Bergamotene synthase mutant and application thereof

Abstract

The invention discloses a bergamot alkene synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and bioengineering. According to the method, bergamotene synthase SanSyn which is obtained from plant clausena lansium and can catalyze FPP to generate alpha-trans-bergamotene is taken as an object, semi-rational design and site-specific mutagenesis are carried out on the bergamotene synthase SanSyn, a series of obtained SanSyn mutants are remarkably improved in the aspect of FPP substrate conversion efficiency, and the optimal bergamotene synthase mutant SanSynI273C-L514I has the advantages that the optimal bergamotene synthase mutant SanSynI273C-L514I can be used for catalyzing the FPP to generate alpha-trans-bergamotene; the unit cell productivity of the alpha-trans-bergamotene generated by catalyzing FPP is 6.9 times of that of a wild type, and the yield is 6.8 times of that of the wild type and reaches 43.58 mg / L. According to the bergamot alkene synthase mutant, the performance of alpha-trans-bergamot alkene synthesized by microorganisms is enhanced, and an effective technical route is provided for green production of industrial high-purity bergamot alkene.

Description

Technical Field

[0001] The invention belongs to the technical fields of enzyme engineering and bioengineering, and particularly relates to a bergapten synthase mutant and application thereof in the biosynthesis of bergapten. Background Art

[0002] Sesquiterpenes are naturally occurring volatile compounds composed of three isoprene units. They are released by diverse organisms but are most commonly found in a variety of plants. As the largest subclass of terpenes, sesquiterpenes have a wide range of applications in the pharmaceutical, biofuel, flavor, and fragrance industries. Bergamotenes are natural bicyclic sesquiterpenes whose unique aroma and diverse biological activities make them important for applications in flavors, perfumes, cosmetics, and medicine. Furthermore, bergamotenes play a role in chemical communication and defense signals within ecosystems, acting as alarm pheromones for insects and repelling herbivorous pests, thus offering potential value for agricultural pest management.

[0003] Among bergamotene compounds, there are two important structural isomers: α-trans / cis-bergamotene and β-trans / cis-bergamotene, which differ only in the position of the double bond. α-Bergamotene, with its sweet citrus aroma and excellent olfactory persistence, is widely used in perfumery and high-end skincare formulations. α-Bergamotene is also used as a natural flavoring agent in foods and beverages, often found in wines, juices, and honey. Its refreshing, fruity aroma can significantly enhance the product's texture. More importantly, α-trans-bergamotene (α-trans-bergamotene / exo-α-bergamotene) is abundant in essential oils and cold-pressed oils from various citrus species, particularly predominating in lemon and bergamot essential oils. In traditional aromatherapy and folk herbal medicine, oils high in α-trans-bergamotene are often used to alleviate chronic intestinal inflammation and related symptoms, such as diarrhea and mucosal damage. Furthermore, α-trans-bergamotene's high volatility and mild irritation make it an irreplaceable aromatic restorative agent in high-end aromatherapy formulations and functional skincare products. Due to its versatility and safety in natural products, α-trans-bergamotene is becoming a hot topic for research and application in fragrances, cosmetics, and even pharmaceuticals.

[0004] Bergamotenes and their derivatives are typically present at extremely low concentrations in natural essential oils or plant tissues. These complex mixed systems make obtaining high-purity bergamotenes through traditional separation methods such as fractionation or chromatography time-consuming and labor-intensive, and the yield is often insufficient to support industrialization needs. Although chemical synthesis routes can achieve the chemical preparation of bergamotenes by constructing a bicyclic skeleton, such methods are generally cumbersome, environmentally unfriendly, and prone to the generation of structural isomers, which imposes additional purification burdens and safety risks. With the rapid development of synthetic biology, researchers have identified a variety of terpene synthases and achieved heterologous expression in plant systems in order to build sustainable biosynthetic platforms. However, most of these studies have focused on in vivo regulation in plants, and there are still only a few reports on the efficient production of bergamotenes using microbial hosts (such as yeast or Escherichia coli). When the sesquiterpene synthases SaSSy (from Santalum album) and SanSyn (from Clausena lansium) were heterologously expressed in Saccharomyces cerevisiae, while bergamotene and santalene and their alcohols were simultaneously detected, the bergamotene content in the total product was extremely low, severely restricting subsequent isolation and application development. To date, there have been no reports of systematic enzyme engineering optimization targeting this product distribution, and the proportion and yield of bergamotene remain low. Furthermore, Wen et al. first identified an enzyme specifically catalyzing the synthesis of (+)-α-trans-bergamotene from the genome of the marine fungus Nectria sp. HLS206 and conducted preliminary functional validation in yeast, but its production data have not yet been released, indicating that there is still significant room for improvement in this field. It can be seen from this that discovering or designing bergapten synthases with high catalytic efficiency and strong product specificity, or conducting targeted modification of existing bergapten synthases (SaSSy / SanSyn) to significantly increase the yield of bergapten, has become a key technical path to solve the bottleneck of efficient biological production of bergapten. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a bergamotene synthase mutant, aiming to improve the catalytic ability of bergamotene synthase to farnesyl pyrophosphate (FPP).

[0006] Another object of the present invention is to provide the use of the bergapten synthase mutant in the efficient biosynthesis of α-trans-bergapten.

[0007] The technical problem to be solved by the present invention is that the production efficiency of α-trans-bergapten when wild-type bergapten synthase is catalyzed using farnesyl pyrophosphate (FPP) as a substrate is relatively low, making it difficult to meet the production requirements for industrial purification and subsequent applications. To this end, the present invention significantly increases bergapten yield by designing and screening a series of bergapten synthase mutants, providing new ideas and examples for the efficient biosynthesis of bergapten by microorganisms.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A santalene synthase mutant, wherein the amino acid sequence of the santalene synthase mutant is SEQ ID No. 2 obtained by any one of the following mutations:

[0010] R263F, I273C, I273V, I294G, L372T, L514A, L514I, I273C-L514A, I273C-L514I, I273V-L514A, or I273V-L514I.

[0011] The santalene synthase is derived from the plant Clausena lansium, the GenBank number of the amino acid sequence thereof is ADR71055.1, and the GenBank number of the encoding gene thereof is HQ452480.1.

[0012] In the present invention, the bergamotene 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.

[0013] The present invention also relates to a bergapten synthase mutant, whose amino acid sequence is obtained by mutation of one or two of amino acids 263, 273, 294, 372, and 514 in SEQ ID No. 2; further, the bergapten synthase mutants are SanSyn_R263F, SanSyn_I273V, SanSyn_I273C, SanSyn_I294G, SanSyn_L372T, SanSyn_L514I, SanSyn_L514A, SanSyn_I273C-L514A, SanSyn_I273C-L514I, SanSyn_I273V-L514A, and SanSyn_I273V-L514I;

[0014] Furthermore, the bergamotene synthase mutant has an amino acid sequence as shown in SEQ ID No. 2, wherein the 273rd amino acid is mutated from isoleucine I to valine C, and the 514th amino acid is mutated from leucine L to isoleucine I. The specific amino acid sequence is shown in SEQ ID No. 3.

[0015] Preferably, in the bergapten synthase mutant, the gene sequence encoding the amino acid sequence shown in SEQ ID No. 3 is shown in SEQ ID No. 4.

[0016] The present invention provides a gene encoding the bergapten synthase mutant.

[0017] The present invention provides a recombinant expression vector containing the gene encoding the SanSyn mutant, and provides a method for producing bergamotene (especially α-trans bergamotene) by introducing the recombinant expression vector into an engineered strain of Saccharomyces cerevisiae.

[0018] 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.

[0019] 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.

[0020] As used herein, the terms "vector" and "expression vector" have the meanings commonly understood by those skilled in the art. A "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 connected to a 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 bergamotene synthase mutant gene in the present invention.

[0021] 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.

[0022] In the present invention, the mutant-related biological material is any one or more combinations of the following biological materials:

[0023] (a) an expression cassette containing the above-mentioned encoding gene;

[0024] (b) a recombinant expression vector containing the above-mentioned encoding gene;

[0025] (c) a recombinant expression vector containing the expression cassette described in (a);

[0026] (d) recombinant expression cells containing the above encoding genes;

[0027] (e) a recombinant expression cell containing the expression cassette described in (a);

[0028] (f) A recombinant expression cell containing the recombinant expression vector described in (b) or (c).

[0029] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a YEp series plasmid, etc., preferably a YEp352 plasmid.

[0030] 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.

[0031] The present invention provides an application of the above mutant, encoding gene, and mutant-related biological materials in preparing a bergapten synthase mutant.

[0032] The present invention provides the use of the above-mentioned mutant, encoding gene, and mutant-related biological materials in the synthesis of bergamotene (especially α-trans bergamotene); further provides the use of the above-mentioned mutant, encoding gene, and mutant-related biological materials in the synthesis of bergamotene (especially α-trans bergamotene); and further provides the use of the above-mentioned mutant, encoding gene, and mutant-related biological materials in the biosynthesis of bergamotene (especially α-trans bergamotene).

[0033] The present invention provides a method for producing bergamotene (especially α-trans-bergamotene), comprising the step of fermenting the above-mentioned recombinant expression cell.

[0034] The specific steps include:

[0035] fermenting the recombinant expression cells and collecting the fermentation product;

[0036] The bergamotene (especially α-trans-bergamotene) in the fermentation product is extracted.

[0037] The specific method is as follows: using Saccharomyces cerevisiae as a host strain, the gene expression vector containing the SanSyn mutant described in the present invention is transformed into the host cell to obtain an engineered Saccharomyces cerevisiae strain. The engineered strain is inoculated into a uracil-deficient culture medium for fermentation, and a two-phase fermentation technique is used, with 20% n-dodecane added as the second phase to promote the extraction of α-trans-bergamotene from the aqueous phase. Specific reaction conditions, such as the composition of the culture medium and the amount of the 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, a sample of the organic phase is obtained, and the content of α-trans-bergamotene in the fermentation broth of different engineered strains can be detected using conventional gas chromatography detection methods in the art.

[0038] 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.

[0039] The recombinant Saccharomyces cerevisiae strain constructed with the SanSyn_I273C-L514I mutant had a yield of 43.58 mg / L of its product α-trans bergamotene after 48 hours of fermentation in uracil-deficient SD medium. The production capacity of its large unit cell was 6.9 times that of the wild-type bergamotene synthase-expressing strain, significantly improving the biosynthesis efficiency of bergamotene.

[0040] The present invention has the following advantages and effects compared to the prior art:

[0041] (1) The present invention uses SanSyn, a bergamotene synthase obtained from the plant Clausena lansium, which can catalyze farnesyl pyrophosphate (FPP) to produce α-trans-bergamotene, as the target. Through semi-rational design and site-directed mutagenesis, a series of SanSyn mutants obtained by the present invention have significantly improved the FPP substrate conversion efficiency. Among them, the optimal bergamotene synthase mutant, SanSyn_I273C-L514I, catalyzes FPP to produce α-trans-bergamotene with a unit cell production capacity 6.9 times that of the wild type and a yield 6.8 times that of the wild type, reaching 43.58 mg / L. The bergamotene synthase mutant of the present invention enhances the performance of microorganisms in synthesizing α-trans-bergamotene, providing an effective technical route for the green production of high-purity bergamotene in industrialization.

[0042] (2) The mutation strategy successfully verified in the present invention on bergamotene synthase SanSyn can provide a reference model for other sesquiterpene synthases (such as bergamote synthase SaSSy, etc.), has good versatility and scalability, and provides a paradigm for the efficient biosynthesis of various natural products in the future.

[0043] (3) Through systematic enzyme modification and high-throughput screening, the present invention breaks through the bottleneck of the existing method of only occasionally obtaining low-abundance by-products, and provides a practical technical path for the industrial production of bergamotene. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the reaction diagram for the production of α-trans-bergamotene by Saccharomyces cerevisiae fermentation.

[0045] Figure 2 This is the gas chromatogram of α-trans-bergamotene in the fermentation product.

[0046] Figure 3 This is the mass spectrometry detection diagram of α-trans-bergamotene in the fermentation product.

[0047] Figure 4 Schematic diagram of the results of α-trans-bergamotene production in fermentation products. DETAILED DESCRIPTION

[0048] 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.

[0049] It is worth noting that the experimental methods in the following examples, unless otherwise specified, are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The reagents, materials, and instruments used, unless otherwise specified, are all commercially available conventional products.

[0050] The reaction flow chart of α-trans-bergamotene catalyzed by bergamotene synthase SanSyn in the present invention is as follows: Figure 1 shown.

[0051] The Saccharomyces cerevisiae PL00 used in the examples is disclosed in the document “CN118792290A, an α-farnesene synthase AoFS from alpinia oxyphylla and its application”.

[0052] Example 1 Construction of a starting strain producing α-trans-bergamotene

[0053] The codon-optimized SanSyn gene was synthesized at Sangon Biotech, as shown in SEQ ID No. 1. Using our laboratory's plasmid YEp352 (shuttle plasmid, 2μ, URA, Addgene, USA, containing the CCW12 promoter and the CYC1 terminator) as the starting vector, the amplified SanSyn gene fragment was inserted into the YEp352 vector digested with NdeI and XhoI, labeled YEp352-SanSyn. The fragment was then transformed into E. coli DH5α competent cells and verified by colony PCR and sequencing to obtain the recombinant plasmid YEp352-SanSyn that correctly encodes the SanSyn gene. Subsequently, Saccharomyces cerevisiae PL00 competent cells were obtained using the Frozen-EZ Yeast Transformation II Kit (purchased from Zymo). The recombinant plasmid YEp352-SanSyn was then transformed into the Saccharomyces cerevisiae PL00 competent cells to obtain the recombinant strain PL00 / YEp352-SanSyn.

[0054] Example 2 Construction of strains containing different bergamotene synthase mutants

[0055] Through simulation analysis of the protein structure of bergamotene synthase SanSyn, it was determined that the mutation sites were the 273rd amino acid from isoleucine I to cysteine ​​C or valine V, and the 514th amino acid from leucine L to alanine A or isoleucine I.

[0056] SanSyn mutants were constructed using homologous recombination. PCR was used as a template for site-directed mutagenesis, resulting in plasmids encoding the SanSyn mutant gene. The mutations at the following sites were identified: R263F, I273C, I273V, I294G, L372T, L514A, L514I, I273C-L514A, I273C-L514I, I273V-L514A, and I273V-L514I.

[0057] The primers used in PCR are as follows (lowercase letters are substituted codons):

[0058] BB-F: 5'-CAAGCTGTGACCGTCTCCGGGAGC-3';

[0059] BB-R: 5'-GCTCCCGGAGACGGTCACAGCTTG-3';

[0060] R263F-F: 5'-GAAGTTGCCATACATTttcGATAGAGTCGTC-3';

[0061] R263F-R: 5'-GACGACTCTATCcaaAATGTATGGCAACTTC-3';

[0062] I273C-F: 5'-TCGAATTGTATTTCTGGtgtTTGGTTGGTGTTAG-3';

[0063] I273C-R: 5'-CTAACACCAACCAAacaCCAGAAATACAATTCGA-3';

[0064] I273V-F: 5'-TCGAATTGTATTTCTGGgttTTGGTTGGTGTTAG-3';

[0065] I273V-R: 5'-CTAACACCAACCAAcaaCCAGAAATACAATTCGA-3';

[0066] I294G-F: 5'-TATCTAAGATTggtTGTTTAGAAACTTTAGTTGATGA-3';

[0067] I294G-R: 5'-TCATCAACTAAAGTTTCTAAACAaccAATCTTAGATA-3';

[0068] L372T-F: 5'-AGATCTTTTCAAGAAactGTTATGAAGTACTTTTGT-3';

[0069] L372T-R: 5'-ACAAAAGTACTTCATAACagtTTCTTGAAAAGATCT-3';

[0070] L514A-F: 5'-TACAAGTTATTTTAGATgctTCACGTTCTGCTGA-3';

[0071] L514A-R: 5'-TCAGCAGAACGTGAcgaATCTAAAATAACTTGTA-3';

[0072] L514I-F: 5'-TACAAGTTATTTTAGATattTCACGTTCTGCTGA-3';

[0073] L514I-R: 5'-TCAGCAGAACGTGAtaaATCTAAAATAACTTGTA-3'.

[0074] Taking the construction of single-point mutation plasmid YEp352-SanSyn_I273C as an example, the specific implementation plan is as follows: using plasmid YEp352-SanSyn as a template, the upstream fragment SI273C-1 and the downstream fragment SI273C-2 of the recombinant vector are amplified using the BB-F / I273C-R primer pair and the I273C-F / BB-R primer pair respectively. The amplified SI273C-1 and SI273C-2 fragments were recombined using the ELISA II recombination cloning kit to obtain a recombinant product. The recombinant product was transformed into E. coli DH5α competent cells and verified by colony PCR and plasmid sequencing to obtain the plasmid YEp352-SanSyn_I273C containing the gene encoding the bergamotene synthase mutant SanSyn-I273C.

[0075] Taking the construction of the double-point mutation plasmid YEp352-SanSyn_I273C-L514A as an example, the specific implementation plan is as follows: Using the plasmid YEp352-SanSyn as a template, the BB-F / I273C-R primer pair, the I273C-F / L514A-R primer pair, and the L514A-F / BB-R primer pair were used to amplify the recombinant vector fragments SICLA-1, SICLA-2, and SICLA-3. The amplified SICLA-1, SICLA-2, and SICLA-3 fragments were recombined using the ELISA II recombination cloning kit to obtain a recombinant product. The recombinant product was transformed into E. coli DH5α competent cells and verified by colony PCR and plasmid sequencing. The plasmid YEp352-SanSyn_I273C-L514A, containing the gene encoding the bergapten synthase mutant SanSyn_I273C-L514A, was obtained.

[0076] Then, the recombinant plasmids (YEp352-SanSyn_R263F, YEp352-SanSyn_I273C, YEp352-SanSyn_I273V, YEp352-SanSyn_I294G, YEp352-SanSyn_L372T, YEp352-SanSyn_L514A, YEp352-SanSyn_L514I, YEp352-SanSyn_I273C-L514A, YEp352-SanSyn_I273C-L514I, YEp352-SanSyn_I273V-L514A, YEp352-SanSyn_I273V-L514I) were transformed into Saccharomyces cerevisiae PL00 competent cells, respectively, to obtain recombinant strains containing genes encoding bergapten synthase mutants.

[0077] Example 3 Shake flask fermentation of the recombinant strain producing α-trans-bergamotene and product detection

[0078] Single clones of the recombinant Saccharomyces cerevisiae strains constructed in Examples 1 and 2 were selected and inoculated into 3 mL of SD-ΔUra liquid medium and cultured overnight in a shaker at 30°C and 220 rpm. 600 The strain was inoculated into a shake flask containing 10 mL of SD-ΔUra liquid medium containing 2 mL of n-dodecane and fermented for 48 h. Three biological replicates were set for each recombinant strain.

[0079] 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 to 70°C at a rate of 20°C / min, held for 1 minute, then increased to 160°C at a rate of 3°C / min, and finally to 300°C at a rate of 20°C / min, for a total of 42 minutes.

[0080] The results of gas chromatography and mass spectrometry detection of the recombinant strain expressing the bergapten synthase mutant SanSyn_I273C-L514I are as follows: Figure 2 and Figure 3 As shown in Figure 2, the peak at 22.83 min is α-trans-bergamotene. The fermentation results of strains expressing different bergamotene synthase mutants are shown in Figure 2. Figure 4The fermentation product, α-trans-bergamotene, produced by eight recombinant strains, all showed varying degrees of improvement in their unit cell productivity and yield. Among them, the recombinant strain expressing the optimal combination of SanSyn_I273C-L514I mutants increased its α-trans-bergamotene production by 6.8 times, reaching 43.58 mg / L. This invention successfully achieves efficient production of bergamotene in microorganisms through enzyme engineering of bergamotene synthase, providing important technical support for the industrial preparation and application promotion of this compound.

[0081] Table 1: Comparison of the effects of bergamotene synthase with mutations compared to SEQ ID No. 2

[0082]

[0083] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A bergamotene synthase SanSyn mutant, characterized in that: The amino acid sequence of the bergamotene synthase SanSyn mutant is obtained by any one of the following mutations of SEQ ID No. 2: R263F, I273C, I273V, I294G, L372T, L514A, L514I, I273C-L514A, I273C-L514I, I273V-L514A, or I273V-L514I.

2. The bergapten synthase SanSyn mutant according to claim 1, characterized in that: The bergapten synthase SanSyn mutants are SanSyn_R263F, SanSyn_I273V, SanSyn_I294G, SanSyn_L372T, SanSyn_L514I, SanSyn_I273C-L514I, SanSyn_I273V-L514A, and SanSyn_I273V-L514I; Among them, the amino acid sequence of the mutant SanSyn_I273C-L514I is shown in SEQ ID No.

3.

3. A gene encoding the bergapten 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_I273C-L514I is shown in SEQ ID No.

4.

5. The biomaterial related to the bergapten 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; further, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C strain or S.cerevisiae PL00 strain.

8. Use of the bergamotene 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 7, characterized in that: One of the following applications: 1) Application in the preparation of bergamotene synthase mutants; 2) Application in the synthesis of bergamotene.

9. A method for producing bergamotene, characterized in that: The method comprises the step of fermenting the recombinant expression cell according to any one of claims 5 to 7.

10. The method according to claim 9, characterized in that: fermenting the recombinant expression cell according to any one of claims 5 to 7, and collecting the fermentation product; The bergamotene in the fermentation product is extracted and obtained.

Images