(-)-alpha-bisabolol synthase mutant and application thereof

Abstract

The invention discloses a (-)-alpha-bisabolol synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. According to the invention, (-)-alpha-bisabolol synthase BOS from cynara scolymus is taken as an object, based on sequence conservative analysis and semi-rational design, a series of BOS mutants obtained by the invention are obviously improved in the aspect of FPP substrate conversion efficiency, and the optimal (-)-alpha-bisabolol synthase BOSG119W-H151Y-F282Y-H302Q has the advantages that the conversion efficiency of the FPP substrate is obviously improved, and the optimal (-)-alpha-bisabolol synthase BOSG119W-H151Y-F282Y-H302Q is obviously improved; the yield of the generated (-)-alpha-bisabolol is 16.7 times that of a wild type, and the yield exceeds 2g / L. The (-)-alpha-bisabolol synthase mutant enhances the performance of microbial synthesis of (-)-alpha-bisabolol, and provides an effective technical route for green production of industrial high-purity (-)-alpha-bisabolol.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and bioengineering, and particularly relates to a (-)-a-bisabolol synthase mutant and its application in biosynthesis of (-)-a-bisabolol. BACKGROUND

[0002] Bisabolol (a-Bisabolol) is a high-value sesquiterpene alcohol with a cyclic structure. It has become an indispensable core ingredient in high-end cosmetics, skin care products and pharmaceutical preparations due to its excellent anti-inflammatory, antibacterial, anti-irritating and soothing repair activities. With the continuous growth of global market demand, the traditional production method relying on plant extraction is difficult to meet the needs of large-scale production stably and economically due to problems such as planting cycle, regional climate and extraction purity.

[0003] The complex chiral structure of bisabolol further restricts the feasibility of its chemical synthesis path. Traditional chemical synthesis usually has a long process, needs to use a large amount of organic solvents and reagents, and has poor atom economy. More importantly, such methods are difficult to precisely control the chiral center, and are easy to generate racemic mixture, resulting in reduced biological activity of the target product. In fact, only specific enantiomer configuration (such as (-)-a-bisabolol) has ideal biological activity, and (-)-a-bisabolol (left-handed configuration) from natural sources is significantly superior to racemic chemical synthesis in activity and commercial value. Therefore, stereochemical control is a key factor to determine the economy of different production paths and the applicability of products.

[0004] With the development of synthetic biology, microbial heterologous synthesis is considered as an innovative way to achieve sustainable production of bisabolol. In this system, bisabolol synthase, as the key rate-limiting enzyme in the biosynthesis pathway, is responsible for catalyzing the cyclization of the universal precursor farnesyl pyrophosphate (FPP) to generate bisabolol. At present, researchers have successfully introduced natural bisabolol synthase genes from plants such as German chamomile into hosts such as Escherichia coli or yeast, and have initially achieved heterologous synthesis of bisabolol. However, the industrial application of this technical route still faces a fundamental bottleneck: the low catalytic activity of natural bisabolol synthase, which leads to low microbial synthesis efficiency and high production cost.

[0005] Therefore, enzyme engineering of natural bisabolol synthase has become a key breakthrough to improve the efficiency of the entire biosynthesis pathway and promote its industrialization. Through directed evolution or rational design means, mutations are made to the active center, substrate channel or key amino acid residues of bisabolol, aiming to directly improve its catalytic activity, thermal stability and product specificity, which is a core strategy to solve the above problems from the source.

[0006] The present application is based on this urgent technical need, by constructing a series of bisabolol synthase mutants, significantly improve its catalytic performance, for the realization of efficient, economic, sustainable microbial production of bisabolol laid a solid foundation. SUMMARY

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a (-)-alpha-bisabolol synthase mutant, which aims to improve the catalytic ability of (-)-alpha-bisabolol synthase to farnesyl pyrophosphate (FPP).

[0008] Another purpose of the present application is to provide the above-mentioned (-)-alpha-bisabolol synthase mutant in the efficient biosynthesis of (-)-alpha-bisabolol.

[0009] The technical problem to be solved by the present application is that the generation efficiency of (-)-alpha-bisabolol is low when wild-type (-)-alpha-bisabolol synthase catalyzes farnesyl pyrophosphate (FPP) as a substrate, which is difficult to meet the yield demand of industrial purification and subsequent application. Therefore, the present application designs and screens a series of (-)-alpha-bisabolol synthase mutants, greatly improves the yield of (-)-alpha-bisabolol, and provides a new idea and case for the efficient biosynthesis of (-)-alpha-bisabolol by microorganisms.

[0010] The purpose of the present application is achieved by the following technical solutions: A (-)-alpha-bisabolol synthase (Bisabolol synthase) mutant, the amino acid sequence of the (-)-alpha-bisabolol synthase mutant is SEQ ID No. 2, which is obtained by mutation of any one of the following: G119W, H151Y, F282Y, H302Q, A524P.

[0011] The (-)-alpha-bisabolol synthase (Bisabolol synthase) is derived from the plant Cynara scolymus (Cynara scolymus) Cynara cardunculus var. scolymus ), the sequence GenBank number of which is XP_024994640.1.

[0012] In the present application, the (-)-alpha-bisabolol synthase is BOS, the nucleotide sequence of which after codon optimization is shown in SEQ ID No. 1, which contains 1710 nucleotides; its amino acid sequence is shown in SEQ ID No. 2, which contains 569 amino acids. Further modification is made on the basis of BOS.

[0013] The application also relates to a (-)-alpha-bisabolol synthase mutant, which is obtained by mutating one or two or three or four of the 119th, 151st, 282nd, 302nd and 524th amino acids in the amino acid sequence of SEQ ID No. 2; further, the (-)-alpha-bisabolol synthase mutant is BOS_G119W, BOS_H151Y, BOS_F282Y, BOS_H302Q, BOS_A524P, BOS_G119W-H151Y, BOS_G119W-F282Y, BOS_G119W-H302Q, BOS_G119W-A524P, BOS_H151Y-F282Y, BOS_H151Y-H302Q, BOS_F282Y-H302Q, BOS_G119W-H151Y-F282Y, BOS_G119W-H151Y-H302Q, BOS_G119W-H151Y-A524P, BOS_G119W-H151Y-F282Y-H302Q, BOS_G119W-H151Y-F282Y-A524P. Further, the (-)-alpha-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q is obtained by mutating the 119th amino acid in the amino acid sequence of SEQ ID No. 2 from glycine G to tryptophan W, the 151st amino acid from histidine H to tyrosine Y, the 282nd amino acid from phenylalanine F to tyrosine Y, and the 302nd amino acid from histidine H to glutamine Q, and the specific amino acid sequence is shown in SEQ ID No. 3.

[0014] Preferably, in the (-)-alpha-bisabolol synthase mutant, the gene sequence encoding the amino acid sequence shown in SEQ ID No. 3 is shown in SEQ ID No. 4.

[0015] In the application, a gene encoding the above (-)-alpha-bisabolol synthase mutant is provided.

[0016] In the application, a recombinant expression vector containing the above BOS mutant gene encoding gene is provided, and a method for preparing (-)-alpha-bisabolol by introducing the recombinant expression vector into a Saccharomyces cerevisiae engineering strain is provided.

[0017] The term "mutation" used herein is used to represent that at least one nucleotide or amino acid in the BOS mutant gene or amino acid sequence is different from the BOS starting sequence for comparison, and the mutation of the enzyme can be realized by site-directed mutation through conventional methods in the art.

[0018] The term "nucleic acid molecule" used herein has the meaning commonly understood by one of ordinary skill in the art, and the nucleic acid molecule can be a polynucleotide including the one shown as SEQ ID No. 1, or a polynucleotide further including additional coding and / or non-coding sequences.

[0019] The term "vector" or "expression vector" used herein has the meaning commonly understood by one of ordinary skill in the art, and "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is referred to as an expression vector. In the process of expressing a foreign gene using S. cerevisiae, the foreign gene to be expressed is first ligated to a vector. The vector can be various conventional vectors in the art, such as commercially available plasmids, bacteriophages, viral vectors, etc. The plasmid YEp352 is preferred as the vector for expressing the mutant gene of (-)-α-bisabolol synthase in the present application.

[0020] In the present application, a recombinant expression cell comprising the vector of the present application is provided. The recombinant expression cell can be prepared by transforming the recombinant expression vector of the present application into a host cell. The host cell can be various conventional host cells in the art, provided that the recombinant expression vector is stably replicated and passed down, and the gene carried thereby is effectively expressed. S. cerevisiae is preferred as the host cell in the present application. Saccharomyces cerevisiae ), more preferably S. cerevisiae CEN.PK2-1C strain or S. cerevisiae PLOO strain.

[0021] In the present application, the biological material related to the above mutant is any one or a combination of the following biological materials: (a) an expression cassette comprising the above-mentioned coding gene; (b) a recombinant expression vector comprising the above-mentioned coding gene; (c) a recombinant expression vector comprising the expression cassette described in (a); (d) a recombinant expression cell comprising the above-mentioned coding gene; (e) a recombinant expression cell comprising the expression cassette described in (a); (f) a recombinant expression cell comprising the recombinant expression vector described in (b) or (c).

[0022] Further, the starting vector of the recombinant expression vector described in (b) and (c) is a plasmid of YEp series, etc.; preferably, YEp352 plasmid.

[0023] Further, the host cell of the recombinant expression cell described in (d), (e), and (f) is selected from eukaryotes, etc.; the eukaryotes include Saccharomyces (S. cerevisiae, S. pombe, etc.), etc. Saccharomyces) and the like. More specifically, the eukaryote is Saccharomyces cerevisiae (S. cerevisiae) Saccharomyces cerevisiae ), specifically Saccharomyces cerevisiae S. cerevisiae CEN.PK2-1C or S.cerevisiae PLOO strain.

[0024] In the present application, the use of the above-mentioned mutant, the encoding gene, and the biological material related to the mutant in the preparation of (-)-a-bisabolol synthase mutant is provided.

[0025] In the present application, the use of the above-mentioned mutant, the encoding gene, and the biological material related to the mutant in the synthesis of (-)-a-bisabolol is provided; further, the use in the biosynthesis of (-)-a-bisabolol is provided.

[0026] In the present application, a method for producing (-)-a-bisabolol is provided, which comprises the step of fermenting the above-mentioned recombinant expression cell.

[0027] Specifically, the method comprises the following steps: fermenting the above-mentioned recombinant expression cell, and collecting the fermentation product; extracting (-)-a-bisabolol in the fermentation product.

[0028] Specifically, the method is as follows: using S. cerevisiae as the host strain, transforming the BOS mutant gene expression vector comprising the BOS mutant gene of the present application into the host cell to obtain S. cerevisiae engineering bacteria. The engineering strain is inoculated into YPD medium for fermentation, and at the same time, the biphasic fermentation technology is used, and 20% n-dodecane is added as the second phase to promote the extraction of (-)-a-bisabolol from the aqueous phase. The specific reaction conditions such as the composition of the culture medium, the amount of the 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 sample of the organic phase is obtained, and the content of (-)-a-bisabolol in the fermentation broth of different engineering strains can be detected by the conventional gas chromatography detection method in the art.

[0029] The bioreactor used is selected in principle to be an apparatus that can ensure the proliferation of living cells and biochemical reactions of enzymes, such as a small-scale shaking flask, a fermentation tank, etc. The culture medium used is mainly the culture medium commonly used in the art which is suitable for the growth of S. cerevisiae, and the YPD culture medium is preferred in the present application. The fermentation temperature is selected in principle to be a temperature at which the cells maintain good growth and the enzymes maintain good catalytic performance. In a specific embodiment, the preferred fermentation temperature is 30°C.

[0030] The recombinant Saccharomyces cerevisiae strain constructed from the BOS_G119W-H151Y-F282Y-H302Q mutant produced a product (-)-α-bisabolol with a yield of 2005.13 mg / L after 48 h of fermentation, which is 16.7 times that of the wild-type (-)-α-bisabolol synthase expression strain, significantly improving the biosynthetic efficiency of (-)-α-bisabolol.

[0031] The present invention has the following advantages and effects compared with the prior art: (1) This invention uses the plant artichoke ( Cynara cardunculus var. scolymus Using the (-)-α-bisabolol synthase BOS, which catalyzes the conversion of farnesyl pyrophosphate (FPP) to (-)-α-bisabolol obtained in [the study], as the research object, this invention, through semi-rational design and site-directed mutagenesis, obtained a series of BOS mutants that significantly improved the FPP substrate conversion efficiency. Among them, the optimal (-)-α-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q, catalyzed the production of (-)-α-bisabolol from FPP to the wild type, reaching a yield of 2005.13 mg / L, which is 16.7 times that of the wild type. The (-)-α-bisabolol mutants of this invention enhance the performance of microbial synthesis of (-)-α-bisabolol, providing an effective technical route for the green industrial production of high-purity (-)-α-bisabolol.

[0032] (2) The mutation strategy successfully verified in (-)-α-bisabolol synthase BOS in this invention can provide a reference model for other sesquiterpene synthases, with good universality and scalability, and provide a paradigm for the efficient biosynthesis of a variety of natural products in the future.

[0033] (3) Through systematic enzyme modification and high-throughput screening, this invention breaks through the bottleneck of only being able to obtain low-abundance byproducts by chance, and provides a practical and feasible technical path for the industrial production of (-)-α-bisabolol. Attached Figure Description

[0034] Figure 1 This is a reaction diagram of the fermentation of Saccharomyces cerevisiae to produce (-)-α-bisabolol.

[0035] Figure 2 This is a gas chromatogram of (-)-α-bisabolol in the fermentation product of a recombinant strain expressing the (-)-α-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q.

[0036] Figure 3This is a mass spectrometry image of (-)-α-bisabolol in the fermentation product of a recombinant strain expressing the bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q.

[0037] Figure 4 This is a schematic diagram showing the yield of (-)-α-bisabolol in the fermentation products. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. It is worth noting that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Unless otherwise specified, the reagents, materials and instruments used can be regarded as conventional products that can be obtained commercially.

[0039] The reaction flow diagram of the synthesis of (-)-α-bisabolol catalyzed by (-)-α-bisabolol synthase BOS in this invention is as follows: Figure 1 As shown.

[0040] The Saccharomyces cerevisiae PL00 used in the examples is disclosed in the literature “CN118792290A, an α-farnesene synthase AoFS of a source of intelligence and its application”.

[0041] Example 1: Construction of the starting strain producing (-)-α-bisabolol The codon-optimized (-)-α-bisabolol synthase BOS gene was synthesized at Sangon Biotech. Its nucleotide sequence is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2. Using our laboratory's plasmid YEp352 (Addgene, containing the promoter CCW12 and terminator CYC1) as the starting vector, the amplified BOS gene fragment was inserted into… NdeI and XhoI The double-digested YEp352 vector was labeled as YEp352-BOS. Then it was transformed into... E. coli In DH5α competent cells, and after verification by colony PCR and sequencing, the recombinant plasmid YEp352-BOS, which correctly encodes the BOS gene, was obtained. Subsequently, Saccharomyces cerevisiae PL00 competent cells were obtained using the Frozen-EZ Yeast Transformation II Kit (purchased from Zymo), and the recombinant plasmid YEp352-BOS was transformed into Saccharomyces cerevisiae PL00 competent cells to obtain the recombinant strain PL00 / YEp352-BOS.

[0042] Construction of Example 2 different (-)-a-bisabolol synthase mutant strains Through the sequence (SEQ ID No. 2) of (-)-a-bisabolol synthase BOS conservative analysis and semi-rational design, the mutation sites are determined as follows: the 119th amino acid is mutated from glycine G to tryptophan W, the 151st amino acid is mutated from histidine H to tyrosine Y, the 282nd amino acid is mutated from phenylalanine F to tyrosine Y, the 302nd amino acid is mutated from histidine H to glutamine Q, and the 524th amino acid is mutated from alanine A to proline P.

[0043] The BOS mutant is constructed by homologous recombination. The obtained recombinant plasmid YEp352-BOS is used as a template for site-directed mutagenesis by PCR technology to obtain a plasmid containing a BOS mutant gene. The mutation sites are as follows: G119W, H151Y, F282Y, H302Q, A524P, G119W-H151Y, G119W-F282Y, G119W-H302Q, G119W-A524P, H151Y-F282Y, H151Y-H302Q, F282Y-H302Q, G119W-H151Y-F282Y, G119W-H151Y-H302Q, G119W-H151Y-A524P, G119W-H151Y-F282Y-H302Q, G119W-H151Y-F282Y-A524P.

[0044] The primers used in PCR are as follows (lowercase for replaced codons): BB-F: 5'-CAAGCTGTGACCGTCTCCGGGAGC-3'; BB-R: 5'-GCTCCCGGAGACGGTCACAGCTTG-3'; G119W-F: 5'-GGACACCAGtggATTCATAATAACAATG-3'; G119W-R: 5'-ATTATGAATccaCTGGTGTCCATAAGTAC-3'; H151Y-F: 5'-AAGAACtatATGGATGAAAAGGGCAATCTG-3'; H151Y-R: 5'-GATTGCCCTTTTCATCCATataGTTCTTAAAAATTTCTG-3'; F282Y-F: 5'-CAAGTTACCTtacGTGAGAGATCG-3'; F282Y-R: 5'-CGATCTCTCACgtaAGGTAACTTG-3'; H302Q-F: 5'-GAACCCcagCACAGTAGATCTAG-3'; H302Q-R: 5'-TCTACTGTGctgGGGTTCAAAATAGATA-3'; A524P-F: 5'-GAGGCCAACAGCAGTGccaTTCCCATTAGTTATG-3'; A524P-R: 5'-CATAACTAATGGGAAtggCACTGCTGTTGGCCTC-3'; For example, taking the plasmid YEp352-BOS_G119W with a single-point mutation as an example, the specific implementation is as follows: taking the plasmid YEp352-BOS as a template, using the primer pair BB-F / G119W-R and the primer pair G119W-F / BB-R respectively, the upstream fragment G119W-1 and the downstream fragment G119W-2 of the recombinant vector are amplified. The two fragments G119W-1 and G119W-2 obtained by amplification are recombined by using the ClonExpress® II recombination cloning kit to obtain a recombination product. The recombination product is transformed into E. coli DH5α competent cells, and colony PCR verification and plasmid sequencing verification are performed to obtain the plasmid YEp352-BOS_G119W containing the gene BOS_G119W encoding the (-)-a-bisabolol synthase mutant.

[0045] Then the recombinant plasmids (YEp352-BOS_G119W, YEp352-BOS_H151Y, YEp352-BOS_F282Y, YEp352-BOS_H302Q, YEp352-BOS_A524P, YEp352-BOS_G119W-H151Y, YEp352-BOS_G119W-F282Y, YEp352-BOS_G119W-H302Q, YEp352-BOS_G119W-A524P, YEp352-BOS_H151Y-F282Y, YEp352-BOS_H151Y-H302Q, YEp352-BOS_F282Y-H302Q, YEp352-BOS_G119W-H151Y-F282Y, BOS_G119W-H151Y-H302Q, YEp352-BOS_G119W-H151Y-A524P, YEp352-BOS_G119W-H151Y-F282Y-H302Q, YEp352-BOS_G119W-H151Y-F282Y-A524P) were transformed into the competent cells of S. cerevisiae PLOO, respectively, to obtain the recombinant strains containing the gene encoding (-)-a-bisabolol synthase mutant, respectively.

[0046] Shake flask fermentation and product detection of the recombinant strains producing (-)-a-bisabolol in Example 3 Single colonies of the recombinant strains constructed in Example 1 and Example 2 were selected and inoculated into 3 mL YPD liquid medium, and cultured at 30°C in a 220 rpm shaker for overnight. Then, 0.05 of initial OD600 was inoculated into 10 mL YPD liquid medium containing 2 mL n-dodecane, and shake flask fermentation was carried out for 48 h. Three biological replicates were set for each recombinant strain. 600 Shake flask fermentation was carried out by inoculating into 10 mL YPD liquid medium containing 2 mL n-dodecane, and the fermentation was carried out for 48 h. Three biological replicates were set for each recombinant strain.

[0047] The gas chromatograph was Shimadzu GC-2014C, the column was HP-5 column with the specification of 30 m x 0.32 mm x 0.25 μm, the detector was flame ionization detector (FID), and the temperature of the detector and the injector was set to 280°C and 250°C, respectively. The detection process was carried out with nitrogen as the carrier gas, and the split ratio was set to 5:1, and 1 μL of aliquot was injected in split mode. The program was set as follows: the initial temperature was 100°C, and maintained for 5 min, then increased to 280°C at a rate of 20°C / min, and maintained for 3 min, and the whole temperature program was run for 16.943 min.

[0048] The results of gas chromatography and mass spectrometry detection of the recombinant strain expressing (-)-a-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q are as follows:Figure 2 and Figure 3 As shown in FIG. 14, the peak with an elution time of 14.63 min is (-)-a-bisabolol. The fermentation results of different (-)-a-bisabolol synthase mutant strains are shown in Table 1 Figure 4 and Table 1, the yield of (-)-a-bisabolol produced by the fermentation of the 17 recombinant strains producing (-)-a-bisabolol is improved to different degrees. Among them, the recombinant strain expressing the optimal combination BOS_G119W-H151Y-F282Y-H302Q mutant has a yield of (-)-a-bisabolol that is 16.7 times that of the wild type, reaching 2005.13 mg / L. Through enzyme engineering of (-)-a-bisabolol synthase, the present application successfully achieves the efficient production of (-)-a-bisabolol in microorganisms, providing important technical support for the industrialized preparation and application of the compound.

[0049] Table 1: Comparison of the effects of (-)-a-bisabolol synthases with mutations compared with SEQ ID No. 2

[0050] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. (-)-α-bisabolol synthase BOS mutant, characterized in that: The amino acid sequence of the (-)-α-bisabolol synthase BOS mutant is obtained by any one of the following mutations of SEQ ID No. 2: At least one of G119W, H151Y, F282Y, H302Q, and A524P.

2. The (-)-α-bisabolol synthase BOS mutant according to claim 1, characterized in that: The (-)-α-bisabolol synthase BOS mutants are BOS_G119W, BOS_H151Y, BOS_F282Y, BOS_H302Q, BOS_A524P, BOS_G119W-H151Y, BOS_G119W-F282Y, BOS_G119W-H302Q, BOS_G119W-A524P, BOS_H151Y-F282Y, and B... OS_H151Y-H302Q, BOS_F282Y-H302Q, BOS_G119W-H151Y-F282Y, BOS_G119W-H151Y-H302Q, BOS_G119W-H151Y-A524P, BOS_G119W-H151Y-F282Y-H302Q or BOS_G119W-H151Y-F282Y-A524P; The amino acid sequence of the mutant BOS_G119W-H151Y-F282Y-H302Q is shown in SEQ ID No.

3.

3. The gene encoding the (-)-α-bisabolol synthase BOS mutant as described in 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 BOS_G119W-H151Y-F282Y-H302Q is shown in SEQ ID No.

4.

5. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in any one of claims 1 to 2, characterized in that: It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 3 or 4; (b) A recombinant expression vector containing the gene of claim 3 or 4; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant expression cells containing the gene of claim 3 or 4; (e) Recombinant expression cells containing the expression cassette described in (a); (f) Recombinant expression cells containing the recombinant expression vector described in (b) or (c).

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

7. The biomaterial according to claim 5 or 6, characterized in that: The host bacteria of the recombinant expression cells is *Saccharomyces cerevisiae*; further, the *Saccharomyces cerevisiae* is *Saccharomyces cerevisiae*. S. cerevisiae CEN.PK2-1C strain or S. cerevisiae PL00 strain.

8. The application of the (-)-α-bisabolol synthase BOS mutant according to any one of claims 1-2, the gene according to any one of claims 3-4, or the biomaterial according to any one of claims 5-7, characterized in that, For one of the following applications: 1) Application in the preparation of (-)-α-bisabolol synthase mutant; 2) Application in the synthesis of (-)-α-bisabolol.

9. A method for producing (-)-α-bisabolol, characterized in that, It includes the step of fermenting the recombinant expression cells as described in any one of claims 5 to 7.

10. The method according to claim 9, characterized in that: Fermenting the recombinant expression cells as described in any one of claims 5 to 7, and collecting the fermentation products; (-)-α-bisabolol was obtained by extraction from the fermentation product.

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