modification process. The CYP82D61 of Psoralea corylifolia published in a 2015 science article can use (-)-deoxypodophyllotoxin, an important intermediate in downstream biosynthesis, as a substrate to produce (-)-epipodophyllotoxin (Epipodophyllotoxin) with a β-hydroxyl group at the C-4 position. However, the one-step or multi-step reaction that catalyzes the conversion of α-hydroxylation at the C4 position of deoxypodophyllotoxin to (-)-podophyllotoxin is still unknown. Therefore, the ShCYP736A469 gene of the Psoralea corylifolia disclosed in the present invention is a key enzyme gene for the formation of α-hydroxyl group at the C-4 position, and is also the final key step in the analysis of the biosynthetic pathway of podophyllotoxin, which will provide a reference for the rational design and directional transformation of the structural modification enzymes of podophyllotoxin compounds, the development and utilization of biosynthetic pathway elements, and the synthetic biology heterologous efficient production of podophyllotoxin. Before the disclosure of the present invention, there has been no disclosure or report on the C-4 α-hydroxylase gene sequence and its amino acid sequence required for the biosynthesis of the aromatic tetralin-type lignan podophyllotoxin in the Psoralea corylifolia disclosed in the present invention. Summary of the invention

[0004] The purpose of the present invention is to provide a CYP450 gene ShCYP736A469 involved in the lignan biosynthesis pathway in the Podophyllotoxin subfamily plant Podophyllum truncatum, the encoded protein of which can catalyze the α-hydroxylation of (-)-deoxypodophyllotoxin C-4 position in the cooperation of a suitable CYP450 reductase (cytochrome P450 reductase, CPR) to generate (-)-podophyllotoxin.

[0005] The present invention provides a key gene for the biosynthesis pathway of aryl tetralin lignans: ShCYP736A469, whose nucleotide sequence is shown in SEQ ID No. 1 in the sequence table, and a protein encoded by the gene, a protein encoded by the amino acid sequence of SEQ ID No. 2 in the sequence table, a protein having the amino acid residue sequence of SEQ ID No. 2 in the sequence table, 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] The DNA sequence of SEQ ID No. 1 of the present invention consists of 1503 bases and encodes the protein sequence SEQ ID No. 2 consisting of 500 amino acid residues in the sequence table.

[0007] The second-generation transcriptome data showed that the expression levels of the gene ShCYP736A469 cloned in the present invention were relatively high in the roots and stems of the Podophyllotoxin subfamily plant Podophyllum, among which the expression level in the roots that accumulated lignans was significantly higher than that in the aboveground parts such as the stems; and the expression levels in the roots and stems were both upregulated with the induction of jasmonic acid.

[0008] The screening method, expression vector, cell line and host bacteria containing the gene ShCYP736A469 of the present invention, and the use of this gene in the subsequent rational design and directional modification of the aryl tetralin lignan structure modification enzyme, the development and utilization of biosynthetic pathway elements, and the synthetic biology heterologous efficient production of aryl tetralin lignans are also within the protection scope of the present invention. pESC-LEU was selected as the eukaryotic expression vector of Saccharomyces cerevisiae BY4741 for ShCYP736A469 gene, CYP450 reductase TwCPR3 from Tripterygium wilfordii Hook.f. was inserted between the restriction endonuclease BamHI and Sal I sites, and the SEQ ID No.1 gene was cloned between the restriction endonuclease Not I and SpeI sites of the eukaryotic expression vector pESC-LEU to construct a recombinant expression plasmid pESC-LEU::(ShCYP736A469+TwCPR3) with ShCYP736A469 gene; the recombinant expression plasmid was transferred into the yeast strain BY4741 expression host bacteria and galactose was used for induction of expression. (-)-deoxypodophyllotoxin was used as a substrate for feeding verification of function, the reaction product was extracted with ethyl acetate, and the product was analyzed by LC-MS to compare the retention time and mass spectrometry fragmentation rules with the standard. The results of LC-MS analysis show that (-)-deoxypodophyllotoxin generates a positive ion peak of [M+H]+=415.13794 under the catalysis of ShCYP736A469 enzyme. By analyzing the molecular ion peak of the product and comparing it with the peak of the podophyllotoxin standard compound under the same liquid phase conditions, 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 α-hydroxylation of (-)-deoxypodophyllotoxin C-4 to form (-)-podophyllotoxin, which has important theoretical and practical significance for the rational design and directional transformation of subsequent podophyllotoxin and subsequent aryl tetralin lignin structure modification enzymes and the development and utilization of biosynthetic pathway elements. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1The bioinformatics analysis diagram of the protein structure encoded by the ShCYP736A469 gene in Example 3, wherein Figure A is the transmembrane structure and analysis of the protein encoded by the ShCYP736A469 gene, and Figure B is the AlphaFold3 tertiary structure prediction of the protein encoded by the ShCYP736A469 gene; Instructions attached Figure 2 The expression of ShCYP736A469 gene in different tissues of Psoralea corylifolia and different jasmonic acid induction times in Example 3; Figure 3 The LC-MS analysis of the reaction products of the ShCYP736A469 gene-encoded protein substrate feeding reaction in Example 4, wherein Figure A is the liquid phase diagram of the ion range extraction (mass range = 390-420) of the ShCYP736A469 catalytic reaction: a. Liquid phase diagram of the substrate deoxypodophyllotoxin standard; b. Liquid phase diagram of the product podophyllotoxin standard; c. Liquid phase diagram of the extraction product of the recombinant expression vector pESC-Leu-TwCPR3 yeast substrate feeding reaction; d. Liquid phase diagram of the extraction product of the recombinant expression plasmid pESC-LEU::(ShCYP736A469+TwCPR3) yeast substrate feeding reaction. Figure B is the corresponding mass spectrum of the extraction product detected by LC-MS of the recombinant expression plasmid pESC-LEU::(ShCYP736A469+TwCPR3) yeast substrate feeding experiment. Instructions attached Figure 4 This is a simplified schematic diagram of the biosynthetic pathway of (-)-podophyllotoxin produced by the protein encoded by the ShCYP736A469 gene in Example 4, catalyzing (-)-deoxypodophyllotoxin. DETAILED DESCRIPTION

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

[0010] Through the transcriptome database constructed in this experiment at different times and in different tissues after jasmonic acid induction, CYP450 genes with higher expression in roots than in stems and upregulated with jasmonic acid induction were screened; based on the amino acid sequence of CYP82D61, a functional gene that is known to generate β-hydroxyl at the C-4 position of (-)-deoxypodophyllotoxin, similarity comparison was performed among the CYP450 annotated in the transcriptome to screen genes; colinearity analysis was performed between candidate CYP450 and known genes downstream of the podophyllotoxin pathway to screen candidate genes. Through the above screening methods, 20 candidate genes were screened from a total of 271 CYP450s 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 Psoralea corylifolia roots induced with jasmonic acid was extracted using the Eastep super total RNA extraction kit from Shanghai Promega Company, and the 5' and 3' ends of the synthesized first-strand cDNA were amplified using the SMARTer RACE 5' / 3'Kit kit from TAKARA to obtain the first-strand cDNA of Psoralea corylifolia.

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

[0015] According to the second and third generation transcriptome data of Taoerqi, 20 candidate CYP450 gene information was screened, ORF regions were searched, full-length gene primers were designed, and cDNA was used as a template for amplification. Agarose gel electrophoresis showed that a specific fragment appeared at about 1500bp. The target fragment was recovered by agarose gel recovery kit (Thermo) and cloned into pEASY-BluntZero vector (Transgen). Positive clones were identified and sequenced (Beijing Ruiboxingke) for the construction of expression vectors.

[0016] Example 3: Bioinformatics analysis, tissue expression and induced expression analysis of ShCYP736A469 gene

[0017] 1. Bioinformatics analysis of ShCYP736A469 gene sequence

[0018] The full-length open reading frame (ORF) of the podophyllotoxin biosynthesis pathway CYP450 gene ShCYP736A469 of the present invention is 1503 bp in length and encodes 500 amino acids. The detailed sequence is shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence table. The full-length open reading frame of ShCYP736A469 is searched for homology in the NCBI database using the BLAST program. The comparison analysis of the gene at the amino acid level shows that the amino acid sequence of the protein encoded by the ShCYP736A469 gene of Psoralea corylifolia has a low homology with other species, and has a maximum similarity of about 58.85% with the Vitis vinifera CYP450 gene. The results of the primary structure of the protein showed that the amino acid sequence of ShCYP736A469 has a highly conserved proline-rich region located between 32 and 35 amino acids in the eukaryotic CYP450, and the downstream is the catalytic functional region of ShCYP736A469; the results of the protein transmembrane domain showed that the 1-23 residues of the ShCYP736A469 protein are hydrophobic structures located outside the membrane, and the rest are located inside the membrane, and it is a soluble protein; the results of the AlphaFold3 tertiary structure prediction showed that the N-terminus of the protein also has a rigid helix anchoring the endoplasmic reticulum membrane, and the rest of the region is similar to the known CYP450 protein. The protein with the highest similarity to the ShCYP736A469 protein in the PDB database is the CYP76AH1 protein from Salvia miltiorrhiza, with a similarity of 30.69% (attached to the instruction manual). Figure 1 ).

[0019] 2. Analysis of tissue expression and induced expression of ShCYP736A469 gene

[0020] Analysis of the relative expression FPKM value of the ShCYP736A469 gene in the second-generation transcriptome data showed that the expression level of the ShCYP736A469 gene in the roots of Psoralea corylifolia was significantly higher than that in the stems, indicating that the roots of Psoralea corylifolia were the main enrichment site of podophyllotoxin; the expression level of the ShCYP736A469 gene induced by jasmonic acid slowly increased after a short downregulation and eventually exceeded the initial expression level, indicating that jasmonic acid can upregulate the expression level of genes in the biosynthesis pathway of podophyllotoxin and induce the promotion of podophyllotoxin biosynthesis (see Appendix to the instruction manual). Figure 2 ).

[0021] Example 4: Eukaryotic expression and functional analysis of ShCYP736A469 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 cloned open reading frame of the Psoralea corylifolia 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::(ShCYP736A469+TwCPR3), which was then identified by PCR and restriction digestion.

[0024] 2. Inducible expression of ShCYP736A469 in Psoralea corylifolia

[0025] The recombinant plasmid pESC-LEU::(ShCYP736A469+TwCPR3) was transferred into the Saccharomyces cerevisiae expression strain BY4741 by chemical transformation, and galactose was used to induce expression. The resulting expression system was used to detect activity in substrate feeding experiments.

[0026] 3. Activity analysis of ShCYP736A469 in Psoralea corylifolia

[0027] After one day of induction, the substrate (-)-deoxypodophyllotoxin was added to the bacterial solution at a final concentration of 20 μM, and the expression was induced for another 2 days. An equal volume of ethyl acetate was added to the bacterial solution to extract the product, and LC-MS analysis was performed. The LC-MS instrument was Thermo QExactive HF, the mobile phase was 0.2% formic acid water-acetonitrile, the flow rate was 0.4 ml / min, the column temperature was 30°C, and the gradient elution was performed in the following proportions (B%): 0 min-25%, 18 min-50%, 19 min-100%, 20 min-100%, 21 min-25%, and the equilibrium time was 2 min. The results showed that, with (-)-deoxypodophyllotoxin as substrate, the catalytic group containing the ShCYP736A469 gene recombinant plasmid pESC-LEU::(ShCYP736A469+TwCPR3) had a new substance produced at 415.13794 (m / z) in the total ion diagram compared with the empty vector control and other gene expression vector reaction products, which was consistent with the liquid phase diagram and mass spectrum of podophyllotoxin (attached to the instruction manual). Figure 3 ), the product was identified as (-)-podophyllotoxin. And from the liquid phase spectrum, it is known that under the current experimental conditions, the substrate deoxypodophyllotoxin is basically completely converted into podophyllotoxin. This shows that the protein encoded by the ShCYP736A469 gene can catalyze (-)-deoxypodophyllotoxin to produce (-)-podophyllotoxin in the metabolic pathway of podophyllotoxin compounds, and the efficiency is relatively high (see the attached manual). Figure 4 ).

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 podophyllotoxin compounds.

8. The use according to claim 7, wherein the aryltetralin lignan compound is (-)-podophyllotoxin 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 by the polyamino acid sequence.

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