A santalene synthase mutant with multiple mutation sites and preparation method thereof

Abstract

The present invention relates to a mutant enzyme of santalene synthase and its application in synthesizing santalene. Specifically involved mutant enzymes are SanSyn (S533A), SanSyn (S533Q), and SanSyn (Q527A & S533A), which are respectively mutated to alanine, glutamine, glutamine at position 533, and mutated to alanine at position 527 and serine at position 533. A method for synthesizing α-santalene in host Escherichia coli DH5α using glucose as a carbon source mainly includes constructing recombinant plasmids pETDuet-SanSyn (S533A), pETDuet-SanSyn (S533Q), pETDuet-SanSyn (Q527A & S533A), and pMVA; constructing a recombinant strain of Escherichia coli, and further fermenting and culturing. The technical solution involved in the present invention significantly increases the yield of α-santalene, and the protein soluble expression is also improved. It lays a foundation for further metabolic engineering of Escherichia coli to produce α-santalene. Provide a reference for the modification of terpene synthase.

Description

[0001] This application is a divisional application. The Chinese national application number of the original application is 202111085704.X, the application date is September 16, 2021, and the name of the invention is “A santalene synthase mutant and its preparation method”. Technical Field

[0002] The invention belongs to the technical field of enzyme engineering, and particularly relates to a santalene synthase mutant and a preparation method thereof. Background Art

[0003] Santalene is a sesquiterpene and a precursor of santalol. Santalene and santalol are the main components of sandalwood essential oil. Sandalwood essential oil is mostly used in cosmetics and fragrances, and has good antibacterial, antioxidant and anti-tumor pharmacological activities. In addition, sandalwood essential oil has low toxicity in animals and is non-mutagenic. It is considered a safe food additive in many European and American countries.

[0004] At present, sandalwood essential oil is mainly obtained by plant extraction, but due to the harsh growth conditions and long growth period of sandalwood, the sandalwood oil content in the trees is scarce, the separation process is complicated and cumbersome, and the extraction is difficult. It cannot meet market demand and will cause a large number of sandalwood trees to be cut down. In addition, although studies have reported that bromocamphor is used as a substrate and santalin is obtained through an eight-step chemical catalytic reaction, the chemical reaction conditions are harsh, the cost is high, and the path is cumbersome. In order to obtain a pure product, the intermediate reaction products need to be separated and purified again and again, which is not an ideal and effective means for large-scale production of santalin. Therefore, the heterologous biosynthesis of santalin and santalol by microbial cell factories provides a new sustainable and green production method for terpenoid compounds with scarce resources and high costs.

[0005] The key steps of terpene biosynthesis include the cyclization of acyclic GPP, FPP, GGPP into monoterpenes, sesquiterpenes or diterpenes by terpene synthases (or cyclases). Santalene synthase (SanSyn) from yellow peel belongs to a type of plant terpene synthase, and these enzymes all contain a similar conserved domain DDxxD ("x" represents any amino acid). At present, the catalytic efficiency, substrate specificity and stability of terpene synthases limit their utilization. We break this limitation by molecular modification of terpene synthases. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a santalene synthase mutant with multiple mutation sites.

[0009] To solve the above technical problems, the present invention provides the following technical solution: the 533rd serine of the wild-type SanSyn enzyme whose nucleotide sequence is shown in SEQ ID NO.1 is mutated to alanine or glutamine to obtain mutant S533A or S533Q;

[0010] The glutamine at position 527 and the serine at position 533 of the SanSyn enzyme whose nucleotide sequence is shown in SEQ ID NO.1 are mutated to alanine to obtain the mutant Q527A&S533A;

[0011] Wherein, the wild-type SanSyn enzyme is derived from the plant Clausenalansium.

[0012] As a preferred embodiment of the santalene synthase mutant with multiple mutation sites of the present invention, wherein:

[0013] The santalene synthase mutant with multiple mutation sites also includes:

[0014] The nucleotide sequence of the mutant S533A is shown in SEQ ID NO.3;

[0015] The nucleotide sequence of the mutant S533Q is shown in SEQ ID NO.4;

[0016] The nucleotide sequence of the mutant Q527A&S533A is shown in SEQ ID NO.5.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a santalene synthase mutant with multiple mutation sites, comprising:

[0018] The wild-type SanSyn enzyme gene with a nucleotide sequence as shown in SEQ ID NO.2 is connected to the plasmid pETDuet-tac to obtain the recombinant plasmid pETDuet-tac-SanSyn, wherein the double restriction sites of the connection are NcoI and BamHI, respectively, and the nucleotide sequence of the plasmid pETDuet-tac is shown in SEQ ID NO.6;

[0019] The nucleotide sequences of the designed mutation primers are respectively as shown in SEQ ID NO.7 to SEQ ID NO.11: mutation primers S533A-F, S533-R, S533Q-F, Q527A&S533A-F, Q527A&S533A-R;

[0020] Wherein, the primers S533A-F and S533-R are used to obtain the mutant S533A;

[0021] Primers S533Q-F and S533-R were used to obtain mutant S533Q;

[0022] Primers Q527A&S533A-F and Q527A&S533A-R were used to obtain mutant Q527A&S533A;

[0023] PCR amplification was performed using mutation primers and plasmid pETDuet-tac-SanSyn as a template to obtain mutation products at different sites, which were transformed into competent cells of the host cell Escherichia coli TOP 10, and the santalene synthase SanSyn mutant expression strain was screened and induced to express, thereby obtaining santalene synthase mutants with multiple mutation sites.

[0024] Beneficial effects of the present invention:

[0025] After 3 days of post-induction fermentation culture, the α-santalene concentration of the S533A mutant strain reached 1028 mg / L, which was 1.9 times higher than that of the non-mutated strain. The final α-santalene concentration of the S533Q mutant strain reached 959 mg / L, and the α-santalene concentration of the Q527A&S533A double-point mutant strain reached 815 mg / L, which were all higher than those of the non-mutated strain. The soluble protein expression of the SanSyn mutant S533Q was also improved.

[0026] The invention provides a recombinant strain containing the santalene synthase encoding gene, and increases the accumulation amount of alpha-santalene and the expression of soluble protein by site-directed mutation of the SanSyn gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0028] Figure 1 This is a three-dimensional structure simulation diagram of the SanSyn enzyme.

[0029] Figure 2 It is the α-santalene production of the recombinant bacteria in Example 2 at 24h, 48h and 72h.

[0030] Figure 3 The recombinant bacteria in Example 2 include a plasmid.

[0031] Figure 4 The relative expression of soluble protein of the santalene synthase mutant in Example 4. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] The primers used in the present invention were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0036] The DMT enzyme used in the present invention was purchased from Beijing Quanshijin Biotechnology Co., Ltd. The PrimeSTARMax used was purchased from Takara. The gel recovery kit Axygen DNA was purchased from Aisjin Biotechnology Co., Ltd. All Western Blot experiment related reagents were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0037] The invention provides a method for producing alpha-santalene, which specifically comprises the following steps: using Escherichia coli as a host, recombinantly expressing a gene encoding a santalene synthase and a mutant thereof to obtain a recombinant bacterium; activating the recombinant Escherichia coli with a seed culture medium and then transferring the recombinant Escherichia coli into a fermentation culture medium, culturing the recombinant Escherichia coli at 37°C and a rotation speed of 200 rpm, adding IPTG to induce enzyme expression when OD600 reaches 1.5, and making the recombinant bacterium produce alpha-santalene with glucose as a substrate.

[0038] Other raw materials used, unless otherwise specified, are commercially available.

[0039] Example 1

[0040] Establishment of SanSyn enzyme tertiary structure simulation model:

[0041] The homology modeling tool Swiss Model was used to perform homology modeling on SanSyn with the santalene synthase SaSS (PDB: 5ZZJ) from Santalumalbum as the reference model. After the model was evaluated, a reliable three-dimensional structural model was obtained. The three-dimensional structural model is shown in the figure Figure 1 shown.

[0042] Example 2

[0043] Construction of site-directed mutagenesis library:

[0044] Using PCR technology, the plasmid pETDuet-SanSyn expressing the wild-type SanSyn gene was used as a template to perform site-directed mutagenesis on positions 19, 408, 442, 473, 527 and 533 of the SanSyn enzyme.

[0045] Design mutation primers. Forward and reverse primers are PCR upstream and downstream primers designed accordingly according to different mutation sites. The specific primer information is shown in Table 1.

[0046] Table 1 Primers used to obtain SanSyn mutants

[0047]

[0048]

[0049] The primer pair F19Y-F and F19Y-R were used to obtain mutant F19Y, and the amino acid sequence of mutant F19Y was SEQ ID NO.12.

[0050] The primer pair I408L-F and I408L-R were used to obtain mutant I408L, and the amino acid sequence of mutant I408L was SEQ ID NO.13.

[0051] The primer pair F442S-F and F442S-R were used to obtain the mutant F442S, and the amino acid sequence of the mutant F442S was SEQ ID NO.14.

[0052] Primer pair V473A-F and V473A-R were used to obtain mutant V473A, and the amino acid sequence of mutant V473A was SEQ ID NO.15.

[0053] Primer pair Q527A-F and Q527A-R were used to obtain mutant Q527A, and the amino acid sequence of mutant Q527A was SEQ ID NO.16.

[0054] The primer pair S533A-F and S533-R was used to obtain mutant S533A, and the nucleotide sequence of mutant S533A was SEQ ID NO.3.

[0055] The primer pair S533Q-F and S533-R was used to obtain mutant S533Q, and the nucleotide sequence of mutant S533Q was SEQ ID NO.4.

[0056] The primer pair Q527A&S533A-F and Q527A&S533A-R were used to obtain mutant Q527A&S533A. The nucleotide sequence of mutant Q527A&S533A is SEQ ID NO.5.

[0057] The PCR reaction system was: 25 μL of PrimeSTARMax (purchased from Takara), 1 μL each of the forward primer, reverse primer, and template DNA, and 22 μL of sterile water.

[0058] The PCR amplification program was as follows: pre-denaturation at 98°C for 5 min, followed by denaturation at 98°C for 10 s, annealing for 20 s, annealing temperature as shown in Table 1, 25 cycles at 72°C for 45 s, and finally incubation at 72°C for 5 min. The recovered fragment was a band of about 7000 bp.

[0059] The above fragment samples were subjected to template removal reaction, and the template removal reaction system was 1 μL of DMT enzyme and 9 μL of purified product, and the reaction was carried out at 37° C. for 1 h.

[0060] Phosphorylation was carried out at 37°C for 3 h. The phosphorylation reaction system included 1 μL T4 Polynucleotide Kinase, 1 μL 10×T4 Polynucleotide Kinase Buffer, 1 μL ATP, and 7 μL sample. After phosphorylation, the enzyme was inactivated at 70°C for 5 min.

[0061] The ligation reaction was carried out at 16°C overnight. The ligation system included 1 μL T4 DNA Ligase, 1 μL 10×T4 DNA LigaseBuffer, and 8 μL sample.

[0062] Add all 10 μL of the ligation product to the competent cells of E. coli TOP10, mix thoroughly, place on ice for 5 minutes, and then heat shock the mixed bacterial solution and plasmid in a 42°C water bath for 90 seconds. After the heat shock is completed, place it in ice water for 5 minutes, add 1 mL of SOC medium, mix well, and recover at 37°C for 45 minutes. Then apply the bacterial solution to the LB plate containing ampicillin resistance and culture at 37°C overnight. Pick the transformants on the plate and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0063] The target gene fragment was recovered using the Axygen DNA gel recovery kit, and the specific steps were as follows:

[0064] (1) Cut the agarose gel containing the target DNA and calculate the gel weight, with one weight being one gel volume (100 mg = 100 μL).

[0065] (2) Add three gel volumes of Buffer DE-A, mix well, and heat at 75°C with intermittent mixing until the gel blocks are completely melted.

[0066] (3) Add 0.5 volume of Buffer DE-B to Buffer DE-A and mix well. The solution will turn yellow after mixing.

[0067] (4) The mixed yellow solution was transferred to a preparation tube, centrifuged at 12000 rpm for 1 min, and the filtrate was discarded.

[0068] (5) Place the preparation tube back into the centrifuge tube, add 700 μL Buffer W1, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0069] (6) Place the preparation tube back into the centrifuge tube, add 700 μL Buffer W2, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; wash once more with Buffer W2 in the same way. Discard the filtrate.

[0070] (7) Place the preparation tube back into the centrifuge tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0071] (8) Move the preparation tube into a new 1.5 mL centrifuge tube, add 20 μL of Eluent to the center of the preparation tube, let stand at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min. The target gene fragment can be obtained.

[0072] Example 3

[0073] Construction of recombinant E. coli strains:

[0074] The plasmid containing santalene synthase and its mutants and plasmid pMVA constructed in Specific Example 1 were transformed into Escherichia coli DH5α (see Construction of Plasmids Figure 3), pick three single colonies on the plate of the strain, inoculate them into 5mL of liquid LB with ampicillin and chloramphenicol resistance, culture overnight at 37°C, and then inoculate them into 50mL of TB medium with ampicillin and chloramphenicol resistance (the culture medium components include 20g / L glucose, 12g / L tryptone, 24g / L yeast extract, 9.4g / L potassium dihydrogen phosphate, 2.2g / L potassium dihydrogen phosphate), and then culture in a shaker at 37°C and 200rpm for 3-4h. After the OD600 value of the recombinant Escherichia coli reaches about 1.5, add the inducer IPTG for induction, and its working concentration is 0.5mM. At the same time, add 5mL of isopropyl myristate to extract the target product α-santalene. The fermentation process is cultured in a shaker at 25°C and 200rpm for 72h. Samples are taken every 24h, and the concentration of α-santalene is determined by gas chromatography. The results are shown in Figure 2 .

[0075] Depend on Figure 2 It can be seen that after 3 days of fermentation culture after induction, the concentration of α-santalene in the S533A mutant strain reached 1028 mg / L, which was 1.9 times higher than that of the non-mutated strain. The concentration of α-santalene in the Q527A mutant strain finally reached 933 mg / L, the final concentration of α-santalene in the S533Q mutant strain reached 959 mg / L, and the concentration of α-santalene in the Q527A&S533A double-point mutant strain was 815 mg / L. All of them were higher than the concentration of the non-mutated strain.

[0076] Example 4

[0077] Western Blot analysis of santalene synthase and its mutant proteins:

[0078] According to the experimental steps of the present invention, plasmids pET28a-SanSyn-6His, pET28a-SanSyn(S533A)-6His, pET28a-SanSyn(S533Q)-6His and pET28a-SanSyn(Q527A)-6His were constructed respectively.

[0079] The construction of the above plasmid is divided into two steps. First, add a 6xHis tag to the C-terminus of each santalene synthase, use pETDuet-SanSyn, pETDuet-SanSyn (S533A), pETDuet-SanSyn (S533Q), and pET28a-SanSyn (Q527A) as vectors, design forward primer: 5'-CATCACCATCATCACCACTAAGGATC-3'; reverse primer: 5'-ATCATCTAATTTAACCGGATCTTTCAGC-3'. The PCR reaction amplification program is: 98°C pre-denaturation for 5min; then denaturation at 98°C for 10s, annealing for 20s, annealing temperature of 59°C, 72°C for 45s for 25 cycles, and finally 72°C for 5min. Recover the band of 7000bp-8000bp, and the specific steps of template removal, phosphorylation reaction, ligation, and heat shock conversion are described in Example 2.

[0080] Secondly, the santalene synthase (SanSyn) and its mutant genes containing a 6xHis tag at the C-terminus were inserted between the NcoⅠ and BamHI sites of the plasmid pET28a.

[0081] Double enzyme digestion system: 36 μL of gene fragment, 5 μL of 10xK Buffer, 5 μL of BSA, 2 μL of NcoⅠ enzyme, 2 μL of BamHI enzyme, 37°C, 3 h, recovery of vector pET28a and SanSyn gene fragment. The recovery steps are the same as those in Example 2.

[0082] Then, the vector pET28a and the SanSyn gene fragment were connected by Ligase. The connection system included 4 μL of the vector fragment and the target fragment, 1 μL of T4 DNA Ligase, and 1 μL of T4 DNA Ligase Buffer. The connection was carried out at 16°C overnight.

[0083] The ligated fragments were transformed into E. coli BL21 (DE3). A single colony was picked from a kanamycin-resistant plate and inoculated into 5 mL of liquid LB with kanamycin resistance, cultured overnight at 37°C, inoculated into 100 mL of LB with kanamycin resistance (inoculation volume 1%), cultured in a shaker at 37°C and 200 rpm for 2-3 hours, and after the OD=600 value of the recombinant E. coli reached 0.6-0.8, the inducer IPTG was added for induction, the working concentration of which was 0.5 mM, and then cultured in a shaker at 20°C and 180 rpm for 20 hours.

[0084] To prepare protein samples, take a certain amount of bacterial solution, centrifuge and discard the supernatant, rinse the bacteria twice with PBS buffer, then add 100 μL of cell lysis solution to each sample, place it on ice for 40 minutes, centrifuge it at 12000 rpm, 4°C for 10 minutes, and aspirate the supernatant into a new EP tube, which is the total cell protein.

[0085] The protein concentration was determined by the BCA method using the BCA protein concentration determination kit from Shanghai Jierui Bioengineering Co., Ltd. Reagent A and reagent B were mixed at a ratio of 50:1, the reaction system was 200 μL reagent + 20 μL protein sample, reacted at 37°C for 30 min, and the absorbance value A was measured at an absorbance of 562 nm. The protein concentration (μg / μL) was calculated according to the formula C=(A-0.0131) / 2.378, and the protein sample was adjusted to a consistent concentration with PBS solution. The sample loading was 20-50 μg, and the sample loading was ~20 μL.

[0086] For SDS-PAGE gel electrophoresis, use 10% SDS-PAGE gel to run the gel, load 20-50μg, load ~20μL. After the electrophoresis, remove the gel and place it in the pre-cooled transfer solution. Cut the PVDF membrane to the same size as the separation gel, activate it in methanol for about 15 seconds, and then wash it with the transfer solution.

[0087] Transfer, cut the PVDF membrane to the same size as the separation gel, activate it in methanol for about 15 seconds, and then wash it with transfer solution. Make a "sandwich" electrotransfer device with filter paper, sponge pad and gel, and place it in the transfer tank in the order of negative electrode, sponge, filter paper, separation gel, PVDF membrane, filter paper, sponge, and positive electrode, ensuring precise alignment between the layers and no bubbles. Immerse it in the transfer tank filled with transfer solution, add ice packs to cool it down, and transfer the membrane at a constant current of 350mA for 1 hour.

[0088] After the transfer is completed, remove the PVDF membrane and place it in a blocking solution containing 5% skim milk (prepared in TBST), with the front side facing down, and incubate at room temperature with shaking for 1 hour.

[0089] Antibody incubation and membrane washing: 1) Wipe the membrane dry, seal it with PE gloves, cut it to the appropriate size according to the position of the marker, and mark it; 2) Primary antibody incubation: dilute the monoclonal antibody with TBST (SanSyn and its mutant proteins use Histag mouse monoclonal antibody, and the internal reference GAPDH protein uses IgG antibody), place the PVDF membrane in the prepared primary antibody solution, and rotate and bind at 4°C overnight; 3) Membrane washing: take out the PVDF membrane the next day and wash it 3 times with TBST, each time for 10 minutes. 4) Secondary antibody incubation: dilute the corresponding horseradish peroxidase-labeled IgG with TBST solution, place the PVDF membrane in it (the operation is similar to the primary antibody incubation), and incubate at room temperature for 2 hours; 5) Membrane washing: wash the membrane 3 times with TBST, each time for 10 minutes.

[0090] For color development, prepare the A and B solutions in the ECL chemiluminescent solution (kit) in a ratio of 1:1, add them to the PVDF membrane, and react for 1 minute under light-proof conditions or longer according to actual conditions. The color development solution should be added and used immediately, and a chemiluminescent instrument should be used to take pictures after adding. The experimental results are shown in Figure 4 .

[0091] The solution formula is 10× transfer solution: Tris 15.18g, glycine 72.08g, configured into 500mL solution; 1× wet transfer solution: 100mL 10× transfer solution, 200mL methanol, distilled water to 1000mL; blocking solution is 5% skim milk powder: 1% skim milk powder dissolved in 20mL TBST. Western Blot experiment related reagents: PVDF membrane, 6xHis tag and GAPDH primary and secondary antibodies, 10xTBST, skim milk powder, ECL chemiluminescent solution were purchased from Shanghai Biotech Biotechnology Co., Ltd.

[0092] Western Blot experiments of santalene synthase and its mutants showed that the soluble protein expression of S533Q and Q527A single mutations was increased compared with the wild-type santalene synthase (the relative protein expression of the wild-type santalene synthase was 100%), and the soluble protein expression of the Q527A mutant was increased by 1.2 times.

[0093] The invention provides a method for producing alpha-santalene, which specifically comprises the following steps: using Escherichia coli as a host, recombinantly expressing a gene encoding a santalene synthase and a mutant thereof to obtain a recombinant bacterium; activating the recombinant Escherichia coli with a seed culture medium and then transferring the recombinant Escherichia coli into a fermentation culture medium, culturing the recombinant Escherichia coli at 37°C and a rotation speed of 200 rpm, adding IPTG to induce enzyme expression when OD600 reaches 1.5, and making the recombinant bacterium produce alpha-santalene with glucose as a substrate.

[0094] The α-santalene concentration of the S533A mutant strain reached 1028 mg / L after 3 days of fermentation after induction, which was 1.9 times higher than that of the non-mutated strain. The final α-santalene concentration of the S533Q mutant strain reached 959 mg / L, and the α-santalene concentration of the Q527A&S533A double-point mutant strain was 815 mg / L, which was higher than that of the non-mutated strain.

[0095] The present invention provides a recombinant strain containing the santalene synthase encoding gene, and increases the accumulation of α-santalene by site-directed mutation of the SanSyn gene. Western Blot experiments show that the soluble protein expression of the mutant S533Q of SanSyn is also increased.

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A santalene synthase mutant, characterized in that: The santalene synthase mutant is obtained by mutating the 533rd serine of the wild-type SanSyn enzyme shown in SEQ ID NO.1 to alanine or glutamine to obtain mutant S533A or S533Q; or the glutamine at position 527 and the serine at position 533 of the SanSyn enzyme whose amino acid sequence is shown in SEQ ID NO.1 are both mutated to alanine to obtain the mutant Q527A & S533A; Wherein, the wild-type SanSyn enzyme is derived from the plant yellow peel ( Clausena lansium ).

2. The santalene synthase mutant according to claim 1, characterized in that: The nucleotide sequence of the mutant S533A is shown in SEQ ID NO. 3; the nucleotide sequence of the mutant S533Q is shown in SEQ ID NO. 4; and the nucleotide sequence of the mutants Q527A & S533A is shown in SEQ ID NO.

5.

3. The method for preparing the santalene synthase mutant according to claim 2, characterized in that: The preparation method comprises: The wild-type SanSyn enzyme gene with a nucleotide sequence as shown in SEQ ID NO.2 is connected to the plasmid pETDuet-tac to obtain the recombinant plasmid pETDuet-tac-SanSyn, wherein the double restriction sites of the connection are NcoI and BamHI, respectively, and the nucleotide sequence of the plasmid pETDuet-tac is shown in SEQ ID NO.6; The nucleotide sequences of the designed mutation primers are respectively as shown in SEQ ID NO.7 to SEQ ID NO.11: mutation primers S533A-F, S533-R, S533Q-F, Q527A&S533A-F, Q527A&S533A-R; Wherein, the primers S533A-F and S533-R are used to obtain the mutant S533A; Primers S533Q-F and S533-R were used to obtain mutant S533Q; Primers Q527A&S533A-F and Q527A&S533A-R were used to obtain mutant Q527A & S533A; PCR amplification was performed using mutation primers and plasmid pETDuet-tac-SanSyn as a template to obtain mutation products at different sites, which were transformed into competent cells of the host cell Escherichia coli TOP 10, and the santalene synthase SanSyn mutant expression strain was screened and induced to express, thereby obtaining santalene synthase mutants with multiple mutation sites.

4. Use of the santalene synthase mutant according to claim 1 or 2 in the preparation of α-santalene.

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