Rhizoma corydalis triterpenoid synthase CyOSC as well as coding gene and key catalytic site thereof

Abstract

The invention relates to the field of plant gene and protein engineering, and aims to analyze a pentacyclic triterpene component skeleton in rhizoma corydalis of papaveris family to form a key enzyme CyOSC and analyze and clarify key amino acid residues in encoded protein. The invention discloses a triterpenoid synthase gene sequence and an amino acid sequence required by synthesis of a 3beta-hydroxy-oleanane-11, 13-diene-28-acid biological skeleton in rhizoma corydalis for the first time, primary structural analysis is performed on two homologous positive genes obtained by cloning, a key site 62 influencing CyOSC activity is found for the first time, and the gene sequence and the amino acid sequence of the triterpenoid synthase gene sequence can be used for synthesizing the 3beta-hydroxy-oleanane-11, 13-diene-28-acid biological skeleton. According to the present invention, the 3 beta-hydroxy-oleanane-11, 13-diene-28-acid is used as the enzyme source, and the CyOSC is subjected to rational design and directional modification to further excavate the key sites affecting the activity or the function of the enzyme, such that the basis is provided for the subsequent OSC mechanism research of other species sources, and the complete analysis of the 3 beta-hydroxy-oleanane-11, 13-diene-28-acid biosynthesis pathway is easily achieved; and a reference is provided for heterologous efficient generation of the pentacyclic triterpenoids as a gene element.

Description

Technical Field

[0001] The invention belongs to the field of plant gene and protein engineering, and relates to an OSC gene involved in triterpene synthesis in a plant of the subfamily Papaveraceae, and a coded product and application thereof. The OSC gene related to the biosynthesis of triterpene components in the Corydalis yanhusuo is screened by analyzing a genome library of a Corydalis yanhusuo plant of the genus Papaveraceae, and the function of the candidate gene is identified by heterologous expression in an ERG-deficient saccharomyces cerevisiae system. Semi-rational design mutation of amino acids near a pocket is performed by docking proteins with substrates, and DLKcat calculation prediction is used to assist mutation design, and a key catalytic site and catalytic mechanism of OSC are clarified by mutation experiments. Background Art

[0002] The subfamily of Papaver includes Corydalis, Papaver, Eupatorium, and Macleaya, and its active ingredients include alkaloids, triterpenes, and organic acids. Corydalis was first recorded as "Yanhusuo" in the "Leigong Paozhi Lun" of the Northern and Southern Dynasties. It was called Xuanhusuo in the ancient herbal books of the subsequent dynasties. It was renamed the well-known Corydalis in the "Kaibao Materia Medica" of the Song Dynasty, specifically referring to the dried tuber of Corydalis yanhusuo (W.T. Wang), a plant of the Poppy family. As one of the famous "Eight Zhejiang Flavors", it has the effects of relieving pain, promoting blood circulation, and promoting qi. Corydalis yanhusuo is rich in benzylisoquinoline alkaloids. With the separation and research of the chemical components of Corydalis yanhusuo, the triterpenes have also been paid attention to. The component 3β-hydroxy-oleanane-11,13-diene-28-acid can be isolated and identified in Corydalis yanhusuo. Currently, there are few related studies on this compound, but it is clear that as a pentacyclic triterpenoid compound that is widely present in nature, it also has a corresponding biosynthetic pathway and specific catalytic enzyme in Corydalis yanhusuo.

[0003] Based on the skeleton structure of the chemical component 3β-hydroxy-oleanane-11,13-diene-28-acid, it can be subdivided into β-amyrin-type pentacyclic triterpenoid compounds. The biosynthetic pathways of this type of compound have been identified in a variety of medicinal plants and model plants and can be simply divided into three parts: (1) The mevalonic acid (MVA) pathway and the methylerythritol-4-phosphate (MEP) pathway synthesize the common precursor 2,3-oxidosqualene. (2) 2,3-oxidosqualene is converted into pentacyclic triterpenoid components with different skeleton conformations under the action of 2,3-oxidosqualene cyclases (OSC). (3) The pentacyclic triterpenoid skeleton is post-modified by cytochrome P450 (CYP450) and glycosyltransferase (GT) to form various pentacyclic triterpenoid components. Summary of the invention

[0004] The invention provides genes CyOSC1 and CyOSC2 in Corydalis yanhusuo of the subfamily Papaveraceae, wherein the encoded proteins are involved in the biosynthesis of triterpenoid compounds, and the encoded proteins can catalyze 2,3-oxidized squalene to form beta-amyrin, and the primary structure comparison between CyOSC1 and CyOSC2 only shows a difference in amino acids at 62 positions, resulting in a 3-fold difference in activity. Further, molecular docking and mutation tests are performed to determine the key sites that affect the activity and function of CyOSC1.

[0005] In the present invention, the protein is involved in the synthesis of corydalis yanhusuo triterpene compounds, especially the key enzyme for the synthesis of the component 3β-hydroxy-oleanane-11,13-diene-28-acid, which are respectively named corydails yanhusuo triterpene synthase 1 (CyOSC1) and corydails yanhusuo triterpene synthase 2 (CyOSC2). The corydalis yanhusuo triterpene synthase (CyOSC1 and CyOSC2) has a protein encoded by the amino acid sequence of SEQ ID No. 2 in the sequence table, an amino acid residue sequence of SEQ ID No. 2 in the sequence table, and a peptide having the same function as the amino acid sequence shown in SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acids.

[0006] Another object of the present invention is to provide a polynucleotide sequence encoding the Tripterygium wilfordii triterpenoid synthase protein, as shown in SEQ ID No. 1 in the sequence table. The full-length polynucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed for the open reading frame sequence according to the relevant nucleotide sequence disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. Corydalis yanhusuo is a tetraploid plant with a high heterozygosity, and its genes are complex. The CyOSC1 and CyOSC2 base sequences in the present invention have a high homology of 99.78%, and the amino acid sequences of the two have a high homology of 99.87% due to degenerate codons.

[0007] The DNA sequence of SEQ ID No. 1 of the present invention consists of 2289 bases and encodes the protein sequence SEQ ID No. 2 consisting of 762 amino acid residues in the sequence table.

[0008] Another object of the present invention is to provide an expression vector, which comprises a polynucleotide encoding the Corydalis yanhusuo triterpene synthase protein or its variant described in the present invention, and the expression vector comprises 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, the host cell comprising the polynucleotide molecules encoding the CyOSC1 and CyOSC2 proteins or their variants described in the present invention, or comprising the expression vector described 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 yeast cells or plant cells.

[0010] The screening method, expression vector, cell line and host bacteria containing the gene CyOSC of the present invention and the rational design and directional transformation of oxidized squalene cyclase using the gene are also within the protection scope of the present invention. The corydalis triterpene synthase of the present invention can be developed and utilized as an OSC element, and at the same time promotes the full analysis of the biosynthetic pathway of 3β-hydroxy-oleanane-11,13-diene-28-acid. pYES2 is selected as the eukaryotic expression vector of CyOSC gene-deficient yeast GIL77, and SEQ ID No.1 and related homologous genes and mutant genes are cloned between the restriction endonuclease EcoR I and Not I sites of the eukaryotic expression vector pYES2, and the expression of the gene is started by using the Gal1 promoter (Gal1 promoter); the gene is transferred into the yeast strain GIL77 expression host bacteria and galactose is used to induce expression. GIL77 is an ERG7-deficient yeast strain that endogenously produces 2,3-oxidosqualene. CyOSC is heterologously expressed in yeast. The reaction product is extracted with n-hexane. The retention time and mass spectrometry fragmentation rules of the product and the standard are compared by GC-MS analysis. The GC-MS analysis results show that 2,3-oxidosqualene generates a characteristic ion peak M / Z=218 under the catalysis of CyOSC enzyme. By analyzing the molecular ion peak of the product and comparing it with the peak of the β-amyrin standard compound under the same gas phase conditions, it is believed that the product is β-amyrin. The research results show that the gene related to the synthesis of pentacyclic triterpenoid compounds of the present invention has the characteristic structural domain of OSC gene. The results of yeast heterologous expression experiments prove that the gene can catalyze 2,3-oxidosqualene to form β-amyrin. The product and primary structure analysis of homologous genes and mutants determine the key sites affecting the corydalis triterpene synthase, which has theoretical and practical significance for the subsequent analysis of the catalytic mechanism of CyOSC and the development and utilization of the biosynthetic pathway elements of oleanane-type pentacyclic triterpenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Instructions attached Figure 1This is a diagram showing the chromosome localization of the CyOSC gene in Corydalis yanhusuo and the gene family analysis in Example 3.

[0012] Instructions attached Figure 2 These are bioinformatics analysis diagrams of the protein structure encoded by the CyOSC gene in Example 3, wherein Figure A is the transmembrane structure and analysis of the protein encoded by the CyOSC gene, and Figure B is the prediction of the tertiary structure of the AlphaFold3 protein encoded by the CyOSC gene.

[0013] Instructions attached Figure 3 The GC-MS analysis diagram of the heterologous expression product extraction of the CyOSC gene encoded protein GIL77 in Example 4, Figure A is the gas phase diagram of the CyOSC catalytic reaction ion extraction (M / Z=218): a. standard gas phase diagram of the product β-amyrin; b. sample gas phase diagram of CyOSC1; c. sample gas phase diagram of CyOSC2; d. sample gas phase diagram of the blank vector pYES2; Figure B is the corresponding mass spectrum of the GC-MS product extraction of the recombinant plasmid pYES2::CyOSC: a. standard mass spectrum of β-amyrin; b. sample mass spectrum of CyOSC1; c. sample mass spectrum of CyOSC2.

[0014] Instructions attached Figure 4 Bar graphs showing the types and yields of the extracted products (M / Z=218) of the heterologous expression product of the CyOSC gene mutant-encoded protein GIL77 in Example 5: A. Bar graph showing the results of the 62-position residue mutation; B. Bar graph showing the results of the mutation sites that affect the catalytic specificity; C. Bar graph showing the results of the mutation sites that affect the catalytic activity. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below with reference to the accompanying drawings and examples. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0016] Example 1: Screening of triterpene synthase CyOSC gene in Corydalis yanhusuo

[0017] According to the relevant annotation information in the genome, a total of 8 genes annotated as β-amyrin synthase were screened. The length of the open reading frame (ORF) of the candidate genes was checked using the ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) website, and the ORF length greater than 2000bp was further selected as candidate genes. Finally, 3 full-length β-amyrin synthase candidate genes were screened.

[0018] Example 2: Cloning of the full-length cDNA sequence of Corydalis triterpenoid synthase

[0019] 1. Extraction of total RNA from Corydalis yanhusuo and acquisition of the first strand of cDNA

[0020] Four frozen portions of Corydalis tissue were ground into powder for RNA extraction, and total RNA was extracted using RNAprep PurePlantPlus Kit (Tiangen Biotechnology Co., Ltd., Beijing, China) according to the instructions of the kit. The integrity of RNA was identified by 1.2% agarose gel electrophoresis, and the purity of RNA was determined by OD260 / OD280. The purity of RNA was determined by Nanodrop One (ThermoFisher Scientific, Massachusetts, USA). The experimental results showed that the extracted RNA samples were of good quality. cDNA was extracted using FastKing cDNA First Strand Synthesis Kit (Tiangen, Beijing, China) according to the instructions.

[0021] 2. Cloning and Sequencing of Full-length cDNA

[0022] According to the screening of 3 candidate CyOSC gene information from the genome data of Corydalis yanhusuo, full-length gene primers were designed according to the ORF region, and cDNA was used as a template for amplification. Agarose gel electrophoresis showed that a specific fragment appeared at about 2000bp. The target fragment was recovered by agarose gel recovery kit (Thermo) and cloned into the pEASY-Blunt Zero vector (Transgen). The positive clones were identified and sequenced (Beijing Ruiboxingke) for the construction of expression vectors. Corydalis yanhusuo is a tetraploid with 32 chromosomes and a high heterozygosity of about 1.69%. A pair of primers can be used to clone multiple different genes with extremely high homology, and a total of 7 genes were cloned.

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

[0024] 1. Bioinformatics Analysis of CyOSC Gene

[0025] The invention relates to a full-length open reading frame of a CyOSC gene formed by pentacyclic triterpenoid compounds in Corydalis yanhusuo, which has a length of 2289 bp and encodes 762 amino acids. The detailed sequence is shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence table. The position of the triterpene synthase CyOSC gene on the chromosome is checked by using chromosome assembly information, and it is found that three candidate genes are located on three chromosomes respectively. The full-length open reading frame of CyOSC is subjected to homology search in the NCBI database by using the BLAST program. The gene is compared and analyzed at the amino acid level and shows a high homology with the beta-amyrin synthase of the Proteaceae family, with a similarity of 82.69%. The OSC genes of different species that have been published are used for tree building analysis. (Instruction manual attached Figure 1 )

[0026] 2. Structural sequence analysis of CyOSC gene

[0027] The results of the primary structure of the protein showed that CyOSCs all contained a highly conserved SDCTAE motif, which is related to the binding of proteins and substrates, and had a repetition of the QW motif, which is of great significance for enhancing the stability of protein structure and carbon cation intermediates. In addition, all CyOSCs contained a conserved MWCXXR motif, which is involved in the formation of β-amyrin. The results of the protein transmembrane domain showed that CyOSC protein had no transmembrane domain and was a soluble protein. In order to further understand the protein structure information, Alphafold3 was used to predict the tertiary structure of the protein. (Attached to the instruction manual Figure 2 )

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

[0029] 1. Construction of yeast expression vector

[0030] The yeast eukaryotic expression vector pYES2 was cut with restriction endonucleases EcoR I and Not I. Homologous primers with EcoR I and Not I restriction sites were designed to amplify the open reading frame of the CyOSC gene of Corydalis yanhusuo triterpene synthase by PCR, and then inserted into the yeast surface vector between EcoR I and Not I sites using homologous recombinase (Transgen) to obtain the recombinant plasmid pYES2::CyOSC, which was transformed into Escherichia coli Trans-T1 competent cells (Transgen). After the cells were cultured on inverted plates for 24 hours, the bacterial liquid was identified by PCR and sequenced for verification (Beijing Ruiboxingke).

[0031] 2. Heterologous Expression in Yeast

[0032] The plasmid was extracted from the successfully verified positive bacterial solution, and transformed into the lanosterol-deficient Saccharomyces cerevisiae GIL77 (gal2hem3-6 erg7 ura3-167) by the lithium acetate method. The plasmid transformation was verified by inverting and culturing on SD-Ura (2% Glu) plates at 37°C for 3 days. The well-growing bacteria were picked and cultured overnight with 5mL SD-Ura (2% Glu). The final OD600 was inoculated into a 100mL shaking flask containing 20mL SD-Ura (2% Glu). The culture was expanded for 24h on a shaking table (30°C, 200rpm), and the same volume of culture medium SD-Ura (2% Gala) was replaced. The culture was fermented for 48h and the bacteria were collected by centrifugation (4000g, 5min).

[0033] 3. Product extraction and detection

[0034] The yeast cells were resuspended in 10 mL of 20% KOH / 50% EtOH and placed in a boiling water bath for 10 min to lyse the cells. The extracts were collected and concentrated three times with an equal volume of n-hexane. The yeast extract was reconstituted in n-hexane and analyzed by GC / MS. An Agilent 7000 gas chromatograph and a DB-5MS capillary column (15 m×250 μm, 0.1 μm) were used. Then 1 μL of the concentrated organic phase was injected at a flow rate of 1 mL min-1 of He. The temperature program was 50°C for 1 min, then a gradient temperature increase of 50°C min-1 from 50°C to 260°C, and then a temperature increase of 1°C / min to 272°C and maintained for 4 min. The temperature of the ion trap was 230°C and the electron energy was 70 eV. The spectra were recorded in the range of 10 to 550 m / z. All prominent peaks in the GC chromatogram were identified by comparison with the database and standards.

[0035] The results showed that the recombinant plasmid containing the corydalis triterpene synthase genes CyOSC1 and CyOSC2 had a new peak in the extracted ion chromatogram at M / Z: 218 compared with the blank vector pYES2, and the peak time was consistent with that of the β-amyrin standard. By comparing the mass spectra of the peak time, it was found that the fragment ion peaks could match, and it was considered that it had the function of catalyzing 2,3-oxidized squalene to generate β-amyrin. (Attached to the instruction manual Figure 3 ) Further quantitative analysis revealed that although the two enzymes differed in only one amino acid site, their activities were very different. (CyOSC1: G62, CyOSC2: R62; CyOSC1: CyOSC2 = 3.05)

[0036] Example 5: Mutation design and key site exploration

[0037] 1. Mutation Design

[0038] The sequence prediction protein structure of CyOSC1 was submitted using Alphafold3 (https: / / golgi.sandbox.google.com / ), and the small molecules of 2,3-squalene oxide (CID: 5459811) and β-amyrin (CID: 73145) were downloaded from Pubchem (PubChem (nih.gov)), and the structures were optimized using Phenix elBow. The molecular docking of substrate, product and protein was performed using Autodockvina to determine the substrate. The amino acid residues in the range were artificially rationally designed and mutated through literature research and molecular calculation. The MWCXXR conserved motif (Trp261, Tyr263) involved in the formation of β-amyrin in the pocket and the DCATE motif (Asp488) involved in substrate recognition were selected. At the same time, this study performed alanine / phenylalanine scanning on the amino acids in the swingable loop region in the pocket (Gly372, Val374, Thr552, Trp537). Based on the literature binding free energy, it was speculated that K376 hindered the reaction, and the sites (Leu712, Glu718, Gly720) were predicted and designed by the Kcat method. By comparing the OSC gene family from multiple species, the evolutionary relationship of the key amino acid site 62 was checked, and a mutual mutation experiment was performed.

[0039] 2. Construction of mutants

[0040] According to the instructions of the Fast Mutagenesis System kit, mutant primers were designed, and recombinant plasmid pYES2:CyOSC1 was used as a cloning template for PCR amplification. After taking 5 μL of the above PCR product and confirming the correct size of the amplified band by 1% agarose gel electrophoresis, 0.8 μL of DMT enzyme was added to the remaining system, mixed well, incubated at 37 ° C in a PCR instrument for 1 hour to remove the original plasmid template (methylated plasmid template), and transformed into the colon Trans-T1 competent cells (Transgen), inverted plate culture cells for 24 hours, and then PCR identification of the bacterial solution and sequencing verification (Beijing Ruibo Xingke).

[0041] 3. Exploration of key sites

[0042] The mutants were tested and analyzed according to Example 4 (see Appendix Figure 4 ) found that D488 in DCATE is the key to CyOSC1 catalyzing 2,3-oxidized squalene to form β-amyrin, which is consistent with the previous docking results. The negatively charged Asp binds to the oxygen atom of squalene, thus initiating the reaction. When mutated to the non-polar amino acid Ala, CyOSC1 D488A Completely lost activity. In the conserved motif MWCYXR, mutations to Trp261 and Tyr263 resulted in CyOSC1 W261F The change from a monofunctional enzyme to a bifunctional enzyme allowed the production of lupeol in addition to β-amyrin, which is consistent with previous studies, but the overall activity of the enzyme also decreased. Y263F and CyOSC1 Y263HThe activity of all of them was lost, indicating that Tyr263 may be a conserved motif, and the hydroxyl group on its side chain may play a key role in the catalytic reaction of CyOSC1. G372A ,CyOSC V374A ,CyOSC1 T552A ,CyOSC1 W537F ), there was no increase in enzyme activity, but rather a decrease in enzyme activity or even a loss of activity. This suggests that for CyOSC1, the loop domain near the pocket is relatively conservative, which may be related to the stable morphology of the protein. After the amino acid is reduced, the movable range of the loop increases, which is not conducive to the maintenance of protein homeostasis and leads to a decrease in enzyme activity.

[0043] In addition, the reference free energy was found to replace the hindered reaction site Lys with a smaller side chain (CyOSC K376A , CyOSC K376N ), resulting in the loss of activity of CyOSC1. It is speculated that Lys, as a positively charged amino acid, has a promoting effect on the reaction, so replacing it with a hydrophobic amino acid or a hydrophilic neutral amino acid prevents the reaction from proceeding and thus inactivates it. cat By calculation, rational design was performed for sites 712, 718, and 720 (CyOSC L712Q , CyOSC L712K , CyOSC1 L712H , CyOSC1 E718M , CyOSC1 G720W ), in which CyOSC1 was found E718M The activity was slightly improved, while other mutations lost their activity. Analysis found that Met717 and Glu718 were both on the loop. When Glu718 mutated to Met, the two adjacent Mets may further form a disulfide bond, thereby increasing the rigidity of the loop to a certain extent, stabilizing the structure of the protein, and thus increasing the activity of the protein.

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 β-amyrin, lupetane 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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