CN116574700B

CN116574700B - Cannabidiol synthetase mutant and application thereof

Info

Publication number: CN116574700B
Application number: CN202310536412.6A
Authority: CN
Inventors: 戴凌燕; 曹宁; 牛庭莉; 李志江
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2023-05-12
Filing date: 2023-05-12
Publication date: 2023-11-14
Anticipated expiration: 2043-05-12
Also published as: CN116574700A

Abstract

The invention discloses a cannabidiol synthase mutant and application thereof, and relates to the technical field of biology. The invention discloses a cannabidiol synthase mutant CBDAS ^G183V+N482W The amino acid sequence is shown as SEQ ID NO. 2. The invention also discloses a cannabidiol synthase mutant CBDAS ^H114L+C176W The amino acid sequence is shown as SEQ ID NO. 4. The invention screens out the amino acid mutation site which can raise the enzyme activity after the computer prediction and analysis, and designs and obtains the cannabidiol synthase mutant CBDAS ^G183V+N482W And CBDAS ^H114L+C176W The conversion efficiency of catalyzing cannabigerol acid to generate cannabidiol is obviously improved, and a foundation is laid for industrial production and application of cannabigerol acid synthetase mutants and improvement of the yield of biosynthesis of cannabigerol.

Description

Cannabidiol synthetase mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a cannabidiol synthase mutant and application thereof.

Background

The unique metabolite produced by cannabis plants, known as cannabinoids, consists of polyketide and monoterpene substructures. Up to now, over 100 cannabinoids have been isolated from cannabis and their chemical and pharmacological properties have been intensively studied. Among these metabolites, Δ9-Tetrahydrocannabinol (THC) was identified as a psychoactive cannabinoid; while Cannabidiol (CBD) is an isomer of THC with a different ring system, CBD has become attractive as an effective drug for the treatment of refractory childhood epilepsy. It is well known that CBD can reduce the adverse effects of THC on spirit, and has been a hot spot of research in recent years. CBD is also used for several other receptors and channels, so that CBD plays an important role in analgesia, anti-inflammatory, mental disease, anticancer, etc., in addition to epileptic aspects.

Molecular docking is one of the important methods of molecular modeling, which is a computer simulation, essentially a process of recognition between two or more molecules involving spatial shape complementation and energy matching between molecules. Molecular docking makes 2 main predictions: molecular binding mode and binding force intensity. Several successful algorithms have been developed to identify the position and possible conformation of the substrate in the enzyme. Even low resolution information about how the substrate binds to the protein is often sufficient to make a informed guess at which positions mutagenesis is required to achieve the maximum desired effect. The protein-ligand complex obtained after molecular docking can further utilize a computer algorithm to replace specific amino acid residues intentionally and accurately, so that the workload of enzyme modification is greatly reduced.

At present, most domestic methods for obtaining CBDA and CBD from cannabis plants are not long-term methods by extracting Cannabidiol (CBDA) from cannabis plants and decarboxylating the extracted cannabidiol at high temperature to form the CBD. In recent years, the hot trend of synthetic biology is that a great deal of synthesis of rare medicinal materials can be realized through heterologous expression. Therefore, the industrial efficient production of CBDA and CBD using Pichia pastoris as host is necessary. The biosynthetic pathway of CBDA is shown in figure 1, cannabidiol Synthase (CBDAs) catalyzes the production of CBDA from the substrate cannabigerol acid (CBGA). Therefore, there is a need to develop a highly active CBDAS enzyme to increase CBDA and CBD production.

Disclosure of Invention

The invention aims to provide a cannabidiol synthase mutant and application thereof, which are used for solving the problems in the prior art, and the cannabidiol synthase mutant provided by the invention has the advantages that the conversion efficiency of catalyzing cannabidiol to generate cannabidiol is obviously improved, and a foundation is laid for the industrial production and application of the cannabidiol synthase mutant and the improvement of the yield of biosynthesis of cannabidiol.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a cannabidiol synthase mutant CBDAS ^G183V+N482W The amino acid sequence is shown as SEQ ID NO. 2.

The invention also provides a cannabidiol synthase mutant CBDAS ^G183V+N482W The nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

The invention also provides a CBDAS for expressing the cannabidiol synthase mutant ^G183V+N482W Comprises the above cannabidiol synthase mutant CBDAS ^G183V+N482W Is a coding gene of (a).

The invention also provides a recombinant microorganism strain which comprises the mutant CBDAS for expressing the cannabidiol synthase ^G183V+N482W Is a recombinant plasmid of (a).

The invention also provides a cannabidiol synthase mutant CBDAS ^H114L+C176W The amino acid sequence is shown as SEQ ID NO. 4.

The invention also provides a cannabidiol synthase mutant CBDAS ^H114L+C176W The nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.

The invention also provides a CBDAS for expressing the cannabidiol synthase mutant ^H114L+C176W Comprises the above cannabidiol synthase mutant CBDAS ^H114L+C176W Is a coding gene of (a).

The invention also provides a recombinant microorganism strain which comprises the mutant CBDAS for expressing the cannabidiol synthase ^H114L+C176W Is a recombinant plasmid of (a).

The invention also provides the cannabidiol synthase mutant CBDAS ^G183V+N482W Or cannabidiol synthase mutant CBDAS ^H114L+C176W In improving the conversion efficiency of cannabidiol acid from cannabigerol acidIs used in the application of (a).

The invention also provides a method for improving the yield of cannabidiol extracted from cannabis plants, which comprises the steps of utilizing the cannabidiol synthase mutant CBDAS ^G183V+N482W Or cannabidiol synthase mutant CBDAS ^H114L+C176W Catalyzing cannabigerol acid in cannabis plants to generate cannabidiol acid, thereby forming cannabidiol.

The invention discloses the following technical effects:

according to the invention, hemp leaves with high CBD content are used as materials, a rational design strategy is adopted, and through a method of homologous modeling, enzyme and substrate molecule butt joint and amino acid mutation site screening, transformation is carried out according to the affinity strength of CBD synthetase (CBDAS) and a substrate, and then the mutant enzymes are subjected to plasmid construction and then are subjected to heterologous expression in pichia pastoris, so that the mutant enzymes with improved catalytic activities of the two enzymes are obtained. After the enzyme activity is measured, the catalytic effect of the two mutant enzymes is found to be in accordance with the molecular docking prediction result. When CBGA in crude extract of hemp leaves is used as substrate, mutant CBDAS ^G183V+N482W Catalytic production of 75.7ng/mL CBDA and 50.76ng/mL CBD; when CBGA standard is used as a substrate, the mutant CBDAS ^G183V+N482W Catalytic production of 36.52ng/mL CBDA and 42.92ng/mL CBD. The recombinant expression mutant has stable effect and remarkably improved catalytic activity compared with the wild type.

According to the invention, the rational design strategy is utilized, and after computer prediction and analysis, the amino acid mutation site capable of improving the enzyme activity is screened out, so that the experiment time and the cost are greatly saved. The invention provides a certain theoretical basis for improving the affinity of the enzyme and the substrate, and lays a foundation for industrial production and application of CBDAS recombinase and improving the yield of biosynthesis CBD.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a biosynthetic pathway of CBDA;

FIG. 2 is a graph showing the spatial relative positions and interactions of CBGA and CBDAS enzymes; wherein A is a molecular docking two-dimensional plan view, and red arrows indicate a substrate CBGA; b is a three-dimensional graph of CBGA and CBDAS enzyme after being butted;

FIG. 3 is a mutant CBDAS ^H114L+C176W And CBDAS ^G183V+N482W Molecular docking three-dimensional and two-dimensional maps; wherein A and C are wild-type and mutant CBDAS respectively ^H114L+C176W A three-dimensional graph after molecular docking; b and D are wild-type and mutant CBDAS, respectively ^G183V+N482W A three-dimensional graph after molecular docking; E-G is wild type, CBDAS ^H114L+C176W And CBDAS ^G183V+N482W A two-dimensional graph after molecular docking;

FIG. 4 is colony PCR of a mutant yeast high copy positive recombinant; m: DS2000 Maker;1-2: pPIC9K-CBDAS ^H114L+C176W 、pPIC9K-CBDAS ^G183V+N482W Positive plasmids of (a); 3: a negative control; lanes 4-9: different colony PCR results after 6mg/mL screening for the two mutants;

FIG. 5 is a diagram showing the case where wild type and mutant catalyze the same mass of CBGA to produce CBDA and CBD; wherein, a diagram: the reaction conditions of the wild type and the mutant in the crude extract of CBGA leaves; b, drawing: wild type and mutant response conditions in CBGA standards; CK: blank control with only substrate and no enzyme solution; WT: the wild CBDAS recombinant protein solution prepared in the example 4; h114L+C176W and G183V+NA82W correspond to the mutant CBDAS prepared in example 4, respectively ^G183V+N482W And mutant CBDAS ^H114L+C176W Recombinant protein solution.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The medium formulation used in the following examples was as follows:

LB medium: 10g/L tryptone, 5g/L yeast extract, 5g/L sodium chloride and 12g/L agar.

MD medium: agar 20g/L, glucose 20g/L, amino-free yeast nitrogen source (YNB) 13.4g/L, biotin 4X 10 ^-4 mL/L。

BMGY medium: tryptone 20g/L, yeast extract 10g/L, dipotassium hydrogen phosphate 3.94g/L, dipotassium hydrogen phosphate 12g/L, glycerol 20mL/L, amino-free yeast nitrogen source (YNB) 13.4g/L, biotin 4X 10 ^-4 mL/L。

BMMY medium: tryptone 20g/L, yeast extract 10g/L, dipotassium hydrogen phosphate 3.94g/L, dipotassium hydrogen phosphate 12g/L, amino-free yeast nitrogen source (YNB) 13.4g/L, biotin 4X 10 ^-4 mL/L。

Note that: the Kana and Amp antibiotics were filtered and sterilized and then added to the sterilized LB medium at a volume ratio of 1:1000.

The vector pPIC9K used in the following examples was purchased from wuhan vast biotechnology limited; the pichia competent cell was pichia (pichia pastoris) GS115, purchased from the high-feather biotechnology company, shanghai.

Example 1

1. Protein preparation and molecular docking

The CBDAS sequence (OP 627098) was input to the I-TASSER website to build a three-dimensional Model, and the Model 1 three-level structure Model predicted by I-TASSER was quality-assessed by 3 Model quality assessment software provided by Model Quality Assessment programs (MQAS) SAVES v 5.0. The cannabigerol acid (CBGA) three-dimensional structure is derived from PubCHem (PubCHem CID: 6449999). After the three-dimensional structure is prepared, molecular docking is performed:

(1) The three-dimensional structures of the proteins Model 1 and CBGA are pretreated by using Discovery Studio 2016 software to remove water molecules, hydrogenate and the like, and are respectively defined as a receptor and a ligand.

(2) Defining binding sites of receptors based on protein cavities, selecting optimal results, clicking CDOCKER docking modes in a molecular docking module, and setting a RMSD threshold value asTo ensure as diverse a docking conformation as possible.

(3) After the operation is finished, the information about the type, distance and the like of intermolecular interaction between the receptor and the ligand is displayed, a two-dimensional plane diagram is generated, and finally, the drawing is drawn by using PyMOL, as shown in figure 2.

2. Virtual amino acid mutation site selection

The most reasonable mutation strategy is obtained, and the most suitable mutation combination is obtained based on the interaction of the amino acid residues at the mutation site and the substrate in 20 common amino acids.

(1) The optimal docking results were introduced into Discovery Studio 2016 software, and the interaction force between receptor and ligand was analyzed to remove water molecules.

(2) The Macromolecules-preparation Protein is unfolded, and the clear Protein is clicked to pretreat the Protein structure.

(3) Spreading the formulation-Change Forcefield, clicking on the applied force field, and applying the CHARMM force field to the protein.

(4) After the above treatment, the ligand is selectedAll amino acids within were used as subsequent mutation points.

(5) Click Calculate Mutation Energy (Binding) module, virtual amino acid mutation of the receptor-ligand complex based on interaction force, determination of key amino acids in the active site, and amino acids capable of improving affinity as mutation targets, and prediction of optimal amino acid mutation combinations using Predict Stabilizing Mutations module.

(6) The obtained mutant CBDAS is screened ^H114L+C176W And CBDAS ^G183V+N482W And (3) re-modeling the protein again, then re-carrying out molecular docking, and using PyMOL software for observing the structure of the enzyme, so as to obtain information such as the action site, the distance and the like between the enzyme and the substrate (the enzyme is CBDAS, the substrate is CBGA), and marking the interaction between the enzyme and the catalytic site of the substrate, as shown in figure 3.

3. Results

According to FIG. 2, the ligand CBGA has hydrogen bonding with Cys176, asn482 and Ser116, etc., and forms additional hydrogen bonding (Van der Waals forces) with Asp115, gly174, trp443, gly183, arg484, his184, gly184 and Ile382, etc., thereby further stabilizing the molecular conformation. The hydrophobic interaction of CBGA with His69, phe380, his114, tyr483 and Val179 etc. to form Pi-Alkyl, pi-PiT bonds may be involved in the enzymatic substrate reaction.

As shown in FIG. 3, it was found by analysis of the force and bond length between the enzyme and the substrate that Cys at position 176 and H at position 114When the is bit is combined with mutation, the hydroxy group of the 176 bit Trp docking ligand is changed into carbonyl group, and the formed hydrogen bond length is formed byShortened to->Leu at position 114 still acts on the benzene ring of the ligand to form a force Pi-Alkyl with a bond length of +.>Shortened to->(A and C in FIG. 3), bond length shortens interaction force enhancement, which may be responsible for the increased affinity of the enzyme to the substrate. Mutant CBDAS ^G183V+N482W After re-docking, the amino acid residues around the substrate CBGA are greatly changed, and Van der Waals force and hydrogen bonding between the Val at 183 th and Trp at 482 th and the ligand are changed into hydrophobic effects; the hydroxy group acting on Trp at position 482 and the ligand is changed into methyl, the bond is lengthened, and Val at position 183 and two methyl groups of the ligand generate acting force, the bond length is increased by +.>Shortened to->(B and D in FIG. 3). Viewing mutant CBDAS from two-dimensional plan ^H114L+C176W And CBDAS ^G183V+N482W More potent than wild-type CBGA (E, F and G in figure 3).

Example 2

Obtaining of wild type plasmid (pPIC 9K-CBDAS):

(1) CBDAS gene specific primers were designed using Primer3 plus online website. PCR amplification was performed using the laboratory-stored cannabis genome as a template using high-fidelity polymerase. PCR reaction system: 5 XSF Buffer 10. Mu.L, dNTPs 1. Mu.L, primer 1 and primer 4 (see Table 1) each 2. Mu.L, genomic DNA up to 100ng, were fixed to a volume of 50. Mu.L with deionized water, and were reconstituted in parallel with a tube of Phanta Super-Fidelity DNApolymerase 0.5.5. Mu.L. The PCR procedure was set at 95℃in 2min,95℃for 10s,55℃for 30s,72℃for 1min for 30s for 32 cycles, and stored at 72℃in 5min at 4 ℃. After electrophoresis on a 1% agarose gel, the PCR product was spotted in an amount of 100. Mu.L, at a constant pressure of 110V for 25min. The results were recorded using a gel imager and cut into gel, and purified according to the gel recovery kit instructions.

(2) The plasmid pPIC9K and the purified CBDAS target fragment are respectively subjected to enzyme digestion by EcoRI and NotI, and the system is as follows: pPIC9K (or CBDAS purified product) has a mass of 1 μg,10×Cutsmart Buffer 2 μL, ecoRI and NotI restriction enzymes 1 μL each, make up to 20 μL with deionized water, react at 37deg.C for 3h, electrophorese the digested products, and then cut gel and recover. Addition of T to PCR vials ₄ Ligase Buffer 2μL，T ₄ Ligase1 mu L, pPIC9K and CBDAS target fragment mass ratio after enzyme digestion and purification reaches 1:5, and deionized water is used for constant volume to 20 mu L. The reaction is carried out for more than 3 hours at 25 ℃ for enzyme linking.

(3) Half of the ligation solution was added to 50 μldh5α competence for transformation, the procedure was: ice bath 20min,42 ℃ heat shock 90s, immediately ice bath 3min, adding 700 u L LB at 37 ℃,190rpm shaking to 45-60min. After centrifugation at 6000rpm for 1min, the excess supernatant was discarded, leaving only about 200. Mu.L of resuspended cells. After mixing, the mixture was uniformly coated on LB plates with Amp resistance. Inverted overnight at 37℃in a thermostated incubator. After obvious single colony is grown, performing colony PCR verification by using the primer 1 and the primer 12, picking 3 shaking bacteria from the colony conforming to the size of the strip, and taking a proper amount of bacterial liquid for sequencing.

Obtaining mutant target fragment:

specific primers were designed using Primer3 plus online website. The overlap extension PCR technique is adopted, pPIC9K-CBDAS plasmid is used as a template, high-fidelity polymerase is selected for amplifying the target fragment, 5 XSF Buffer 10 mu L and dNTPs 1 mu L are added into a PCR small tube, 2 mu L of each of primer 1 and primer 2 (see table 1) are added, the genome DNA reaches 100ng, deionized water is used for constant volume to 50 mu L, and Phanta Super-Fidelity DNApolymerase 0.5.5 mu L is added for amplifying. PCR amplification procedure: 95 ℃,2min,95 ℃,10s,55 ℃,30s,72 ℃,1min and 30s, for 32 cyclesStored at 72℃for 5min at 4 ℃. After electrophoresis on a 1% agarose gel, the PCR product was spotted in an amount of 50. Mu.L, at a constant pressure of 110V for 30min. Recording the result by using a gel imager, cutting the gel, and purifying according to the specification of a gel recovery kit to obtain a G183V-1 fragment; as above, primers 3 and 4 (see Table 1) were amplified and purified to obtain the G183V-2 fragment; the G183V single-fragment was obtained by amplification purification using primers 1 and 4 (see Table 1) using the G183V-1 and G183V-2 fragments as templates. The G183V single-bulge fragment was used as a template, and primers 1 and 5 (see Table 1) were used for amplification to obtain a G183V+N482W-1 fragment; primers 4 and 6 (see Table 1) were amplified to obtain the G183V+N482W-2 fragment; then using two fragments as templates, amplifying and purifying by using primers 1 and 4 (see Table 1) to finally obtain CBDAS ^G183V+N482W And (5) double-protruding fragments, and sequencing and verification.

Example 3

The H114L-1 fragment was obtained by using primers 1 and 7 (see Table 1) and the H114L-2 fragment was obtained by amplification and purification of primers 8 and 4 (see Table 1) in the same manner as in example 2; the H114L single-protruding fragment was obtained by amplification and purification using primers 1 and 4 (see Table 1) using the H114L-1 and H114L-2 fragments as templates. The H114L single-protruding fragment is used as a template, and primers 1 and 9 (see Table 1) are used for amplification to obtain a H114L+C176W-1 fragment; primers 4 and 10 (see Table 1) were amplified to give the H2L+C176W-2 fragment; then using the purified products of the two fragments as templates, amplifying and purifying by using the primers 1 and 4 (see Table 1) to finally obtain the CBDAS ^H114L+C176W And (5) double-protruding fragments, and sequencing and verification.

The overlapping extension PCR is utilized to change the amino acid caused by single or multiple base differences in the CBDAS sequence, and the sequencing result is correct, thus forming the CBDAS ^G183V+N482W And CBDAS ^H114L+C176W Two mutant fragments.

CBDAS ^G183V+N482W And CBDAS ^H114L+C176W The sequence of the fragment is as follows, wherein the underlined positions in the sequence are the post-translational amino acid correspondence changes following the base mutation:

CBDAS ^G183V+N482W nucleotide sequence of (SEQ ID NO. 1):

ATGAAGTACTCAACATTCTCCTTTTGGTTTGTTTGCAAGATAATATTTTTCTTTTTCTCATTCAATATCCAAACTTCCATTGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAACAAATCTAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAATCTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCTCATATCCAAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTGGTCATGATTCTGAGGGCATGTCCTACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAACATGCGTTCAATCAAAATAGATGTTCATAGCCAAACTGCATGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTTGGCTGCTGGGTATTGCCCTACTGTTTGCGCAGGTGtACACTTTGGTGGAGGAGGCTATGGACCATTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATGGAAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGCAGAAAGCTTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTTTAGTGTTAAAAAGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGAATAAGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATTGATACTATCATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTTTGCTTGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGTCCAAATTTTGGAAAAATTATATGAAGAAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGAGATTTCTGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGCTGGGAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTCCTTATGTGTCCCAAAATCCAAGATTGGCATATCTCTGGTATAGAGACCTTGATATAGGAATAAATGATCCCAAGAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATTTTGACAGGCTAGTAAAAGTGAAAACCCTGGTTGATCCCAATAATTTTTTTAGAAACGAACAAAGCATCCCACCTCTTCCACGGCATCGTCATTAA。

CBDAS ^G183V+N482W amino acid sequence (SEQ ID NO. 2):

MKYSTFSFWFVCKIIFFFFSFNIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYCPTVCAGVHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLWYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH*。

CBDAS ^H114L+C176W nucleotide sequence of (SEQ ID NO. 3):

ATGAAGTACTCAACATTCTCCTTTTGGTTTGTTTGCAAGATAATATTTTTCTTTTTCTCATTCAATATCCAAACTTCCATTGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAACAAATCTAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAATCTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCTCATATCCAAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTGGTCTTGATTCTGAGGGCATGTCCTACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAACATGCGTTCAATCAAAATAGATGTTCATAGCCAAACTGCATGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTTGGCTGCTGGGTATTGGCCTACTGTTTGCGCAGGTGGACACTTTGGTGGAGGAGGCTATGGACCATTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATGGAAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGCAGAAAGCTTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTTTAGTGTTAAAAAGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGAATAAGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATTGATACTATCATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTTTGCTTGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGTCCAAATTTTGGAAAAATTATATGAAGAAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGAGATTTCTGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGCTGGGAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTCCTTATGTGTCCCAAAATCCAAGATTGGCATATCTCAATTATAGGGACCTTGATATAGGAATAAATGATCCCAAGAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATTTTGACAGGCTAGTAAAAGTGAAAACCCTGGTTGATCCCAATAATTTTTTTAGAAACGAACAAAGCATCCCACCTCTTCCACGGCATCGTCATTAA。

CBDAS ^H114L+C176W amino acid sequence (SEQ ID NO. 4):

MKYSTFSFWFVCKIIFFFFSFNIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGLDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYWPTVCAGGHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH*。

example 4

Construction and transformation of yeast expression vectors:

(1) CBDAS successfully sequenced example 2 ^G183V+N482W Fragment, CBDAS ^H114L+C176W The fragment and pPIC9K vector were digested with NotI and EcoRI restriction enzymes, respectively, and subjected to 1% agarose gel electrophoresis, followed by gel cutting and recovery. pPIC9K carrier and CBDAS after glue recovery ^G183V+N482W The mass ratio of the fragment-purified product reaches about 1:5, T ₄ Ligase Buffer 2μL，T ₄ Ligase 1. Mu.L, deionized water was made up to 20. Mu.L. The reaction was carried out at 25℃for 3h. Adding 10 mu L of the connecting solution into 50 mu L of escherichia coli DH5 alpha competence, and immediately carrying out ice bath for 20min;42 ℃ for 90s; after 3min of ice bath, shake to 45min at 37℃and 190 rpm. After centrifugation at room temperature for 2min, about 200. Mu.L of the supernatant was left to resuspend the cells, and the cells were spread evenly on LB plates containing Amp and incubated at 37℃for 16h. Colony PCR was performed on the single clone on the plates by using primers 1 and 12 (see Table 2) on every other day, positive recombinants were identified, and bacterial solutions were sent for sequencing verification. CBDAS ^H114L+C176W The construction method of the fragment is the same as above. Respectively extracting to obtain recombinant plasmids pPIC9K-CBDAS after sequencing verification ^H114L+C176W And pPIC9K-CBDAS ^G183V+N482W 。

(2) Recombinant plasmid pPIC9K-CBDAS ^H114L+C176W And pPIC9K-CBDAS ^G183V+N482W Linearization was performed with SacI restriction enzymes, respectively, followed by the following operations, respectively: 1 mu g of linearized recombinant plasmid is mixed into every 100 mu L of pichia pastoris competence, and after the mixture is uniformly mixed and added into an electric shock cup for 5min,1500V and 5ms of ice bath for power down, 600 mu L of 1M precooling is immediately addedSorbitol was mixed and incubated at 30℃for 1h. Centrifuge at room temperature for 2min, and leave 500. Mu.L of supernatant to resuspend the bacterial cells. 200. Mu.L of the bacterial liquid was spread on an MD plate and incubated at 30℃for 3d. The colonies on the plates were rinsed with 5mL deionized water, 200. Mu.L of the bacterial liquid was sequentially spread on YPD plates of different G418 concentrations (1 mg/mL, 2mg/mL, 4mg/mL, 6 mg/mL), cultured for 3d at 30℃and screened for high copy number yeast recombinants.

Screening of Yeast Positive recombinants:

selecting a monoclonal on a YPD plate, and releasing a yeast genome by adopting a high-temperature ethyl acetate method, wherein the method comprises the following steps of: selecting a proper amount of thalli by using a gun head, placing the thalli in 100 mu L of ethyl acetate, fully swirling the thalli, and heating the thalli to 100 ℃ until the ethyl acetate is volatilized; then 50 mu L of deionized water is added, vortex is carried out fully, heating is carried out for 5min at 100 ℃, and single colony DNA extracting solution of yeast is obtained and is used as a template for PCR reaction. The reaction system is as follows: 2×Taq Max 7.5. Mu.L, primers 1 and 12 (see Table 1) each 2. Mu.L, yeast single colony extract 2. Mu.L, deionized water was added to 15. Mu.L. PCR reaction procedure: 95 ℃ for 3min;95 ℃,20s,54 ℃,30s,72 ℃,2min10s,32 cycles; stored at 72℃for 5min at 4 ℃. The PCR products were identified by 1% agarose gel electrophoresis as shown in FIG. 4. The positive recombinant band is 2002bp, and a positive single colony is selected for glycerol cryopreservation.

According to FIG. 4, the CBDAS ^G183V+N482W And CBDAS ^H114L+C176W The mutant fragment is successfully inserted into a yeast expression vector, and PCR amplification is carried out by using a primer 12 on the yeast expression vector and a primer 1 designed by a CBDAS gene sequence, and the size of a strip accords with a positive control, which shows that pPIC9K-CBDAS ^H114L+C176W 、pPIC9K-CBDAS ^G183V+N482W The plasmids have been integrated into the chromosomes of pichia pastoris GS115, respectively.

TABLE 1 primers used in the present invention

Note that: the underline position being the joint

Induction expression of recombinant proteins:

selecting positive recombinants on a flat plate, and respectively separating wild CBDAS and mutant CBDAS ^H114L+C176W And mutant CBDAS ^G183V+N482W Inoculated in YPD liquid medium and cultured overnight at 30℃and 190 rpm. The next day was inoculated at 1% into 20mL of MGY medium and incubated at 30℃and 190rpm to OD ₆₀₀ At a value of 2-6, the number of yeasts is counted by a hemocytometer, and after the collection of the bacteria, the bacteria are resuspended to a bacterial count of about 8.0X10 with BMMY medium (pH 6.0) ⁹ Per mL, further cultured at 30℃and 190rpm, supplemented with 1% methanol by volume every 24 hours, and continuously induced for 48 hours, 3 replicates per group. Collecting thallus, adding appropriate amount of bacterial liquid, ultrasonic crushing for 25min, centrifuging to collect supernatant, and concentrating with ultrafilter tube to obtain wild CBDAS and mutant CBDAS ^G183V ^+N482W And mutant CBDAS ^H114L+C176W The recombinant protein solution was used for the detection of the catalytic activity after measuring the protein concentrations of 98.16. Mu.g/mL, 103.57. Mu.g/mL and 95.82. Mu.g/mL, respectively.

Example 5

CBDAS enzyme activity assay:

taking dried leaves of hemp varieties with high CBGA content, adding 50mL of methanol into each 0.1g, ultrasonically crushing for 30min, centrifuging for 20min at 4 ℃ to collect supernatant, removing impurities by using a 0.22 mu m filter membrane to obtain a CBGA crude extract with the concentration of 28.6 mu g/mL, and preserving at-20 ℃ for later use.

100. Mu.L (containing 2.86. Mu.g CBGA) of crude leaf extract, 0.5. Mu.L of TritionX-100, example 4 ultrafiltered concentrated recombinant protein solution (wild-type CBDAS, mutant CBDAS) ^G183V+N482W Or mutant CBDAS ^H114L ^+C176W ) 25 μg, and the volume was fixed to 500 μl with 0.1M sodium citrate buffer (pH 5.0). After reaction at 37℃for 12h, the reaction was terminated and each set was repeated three times, and the reaction mixture was centrifuged for 3min, and the supernatant was filtered through a 0.22 μm filter for HPLC detection.

Example 6

Adding CBGA standard substance, 0.5 mu LTrition X-100 and the recombinant protein solution after ultrafiltration concentration in example 4 into a 1.5mL centrifuge tube(wild-type CBDAS, mutant CBDAS) ^G183V+N482W Or mutant CBDAS ^H114L+C176W ) 25 μg, was then fixed to 500 μl (total concentration of CBGA 15.9 μΜ) with 0.1M sodium citrate buffer (pH 5.0). After reaction at 37℃for 12h, the reaction was terminated and each set was repeated three times, and the reaction mixture was centrifuged for 3min, and the supernatant was filtered through a 0.22 μm filter for HPLC detection.

CBDA and CBD content determination:

samples prepared from the reaction solutions obtained in the enzymatic reactions of examples 5 and 6 were subjected to CBDA and CBD product identification using an agilent high performance liquid chromatography apparatus, in which the liquid chromatography conditions were: chromatographic column: shimadzu sil-16C18 column (150 mm. Times.4.6 mm. Times.3 μm), column temperature: 30 ℃; mobile phase: a is aqueous solution containing 0.1% formic acid, B is acetonitrile containing 0.1% formic acid; isocratic elution: 25% A,75% B, retention time 30min; ultraviolet detector: 230nm; flow rate: 0.7mL/min; sample injection amount: 10 mu L. Analysis was performed with no enzyme solution, only substrate as control, and CBDA, CBD and CBGA standards as references. The results of the reaction of examples 5 and 6 to give the reaction liquid are shown in FIG. 5.

As shown in FIG. 5, when the reaction was carried out using CBGA in crude extract of hemp leaves as a substrate, under 100. Mu.L (containing 2.86. Mu.g CBGA) of crude extract of leaves, small amounts of CBD were detected in CK group, and it was found that the enzyme activities obtained by computer screening were higher than those of wild type, wherein the mutant CBDAS ^H114L+C176W Producing 63.08ng/mL and 43.98ng/mL CBDA and CBD 20.81% and 11.85% higher than wild-type; mutant CBDAS ^G183V+N482W CBDA and CBD of 75.7ng/mL and 50.76ng/mL were produced 37.09% and 29.13% higher than wild-type (A in FIG. 5). To reduce the interference of other substances in the crude extract of hemp leaves on the reaction, the wild-type and mutant CBDAS were further determined ^H114L+C176W And CBDAS ^G183V+N482W The same mass of CBGA standard is used as a substrate, and the conditions that the substrate CBGA is catalyzed by the recombinase to generate CBDA and CBD are analyzed after the reaction is carried out for 12 hours. As in FIG. 5B, mutant CBDAS at 15.9. Mu.M (2.86. Mu.g CBGA) ^H114L+C176W The content of the catalytic CBDA and the content of the CBD are respectively 15.69 percent and 8.37 percent higher than that of the wild type; mutant CBDAS ^G183V+N482W Catalytic formation of CBDA and content of CBD32.87% and 22% higher than the wild type, respectively.

Final determination of mutant CBDAS ^H114L+C176W And CBDAS ^G183V+N482W The enzyme activity of the polypeptide is higher than that of a wild type, and the polypeptide accords with the predicted result of molecular docking. After the reaction under the condition of containing the same mass of CBGA, the catalytic activity of the recombinase in the crude extract of hemp leaves is higher than that of a CBGA standard, but the CBD synthesis in the crude extract is less than that of CBDA, and the effect of other components in the crude extract is probably caused, and the specific reasons are still to be further studied.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. Cannabis diphenolic acid synthetase mutant CBDAS ^G183V+N482W The amino acid sequence is shown as SEQ ID NO. 2.

2. A cannabidiol synthase mutant CBDAS as claimed in claim 1 ^G183V+N482W The coding gene of (2) is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

3. A recombinant plasmid comprising the cannabidiol synthase mutant CBDAS of claim 2 ^G183V+N482W Is a coding gene of (a).

4. A recombinant microorganism strain comprising the recombinant plasmid of claim 3.

5. Cannabis diphenolic acid synthetase mutant CBDAS ^H114L+C176W The amino acid sequence is shown as SEQ ID NO. 4.

6. A method as claimed in claim 5Cannabidiol synthase mutant CBDAS ^H114L+C176W The coding gene of (2) is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.

7. A recombinant plasmid comprising the coding gene of claim 6.

8. A recombinant microorganism strain comprising the recombinant plasmid of claim 7.

9. A cannabidiol synthase mutant CBDAS as claimed in claim 1 ^G183V+N482W Or the cannabidiol synthase mutant CBDAS of claim 5 ^H114L+C176W The application in improving the conversion efficiency of cannabidiol acid generated by cannabigerol acid.

10. A method for increasing the yield of cannabidiol from cannabis plants comprising utilizing the cannabidiol synthase mutant CBDAS of claim 1 ^G183V+N482W Or the cannabidiol synthase mutant CBDAS of claim 5 ^H114L+C176W Catalyzing cannabigerol acid in cannabis plants to generate cannabidiol acid, thereby forming cannabidiol.