A vincamine synthetase and its encoding gene and use thereof

Abstract

The application discloses a vinblastine synthase, an encoding gene thereof and an application, and belongs to the technical field of genetic engineering. By using transcriptome data of Vinca minor, a 2OGD enzyme, namely, vinblastine synthase Vm15G000668, is screened and obtained, and in-vitro purification finds that the vinblastine synthase Vm15G000668 can specifically catalyze conversion of longchun dimethylbiguanide into vinblastine, and a key catalytic step of vinblastine biosynthesis is analyzed. The application provides a high-efficiency and specific vinblastine synthetic biocatalyst, and provides technical support for large-scale and green preparation of vinblastine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a vinblastine synthase, its encoding gene, and its applications. Background Technology

[0002] Vinca minor, a perennial herbaceous plant belonging to the genus Vinca in the family Apocynaceae, is rich in the important alkaloid vinblastine. Vinblastine, as a natural cerebral vasodilator, plays a crucial role in the treatment of cerebral arteriosclerosis, cerebral infarction, and other diseases, possessing extremely high medicinal value. Vinblastine is a monoterpenoid indole alkaloid. Currently, research on monoterpenoid indole alkaloids has largely focused on periwinkles in the genus Vinca. The synthetic pathways of monoterpenoid indole alkaloids such as vincristine and vinblastine in this plant have been elucidated, but the biosynthetic pathways of monoterpenoid indole alkaloids (MIAs) in the genus Vinca minor remain unclear. As a typical representative of the genus Vinca minor, research on its vinblastine biosynthetic pathway will fill a gap in this field.

[0003] Currently, there are three main ways to obtain vinblastine: First, direct extraction from plants. However, the content of vinblastine in plants is extremely low, and it depends on the large-scale cultivation of plants. It is limited by factors such as growth cycle and environmental conditions, resulting in low extraction efficiency and high cost. Second, chemical synthesis. This method involves complex steps, multiple oxidation, reduction, and cyclization reactions, harsh reaction conditions, many byproducts, difficulty in controlling product purity, and significant environmental pollution. Third, biosynthesis using microbial cell factories or in vitro enzyme catalysis systems. This method has advantages such as mild conditions, high specificity, and environmental friendliness, making it an ideal direction for future large-scale preparation.

[0004] To overcome the limitations of traditional extraction and chemical synthesis methods, recent research has focused on elucidating the biosynthetic pathway of vincaine, aiming to discover key genes or transcription factors to achieve efficient vincaine biosynthesis. In 2020, Vincent's team used transcriptome data from the roots, old leaves, and new leaves of *Catharanthus chinensis* (exposed to low or high light conditions, respectively) to clone a tabersonine / vincadifformine 16-O-methyltransferase (Vm16OMT) isoform through homology search. Functional verification showed that this enzyme can catalyze the methylation of 16-hydroxy derivatives of tabersonine, vincadifformine, and lochnericine. Furthermore, the team combined Oxford Nanopore long-read sequencing and Illumina short-read sequencing technologies to successfully assemble the first contig-level genome of *Catharanthus chinensis*, and identified gene clusters involved in the synthesis of monoterpenoid indole alkaloids through homology analysis and metabolomics screening. Based on collinearity analysis, vincadifformine-16-hydroxylase (Vm16H) was further screened and identified, providing important clues for elucidating the vinca amine biosynthesis pathway.

[0005] Currently, the academic community generally believes that vincadifformine is an important precursor in the biosynthesis of vincamine. Studies have shown that vincadifformine is most abundant in the young leaves of *Catharanthus chinensis*, and that methyl jasmonic acid induction promotes the conversion of vincadifformine to 9-methoxyvincamine, leading to a decrease in its content. Although an α / β-hydrolase catalyzing the production of vincadifformine has been identified in *Catharanthus chinensis*, no enzyme with similar function has been found in *Catharanthus chinensis*. Furthermore, 16-methoxytabersonine, an analogue of 16-methoxyvincadifformine (different from vincadifformine only at the 8-carbon double bond), has been reported to be catalyzed by cytochrome P450 (CYP71D1, 16-methoxytabersonine 3-oxidase, 16T3O) to generate a vincamine-like skeleton, suggesting that homologs of T3O may be involved in the formation of vincamine-related alkaloids, providing an important reference for future research.

[0006] However, the existing technology does not report the functional enzymes and related encoding genes that catalyze the conversion of vinblastine to vinblastine, which cannot meet the requirements of key steps in the biosynthesis of vinblastine, making it difficult to construct a complete biosynthetic pathway for vinblastine.

[0007] Therefore, how to provide an enzyme that can specifically catalyze the conversion of vinblastine to vinblastine and its encoding gene is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a vinblastine synthase, its encoding gene, and its applications.

[0009] In existing technologies, the key steps in the biosynthetic pathway of vinblastine are not yet clearly defined. The lack of functional enzymes and their encoding genes that specifically catalyze the conversion of metformin into vinblastine makes it difficult to efficiently prepare vinblastine through biosynthesis. Methods relying on chemical synthesis or plant extraction suffer from cumbersome procedures, high costs, low product purity, and poor environmental friendliness. This invention aims to identify the key catalytic enzyme (vinblastine synthase) and its encoding gene in the biosynthesis of vinblastine, providing a biological tool for efficient in vitro catalysis of vinblastine synthesis, thus overcoming the technical bottleneck of missing key enzymes in vinblastine biosynthesis. This invention plays an important role in the biosynthesis and metabolic regulation of vinblastine in *Catharanthus roseus*, as well as in the genetic breeding of this medicinal plant.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] A vinblastine synthase, the amino acid sequence of which is shown in SEQ ID No. 1.

[0012] The nucleotide sequence of the gene encoding the aforementioned vinblastine synthase is shown in (a), (b), or (c):

[0013] (a) The nucleotide sequence shown in SEQ ID No. 2;

[0014] Or (b) a nucleotide sequence that can hybridize with the complementary nucleotide sequence of SEQ ID No. 2 under stringent hybridization conditions, wherein the protein encoded by the nucleotide still has the function or activity of vinblastine synthase;

[0015] Or (c) a nucleotide sequence that has at least 80% homology with the nucleotide sequence of SEQ ID No. 2, and the protein encoded by the nucleotide still has the function or activity of vinblastine synthase.

[0016] Chimeric genes or expression cassettes containing the above-mentioned coding genes.

[0017] Recombinant expression vectors containing the above-mentioned coding genes or containing the above-mentioned chimeric genes or expression cassettes.

[0018] Recombinant host cells containing the aforementioned recombinant expression vector.

[0019] The above-mentioned vinblastine synthase is used as a catalytic enzyme in the biosynthesis of vinblastine.

[0020] Furthermore, the substrate was metformin.

[0021] The application of the aforementioned coding genes, chimeric genes, or expression cassettes in the biosynthesis of vinblastine.

[0022] A method for the in vitro synthesis of vinblastine uses the aforementioned vinblastine synthase as a catalyst and vinblastine metformin as a substrate in a solution of 2-oxoglutaric acid and Fe... 2+ The reaction is catalyzed in the presence of ascorbic acid and NaDPH.

[0023] As used in this article, the term "vinblastine synthase" refers to a 2-oxoglutarate-dependent dioxygenase (2OGD enzyme) derived from the periwinkle, which has the specific function of catalyzing the activation reaction of vinblastine substrate to produce vinblastine.

[0024] As used in this article, the term "2OGD enzyme (2-oxoglutarate-dependent dioxygenase)" refers to a class of enzymes that use 2-oxoglutarate and molecular oxygen as cofactors to catalyze oxidative modification reactions of various biomolecules and are widely involved in metabolic processes such as the biosynthesis of natural products.

[0025] As used herein, the term "vincadifformine" refers to a key precursor substrate in the vincaamine biosynthesis pathway, which can be converted into vincaamine by vincaamine synthase. CAS No.: 3247-10-7, structural formula as follows:

[0026]

[0027] As used in this article, the term "vinblastine" refers to an alkaloid with important medicinal value, commonly used in the treatment of diseases related to improving cerebral circulation and protecting nerve cells. CAS No.: 1617-90-9, structural formula as follows:

[0028]

[0029] As used herein, the term "in vitro recombinant expression" refers to the technique of cloning a target gene into a suitable expression vector, transferring it into a host cell (in this invention, the Escherichia coli strain is BL21(DE3), and the vector is PET28a), and obtaining a recombinant protein by inducing expression in the host cell.

[0030] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) Solved the technical bottleneck of missing key enzymes in vinblastine biosynthesis: For the first time, this invention identified vinblastine synthase, which can specifically catalyze the conversion of vinblastine metformin into vinblastine, from the small vine periwinkle. The encoding gene was identified, filling the gap in the key steps of vinblastine biosynthesis in the prior art and providing a core tool for the complete construction of the vinblastine biosynthesis pathway.

[0032] (2) Verification by in vitro recombinant expression in Escherichia coli showed that the vinca synthase can efficiently recognize vinca metformin substrate and specifically catalyze its conversion into vinca amine, thus providing a guarantee for the efficient preparation of vinca amine.

[0033] (3) Achieve large-scale preparation of enzymes: Using the Escherichia coli heterologous expression system, recombinant vinblastine synthase can be prepared rapidly and in large quantities at low cost, thus reducing costs.

[0034] (4) Based on the vinblastine synthase and its encoding gene of the present invention, microbial cell factories or in vitro enzyme catalysis systems can be constructed to realize the biosynthesis of vinblastine, which is in line with the development trend of green production and solves many defects of traditional plant extraction and chemical synthesis. It can also be used as a tool enzyme for the synthesis and screening of vinblastine derivatives, providing technical support for the research and development of related drugs. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0036] Figure 1 This is a heatmap of transcriptome screening in Example 1 of the present invention.

[0037] Figure 2 This is an SDS-PAGE electrophoresis image from Example 2 of the present invention, where M: protein marker, 1: supernatant after PET28a empty-load induced disruption, 2: supernatant after PET28a-Vm15G000668 induced disruption, 3: PET28a-Vm15G000668 (third tube eluted with 250mM imidazole), 4: PET28a-Vm15G000668 (second tube eluted with 250mM imidazole), 5: PET28a-Vm15G000668 (first tube eluted with 250mM imidazole), 6: precipitate after PET28a-Vm15G000668 induced disruption, and 7: PET28a-Vm15G000668 flow.

[0038] Figure 3 The results are LC-MS / MS results from Example 3 of this invention.

[0039] Figure 4 These are secondary ion fragments of the product in the catalytic system of Example 3 of this invention.

[0040] Figure 5 This refers to the secondary ion fragments of vinblastine standard in Example 3 of this invention.

[0041] Figure 6 This is the standard curve used to calculate the yield in Embodiment 6 of the present invention.

[0042] Figure 7 The results are the enzyme kinetics results in Example 6 of this invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] Candidate screening of vinblastine synthase

[0046] To screen candidate genes for vinca synthase, this invention first obtained the Hidden Markov Model (HMM) file corresponding to the 2OGD family (PF03171) from the Pfam database (http: / / pfam.xfam.org / ), and performed homology searches on the 2OGD genes in the *Catharanthus chinensis* genome using HMMERv3.2.1 software. Subsequently, the candidate protein sequences obtained from the search were validated for conserved domains using the NCBI Conserved Domain Database (CDD, https: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi) to ensure the structural conservation of the target sequences. Simultaneously, based on multi-tissue / treatment transcriptome data covering shoot tips, roots, flowers, old leaves, young leaves, light-treated young leaves, and dark-treated young leaves, gene expression quantification analysis was performed using the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) method to extract the expression levels of all 2OGD family genes. The transcriptome screening heatmap is shown below. Figure 1As shown, the above-mentioned gene was further co-expressed with upstream genes of the vinblastine biosynthesis pathway identified through homology comparison with periwinkle, and candidate genes with FPKM>10 and co-expression correlation coefficient>0.8 were finally screened. Vinblastine synthase Vm15G000668 was obtained by this method.

[0047] The nucleotide sequence of vinblastine synthase Vm15G000668 is as follows:

[0048] SEQ ID No. 2.

[0049] The amino acid sequence of vinblastine synthase Vm15G000668 is as follows:

[0050] MDSEGIKLPIIDFTSPDLKPGTPIWDTVKLQVKKALEEFGCFEALFDKITKEIRKSLFEGLAELFDLPFETKMKNSSENRLTSYAGPKQYPMAPLFESMGVEEPTLLQNVESLSKLMWPQGNSAFSKTMQSYSEKLSELDKIVRRMVIESLGLEKYLDE HMNSTKHVLRILKYKGPQTSETEVGLFPHTDTGIISILHQNQVKGLEVQTKDGHWIRLNSSPNSFVVMIGNSLRAWTNGRLHAPYHRVMMSGNESRYSAALFTIPKDDYTIKVPDELVDEEHPLLFKPFDHMGFVAFSTSKPAQKGQDILKDYCGI, SEQ ID No.1.

[0051] Example 2

[0052] Amplification and purification of vinblastine synthase Vm15G000668

[0053] ①RNA extraction

[0054] 1. Material preparation: Select vigorous small-vine periwinkle plants, collect their leaf tissues, quick-freeze them with liquid nitrogen, grind them into powder, and use them for total RNA extraction.

[0055] 2. RNA extraction:

[0056] The extraction was performed using a plant total RNA extraction kit (TIANGEN, DP441). The specific steps are as follows:

[0057] (1) Weigh about 100 mg of periwinkle leaf tissue and grind it into powder in sufficient liquid nitrogen. Add 500 μL of lysis buffer SL (with added β-mercaptoethanol) and immediately vortex to mix.

[0058] (2) Centrifuge at 12000 rpm for 2 min.

[0059] (3) Transfer the supernatant to the filter column CS that has been placed in the collection tube, centrifuge at 12000 rpm for 2 min, and carefully aspirate the supernatant from the tube into a new RNase-Free centrifuge tube. Do not let the pipette tip touch the precipitate.

[0060] (4) Slowly add 200 μL of anhydrous ethanol and mix well. Transfer the solution and precipitate in the tube into the adsorption column CR3. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the tube, and return the adsorption column CR3 to the collection tube.

[0061] (5) Add 350 μL of RW1 (protein removal solution) to the adsorption column CR3, centrifuge at 12000 rpm for 15 seconds, and discard the waste liquid.

[0062] (6) Add 80 μL of DNase I working solution (prepared from 10 μL of DNase I stock solution and 70 μL of RDD buffer) to the center of the CR3 adsorption column and let it stand at room temperature for 15 min.

[0063] (7) Add 350 μL of RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 15 seconds, and discard the waste liquid.

[0064] (8) Add 500 μL of RW to the CR3 adsorption column, centrifuge at 12000 rpm for 15 seconds, and then discard the waste liquid. (Repeat step 8)

[0065] (9) After centrifuging at 12000 rpm for 2 min, the adsorption column CR3 was placed into a brand new RNase-Free centrifuge tube. 30 RNase-Free ddH2O (freshly sterilized) was added dropwise to the center of the adsorption membrane. The tube was left to stand at room temperature for 2 min, centrifuged at 12000 rpm for 1 min, and then the process was repeated once more to obtain the extracted RNA solution.

[0066] (10) Take 1 μL of the RNA solution obtained in the previous step and perform agarose gel electrophoresis to detect whether the RNA has been degraded. Use Nanadrop2000 to determine the ratio of OD260 / OD280 to detect the final RNA purity.

[0067] ②Reverse transcription to synthesize cDNA

[0068] The reaction system was prepared according to the instructions using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A), and the procedure was as follows:

[0069] 1. Mix1 reaction solution preparation (10 μL): dNTP Mixture 1 μL, Oligo dT Primer 1 μL, Total RNA 1 μg, RNase-free ddH2O up to 10 μL.

[0070] 2. Keep the metal bath at 65℃ for 5 minutes, then immediately place it on ice to cool.

[0071] 3. Preparation of Mix2 reaction solution (10μL): PrimeScript II RTase 1 μL, 5×PrimeScript II Buffer 4 μL, RNase Inhibitor 0.5 μL, RNase-free ddH2O 4.5 μL.

[0072] 4. Mix Mix1 and Mix2 reaction solutions thoroughly.

[0073] 5. After amplification by setting the PCR reaction at 42℃ for 60 min and 95℃ for 5 min, cool on ice and reverse transcribe to obtain cDNA solution, which can be stored at -20℃ or -80℃ for long-term storage.

[0074] ③ Target gene amplification

[0075] Based on the CDS sequence of the candidate vinblastine synthase Vm15G000668, homologous arm primers were designed using Snapgene 6.0.2. The vector used was PET28a, and the restriction sites were selected as 5' BamHI and 3' EocRI, as follows:

[0076] pET28a-Vm15G000668-F: 5'-cagcaaatgggtcgcggatccATGGATTCTGAAGGCATAAAGCTT-3', SEQ ID No. 3;

[0077] pET28a-Vm15G000668-R: 5'-ttgtcgacggagctcgaattcTTAGATGCCACAATAGTCCTTTAGAAT-3', SEQ ID No. 4.

[0078] After obtaining cDNA by reverse transcription of total RNA from periwinkle leaves, the target gene was amplified using KOD One™ PCR MasterMix (TOYOBO, KMM-101 / 201). The PCR reaction system was prepared as follows: 25 μL KOD One™ PCR Master Mix, 2 μL cDNA, 1.5 μL Reverse Primer (10 μM), 1.5 μL Forward Primer (10 μM), and up to 50 μL ddH2O.

[0079] PCR amplification reaction conditions: 94℃ for 2 min, 40 cycles (98℃ for 10 s, 60℃ for 5 s, 68℃ for 15 s), 72℃ for 1 min, 4℃ ∞.

[0080] After running the PCR product on a 1% TAE gel at 150V for 20 min, the portion containing the target gene band was excised and the target fragment was recovered.

[0081] ④ linearization of pET28a plasmid

[0082] The extracted pET28a plasmid was double-digested using Takara's Quickcut enzyme at the BamHI and EcoRI sites. The enzyme digestion preparation system consisted of: plasmid ≤8 μg, restriction enzyme 1 (BamHI) 2 μL, restriction enzyme 2 (EcoRI) 2 μL, reaction buffer (10×) 5 μL, and ddH2O up to 50 μL.

[0083] The reaction was first carried out at 30℃ for 0.5 h, and then at 37℃ for 0.5 h. The enzyme digestion products were detected by agarose gel electrophoresis, and the target band was recovered.

[0084] ⑤ Agarose gel electrophoresis and recovery

[0085] The PCR product and linearized vector were simultaneously run on an agarose gel (1% TAE) at 150V for 20 min. The band containing the target gene and the restriction enzyme vector was carefully cut out and recovered from the gel. The specific operation is as follows:

[0086] 1. Add 200 μL of PE solution to the cut gel block and mix the gel and sol liquid in a water bath at 25~30℃ for 5~10 min until it is uniform and free of residual gel blocks.

[0087] 2. Add the liquid from step 1 to the adsorption column, let stand for 2 minutes, centrifuge for 1 minute, and discard the waste liquid in the collection tube.

[0088] 3. Add 600 μL of PW washing buffer to the CA5 adsorption column, centrifuge for 1 min, and discard the waste liquid. Repeat step 3.

[0089] 4. Centrifuge for 2 minutes, then let stand at room temperature for 3-5 minutes to allow the PW rinsing solution to dry as much as possible.

[0090] 5. Add 30 μL of double-distilled water to the center of the adsorption membrane, let it stand at room temperature for 2 min, then centrifuge for 2 min. Collect the DNA solution into a clean centrifuge tube and repeat the above operation once.

[0091] 6. Take 1 μL for concentration detection (Nanodrop2000).

[0092] ⑥ Homologous recombination

[0093] The obtained insert fragment with homologous sequence was homologously recombinated with the enzyme-digested linearized vector using the ClonExpress® II One Step Cloning Kit (product code: C112) from Nanjing Vyzeme Biotechnology Co., Ltd. to obtain the expression vector. The operation steps are as follows:

[0094] 1. Prepare the reaction system in a low-temperature environment on ice: linearized vector X μL, insert fragment (target gene) Y μL, 5×CEⅡ buffer 4 μL, ExnaseⅡ 2 μL, ddH2OUp to 20 μL.

[0095] X (cloning vector usage) = [0.02 × number of cloning vector base pairs] ng;

[0096] Y (insertion fragment usage) = [0.04 × number of insertion fragment base pairs] ng.

[0097] 2. Use a micropipette to gently aspirate and blow into the tube to mix evenly, then centrifuge at low speed for a short time to collect all the liquid at the bottom of the centrifuge tube.

[0098] 3. Connect at 37℃ for 30 minutes; then cool to 4℃ or immediately place in an ice box to cool.

[0099] 4. Transformation of DH5α competent cells: This process uses DH5α competent cells (product code: ZC101) from Beijing Zhuangmeng International Biotechnology Co., Ltd. First, thaw 100 μL of competent cells stored at -80℃ on ice, add 10 μL of the recombinant plasmid obtained above, gently tap the outer wall of the centrifuge tube to mix, and incubate on ice for 30 min.

[0100] 5. Incubate in a 42℃ water bath for 90 seconds, then quickly transfer to ice and let stand for 2 minutes.

[0101] 6. Take 600 μL of antibiotic-free empty LB medium and activate it in a shaker set at 37℃ and 110 rpm for 50-60 min.

[0102] 7. Using a sterile spreader, spread 100 μL of bacterial culture evenly onto LB solid medium containing the corresponding antibiotic resistance, and incubate at 37°C for 12 h.

[0103] 8. Pick a single colony from the surface of the solid culture medium in the laminar flow hood and verify the transformation results using 2×Taq PCR Master Mix (product code: PC0901):

[0104] (1) Prepare the reaction system in a clean EP tube (200 μL): 5 μL of 2×Taq PCR Master Mix, 0.2 μL of Reverse Primer (10 μM), 0.2 μL of Forward Primer (10 μM), and 4.6 μL of ddH2O.

[0105] (2) Select single colonies of Escherichia coli after transformation culture (10 single colonies were selected from each solid culture medium for PCR verification experiment). The PCR reaction program was 94℃ for 3 min; 30 cycles: 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2.5 min; 72℃ for 5 min, 4℃ for ∞.

[0106] (3) After agarose gel electrophoresis, three positive clones were selected, cultured and plasmids were extracted and sent for Sanger sequencing.

[0107] ⑦ Expression and purification of recombinant vinblastine synthase

[0108] 1. Transform the correctly sequenced recombinant expression vector containing the target gene and the empty vector into competent cells of expression strain BL21(DE3), respectively, plate them on LB agar containing kanamycin, and incubate overnight at 37°C. Pick single colonies and screen positive clones by colony PCR to verify that the target gene is correctly inserted into the expression vector.

[0109] 2. Pick the transformed single clones from step 1 and put them into 5 mL of LB liquid medium. Place them in a constant temperature shaking incubator set at 37℃ and 200 rpm for 12 h.

[0110] 3. Take 500 μL of the pre-cultured bacterial solution from step 2 into 50 mL of freshly prepared culture medium (diluted 100 times), set the parameters to 37℃ and 200 rpm, and incubate for 4-5 h; use an ELISA reader to measure the OD600 value. When the result is 0.6-0.8, add IPTG (final concentration 0.5 mM), and then place it in a constant temperature shaker at 16℃ and 110 rpm for induction expression.

[0111] 4. Protein purification: Recombinant vinblastine synthase in the supernatant was purified using a Ni-NTA affinity chromatography column. The protein was washed with a gradient of TBS buffers containing 20 mM, 50 mM, and 100 mM imidazole, followed by elution with TBS buffer containing 250 mM imidazole. The corresponding protein solutions were collected, and protein purity was verified by SDS-PAGE electrophoresis. Figure 2 We obtained high-purity recombinant vinblastine synthase.

[0112] Example 3

[0113] catalytic activity verification

[0114] Reaction system construction: In a 200 μL reaction system, add 250 mM Tris-HCl buffer (pH 7.5), 0.5 mM metformin (substrate), 1 mM 2-oxoglutaric acid, 1 mM FeSO4, 1 mM NADPH, and 7.5 mM ascorbic acid (Fe2+). 2+ It is the core metal cofactor of the 2-OGDD family of enzymes. 250mM Tris-HCl is used to stabilize pH. NADPH, as a reducing coenzyme, is a key reducing equivalent donor in this system. Ascorbic acid is a classic antioxidant additive for this type of enzymatic reaction. 2-OG provides reactive oxygen species and reducing equivalents for the oxidation of the substrate.

[0115] The four processing groups are set up as follows:

[0116] ① Add 100 μL of the supernatant (vincible amine synthase before purification) obtained from PET28a-Vm15G000668 after induction and lysis in step 3 of Example 2 to the above reaction system;

[0117] ② Add 10 μg of the purified recombinant vinblastine synthase obtained in step 4 of Example 2 to the above reaction system;

[0118] ③ No enzymes are added to the above reaction system;

[0119] ④ Add 100 μL of the supernatant of PET28a after unloaded induced fragmentation prepared in Example 2 to the above reaction system.

[0120] Reaction conditions: Incubate the reaction system in a shaker at 30°C for 2 hours, add 200 μL of methanol to terminate the reaction, centrifuge at 12000 rpm for 3 minutes, and collect the supernatant.

[0121] The reaction product was detected using liquid chromatography-mass spectrometry (LC-MS / MS), with a standard vinblastine sample as a control. The product was confirmed as vinblastine by retention time and mass spectrometry peaks. Specific parameters are as follows:

[0122] Separation was performed using a Waters ultra-high performance liquid chromatography (UHPLC) column (1.7 μm particle size, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B) at a flow rate of 300 μL / min. The gradient elution conditions were set as follows: 0–3 min, 5% phase B; 3–12 min, phase B linearly increased from 5% to 30%; 12–15 min, phase B linearly increased from 30% to 95%; 15–18 min, phase B remained at 95%; 18–21 min, phase B linearly decreased from 95% to 5%; 21–24 min, phase B remained at 5%. Mass spectrometry parameters in electrospray ionization (ESI) mode were set as follows: sheath gas pressure 50 psi, auxiliary gas pressure 10 psi, purge gas pressure 0 psi; ion transfer tube temperature 320 °C, vaporization chamber temperature 320 °C; positive ion spray voltage 3.5 kV. The analysis was performed using XCalibur™ software (version 4.4.16.14).

[0123] LC-MS / MS results are as follows Figure 3 As shown ( Figure 3 From top to bottom: Treatment ①, Treatment ②, Vinpocetine standard, Treatment ③, Treatment ④ (LC-MS / MS results). Secondary ion fragments of the product in the catalytic system of Treatment ② are shown below. Figure 4 As shown. Secondary ion fragments of vinblastine standard, such as... Figure 5 As shown. Figure 3 The results of treatment ② show two peaks, indicating the formation of two compounds with a mass of 355.20. The first peak (retention time 12.41 min) has a retention time close to that of the peak corresponding to the vinblastine standard (12.33 min), and the secondary ion fragments show similar characteristics. Figure 4 , Figure 5 The enzyme-catalyzed product has the same fragments as the vinblastine standard, which proves that the first compound in the doublet is vinblastine. The second compound should be an epimer of it.

[0124] The synthetic pathway reaction formula is as follows:

[0125]

[0126] Example 4

[0127] Investigating the optimal temperature for enzyme-catalyzed reactions

[0128] In a catalytic reaction using metformin as a substrate, the pH of the catalytic reaction buffer was kept constant at 7.0, and the enzyme's catalytic activity was tested by reacting at different temperatures (20, 25, 30, 35, 40, 45, 50℃) for 2 h. The sample loading system is as follows:

[0129] Table 1. Sampling System

[0130]

[0131] After the reaction is complete, add an equal volume of methanol solution to terminate the reaction, filter and load the sample.

[0132] The reaction products were detected using liquid chromatography-mass spectrometry (LC-MS / MS). The peak areas of the products under different experimental conditions were normalized to the maximum peak area among all samples, thereby calculating and characterizing the relative enzyme activity.

[0133] The results are shown in the table below.

[0134] Table 2 Effect of temperature on relative enzyme activity

[0135]

[0136] This invention uses metformin as a reaction substrate to determine the catalytic activity of vinblastine synthase Vm15G000668 in the temperature range of 20-50℃. The results show that when the pH of the enzymatic reaction with Vm15G000668 is fixed at 7.0, the catalytic activity of the enzyme gradually increases with increasing reaction temperature, starting from 20℃. The catalytic activity reaches its maximum at 35℃, and decreases with further increases in reaction temperature.

[0137] Example 5

[0138] Investigating the optimal pH for enzyme-catalyzed reactions

[0139] In the catalytic reaction using vinblastine as a substrate, the immobilized enzyme catalytic reaction temperature was 35℃. After reacting for 2 h in different pH buffer systems (3.0, 4.0, 5.0, (citric acid-sodium citrate buffer), 6.0, 7.0, 8.0, 9.0 (Tris-HCl)), the sample was treated using the above method, and the content of vinblastine generated in the catalytic reaction product was detected by LC-MS / MS to obtain the optimal reaction pH. The reaction system is as follows:

[0140] Table 3 Reaction System

[0141]

[0142] After the reaction is complete, add an equal volume of methanol solution to terminate the reaction, filter and load the sample.

[0143] The detection and data processing methods are the same as above.

[0144] The results are shown in the table below.

[0145] Table 4 Effect of pH on relative enzyme activity

[0146]

[0147] This invention uses metformin as a reaction substrate to determine the catalytic activity of vinblastine synthase Vm15G000668 in the pH range of 3.0–9.0. The results show that when the reaction temperature of vinblastine synthase Vm15G000668 is fixed at 35°C, the catalytic activity of the enzyme gradually increases with increasing pH, starting from pH 3.0. The catalytic activity reaches its maximum at pH 7.0, and decreases with further increases in pH.

[0148] Example 6

[0149] Enzyme kinetic parameter determination

[0150] Plot a standard curve with peak area as the y-axis and vinblastine concentration as the x-axis, as shown below. Figure 6 As shown. Used for subsequent calculations of vinblastine yield.

[0151] Under optimal temperature and pH conditions, with other components fixed, the experimental method was the same as above. The yield of vinblastine produced from different concentrations of metformin (0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0 mM) catalyzed by vinblastine synthase Vm15G000668 was detected by LC-MS. The reaction system is as follows:

[0152] Table 5 Reaction System

[0153]

[0154] After the reaction was complete, an equal volume of methanol solution was added to terminate the reaction. The mixture was then evaporated to dryness under nitrogen, reconstituted with 250 μL of methanol, filtered, and loaded onto the sample.

[0155] The original peak area results are shown in Table 6.

[0156] Table 6 Original Peak Area Results

[0157]

[0158] Substitute the original peak area value into Figure 6 The standard curve shown provides the yield of vinblastine at different substrate concentrations. The rate of vinblastine formation (nmol·min⁻¹) can then be calculated from the product yield / time. - ¹·mg - ¹), the results are as follows.

[0159] Table 7 Reaction rate results (nmol·min) - ¹·mg - ¹)

[0160]

[0161] Nonlinear fitting was performed using the Michaelis-Menten equation kinetics in GraphPad Prism 8 software to calculate the values ​​of Km and Vmax (with the rate of vinblastine generation as the y-axis and the concentration of vinblastine metformin as the x-axis).

[0162] The results are as follows Figure 7 As shown in Table 8.

[0163] Table 8 Enzyme kinetic parameters

[0164]

[0165] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0166] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A longifolene synthase, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the vincamine synthase according to claim 1, characterized in that, Its nucleotide sequence is shown in (a), (b), or (c): (a) The nucleotide sequence shown in SEQ ID No. 2; Or (b) a nucleotide sequence that can hybridize with the complementary nucleotide sequence of SEQ ID No. 2 under stringent hybridization conditions, wherein the protein encoded by the nucleotide still has the function or activity of vinblastine synthase; Or (c) a nucleotide sequence that has at least 80% homology with the nucleotide sequence of SEQ ID No. 2, and the protein encoded by the nucleotide still has the function or activity of vinblastine synthase.

3. A chimeric gene or expression cassette containing the encoding gene of claim 2.

4. A recombinant expression vector containing the coding gene of claim 2 or the chimeric gene or expression cassette of claim 3.

5. A recombinant host cell containing the recombinant expression vector of claim 4.

6. The use of the vinblastine synthase according to claim 1 as a catalytic enzyme in the biosynthesis of vinblastine.

7. Use according to claim 6, wherein The substrate was metformin.

8. The use of the encoding gene of claim 2, the chimeric gene of claim 3, or the expression cassette in the biosynthesis of vinblastine.

9. A method for in vitro synthesis of vinblastine, characterized in that, The catalytic reaction is carried out using the vincamine synthase of claim 1 as catalyst, with vincaminedimethylglycinate as substrate, in the presence of 2-oxoglutaric acid, Fe 2+ , ascorbic acid and NaDPH.

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