A (-)-a-bisabolol synthase mutant and use thereof

Abstract

The application discloses a kind of (-) -alpha-bisabolol synthase mutant and its application, belong to enzyme engineering and microbial engineering technical field.The application takes (--) -alpha-bisabolol synthase BOS of chrysanthemum source as object, based on sequence conservation analysis and semi-rational design, a series of BOS mutants obtained in the application are significantly improved in FPP substrate conversion efficiency, wherein, optimal (--) -alpha-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q, the yield of (-) -alpha-bisabolol is 16.7 times of wild type, yield is more than 2g / L, the (--) -alpha-bisabolol synthase mutant of the application enhances the performance of microorganism synthesis (--) -alpha-bisabolol, provides effective technical route for green production of industrial high-purity (--) -alpha-bisabolol.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and bioengineering technology, and specifically relates to a (-)-α-bisabolol synthase mutant and its application in the biosynthesis of (-)-α-bisabolol. Background Technology

[0002] α-Bisabolol is a high-value sesquiterpene alcohol with a cyclic structure. Due to its outstanding anti-inflammatory, antibacterial, anti-irritant, and soothing / repairing activities, it has become an indispensable core ingredient in high-end cosmetics, skincare products, and pharmaceutical preparations. With the continued growth of global market demand, traditional production methods relying on plant extraction are limited by factors such as planting cycles, regional climate, and extraction purity, making it difficult to stably and economically meet the demands of large-scale production.

[0003] The complex chiral structure of bisabolol further restricts the feasibility of its chemical synthesis route. Traditional chemical synthesis is usually lengthy, requiring large amounts of organic solvents and reagents, resulting in poor atom economy. More importantly, these methods struggle to precisely control the chiral center, easily generating racemic mixtures that reduce the bioactivity of the target product. In fact, only specific enantiomers (such as (-)-α-bisabolol) possess ideal bioactivity, and naturally derived (-)-α-bisabolol (levorotatory configuration) is significantly superior to the chemically synthesized racemate in both activity and commercial value. Therefore, stereochemical control becomes a key factor determining the economics and applicability of different production routes.

[0004] With the development of synthetic biology, heterologous synthesis by microorganisms has been identified as an innovative approach to achieve the sustainable production of bisabolol. In this system, bisabolol synthase, as a key rate-limiting enzyme in the biosynthetic pathway, is responsible for catalyzing the cyclization of the universal precursor farnesyl pyrophosphate (FPP) to bisabolol. Currently, researchers have successfully introduced natural bisabolol synthase genes derived from plants such as German chamomile into hosts such as Escherichia coli or yeast, achieving preliminary heterologous synthesis of bisabolol. However, the industrial application of this technology still faces a fundamental bottleneck: the low catalytic activity of natural bisabolol synthase leads to low microbial synthesis efficiency and high production costs.

[0005] Therefore, enzyme engineering modification of natural bisabolol synthase has become a key breakthrough for improving the efficiency of the entire biosynthetic pathway and promoting its industrialization. Mutating the active site, substrate channel, or key amino acid residues of bisabolol through directed evolution or rational design aims to directly enhance its catalytic activity, thermal stability, and product specificity, representing a core strategy for fundamentally solving these problems.

[0006] Based on this urgent technological need, this invention significantly improves the catalytic performance of bisabolol synthase by constructing a series of bisabolol synthase mutants, laying a solid foundation for the efficient, economical and sustainable microbial production of bisabolol. Summary of the Invention

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

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

[0009] The technical problem this invention aims to solve is that the production efficiency of (-)-α-bisabolol by wild-type (-)-α-bisabolol synthase using farnesyl pyrophosphate (FPP) as a substrate is low, making it difficult to meet the yield requirements for industrial purification and subsequent applications. To address this, this invention designs and screens a series of (-)-α-bisabolol synthase mutants, significantly increasing the yield of (-)-α-bisabolol and providing a new approach and case study for the efficient microbial biosynthesis of (-)-α-bisabolol.

[0010] The objective of this invention is achieved through the following technical solution: A (-)-α-bisabolol synthase mutant, wherein the amino acid sequence of the (-)-α-bisabolol synthase mutant is obtained by any one of the following mutations as shown in SEQ ID No. 2: At least one of G119W, H151Y, F282Y, H302Q, and A524P.

[0011] The (-)-α-bisabolol synthase is derived from the plant artichoke ( Cynara cardunculus var. scolymus Its sequence number in GenBank is XP_024994640.1.

[0012] In this invention, the (-)-α-bisabolol synthase is BOS, whose codon-optimized nucleotide sequence is shown in SEQ ID No. 1, containing a total of 1710 nucleotides; its amino acid sequence is shown in SEQ ID No. 2, containing a total of 569 amino acids. Further modifications are made based on BOS.

[0013] This invention also relates to a (-)-α-bisabolol synthase mutant, the amino acid sequence of which is obtained by mutation at one, two, three, or four sites of amino acids 119, 151, 282, 302, and 524 of SEQ ID No. 2; further, the (-)-α-bisabolol synthase mutants are BOS_G119W, BOS_H151Y, BOS_F282Y, BOS_H302Q, BOS_A524P, BOS_G119W-H151Y, BOS_G119W-F282Y, BOS_G119W-H302Q, and BOS_G119W. -A524P, BOS_H151Y-F282Y, BOS_H151Y-H302Q, BOS_F282Y-H302Q, BOS_G119W-H151Y-F282Y, BOS_G119W-H 151Y-H302Q, BOS_G119W-H151Y-A524P, BOS_G119W-H151Y-F282Y-H302Q, BOS_G119W-H151Y-F282Y-A524P; Furthermore, the (-)-α-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q has the following amino acid sequence: amino acid position 119 is mutated from glycine (G) to tryptophan (W), amino acid position 151 is mutated from histidine (H) to tyrosine (Y), amino acid position 282 is mutated from phenylalanine (F) to tyrosine (Y), and amino acid position 302 is mutated from histidine (H) to glutamine (Q). The specific amino acid sequence is shown in SEQ ID No. 3.

[0014] Preferably, in the (-)-α-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 this invention, a gene encoding the above-mentioned (-)-α-bisabolol synthase mutant is provided.

[0016] This invention provides a recombinant expression vector containing the above-mentioned BOS mutant encoding gene, and provides a method for producing (-)-α-bisabolol by introducing the recombinant expression vector into an engineered strain of Saccharomyces cerevisiae.

[0017] As used herein, the term "mutation" refers to a mutation in the BOS mutant gene or amino acid sequence that differs from the BOS starting sequence being compared, and the mutation of the enzyme can be achieved by site-directed mutagenesis using methods conventional in the art.

[0018] As used herein, the term “nucleic acid molecule” has the meaning commonly understood by one of ordinary skill in the art. A nucleic acid molecule may include polynucleotides such as those shown in SEQ ID No. 1, or may include polynucleotides that also include additional coding and / or non-coding sequences.

[0019] 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 medium into which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. In the process of expressing a foreign gene using *Saccharomyces cerevisiae*, the foreign gene to be expressed must first be ligated to a vector. The vector can be any conventional vector in the art, such as commercially available plasmids, bacteriophages, viral vectors, etc. This invention preferably uses plasmid YEp352 to express the (-)-α-bisabolol synthase mutant gene.

[0020] The invention provides recombinant expression cells containing the vector of the present invention. These recombinant expression cells can be prepared by transforming the recombinant expression vector of the present invention into host cells. The host cells can be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated and passaged, and the gene carried by it can be effectively expressed. The present invention preferably uses *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), more S. cerevisiae CEN.PK2-1C strain or S. cerevisiae PL00 strain.

[0021] In this invention, the aforementioned mutant-related biomaterials are any one or more combinations of the following biomaterials: (a) An expression cassette containing the above-mentioned encoded genes; (b) Recombinant expression vectors containing the above-mentioned coding genes; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant expression cells containing the above-encoded genes; (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).

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

[0023] Furthermore, the host bacteria of the recombinant expression cells described in (d), (e), and (f) are selected from eukaryotes, etc.; the eukaryotes include the genus *Saccharomyces* (Saccharomyces). SaccharomycesMore specifically, the eukaryote is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae Specifically, it can be brewing yeast. S. cerevisiae CEN.PK2-1C or S.cerevisiae PL00 strain.

[0024] This invention provides an application of the above-mentioned mutant, encoding gene, and mutant-related biological materials in the preparation of (-)-α-bisabolol synthase mutant.

[0025] This invention provides the application of the above-mentioned mutant, encoding gene, and mutant-related biological material in the synthesis of (-)-α-bisabolol; and further its application in the biosynthesis of (-)-α-bisabolol.

[0026] This invention provides a method for producing (-)-α-bisabolol, comprising the step of fermenting the above-mentioned recombinant expression cells.

[0027] Specifically, the steps include the following: Ferment the above-mentioned recombinant expression cells and collect the fermentation products; (-)-α-bisabolol was obtained by extraction from the fermentation product.

[0028] The specific method is as follows: Using *Saccharomyces cerevisiae* as the host strain, the expression vector containing the BOS mutant gene described in this invention is transformed into the host cells to obtain engineered *Saccharomyces cerevisiae* strains. The engineered strains are inoculated into YPD medium for fermentation, simultaneously employing a two-phase fermentation technique with the addition of 20% n-dodecane as the second phase to promote the extraction of (-)-α-bisabolol from the aqueous phase. Specific reaction conditions, such as the composition of the culture medium and the 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 preferred fermentation time is 48 hours. After the reaction is completed, an organic phase sample is obtained, and the content of (-)-α-bisabolol in the fermentation broth of different engineered strains can be detected using conventional gas chromatography methods in the art.

[0029] The bioreactor used is, in principle, selected from equipment capable of ensuring cell proliferation and biochemical reactions by living cells and enzymes, such as small shake flasks or fermenters. The culture medium used is primarily a commonly used medium suitable for the growth of *Saccharomyces cerevisiae*, with YPD medium being preferred in this invention. The fermentation temperature is preferably selected to maintain good cell growth and optimal enzyme catalytic performance. In one specific embodiment, a fermentation temperature of 30°C is preferred.

[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] Example 2: Construction of strains containing different (-)-α-bisabolol synthase mutants Through conservation analysis of the sequence (SEQ ID No. 2) of (-)-α-bisabolol synthase BOS and semi-rational design, the mutation sites were determined to be: amino acid 119, which was mutated from glycine (G) to tryptophan (W); amino acid 151, which was mutated from histidine (H) to tyrosine (Y); amino acid 282, which was mutated from phenylalanine (F) to tyrosine (Y); amino acid 302, which was mutated from histidine (H) to glutamine (Q); and amino acid 524, which was mutated from alanine (A) to proline (P).

[0043] The BOS mutant was constructed using homologous recombination. Using PCR, the recombinant plasmid YEp352-BOS was used as a template for site-directed mutagenesis to obtain a plasmid containing the gene encoding the BOS mutant. 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-H30 2Q, G119W-H151Y-A524P, G119W-H151Y-F282Y-H302Q, G119W-H151Y-F282Y-A524P.

[0044] The primers used for PCR are as follows (lowercase letters indicate the 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'; Taking the construction of the single-point mutation plasmid YEp352-BOS_G119W as an example, the specific implementation plan is as follows: Using plasmid YEp352-BOS as a template, the upstream fragment G119W-1 and the downstream fragment G119W-2 of the recombinant vector are amplified using primer pairs BB-F / G119W-R and G119W-F / BB-R, respectively. The amplified G119W-1 and G119W-2 fragments are recombined using the ClonExpress® II Recombinant Cloning Kit to obtain the recombinant product. The recombinant product is then transformed into... E. coli DH5α competent cells were used and colony PCR and plasmid sequencing were performed to verify the results, and plasmid YEp352-BOS_G119W containing the gene encoding the (-)-α-bisabolol synthase mutant gene BOS-G119W was obtained.

[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-H) were respectively... Recombinant strains containing the gene encoding (-)-α-bisabolol synthase mutant were obtained by transforming YEp352Q, YEp352-BOS_F282Y-H302Q, YEp352-BOS_G119W-H151Y-F282Y, BOS_G119W-H151Y-H302Q, YEp352-BOS_G119W-H151Y-A524P, YEp352-BOS_G119W-H151Y-F282Y-H302Q, and YEp352-BOS_G119W-H151Y-F282Y-A524P into Saccharomyces cerevisiae PL00 competent cells.

[0046] Example 3: Shake-flask fermentation and product detection of recombinant strains producing (-)-α-bisabolol Single clones of the recombinant strains constructed in Examples 1 and 2 were selected and inoculated into 3 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm in a shaker. Subsequently, the culture was performed with an initial OD of 0.05. 600 The inoculum was transferred to a shake flask containing 2 mL of n-dodecane in 10 mL of YPD liquid medium and fermented for 48 h. Each recombinant strain was divided into three biological replicates.

[0047] The gas chromatograph was a Shimadzu GC-2014C, with an HP-5 column measuring 30m × 0.32mm × 0.25μm. The detector was a flame ionization detector (FID), and the detector and injector temperatures were set to 280℃ and 250℃, respectively. Nitrogen was used as the carrier gas, with a split ratio of 5:1, and 1μL aliquots were injected in split mode. The program was as follows: initial temperature 100℃, held for 5 min, then increased to 280℃ at a rate of 20℃ / min, held for 3 min. The total temperature program duration was 16.943 min.

[0048] The gas chromatography and mass spectrometry results of the recombinant strain expressing the (-)-α-bisabolol synthase mutant BOS_G119W-H151Y-F282Y-H302Q are as follows: Figure 2 and Figure 3 As shown, the peak eluted at 14.63 min is (-)-α-bisabolol. Fermentation results for different (-)-α-bisabolol synthase mutant strains are shown in [the table / reference needed]. Figure 4 As shown in Table 1, the yield of (-)-α-bisabolol, a fermentation product, was increased to varying degrees in all 17 recombinant strains producing (-)-α-bisabolol. Among them, the recombinant strain expressing the optimal mutant combination BOS_G119W-H151Y-F282Y-H302Q achieved a (-)-α-bisabolol yield 16.7 times that of the wild type, reaching 2005.13 mg / L. This invention successfully achieved the efficient production of (-)-α-bisabolol from microorganisms by modifying (-)-α-bisabolol synthase through enzyme engineering, providing important technical support for the industrial preparation and application of this compound.

[0049] Table 1: Comparison of the effects of (-)-α-bisabolol synthase with mutation compared to SEQ ID No. 2

[0050] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. (-)-α-bisabolol synthase BOS mutant, characterized by: The (-)-α-bisabolol synthase BOS mutant is obtained by mutation of SEQ ID No. 2, specifically: 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_H 151Y-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.

2. The gene encoding the (-)-α-bisabolol synthase BOS mutant as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the mutant BOS_G119W-H151Y-F282Y-H302Q is shown in SEQ ID No.

4.

4. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biological material is an expression cassette containing the gene described in claim 2 or 3.

5. The biomaterial related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biomaterial is a recombinant expression vector containing the gene described in claim 2 or 3.

6. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biomaterial is a recombinant expression vector containing the expression cassette of claim 4.

7. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biological material is a recombinant expression cell containing the gene described in claim 2 or 3.

8. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biomaterial is a recombinant expression cell containing the expression cassette of claim 4.

9. The biomaterials related to the (-)-α-bisabolol synthase BOS mutant as described in claim 1, characterized in that: The biological material is a recombinant expression cell containing the recombinant expression vector of claim 5 or 6.

10. The biomaterial according to claim 5 or 6, characterized in that: The starting vector for the recombinant expression vector is a plasmid from the YEp series.

11. The biomaterial according to any one of claims 7 to 9, characterized in that: The host bacteria of the recombinant expression cells are selected from eukaryotes.

12. The biomaterial according to claim 11, characterized in that: The host bacteria of the recombinant expression cells is Saccharomyces cerevisiae.

13. The biomaterial according to claim 12, characterized in that: The brewing yeast is brewing yeast. S. cerevisiae CEN.PK2-1C strain or S. cerevisiae PL00 strain.

14. The use of the gene according to any one of claims 2 to 3 or the biological material according to any one of claims 4 to 13 in the preparation of (-)-α-bisabolol synthase mutant.

15. The use of the (-)-α-bisabolol synthase BOS mutant of claim 1, the gene of any one of claims 2 to 3, or the biological material of any one of claims 4 to 13 in the synthesis of (-)-α-bisabolol.

16. 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 7-9 and 11-13.

17. The method according to claim 16, characterized in that: Fermenting the recombinant expression cells as described in any one of claims 7-9 and 11-13, and collecting the fermentation products; (-)-α-bisabolol was obtained by extraction from the fermentation product.

Images