Gene, carrier, protein and application of split-ring loganin synthase in uncaria rhynchophylla

Abstract

The invention provides a gene, a carrier, a protein and application of split-ring loganin synthase in uncaria rhynchophylla, and belongs to the technical field of gene engineering. The invention provides gene sequences and coding amino acid sequences of three UrCYP72A enzyme elements in uncaria rhynchophylla. The host cell constructed by the invention can induce expression of UrCYP72A enzyme, and the three proteins can catalyze oxidative cleavage of loganin C7-C8 to generate iridoid compound biosynthesis key precursor split-ring loganin. According to the present invention, the key residues participating in the combination of the loganin CYP72A1177, the CYP72A1178 and the CYP72A1179 are analyzed through molecular docking and rational mutation, and the catalytic potential of the loganin CYP72A1177, the CYP72A1178 and the CYP72A1179 is improved by widening the substrate inlet and enhancing the substrate binding force; the key precursor split-ring loganin of the rhynchophylline can be prepared by serially connecting UrLAMT and UrCYP72s genes in the body of the nicotiana benthamiana.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a Uncaria rhynchophylla (Miq.) Miq. ex Havil. midcycle ring loganin synthase gene, vector, protein and application. BACKGROUND

[0002] Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a liana plant of Rubiaceae, and its stem branches with hooks are used as medicine and have good medicinal value. The plant is rich in various chemical components, including alkaloids, flavones, triterpenes, organic acids and the like. Among the numerous chemical components, alkaloids occupy a dominant position, mainly including uncarine, dehydrogenated uncarine, isounarine and isodehydrogenated uncarine, etc., among which uncarine and isounarine are the most abundant, reaching 28.9% and 14.7% of the total alkaloids respectively, and are the medicinal material basis of Uncaria rhynchophylla. Uncarine is applied in the treatment of cardiovascular and central nervous system diseases in clinic, and shows multiple pharmacological effects such as lowering blood pressure, sedation, anti-arrhythmia, anti-epilepsy, anti-convulsion, anti-tumor and neuroprotection.

[0003] Uncaria rhynchophylla is rich in indole alkaloids, and the biosynthesis of this kind of compounds starts from two independent metabolic pathways. In the tryptamine synthesis pathway, tryptophan decarboxylase catalyzes the conversion of tryptophan into tryptamine. In the iridoid pathway, loganin is converted into secologanin through two steps of loganic acid-O-methyltransferase and secologanin synthase. Subsequently, the products of the two pathways are condensed into loganin under the catalysis of strictosidine synthase. As a key metabolic precursor, loganin undergoes a series of enzymatic reactions to ultimately generate uncarine and isounarine. Among them, secologanin in the iridoid pathway is one of the key precursors of the upstream synthesis pathway of uncarine. The biosynthesis of secologanin needs the continuous action of nine enzymes, namely geraniol 8-hydroxylase (G8H), 8-hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), iridoid oxidase (IO), 7-deoxyloganic acid synthase (7-DLS), 7-deoxyloganic acid glucosyltransferase (7-DLGT), 7-deoxyloganic acid hydroxylase (7DLH), loganic acid-O-methyltransferase (LAMT) and secologanin synthase (SLS), and SLS is a key step for comprehensively analyzing the iridoid synthesis metabolism in Uncaria rhynchophylla.

[0004] The first SLS family member, CYP72A1, was discovered in Catharanthus roseus in 1992. This enzyme has been shown to catalyze the C-C bond cleavage of the cyclopentane ring of strychnine, converting strychnine to strychnine, a process crucial in the biosynthesis of vincristine and vinblastine. In recent years, with the continued deepening of research, the functions of CYP72 family members have gradually been revealed. In 2019, CaCYP72A565 / 610, discovered in Camptotheca acuminata, was shown to possess bifunctional properties. CaCYP72As catalyze the selective hydroxylation of 7-deoxystrychnine at the C-7 position, followed by cleavage of the carbon-carbon (C-C) bond between C-7 and C-8 of the iridoid glycoside, generating the cyclopentane-opened iridoid glycoside. In 2020, NnCYP72A1, identified in Nothapodytes nimmoniana, was found to possess secoxyloganine synthase activity. In 2021, OeOMES and OeSXS were discovered in Olea europaea. Both enzymes catalyze the cleavage of C-C bonds, converting 7-epi-loganin to oleoside methyl ester (OeOMES) and secoxy-loganin (OeSXS), respectively, with ketologanin as the common intermediate. However, the complete metabolic pathway for the biosynthesis of rhynchophylline in Uncaria rhynchophylla has not yet been elucidated. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a gene, vector, protein and application of schizoside synthase in Uncaria rhynchophylla. By using the Uncaria rhynchophylla gene library, the CYP72A family enzyme elements in Uncaria rhynchophylla were mined and optimized, thereby obtaining the CYP72A enzyme gene in Uncaria rhynchophylla and the corresponding protein encoded by the gene. The present invention constructs a plasmid of the gene and transforms the expression plasmid into Saccharomyces cerevisiae WAT11 host cells, and uses the host cells to induce expression of three CYP72A microsomal proteins, CYP72A1177, CYP72A1178 and CYP72A1179. It is found that all three microsomal proteins show C7-C8 oxidative cleavage activity on strinoside, a key precursor in the iridoid biosynthesis pathway, and can be used to prepare schizoside. By combining molecular docking and site-directed mutagenesis techniques, the key residues for substrate-directed stabilization of the three proteins, CYP72A1177, CYP72A1178 and CYP72A1179, are determined, and their catalytic activities are improved. By tandem connection of enzyme elements, strychnine was successfully prepared in tobacco.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a secoyrin synthase gene, wherein the secoyrin synthase gene includes CYP72A1177, CYP72A1178 and CYP72A1179;

[0008]

[0009]

[0010]

[0011] The present invention also provides a protein encoded by the strychnine synthase gene. The amino acid sequence of the protein encoded by CYP72A1177 is shown in SEQ ID NO.4, specifically MEMDITHQAIAATCFLALFAWAWRVLNWAWFKPKRIEKRLRQQGFRGNPYRFLVGDVKESGMMLQEAMSKPIPLNNDIVPRIMPHIDKTIRTYGKNSFTWMGRIPRIHVMDPDLIREVLTHSNKFMKNFDVHNPLVKFLLTGVGSFEGEKWSRHRRIISPAFTLEKLKTMLPAFAVSYDDLLRKWGQTATREGSVEVDIFPTFDVLTSDVISKVAFGSTYEEGGKIFQLLKELMELTIETMRDVYIPGLSLLPTKR NKRMKEINKEITDMLRNIISKRVKAMKAGEPSEDDLLGVLLESNYREIQKQGNNNNVGMTIDDVIEECKLFYFAGQETTGVLLTWTTILLSKHPEWQERAREEVMQAFGKNKPEFEKLNHLKYVSMILYEVLR LYPPVIDLTKIVHEDTKLGPYTVPAGTQIMLPTVILHREKSIWGEDAMEFNPGRFADGVANATKNQVTYLPFSWGPRVCLGQNFALLQAKLGLAMILQRFSFNVSPSYAHAPYTILTLQPQLGSHVIYRKLE;

[0012] 所述CYP72A1178编码蛋白质的氨基酸序列如SEQ ID NO.5所示,具体为MEIAYNPLAVFSSCIFLLMLVLAWRVFNWIWLTPKKMEKQLKEQGLRGNPYKLLYGDFKEISTLFNEVHSKPVNLSDDFVPRVIPHFYEAVKKYGKNTYLWLGPKPAVVIMDPEHIREVTQKIYIFEKPRNNPLAKLLAQGLVNHDGDKWAKHRKLINPAFHVEKLKHMLPSLYTCASEMLSKWEETVSSNGSSELDVWPDLQTLTCDAISRTAFGSNYEEGTRIFELQREQAEHVIKAVQSVYIPGWRFLPTKMNRRMKQIAKDVQDSIGQIIDSRLKAMKTGEACDNDLLGILLESNSKEIDNHGNKDFGMTIREVIEECKLFYFAGQETTSVLLVWTMVLLSRYPDWQARAREEVLQLFGTNKPDFDGLNHLKLVTMILHEVLRLYPPLPVLTRRAAKETQLGNLTLPDQVLVSLPAMLLHYDPEIWGDDVKEFNPERFADGVSNATKGQVAFFPFGWGPRICIGQNFAMLEAKLAMAMILQRFSFELSPSYSHAPRAVVTLQPQYGAHLILHKL;

[0013] The amino acid sequence of the CYP72A1179 encoded protein is shown as SEQ ID NO. 6, in particular, MDILYSIVAVVSTAFVLVQAWRILNWAWFKPKKLEKCLRQQGFRGNSYKLVFGDMKETVEMIQEAKSKPINFTNDIIPRVMPFVDKTIKTYGENSYTWAGPMPGLLLTDPELIKEVLNKSFNYLKPPGNPLTKLLATGLVNYETDKWAKHRKLINPAFHLEKVKLMLPAFYLSCSEMLSKWEEITSAKGSCELDVWPSIQTLTSDAISRTAFGSNYKEGQKIFELQKEQAELIIQAAQSLYVPGWRFVPTKRNKRMKEIFKEVKSLVMDMINNRVKRMRAGEAKNDDLLDILLESNFKEIQQGGDKKFGMSLDEVIEECKLFYFAGQETTSVLLVWTLILLSKHLDWQERARDEVQQVFGNRKPEFEELNHLKVITMIFNEVLRLYPPGVMLGRMTTETIKLGEFTLPAGVLLLMSALLLHHDTKTWGDDANEFKPERFSEGILKATKGQLTYFPFGWGPRICIGQNFAMLEAKLALAMILQRFSFEISPLYAHAPHTIITLQPQHGAQLVLRKL.

[0014] The application further provides a recombinant expression vector comprising a target gene and a pYES2 / CT expression vector, wherein the target gene comprises CYP72A1177, CYP72A1178 and CYP72A1179.

[0015] The application further provides a transformant comprising the recombinant expression vector and a WAT11 Saccharomyces cerevisiae competent cell.

[0016] The application further provides application of the said seco-strychnine glycoside synthase gene, the said protein, the said recombinant expression vector or the said transformant in preparation of seco-strychnine.

[0017] The application further provides a seco-strychnine glycoside synthase recombinant microsomal protein, and a preparation method of the seco-strychnine glycoside synthase recombinant microsomal protein comprises the following steps:

[0018] 1) CYP72A1177, CYP72A1178, and CYP72A1179 were respectively connected to the pYES2 / CT expression vector to obtain the pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 recombinant expression vectors;

[0019] 2) The pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 recombinant expression vectors were respectively introduced into WAT11 Saccharomyces cerevisiae competent cells to obtain WAT11 recombinant Saccharomyces cerevisiae strains expressing CYP72A1177, CYP72A1178, and CYP72A1179;

[0020] 3) The WAT11 recombinant Saccharomyces cerevisiae strains expressing CYP72A1177, CYP72A1178, and CYP72A1179 were inoculated into SC-Ura liquid medium for expansion, transferred to YPGal liquid medium for induction culture, centrifuged, and the supernatant discarded. The cells were collected and disrupted using sterile ceramic beads to extract the recombinant microsomal protein of strychnine synthase.

[0021] The present invention also provides a recombinant microsomal mutant protein of schizoside synthase, including CYP72A1177-D396E

[0022] , CYP72A1178-F327G, CYP72A1178-P393V, CYP72A1179-L321G, CYP72A1179-V390I, CYP72A1177-T334G+D396E, CYP72A1178-F327G+P393V, and CYP72A1179-L321G+V390I;

[0023] The CYP72A1177-D396E is a mutation of aspartic acid at position 396 of the amino acid sequence shown in SEQ ID NO. 4 to glutamic acid;

[0024] The CYP72A1178-F327G is a mutation of the phenylalanine at position 327 of the amino acid sequence shown in SEQ ID NO.5 to glycine;

[0025] The CYP72A1178-P393V is a mutation of the proline at position 393 of the amino acid sequence shown in SEQ ID NO.5 to valine;

[0026] The CYP72A1179-L321G is that leucine at the 321th position of the amino acid sequence shown in SEQ ID NO. 6 is mutated into glycine;

[0027] The CYP72A1179-V390I is that valine at the 390th position of the amino acid sequence shown in SEQ ID NO. 6 is mutated into isoleucine;

[0028] The CYP72A1177-T334G+D396E is that threonine at the 334th position of the amino acid sequence shown in SEQ ID NO. 4 is mutated into glycine and that aspartic acid at the 396th position of the amino acid sequence shown in SEQ ID NO. 4 is mutated into glutamic acid;

[0029] The CYP72A1178-F327G+P393V is that phenylalanine at the 327th position of the amino acid sequence shown in SEQ ID NO. 5 is mutated into glycine and that proline at the 393th position of the amino acid sequence shown in SEQ ID NO. 5 is mutated into valine;

[0030] The CYP72A1179-L321G+V390I is that leucine at the 321th position of the amino acid sequence shown in SEQ ID NO. 6 is mutated into glycine and that valine at the 390th position of the amino acid sequence shown in SEQ ID NO. 6 is mutated into isoleucine.

[0031] The application also provides application of the said secologanin synthase recombinant microsomal protein or the said secologanin synthase recombinant microsomal mutant protein in preparation of secologanin.

[0032] Preferably, the reaction temperature in the process of preparing secologanin is 20-60 DEG C, the reaction time in the process of preparing secologanin is 0.5-8h, the pH value in the process of preparing secologanin is 6-8, and the NADPH concentration in the process of preparing secologanin is 50-1000 μM.

[0033] The application also provides application of the said secologanin synthase recombinant microsomal protein or the said secologanin synthase recombinant microsomal mutant protein in combination with Uncarine-O-methyltransferase in preparation of secologanin.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] The application provides gene sequences and coding amino acid sequences of three UrCYP72A enzyme elements in Uncaria, plasmids containing the genes and Saccharomyces cerevisiae WAT11 host cells containing the expression plasmids.

[0036] The host cell constructed by the application can induce expression of UrCYP72A enzyme, and three proteins CYP72A1177, CYP72A1178 and CYP72A1179 can all catalyze C7-C8 oxidative cleavage of loganin to generate a key precursor of biosynthesis of seciiridoid compound, i.e., seco-loganin.

[0037] The application analyzes key residues involved in binding of loganin to CYP72A1177, CYP72A1178 and CYP72A1179 through molecular docking and rational mutation, and improves catalytic potential of CYP72A1177, CYP72A1178 and CYP72A1179 by widening a substrate entrance and enhancing substrate binding force.

[0038] The application can prepare a key precursor of rhynchophylline, i.e., seco-loganin, by connecting UrLAMT and UrCYP72s genes in tandem in Nicotiana benthamiana, and through two-step continuous enzymatic reaction. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a multiple sequence alignment of UrCYP72 proteins and characterized SLS proteins;

[0040] Figure 2 is a phylogenetic tree of UrCYP72 and other characterized CYP72 family proteins in plants;

[0041] Figure 3 is a cluster analysis of UrCYP72 gene expression;

[0042] Figure 4 is a quantitative analysis of UrCYP72 gene;

[0043] Figure 5 is electrophoresis and enzyme digestion verification of pYES2 / CT-UrCYP72A recombinant plasmid (M: DNA Marker (250bp-15000bp); Lane 1. pYES2 / CT-CYP72A1178; Lane 2. pYES2 / CT-CYP72A1178 enzyme digestion product; Lane 3. pYES2 / CT-CYP72A1177; Lane 4. pYES2 / CT-CYP72A1177 enzyme digestion product; Lane 5. pYES2 / CT-CYP72A1179; Lane 6. pYES2 / CT-CYP72A1179 enzyme digestion product);

[0044] Figure 6 is colony PCR verification of UrCY72A gene (M: DNA Marker (100bp-2000bp); 1. CYP72A1178; 2. CYP72A1177; 3 CYP72A1179).

[0045] Figure 7 It is the target UrCYP72A protein Western blot (M: Protein Marker (18kDa~94kDa); 1.CYP72A1178; 2.CYP72A1177; 3.CYP72A1179);

[0046] Figure 8 EIC of the reaction mixture of UrCYP72A microsomal protein with strychnine as substrate (A. EIC of the reaction mixture of the enzymatic assay of UrCYP72As; B. EIC of the reaction mixture of the enzymatic assay of UrCYP72As in tobacco);

[0047] Figure 9 The MS is the secondary spectrum of the standard and C-C bond cleavage products (A. strychnine standard; B. split-ring strychnine standard; C. oxidative cleavage products of strychnine by CYP72A1177; D. oxidative cleavage products of strychnine by CYP72A1178; E. oxidative cleavage products of strychnine by CYP72A1179);

[0048] Figure 10 This is the MS / MS spectrum of the C-C bond cleavage products of UrCYP72A in Nicotiana benthamiana using strychnine as a substrate (A. strychnine standard; B. split-ring strychnine standard; C. oxidative cleavage products of strychnine by CYP72A1177; D. oxidative cleavage products of strychnine by CYP72A1178; E. oxidative cleavage products of strychnine by CYP72A119);

[0049] Figure 11 It is strychnine 1 H and 13 C NMR spectrum (A. 1 H spectrum; B. 13 C spectrum);

[0050] Figure 12 It is the oxidative cleavage product of strychnine 1 H and 13 C NMR spectrum (A. 1 H spectrum; B. 13 C spectrum);

[0051] Figure 13 Optimization of reaction conditions for CYP72A1177 microsomal protein (A. reaction temperature, B. reaction time, C. reaction pH, D. NADPH concentration, different letters (a, b, c) indicate significant differences (P < 0.05));

[0052] Figure 14Optimization of reaction conditions for CYP72A1178 microsomal protein (A. reaction temperature, B. reaction time, C. reaction pH, D. NADPH concentration, different letters (a, b, c) indicate significant differences (P < 0.05));

[0053] Figure 15 Optimization of reaction conditions for CYP72A1179 microsomal protein (A. reaction temperature, B. reaction time, C. reaction pH, D. NADPH concentration, different letters (a, b, c) indicate significant differences (P < 0.05));

[0054] Figure 16 Characterization of the kinetics of target CYP72 microsomal proteins (A. Steady-state kinetics of CYP72A1177 on strychnine; B. Steady-state kinetics of CYP72A1178 on strychnine; C. Steady-state kinetics of CYP72A1179 on strychnine, different letters (a, b, c) indicate significant differences (P < 0.05));

[0055] Figure 17 The substrate binding pockets of the UrCYP72A wild-type protein and mutant proteins (A. substrate binding pocket of UrCYP72A1177 and strychnine; B. substrate binding pocket of UrCYP72A1178 and strychnine; C. substrate binding pocket of UrCYP72A1179 and strychnine. D. substrate binding pocket of the UrCYP72A1177-D396E mutant; E. substrate binding pocket of the UrCYP72A1178-P393V mutant; F. substrate binding pocket of the UrCYP72A1179-V390I mutant);

[0056] Figure 18 These are the substrate entrances of the CYP72A wild-type and mutant proteins (A. substrate entrance of the CYP72A1177 wild-type protein, B. substrate entrance of the CYP72A1177-T334G mutant protein, C. substrate entrance of the CYP72A1178 wild-type protein, D. substrate entrance of the CYP72A1178-F327G mutant protein, E. substrate entrance of the CYP72A1179 wild-type protein, F. substrate entrance of the CYP72A1179-L321G mutant protein);

[0057] Figure 19Relative enzyme activities of CYP72A wild-type and mutant proteins (A. relative enzyme activity of CYP72A1177 mutant; B. relative enzyme activity of CYP72A1178 mutant; C. relative enzyme activity of CYP72A1179 mutant. Each value represents the mean ± standard deviation (n = 3). Different letters (a, b, c) indicate significant differences at P < 0.05);

[0058] Figure 20 This is the EIC plot of the leaf extract of Nicotiana benthamiana containing UrLAMT and UrCYP72 in tandem;

[0059] Figure 21 This is the secondary mass spectrometry analysis of the tandem products of UrLAMT and UrCYP72 in tobacco (A. Strychnine standard; B. Seco-strychnine standard; C. The tandem product of UrLAMT and CYP72A1177; D. The tandem product of UrLAMT and CYP72A1178; E. The tandem product of UrLAMT and CYP72A1179);

[0060] Figure 22 Figure 2 is a graph of the strychnine content of tandem UrLAMT and UrCYP72 mutants in Nicotiana benthamiana. DETAILED DESCRIPTION

[0061] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1 Mining, conserved domains and phylogenetic analysis of target UrCYP72A elements

[0063] Based on the conserved sequence of CYP72, 21 complete gene sequences encoding CYP72 were obtained. Sequences were then compared for similarity using DNAMAN software. Sequences with a similarity exceeding 95% were considered duplicates, and redundant candidate sequences were removed, resulting in seven candidate sequences. Homology analysis was performed at NCBI, screening for candidate sequences with a similarity exceeding 60% with characterized sequences. Ultimately, three CYP72 candidate sequences (CYP72-Ur_transcript26190 / f104p0 / 1922, CYP72-Ur_transcript27775 / f2p0 / 1871, and CYP72-Ur_transcript27898 / f4p0 / 1826) were identified. The three UrCYP72s were officially named CYP72A1177, CYP72A1178, and CYP72A1179, respectively. Five amino acid sequences with verified functions, namely CYP72A1 (L10081.1), CYP72A610 (MH763570.1), CYP72A565 (MH763569.1), CrSLS2 (KF415117.1) and OpSLS1 (MT942678.1), were selected and the conservation of CYP2A1177, CYP2A1178 and CYP2A1179 was analyzed using ESPript 3.0 software.

[0064] Experimental results: Figure 1 The results showed that the proteins encoded by UrCYP2A1177, UrCYP2A1178, and UrCYP2A1179 genes all contain 10 CYP450 characteristic conserved domains, including the heme-binding region, K helix, O2-binding region, I / V / LPK / AG, proline-rich region, and EXXR, and have typical characteristic sequences of plant CYP72 family proteins.

[0065] Twenty-two CYP72 and seven DL7H proteins were selected from the NCBI database and, together with the seven Uncaria rhynchophylla CYP72A elements screened previously, a phylogenetic tree was constructed using the neighbor-joining method.

[0066] Experimental results: Figure 2As shown in the figure, the newly discovered UrCYP72 (CYP72A1177, CYP72A1178, and CYP72A1179) and DL7H are distributed in different clades, indicating that they have functional differences. Among them, CYP72A1178 and CYP72A1179 are clustered in the same clade. The neighboring genes of CYP72A1178 are other CYP72 candidate genes in Uncaria rhynchophylla (UrCYP72A_transcript29832 / f5p0 / 1690, UrCYP72A_transcript28249 / f7p0 / 1831), while the neighboring genes of CYP72A1179 are the strychnine synthase identified in Snakeroot brevis (OpSLS6 MT942683.1) and the Uncaria rhynchophylla CYP72 candidate gene (UrCYP72A_transcript26554 / f2p0 / 1938), indicating that UrCYP72A1178 and UrCYP72A1179 may be related to the biosynthesis of strychnine. CYP72A1177 is located in a separate branch, and its neighboring genes include schizoside synthases (CrCYP72A1 L10081.1 and OpSLS1MT942678.1) identified from schizoside and Catharanthus roseus. These schizoside synthases exhibit oxidative cleavage activity at C7-C8 of the iridoid backbone, suggesting that CYP72A1177 may have a similar function. Based on phylogenetic analysis and the functional characteristics of neighboring genes, CYP72A1177, CYP72A1178, and CYP72A1179 may possess site-specific C-C bond cleavage activity.

[0067] Example 2 Expression cluster analysis and qPCR analysis of target UrCYP72A gene elements

[0068] To explore the expression pattern of UrCYP72 gene and its correlation with rhynchophylline biosynthesis, the target UrCYP72A gene elements and the expression level data (FP KM) of the bait gene were extracted from the transcriptome database of different parts of Uncaria rhynchophylla (BioProject No: PRJNA1127859), and expression cluster analysis was performed using Multi Experiment Viewer 4.9 software.

[0069] Experimental results: Figure 3As shown in the transcriptome analysis, the UrCYP72 family genes CYP72A1178 (Ur-transcript27775 / f2p0 / 1871), CYP72A1177 (transcript26190 / f104p0 / 1922), and CYP72A1179 (transcript27898 / f4p0 / 1826) clustered with the well-characterized gene UrSTR (UrSTR-transcript35843 / f2p0 / 1260) involved in rhynchophylline biosynthesis. The clustering pattern of these genes suggests that they are involved in the biosynthesis of iridoids, precursors of rhynchophylline synthesis.

[0070] Total RNA was extracted using the RNA prep purer Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Beijing), and trace DNA was degraded with DNAase. Single-stranded cDNA was prepared using the Goldenstar™ RT6 cDNA Synthesis Mix Kit (Beijing Qingke Biotechnology Co., Ltd., Beijing). The target gene UrCYP72A was cloned from Uncaria rhynchophylla cDNA using the pLB Zero Background Rapid Cloning Kit (Tiangen Biochemical Technology Co., Ltd., Beijing). The resulting gene fragment was sequenced by Chengdu Qingke. The cloning primers are shown in Table 1. RT-qPCR was used to examine the tissue expression characteristics of the UrCYP72 target gene and analyze its expression patterns in three tissues. The fluorescence quantitative primers are shown in Table 2.

[0071] Table 1 UrCYP72A cloning primers

[0072]

[0073]

[0074] Table 2 Fluorescence quantitative PCR primers

[0075]

[0076] Experimental results: Figure 4 As shown in the results, UrCYP72A1177 was expressed at a relatively high level in the stem, while UrCYP72A1178 and UrCYP72A1179 were highly expressed in the root. These results indicate that UrCYP72A1177 is more actively expressed in the stem, while UrCYP72A1178 and UrCYP72A1179 are more vigorously expressed in the root.

[0077] To confirm the existence of these genes, the target genes CYP72A1177, CYP 72A1178 and CYP72A1179 were successfully cloned using the cDNA of Uncaria rhynchophylla as a template. The cloned sequences were consistent with the transcriptome annotation sequences transcript26190 / f104p0 / 1922, Ur-transcript27775 / f2p0 / 1871 and transcript27898 / f4p0 / 1826.

[0078] Example 3 Construction and Verification of the Target UrCYP72A Gene Element Recombinant Expression Vector

[0079] After codon optimization, the CYP72A1177, CYP72A1178, and CYP72A1179 gene sequences were constructed into the pYES2 / CT-UrCYP 72A expression vector. The recombinant expression vectors were transformed into TOP10 competent cells. The recombinant bacterial suspension was cultured overnight at 37°C in LB medium. The recombinant plasmids pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 were extracted using a plasmid extraction kit. The recombinant plasmids were verified by double digestion with Kpn I and Not I at 37°C for 10 min.

[0080] Experimental results: Figure 5 As shown, the size of the enzyme digestion product bands is consistent with the length of the three target CYP72A genes, indicating that the target CYP72A gene recombinant plasmid was successfully constructed. Sequencing results showed that the target CYP72A gene had no base mutations and could be used for subsequent protein heterologous expression experiments.

[0081] Competent cells were prepared from a logarithmic-growth culture of Saccharomyces cerevisiae (WAT11). Sequencing-verified recombinant plasmids pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 were electroporated into WAT11 competent cells using an electroporator. The pYES2 / CT empty vector was also transformed as a control. The cells were recovered in 1 mL of sterile, antibiotic-free YPDA liquid medium at 30°C with shaking at 180 rpm for 2 hours. The cells were centrifuged at 3000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells were resuspended and washed in 1 mL of sterile water three times. Finally, the cells were resuspended in 200 μL of sterile water, plated on SC-Ura nutrient-deficient medium, and cultured at 30°C for 72 hours. Single WAT11 colonies were selected for colony PCR. The PCR program settings were: 95°C, 10 min; 30 cycles of denaturation-annealing-extension (95°C, 30 s; 57°C, 30 s; 72°C, 1 min); and incubation at 12°C. PCR verification primers are shown in Table 3.

[0082] Table 3 Colony PCR verification primers

[0083]

[0084]

[0085] Experimental results: Figure 6 As shown, the size of the PCR product was consistent with the expected size, indicating that the WAT11 recombinant Saccharomyces cerevisiae strain with three CYP72A genes was successfully constructed.

[0086] Example 4 Preparation and Expression Verification of Target UrCYP72A Gene Element Microsomal Protein

[0087] Use a sterile toothpick to pick a positive single colony and inoculate it into 15 mL of SC-Ura liquid medium. Cultivate with shaking at 30°C, 180 rpm for 24 hours. Inoculate 1 mL of the above bacterial solution into fresh 50 mL of SC-Ura liquid medium. Cultivate with shaking at 30°C, 180 rpm for 48 hours. Centrifuge at 3000 rpm for 5 minutes at room temperature, discard the supernatant, and collect the cells. Add 50 mL of sterile water to resuspend and wash the cells. Centrifuge at 3000 rpm for 5 minutes at room temperature, discard the supernatant, and collect the cells. Repeat three times. Resuspend the washed cells in 5 mL of YPGal liquid medium and transfer them to 100 mL of YPGal liquid medium. Cultivate at 30°C, 180 rpm, and induce for 24 hours. Centrifuge at 3500 g for 5 minutes at 4°C, discard the supernatant, and collect the cells. Resuspend the cells (0.5 g of cells / mL) in pre-cooled TESB buffer (50 mM Tris, 1 mM EDTA, 0.6 M sorbitol, pH 7.4), add sterile ceramic beads with a diameter of 0.5 mm (1000 ceramic beads / mL of bacterial solution per 1 mL of bacterial solution) until the cells are submerged, and let stand on ice for 5 minutes. Use a three-dimensional cryo-grinder (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai) with an oscillation speed of 20 m / s, a crushing time of 30 seconds, and a cooling time of 1 minute to continuously crush 10 times. Repeat the operation three times, pipette and blow to mix, and take an appropriate amount of supernatant for microscopic examination. If there is no intact cell structure visible to the naked eye, it can be used to extract microsomal proteins. Dilute the supernatant 2-3 times with TESB buffer, centrifuge at 12000 rpm, 4°C for 20 minutes, and collect the crushed supernatant into a new centrifuge tube. 1.5 M NaCl and solid PEG-4000 were added to final concentrations of 0.15 M and 0.1 g / mL, respectively, and microsomal proteins were precipitated on ice for 40 min. The microsomal proteins were collected by centrifugation at 12,000 rpm and 4°C for 30 min, weighed, and resuspended in TEG buffer (50 mM Tris, 1 mM EDTA, 20% glycerol). The extinction coefficient (ε) of the UrCYP72A protein was calculated using ExPasy ProtParam.280nm The concentration of UrCYP72A protein was determined by the extinction coefficient method. The expression of UrCYP72A in WAT11 Saccharomyces cerevisiae was verified by Western-blot.

[0088] Experimental results: Figure 7 Western blot results showed that all three target CYP72A recombinant proteins were successfully expressed, and the band sizes were consistent with the expected target band sizes. The target CYP72A recombinant microsomal proteins were frozen at -80°C until use.

[0089] Example 5 Determination of UrCYP72A Enzyme Activity in Vitro and in Tobacco

[0090] A 50 mM substrate stock solution was prepared by dissolving a standard of strychnine (Sichuan Weikeqi Biotechnology Co., Ltd., Chengdu) in dimethyl sulfoxide. A 500 μL in vitro enzymatic reaction system consisting of 100 μM strychnine, 500 μM NADPH, and 15 μL UrC YP72A microsomal protein was added to 500 μL of 100 mM Tris-HCl buffer (pH 7.5) and incubated at 30°C for 1 hour. Empty microsomal protein was also used as a control. After completion, the reaction was terminated with 500 μL of ethyl acetate and the product was extracted twice. The organic layer was transferred, dried under nitrogen, dissolved in 500 μL of chromatography-grade methanol, filtered through a 0.45 μm organic filter, and stored at 4°C. Each reaction was repeated three times. The UrCYP72A gene was inserted into the pEAQ-HT vector plasmid using the Age I and Xho I sites to construct the recombinant plasmid pEAQ-HT-UrCYP72A. According to the manufacturer's instructions, the recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 competent cells, and the obtained GV3101 strains were cultured in LB medium (50 μg / mL rifampicin, 50 μg / ml kanamycin) to OD 600 =0.6. The cell pellet was collected by centrifugation, resuspended in infiltration buffer (10 mM MES, 200 μM Acetosyring one, 10 mM MgCl2, pH 5.6), and diluted to OD 600=0.6. The UrCYP72A gene was transiently expressed in 5-week-old Nicotiana benthamiana leaves. The resuspension was infiltrated onto the underside of the leaves using a syringe. Three days later, a substrate injection solution (containing 100 μM strychnine and 500 μM NADPH) was prepared and infiltrated into the same leaves using a syringe. After two days of incubation at room temperature, the leaves were harvested, ground into a fine powder using liquid nitrogen, and extracted with 500 μL of methanol for 30 minutes at room temperature. The mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected and filtered through a 0.45 μm filter membrane to obtain a sample for subsequent analysis. Tobacco leaves were transformed with Agrobacterium carrying the pEAQ-HT empty plasmid, and the same procedure was used as a negative control. The reaction mixture and tobacco leaf extract were monitored using an Agilent G6125B liquid chromatography-mass spectrometry system. Analysis was performed using an SB-C18 (2.1×50 mm, 1.8 μm) column at 35.0°C. Mobile phase A consisted of 0.1% formic acid solution, and mobile phase B consisted of chromatography-grade acetonitrile. The injection volume was 5 μL, and the flow rate was 0.2 mL / min. The elution profile was as follows: 0–2 min, 5–10% (Phase B); 2–4 min, 10–20% (Phase B); 4–7 min, 20–50% (Phase B); 7–10 min, 50–80% (Phase B); 10–13 min, 80–98% (Phase B); 13–13.5 min, 98–5% (Phase B); and 13.5–18 min, 5–5% (Phase B). Mass spectrometry analysis conditions were: API-ES ionization mode; positive ionization mode; capillary voltage: 3.0 kV (+); drying gas: nitrogen (>99.999% purity); gas temperature: 350°C; gas flow rate: 12.0 L / h; nebulizer pressure: 50 psig; quadrupole temperature: 350°C; collection molecular weight range: 50-1000 Da; fragmentor voltage: 10 eV. Identification of the target product was based on its molecular weight. The target product was analyzed by secondary mass spectrometry using a Xevo G2-XSQTOF (Waters, USA) mass spectrometer. The mass spectrometry analysis conditions were as follows: the ion source was an electrospray ionization source (ESI); the ionization mode was positive ion; the capillary voltage was 2.5 kV (+) and 2.5 kV (-); the cone voltage was 40 V; the drying gas was N2 with a purity of >99.999%; the desolvation temperature was 400°C; the desolvation flow rate was 800 L / h; the cone backflush flow rate was 50 L / h; the heating temperature was 120°C; the full scan detection molecular weight range was 50-1000 Da; the leucine-enkephalin concentration was 1.0 ng / μL, and the flow rate was 20 μL / min as the lock mass to ensure that the error was <10 ppm.

[0091] Experimental results: The extracted ion current (EIC) of the microsomal enzyme catalyzed reaction solution is shown in Figure 8A in Figure 1 was detected in the reaction mixtures of CYP72A1177, CYP72A1178, and CYP72A1179 enzymes, and a C—C bond cleavage product (m / z 389.4) 2 Da lower than that of strychnine (m / z 391.4) was detected. 17 H 26 O 10 , m / z 389.2659), CYP72A1178(C 17 H 26 O 10 , m / z 389.2619) and CYP72A1179 (C 17 H 26 O 10 , m / z 389.2417) of the C-C bond cleavage product ( Figure 9 -162Da (-C6H 11 O5), -18Da (H2O) and -32Da (-CH3OH), generating the corresponding intermediate ions m / z 227.9839 [M-162] + 、m / z 209.0179[M-162-18] + and m / z 177.7639 [M-162-18-32] + Subsequently, characteristic fragments m / z 151.0265 [M-162-18-32-26] generated by the removal of vinyl groups (-26Da, -CH=CH2) and -28Da (-C=O) were detected. + and m / z 149.0196 [M-162-18-32-28] + This process showed the presence of characteristic aldehyde and vinyl groups, and by comparing the spectrum with the standard of secoyrin, the product was confirmed to be secoyrin.

[0092] When loganin was injected as a substrate into tobacco leaves carrying CYP72A1177, CYP72A1178, and CYP72A1179, the same -2 Da loganin C-C bond cleavage product (m / z 389.4) was observed ( Figure 8 B in the figure), and their MS / MS spectra are identical to those of the strychnine standard ( Figure 10 These results confirmed that the newly discovered CYP72A1177, CYP 72A1178, and CYP72A1179 also exhibited strychnine C-C bond cleavage activity in tobacco.

[0093] Example 6 Structural Identification of CC Cleavage Products of CYP72A1177, CYP72A1178, and CYP72A1179

[0094] The WAT11 strain, which harbors UrCYP72A, was used for scale-up experiments. The total reaction volume was 500 mL, and the final substrate concentration was 100 μM. The yeast was cultured at 30°C, 180 rpm for 48 h. The supernatant was collected by centrifugation and extracted three times with 500 mL of ethyl acetate. The extracts were combined and the ethyl acetate was removed using a rotary evaporator. The C-C bond cleavage product of strychnine was purified using a semi-preparative LC3050N HPLC column. The column was a Cosmosil 5C18-MS-II (10 ID × 250 mm) packed column at a flow rate of 3.0 mL / min. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile. Gradient elution conditions were as follows: 0-10 min, 5-20% (Phase B); 10-30 min, 20-80% (Phase B); 30-32 min, 80-98% (Phase B); 32-37 min, 98-98% (Phase B); 37-39 min, 98-5% (Phase B); 39-45 min, 5-5% (Phase B). The detection wavelength was set at 235 nm. Finally, the purified C-C bond cleavage product was dissolved in DMSO-d6 and structural analysis was performed using an Advance III HD NMR spectrometer.

[0095] Experimental results: Figure 12 shown. 1H NMR (600MHz, DMSO-d6): δ9.61 (1H, s, -CHO), 7.48 (1H, dd, J = 8.9, 1.7Hz, H-3), 5.82 (1H, d, J = 7.8Hz, H-1), 5.57–5.48 (1H, m, H-8), 5.40 (1H, d, J =4.6Hz, H-1'), 5.33–5.17 (2H, m, H-10), 5.08 (1H, d, J = 5.3Hz, 4'OH), 4.98 (2H, dd, J = 15.4, 5.3Hz, 2'OH, 3'OH), 4.61–4.54 (1H, m, 6'OH), 4.50 (1 H, d, J=7.9Hz, H-2'), 3.69–3.63 (1H, m, H-5), 3.59 (3H, d, J=5.3Hz, -OCH3), 3.45–3.38 (1H, m, H-3'), 3.17–3.09 (2H, m, H-6'a, H-6'b), 3.01 (1H , dd, J=8.8, 4.4Hz, H-4'), 2.96 (1H, d, J=5.3Hz, H-5'), 2.67–2.63 (1H, m, H-9), 2.60 (1H, d, J=8.7Hz, H-6a), 2.42 (1H, dd, J=16.8, 6.0Hz, H-6b). 13 C NMR (150 MHz, DMSO-d6): δ 206.11 (C-7), 176.05 (C-11), 161.87 (C-3), 143.20 (C-8), 129.22 (C-10), 117.70 (C-4), 108.07 (C-1'), 104.85 (C-1), 86.84 (C-5'), 86.14 (C-3'), 82.50 (C-2'), 79.50 (C-4'), 70.60 (C-6'), 60.56 (C-11, -OC H3), 53.10 (C-9), 52.87 (C-6), 9.61 (C-5). Compared with the substrate spectrum, δ H The new signal at 9.61 (1H, s, -CHO) is the aldehyde proton signal, δ H 5.57-5.48 (1H, m, H-8) and δ H The new signal at 5.33-5.17 (2H, m, H-10) is the olefin proton signal. At the same time, the C7 hydroxyl signal (δ H 4.48–4.37) and C7 (δ H 4.12–3.94), C8(δ H 1.43), C10(δ H0.98) The alkyl proton signal disappears, indicating that the C7-C8 bond undergoes oxidative cleavage and ring opening. 13 In the C-NMR spectrum, the number of carbon atoms remains unchanged compared to the substrate spectrum, but a new aldehyde carbon signal δ C 206.11 (C-7) and new alkenyl carbon signal δ C 143.20 (C-8) and δ C 129.22 (C-10), which also indicates that C7-C8 of strychnine is oxidatively broken, which is consistent with the spectrum of split strychnine reported in the literature. Figure 1 The oxidation product was finally confirmed to be secoyrin.

[0096] Example 7 Catalytic Performance of Target UrCYP72A Protein

[0097] A series of strychnine standard solutions (0.50, 1.00, 2.00, 4.00 and 6.00 μM) were prepared, and the standard solutions were analyzed using the above-mentioned LC-MS method to establish a standard curve of peak area and concentration for quantitative analysis of the product. The optimal reaction conditions for the target UrCYP72A were determined based on a single-factor experiment, including reaction time (0.5-8h), temperature (20-60°C), pH (6.0-8.0) and NADP H concentration (50μM–1000μM). The kinetic characterization of the UrCYP72A enzyme element was carried out under the optimized reaction conditions, and a reaction system containing a series of strychnine substrate concentrations (50-1000μM) was configured. The reaction was carried out for 10min, 20min, 40min, 60min, and 80min, respectively. The initial reaction velocity was measured, and a velocity-substrate concentration curve was constructed. The Michaelis-Menten equation was fitted using the Michaelis-Menten method of Origin 2021 to generate V max and K m Value, V max and K m Used to evaluate catalytic performance.

[0098] Experimental results: Figures 13-15 The reaction temperature, optimal reaction time, pH value and NADPH concentration of CYP72A1177, CYP72A1178 and CYP72A1179 were 30°C, 1 h, pH 7.0 and 500 μM, respectively.

[0099] like Figure 16 As shown, the K of CYP72A1177 mThe value of CYP72A1177 (96.24 ± 17.44 nM) was significantly lower than that of CYP72A1178 (131.64 ± 12.33 nM) and CYP72A1179 (127.17 ± 10.60 nM), indicating that it has a higher affinity for strychnine. max Value (64.56±2.61nM min -1 ) was also significantly higher than that of CYP72A1179 (57.49±1.14nM min -1 ) and CYP72A1178 (49.26±1.22 nM min -1 ), indicating that its catalytic efficiency is better. Comprehensive analysis showed that the catalytic performance of CYP72A1178 and CYP72A1179 for strychnine is relatively similar, while CYP72A1177 is the best in terms of substrate affinity and catalytic performance.

[0100] Example 8 Analysis of the UrCYP72A substrate binding pocket and rational mutation

[0101] Three-dimensional structural models of CYP72A1177, CYP72A1178, and CYP72A1179 were constructed using AlphaFold 3. The molecular structure of loganin was downloaded from PubChem. Loganin was docked into the binding pockets of CYP72A1177, CYP72A1178, and CYP72A1179 using AutoDock 4.2.6, and the CYP72A-loganin complex was visualized using PyMOL software (version 2.5.0) to identify the key amino acid residues involved in CYP72A substrate binding.

[0102] Experimental results: Figure 17 In UrCYP72A1177, strychnine is bound in a hydrophobic core around the heme group, which consists of 9 amino acids, including LYS-127, GLY-144, THR-334, VAL-394, ILE-395, ASP-396, LEU-397, THR-398, THR-506 ( Figure 17 A in Figure 1). The C11 carbonyl group of strychnine forms a hydrogen bond with LYS-127. ILE-395, ASP-396 and THR-398 form five hydrogen bonds with the 6'-OH, 4'-OH and 3'-OH groups of the glucose group of strychnine, respectively The distance between C7-C8 of strychnine and the iron in the heme center is This is the ideal spatial distance for CC bond cleavage reaction, which is conducive to the occurrence of CC bond cleavage reaction. Figure 17B) in Figure 1 shows that strychnine is bound to a hydrophobic pocket around the heme group and consists of 14 amino acids, including LYS-128, LEU-138, ALA-139, VAL-143, ASN-144, LEU-324, PHE-327, ALA-328, THR-332, LEU-392, VAL-394, LEU-395, THR-396, and ARG-397. LYS-128, THR-396, and ARG-397 form five hydrogen bonds with the 2'-OH, 4'-OH, and 3'-OH groups on the glucose group of strychnine, respectively. The distance between C7-C8 of strychnine and the iron in the heme center is This is also the ideal spatial distance for the C-C bond cleavage reaction. In UrCYP72A1179, strychnine is bound in a hydrophobic core around the heme group, which consists of 13 amino acids, including LYS-125, VAL-140, LEU-321, PHE-324, ALA-325, THR-329, GLY-389, VAL-390, MET-391, LEU-392, GLY-393, ARG-394, ILE-500 ( Figure 17 C in Figure 1). LYS-125, MET-391, and GLY-393 form five hydrogen bonds with the 2'-OH, 6'-OH, 4'-OH, and 3'-OH groups of the glucose moiety of strychnine. The distance between C7-C8 of strychnine and the iron in the heme center is This is the ideal spatial distance for C-C bond cleavage reactions. The distance between C7-C8 and the heme center in UrCYP72A1177 is shorter than that in UrCYP72A1178 and UrCYP72A1179, which leads to UrCYP72A1177 showing better catalytic performance towards strychnine.

[0103] Mutation sites were designed based on molecular docking results. The UrCYP72A mutant gene sequence was synthesized by Shanghai Bioengineering and inserted between the Kpn I and Not I sites of the pYES2 / CT plasmid. UrCYP72A mutant proteins were prepared according to the above-described method, and enzymatic activity of the UrCYP72A mutants was assayed. Relative enzyme activity was calculated to evaluate the catalytic performance of the UrCYP72A mutants.

[0104] Experimental results: We speculate that LYS127, ILE395, ASP-396 and THR-398 in CYP72A1177, LYS128, THR-396 and ARG-397 in CYP72A1178, and LYS125, MET-391 and GLY-393 in CYP72A1179 are key residues for substrate directional stabilization. Mutation experiments revealed that compared with wild-type UrCYP72A1177, UrCYP72A1178, and UrCYP72A1179 recombinant proteins, the relative enzyme activities of mutants CYP72A1177-K127A, CYP72A1177-I395A, CYP72A1177-D396A, and CYP72A1177-T398A decreased by 91.5%, 94.3%, 96.2%, and 89.5%, respectively. The relative enzyme activities of mutants CYP72A1178-K128A, CYP72A1178-T396A, and CYP72A1178-R397A decreased by 98.5%, 94.1%, and 92.4%, respectively. The relative enzyme activities of mutants CYP72A1179-K125A, CYP72A1179-M391A, and CYP72A1179-G393A decreased by 96.5%, 98.2%, and 86.3%, respectively. Figure 19 ), these findings confirmed our hypothesis. To improve their enzyme activity, rational mutations were performed on residues at the substrate entrance and residues adjacent to the hydroxyl group of the substrate that is not bound to form hydrogen bonds. Three different residues, THR-334 in CYP72A1177, PHE-327 in CYP72A1178, and LEU-321 in CYP72A1179, are located at the substrate entrance ( Figure 18 ), the T334G mutation in CYP72A1177 resulted in a narrowing of the substrate-binding pocket entrance and a 13.2% decrease in enzyme activity; the F327G mutation in CYP72A1178 resulted in a widening of the substrate-binding pocket entrance and a 24.1% increase in enzyme activity; the L321G mutation in CYP72A1179 resulted in a widening of the substrate-binding pocket entrance and an 18.6% increase in enzyme activity. These results indicate that the wider substrate entrances in CYP72A1177, CYP72A1178, and CYP72A1179 facilitate the insertion of strychnine.

[0105] In CYP72A1177, the mutation CYP72A1177-D396E reduces the distance between the substrate strychnine C7-C8 and the iron in the heme center. But the number of hydrogen bonds decreases The relative enzyme activity increased by 21.5% ( Figure 17D). In the mutant CYP72A1178-P393V, Pro-393 is replaced by VAL, which reduces the distance between the substrate strychnine C7-C8 and the iron in the heme center. Increased number of hydrogen bonds The relative enzyme activity increased by 42.6% ( Figure 17 E). In the mutant CYP72A1179-V390I, VAL-390 is replaced by ILE, which reduces the distance between the substrate strychnine C7-C8 and the iron in the heme center. The number of hydrogen bonds remains unchanged The relative enzyme activity increased by 25.4% ( Figure 17 To further improve the activity of the target enzyme CYP72As, the catalytic potential was enhanced by combining mutations at two sites ( Figure 18 The T334G+D396E mutations in CYP72A1177 resulted in a 14.6% increase in enzyme activity. The F327G+P393V mutations in CYP72A1178 resulted in a 62.7% increase in enzyme activity. The L321G+V390I mutations in CYP72A1179 resulted in a 37.3% increase in enzyme activity.

[0106] In summary, the wider substrate entrances of CYP72A1177, CYP72A1178, and CYP72A1179 facilitate the insertion of strychnine. Furthermore, the increased hydrogen bonding of strychnine leads to enhanced enzyme activity, and the reduced distance between the C7-C8 region of strychnine and the iron in the heme center also contributes to enhanced enzyme activity. This suggests that through rational mutational engineering, the catalytic performance of strychnine synthase can be significantly improved.

[0107] Example 9: Experimental Study on the Tandem Connection of UrLAMT and UrCYP72A Enzyme Elements in Nicotiana benthamiana

[0108] Construct the pEAQ-HT-UrLAMT recombinant plasmid and insert the UrLAMT target gene into the Age I and Xho I restriction sites of the plasmid pEAQ-HT. Transform the recombinant plasmid and the empty plasmid into GV3101 competent cells by heat shock method, spread the culture, and pick a single clone to culture until OD 600 =0.6-0.8, the cells were collected by centrifugation and resuspended in infiltration buffer (10 mM MES, 200 μM acetosyringone, 10 mM MgCl2, pH = 5.6) to OD 600=0.6. The resuspension buffer of the UrLAMT strain and the different UrCYP72 strains constructed above was mixed at a 1:1 ratio and injected into 5- to 7-week-old Nicotiana benthamiana leaves. The leaves were incubated in the dark for 24 hours and then in the light for 48 hours. The substrates strychnine (100 μmol / L) and SAM (200 μmol / L) were infiltrated into the same tobacco leaves. On the third day, the tobacco leaves were ground into a fine powder using liquid nitrogen, decolorized with n-hexane (0.1 g of powder), and ultrasonically extracted with methanol (500 μL) for 20 minutes. The extracts were centrifuged for 5 minutes, the supernatant collected, dried with nitrogen, and filtered through an organic phase filter (0.22 μm). The reaction was repeated three times, with the pEAQ-HT empty plasmid as a negative control. Sample analysis and product identification were performed using the aforementioned methods.

[0109] Experimental results: When UrLAMT was co-expressed with CYP72A1177, CYP72A1178, and CYP72A1179 in tobacco, and the substrates strychnine, SAM, and NADPH were injected, strychnine (m / z 389.4) was detected in tobacco leaves at the same retention time as in vitro reaction ( Figure 20 ), its cleavage pattern ( Figure 21 C, D and E) are consistent with the standard of strychnine ( Figure 21 B). When UrLAMT+CYP72A1177 was co-expressed, the content of the product strychnine in tobacco leaves was 2.02 μg / g, while the content of the product strychnine in tobacco leaves of UrLAMT+CYP72A1178 and UrLAMT+CYP72A1179 were 0.42 μg / g and 1.22 μg / g, respectively, which were lower than those of UrLAMT+CYP72A1177 ( Figure 22 The key precursor of rhynchophylline, schizotyrin, can be prepared through a two-step enzymatic cascade reaction in tobacco. Under identical conditions, UrLAMT was co-expressed with the combined mutants CYP72A1177 (D396E), CYP72A1178 (F327G+P393V), and CYP72A1179 (L321G+V390I) in tobacco. A product (m / z 389.4) with a retention time consistent with that of a schizotyrin standard was detected in tobacco leaves. The contents of strychnine products in tobacco leaves of UrLAMT+CYP72A1177(D396E), UrLAMT+CYP72A1178(F327G+P393V) and UrLAMT+CYP72A1179(L321G+V390I) were 3.35μg / g, 2.18μg / g and 1.99μg / g, respectively, among which UrLAMT+CYP72A1177(D396E) had the highest content.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A secoyrin synthase gene, characterized in that: The secosinoside synthase genes include CYP72A1177, CYP72A1178 and CYP72A1179; The nucleotide sequence of CYP72A1177 is shown in SEQ ID NO.1; The nucleotide sequence of CYP72A1178 is shown in SEQ ID NO.2; The nucleotide sequence of CYP72A1179 is shown in SEQ ID NO.

3.

2. The protein encoded by the secoyrin synthase gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by CYP72A1177 is shown in SEQ ID NO.4; The amino acid sequence of the protein encoded by CYP72A1178 is shown in SEQ ID NO.5; The amino acid sequence of the protein encoded by CYP72A1179 is shown in SEQ ID NO.

6.

3. A recombinant expression vector, characterized in that: The invention comprises target genes and a pYES2 / CT expression vector, wherein the target genes include CYP72A1177, CYP72A1178 and CYP72A1179.

4. A transformant, characterized in that The method comprises the recombinant expression vector according to claim 3 and WAT11 Saccharomyces cerevisiae competent cells.

5. Use of the secoyrin synthase gene according to claim 1, the protein according to claim 2, the recombinant expression vector according to claim 3 or the transformant according to claim 4 in the preparation of secoyrin.

6. A recombinant microsomal protein of secoyrin synthase, characterized in that: The preparation method of the secoyrin synthase recombinant microsomal protein comprises the following steps: 1) CYP72A1177, CYP72A1178, and CYP72A1179 were respectively connected to the pYES2 / CT expression vector to obtain the pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 recombinant expression vectors; 2) The pYES2 / CT-CYP72A1177, pYES2 / CT-CYP72A1178, and pYES2 / CT-CYP72A1179 recombinant expression vectors were respectively introduced into WAT11 Saccharomyces cerevisiae competent cells to obtain WAT11 recombinant Saccharomyces cerevisiae strains expressing CYP72A1177, CYP72A1178, and CYP72A1179; 3) The WAT11 recombinant Saccharomyces cerevisiae strains expressing CYP72A1177, CYP72A1178, and CYP72A1179 were inoculated into SC-Ura liquid medium for expansion, transferred to YPGal liquid medium for induction culture, centrifuged, and the supernatant discarded. The cells were collected and disrupted using sterile ceramic beads to extract the recombinant microsomal protein of strychnine synthase.

7. A recombinant microsomal mutant protein of secoyrin synthase, characterized in that: These include CYP72A1177-D396E, CYP72A1178-F327G, CYP72A1178-P393V, CYP72A1179-L321G, CYP72A1179-V390I, CYP72A1177-T334G+D396E, CYP72A1178-F327G+P393V, and CYP72A1179-L321G+V390I; The CYP72A1177-D396E is a mutation of aspartic acid at position 396 of the amino acid sequence shown in SEQ ID NO. 4 to glutamic acid; The CYP72A1178-F327G is a mutation of the phenylalanine at position 327 of the amino acid sequence shown in SEQ ID NO.5 to glycine; The CYP72A1178-P393V is a mutation of the proline at position 393 of the amino acid sequence shown in SEQ ID NO.5 to valine; The CYP72A1179-L321G is a mutation of the leucine at position 321 of the amino acid sequence shown in SEQ ID NO.6 to glycine; The CYP72A1179-V390I is a mutation of the valine at position 390 of the amino acid sequence shown in SEQ ID NO.6 to isoleucine; The CYP72A1177-T334G+D396E is a mutation of the threonine at position 334 of the amino acid sequence shown in SEQ ID NO. 4 to glycine and the mutation of the aspartic acid at position 396 of the amino acid sequence shown in SEQ ID NO. 4 to glutamic acid; The CYP72A1178-F327G+P393V is a mutation of the phenylalanine at position 327 of the amino acid sequence shown in SEQ ID NO.5 to glycine and a mutation of the proline at position 393 of the amino acid sequence shown in SEQ ID NO.5 to valine; The CYP72A1179-L321G+V390I is a mutation of the leucine at position 321 of the amino acid sequence shown in SEQ ID NO. 6 to glycine and a mutation of the valine at position 390 of the amino acid sequence shown in SEQ ID NO. 6 to isoleucine.

8. Use of the recombinant microsomal protein of secoyrin synthase according to claim 6 or the recombinant microsomal mutant protein of secoyrin synthase according to claim 7 in the preparation of secoyrin.

9. The use according to claim 8, characterized in that The reaction temperature in the process of preparing strychnine is 20-60° C., the reaction time in the process of preparing strychnine is 0.5-8 h, the pH value in the process of preparing strychnine is 6-8, and the NADPH concentration in the process of preparing strychnine is 50-1000 μM.

10. Use of the recombinant microsomal protein of secoyrin synthase according to claim 6 or the recombinant microsomal mutant protein of secoyrin synthase according to claim 7 in combination with Uncaria rhynchophylla strychnine-O-methyltransferase in the preparation of secoyrin.

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