Cannabidiolic acid synthase mutant, high-yield recombinant enzyme pichia pastoris strain and application

Abstract

The application discloses a cannabidiolic acid synthase mutant, a high-yield recombinant enzyme Pichia pastoris strain and application, and relates to the technical field of biology.The amino acid sequence of the cannabidiolic acid synthase mutant is shown as SEQ ID NO.1.The mutant CBDAS G183V‑N482W is used as research material, residues around an active site and a glycosylation site are systematically modified, and a key mutant site capable of significantly improving the enzyme activity of the cannabidiolic acid synthase mutant CBDAS G183V‑N482W is screened by combining domain function analysis and computer-aided calculation.Finally, the multiple mutant CBDAS H114E‑S116A‑C176Y‑G183V‑N328Q‑N482W shows excellent catalytic performance and can effectively improve the conversion efficiency of catalyzing cannabigerolic acid to generate cannabidiolic acid.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a cannabidiol synthase mutant, a high-yield recombinant enzyme Pichia pastoris strain, and their applications. Background Technology

[0002] The cannabis plant contains over 100 different cannabinoids, with cannabidiol (CBD) being the second most abundant after tetrahydrocannabinol (THC). Studies have reported that CBD possesses anti-epileptic, anticonvulsant, anti-anxiety, antipsychotic, anticancer, and antirheumatic effects. Recent research shows that CBD also has functions such as regulating fear memory, protecting the skin, and anti-inflammation. Unlike THC, CBD is non-psychoactive and has a higher safety profile. In recent years, CBD has been widely used in the healthcare field. With the expansion of the CBD market, the demand for CBD production has also increased. Currently, the main synthetic routes for cannabidiol are direct extraction from the cannabis plant and chemical synthesis. However, these methods are limited in terms of time and cost, and also pose certain environmental pollution risks. Therefore, biosynthetic methods have received widespread attention in recent years.

[0003] However, research on the modification and heterologous expression of cannabidiol synthase (CBDAS) is limited. Cannabidiol (CBD) has been confirmed to be produced from cannabidiol acid (CBDA) under natural conditions through decarboxylation, and CBDA is produced from cannabidiol acid via CBDAS catalysis. Therefore, CBDAS is a key enzyme in cannabidiol synthesis. Developing highly active CBDAS enzymes is beneficial for improving the conversion efficiency of cannabidiol acid to CBDA, thereby increasing the yield of biosynthesized cannabidiol. Summary of the Invention

[0004] The purpose of this invention is to provide a cannabidiol synthase mutant, a high-yield recombinant enzyme Pichia pastoris strain, and their applications, to solve the problems existing in the prior art. This cannabidiol synthase mutant exhibits excellent catalytic performance and can effectively improve the conversion efficiency of cannabidiol from cannabidiol.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a cannabidiol synthase mutant CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W The amino acid sequence is shown in SEQ ID NO.1.

[0007] This invention also provides the above-mentioned cannabidiol synthase mutant CBDAS. H114E-S116A-C176Y-G183V-N328Q-N482W The encoding gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant plasmid comprising the above-described encoding gene.

[0009] Furthermore, the recombinant plasmid is obtained by ligating the coding gene into an expression vector; the expression vector is a pPIC9K vector.

[0010] The present invention also provides a Pichia pastoris strain that produces a high-yield cannabidiol synthase mutant, comprising the above-mentioned recombinant plasmid.

[0011] This invention also provides a method for constructing the above-mentioned high-yield cannabidiol synthase mutant Pichia pastoris strain, comprising the following steps:

[0012] The coding gene shown in SEQ ID NO.2 was ligated into an expression vector to obtain a recombinant plasmid;

[0013] The recombinant plasmid was introduced into Pichia pastoris competent cells twice in succession, and positive transformants were obtained by screening. These were the Pichia pastoris strains that produced the high-yield cannabidiol synthase mutant.

[0014] Furthermore, the expression vector is the pPIC9K vector.

[0015] This invention also provides the above-mentioned encoding gene, recombinant plasmid, or Pichia pastoris strain for preparing the above-mentioned cannabidiol synthase mutant CBDAS. H114E-S116A-C176Y-G183V-N328Q-N482W Applications in [the field].

[0016] This invention also provides a CBDAS mutant that enhances cannabidiol synthase. G183V-N482W Methods for determining the enzyme activity of the cannabidiol synthase mutant CBDAS G183V-N482W The amino acid sequence is shown in SEQ ID NO.3; the method includes processing the cannabidiol synthase mutant CBDAS. G183V-N482W The steps involve mutating amino acid position 114 to glutamic acid, amino acid position 176 to tyrosine, amino acid position 328 to glutamine, and amino acid position 116 to alanine.

[0017] The present invention also provides a method for increasing the yield of cannabidiol extracted from cannabis plants, comprising using the aforementioned cannabidiol synthase mutant CBDAS. H114E-S116A-C176Y-G183V-N328Q-N482W The process of catalyzing the conversion of cannabidiol acid in cannabis plants into cannabidiol acid, which in turn forms cannabidiol.

[0018] The present invention discloses the following technical effects:

[0019] This invention uses the cannabidiol synthase mutant CBDAS G183V-N482WTo develop research materials, residues and glycosylation sites surrounding the active site were systematically modified. Combined with domain functional analysis and computer-aided calculations, mutants of cannabidiol synthase (CBDAS) that could significantly enhance the synthesis of cannabidiol synthase were screened. G183V-N482W The key mutation sites for enzyme activity, ultimately yielding the multiple mutant CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W It exhibits excellent catalytic performance.

[0020] In terms of the expression system, this invention uses Pichia pastoris GS115 as the host strain and pPIC9K as the expression vector, and successfully achieves efficient transformation of recombinant enzymes, obtaining a high-yield Pichia pastoris engineered strain of CBDAS.

[0021] Compared with existing technologies, this invention significantly improves the heterologous expression yield of CBDAS and enhances its catalytic efficiency on crude cannabis leaf extract substrates. This not only increases the added value of cannabis byproducts but also endows the technology with significant market application prospects and economic value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Diagram showing the conserved domains of the CBDAS protein;

[0024] Figure 2 Two-dimensional planar diagram (A) and three-dimensional diagram (B) showing the molecular docking of CBDAS enzyme and cofactor FAD;

[0025] Figure 3 For CBDAS G183V-N482W Two-dimensional planar diagram of molecular docking with CBGA;

[0026] Figure 4 For CBDAS G183V-N482W Three-dimensional diagram of molecular docking with CBGA;

[0027] Figure 5 For CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W Two-dimensional planar diagram of molecular docking with CBGA;

[0028] Figure 6 For CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W Three-dimensional diagram of molecular docking with CBGA;

[0029] Figure 7The results represent the identification of high-copy-value-positive transformants of recombinase; where M: Marker; 1: Water; 2: GS115-pPIC9K-CBDAS G183V-N482W ;3-4: GS115-pPIC9K-CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W ;

[0030] Figure 8 The results of identification of positive transformants after re-transformation; where M: Maker; 1: water; 2: GS115-pPIC9K-CBDAS G183V-N482W ;3-4: GS115-pPIC9K-CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W ;

[0031] Figure 9 The results are Western blot validations of the recombinant protein; where M: Marker; 1: Water; 2: GS115-pPIC9K-CBDAS G183V-N482W ;3-4: GS115-pPIC9K-CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W ;

[0032] Figure 10 A statistical graph showing the yields of CBDA(A) and CBD(B) catalyzed by using the recombinant enzyme generated from a single conversion as the catalytic enzyme;

[0033] Figure 11 This is a statistical graph showing the yields of CBDA(A) and CBD(B) catalyzed by recombinases generated from single and double transformations, respectively; where 1 represents CBDAS. G183V-N482W ;2 represents CBDAS H114E -S116A-C176Y-G183V-N328Q-N482W . Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] This invention addresses the mutant CBDAS G183V-N482W The stability and enzyme activity were increased by modifying the glycosylation sites and homologously substituting residues near the active site. The function of the CBDAS domain was analyzed, and molecular docking technology was used to modify the domain, increasing its binding affinity to the cofactor FAD, thereby promoting the catalysis of CBGA. The resulting mutants were expressed in vitro using Pichia pastoris as a chassis, and the recombinant yeast strains were then transformed a second time to obtain Pichia pastoris strains with high CBDAS activity and high CBDAS production. Details are as follows:

[0040] Example 1

[0041] 1. Materials and Methods

[0042] 1.1 Test Materials

[0043] 1.1.1 Strains and Plasmids

[0044] Recombinant plasmid pPIC9K-CBDAS containing wild-type gene WT And the recombinant plasmid pPIC9K-C BDAS containing the mutant gene. G183V-N482W The construction method has been disclosed in patent CN202310536412.6; recombinant plasmid pPIC9K-CBDAS H 114E-S116A-C176Y-G183V-N328Q-N482WThe construction method refers to CN202310536412.6; the vector pPIC9K was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the competent cells of Pichia pastoris were Pichia pastoris GS115, purchased from Shanghai Angyu Biotechnology Co., Ltd.

[0045] 1.1.2 Main Reagents and Culture Media

[0046] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride and 12 g / L agar;

[0047] MD medium: agar 20 g / L, glucose 20 g / L, amino-free nitrogen source 13.4 g / L and biotin 4 × 10⁻⁶ -4 mL / L;

[0048] BMGY medium: tryptone 20 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 3.94 g / L, dipotassium hydrogen phosphate 12 g / L, glycerol 20 mL / L, amino-free nitrogen source 13.4 g / L, and biotin 4 × 10⁻⁶. -4 mL / L.

[0049] BMMY medium: tryptone 20 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 3.94 g / L, dipotassium hydrogen phosphate 12 g / L, amino-free nitrogen source 13.4 g / L, and biotin 4 × 10⁻⁶ -4 mL / L.

[0050] Note: After preparing a 50 mg / mL aqueous solution of Kana or Amp antibiotics, filter it to sterilize it, and then add it to the sterilized LB medium at a volume ratio of 1:1000.

[0051] Antibiotic G418, Beijing Solarbio Science & Technology Co., Ltd.; Nucleic acid molecular weight marker, protein molecular weight standard, and loading buffer, Beyotime Biotechnology Co., Ltd.; Gel extraction kit, Omega Bio-Tek; Plasmid extraction kit, Tiangen Biotech (Beijing) Co., Ltd.; Other related reagents were all commercially available analytical grade.

[0052] 1.1.3 Primer Design

[0053] Specific primers were designed using the Primer3 plus online website (https: / / www.primer3plus.com / ), and CBDAS-F primers were designed based on the CBDAS gene sequence specificity. CCGGAATTC ATGAAGTACTCCACTTTCTCTTTCTG(SEQ IDNO.5); CBDAS-R: AAGGAAAAAAGCGGCCGCATGTCTATGTCTTGGCAAAGGAG (SEQ ID NO. 6). Specific primers AOX-F: GACTGGTTCCAATTGACAAGC (SEQ ID NO. 7); AOX-R: GGCAAATGGCATTCTGACAT (SEQ ID NO. 8) were designed based on the pPIC9K vector sequence. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0054] 1.2 Test Methods

[0055] 1.2.1 Regarding CBDAS G183V-N482W Transformation strategy

[0056] Conserved domains of the CBDAS protein were analyzed using Smart (http: / / smart.embl-heidelberg.de / ) and NCBI's CD-search (https: / / www.ncbi.nlm.nih.gov / cdd / ) online software. The three-dimensional structure of CBDAS was constructed using the Alpha Fold 3 online website. The three-dimensional structure of the FAD cofactor was obtained from ChEBI (CHEBI:57692). Molecular docking and mutation site screening were performed using Discovery Studio 2016 software.

[0057] The modification sites were determined based on the changes in enzyme activity after replacing the CBDAS glycosylation sites one by one and homologous substitution of residues around the active site, as described by Bastian Zirpel et al. (2018).

[0058] 1.2.2 Mutant-Substrate Interaction Analysis

[0059] The mutant and substrate were re-coupled using Discovery Studio 2016 software. Based on the coupling results, a planar diagram was drawn using PyMOL plotting software to analyze their interaction forces and binding sites.

[0060] 1.2.3 Transformation of Pichia pastoris with recombinant enzymes and screening of high-copy-positive transformants

[0061] Will contain pPIC9K-CBDAS WT pPIC9K-CBDAS G183V-N482W pPIC9K-CBDAS H114E -S116A-C176Y-G183V-N328Q-N482W DH5α plasmid, coated on a substrate containing Amp +The cells were cultured overnight at 37°C on LB agar plates. Single colonies were picked and cultured overnight in 6 mL of LB liquid medium. The next day, plasmids were extracted using a plasmid extraction kit. The extracted plasmids were linearized with restriction endonuclease SacI, and the linearized plasmids were recovered using a product purification kit. The plasmids were then electroporated into Pichia pastoris GS115 competent cells at 2 kV for 5 ms. After electroporation, 600 μL of pre-chilled 1 M sorbitol was quickly added, and the cells were incubated at 30°C for 1 h. The transformed yeast cells were plated on MD agar plates and cultured until single colonies appeared. Single colonies were washed with 3 mL of ddH2O and plated onto YPD solid medium containing 2 mg / mL, 4 mg / mL, and 6 mg / mL G418 concentrations for high copy number selection to express the target protein. After 3-5 days of culture, single colonies from the medium with the highest G418 concentration were picked for PCR verification.

[0062] 1.2.4 Construction and validation of high-yield CBDAS yeast expression strain

[0063] (1) Preparation of competent yeast cells containing CBDAS

[0064] Will contain pPIC9K-CBDAS G183V-N482W pPIC9K-CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W The recombinant Pichia pastoris was inoculated into 3 ml LYPD medium and cultured overnight. The following day, it was inoculated at 1% into 50 ml LYPD liquid medium, and cultured to OD... 600 Centrifuge at 5000 rpm for 15 min at 4℃, discard the supernatant, and resuspend in 40 mL of pre-chilled sterile water. Centrifuge for 15 min, discard the supernatant, and resuspend in 20 mL of pre-chilled sterile water. Centrifuge for 10 min, discard the supernatant, and resuspend in 3 mL of pre-chilled 1M sorbitol. Centrifuge for 5 min, discard the supernatant, and resuspend in 300 μL of pre-chilled 1M sorbitol. Aliquot into 100 μL tubes.

[0065] (2) The GS115-pPIC9K-CBDAS recombinant bacteria were transformed again.

[0066] Will contain pPIC9K-CBDAS G183V-N482W pPIC9K-CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W DH5α plasmid, coated on a substrate containing Amp + The culture was carried out overnight at 37°C on LB agar plates. Single colonies were picked from the plates and cultured for 116 h. Plasmids were then extracted using a plasmid extraction kit, linearized with restriction endonuclease SacⅠ, and the product was recovered using a product purification kit.

[0067] Linearized plasmids were electroporated into competent Pichia pastoris cells containing CBDAS. Screening was performed using YPD solid medium with G418 concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, and 10 mg / mL. After 3-5 days of incubation, single colonies from the medium with the highest G418 concentration were picked for PCR verification.

[0068] 1.2.5 Induced Expression of Recombinant Proteins

[0069] Selected recombinant bacteria that tested positive were inoculated into YPD liquid medium and cultured overnight at 30°C and 190 rpm / min. The activated bacterial solution was then inoculated at 1% onto 20 mL of BMGY medium and cultured at 30°C and 190 rpm / min until OD (digesterone) was reached. 600 When the value is 2, the bacterial cells are collected by centrifugation at 5000 rpm for 10 min, and resuspended in BMMY medium until the initial OD value is reached. 600 When the concentration is 1, take 20 mL and culture in a shake flask. Add methanol every 24 hours until the final concentration is 1.0%, and culture for 48 hours. Collect the bacterial pellet and add 20 mL of lysis buffer. Sonicate the pellet in an ice bath. After lysis, centrifuge at 5000 rpm for 10 min at 4°C. Collect the supernatant and concentrate it through a 30 kDa ultrafiltration tube. Finally, collect the protein solution in the ultrafiltration tube and measure it using a BCA protein concentration kit. Store at 4°C for later use.

[0070] 1.2.6 Western blot identification

[0071] After SDS-PAGE electrophoresis of the prepared protein sample, the stacking gel portion is removed. A PVDF membrane of the same size as the protein gel is cut off and soaked in methanol for 1-2 minutes. The prepared filter paper, membrane, and protein gel are then immersed in pre-cooled transfer buffer and stacked in a "sandwich" configuration, with the protein gel near the cathode and the PVDF membrane near the anode. Excess buffer is blotted off, the transfer tank is capped, and the membrane is transferred using a semi-dry transfer apparatus at a constant voltage of 15V for 40 minutes. The membrane is blocked with 5% skim milk powder (prepared with TBST) and incubated on a horizontal shaker at room temperature for 2 hours. The PVDF membrane is washed twice with TBST and incubated overnight at 4°C with Anti-His tag primary antibody (diluted 1:5000 with TBST). The membrane is washed 6 times with TBST for 5 minutes each time. HRP-labeled rabbit anti-mouse IgG secondary antibody (diluted 1:4000 with TBST) is transferred and incubated at room temperature for 2 hours. The membrane is washed 6 times with TBST for 5 minutes each time. Finally, the bands were detected using an ECL high-sensitivity chemiluminescence detection kit and an imaging analyzer.

[0072] 1.2.7 Enzyme activity assay

[0073] Preparation of crude CBGA leaf extract: Take 0.1g of dried cannabis leaves, add 50mL of methanol, sonicate for 30min, centrifuge at 4℃ for 20min to collect the supernatant, and then remove impurities with a 0.22μm filter membrane to obtain the crude CBGA leaf extract with a CBGA concentration of 30.94μg / mL.

[0074] The crude extract of CBGA leaves (CBGA concentration of 30.94 μg / mL) was used as the reaction substrate to determine the protein activity of the recombinase. The pPIC9K empty vector was used as a blank control. The reaction system is shown in Table 1.

[0075] Table 1 Enzyme activation reaction system

[0076]

[0077] 1.2.8 Liquid Chromatography Reaction Conditions

[0078] Chromatographic column: Shimadzu sil-16 C18 column (150 mm × 4.6 mm × 3 μm), column temperature: 30 ℃; mobile phase: A is an aqueous solution containing 0.1% formic acid, B is acetonitrile containing 0.1% formic acid; isocratic elution: 25% A, 75% B, retention time 30 min; UV detector: 230 nm; flow rate: 0.7 mL / min; injection volume: 10 μL.

[0079] 1.2.9 Data Processing

[0080] GraphPad Prism 8.0 was used for data analysis and plotting of liquid chromatography data. SPSS 24.0 was used for one-way ANOVA and Duncan's method was used for significance analysis (P < 0.05, P < 0.01). All data were obtained by repeating the experiment three times.

[0081] 2 Results and Analysis

[0082] 2.1 Analysis of molecular docking and mutant amino acid site results of CBDAS enzyme

[0083] The conserved domains of the CBDAS protein were analyzed using Smart and NCBI's CD-search online websites, and the results were consistent. Figure 1As shown, the BBE domain, spanning amino acid range 479-537, is present in berberine bridges and berberine-bridge-like enzymes, participating in the biosynthesis of many isoquinoline alkaloids. The 81-218 domain family comprises various enzymes with FAD as a cofactor, most of which are similar to oxidoreductases. Some articles report that both the BBE and FAD domains bind to the FAD cofactor and promote the catalytic conversion of CBGA to CBDA by CBDAS. To understand the mechanism of action of CBDAS enzymes and the cofactor FAD, the FAD substrate was docked into the binding cavity of the enzyme with geometric complementarity using CODOCKER in Discovery Studio. During docking, the ligand molecule was placed at the protein's active site. The interaction between the ligand and protein was evaluated in real-time according to the principles of geometric complementarity, energy complementarity, and chemical environment complementarity. The optimal binding mode between the two molecules was found, resulting in a two-dimensional plane diagram of the protein-ligand docking. This diagram was then plotted using PyMOL software, and the results are shown below. Figure 2 Then, using Discovery Studio's Calculate Mutation Energy (Binding) to perform virtual amino acid mutations on the protein-ligand complex, it was determined that the combination of the mutation of histidine (His, H) at position 114 to glutamic acid (Glu, E) and the mutation of cysteine ​​(Cys, C) at position 176 to tyrosine (Tyr, y) resulted in the greatest reduction.

[0084] The CBDAS glycosylation mutation site, namely the 328th amino acid asparagine (Asn, N), was mutated to glutamine (Gln, Q). The modification sites of the residues around the active site were determined, and the 116th amino acid serine (Ser, S) was mutated to alanine (Ala, A).

[0085] Finally, the mutant CBDAS was obtained. H114E-S116A-C176Y-G183V-N328Q-N482W (The amino acid sequence is shown in SEQ ID NO.1), and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO.2.

[0086] The mutant gene was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0087] CBDAS mutant H114E-S116A-C176Y-G183V-N328Q-N482W The amino acid sequence (SEQ ID NO.1):

[0088] MKYSTFSFWFVCKIIFFFFSFNIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGEDAEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYYPTVCAGVHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMQKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLWYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH*。

[0089] Mutant CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W The nucleotide sequence of the encoding gene (SEQ ID NO.2):

[0090] ATGAAGTACTCCACTTTCTCTTTCTGGTTTGTTTGTAAGATCATCTTCTTCTTCTTCTCCTTCCAACATCCAAACTTCTATCGCTAACCCAAGAGAAAATTTCTTGAAGTGTTTCTCCCAGTACATCCCCAAATAATGCTACTAATCTGAAGCTGGTTTACACTCAAAACAACCCATTGTACATGTCTGTTTTGAACTCTACTATCCACAACTTGAGATTCACTTCTGATACTACTCCTAAGCCTTTGGTTATTGTTACTCCTTCTCATGTTTCCCATATCCAAGGTACTATTTTGTGTTCTAAGAAGGTCGGTTTGCAAATTAGAACTAGATCTGGTGGTGAAGATGCTGAAGGTATGTCTTACATTTCCCAAGTTCCTTTCGTTATCGTTGATTTGAGAAACATGAGATCCATCAAGATCGATGTTCATTCTCAAACTGCTTGGGTTGAAG CTGGTGCTACTTTGGGTGAAGTTTATTACTGGGTTAACGAAAAGAACGAGAACTTGTCTTTGGCTGCTGGTTACTACCCTACTGTTTGTGCTGGTGTTCATTTCGGTGGTGGTGGTTACGGTCCATTGATGAGAAACTATGGTTTTGGCTGCTGATAACATTATCGATGCTCATTTGGTTAACGTCCATGGTAAGGTTTTGGATAGAAAGTCTATGGGTGAAGATT TGTTCTGGGCTTTGAGAGGTGGTGGTGCTGAATCTTTCGGTATTATTGTTGCTTGGAAGATCAGATTGGTCGCTGTTCCTAAATCTACTATGTTCTCTGTTAAGAAGATCATGGAGATCCATGAATTGGTTAAGTTGGTTAACAAGTGGCAAAACATTGCTTACAAGTACGATAAGGACTTGTTGTTGATGACTCATTTCATCACTAGAAACATCACCGATAACCAAGGTAAGAACAAGACTGCTATTCATACTTACTTCTCCTCTGTTTTCCTGGGTGGTGTTGATTCTTTGGTTGATTTGATGCAAAAGTCCTTCCCTGAATTGGGTATTAAAAAGACTGACTGTAGACAGTTGTCCTGGATTGATACTATTATCTTCTACTCCGGTGTCGTTAATTACGATACTGATAATTTCAACAAGGAGATCCTGTTGGATAGATCTGCTGGTCAAAACGGTGCTTTTAAGATTAAGTTGGACTACGTTAAGAAGCCTATCCCTGAATCTGTTTTCGTTCAAATTCTGGAAAAGCTGTACGAAGAAGATATCGGTGCTGGTATGTATGCTTTGTATCCTTACGGTGGTATTATGGATGAAATCTCTGAATCTGCTATCCCATTTCCTCATAGAGCTGGTATTTTGTATGAGTTGTGGTACATTTGCTCCTGGGAAAAACAAGAAGATAACGAAAAGCACCTGAACTGGATTAGAAATATCTACAACTTCATGACCCCCTATGTTTCTCAAAATCCTAGATTGGCTTACTTGTGGTATAGAGATTTGGATATCGGTATCAACGACCCTAAAAACCCTAACAACTACACTCAAGCTAGAATTTGGGGTGAAAAGTATTTCGGTAAGAACTTCGATAGACTGGTTAAAGTTAAGACCTTGGTTGATCCAAACAACTTCTTTAGAAACGAGCAATCTATCCCTCCTTTGCCAAGACATAGACAT。

[0091] Amino acid sequence of mutant CBDAS G183V-N482W (SEQ ID NO.3):

[0092] MKYSTFSFWFVCKIIFFFFSFNIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYCPTVCAGVHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLWYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH*。

[0093] Mutant CBDAS G183V-N482W The nucleotide sequence of the encoding gene (SEQ ID NO.4):

[0094]

[0095] 2.3 Analysis of the interaction results between mutants and substrates

[0096] The identified mutant CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W and CBDAS G183V-N482W Molecular docking with CBGA was performed, and then PyMOL software was used to observe the enzyme structure, obtaining information such as the interaction site and distance between the enzyme and the substrate. Figures 3-6 The interaction forces between the enzyme and substrate catalytic sites were labeled. After docking, it was found that the molecular forces between the mutant and the substrate were mainly hydrogen bonds, van der Waals forces, and hydrophobic interactions (including Pi-Alkyl and Pi-Pi T-Shaped). Analysis of the interaction forces and bond lengths between the enzyme and substrate revealed that the hydrogen bond length formed by the Trp docking ligand at position 176 increased from […]. shortened to An interaction force, Pi-Alkyl, is added between the ligand and the ligand, with a bond length of [value missing]. The interaction force between Tyr at position 483 and the ligand changes from a Pi-Pi T-shaped to a Pi-Pi stacked structure, and the bond length changes from... shortened to The Alkyl bond between the protein and substrate at position 184 increased. The interaction force between the Trp bond at position 482 and the substrate increased while remaining constant. Therefore, the increased amino acid interactions and shortened bond lengths between the protein and substrate may be the reason for the enhanced enzyme-substrate affinity. Subsequent enzyme activity assays were performed to examine whether the enzyme activity was related to these factors.

[0097] 2.4 Identification of high-copy-rate recombinase positive transformants

[0098] Colonies from the highest concentration of G418 plates were selected, and PCR templates were obtained using the high-temperature ethyl acetate method. PCR amplification and verification were performed using primers CBDAS-F designed based on CBDAS and AOX-R designed based on the pPIC9K vector terminator fragment. Results were analyzed based on electrophoresis results. Figure 7 This demonstrates that the linearized recombinant plasmid has been successfully integrated into the Pichia pastoris genome.

[0099] 2.5 Identification of positive transformants after re-transformation

[0100] Using colonies from YPD medium at concentrations of 2 mg / mL, 4 mg / mL, and 6 mg / mL G418 as references, screening was performed using 10 mg / mL G418. Colony PCR was conducted using the experimental method described in section 2.4, and the results are shown below. Figure 8 .

[0101] 2.6 Western blot validation of recombinant proteins

[0102] According to the Western blot results ( Figure 9 The results showed that the recombinant protein CBDAS was expressed normally in Pichia pastoris, with a specific band at approximately 70 kDa, while the empty vector was not expressed in Pichia pastoris.

[0103] 2.7 Enzyme activity assay of recombinant protein

[0104] CBDA and CBD products were detected by HPLC analysis, and the yields of CBDA and CBD were calculated using standard curves. The HPLC analysis results of CBDA and CBD produced by the recombinase generated after a single conversion are shown in [Figure number missing]. Figure 10 CBDAS H114E -S116A-C176Y-G183V-N328Q-N482W CBDA production was significantly higher than CBDAS. G183V-N482W CBDAS H114E -S116A-C176Y-G183V-N328Q-N482W It produced 71.543 ng / mL CBDA and 75.163 ng / mL CBD, CBDAS G183V-N482W It produced 60.297 ng / mL CBDA and 65.059 ng / mL CBD. (CBDAS) H114E-S116A-C176Y-G183V-N328Q-N482W The ratio of CBDA to CBD in the production of CBDAS G183V-N482W These figures are 11.87% and 11.53% higher.

[0105] CBDAS after two transformations G183V-N482W The resulting CBDA and CBD concentrations were 67.128 ng / mL and 76.852 ng / mL, respectively, which was higher than that of a single CBDAS conversion. G183V-N482W Increased by 11.13% and 11.81% after two conversions of CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W CBDA and CBD were produced at concentrations of 78.523 ng / mL and 95.075 ng / mL, respectively, representing increases in yield of 9.77% and 12.65% compared to a single conversion. Figure 11 ).

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cannabidiol synthase mutant CBDAS H114E-S116A-C176Y-G183V-N328Q-N482W Its characteristics are, The amino acid sequence is shown in SEQ ID NO.

1.

2. A cannabidiol synthase mutant CBDAS as described in claim 1 H114E-S116A-C176Y-G183V-N328Q-N482W The encoding gene, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, Includes the coding gene as described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid is obtained by ligating the coding gene into an expression vector; the expression vector is a pPIC9K vector.

5. A Pichia pastoris strain that produces a high-yield cannabidiol synthase mutant, characterized in that, Includes the recombinant plasmid as described in claim 3.

6. A method for constructing a Pichia pastoris strain with a high cannabidiol synthase-producing mutant as described in claim 5, characterized in that, Includes the following steps: The coding gene shown in SEQ ID NO.2 was ligated into an expression vector to obtain a recombinant plasmid; The recombinant plasmid was introduced into Pichia pastoris competent cells twice in succession, and positive transformants were obtained by screening. These were the Pichia pastoris strains that produced the high-yield cannabidiol synthase mutant.

7. The construction method according to claim 6, characterized in that, The expression vector is the pPIC9K vector.

8. A gene encoding as described in claim 2, a recombinant plasmid as described in claim 3 or 4, or a Pichia pastoris strain as described in claim 5, used to prepare the cannabidiol synthase mutant CBDAS as described in claim 1. H114E-S116A-C176Y -G183V-N328Q -N482W Applications in [the field].

9. A mutant CBDAS that enhances cannabidiol synthase G183V-N482W A method for determining enzyme activity, characterized in that, The cannabidiol synthase mutant CBDAS G183V-N482W The amino acid sequence is shown in SEQ ID NO.3; The method includes processing the cannabidiol synthase mutant CBDAS. G183V-N482W The steps involve mutating amino acid position 114 to glutamic acid, amino acid position 176 to tyrosine, amino acid position 328 to glutamine, and amino acid position 116 to alanine.

10. A method for increasing the yield of cannabidiol extracted from cannabis plants, characterized in that, Including the cannabidiol synthase mutant CBDAS as described in claim 1 H114E-S116A-C176Y-G183V-N328Q-N482W The process of catalyzing the conversion of cannabidiol acid in cannabis plants into cannabidiol acid, which in turn forms cannabidiol.

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