Santalene synthase mutant and application thereof

Abstract

The invention discloses a santalene synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. Different santalene synthase mutants are screened through bioinformatics analysis, I402C mutation enables the ratio of alpha configuration in the product santalene to be 54.2%, the ratio of beta configuration in the product santalene to be 32.0%, and the configuration ratio accords with the configuration ratio of natural sandalwood essential oil. The mutation of P460A and P460S enables the beta configuration proportion in the product santalene to exceed 50%, which is reported for the first time in China. The configuration proportion of the natural sandalwood essential oil and the sandalene containing more beta configuration proportions are realized, possibility is provided for producing sandalwood essential oil with different compositions according to specific requirements in industrial production, and the method has a good application prospect. Meanwhile, the biological preparation method of the santalene has the advantages of simplicity and convenience in operation, environmental friendliness, mild reaction conditions and the like, and has a good application prospect in the field of production of the santalene.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering and microbial engineering, and relates to a mutant of a santalene synthase (SanSyn) having an amino acid sequence such as that shown in SEQ ID No. 3 in a sequence table, and the application of a recombinant expression cell thereof in the biological preparation of santalene with different configuration ratios, and relates to a nucleic acid sequence encoding the enzyme, a recombinant expression vector comprising the encoding nucleic acid sequence, and a method for preparing santalene. Background Art

[0002] Sesquiterpenoids are 15-carbon compounds composed of 3 isoprenoid units, belonging to one of the terpenoid family, most of which have strong aroma and biological activity, and are widely used in food, cosmetics, and pharmaceutical industries. Both santalene and santalol belong to sesquiterpenoids, and santalene is the precursor of santalol synthesis, and the two are the main components of sandalwood essential oil. Sandalwood essential oil has a pleasant and lasting fragrance, is one of the most popular natural spices in the world, and is widely used in the cosmetics industry and the food industry. In addition, sandalwood essential oil also has many pharmacological activities with health benefits, such as antioxidant, antibacterial, antiviral, anticancer activity, etc., and has great economic value. At present, sandalwood essential oil is mainly obtained by steam distillation from the heartwood and roots of sandalwood trees, but sandalwood trees need to grow for 30 years to extract sandalwood essential oil, and the sandalwood essential oil extraction rate of mature sandalwood trees is only between 2.5% and 6%, which is far from meeting the growing market demand for sandalwood products. The process of synthesizing santalene and santalol by chemical synthesis is complex, the reaction conditions are harsh, the cost is high, the product purity is low, and the price of natural sandalwood essential oil remains high. Compared with traditional plant extraction and chemical synthesis methods, the method of biosynthesis by constructing microbial cell factories provides the possibility for the green, efficient and low-cost industrial production of sesquiterpenes.

[0003] So far, more than 200 components have been identified from sandalwood essential oil, mainly composed of sesquiterpenoids, such as (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol, (Z)-α-exo-bergamoto, α-santalene, β-santalene, α-exo-bergamotene, and epi-β-santalene. Among them, (Z)-α-santalol and (Z)-β-santalol are the two most critical components that affect the biological activity and fragrance of sandalwood essential oil. α-santalol has a slight woody smell, similar to the smell of cedar wood and α-cedrene; while the abundance of β-santalol is usually low, but the aroma is stronger than α-santalol. People believe that the main healing effect of sandalwood is attributed to α-santalol, while the rich and heavy woody aroma of sandalwood mainly comes from β-santalol. According to the quality control index established by the International Organization for Standardization (ISO 3518:2002), the content of α-santalol and β-santalol in natural sandalwood essential oil must be within the range of 41-55% and 16-24% respectively.

[0004] As mentioned above, the composition ratio of sandalwood essential oil determines the flavor and quality of sandalwood essential oil, and the reasonable control of product ratio is the difficulty in constructing a heterologous biosynthesis system of sandalwood essential oil. In the biosynthesis pathway of santalol, the main component of sandalwood essential oil, in Saccharomyces cerevisiae, santalene synthase is not only the rate-limiting enzyme of the pathway, but also the key enzyme that determines the configuration ratio of santalol. The catalytic enzyme CYP450 (Cytochrome P450 monooxygenase) from ene to alcohol shows similar preferences for substrates of different configurations. The two types of santalene synthases currently in use are the SanSyn enzyme from Clausena lansium and the SaSSy enzyme from S. album. SaSSy is a product-mixed enzyme, and its products include four different configurations of santalol. Zha et al. introduced SaSSy enzyme and CYP450 / CPR into Saccharomyces cerevisiae, and the α and β configuration ratios of its product santalol were 35.7±1.1% and 17.8±0.8%, respectively. The configuration ratio of santalol did not meet the ISO standard. Unlike the SaSSy enzyme with mixed products, SanSyn is a santalene synthase with α-specific product configuration. It uses (E,E)-FPP as a substrate, and its product is mainly α-santalene, accompanied by a trace amount of α-bergamotene. Zha et al. proposed that the lack of unoccupied space in the active pocket of SanSyn is the key factor that restricts its intermediate conformation and the α-specificity of the product. They introduced the mutant SanSyn into Saccharomyces cerevisiae. F411VThe ratio of α and β configurations of the product santalene was 57.2% and 28.6%, and after further introduction into CYP450, the ratio of α and β configurations of the product was 43.4% and 22%, which met the ISO standard, but the activity of the enzyme was greatly reduced after mutation. In addition, Styles et al. reported that the β configuration of the santalene product was greater than the α configuration after the CiCassy enzyme from Cinnamomumcamphora was mutated to N267S / N267L, but the yield was not reported.

[0005] Enzyme engineering has been proven to be an effective method to optimize enzyme performance. We modified the configuration ratio of the product santalene by molecular modification of the enzyme, and finally obtained a strain of Saccharomyces cerevisiae with an ideal ratio of santalene products to meet the needs of large-scale biosynthesis of sandalwood essential oil in the future. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a santalene synthase mutant.

[0007] Another object of the present invention is to provide use of the above-mentioned santalene synthase mutant in the preparation of santalene.

[0008] The technical problem to be solved by the present invention is to provide a series of santalene synthase mutants with better catalytic activity in terms of product ratios, in view of the fact that santalene synthase is a key enzyme for the biosynthesis of santalene santalol and a key enzyme for determining the configuration ratio of santalol, but its product configuration ratio is single.

[0009] Enzymes whose product configuration ratios meet ISO standards make the products closer to the components of natural sandalwood essential oil. In addition, enzymes with higher specificity for β-santalene are desirable because β-santalol has a strong and heavy smell and is considered to give sandalwood its most important smell. In industrial production, it is ideal to be able to control the product ratio of the two main products of santalene synthase according to specific needs.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A santalene synthase mutant, the amino acid sequence of the santalene synthase mutant is shown in SEQ ID No.3, wherein at least one site marked as "X" in SEQ ID No.3 and the amino acid residue of the corresponding site in SEQ ID No.2 are mutated.

[0012] The santalene synthase is derived from the plant Clausena lansium, the GenBank number of the amino acid sequence is ADR71055.1, and the GenBank number of the encoding gene is HQ452480.1.

[0013] In the present invention, the santalene synthase is SanSyn, whose nucleotide sequence after codon optimization is shown in SEQ ID NO.1, which contains 1656 nucleotides in total; and its amino acid sequence is shown in SEQ ID No.2, which contains 551 amino acids in total. Further modification is performed on the basis of SanSyn.

[0014] It has been found in particular that mutations at the following positions of SEQ ID No. 2 are advantageous for obtaining mutants with altered product ratios: 269, 273, 402, 460, 503, 514, 529. In a preferred embodiment, good results have been achieved with SanSyn compared to SEQ ID No. 2 at one of the following positions: 269, 273, 402, 460, 503, 514, 529. It is to be noted that in principle there is no limit to the number of mutated amino acid residues in SanSyn, provided that the enzyme retains sufficient catalytic properties as a santalene synthase.

[0015] Preferably, the santalene synthase mutants are SanSyn_L269A, SanSyn_I273S, SanSyn_I402C, SanSyn_P460S, SanSyn_P460A, SanSyn_P460T, SanSyn_I503A, SanSyn_L514C, and SanSyn_F529V.

[0016] More preferably, the santalene synthase mutant is SanSyn_I402C, i.e., mutant I402C in which isoleucine at position 402 is mutated to cysteine, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence is shown in SEQ ID NO.5; SanSyn_P460A, i.e., mutant P460A in which proline at position 460 is mutated to alanine, and the amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence is shown in SEQ ID NO.7. SanSyn_P460S, i.e., mutant P460S in which proline at position 460 is mutated to serine, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence is shown in SEQ ID NO.9.

[0017] The present invention provides a gene encoding the above-mentioned santalene synthase mutant.

[0018] The present invention provides a recombinant expression vector containing the gene encoding the SanSyn mutant, and provides a method for preparing santalene by introducing the recombinant expression vector into an engineering strain of Saccharomyces cerevisiae.

[0019] According to the present invention, experiments have confirmed that the mutant of santalene synthase SanSyn described in the present invention has an ideal change in product ratio compared to the SanSyn with product configuration α specificity that has been reported and widely used in biosynthesis. The experimental results show that under the same conditions, the recombinant strain constructed using the SanSyn mutant of SEQ ID No.4 described in the present invention has a product santalene configuration ratio that conforms to the configuration ratio of natural sandalwood essential oil; the recombinant strain constructed using the SanSyn mutants of SEQ ID No.6 and SEQ ID No.8 described in the present invention has a product santalene with a β configuration accounting for more than 50%, which is rarely reported.

[0020] The term "mutation" as used herein means that at least one nucleotide or amino acid in the SanSyn mutant gene or amino acid sequence is different from the SanSyn starting sequence for comparison. The mutation of the enzyme can be achieved by site-directed mutagenesis using conventional methods in the art.

[0021] The term "nucleic acid molecule" used in this article has the meaning generally understood by ordinary technicians in the field. A nucleic acid molecule can be a polynucleotide including a polynucleotide as shown in NCBI Accession No. HQ452480.1 or SEQ ID No. 1, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0022] The terms "vector" and "expression vector" used herein have the meanings commonly understood by those of ordinary skill in the art. "Vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. In the process of using Saccharomyces cerevisiae to express foreign genes, the foreign genes to be expressed must first be connected to the vector. The vector can be any conventional vector in the art, such as commercially available plasmids, phages, viral vectors, etc. The preferred plasmid YEp352 of the present invention expresses the santalene synthase mutant gene.

[0023] In the invention, a recombinant expression cell comprising the vector of the present invention is provided. The recombinant expression cell can be obtained by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated and propagated, and the gene carried by the recombinant expression vector can be effectively expressed. The present invention preferably uses Saccharomyces cerevisiae, and more preferably S.cerevisiae CEN.PK2-1C strain or S.cerevisiae PL00 strain.

[0024] In the present invention, the mutant-related biological material is any one or more combinations of the following biological materials:

[0025] (a) an expression cassette containing the above encoding gene;

[0026] (b) a recombinant expression vector containing the above encoding gene;

[0027] (c) a recombinant expression vector containing the expression cassette described in (a);

[0028] (d) a recombinant expression cell containing the above encoding gene;

[0029] (e) a recombinant expression cell containing the expression cassette described in (a);

[0030] (f) A recombinant expression cell containing the recombinant expression vector described in (b) or (c).

[0031] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a YEp series plasmid, etc.; preferably, it is a YEp352 plasmid.

[0032] Furthermore, the host bacteria of the recombinant expression cells in (d), (e), and (f) are selected from eukaryotic organisms, etc. The eukaryotic organisms include Saccharomyces, etc. More specifically, the eukaryotic organism is Saccharomyces cerevisiae, and specifically can be Saccharomyces cerevisiae CEN.PK2-1C or S.cerevisiae PL00 strains.

[0033] The present invention provides an application of the mutant, encoding gene, and mutant-related biological materials in the synthesis of santalene; and further provides an application in the biosynthesis of santalene.

[0034] The specific method is to use Saccharomyces cerevisiae as a host, transform the gene expression vector containing the SanSyn mutant of the present invention into the host, and obtain an engineered Saccharomyces cerevisiae strain. The engineered strain is inoculated into a uracil-deficient culture medium for fermentation, and 20% n-dodecane is added for biphasic fermentation. Specific reaction conditions such as substrate concentration, pH, culture medium composition, amount of recombinant expression transformant, etc. can be selected according to the conventional conditions of such reactions in the art. The fermentation can be carried out under shaking or stirring conditions. The fermentation time is preferably 48 hours. After the reaction is completed, the content of different configurations of santalene in the reaction mixture can be obtained according to the conventional gas chromatography detection method in the art.

[0035] In principle, the bioreactor used is selected to ensure that living cells and enzymes can carry out cell proliferation and biochemical reactions, such as small shake flasks, fermentation tanks and other equipment. The culture medium used is mainly a culture medium suitable for the growth of brewer's yeast commonly used in the art, and the present invention preferably uses a uracil-deficient culture medium. In principle, the fermentation temperature is preferably selected to keep the cells growing well and the enzymes maintaining good catalytic performance. In a specific embodiment, the preferred fermentation temperature is 30°C.

[0036] The recombinant strain constructed by the SanSyn_I402C mutant has a santalinium configuration ratio in the product santalin that is consistent with the configuration ratio of natural sandalwood essential oil; while the recombinant strains constructed by the SanSyn_P460A and SanSyn_P460S mutants have a santalinium configuration ratio of more than 50%, which is rarely reported.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] The present invention transforms SanSyn through protein engineering principles, aiming to change the configuration ratio of its product santalene. The present invention screened different santalene synthase mutants through bioinformatics analysis, among which the I402C mutation makes the α configuration account for 54.3% and the β configuration account for 32.0% in the product santalene, and the configuration ratio is consistent with the configuration ratio of natural sandalwood essential oil. The P460A and P460S mutations make the β configuration account for more than 50% in the product santalene, which is the first report in China; the configuration ratio of natural sandalwood essential oil and santalene containing more β configuration ratio are achieved, which provides the possibility of producing sandalwood essential oils of different compositions according to specific needs in industrial production, and has good application prospects. At the same time, the biological preparation method of santalene described in the present invention has the advantages of simple operation, environmental friendliness, mild reaction conditions, etc., and has good application prospects in the field of santalene production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the reaction diagram for the production of santalene and santalol by fermentation of Saccharomyces cerevisiae.

[0040] Figure 2 These are the mass spectrometry results of α-santalene in fermentation products.

[0041] Figure 3 These are the mass spectrometry results of β-santalene in fermentation products. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0043] It is worth noting that any process not specifically described below can be implemented or understood by those skilled in the art with reference to the prior art. The reagents, materials and instruments used, unless otherwise specified, can be regarded as conventional products available from commercial channels.

[0044] The reaction flow chart of SanSyn catalyzing santalene in the present invention is as follows: Figure 1 shown.

[0045] Example 1 Construction of santalene synthase starting strain

[0046] The codon-optimized SanSyn gene shown in SEQ ID NO.1 was synthesized at Sangon Biotech, and the plasmid YEp352 purchased from Invitrogen was used as the starting vector. The obtained gene was ligated with the YEp352 plasmid after double digestion with NdeⅠ and XhoⅠ. 10 μL of the ligation product was added to 20 μL of E. coli DH5α competent cells, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and then quickly placed on ice for 5 minutes; then 800 μL of LB liquid medium without resistance was added, and incubated at 37°C and 220 rpm for 1 hour; after centrifugation, 500 μL of supernatant was poured out, and the remaining 300 μL of bacterial liquid was taken after resuspending and coated on a LB solid plate containing 100 μg / mL ampicillin, and cultured at 37°C for 12-16 hours; after the transformants grew, the transformants were picked on the plate for colony PCR to verify whether the size of the target band was correct. Then, the correctly verified transformant bacteria were picked and inoculated into LB liquid culture medium containing 100 μg / mL ampicillin for culture. After culturing at 37°C and 220 rpm for 12-16 hours, the plasmid was extracted, and the extracted plasmid was subjected to restriction enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant plasmid YEp352-SanSyn containing the gene encoding SanSyn was obtained. The obtained recombinant plasmid YEp352-SanSyn was transformed into Saccharomyces cerevisiae PL00 competent cells, and the recombinant strain PL00 / YEp352-SanSyn containing the gene encoding SanSyn was obtained.

[0047] Among them, Saccharomyces cerevisiae PL00 is disclosed in the document "CN118792290A, an α-farnesene synthase AoFS from a nootropic source and its application".

[0048] Example 2 Construction of santalene synthase mutant strain

[0049] The SanSyn mutant was constructed by homologous recombination method commonly used in the field of molecular cloning. The recombinant plasmid YEp352-SanSyn was used as a template for site-directed mutagenesis using PCR technology to obtain a plasmid containing the gene encoding the SanSyn mutant. The mutation sites are as follows: L269A, I273S, I402C, P460S, P460A, P460T, I503A, L514C, and F529V.

[0050] The primers used for PCR are as follows (lower case indicates the replaced codons):

[0051] 352-Tong-F: 5'-CAAGCTGTGACCGTCTCCGGGAGC-3';

[0052] 352-Tong-R: 5'-GCTCCCGGAGACGGTCACAGCTTG-3';

[0053] L269A-F: 5'-GTGATAGAGTCGTCGAAgcaTATTTCTGGATCTT-3';

[0054] L269A-R: 5'-AAGATCCAGAAATAtgcTTCGACGACTCTATCAC-3';

[0055] I273S-F: 5'-TCGAATTGTATTTCTGGtctTTGGTTGGTGTTAG-3';

[0056] I273S-R: 5'-CTAACACCAACCAAagaCCAGAAATACAATTCGA-3';

[0057] I402C-F: 5'-CCGTTTCCTTGAGATCCtgtGGTTTCTTGCCA-3';

[0058] I402C-R: 5'-TGGCAAGAAACCacaGGATCTCAAGGAAACGG-3';

[0059] P460S-F: 5'-AAAAAAGAGAACATTCTtctTCCGCTATTG-3';

[0060] P460S-R:5'-CAATAGCGGAagaAGAATGTTCTCTTTTTT-3';

[0061] P460A-F:5'-AAAAAAGAGAACATTCTgcaTCCGCTATTG-3';

[0062] P460A-R:5'-CAATAGCGGAtgcAGAATGTTCTCTTTTTT-3';

[0063] P460T-F:5'-AAAAAAGAGAACATTCTacgTCCGCTATTG-3':

[0064] P460T-R:5'-CAATAGCGGAcgtAGAATGTTCTCTTTTTT-3';

[0065] I503A-F:5'-TGTTGAATCCAATGGCTgcaCCATTGCCTTTGTT-3':

[0066] I503A-R:5'-AACAAAGGCAATGGtgcAGCCATTGGATTCAACA-3':

[0067] L514C-F:5'-TACAAGTTATTTTAGATtgtTCACGTTCTGCTGA-3';

[0068] L514C-R:5'-TCAGCAGAACGTGAacaATCTAAATAACTTGTA-3';

[0069] F529V-F:5'-GTAACGCTCAAGATAGAgtaACTCATTCTACTAT-3';

[0070] F529V-R:5'-ATAGTAGAATGAGTtacTCTATCTTGAGCGTTAC-3';

[0071] The upstream primer F and the universal primer 352-Tong-R were used to amplify the upstream fragment, and the downstream primer R and the universal primer 352-Tong-F primer pair were used to amplify the downstream fragment. The PrimeSTAR Max enzyme was used to PCR amplify the target fragment, and the amplified upstream fragment and downstream fragment were homologously recombined and connected using the ClonExpress II recombination cloning kit (purchased from Nanjing Novozyme). Then the recombinant product was transformed into E. coli DH5α competent cells and colony PCR verification and plasmid extraction were performed. If the sequencing verification was correct, the recombinant plasmids containing the genes encoding the santalene synthase mutants were obtained, and the obtained recombinant plasmids were transformed into the PL00 competent cells of Saccharomyces cerevisiae to obtain the recombinant strains containing the genes encoding the santalene synthase mutants.

[0072] Example 3 Shake flask fermentation and product detection of santalene synthase mutant strain

[0073] The recombinant Saccharomyces cerevisiae strains constructed in Example 1 and Example 2 were selected for shake flask fermentation. A single colony was inoculated into 5 mL of SD-ΔUra liquid medium and cultured at 30°C in a shaker at 220 rpm for 16 h. 600 The concentration of 0.05 was transferred to 10 mL SD-ΔUra liquid medium containing 20% ​​n-dodecane for shake flask fermentation. Three parallel cultures were set for each strain, and the fermentation time was 48 h.

[0074] After the catalytic reaction is completed, the upper organic phase is taken into a 2 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. 500 μL of the upper organic phase is drawn with a 1 mL syringe, filtered through a 0.22 μm sterile filter head, and the product is detected by GC.

[0075] The model of the gas chromatograph is Shimadzu GC-2014C, the chromatographic column is HP-5 column, the specifications are 30m×0.32mm×0.25μm, the detector is flame ionization detector (FID), and the temperature of the detector and the injector are set to 280℃ and 250℃ respectively. The detection process uses nitrogen as the carrier gas, sets a split ratio of 5:1, and injects 1μL of aliquots in split mode. The setting program is as follows: the initial temperature of the column box is 50℃, maintained for 3min, heated to 70℃ at a rate of 20℃ / min, maintained for 1min, then heated to 160℃ at a rate of 3℃ / min, and then heated to 300℃ at a rate of 20℃ / min, and the total time is 42min.

[0076] GC analysis results are as follows Figure 2-3As shown in Table 1, the product ratios of the mutant strains have changed. Among them, the I402C mutation makes the α configuration account for 54.3% and the β configuration account for 32.0% in the product santalene, and the configuration ratio is consistent with the configuration ratio of natural sandalwood essential oil; the P460S mutation makes the α configuration account for 29.3% and the β configuration account for 53.1% in the product santalene, and the P460A mutation makes the α configuration account for 30.9% and the β configuration account for 50.1% in the product santalene. The β configuration accounts for more than 50% in the santalene product, which is the first report in China. The various santalene synthase mutants screened by the present invention realize different product preferences of santalene synthase, among which the I402C mutation enables the santalene synthase to produce the configuration ratio of natural sandalwood essential oil, and the P460A and P460S mutations enable the santalene synthase to produce santalene containing more than 50% β configuration ratio, which provides the possibility of producing sandalwood essential oils of different compositions according to specific needs in industrial production, and also provides insights for the engineering transformation of other product configuration mixed enzymes.

[0077] Table 1: Comparison of the ratio of santalene synthase products with single modification compared with SEQ ID NO.2

[0078]

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A santalene synthase SanSyn mutant, characterized in that: The amino acid sequence of the santalene synthase SanSyn mutant is shown in SEQ ID No.3, wherein at least one of the sites marked as "X" in SEQ ID No.3 and the amino acid residue of the corresponding site in SEQ ID No.2 are mutated.

2. The santalene synthase SanSyn mutant according to claim 1, characterized in that: The santalene synthase SanSyn mutants are SanSyn_L269A, SanSyn_I273S, SanSyn_I402C, SanSyn_P460S, SanSyn_P460A, SanSyn_P460T, SanSyn_I503A, SanSyn_L514C, and SanSyn_F529V; Among them, the amino acid sequence of the mutant SanSyn_I402C is shown in SEQ ID No.4, the amino acid sequence of SanSyn_P460A is shown in SEQ ID No.6, and the amino acid sequence of SanSyn_P460S is shown in SEQ ID No.

8.

3. A gene encoding the santalene synthase SanSyn mutant according to any one of claims 1 to 2.

4. The gene according to claim 3, characterized in that: The nucleotide sequence of the gene encoding the mutant SanSyn_I402C is shown in SEQ ID No.5, the nucleotide sequence of the gene encoding the mutant SanSyn_P460A is shown in SEQ ID No.7, and the nucleotide sequence of the gene encoding the mutant SanSyn_P460S is shown in SEQ ID No.

9.

5. The biomaterial related to the santalene synthase SanSyn mutant according to any one of claims 1 to 2, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 3 or 4; (b) a recombinant expression vector containing the gene according to claim 3 or 4; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant expression cell containing the gene according to claim 3 or 4; (e) a recombinant expression cell containing the expression cassette described in (a); (f) A recombinant expression cell containing the recombinant expression vector described in (b) or (c).

6. The biomaterial according to claim 5, characterized in that: The starting vector of the recombinant expression vector in (b) and (c) is a plasmid of the YEp series; The host bacteria of the recombinant expression cells in (d), (e) and (f) are selected from eukaryotic organisms.

7. The biomaterial according to claim 5 or 6, characterized in that: The host bacteria of the recombinant expression cell is Saccharomyces cerevisiae.

8. The biomaterial according to claim 7, characterized in that: The brewer's yeast is a brewer's yeast S.cerevisiae CEN.PK2-1C strain or a S.cerevisiae PL00 strain.

9. Use of the santalene synthase SanSyn mutant according to any one of claims 1 to 2, the gene according to any one of claims 3 to 4, or the biomaterial according to any one of claims 5 to 8 in synthesizing santalene.

10. The use according to claim 9, characterized in that: Application in biosynthesis of santalene.

Images