Cannabinoid phenolic acid synthase mutant and application thereof

Abstract

The invention discloses a cannabinoid phenolic acid synthase mutant and application thereof, and relates to the technical field of biology. The amino acid sequence of the cannabinoid phenolic acid synthase mutant is as shown in SEQ ID NO. 4 or SEQ ID NO. 6. Amino acid mutation sites capable of improving the enzyme activity are screened out through a bioinformatics method, the cannabinoid phenolic acid synthase mutants CBCASQ106Y + E534R and CBCASQ124L + E534K are designed, and the conversion efficiency of catalyzing cannabinoid phenolic acid to generate cannabinoid phenolic acid is remarkably improved through the cannabinoid phenolic acid synthase mutants CBCASQ106Y + E534R and CBCASQ124L + E534K. The invention lays a foundation for industrial production and application of the cannabinoid phenolic acid synthetase mutant and improvement of the yield of biosynthesized cannabinoid phenol.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a cannabichromenic acid synthase mutant and an application thereof. Background Art

[0002] Cannabinoids are a type of secondary metabolites unique to cannabis and are also the main active substances in cannabis. Cannabinoids are widely used in food, medicine, and medical industries because they can alleviate the symptoms of certain diseases, such as epilepsy, chronic pain, and inflammation. Among the more than 140 cannabinoids that have been discovered, cannabichromene (CBC), which has the third highest content, has attracted attention due to its medical potential and lack of psychoactive properties. Studies have shown that CBC is more effective in anti-anxiety than cannabidiol (CBD), and CBC has the potential to work synergistically with other cannabinoids. Studies have shown that CBC has a stronger analgesic effect when acting together with Δ9-tetrahydrocannabinol (THC) than when THC acts alone.

[0003] The directed evolution technology of enzymes refers to the introduction of mutations into the amino acid sequence that makes up the protein and the screening of mutants to obtain mutants that meet expectations, such as improved enzyme activity, improved substrate specificity, and improved enzyme thermal stability. Molecular docking refers to the technology of combining two or more molecules from the perspectives of spatial conformation and energy complementarity through computer simulation. In the process of docking enzymes and substrate molecules, it can be analyzed which amino acids can be changed to enhance the force of the enzyme-substrate docking site and reduce the steric hindrance effect during the substrate docking process, thereby enhancing the affinity of the enzyme-substrate binding. Generally speaking, enzymes with better activity have stronger affinity with the corresponding substrates.

[0004] The Pichia pastoris expression system has become a widely used eukaryotic expression system for exogenous protein expression due to its low culture cost, short induction cycle, high protein expression level, no inclusion body formation, and protein post-translational modification. Currently, more than a thousand heterologous proteins have been successfully expressed in Pichia pastoris.

[0005] At present, most of the domestic extraction of cannabis is to extract cannabidiol acid (CBDA). At this time, there are still many components in the waste after extraction, including a certain amount of cannabidiol acid (CBGA), and CBGA can generate cannabidiol acid (CBCA) under the action of cannabidiol acid synthase (CBCAS). CBCA will be converted into CBC under natural conditions. If this part of CBGA can be used to synthesize CBC in a directed manner, the utilization value of cannabis by-products when extracting CBDA can be effectively improved. CBCAS enzyme has been successfully expressed in the Pichia pastoris expression system, but its low catalytic activity limits the generation of CBC. Therefore, it is necessary to develop a highly active CBCAS enzyme to increase the yield of CBC. Summary of the invention

[0006] The object of the present invention is to provide a cannabigerolic acid synthase mutant and its application to solve the problems existing in the above-mentioned prior art. The cannabigerolic acid synthase mutant provided by the present invention can effectively improve the conversion efficiency of cannabigerol acid to cannabigerolic acid.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a cannabigerolic acid synthase mutant, and the amino acid sequence of the cannabigerolic acid synthase mutant is as shown in SEQ ID NO.4 or SEQ ID NO.6.

[0009] The present invention also provides a coding gene for the above-mentioned cannabigerolic acid synthase mutant.

[0010] Further, when the amino acid sequence of the cannabigerolic acid synthase mutant is as shown in SEQ ID NO.4, the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3;

[0011] When the amino acid sequence of the cannabigerolic acid synthase mutant is as shown in SEQ ID NO.6, the nucleotide sequence of the coding gene is as shown in SEQ ID NO.5.

[0012] The present invention also provides a recombinant expression vector, including the above-mentioned coding gene.

[0013] The present invention also provides a recombinant microbial strain, including the above-mentioned recombinant expression vector.

[0014] Further, the recombinant microbial strain is recombinant Pichia pastoris.

[0015] The present invention also provides the application of the above-mentioned coding gene, recombinant expression vector or recombinant microbial strain in the preparation of a cannabigerolic acid synthase mutant.

[0016] The present invention also provides a method for preparing a cannabigerolic acid synthase mutant, including the steps of fermenting and culturing the above-mentioned recombinant microbial strain to obtain bacterial cells, and then performing cell wall breaking and extraction treatment to prepare the cannabigerolic acid synthase mutant.

[0017] The present invention also provides the application of the above-mentioned cannabigerolic acid synthase mutant in improving the conversion efficiency of cannabigerol acid to cannabigerolic acid.

[0018] The present invention also provides a method for increasing the yield of cannabigerol extracted from cannabis plants, including the steps of using the above-mentioned cannabigerolic acid synthase mutant to catalyze cannabigerol acid in cannabis plants to generate cannabigerolic acid, and then generating cannabigerol through a decarboxylation reaction.

[0019] The present invention discloses the following technical effects:

[0020] By using bioinformatics methods, the present invention screened out amino acid mutation sites that can increase enzyme activity and designed a mutant of cannabinolic acid synthase, CBCAS Q106Y+E534R and CBCAS Q124L+E534K , and the conversion efficiency of catalyzing cannabinolic acid to form cannabigerolic acid was significantly improved. The present invention lays a foundation for the industrial production and application of the mutant of cannabinolic acid synthase and for increasing the yield of biosynthetic cannabigerolic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a schematic diagram of the tertiary structure of CBCAS and the docking sites and interaction forces between CBCAS and substrate molecules; wherein, A: the tertiary structure of the CBCAS protein; B: the two-dimensional plan view of the docking sites and interaction forces between CBCAS and substrate molecules; C: the pymol visualization schematic diagram of the docking sites and interaction forces between CBCAS and substrate molecules;

[0023] Figure 2 It is a schematic diagram of the docking sites and interaction forces between wild-type CBCAS and mutants and substrate molecules;

[0024] Figure 3 It is the detection result of yeast colony PCR after introducing the CBCAS mutant; wherein, M: DS2000 Marker; 1: positive control with the pPIC9K-CBCAS plasmid as the template; 2: negative control without adding template DNA; 3: mutant CBCAS Q106Y +E534R ; 4: mutant CBCAS Q124L+E534K ;

[0025] Figure 4 It is the result diagram of Western blot for verifying the protein expression of the mutant; wherein, M: colored prestained protein Marker; 1: negative control with the pPIC9K plasmid as the template; 2: mutant CBCAS Q106Y+E534R ; 3: mutant CBCAS Q124L +E534K ;

[0026] Figure 5It is a result diagram for the determination of CBCAS enzyme activity; among them, pPIC9K: the control group of the crude protein extract transfected with the empty vector pPIC9K; WT: the control group adding wild-type CBCAS; Q106Y+E534R: mutant CBCAS Q106Y+E534R ; Q124L+E534K: mutant CBCAS Q124L+E534K . Detailed implementation manners

[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0031] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, meaning including but not limited to.

[0032] Example 1

[0033] 1. Materials and methods

[0034] 1.1 Materials and instruments

[0035] The culture media related to the present invention are as follows:

[0036] LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 5 g / L sodium chloride, and 12 g / L agar;

[0037] MD medium: 20 g / L agar, 20 g / L glucose, 13.4 g / L nitrogen-free nitrogen source, and 4×10 -4 mL / L;

[0038] BMGY medium: 20 g / L tryptone, 10 g / L yeast extract powder, 3.94 g / L dipotassium hydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 20 mL / L glycerol, 13.4 g / L nitrogen-free nitrogen source, and 4×10 -4 mL / L.

[0039] BMMY medium: 20 g / L tryptone, 10 g / L yeast extract powder, 3.94 g / L dipotassium hydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 13.4 g / L nitrogen-free nitrogen source, and 4×10 -4 mL / L.

[0040] Note: The Amp antibiotic was prepared into a 50 mg / mL aqueous solution, filtered and sterilized, and then added to the sterilized LB medium at a volume ratio of 1:1000.

[0041] The vector pPIC9K used in this experiment was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the Pichia pastoris competent cells were Pichia pastoris GS115, purchased from Shanghai Angyu Biotechnology Co., Ltd.; the cannabis materials were provided by the Economic Crop Research Institute of Heilongjiang Academy of Agricultural Sciences; all primer syntheses and sequencing were completed by Beijing Tsingke Biotechnology Co., Ltd.; the content of CBC in this experiment was detected using an Agilent high-performance liquid chromatography analyzer, and the liquid chromatography conditions were as follows: chromatographic column: Shimadzu sil-16C18 column (150 mm×4.6 mm×3 μm), column temperature: 30°C; mobile phase: A was an aqueous solution containing 0.1% formic acid, B was an acetonitrile solution containing 0.1% formic acid; isocratic elution: 25% A, 75% B, retention time was 20 min; ultraviolet detector: 230 nm; flow rate: 0.7 mL / min; injection volume: 10 μL.

[0042] The nucleotide sequence of the wild-type CBCAS gene is shown in SEQ ID NO.1; the amino acid sequence of the wild-type CBCAS protein is shown in SEQ ID NO.2.

[0043] 1.2 Experimental methods

[0044] 1.2.1 Protein preparation and molecular docking

[0045] The amino acid sequence of CBCAS was input into the AlphaFold2 website (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb) to predict the tertiary structure of CBCAS. The Model1 with the highest AlphaFold2 score was selected for model quality assessment using three model quality assessment software provided by ModelQualityAssessment programs (MQAPS) SAVES v6.1. The three-dimensional structure of CBGA was obtained from PubChem (PubChem CID: 6449999). After preparing the above three-dimensional structures, molecular docking was performed:

[0046] (1) Use the Discovery Studio 2019 software to first preprocess the three-dimensional structures of protein Model1 and CBGA, such as removing water molecules and adding hydrogen, and define them as the receptor and ligand respectively.

[0047] (2) Define the binding site for the receptor based on the protein cavity, select the optimal result, click the CDOCK ER docking method in the molecular docking module, and set the RMSD threshold to to ensure that the docking conformations are as diverse as possible.

[0048] (3) After the operation is completed, display the relevant information such as the types and distances of intermolecular interactions between the receptor and the ligand, generate a two-dimensional plan view, and finally draw the graph using PyMOL.

[0049] 1.2.2 Virtual Amino Acid Mutation Site Screening

[0050] To obtain the most reasonable mutation strategy, among the 20 common amino acids, the most suitable mutation combination was obtained based on the interaction between the amino acid residue at the mutation site and the substrate.

[0051] (1) Import the docking result with the best conformation in the Discovery Studio 2019 software, analyze the intermolecular forces between the receptor and the ligand, and first remove the water molecules.

[0052] (2) Expand Macromolecules - Prepare Protein, and click Clean Protein to preprocess the protein structure.

[0053] (3) Expand Simulation - Change Forcefield, click Apply Forcefield, and assign the CHARMm force field to the protein.

[0054] (4) After the above treatment, all amino acids within the range of the selected ligand are used as subsequent mutation sites.

[0055] (5) Click on the Calculate Mutation Energy (Binding) module to perform virtual amino acid mutations on the protein-ligand complex based on the interaction forces, determine the key amino acids in the active site, and the amino acids that can improve the affinity as the mutation targets, and use the Predict Stabilizing Mutations module to predict the best amino acid mutation combinations.

[0056] (6) Re-simulate the obtained mutants CBCAS Q106Y+E534R and CBCAS Q124L+E534K proteins, and then perform molecular docking again. Use the PyMOL software to observe the enzyme structure.

[0057] 1.2.3 Obtaining the mutant sequences

[0058] Using the CBCAS sequence (SEQ ID NO.1) optimized according to the codon preference of Pichia pastoris as a template, the mutant DNA fragments were obtained by the overlap extension PCR technique. The specific method is as follows: Design specific primers 1 and 4 (see Table 1) using the Primer 3plus online website. Add 10 μL of 5×SF Buffer, 1 μL of dNTPs, 2 μL of each of primers 1 and 4, 2 μL of cannabis genomic DNA, and make up the volume to 50 μL with deionized water. Add 1 μL of Phanta Super-Fidelity DNA polymerase for amplification. PCR amplification program: Pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 10 s, annealing at 56 °C for 30 s, extension at 72 °C for 2 min, for a total of 32 cycles; extension at 72 °C for 5 min, and store at 4 °C. After the PCR products were electrophoresed on a 1% agarose gel, the gel at the position of the target band was cut off and purified according to the instructions of the gel extraction kit to obtain the Q106Y-1 fragment. Similarly, use primers 2 and 3 to amplify the Q106Y-2 fragment with the CBCAS sequence as a template. Using the Q106Y-1 and Q106Y-2 fragments as templates, amplify with primers 1 and 2 to obtain the Q106Y single mutant fragment. Then, using the Q106Y single mutant fragment as a template, use primers 1 and 6, and primers 2 and 5 for amplification respectively to obtain the Q106Y+E534R-1 and Q106Y+E534R-2 fragments. Use primers 1 and 2 for overlap extension PCR with these two fragments as templates, and after purification, obtain the mutant CBCAS Q106Y+E534R .

[0059] Using the same method, the CBCAS sequence was amplified with high fidelity using primer 1 and primer 8, and primer 2 and primer 7 to obtain Q124L-1 and Q124L-2. After purification by overlap extension PCR using primer 1 and primer 2, the Q124L single mutant fragment was obtained. The Q124L fragment was amplified using primer 1 and primer 10, and primer 2 and primer 9 to obtain the Q124L+E534K-1 and Q124L+E534K-2 fragments. Using these two fragments as templates, overlap extension PCR was performed using primer 1 and primer 2, and after purification, the mutant CBCAS was obtained. Q124L+E534K fragment.

[0060] The primers used for vector construction and identification in the present invention are shown in Table 1.

[0061] Table 1 Primers used for vector construction and identification

[0062]

[0063] Note: The underlines are the adapters of the primers.

[0064] 1.2.4 Construction and transformation of mutant yeast expression vector

[0065] The empty vector pPIC9K and the mutant CBCAS were double digested with the restriction enzymes EcoR I and Not I. After electrophoresis of the digestion products on a 1% agarose gel, the gel at the position of the target band was cut off and gel recovery was performed according to the instructions of the gel recovery kit. The digestion products of the empty vector pPIC9K and the mutant CBCAS were ligated using T4 DNA ligase at a mass ratio of 1:5. 10 μL of the ligation product was transferred into competent Escherichia coli DH5α cells by heat shock method. Positive screening was performed using LB solid medium containing ampicillin, and the positive monoclonal colonies screened were subjected to colony PCR and sequencing verification to determine that the recombinant plasmid pPIC9K-CBCAS was successfully constructed. The pPIC9K-CBCAS plasmid was constructed using the same method. Q106Y+E534R After electrophoresis of the digestion products on a 1% agarose gel, the gel at the position of the target band was cut off and gel recovery was performed according to the instructions of the gel recovery kit. The digestion products of the empty vector pPIC9K and the mutant CBCAS were ligated using T4 DNA ligase at a mass ratio of 1:5. 10 μL of the ligation product was transferred into competent Escherichia coli DH5α cells by heat shock method. Positive screening was performed using LB solid medium containing ampicillin, and the positive monoclonal colonies screened were subjected to colony PCR and sequencing verification to determine that the recombinant plasmid pPIC9K-CBCAS was successfully constructed. Q106Y+E534R After electrophoresis of the digestion products on a 1% agarose gel, the gel at the position of the target band was cut off and gel recovery was performed according to the instructions of the gel recovery kit. The digestion products of the empty vector pPIC9K and the mutant CBCAS were ligated using T4 DNA ligase at a mass ratio of 1:5. 10 μL of the ligation product was transferred into competent Escherichia coli DH5α cells by heat shock method. Positive screening was performed using LB solid medium containing ampicillin, and the positive monoclonal colonies screened were subjected to colony PCR and sequencing verification to determine that the recombinant plasmid pPIC9K-CBCAS was successfully constructed. Q106Y+E534R After electrophoresis of the digestion products on a 1% agarose gel, the gel at the position of the target band was cut off and gel recovery was performed according to the instructions of the gel recovery kit. The digestion products of the empty vector pPIC9K and the mutant CBCAS were ligated using T4 DNA ligase at a mass ratio of 1:5. 10 μL of the ligation product was transferred into competent Escherichia coli DH5α cells by heat shock method. Positive screening was performed using LB solid medium containing ampicillin, and the positive monoclonal colonies screened were subjected to colony PCR and sequencing verification to determine that the recombinant plasmid pPIC9K-CBCAS was successfully constructed. Q124L +E534K plasmid.

[0066] The vector pPIC9K-CBCAS was digested with the restriction enzyme Sac I. Q106Y+E534RLinearization: Add 1 μg of linearized recombinant plasmid into 100 μL of Pichia pastoris competent cells, gently pipette to mix well, transfer it into an electroporation cuvette, incubate on ice for 5 min, then perform electroporation at 1500 V and 5 ms using an electroporator. Immediately after electroporation, add 600 μL of pre-cooled 1 M sorbitol, mix well, and incubate statically at 30 °C for 1 h. Centrifuge at 5000 rcf at room temperature for 2 min, and retain 500 μL of the supernatant to resuspend the cells. Take 200 μL of the cell suspension and spread it on an MD plate, then incubate it upside down at 30 °C for 3 d. Rinse the colonies on the plate with 5 mL of deionized water, take 200 μL of the cell suspension and spread it successively on YPD solid plates containing 2 mg / mL, 4 mg / mL, and 6 mg / mL G418, and incubate upside down at 30 °C for 5 d to screen for high-copy yeast recombinants. Pick single yeast colonies on the G418 medium with the highest concentration, break the yeast cell walls using the high-temperature ethyl acetate method, and perform PCR verification and sequencing verification on the broken yeast cells to determine the recombinant plasmid pPIC9K-CBCAS Q106Y+E534R has been successfully transferred into Pichia pastoris cells. Obtain high-copy pPIC9K-CBCAS Q124L+E534K recombinant bacteria using the same method.

[0067] 1.2.5 Induced expression of recombinant protein and Western Blot verification

[0068] Pick the recombinant bacteria pPIC9K-CBCAS Q106Y+E534R and recombinant bacteria pPIC9K-CBCAS Q124L+E534K that are verified as positive on the YPD plate and add them respectively into 3 mL of YPD liquid medium, and incubate overnight at 30 °C and 230 rpm. Add the activated cell suspension into 10 mL of BMGY medium at a volume ratio of 1:100, and incubate at 30 °C and 230 rpm for about 24 h until the OD 600 of the cell suspension is 2 - 6. Centrifuge at 4 °C for 10 min to collect the cell precipitate, resuspend the cells with sterile water, centrifuge at 4 °C for 5 min to remove the supernatant, resuspend the cell precipitate with BMMY medium until the OD of the cell suspension is 1.0, then induce the expression of the recombinant bacteria at 30 °C and 230 r / min, supplement 1% (v / v) methanol every 24 h, and continuously induce for 72 h, with 3 replicates for each group. Add 20 mL of cell lysis buffer to the cell precipitate after induced expression, ultrasonically disrupt for 30 min, centrifuge at 4 °C for 10 min, and take the supernatant, which is the crude extract and is reserved for use.

[0069] The mutant protein was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane using the "sandwich method" for semi-dry transfer. Block with skim milk for 2 h and rinse the surface of the PVDF membrane 8 times with TBST (5 min each time). Transfer the PVDF membrane to TBST containing His antibody (1:5000) and incubate overnight at 4 °C. After shaking and washing 6 times with TBST (5 min each time), transfer it to TBST containing HRP-labeled secondary antibody (1:4000) and incubate at room temperature for 2 h. Shake and wash 8 times with TBST (5 min each time). Finally, detect the bands using an ECL hypersensitive chemiluminescence detection kit and an imaging analyzer.

[0070] 1.2.6 Determination of recombinant protease activity

[0071] Take 0.1 g of hemp leaves with high CBGA content, add 50 mL of methanol and ultrasonically disrupt for 30 min. Detect by high performance liquid chromatography to obtain a crude extract of CBGA at 32.8 μg / mL and store at -20 °C for later use.

[0072] Wild-type CBCAS, mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K Preparation of protein concentrates: Take 20 mL of the crude extracts of wild-type CBCAS, mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K that have been ultrasonically disrupted (the preparation method is the same as 1.2.5), add them to a 30 kD protein ultrafiltration tube, centrifuge at 4 °C and 5000 rpm until there is approximately 500 μL of liquid remaining in the upper tube. Collect the crude extract in the upper tube, detect the concentration using a BCA protein concentration kit, and store at -20 °C for later use.

[0073] Take 50 μg of the protein concentrates of wild-type CBCAS, mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K respectively. Use 50 μL of the crude extract of hemp leaves with high CBGA as the substrate, supplement to 500 μL with 0.1 M citric acid-sodium citrate buffer (pH 5.0), let stand at 30 °C for 12 h, then add 600 μL of methanol to terminate the reaction, invert and mix well, centrifuge at 10000 rcf for 3 min, take the supernatant and filter through a 0.22 μm filter membrane, and use a high performance liquid chromatograph to detect the amount of CBC generated. Each group has three replicates.

[0074] 2 Results and discussion

[0075] 2.1 Protein preparation and molecular docking

[0076] The tertiary structure of CBCAS predicted by AlphaFold2 is as Figure 1As shown. The proteins CBCAS and CBGA were subjected to molecular docking using Discovery Studio 2019 software, and the molecular docking results were plotted using pymol software.

[0077] 2.2 Virtual Amino Acid Mutation Site Screening

[0078] Using Discovery Studio 2019 software to analyze the key amino acid sites and predict the combined key amino acid site mutations, two mutants with lower mutation energy after combination mutations were obtained: mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K . After predicting the tertiary structure of these two mutants using AlphaFold2, they were redocked with the substrate CBGA ( Figure 2 ). From the docking results, it can be seen that in mutant CBCAS Q106Y+E534R , after the glutamine (Gln) at position 106 mutates to tyrosine (Tyr), the interaction between the amino acid at position 106 and the substrate CBGA changes from a hydrogen bond (Conventional HydrogenBond) to an attractive charge interaction (Attractive Charge). After the glutamate (Glu) at position 534 mutates to arginine (Arg), the interaction between position 534 and the substrate increases hydrogen bonds and hydrophobic interactions (π-alkyl, Pi-Alkyl) in addition to the original attractive charge interaction; in mutant CBCAS Q124L+E534K , after the glutamine (Gln) at position 124 mutates to leucine (Leu), the interaction between the amino acid at position 124 and the substrate CBGA changes from a hydrogen bond to a hydrophobic interaction. After the glutamate at position 534 mutates to lysine (Lys), the interaction between position 534 and the substrate changes from the original attractive charge interaction to two hydrophobic interactions. It is speculated that due to the increase in the interaction sites between the enzyme and the substrate, the affinity between the enzyme and the substrate will be enhanced, further enhancing the enzyme activity.

[0079] 2.3 Obtaining CBCAS Mutant Fragments

[0080] Using the wild-type CBCAS gene as a template, the mutants CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K were amplified by overlapping extension PCR. After sequencing, it was confirmed that the amplification of the two mutant fragments was successful. The sequences are as follows, where the underlined positions in the sequences are the amino acid changes corresponding to the base mutations:

[0081] CBCAS Q106Y+E534R Nucleotide sequence of the gene (SEQ ID NO.3):

[0082] ATGAACTGCTCTACCTTCTCCTTCTGGTTCGTCTGTAAGATTATCTTCTTCTTCCTGTCCTTCAACATCCAAATTAGTATCGCTAACCCACAAGAAAACTTCTTGAAATGCTTCTCTGAGTACATCCCAAACAACCCTGCTAATCCAAAGTTCATCTATACTCAACACGACCAATTGTACATGTCTGTTCTTAACTCTACCATCCAAAACTTGAGATTCATCTCTGATACCACTCCAAAACCTCTTGTTATTGTTACTCCATCTAACAACTCCCATATCCAAGCTACTATTCTGTGTTCTAAGAAGGTTGGTCTT T A CAG ACAATCTATCCCTCCATTGCCACCTAGACATCATCATCATCACCATCACCATTAA。

[0083] CBCAS Q106Y+E534R Amino acid sequence of the mutant (SEQ ID NO.4):

[0084] MNCSTFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPANPKFIYTQHDQLYMSVLNSTIQNLRFISDTTPKPLVIVTPSNNSHIQATILCSKKVGL Y IRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEMNENFSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVVVPSKATIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLMLTTHFRTRNITDNHGKNKTTVHGYFSSIFLGGVDSLVDLMNKSFPELGIKKTDCKELSWIDTTIFYSGVVNYNTANFKKEILLDRSAGKKTAFSIKLDYVKKLIPETAMVKILEKLYEEEVGVGMYVLYPYGGIMDEISESAIPFPHRAGIMYELWYTATWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLGKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPNNFFRN R QSIPPLPPRHH。

[0085] CBCAS Q124L+E534K Nucleotide sequence of the gene (SEQ ID NO.5):

[0086] ATGAACTGCTCTACCTTCTCCTTCTGGTTCGTCTGTAAGATTATCTTCTTCTTCCTGTCCTTCAACATCCAAATTAGTATCGCTAACCCACAAGAAAACTTCTTGAAATGCTTCTCTGAGTACATCCCAAACAACCCTGCTAATCCAAAGTTCATCTATACTCAACACGACCAATTGTACATGTCTGTTCTTAACTCTACCATCCAAAACTTGAGATTCATCTCTGATACCACTCCAAAACCTCTTGTTATTGTTACTCCATCTAACAACTCCCATATCCAAGCTACTATTCTGTGTTCTAAGAAGGTTGGTCTTAACATTAGAACCAGATCTGGTGGTCATGATGCTGAAGGTATGTCTTACATTAGT TTGA A G CAATCTATCCCTCCATTGCCACCTAGACATCAT。

[0087] CBCAS Q124L+E534K Amino acid sequence of the mutant (SEQ ID NO.6):

[0088] MNCSTFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPANPKFIYTQHDQLYMSVLNSTIQNLRFISDTTPKPLVIVTPSNNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYIS L VPFVVVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEMNENFSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVVVPSKATIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLMLTTHFRTRNITDNHGKNKTTVHGYFSSIFLGGVDSLVDLMNKSFPELGIKKTDCKELSWIDTTIFYSGVVNYNTANFKKEILLDRSAGKKTAFSIKLDYVKKLIPETAMVKILEKLYEEEVGVGMYVLYPYGGIMDEISESAIPFPHRAGIMYELWYTATWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLGKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPNNFFRN K QSIPPLPPRHH。

[0089] 2. Construction and transformation of the mutant vector

[0090] Since primers 1 and 2 used for amplifying the full-length mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K respectively contain the restriction enzyme cleavage sites of EcoR I and Not I, EcoR I and Not I were used to perform double digestion on the two mutant fragments and the empty vector pPIC9K. The digested products were purified, ligated and successfully transformed into competent cells of Escherichia coli DH5α. After sequencing, pPIC9K-CBCAS Q106Y+E534R and pPIC9K-CBCAS Q124L+E534KThe vector was successfully constructed. Two mutant plasmids were extracted and linearized, and then electrotransformed into competent cells of Pichia pastoris GS115 respectively. It was determined by colony PCR and sequencing verification that Figure 3 is the result of colony PCR of yeast.

[0091] 2.5 Verification of mutant protein expression by Western blot

[0092] The Western blot results showed that mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K proteins could be normally expressed in Pichia pastoris. The sizes of mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K proteins were both about 74 kD, which was consistent with the size of wild-type CBCAS.

[0093] 2.6 Detection of mutant CBCAS enzyme activity by HPLC

[0094] In this invention, the crude yeast protein extract transformed with the pPIC9K vector was used as a negative control. The crude CBGA extract (CBGA content was 32.8 μg / mL) obtained from cannabis leaves with high CBGA content was used as a substrate for mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534K respectively. The enzyme activity was determined by detecting the production amount of CBC using a high-performance liquid chromatograph.

[0095] Since there was a small amount of CBC in the crude CBGA extract, CBC would also be detected when no enzyme solution was added or the crude protein extract of the empty vector bacteria was added. Therefore, the following method was used to calculate the production amount of CBC: Production amount of CBC = CBC content in the treatment group - CBC content in the group without adding enzyme solution.

[0096] It was found that the CBC contents in the groups adding the crude protein extracts of mutant CBCAS Q106Y+E534R and CBCAS Q124L+E534K were both significantly higher than those in other treatment groups. Among them, the production amount of CBC in the mutant CBCAS Q106Y+E534R group was 93.3 ng / mL, which was 37.5% higher than the CBC content in the wild-type CBCAS control group. The production amount of CBC in the mutant CBCAS Q106Y+E534R group was 91.4 ng / mL, which was 34.7% higher than the CBC content in the wild-type CBCAS control group.

[0097] In summary, in this invention, bioinformatics methods were used. By predicting the tertiary structure of wild-type CBCAS enzyme, performing molecular docking with the substrate CBGA, and analyzing the mutation sites, mutant CBCAS Q106Y+E534R and mutant CBCAS Q124L+E534KThe binding to the substrate is enhanced. To verify this conclusion, vectors were constructed for these two CBCAS mutants and introduced into Pichia pastoris cells. After induced expression, enzyme activity assays were performed. In the crude protein extract groups of mutants CBCAS Q106Y+E534R and CBCAS Q124L +E534K , the CBC contents were all significantly higher than those in the wild-type CBCAS group. Among them, the production amount of CBC in the mutant CBCAS Q106Y+E534R group was 93.3 ng / mL; the production amount of CBC in the mutant CBCAS Q106Y+E534R group was 91.4 ng / mL.

[0098] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A cannabidiol acid synthase mutant, characterized in that: The amino acid sequence of the cannabicyclol acid synthase mutant is shown in SEQ ID NO.4 or SEQ ID NO.

6.

2. A gene encoding the cannabidiol acid synthase mutant as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that When the amino acid sequence of the cannabicyclopentyl acid synthase mutant is as shown in SEQ ID NO.4, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.3; When the amino acid sequence of the cannabicyclophenolic acid synthase mutant is as shown in SEQ ID NO.6, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.

5.

4. A recombinant expression vector, characterized in that: Comprising the coding gene described in claim 2 or 3.

5. A recombinant microbial strain, characterized in that: Comprising the recombinant expression vector according to claim 4.

6. The recombinant microbial strain according to claim 5, characterized in that The recombinant microbial strain is a recombinant Pichia pastoris.

7. Use of the coding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the recombinant microbial strain according to claim 5 or 6 in preparing a cannabichromenic acid synthase mutant.

8. A method for preparing a cannabidiol acid synthase mutant, characterized in that: The method comprises the steps of fermenting and culturing the recombinant microbial strain according to claim 6 to obtain bacterial cells, and then subjecting the cells to cell wall breaking and extraction treatment to prepare the cannabidiol acid synthase mutant.

9. Use of the cannabichromenic acid synthase mutant as claimed in claim 1 in improving the conversion efficiency of cannabichromenic acid to cannabichromene.

10. A method for increasing the yield of cannabidiol extracted from cannabis plants, characterized in that: The method comprises the steps of using the cannabicyclopentyl acid synthase mutant described in claim 1 to catalyze cannabicyclopentyl acid in the cannabis plant to generate cannabicyclopentyl acid, and then generating cannabicyclopentyl through a decarboxylation reaction.

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