CN117645937A

CN117645937A - Recombinant saccharomyces cerevisiae, construction method and application

Info

Publication number: CN117645937A
Application number: CN202311491841.2A
Authority: CN
Inventors: 董扬; 杨玲
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2023-11-09
Filing date: 2023-11-09
Publication date: 2024-03-05

Abstract

The invention relates to a recombinant saccharomyces cerevisiae, a construction method and application thereof, and the recombinant saccharomyces cerevisiae has an amino acid sequence shown as SEQ ID NO. 1; the method comprises the following steps: constructing an over-expressed gene fragment cassette1 of IDI1 and tHMG 1; constructing a GES-Erg20F96W-N127W fusion expression gene fragment cassette2; chassis cells were obtained BY fully integrating cassette1 and cassette2 into the Saccharomyces cerevisiae BY4742 genome. The invention has the advantages that: a fully integrated recombinant Saccharomyces cerevisiae strain was developed for the production of geraniol to facilitate future optimization of the later steps of the biosynthetic pathway for other monoterpenes. Compared to plasmid-based systems, fully integrated strains allow for more stable gene expression in the geraniol pathway and more flexible culture conditions.

Description

Recombinant saccharomyces cerevisiae, construction method and application

Technical Field

The invention relates to the field of biotechnology, in particular to a fusion protein, an expression vector, a construction method and application.

Background

The content of the compound in the plant body is not high, and the secondary metabolite monomer is directly obtained from the plantLow rate and complicated separation and purification steps. With the development of synthetic biology, integration of heterologous biosynthesis pathways in engineering bacteria has become an important research tool for the heterologous synthesis of various secondary metabolites. Geraniol (trans-3, 7-dimethyl-2,6-octadien-1-ol, C) ₁₀ H ₁₈ O) is an acyclic monoterpene alcohol, which is widely used in perfume and essence industries because of pleasant smell, and geraniol mainly comes from extraction of natural products of plants, and has low yield. Metabolic engineering of monoterpene production is mainly performed in e.coli, and the yield of most monoterpenes is low. At present, few reports indicate that a small amount of monoterpenes are synthesized in yeast, but the yield is low. The invention aims to develop a recombinant saccharomyces cerevisiae strain for efficiently producing geraniol, and integrates exogenous genes into a host genome, so that the exogenous genes can be stably expressed after being introduced into the host genome.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provide a recombinant saccharomyces cerevisiae, a construction method and application, develop a completely integrated recombinant saccharomyces cerevisiae strain, and be used for producing geraniol so as to facilitate the optimization of the later steps of biosynthesis paths of other monoterpenes. Compared to plasmid-based systems, fully integrated strains allow for more stable gene expression in the geraniol pathway and more flexible culture conditions.

The fusion protein has an amino acid sequence shown as SEQ ID NO. 1.

An expression vector is GES-Erg20 ^F96W-N127W The structure of which is shown in figure 1.

A method of constructing an expression vector, comprising the steps of:

s1, constructing an overexpression gene cassette1 of IDI1 and tHMG 1;

s2, construction of GES-Erg20 ^F96W-N127W Fusion expression gene cassette2;

s3, completely integrating the cassette1 and the cassette2 into a saccharomyces cerevisiae BY4742 genome to obtain an expression vector.

Further, the method further comprises the following steps: fermenting recombinant saccharomyces cerevisiae: selecting recombinant saccharomyces cerevisiae strains 1 and 2, respectively inoculating the recombinant saccharomyces cerevisiae strains into an SD-Ura/LEU auxotroph liquid culture medium, and culturing seed liquid at 30 ℃ for 220r/min for 48 hours; the seed solution was then inoculated at a rate of 1% into 50mL of YPD medium and shake-cultured at 30℃for 3 days at 220 r/min.

Further, the method further comprises the following steps: extracting and detecting recombinant saccharomyces cerevisiae engineering bacteria products: after the shake flask fermentation is finished, the fermentation broth is centrifuged at 3000rpm, the supernatant and the bacterial precipitate are separated, only the supernatant is remained, the supernatant is fully extracted by using equal volume of ethyl acetate, the upper organic phase is sucked, the extraction is repeated for 3 times, and the extracts are combined. Ethyl acetate was distilled from the supernatant using a rotary evaporator to obtain a concentrated extract. The resulting fermented extract was tested using high performance liquid chromatography (fig. 3), liquid phase conditions: the flow rate is 0.8ml/min, the sample injection amount is 5 mu L, the column temperature is 35 ℃, and the maximum absorption wavelength is 210nm; a: acetonitrile, B:0.1% formic acid water, 0min 40% a,60% b;15min 80% A,20% B.

Further, the step S1 includes the steps of:

cloning of S11, IDI1 and tHMG1 genes;

s12, saccharomyces cerevisiae promoter and terminator amplification;

s13, in vitro assembly of IDI1 and tHMG1 over-expressed gene cassette1.

Further, the step S2 includes the steps of:

s21, synthesizing a geraniol synthase gene truncated by gentiana rigescens signal peptide;

s22, saccharomyces cerevisiae promoter and terminator amplification;

s23, in vitro Assembly GES-Erg20 ^F96W-N127W The fusion expression gene cassette2.

8. The method of constructing an expression vector according to claim 3, wherein the step S3 comprises:

s31, preparing cassette1 and cassette2 fragments: plasmid 3 and plasmid 4 were each extracted at 5. Mu.g using the full gold plasmid miniprep kit (Trans, EM 101). Fragment recovery was performed using restriction enzyme SacI introduced at both ends at the time of primer design, and the total volume of the reaction system was 50. Mu.L: 10rCutSmar Buffer 5. Mu.L, 5. Mu.g of plasmid 3 or plasmid 4, sacI-HF 5. Mu.L, 50. Mu.L of water were added and incubated at 37℃for 2h. Fragments of interest, cassette1 and cassette2, were recovered using 1% agarose gel electrophoresis.

S32, BY4742 competent preparation: firstly, strain activation is carried out, strains preserved at-80 ℃ are streaked on YPDA culture medium plates, and the strains are cultured for 2-4 days at 30 ℃. Yeast single colonies were picked and streaked on YPDA medium plates for 3-5mm short lines and incubated at 30℃for 2-4 days. When the single colony of yeast grows to 2mm in diameter, the yeast cells are inoculated into 3mL of YPDA liquid medium and cultured overnight at 30 ℃. The next day was transferred to a flask containing 30-50mL YPDA liquid medium for continued culture, and after OD600 reached 0.4-0.5, centrifugation was performed at 3000rpm for 5min, the supernatant was discarded. The pellet was suspended with 30-50mL of sterile deionized water. Centrifuge at 3000rpm for 5min, discard supernatant. The pellet was resuspended in 1.5mL 1 XLIAC (150. Mu.L 10 XLIAC Solution plus 1350. Mu.L sterile water) and transferred to a 1.5mL centrifuge tube, centrifuged at 3000rpm for 5min, and the supernatant discarded. 1mL of 1 XLiAc was added for resuspension and small volume transformation was split at 100. Mu.L per tube for plasmid transformation. Centrifuging at 3000rpm for 5min, discarding supernatant, and preparing BY4742 competent cells.

S33, cassette1 and cassette2 homologous recombination integrate into the BY4742 genome: 360 μl of the premix was added to 1 competent cell, and the pellet was repeatedly aspirated to thoroughly suspend the yeast cells in the premix. Incubating in a water bath at 30deg.C for 30min, and mixing every 10 min. Heat-beating in a water bath at a temperature of 42 ℃ for 30min, and uniformly mixing every 10 min. Centrifuge at 12000rpm for 15s and discard the supernatant. Resuspended in 1mL YPD and shake cultured at 30℃for 30-60min. Centrifuge at 12000rpm for 15s and discard the supernatant. Adding 0.1-1mL sterile deionized water or 0.9% sodium chloride solution, suspending and precipitating, taking 100 μl of SD-Ura auxotroph coated solid culture medium, and culturing at 30deg.C for 2-4 days. And after the transformant grows out, selecting positive clones by colony PCR, extracting the whole genome by using a fungus genome DNA extraction kit, and carrying out PCR amplification and Sanger sequencing verification on the target region to obtain a recombinant saccharomyces cerevisiae strain I and a recombinant saccharomyces cerevisiae strain II.

Further, the recombinant saccharomyces cerevisiae strain I has the structure as follows:

BY4742 gal80::PTEF1-IDI1-TCYC1-PTEF1-tHMG-TCYC1-LEU2-PTEF1

oye2::PTEF1-tGES-Linker-Erg20F96W-N127W-TPGK1-TADH1-URA3-PTE F1。

use of an expression vector for the production of geraniol.

The invention has the advantages that: the invention constructs the yeast strain for efficiently producing the geraniol by regulating GPP flux and cutting off geraniol synthase of signal peptide. First, the synthesis of GPP was increased by over-expression of key restriction enzymes in the MVA pathway. The platforms developed here can be used at any time for the synthesis of other monoterpenes. While previous strain engineering has greatly improved geraniol production by yeast, these efforts have largely utilized plasmid-based systems to overexpress native cell membrane pathway genes. In this work we developed a fully integrated recombinant s.cerevisiae strain for the production of geraniol to facilitate future optimisation of the further steps of the monoterpene biosynthetic pathway. The fully integrated strain also allows for more stable gene expression in the geraniol pathway and more flexible culture conditions compared to plasmid-based systems.

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is an assembly process diagram of case 1.

Fig. 2 is an assembly process diagram of case 2.

FIG. 3 is a high performance liquid chromatogram of the recombinant Saccharomyces cerevisiae fermentation extract. And (3) injection: a is a geraniol standard substance, B is a fermentation extract of a recombinant saccharomyces cerevisiae strain 1, C is a fermentation extract of a recombinant saccharomyces cerevisiae strain 2, and D is a fermentation extract of a blank BY4742 strain.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The fusion protein, the expression vector, the construction method and the application related in a preferred embodiment of the invention comprise the following steps:

construction of the overexpression genes cassette1 of S1, IDI1 and tHMG1

Cloning of S11, IDI1 and tHMG1 genes: a single colony of Saccharomyces cerevisiae BY4742 was selected and cultured in 50mL of YPD medium at 220r/min for 48h at 30 ℃. Collecting strain at 3,000rpm/5min, extracting BY4742 RNA with fungus RNA extraction kit (Coolaber, RE 781), subjecting the total RNA to agarose gel electrophoresis and Nanodrop 2000 (Thermo) quality detection, and removing contaminating DNA and DNase (Thermo, AM1906, DNA-free Kit DNase Treatment)&Removal), using reverse transcription kit (NEB, E6560,II First Strand cDNA Synthesis Kit) cDNA was obtained (ProtoScript reverse transcription total volume 50. Mu.L: 0.1M DTT 5. Mu.L, 10mM dNTP 2.5. Mu.L, 50mM MgCl 25. Mu.L, 750mM KCl 5. Mu.L, 60. Mu. M Random Primer Mix 5. Mu.L, 5X ProtoScript II buffer 10. Mu.L, protoScript II RT 0.625. Mu.L, RNase Inhibitor 1.25. Mu.L, 1. Mu.g template was added and then water was added to 50. Mu.L, as in Table 1. Primers were designed (sequences shown in Table 2) using cDNA as template and Q5 high fidelity DNA polymerase (NEB, M0492, & gt>High-Fidelity 2X Master mix) amplified IDI1 and tHMG1 groups of BY4742, respectivelyThe procedure is as in Table 3, for fragments (25. Mu.L of Q5 High-Fidelity 2X Master Mix 12.5. Mu.L of the PCR system for the High-Fidelity DNA polymerase, 1.25. Mu.L of the 10. Mu.M forward primer, 1.25. Mu.L of the 10. Mu.M reverse primer, 1. Mu.L of the cDNA template, 9. Mu.L of water). The obtained IDI1 and tHMG1 fragments were ligated into pEASY-Blunt vector (total ligation volume: pEASY-Blunt vector 1. Mu.L and PCR purified product 4. Mu.L, ligation reaction at 25℃for 2 h) respectively. After the ligation was completed, 5. Mu.L of the ligation reaction system was transferred to 50. Mu.L of Trans1-T1 competent cells, 1mL of SOC medium was added, resuscitated at 37℃for 1 hour at 220r/min, and transferred to LB solid medium containing the corresponding resistance of the vector, overnight at 37 ℃. Positive clones were selected for Sanger sequencing using colony PCR (total volume of colony PCR system 20. Mu.L: 2 XTaq PCRMix 10. Mu.L, template 2. Mu.L, forward primer 0.5. Mu.L, reverse primer 0.5. Mu.L, and water 7. Mu.L, procedure as in Table 4). And comparing the sequencing result of the selected positive clone with the genome sequence, wherein the sequence comparison result shows that the similarity of the nucleotide sequence of the clone and the original data is 100%, so that the plasmid 1pEASY-IDI1 and the plasmid 2pEASY-tHMG1 are obtained.

S12, saccharomyces cerevisiae promoter and terminator amplification: the fungal genomic DNA extraction kit (BioFlux, BSC14M 1) extracts the whole genome of BY 4742. IDI1 and tHMG1 were both used with the TEF1 promoter CYC1 terminator. The TEF1 promoter CYC1 terminator fragment of BY4742 was amplified separately using Q5 high fidelity DNA polymerase using genomic DNA of BY4742 as a template.

In vitro assembly of the overexpression genes cassette1 of S13, IDI1 and hmg 1: the assembly was performed using overlap extension PCR for the assembly of short fragments. The small fragments were assembled and the large fragments were ligated using the method of gibbon assembly (NEB, E2611) (fig. 1).

S13a, designing a primer and amplifying fragments, wherein an overlapping region of at least 20bp is needed between two fragments of overlapping extension PCR and Gibbsen assembly, so that the 5' end of the primer needs to comprise an overlapping region of 20-25bp and an extension region of about 10bp, and the sequence of the primer is shown in Table 2. Each fragment was amplified separately using Q5 high fidelity DNA polymerase, including Fra1-1, fra1-2, fra3-1, fra3-2, fra4, fra5, fra6.Fra1-1 and Fra6 are respectively 400bp homologous fragments upstream and downstream of the GAL80 locus of the Saccharomyces cerevisiae BY4742 genome. Fra5 was amplified using plasmid pCT as template. The PCR products were purified using 1% agarose gel electrophoresis.

S13b, using overlap extension PCR, fra1-1 and Fra1-2 were ligated as Fra1, fra3-1 and Fra3-2 were ligated as Fra3. The overlap extension PCR reaction is performed in three steps. The first step of overlapping PCR reaction, without using any primers, relies mainly on (1) the overlapping region generated in PCR amplification for extension, and the two fragments must be added in equimolar amounts. The total volume of the reaction system was 50. Mu.L: q5 High-Fidelity 2X Master mix 25. Mu.L, 100ng/Kb Fragment1, 100ng/Kb Fragment2, and 50. Mu.L were supplemented with water, and the reaction procedure is shown in Table 5. And in the second step, PCR amplification of the fusion fragment, the full length of the fusion fragment is amplified by using Q5 high-fidelity DNA polymerase and a low-concentration primer. The total volume of the reaction system was 50. Mu.L: q5 High-Fidelity 2X Master mix 25. Mu.L, 4. Mu.L of the first step reaction solution, 1. Mu.L of Fragment1 forward primer, 1. Mu.L of Fragment2 reverse primer, and water to 50. Mu.L were added, see reaction procedure 5. The resulting PCR product was purified by 1% agarose gel electrophoresis.

S13c, gibbsen assembly: fra1-6 and linearized pEASY vector fragments were assembled into cassette1 using Gibbson. The total volume of ligation reaction was 20. Mu.L: 2X Gibson Assembly Master Mix 10. Mu.L each of the fragments Fra1-6 (pmoles= (mass of fragment to be added/ng) 1000/(base number 650 daltons)), 0.1 pmoles of the vector, and 20. Mu.L of water were added. After the above reaction system was prepared on ice, the centrifuge tube was placed in a PCR apparatus, incubated at 50℃for 30 minutes, and immediately placed on ice after the completion of the reaction. mu.L of the ligation product was transferred to 50. Mu.L of DH5 competent cells, and positive clones were picked by colony PCR and Sanger sequencing verified (procedure as described above). Finally, plasmid 3 (pEASY-cassette 1) was obtained.

S2、GES-Erg20 ^F96W-N127W Construction of fusion expression Gene cassette2

S21, synthesis of geraniol synthase genes truncated by gentiana rigescens signal peptide: the amino terminus of terpene synthases typically contains a signal peptide for plastid localization that is cleaved after insertion of the protein into the plastid. When plant terpene synthases are expressed in yeast, their signal peptides may affect the enzymatic activity. The present invention contemplates the use of a signal peptide-free GES with higher enzymatic activity. At the same time, the stop codon of GES was removed, and the Linker sequence required for fusion expression was added to the 3' -end (GGTGGTGGTAGC). Finally, the protein expression quantity of the heterologous gene in the host cell is improved through codon optimization. Optimization improves the efficiency of translation initiation and termination, and gene synthesis is completed by Huada genes. After obtaining the fragment, it was cloned into the vector pEASY, as described in 1- (1), and finally the plasmid 4pEASY-tGES was obtained.

S22, saccharomyces cerevisiae promoter and terminator amplification: and (3) using the BY4742 genome DNA extracted from 1- (2) as a template to perform a promoter and a terminator. The TEF1 promoter, PGK1 and ADH1 terminator fragments of BY4742 were amplified using Q5 high fidelity DNA polymerase, respectively, using genomic DNA of BY4742 as a template.

S23、GES-Erg20 ^F96W-N127W In vitro assembly of fusion expressed gene cassette 2: the assembly was performed using overlap extension PCR for the assembly of short fragments. The small fragments were assembled and the large fragments were ligated using the method of gibbon assembly (NEB, E2611) (fig. 2).

S23a, primer design and fragment amplification, wherein the primer sequences are shown in Table 2. Each fragment was amplified separately using Q5 high fidelity DNA polymerase, including Fra7-1, fra7-2, fra8, fra9, fra10-1, fra10-2, fra11, fra12.Fra7-1 and Fra12 are respectively 400bp homologous fragments upstream and downstream of the OYE2 site of the Saccharomyces cerevisiae BY4742 genome. Wherein the template of Fra11 is plasmid pCRCT. The PCR products were purified using 1% agarose gel electrophoresis.

S23b, using overlap extension PCR, fra7-1 and Fra7-2 were ligated as Fra7, fra10-1 and Fra10-2 were ligated as Fra10. The reaction system and the procedure are described in 1- (3) - (b). The resulting PCR product was purified by 1% agarose gel electrophoresis.

S23c, gibbsen assembly: the Fra7-12 fragment was assembled into cassette2 using Gibbsen. The operation method is as described in 1- (3) - (c). Finally, plasmid 4 (pEASY-cassette 2) was obtained.

S3, complete integration of cassette1 and cassette2 into Saccharomyces cerevisiae BY4742 genome

S33, cassette1 and cassette2 homologous recombination integrate into the BY4742 genome: 360. Mu.L of premix (PEG Solution 240. Mu.L, 10 XLiAc Solution 36. Mu.L, carrier DNA 10. Mu.L, cassette1 and cassette2 each 1. Mu.g, and water to 360. Mu.L) was added to 1 competent cell, and the pellet was repeatedly aspirated to thoroughly suspend the yeast cells in the premix. Incubating in a water bath at 30deg.C for 30min, and mixing every 10 min. Heat-beating in a water bath at a temperature of 42 ℃ for 30min, and uniformly mixing every 10 min. Centrifuge at 12000rpm for 15s and discard the supernatant. Resuspended in 1mL YPD and shake cultured at 30℃for 30-60min. Centrifuge at 12000rpm for 15s and discard the supernatant. Adding 0.1-1mL sterile deionized water or 0.9% sodium chloride solution, suspending and precipitating, taking 100 μl of SD-Ura auxotroph solid culture medium (containing 2% glucose), and culturing at 30deg.C for 2-4 days. When the transformant grows out, positive clones are selected BY colony PCR, and the whole genome is extracted BY using a fungus genome DNA extraction kit (BioFlux, BSC14M 1) to carry out PCR amplification and Sanger sequencing verification of a target region, so as to obtain recombinant saccharomyces cerevisiae strains 1 and 2 (BY 4742gal80: PTEF1-IDI1-TCYC1-PTEF1-tHMG-TCYC1-LEU2-PTEF1;

oye2::PTEF1-tGES-Linker-Erg20F96W-N127W-TPGK1-TADH1-URA3-PTEF1)。

s4, fermenting recombinant saccharomyces cerevisiae: recombinant Saccharomyces cerevisiae strains 1 and 2 are selected and respectively inoculated into SD-Ura/LEU auxotroph liquid culture medium, and seed liquid is firstly cultured at 30 ℃ for 220r/min for 48 hours. Subsequently, the seed solution was inoculated into YPD medium (50 mL) at a ratio of 1%, and shake-cultured at 30℃for 3 days at 220 r/min.

S5, extracting and detecting a recombinant saccharomyces cerevisiae engineering bacteria product: after the shake flask fermentation is finished, the fermentation broth is centrifuged at 3000rpm, the supernatant and the bacterial precipitate are separated, only the supernatant is remained, the supernatant is fully extracted by using equal volume of ethyl acetate, the upper organic phase is sucked, the extraction is repeated for 3 times, and the extracts are combined. Ethyl acetate was distilled from the supernatant using a rotary evaporator to obtain a concentrated extract. The resulting fermented extract was tested using high performance liquid chromatography (fig. 3), liquid phase conditions: the flow rate is 0.8ml/min, the sample injection amount is 5 mu L, the column temperature is 35 ℃, and the maximum absorption wavelength is 210nm; acetonitrile, 0.1% of formic acid water, 0min 40% of A and 60% of B;15min 80% A,20% B.

TABLE 1 ProtoScript reverse transcription procedure

TABLE 2 primer sequences used in the present invention

TABLE 3Q5 Hi-Fi DNA polymerase PCR reaction procedure

TABLE 4 colony PCR reaction procedure

TABLE 5 overlap extension PCR reaction procedure

The principles and embodiments of the present invention have been described in detail with reference to specific examples, which are provided to facilitate understanding of the method and core ideas of the present invention; meanwhile, as those skilled in the art will have variations in the scope of the specific embodiments according to the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.

Claims

1. A recombinant saccharomyces cerevisiae is characterized by having an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. The method for constructing recombinant saccharomyces cerevisiae according to claim 1, comprising the steps of:

s1, constructing an overexpression gene cassette1 of IDI1 and tHMG 1;

s2, construction of GES-Erg20 ^F96W-N127W Fusion expression gene cassette2;

s3, completely integrating the cassette1 and the cassette2 into a saccharomyces cerevisiae BY4742 genome to obtain the recombinant saccharomyces cerevisiae.

3. The method for constructing recombinant saccharomyces cerevisiae according to claim 2, further comprising: fermenting recombinant saccharomyces cerevisiae: selecting recombinant saccharomyces cerevisiae strains 1 and 2, respectively inoculating the recombinant saccharomyces cerevisiae strains into an SD-Ura/LEU auxotroph liquid culture medium, and culturing seed liquid at 30 ℃ for 220r/min for 48 hours; the seed solution was then inoculated at a rate of 1% into 50mL of YPD medium and shake-cultured at 30℃for 3 days at 220 r/min.

4. The method for constructing recombinant saccharomyces cerevisiae according to claim 2, further comprising: extracting and detecting recombinant saccharomyces cerevisiae engineering bacteria products: after the shake flask fermentation is finished, the fermentation broth is centrifuged at 3000rpm, the supernatant and the bacterial precipitate are separated, only the supernatant is remained, the supernatant is fully extracted by using equal volume of ethyl acetate, the upper organic phase is sucked, the extraction is repeated for 3 times, and the extracts are combined. Ethyl acetate was distilled from the supernatant using a rotary evaporator to obtain a concentrated extract, and the resulting fermented extract was examined using high performance liquid chromatography, liquid phase conditions: the flow rate is 0.8ml/min, the sample injection amount is 5 mu L, the column temperature is 35 ℃, and the maximum absorption wavelength is 210nm; acetonitrile, 0.1% of formic acid water, 0min 40% of A and 60% of B;15min 80% A,20% B.

5. The method for constructing an expression vector according to claim 1, wherein the step S1 comprises the steps of:

cloning of S11, IDI1 and tHMG1 genes;

s12, saccharomyces cerevisiae promoter and terminator amplification;

s13, in vitro assembling IDI1 and tHMG1 over-expressed gene fragment cassette1.

6. The method for constructing recombinant saccharomyces cerevisiae according to claim 2, wherein the step S2 comprises the following steps:

s22, saccharomyces cerevisiae promoter and terminator amplification;

s23, in vitro Assembly of GES-Erg ^20F96W-N127W The fusion expressed gene fragment cassette2.

7. The method of constructing an expression vector according to claim 3, wherein the step S3 comprises:

s31, preparing cassette1 and cassette2 fragments: plasmid 3 and plasmid 4 were each extracted at 5. Mu.g using the full gold plasmid miniprep kit (Trans, EM 101), and fragment recovery was performed using restriction enzyme SacI introduced at both ends at the time of primer design, and the total volume of the reaction system was 50. Mu.L: 10rCutSmarBuffer 5. Mu.L, 5. Mu.g of plasmid 3 or plasmid 4, sacI-HF 5. Mu.L, water 50. Mu.L, incubation at 37℃for 2h, recovery of the fragments of interest cassette1 and cassette2 using 1% agarose gel electrophoresis;

s32, BY4742 competent preparation: firstly, strain activation is carried out, strains stored at minus 80 ℃ are streaked on a YPDA culture medium plate, culturing is carried out for 2-4 days at 30 ℃, single yeast colony is streaked on the YPDA culture medium plate for 3-5mm short lines, culturing is carried out for 2-4 days at 30 ℃, when the single yeast colony grows to 2mm in diameter, yeast cells are inoculated into 3mL YPDA liquid culture medium, culturing is carried out overnight at 30 ℃, the next day is transferred into a triangular flask containing 30-50mL YPDA liquid culture medium for continuous culturing, when OD600 reaches 0.4-0.5, centrifugation is carried out for 5min at 3000rpm, supernatant is discarded, sediment is suspended with 30-50mL sterile deionized water, centrifugation is carried out for 5min at 3000rpm, 1.5mL of 1 XLIAC (150 mu L10 XLIAC Solution plus 1350 mu L sterile water) is discarded, sediment is transferred into 1.5mL, centrifugation is carried out for 5min at 3000rpm, and supernatant is discarded. Adding 1mL of 1 XLiAc for resuspension, split charging the small-volume transformation into 100 mu L of each tube for plasmid transformation, centrifuging at 3000rpm for 5min, discarding the supernatant, and obtaining BY4742 competent cells;

s33, cassette1 and cassette2 homologous recombination integrate into the BY4742 genome: adding 360 mu L of premix into 1 100 mu L of competent cells, repeatedly blowing and sucking to precipitate, and thoroughly suspending yeast cells in the premix; incubating in a water bath at 30deg.C for 30min, and mixing every 10 min; heat-beating in a water bath at a temperature of 42 ℃ for 30min, uniformly mixing every 10min, centrifuging at 12000rpm for 15s, discarding supernatant, re-suspending with 1mL YPD, shake culturing at a temperature of 30 ℃ for 30-60min, centrifuging at 12000rpm for 15s, and discarding supernatant; adding 0.1-1mL sterile deionized water or 0.9% sodium chloride solution, suspending and precipitating, taking 100 μl of SD-Ura auxotroph coated solid culture medium, and culturing at 30deg.C for 2-4 days; and after the transformant grows out, selecting positive clones by colony PCR, extracting the whole genome by using a fungus genome DNA extraction kit, and carrying out PCR amplification and Sanger sequencing verification on the target region to obtain a recombinant saccharomyces cerevisiae strain I and a recombinant saccharomyces cerevisiae strain II.

8. The method for constructing recombinant s.cerevisiae according to claim 7, wherein the genotypes of the recombinant s.cerevisiae strain I and strain II are:

BY4742 gal80::PTEF1-IDI1-TCYC1-PTEF1-tHMG-TCYC1-LEU2-PTEF1,oye2::PTEF1-tGES-Linker-Erg20F96W-N127W-TPGK1-TADH1-URA3-PTEF1

9. use of the recombinant saccharomyces cerevisiae of claim 2 for the production of geraniol.