Recombinant saccharomyces cerevisiae as well as preparation method and application thereof
By transferring the sesquiterpene synthase gene in the stem of the scented cerevisiae into Saccharomyces cerevisiae and using the expression system of the cerevisiae for heterologous expression, the problem of difficult to improve the yield and quality of trans nervoyant in the volatile oil in the scented volatile oil in the prior art is solved, and efficient and green trans nervoyant production is achieved.
Patent Information
- Application Number
- CN202311574046.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-23
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to effectively improve the yield and quality of trans neroli in aroma-lowering volatile oils, and is limited by the spatiotemporal and spatiotemporal specific expression of sesquiterpene synthase gene.
The sesquiterpene synthase gene of the ceramia ceramia stem was obtained by cloning from the discoloration area of the stem, and transferred it into Saccharomyces ceramia cerevisiae. The eukaryotic expression system of Saccharomyces ceramia cerevisiae was used for heterologous expression, and combined with galactose induction and biphasic fermentation technology, trans tert-ol was obtained.
It realizes the efficient expression of sedanol sesquiterpene synthase in Saccharomyces cerevisiae, and directly recovers trans neroli tertol from the fermentation broth, which improves yield and quality, and provides a sustainable green production path.
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Figure CN120025916A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant brewer's yeast capable of expressing a dalbergia odorifera sesquiterpene synthase gene, and a preparation method and application thereof. Background Art
[0002] The traditional Chinese medicine Dalbergia odorifera is the dried heartwood of the trunk and root of the leguminous plant Dalbergia odorifera T.Chen. It is pungent and warm in nature. It enters the liver and spleen meridians, has the effects of removing blood stasis and stopping bleeding, regulating qi and relieving pain, and is often used in clinical prevention and treatment of cardiovascular diseases. Modern research shows that it has the effects of resisting myocardial hypoxia, improving myocardial function, and resisting thrombosis. In addition, Dalbergia odorifera has also shown efficacy in anti-oxidation, anti-inflammatory, anti-tumor, and anti-pathogenic microorganisms. At present, more than 270 chemical components have been isolated and identified from Dalbergia odorifera, including flavonoids, volatile oils (including sesquiterpenes), phenols, quinones, etc. It is generally believed that the main chemical components in Dalbergia odorifera are volatile oils and flavonoids, accounting for 3.61% to 3.79% and 2.51% to 5.82%, respectively. Among them, trans-nerolidol occupies a dominant position in Dalbergia odorifera volatile oils, and is also the material basis of the efficacy of Dalbergia odorifera in preventing and treating cardiovascular diseases and other diseases. In Dalbergia odorifera, the expression of sesquiterpene synthase genes is regulated by multiple factors, and its expression has temporal and spatial specificity. The composition and content of Dalbergia odorifera volatile oil obtained by different artificial induction methods have certain differences, which limits the application of Dalbergia odorifera volatile oil to a certain extent. Therefore, it is of great significance to mine the sesquiterpene synthase genes from Dalbergia odorifera and clone the Dalbergia odorifera sesquiterpene synthase genes into bacteria or fungi for heterologous expression by genetic engineering methods to improve the yield and quality of Dalbergia odorifera sesquiterpene.
[0003] Nerolidol is a naturally occurring acyclic sesquiterpene alcohol with a special aroma. It is widely found in the volatile oils of plants such as orange blossom, lemongrass, ginger, and dalbergia odorifera. Studies have shown that nerolidol has a wide range of biological activities, such as antibacterial, antioxidant, anti-inflammatory, anti-malarial, anti-cancer, and enhanced skin permeability. In addition, nerolidol has been approved for use in China, the United States, Europe and other countries or regions, and is widely used in cosmetics, detergents, and food additives. However, the content of nerolidol in plants is usually very low. Metabolic engineering of microorganisms such as Escherichia coli and Saccharomyces cerevisiae provides a path for the sustainable and green production of nerolidol. Since Escherichia coli does not have the corresponding terpene synthase metabolic pathway and cannot process and modify proteins, it is necessary to break the Escherichia coli cells, separate and purify the heterologously expressed proteins, and obtain the target product through in vitro enzymatic reactions. For example, in the study by Ye et al. [Ye, et al. (2018). Identification and Characterization of a Novel Sesquiterpene Synthase from Aquilaria Sinensis: An Important Gene for Agarwood Formation. International Journal of Biological Macromolecules, 108, 884–892.], the As-sesTPS gene encoding the sesquiterpene synthase from Aquilaria sinensis was expressed in Escherichia coli BL21 (DE3) in the form of inactive inclusion bodies. After purification and renaturation, it can catalyze farnesyl pyrophosphate (FPP) in vitro to produce nerolidol. Compared with prokaryotes, yeast has a mature eukaryotic expression system. The yeast can accumulate FPP through its own MVA pathway and has compatibility with expressing plant-derived sesquiterpene synthase. Therefore, heterologous expression of sesquiterpene synthase genes using Saccharomyces cerevisiae can simultaneously achieve heterologous expression of sesquiterpene synthase genes and substrate conversion, and can directly separate and recover the desired terpene products from the fermentation broth. For example: Li et al. [Li, et al. (2021). Characterization of Trans-Nerolidol Synthase from Celastrus Angulatus Maxim and Production of Trans-Nerolidol in Engineered Saccharomyces Cerevisiae. Journal of Agricultural and Food Chemistry, 69(7), 2236–2244.].The nerolidol synthase gene from Celastrusangulatus was introduced into the engineered yeast of Saccharomyces cerevisiae. By inducing overexpression of MVA pathway enzymes, optimizing the allocation of carbon sources to the nerolidol synthesis pathway, and adopting batch-fed fermentation, the trans-nerolidol shake flask yield reached 740.13 mg / L and the fermentation tank yield reached 7.01 g / L. Recently, Hong et al. reported the genome of Dalbergia odorifera [Hong, et al. (2020). The chromosome-level draft genome of Dalbergia odorifera. GigaScience. 9, 1-8.]. However, there are no reports on heterologous expression of the terpenoid synthase gene of Dalbergia odorifera. Summary of the invention
[0004] To this end, on the one hand, the present application provides a recombinant Saccharomyces cerevisiae containing the Dalbergia odorifera sesquiterpene synthase gene, which is obtained by transferring the Dalbergia odorifera sesquiterpene synthase gene shown in SEQ ID NO.1 cloned from the color change zone in the stem of Dalbergia odorifera into a competent cell of Saccharomyces cerevisiae, and the gene sequence of SEQ ID NO.1 is described in the sequence list of the specification.
[0005] Preferably, the amino acid sequence of the protein encoded by the Dalbergia odorifera sesquiterpene synthase gene is shown as SEQ ID NO.2, and the amino acid sequence of SEQ ID NO.2 is as described in the sequence table in the specification.
[0006] Preferably, the dalbergia odorifera sesquiterpene synthase gene is DoNES5, the competent cell of the recombinant Saccharomyces cerevisiae is WAT11, and the strain of the recombinant Saccharomyces cerevisiae is DoNES5-WAT11. The recombinant Saccharomyces cerevisiae strain DoNES5-WAT11 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101. Its deposit number is CGMCC No. 29123, the recommended classification name is Saccharomyces cerevisiae, and the deposit date is November 23, 2023.
[0007] On the other hand, the present application provides a method for preparing the recombinant Saccharomyces cerevisiae containing the above-mentioned dalbergia odorifera sesquiterpene synthase gene, which comprises the following steps:
[0008] a) cloning the Dalbergia odorifera sesquiterpene synthase gene shown in SEQ ID NO.1 from the color change region of Dalbergia odorifera stems;
[0009] b) recombining the dalbergia odorifera sesquiterpene synthase gene into the expression vector by seamless connection to obtain a connection product;
[0010] c) transforming the ligated product into a recombinant Saccharomyces cerevisiae strain;
[0011] d) Screening the successfully transformed recombinant Saccharomyces cerevisiae strains.
[0012] Preferably, the dalbergia odorifera sesquiterpene synthase gene in step a) is DoNES5, the expression vector in step b) is pESC-ura, the recombinant Saccharomyces cerevisiae strain in step c) is WAT11, and the recombinant Saccharomyces cerevisiae strain successfully transformed in step d) is DoNES5-WAT11.
[0013] On the other hand, the present application provides a use of the above-mentioned recombinant Saccharomyces cerevisiae in producing sesquiterpenes, which comprises using galactose to induce the recombinant Saccharomyces cerevisiae to express the dalbergia odorifera sesquiterpene synthase gene, and performing biphasic fermentation and recovering a sesquiterpene: trans-nerolidol.
[0014] According to the above technical scheme of the present application, a new recombinant Saccharomyces cerevisiae containing the dalbergia odorifera sesquiterpene synthase gene and its preparation method and use are obtained. The recombinant Saccharomyces cerevisiae can be used to express trans-nerolidol. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention is described below in conjunction with the accompanying drawings, but the present invention is not limited thereto. In the accompanying drawings:
[0016] Figure 1 It is a nucleic acid sequence alignment diagram. Among them, DoNES5 is the gene sequence cloned in this application (SEQ ID NO.1), and novel.1158 is the sequence published by GigaDB. There are 15 base differences between the two. This phenomenon of large-scale nucleotide variation also appeared in our previous studies, such as the comparison of agarwood ASS1, ASS2 and ASS3 genes with published gene sequences [Ran, et al. (2023). Identification of Sesquiterpene Synthase Genes in the Genome of Aquilaria Sinensis and Characterization of an α-Humulene Synthase. Journal of Forestry Research, 34 (4), 1117–1131.]. This indicates that the frequency of single nucleotide variation of sesquiterpene synthase genes at certain sites in plants may be relatively high.
[0017] Figure 2It is an amino acid sequence alignment diagram. Among them, there are 9 different amino acid sequences, namely KN (lysine-asparagine) at position 13, IN (isoleucine-asparagine) at position 50, EK (glutamic acid-lysine) at position 51, RH (arginine-histidine) at position 63, ND (asparagine-aspartic acid) at position 66, GS (glycine-serine) at position 71, TA (threonine-alanine) at position 80, EK (glutamic acid-lysine) at position 103, and LV (leucine-valine) at position 244.
[0018] Figure 3 It is the total ion chromatogram of the GC-MS detection of the fermentation broth of recombinant Saccharomyces cerevisiae. Among them, Figure A is the GC-MS detection gas chromatogram and mass spectrum of the fermentation broth of the recombinant Saccharomyces cerevisiae strain DoNES5-WAT11, Figure B is the GC-MS detection gas chromatogram and mass spectrum of the trans-nerolidol standard (purchased from Bid Pharmaceutical Technology Co., Ltd.), and Figure C is the GC-MS detection total ion chromatogram of the fermentation broth of the Saccharomyces cerevisiae strain control pESC-ura-WAT11. By comparing the mass spectrum of the fermentation product in Figure A with the mass spectrum of the trans-nerolidol standard in Figure B and / or the NIST database, it can be determined that Peak 1 (retention time 30.0min) is trans-nerolidol. Compared with Figure A, no sesquiterpenoids were detected at the same position in Figure C, indicating that the recombinant Saccharomyces cerevisiae strain DoNES5-WAT11 can efficiently express the DoNES5 protein, which is a functional sesquiterpenoid synthase that can utilize farnesyl pyrophosphate (FPP) to produce a major sesquiterpenoid compound, namely trans-nerolidol. DETAILED DESCRIPTION
[0019] In order to more clearly understand the present invention, the present invention is described in detail below by means of preferred embodiments in conjunction with the accompanying drawings, but the present invention is not limited thereby.
[0020] After sequence search, the inventors found the sesquiterpene synthase gene novel.1158 contained in the Dalbergia odorifera genome. The inventors designed specific primers based on the sequence of the sesquiterpene synthase gene novel.1158 and cloned the sesquiterpene synthase gene from the color change region in the stem of Dalbergia odorifera. The sesquiterpene synthase gene cloned from the color change region in the stem of Dalbergia odorifera by the inventors through specific primers has a high similarity with the novel.1158 gene in the Dalbergia odorifera genome reported by Hong et al., but has a difference of 8 base pairs after sequence alignment. At the level of encoded protein, the predicted protein has a difference of 8 amino acids from the novel.1158 protein reported by Hong et al. The inventors believe that the gene is the allele of the novel.1158 gene reported by Hong et al. in different Dalbergia odorifera strains, and the sequence variation is caused by single nucleotide polymorphisms (SNPs) between different individuals, or by base mutations caused by sequence amplification during sequencing. The inventors named the gene DoNES5, and transferred it into Saccharomyces cerevisiae. After galactose induction, the recombinant Saccharomyces cerevisiae expressed trans-nerolidol.
[0021] It should be noted that the scientific terms and test methods mentioned in the present invention but not explained or described in detail have the same meanings and contents as those understood by those skilled in the art.
[0022] The plasmids, endonucleases, PCR enzymes, RNA extraction kits, and DNA gel recovery kits used in the following preferred embodiments are commercially available products, which are mainly purchased from Tiangen Biochemical Technology Co., Ltd., and the specific operations are performed according to the instructions of the kits. The chemical reagents and conventional biochemical experimental consumables used in the embodiments are mainly purchased from Shanghai Shenggong Bioengineering Co., Ltd.
[0023] The routine operation methods such as colony PCR, nucleic acid agarose gel electrophoresis, polyethylene glycol / lithium chloride (PEG / LiAc) transformation, and preparation of competent cells were performed according to the fourth edition of Molecular Cloning: A Laboratory Manual (Fourth Edition). The sequencing of plasmids and DNA products was assisted by Shanghai Shenggong Biotechnology Co., Ltd.
[0024] Example
[0025] Example 1
[0026] 1. Construction of Saccharomyces cerevisiae containing the gene for Dalbergia odorifera sesquiterpenoid synthase
[0027] 1) Extraction of RNA from the discolored area of Dalbergia odorifera stems
[0028] The discolored area in the stem of a 5-year-old Dalbergia odorifera (planted in the greenhouse of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences, Beijing) was cut into small pieces with pruning shears and immediately ground in a mortar pre-cooled with liquid nitrogen. RNA from the stem of Dalbergia odorifera was obtained using a polysaccharide and polyphenol plant total RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) according to the RNA extraction method given in the kit.
[0029] 2) Reverse transcription and gene amplification
[0030] TIANScriptⅡ reverse transcription kit (purchased from Tiangen Biochemical Technology Co., Ltd.) was used for reverse transcription: RNA was used as a template to synthesize the first-strand cDNA by reverse transcription; then, the cDNA was used as a template to amplify the sesquiterpene synthase gene from Dalbergia odorifera using primers DoNES5-F and DoNES5-R (provided by Shanghai Shenggong Biotechnology Co., Ltd.), wherein the sequences of the primers are as follows:
[0031] DoNES5-F:5'-atggatgcaatctatgtcaaagaa-3'
[0032] DoNES5-R:5'-ctaaccagtgacttgaattaattgct-3'
[0033] The amplification program was as follows: denaturation at 95°C for 10 min; 35 temperature cycles, each cycle including denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 2 min; and insulation at 72°C for 10 min.
[0034] The amplified product was electrophoresed, recovered using a gel recovery kit, and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing to verify the correctness of the sequence. The full-length CDS sequence of DoNES5 and the original genome had a consistency of 99.02%, the encoding amino acid sequence had a consistency of 98.24% and a similarity of 99.02%, and the amino acid sequence encoded by the gene did not have frameshift and premature termination, and no mutations occurred in the conserved domains "DDXXD" and "NSE / DTE", so it was considered that the full length of the gene had been successfully obtained.
[0035] 3) Construction of recombinant Saccharomyces cerevisiae expression vector by seamless cloning and ligation
[0036] A plasmid extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) was used to extract the pESC-ura plasmid stored in the laboratory. The plasmid was fully digested with BamH I restriction endonuclease and HindⅢ restriction endonuclease (both purchased from Tiangen Biochemical Technology Co., Ltd.). After the digestion, a linearized vector was formed. The linearized vector was gel-recovered and purified using a universal DNA purification and recovery kit (purchased from Tiangen Biochemical Technology Co., Ltd.).
[0037] Design homologous recombination primers as follows:
[0038] pESC-ura-DoNES5-F:5'-acgtcaaggagaaaaaaccccgatggatgcaatctatgtcaaagaa-3'
[0039] pESC-ura-DoNES5-R:5'-atcttagctagccgcggtaccactaaccagtgacttgaattaattgct-3'
[0040] The homologous recombination primers were used to amplify the coding sequence of the DoNES5 gene, and the amplification program was as follows: denaturation at 95°C for 10 min; 35 temperature cycles, each cycle including denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 2 min; and insulation at 72°C for 10 min.
[0041] The amplified product was recovered using a universal DNA purification and recovery kit. The DoNES5 gene coding sequence recovery product was mixed with the linearized vector recovered by enzyme digestion to form a mixture. According to the instructions of the EasyGeno Single Fragment Recombination Cloning Kit (purchased from Tiangen Biochemical Technology Co., Ltd.), the reaction solution of the kit was added to the mixture to prepare a mixed solution. The mixed solution was placed at 50°C for reaction for 15 minutes. After instantaneous centrifugation, the centrifuge tube was placed on ice, and the reaction product was transformed into DH5α competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.). After colony PCR verification, the bacterial solution was sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing to verify the correctness of the recombinant plasmid construction.
[0042] Competent cells of Saccharomyces cerevisiae strain WAT11 (purchased from Baosai Plasmid Strain Resource Company) were prepared using lithium acetate buffer (TE / LiAc) (purchased from Beijing Coolbo Technology Co., Ltd.). The successfully constructed recombinant plasmid was extracted from Escherichia coli DH5α strain (purchased from Tiangen Biochemical Technology Co., Ltd.) using the Tiangen Plasmid Mini-Extraction Kit, and transformed into competent cells of Saccharomyces cerevisiae strain WAT11 using the polyethylene glycol / lithium acetate (PEG / LiAc) transformation method, and verified by the PCR method. The verified correct strain was named DoNES5-WAT11 strain.
[0043] 2. Inducing DoNES5-WAT11 Saccharomyces cerevisiae to express Dalbergia odorifera sesquiterpenoid synthase and produce sesquiterpenoids
[0044] Yeast uracil-deficient synthetic agar fine powder medium [uracil-deficient / SC-Ura, ELITE-MEDIA] was purchased from ELITE Biotechnology Co., Ltd. to prepare yeast uracil-deficient glucose liquid medium (SC / Ura powder 7.9 g / L, glucose 20 g / L), yeast uracil-deficient glucose solid medium (SC / ura powder 7.9 g / L, glucose 20 g / L, agar 29 g / L), and yeast uracil-deficient galactose liquid medium (SC / ura powder 7.9 g / L, galactose 20 g / L).
[0045] The DoNES5-WAT11 strain was streaked to obtain a single colony, and a positive single colony was picked and placed in 5 mL of the above SC / ura liquid medium (containing 2% glucose), and cultured overnight at 9°C and 200 r / min. 1 mL of the bacterial solution was added to 10 mL of fresh SC / ura liquid medium (containing 2% glucose), where OD 600 The value is about 2.5. Collect the cells by centrifugation and wash with sterile ddH 2 O and washed twice. Take an appropriate amount of bacteria and inoculate them into 50mL of SC-ura medium containing 2% galactose carbon source, and then induce culture at 29°C and 200r / min for 3 days to obtain the culture product. In order to avoid product volatilization, after 12h of fermentation, 5mL of n-dodecane was added to the product for two-phase fermentation. The fermentation broth of the pESC-ura-WAT11 strain (prepared by polyethylene glycol / lithium acetate (PEG / LiAc) conversion method) was used as a blank control.
[0046] 3. Detection of fermentation products of DoNES5-WAT11 Saccharomyces cerevisiae
[0047] The bacterial suspension of DoNES5-WAT11 and the empty vector pESC-ura-WAT11 was poured into a centrifuge tube, centrifuged at 8000 rpm for 10 min, the upper organic phase (n-dodecane) was collected, and anhydrous sodium sulfate was added for dehydration overnight. After centrifugation again, the sample was collected in the inner tube of the injection bottle using a 0.22 μM organic filter membrane (purchased from Beekman Biotechnology Co., Ltd.) and detected using a gas chromatography-mass spectrometer (GC-MS) (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing).
[0048] GC-MS analysis was carried out on a Thermo Fisher Trace ISQ, with a chromatographic column of Agilent DB-5MS, 30 m × 0.32 mm × 0.25 μm. The carrier gas was He, with a flow rate of 1.5 mL / min, an inlet temperature of 250 °C; the transfer line temperature was 250 °C, the injection volume was 1 μL, and the split ratio was 20:1. The temperature programming was as follows: the initial temperature of the column oven was set at 60 °C, heated to 100 °C at a rate of 5 °C / min, then heated to 140 °C at a rate of 2 °C / min and held for 5 min, and then the temperature was further increased to 250 °C at a rate of 10 °C / min and held for 1 min. The ionization mode was EI source, the ionization voltage was 70 eV, the ion source temperature was 250 °C, and the scanning range was 50 - 500 aum.
[0049] The GC-MS detection results are as Figure 3 shown. One main product, trans-nerolidol (as shown in Figure A), was detected in the fermentation broth of the DoNES5-WAT11 strain, while no sesquiterpenoids were detected in the fermentation broth of the blank control group, the pESC-ura-WAT11 strain (as shown in Figure C). This result indicates that the recombinant Saccharomyces cerevisiae strain DoNES5-WAT11 can be induced by galactose to express a functional Dalbergia odorifera sesquiterpene synthase, and can generate a single sesquiterpenoid product, trans-nerolidol, without adding the substrate FPP.
[0050] Industrial Applicability
[0051] The recombinant Saccharomyces cerevisiae strain constructed in the present invention (i.e., DoNES5-WAT11) can use its own metabolic intermediate FPP as a substrate for catalytic reaction to generate trans-nerolidol. This indicates that the recombinant Saccharomyces cerevisiae can produce Dalbergia odorifera sesquiterpene synthase, which can catalyze FPP to form sesquiterpenes. In actual production, this recombinant Saccharomyces cerevisiae can be used for fermentation to obtain trans-nerolidol with various biological activities.
Claims
1. A recombinant Saccharomyces cerevisiae containing a Dalbergia odorifera sesquiterpene synthase gene, which is obtained by transferring the Dalbergia odorifera sesquiterpene synthase gene shown in SEQ ID NO.1 cloned from the color-changing region of Dalbergia odorifera stems into a competent cell of Saccharomyces cerevisiae, wherein the gene sequence of SEQ ID NO.1 is as described in the sequence table of the specification.
2. The recombinant Saccharomyces cerevisiae containing the dalbergia odorifera sesquiterpene synthase gene according to claim 1, wherein the amino acid sequence of the protein encoded by the dalbergia odorifera sesquiterpene synthase gene is shown in SEQ ID NO.2, and the amino acid sequence of SEQ ID NO.2 is as described in the sequence table in the specification.
3. The recombinant Saccharomyces cerevisiae containing the dalbergia odorifera sesquiterpene synthase gene according to claim 1, wherein the dalbergia odorifera sesquiterpene synthase gene is DoNES5, the competent cell of the recombinant Saccharomyces cerevisiae is WAT11, and the strain of the recombinant Saccharomyces cerevisiae is DoNES5-WAT11.
4. A method for preparing a recombinant Saccharomyces cerevisiae containing the dalbergia odorifera synthase gene according to any one of claims 1 to 3, comprising the following steps: a) cloning the Dalbergia odorifera sesquiterpene synthase gene shown in SEQ ID NO.1 from the color change region of Dalbergia odorifera stems; b) recombining the dalbergia odorifera sesquiterpene synthase gene into an expression vector by seamless connection to obtain a connection product; c) transforming the ligated product into a recombinant Saccharomyces cerevisiae strain; d) Screening the successfully transformed recombinant Saccharomyces cerevisiae strains.
5. The method according to claim 4, wherein the dalbergia odorifera sesquiterpene synthase gene in step a) is DoNES5, the expression vector in step b) is pESC-ura, the recombinant Saccharomyces cerevisiae strain in step c) is WAT11, and the recombinant Saccharomyces cerevisiae strain successfully transformed in step d) is DoNES5-WAT11.
6. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3 in producing sesquiterpenes, comprising inducing the recombinant Saccharomyces cerevisiae with galactose to express the dalbergia odorifera sesquiterpene synthase gene, and performing biphasic fermentation and recovering the sesquiterpene: trans-nerolidol.