A santalene synthase mutant and its application

Abstract

The present invention discloses a santalene synthase mutant and its application, belonging to the technical fields of enzyme engineering and microbial engineering. The present invention screened different santalene synthase mutants through bioinformatics analysis, among which the I402C mutation caused the α configuration in the product santalene to account for 54.2% and the β configuration to account for 32.0%, and the configuration ratio is consistent with the configuration ratio of natural sandalwood essential oil. The P460A and P460S mutations caused the β configuration in the product santalene to account for more than 50%, which is the first report in China; the configuration ratio of natural sandalwood essential oil and santalene containing a higher 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.

Description

Technical Field

[0001] The present invention belongs to the technical fields of enzyme engineering and microbial engineering, and relates to a mutant of santalene synthase (SanSyn) having an amino acid sequence as shown in SEQ ID No. 3 in the sequence listing, and the use of a recombinant expression cell thereof in the biological preparation of santalene with different configuration ratios, 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] Sesquiterpenes are 15-carbon compounds composed of three isoprenoid units, belonging to the terpenoid family. Most of them have a strong aroma and biological activity and are widely used in the food, cosmetics, and pharmaceutical industries. Both santalene and santalol belong to the sesquiterpenoid family. Santalene is the precursor for santalol synthesis, and both are the main components of sandalwood essential oil. Sandalwood essential oil has a pleasant and long-lasting aroma and is one of the most popular natural fragrances in the world. It is widely used in the cosmetics and food industries. Furthermore, sandalwood essential oil has many pharmacological activities with health benefits, such as antioxidant, antibacterial, antiviral, and anticancer activities, making it of great economic value. Currently, sandalwood essential oil is mainly obtained by steam distillation from the heartwood and roots of the sandalwood tree. However, sandalwood trees require 30 years of growth to extract sandalwood essential oil, and the extraction rate of sandalwood essential oil from mature sandalwood trees is only between 2.5% and 6%, far from meeting the growing market demand for sandalwood products. Chemical synthesis of santalene and santalol is complex, requires harsh reaction conditions, is costly, and produces low-purity products, leading to high prices for natural sandalwood essential oil. Compared to traditional plant extraction and chemical synthesis methods, biosynthesis through the construction of microbial cell factories offers the potential for green, efficient, and low-cost industrial production of sesquiterpenes.

[0003] To date, over 200 components have been identified in sandalwood essential oil, primarily composed of sesquiterpenes, 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. (Z)-α-santalol and (Z)-β-santalol are the two most critical components influencing the biological activity and aroma of sandalwood essential oil. α-santalol has a slightly woody aroma, similar to cedarwood and α-cedrene; β-santalol, while typically less abundant, has a more intense aroma than α-santalol. It is believed that the primary therapeutic benefits of sandalwood are attributed to α-santalol, while the rich, woody aroma of sandalwood is primarily derived from β-santalol. According to the quality control standards set by the International Organization for Standardization (ISO 3518:2002), the contents of α-santalol and β-santalol in natural sandalwood essential oil must be within the ranges of 41-55% and 16-24%, respectively.

[0004] As previously mentioned, the composition of sandalwood essential oil determines its flavor and quality, and rationally controlling the product ratios is a major challenge in constructing a heterologous biosynthetic system for sandalwood essential oil. In the biosynthesis of santalol, the main component of sandalwood essential oil, in Saccharomyces cerevisiae, santalene synthase is not only the rate-limiting enzyme but also a key enzyme in determining the configurational ratio of santalol. The CYP450 (Cytochrome P450 monooxygenase) enzymes that catalyze the conversion of ene to alcohol exhibit similar preferences for substrates of different configurations. The two most commonly used santalene synthases are SanSyn from Clausena lansium and SaSSy from S. album. SaSSy is a mixed-product 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 mixed-product SaSSy enzyme, 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 for the restriction of its intermediate conformation and the α-specificity of the product. They introduced the mutant SanSyn into Saccharomyces cerevisiae. F411VThe product santalene had an α- and β-configuration ratio of 57.2% and 28.6% respectively. Further introduction into CYP450 reduced the ratio to 43.4% and 22% respectively, meeting ISO standards. However, the enzyme activity was significantly reduced after the mutation. Furthermore, Styles et al. reported that CiCassy, ​​derived from Cinnamomum camphora, produced a greater β-configuration than α-configuration of santalene after the N267S / N267L mutation, but the yield was not reported.

[0005] Enzyme engineering has been proven to be an effective method for optimizing enzyme performance. By molecularly modifying the enzyme, we altered the configuration ratio of the product santalene, ultimately obtaining a strain of Saccharomyces cerevisiae that produces an ideal ratio of santalene products, thereby meeting the future demand for large-scale biosynthesis of sandalwood essential oil. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims 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 and different product ratios, in view of the fact that santalene synthase is a key enzyme in the biosynthesis of santalene and santalol and a key enzyme in determining the configuration ratio of santalol, but its product configuration ratio is single.

[0009] Enzymes with product configuration ratios that 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 rich aroma and is considered to give sandalwood its most important odor. In industrial production, it is more 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 position marked as "X" in SEQ ID No. 3 and the amino acid residue of the corresponding position in SEQ ID No. 2 are mutated.

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

[0013] In the present invention, the santalene synthase is SanSyn, whose codon-optimized nucleotide sequence is shown in SEQ ID NO. 1, comprising a total of 1656 nucleotides; and its amino acid sequence is shown in SEQ ID No. 2, comprising a total of 551 amino acids. Further modifications were made based on SanSyn.

[0014] It has been found that mutations at the following positions in SEQ ID No. 2 are particularly 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 should be noted that, in principle, there is no limit to the number of amino acid residues that can be mutated 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, with an amino acid sequence as shown in SEQ ID NO.4 and a nucleotide sequence as shown in SEQ ID NO.5; SanSyn_P460A, i.e., mutant P460A in which proline at position 460 is mutated to alanine, with an amino acid sequence as shown in SEQ ID NO.6 and a nucleotide sequence as shown in SEQ ID NO.7; and SanSyn_P460S, i.e., mutant P460S in which proline at position 460 is mutated to serine, with an amino acid sequence as shown in SEQ ID NO.8 and a nucleotide sequence as 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 engineered strain of Saccharomyces cerevisiae.

[0019] According to the present invention, experiments have confirmed that the santalene synthase mutant described herein, SanSyn, exhibits a desirable shift in product ratio compared to the previously reported and widely used SanSyn variant, which is specific for the α-configuration. Experimental results show that under identical conditions, recombinant strains constructed using the SanSyn mutant of SEQ ID No. 4 produced santalene with a configuration consistent with that of natural sandalwood essential oil. Recombinant strains constructed using the SanSyn mutants of SEQ ID No. 6 and SEQ ID No. 8 produced santalene with a β-configuration ratio exceeding 50%, a rare finding.

[0020] The term "mutation" as used herein indicates 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. Mutation of the enzyme can be achieved by site-directed mutagenesis using conventional methods in the art.

[0021] The term "nucleic acid molecule" as used herein has the meaning generally understood by those of ordinary skill in the art. A nucleic acid molecule may be a polynucleotide including a polynucleotide as shown in NCBI Accession No. HQ452480.1 or SEQ ID No. 1, or a polynucleotide further including additional coding and / or non-coding sequences.

[0022] As used herein, the terms "vector" and "expression vector" have the meanings commonly understood by those skilled in the art. "Vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is referred to as an expression vector when it allows for the expression of a protein encoded by the inserted polynucleotide. In the process of expressing exogenous genes using Saccharomyces cerevisiae, the exogenous gene to be expressed must first be ligated to the vector. Such vectors can be any conventional vector in the art, such as commercially available plasmids, phages, and viral vectors. Plasmid YEp352 is preferred for expressing the santalene synthase mutant gene in the present invention.

[0023] The present invention provides a recombinant expression cell comprising the vector of the present invention. The recombinant expression cell can be produced by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be any conventional host cell known 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. Saccharomyces cerevisiae is preferred, and S. cerevisiae CEN.PK2-1C or S. cerevisiae PL00 is more preferred.

[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-mentioned encoding gene;

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

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

[0028] (d) recombinant expression cells containing the above encoding genes;

[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 a YEp352 plasmid.

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

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

[0034] The specific method is to use Saccharomyces cerevisiae as a host and transform the gene expression vector containing the SanSyn mutant described in the present invention into the host to obtain an engineered Saccharomyces cerevisiae strain. The engineered strain is inoculated into a uracil-deficient culture medium for fermentation, while simultaneously adding 20% ​​n-dodecane for a two-phase fermentation culture. Specific reaction conditions, such as substrate concentration, pH, culture medium composition, and amount of recombinant expression transformant, can be selected according to conventional conditions for 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 determined using conventional gas chromatography methods in the art.

[0035] In principle, the bioreactor used should be equipment that can ensure viable cells and enzymes undergo cell proliferation and biochemical reactions, such as small shake flasks or fermenters. The culture medium used should primarily be one commonly used in the art suitable for growing Saccharomyces cerevisiae, with uracil-deficient culture medium being preferred in this invention. The fermentation temperature should ideally be selected to maintain optimal cell growth and enzyme catalytic performance. In one specific embodiment, the preferred fermentation temperature is 30°C.

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

[0037] The present invention has the following advantages and effects compared to the prior art:

[0038] The present invention modifies SanSyn through protein engineering principles, aiming to alter the configuration ratio of its product, santalene. Bioinformatics analysis screened different santalene synthase mutants, among which the I402C mutation resulted in a 54.3% α-configuration and a 32.0% β-configuration in the product santalene, a configuration ratio consistent with that of natural sandalwood essential oil. The P460A and P460S mutations increased the β-configuration to over 50% in the product santalene, a first reported domestic finding. This achieved a configuration ratio similar to that of natural sandalwood essential oil and santalene with a higher β-configuration ratio, paving the way for the industrial production of sandalwood essential oils with varying compositions according to specific needs, and thus possessing promising application prospects. Furthermore, the biopreparation method for santalene described in the present invention offers advantages such as ease of operation, environmental friendliness, and mild reaction conditions, and holds great promise for application in the field of santalene production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the reaction diagram for the fermentation of santalene and santalol by 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 will be described in further detail below with reference to the 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 considered 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. Using plasmid YEp352 purchased from Invitrogen as the starting vector, the resulting gene was double-digested with NdeI and XhoI and then ligated. 10 μL of the ligation product was added to 20 μL of E. coli DH5α competent cells. After incubation on ice for 30 minutes, the cells were heat-shocked in a 42°C water bath for 90 seconds and immediately placed on ice for 5 minutes. 800 μL of antibiotic-free LB medium was then added and incubated at 37°C, 220 rpm, for 1 hour. After centrifugation, 500 μL of the supernatant was decanted, the cells were resuspended, and the remaining 300 μL of the bacterial suspension was plated on LB plates containing 100 μg / mL ampicillin and incubated at 37°C for 12-16 hours. Transformants were picked from the plates for colony PCR to verify the correct size of the target band. Then, the correctly verified transformant bacteria were picked and inoculated into LB liquid culture medium containing 100 μg / mL ampicillin for culture. After culture at 37°C and 220 rpm for 12-16 hours, the plasmid was extracted, and the extracted plasmid was subjected to 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 derived from alpinia oxyphylla and its application".

[0048] Example 2 Construction of santalene synthase mutant strain

[0049] SanSyn mutants were constructed using homologous recombination, a method commonly used in molecular cloning. PCR was used as a template for site-directed mutagenesis, resulting in a plasmid encoding the SanSyn mutant gene. The mutations were at the following sites: L269A, I273S, I402C, P460S, P460A, P460T, I503A, L514C, and F529V.

[0050] The primers used for PCR are as follows (lowercase indicates the replaced codon):

[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] Upstream primer F and universal primer 352-Tong-R were used to amplify the upstream fragment, while downstream primer R and universal primer 352-Tong-F were used to amplify the downstream fragment. PCR amplification of the target fragments was performed using PrimeSTAR Max enzyme. The amplified upstream and downstream fragments were then homologously recombined and ligated using the ClonExpress II Recombination Cloning Kit (purchased from Nanjing Novozymes). The recombinant products were then transformed into E. coli DH5α competent cells, verified by colony PCR, and plasmids were extracted. Once confirmed by sequencing, recombinant plasmids containing the genes encoding the santalene synthase mutants were obtained. The resulting recombinant plasmids were then transformed into Saccharomyces cerevisiae PL00 competent cells to obtain 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. Single colonies were seeded in 5 mL of SD-ΔUra liquid medium and cultured at 30°C in a shaker at 220 rpm for 16 h. 600 The strain was transferred to 10 mL SD-ΔUra liquid medium containing 20% ​​n-dodecane at a concentration of 0.05, and shake flask fermentation was carried out. Three replicates 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, and the product is detected by GC.

[0075] The gas chromatograph was a Shimadzu GC-2014C, with an HP-5 column (30 m × 0.32 mm × 0.25 μm). The detector was a flame ionization detector (FID). The detector and injector temperatures were set at 280°C and 250°C, respectively. Nitrogen was used as the carrier gas, with a 5:1 split ratio. A 1 μL aliquot of sample was injected in split mode. The program was as follows: the column oven temperature was initially set at 50°C, held for 3 minutes, then increased at a rate of 20°C / min to 70°C, held for 1 minute, then increased at a rate of 3°C / min to 160°C, and then increased at a rate of 20°C / min to 300°C, for a total of 42 minutes.

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

[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 implementations of the present invention, but the implementations 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 considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A santalene synthase SanSyn mutant, characterized in that: The santalene synthase SanSyn mutant is SanSyn_I402C, SanSyn_P460S or SanSyn_P460A; 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.

2. A gene encoding the santalene synthase SanSyn mutant according to claim 1.

3. The gene according to claim 2, 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.

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

5. The biomaterial according to claim 4, characterized in that: The starting vector of the recombinant expression vectors 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.

6. The biomaterial according to claim 4 or 5, characterized in that: The host bacteria of the recombinant expression cell is Saccharomyces cerevisiae.

7. The biomaterial according to claim 6, characterized in that: The saccharomyces cerevisiae is S. cerevisiae CEN.PK2-1C strain.

8. Use of the santalene synthase SanSyn mutant according to claim 1, the gene according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 7 in synthesizing santalene.

9. The use according to claim 8, characterized in that: Application in the biosynthesis of santalene.

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