CN114107078A

CN114107078A - High-yield valencene genetic engineering bacterium and construction method and application thereof

Info

Publication number: CN114107078A
Application number: CN202111275680.4A
Authority: CN
Inventors: 陈强; 刘登辉; 向景; 刘传春
Original assignee: Hubei Guanzhongtong Technology Co ltd
Current assignee: Hubei Guanzhongtong Technology Co ltd
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-03-01

Abstract

The invention provides a construction method of high-yield valencene gene engineering bacteria, which integrates and expresses an MVA pathway rate-limiting enzyme-truncated HMG-CoA reductase coding gene (tHMG1) and integrates and fuses and expresses an FPP synthase coding gene (ERG20) and a valencene synthetase coding gene (SmVS) in saccharomyces cerevisiae by utilizing a homologous recombination mode so as to enhance the metabolic strength of the MVA pathway and enhance the expression of valencene. Meanwhile, a copper ion induced promoter pCUP1 is used for replacing a squalene synthase coding gene ERG9 promoter, so that an ergosterol competitive pathway is reduced, and the yield of valencene is increased; finally obtaining the genetic engineering strain with high yield of valencene. The shake flask fermentation yield of the valencene of the genetic engineering bacteria can reach about 117mg/L, the fermentation tank yield can reach about 8g/L, and the genetic engineering bacteria completely have a commercial production level and have good industrial application prospects.

Description

High-yield valencene genetic engineering bacterium and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a high-yield valencene gene engineering bacterium and a construction method and application thereof.

Background

Valencene (Valencene), molecular formula: c₁₅H₂₄Valencene, is a sesquiterpene, a major aroma component of citrus fruit and citrus flavors, and is obtained by cold pressing citrus fruit peel. Valencene is widely used in food and cosmetic industries as a perfume, and can be used as a precursor molecule to produce Nootkatone (Nootkatone), wherein Nootkatone is usually extracted from grapefruit by chemical or biochemical oxidation of valencene, and has high economic and medicinal values.

Valencene can be separated and extracted from plants, but the extraction steps are complicated, the quality is closely related to raw materials, and the application of valencene synthesized by a chemical method is greatly limited because the application of valencene scenes is close to the contact of people. In addition, microbial fermentation is also a source of valencene, the valencene produced by the microbial fermentation method is relatively safe, and the production has the advantages of mild conditions, no geographical and climatic influence, easy large-scale production and the like.

At present, the strains for producing the valencene through biological fermentation mainly utilize escherichia coli engineering bacteria and saccharomyces cerevisiae engineering bacteria which are transformed with the valencene synthetase, but the yield is low and is about 10mg/L, and the industrial production level is not achieved. In addition, endotoxin is generated during fermentation production of escherichia coli, separation is not easy in the later period, and the production of valencene which is used for being in close contact with human is in a disadvantage. And the saccharomyces cerevisiae is an internationally recognized safe model strain, and the genetic operation method is mature and has natural advantages. Therefore, the construction of the high-yield and stable valencene saccharomyces cerevisiae production strain has important economic value and social significance.

Disclosure of Invention

The invention aims to provide a high-yield valencene genetic engineering bacterium and a construction method and application thereof, aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a construction method of high-yield Valencene gene engineering bacteria, which comprises the following steps:

step S1, constructing a Saccharomyces cerevisiae MVA pathway related gene expression module: the over-expression tHMG1 module comprises an inducible bidirectional strong promoter pGAL1-10 and an MVA pathway rate-limiting enzyme coding gene tHMG1, wherein the nucleotide sequence of pGAL1-10 is shown as SEQ NO.1, and the nucleotide sequence of tHMG1 is shown as SEQ NO. 2;

step S2, constructing a valencene production related gene expression module: the ERG20-Linker-SmVS module comprises an encoding gene ERG20 of FPP synthase and an encoding gene SmVS of valencene synthetase, wherein the nucleotide sequence of the ERG20 is shown as SEQ NO.3, and the nucleotide sequence of the SMVS is shown as SEQ NO. 4;

step S3, constructing engineering bacteria for knocking out expression of galactose regulatory protein GAL80 gene: integrating the saccharomyces cerevisiae MVA pathway related gene expression module in the step S1 and the valencene production related gene expression module in the step S2 to a galactose regulatory protein GAL80 gene locus, simultaneously knocking out a galactose regulatory protein GAL80 gene to obtain a knocked-out galactose regulatory protein GAL80 gene expression module, transforming the knocked-out galactose regulatory protein GAL80 gene expression module into the constructed saccharomyces cerevisiae genetically engineered bacterium GS-A3, and obtaining the genetically engineered bacterium with the galactose regulatory protein GAL80 gene expression knocked out through multiple genetic engineering operations;

step S4, constructing a squalene synthase pathway down-regulation expression plasmid, wherein the expression plasmid comprises a copper ion-induced promoter pCUP1 replaced by a squalene synthase gene ERG9 promoter, and the nucleotide sequence of pCUP1 is shown as SEQ NO. 5; the squalene synthase gene ERG9 promoter is obtained 450bp before the initiation codon of ERG9 gene, and the nucleotide sequence is shown as SEQ NO. 6;

step S5, transforming the squalene synthase pathway down-regulated expression plasmid of step S5 into the genetic engineering bacteria of the knockout galactose regulatory protein GAL80 gene expression of step S3, sequentially screening antibiotic resistance, and then obtaining the high-yield valencene genetic engineering bacteria through colony PCR verification;

the construction method also comprises two terminators tCYC1 and tERG20, wherein the nucleotide sequence of the terminator tCYC1 is shown as SEQ NO.7, and the nucleotide sequence of the terminator tERG20 is shown as SEQ NO. 8.

Further, in step S1, the method for constructing the over-expressed tmgb 1 module includes the steps of:

step S1, using Saccharomyces cerevisiae 3000B genome DNA as a template, respectively carrying out PCR reaction by using primers of tCYC1-F, tCYC1-R, tHMG1-F, tHMG1-R, pGAL1pGAL10-F and pGAL1pGAL10-R to obtain DNA fragments tCYC1, tHMG1 and pGAL10pGAL 1;

in step S2, the three DNA fragments tCYC1, tHMG1 and pGAL10pGAL1 obtained in step S1 were ligated together by overlap extension PCR reaction using primers tCYC1-F and pGAL1pGAL10-R to obtain an overexpressed tHMG1 module, i.e., tCYC1_ tHMG1_ pGAL10pGAL 1.

Further, in step S2, the method for constructing the ERG20-Linker-SmVS module includes the following steps:

s1, performing PCR reaction by respectively using the genomic DNA of saccharomyces cerevisiae 3000B as a template and ERG20-F and ERG20-Linker-W-R primers, and amplifying to obtain a DNA fragment ERG20_ Linker;

step S2, carrying out PCR reaction by using primers tERG20-W-F and tERG20-R, and amplifying to obtain a DNA fragment tERG 20;

step S3, carrying out PCR reaction by using the plasmid pGZ221 as a template and primers Linker-SmVS-F and SmVS-R, and amplifying to obtain a DNA fragment Linker-SmVS;

step S4, the DNA fragment ERG20_ Linker obtained in step S1, the DNA fragment tERG20 obtained in step S2 and the DNA fragment Linker _ SmVS obtained in step S3 are connected together by performing overlap extension PCR reaction with primers ERG20-F and tERG20-R to obtain a fusion expression ERG20-Linker-SmVS module, namely ERG20_ Linker _ SmVS _ tERG 20.

Further, the Linker comprises glycine and serine, and the combined structure thereof comprises any one of GSG, GGGS and GSGGSG, wherein G corresponds to the nucleic acid sequence GGT, and S corresponds to the nucleic acid sequence TCT.

Further, in step S2, the Linker in the ERG20-Linker-SmVS module is GSGGSG.

Further, in step S3, the method for constructing a module expressing a knockout galactose regulatory protein GAL80 gene includes the steps of:

step S1, using Saccharomyces cerevisiae 3000B genome DNA as a template, respectively using GAL80left-F, GAL80left-R, GAL80right-F and GAL80right-R primers to carry out PCR reaction, and obtaining left and right homologous arms GAL80left and GAL80right of a DNA fragment GAL80 through amplification;

step S2, carrying out PCR reaction by using a plasmid vector pFZ201 as a template and primers Hyg-F and Hyg-R to obtain a hygromycin expression cassette Hyg;

step S3, connecting the over-expressed tHMG1 module and five DNA fragments of the ERG20-Linker-SmVS module, GAL80left, GAL80right and Hyg together by performing overlap extension PCR reaction by using primers GAL80left-F and GAL80right-R to obtain a DNA fragment for knocking out the galactose regulatory protein GAL80 gene expression module;

and step S4, connecting the DNA fragment obtained in the step S3 with a plasmid pMD19-T to obtain a recombinant plasmid vector, linearizing the recombinant plasmid by using a restriction enzyme PmeI, recovering a fragment with a target gene, transforming the fragment into host bacteria Saccharomyces cerevisiae by using a yeast lithium acetate transformation method, sequentially screening antibiotic resistance, and obtaining a positive bacterial colony by colony PCR to obtain the engineering bacteria of which the expression of the galactose regulatory protein GAL80 is knocked out.

Further, in step S3, the host bacteria saccharomyces cerevisiae includes any one of saccharomyces cerevisiae 30000B, saccharomyces cerevisiae s.cerevisiae cen.pk2-1D, saccharomyces cerevisiae BY4741 and saccharomyces cerevisiae GS-A3, and the saccharomyces cerevisiae GS-A3 is deposited in the chinese type culture collection at 2021, 9, 17 days, with the deposit numbers: CCTCC NO: M20211191.

Further, in step S4, the method for constructing the squalene synthase pathway down-regulation expression plasmid comprises the following steps:

s1, taking Saccharomyces cerevisiae 3000B genome DNA as a template, respectively carrying out PCR reaction by using pERG9-left-F, pERG9-left-R, pCUP1-F, pCUP1-R, pERG9-right-F and pERG9-right-R primers, and amplifying to obtain DNA fragments pERG9-left, pCUP1 and pERG 9-right;

step S2, taking the plasmid vector pFZ202 as a template, and carrying out PCR reaction by using primers G418-F and G418-R to obtain a G418 expression cassette G418;

step S3, connecting the four DNA fragments of the DNA fragments pERG9-left, pCUP1 and pERG9-right obtained in step S1 and the expression cassette G418 obtained in step S2 together by performing overlap extension PCR reaction with primers pERG9-left-F and pERG9-right-R to obtain a DNA fragment G418_ pCUP 1/delta pEGR 9;

step S4, connecting the DNA fragment G418_ pCUP 1/delta pEGR9 obtained in step S3 with a plasmid pMD19-T to obtain a squalene synthase pathway down-regulated expression plasmid vector which is marked as pCZ 101.

The invention also provides a high-yield valencene genetic engineering bacterium obtained by adopting the construction method.

The invention also provides a method for producing valencene by using the high-yield valencene gene engineering bacteria, wherein the liquid fermentation medium components of the high-yield valencene gene engineering bacteria comprise 20-50g/L of glucose, 5-10g/L of yeast extract, 6-15g/L of ammonium sulfate, 3-8g/L of potassium dihydrogen phosphate, 5-10g/L of magnesium sulfate heptahydrate, 500mg/L of thiamine 100-containing material, 500mg/L of pyridoxine 100-containing material, 800mg/L of inositol 400-containing material, 20-100mg/L of biotin and 500mg/L of calcium pantothenate 100-containing material.

The technical scheme provided by the invention has the beneficial effects that:

(1) the construction method of the invention utilizes a homologous recombination mode to limit the rate of a mevalonic acid pathway of saccharomyces cerevisiae source driven by a bidirectional strong promoter (pGAL 1-10): the gene elements (ERG20-Linker-SmVS) of the truncated HMG-CoA reductase coding genes tHMG1 and FPP synthase (ERG20) and valencene synthetase (SmVS) fusion protein are integrated into the genome of saccharomyces cerevisiae of the starting strain, the integration site is galactose regulatory protein 80 gene (GAL80), and the galactose regulatory protein 80 gene is knocked out while the target gene is integrated, so that the metabolic strength of a mevalonate metabolic pathway is enhanced, and the expression of valencene is further enhanced. The construction method has the advantages of simplicity, rapidness and high efficiency, large-fragment integration engineering strains can be obtained in 2-3 weeks, and the construction period of the engineering strains is obviously shortened.

(2) Based on the engineering bacteria obtained in the step (1), the copper ion induced promoter pCUP1 is used for replacing a squalene synthase coding gene ERG9 promoter to reduce the expression level of the squalene synthase coding gene ERG9 promoter, so that the FPP flow to ergosterol competition ways can be reduced, and the yield of valencene is further improved; finally obtaining the genetic engineering strain with high yield of valencene. The shake flask fermentation yield of the farnesol of the genetic engineering bacteria obtained by the method can reach about 125mg/L, the fermentation tank yield can reach about 21g/L, and the method completely has a commercial production level and has good industrial application prospect.

(3) The invention can utilize simple culture medium to ferment and produce the valencene, can realize the one-time high-efficiency transformation and integration of polygene, can obviously shorten the construction time of engineering bacteria, and the obtained engineering bacteria can utilize simple carbon sources such as glucose, sucrose and the like to ferment and produce the valencene, thereby having better application prospect.

Drawings

FIG. 1 is a schematic diagram of the biological metabolism principle of producing valencene by Saccharomyces cerevisiae genetically engineered bacteria of the present invention;

FIG. 2 is a schematic structural diagram of a recombinant plasmid pWZ300 of a knockout galactose regulatory protein GAL80 gene expression module constructed in example 1 of the present invention;

FIG. 3 is a schematic structural diagram of a recombinant plasmid pCZ101 constructed in accordance with example 1 of the present invention to downregulate expression of the squalene synthase pathway;

FIG. 4 is a graph comparing the effect of different Linke junctions on valencene production;

FIG. 5 is a graph comparing production of valencene BY different genetically engineered bacteria GS-A3-W4, G30000B-W2, CEN. PK2-1D-W2 and BY 474-W2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings and examples.

The materials and methods used in the course of the study of the present invention were as follows:

the whole gene synthesis, primer synthesis and sequencing in the invention are all completed by Wuhan Tianyi Huayu gene technology company Limited, and the used high fidelity enzyme (PrimeSTAR GXL DNA Polymerase), common Taq enzyme (Premix Taq), pMD19-T Vector and the like are purchased from Wuhan Yongming biotechnology company Limited, and the used restriction enzyme is purchased from Hubei Jingyu biotechnology Limited; saccharomyces cerevisiae 30000B is commercially available.

The molecular biology experiments in the invention include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., which are mainly performed according to molecular cloning experimental guidelines (third edition), J. SammBruk, D.W. Lassel (America), translation of Huangpetang, scientific Press, 2002).

LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 20g/L agar powder;

YPD medium: 10g/L yeast extract, 20g/L tryptone, 20g/L glucose; adding 10% isopropyl myristate during fermentation to prevent product volatilization;

YPD solid culture medium comprising 10g/L yeast extract, 20g/L tryptone, 20g/L glucose, and 20g/L agar powder.

In the following examples, there are also test methods in which specific experimental conditions are not specified, usually according to the conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

As shown in figure 1, the metabolic schematic diagram of biosynthetic valencene, the high-yield valencene gene engineering bacteria constructed by the invention are saccharomyces cerevisiae-derived mevalonate pathway rate-limiting enzyme coding genes tHMG1 driven by bidirectional strong promoters pGAL1-10 by utilizing a homologous recombination mode; the gene element (ERG20-Linker-SmVS) of the fusion protein of the FPP synthase (ERG20) and the valencene synthetase (SmVS) is integrated into the genome of saccharomyces cerevisiae of the starting strain at the site of galactose regulatory protein 80 gene (GAL80), and the galactose regulatory protein 80 gene is knocked out while the target gene is integrated; then the copper ion induced promoter pCUP1 is used to replace the promoter of squalene synthase gene ERG9 in recombinant bacteria to reduce the expression level.

Wherein the nucleotide sequence of the inducible bidirectional strong promoter pGAL1-10 is shown as SEQ NO. 1; the mevalonate pathway rate-limiting enzyme is truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase, the nucleotide sequence of a coding gene tHMG1 is shown as SEQ NO.2, the nucleotide sequence of an FPP synthase (ERG20) coding gene is shown as SEQ NO.3, the nucleotide sequence of a valencene synthetase (SmVS) coding gene is shown as SEQ NO.4, a termination codon TAG at the 3 'end of the sequence shown as SEQ ID NO.3 is removed, and the termination codon TAG is connected with a base sequence for a Linker at the 5' end of the sequence shown as SEQ ID NO. 4; constructing a gene element of ERG20-Linker-SmVS fusion protein; the catalytic product FPP of ERG20, which is the substrate of SmVS, is expressed by the fusion of linker sequences, and the two enzymes are close to each other in space conformation, so that the reaction efficiency from IPP to FPP to valencene can be improved, the waste of FPP is avoided, the FPP is reduced to go to other metabolic branches, and the yield of valencene is improved.

And because squalene synthase has a strong ability to compete for substrates, most of the FPP flows to ergosterol, resulting in a low yield of other terpenoids. Therefore, it is essential to block or down-regulate the ergosterol competitive pathway in order to achieve efficient synthesis of the desired product. However, ERG9 is an essential gene for growth, cannot be knocked out and must be dynamically regulated. Therefore, the copper ion inducible promoter pCUP1 is used for replacing the promoter of the squalene synthase gene ERG9 to reduce the expression level of the squalene synthase gene ERG 9; the nucleotide sequence of the copper ion induced promoter pCUP1 is shown in SEQ NO. 5; the squalene synthase gene ERG9 promoter is selected to be 450bp in front of the initiation codon of ERG9 gene, and the nucleotide sequence is shown as SEQ NO. 6.

In order to more accurately guide the correct combination of each module constructed, two terminators tCYC1 and tERG20 were also used in the construction process, the corresponding nucleotide sequences being SEQ No.7 and SEQ No.8, respectively.

The sequence information of the primers used in examples 1 to 3 and comparative examples 1 to 3 is shown in Table 1:

table 1: primer sequences

Example 1

Constructing a high-yield valencene genetic engineering bacterium GS-A3-W4:

step S1, construction of an overexpressed tHMG1 Module

tCYC1_tHMG1_pGAL10pGAL1

1) Carrying out PCR reaction by using Saccharomyces cerevisiae 3000B genome DNA as a template and primers of tCYC1-F, tCYC1-R, tHMG1-F, tHMG1-R, pGAL1pGAL10-F and pGAL1pGAL10-R respectively to obtain DNA fragments tCYC1, tHMG1 and pGAL10pGAL 1;

2) the three DNA fragments tCYC1, tHMG1 and pGAL10pGAL1 obtained in step S1 were ligated together by overlap extension PCR reaction using primers tCYC1-F and pGAL1pGAL10-R to obtain an overexpressed tHMG1 module, i.e., a tCYC1_ tHMG1_ pGAL10pGAL1 module.

Step S2, construction of fusion expression ERG20-Linker-SmVS module

ERG20_Linker_SmVS_tERG20

1) PCR reaction is carried out by taking Saccharomyces cerevisiae 3000B genome DNA as a template and respectively using ERG20-F and ERG 20-Linker-R primers, and a DNA fragment ERG20_ Linker is obtained by amplification.

2) PCR reaction is carried out by using primers tERG20-W-F and tERG20-R, and a DNA fragment tERG20 is obtained by amplification.

3) And (3) carrying out PCR reaction by using the plasmid pGZ221 as a template and using primers Linker-SmVS-F and SmVS-R, and amplifying to obtain a DNA fragment Linker-SmVS. The plasmid pGZ221 is obtained by obtaining a sequence of sesquiterpene synthetase encoding protein derived from Salvia miltiorrhiza (SmVS, GenBank: A0A1W6GW18.1) through NCBI, sending the sequence to Wuhan Tianyi Huayu Gene science and technology Limited company for codon optimization and gene synthesis, and connecting the SmVS after codon optimization to a puc57 vector.

4) The DNA fragment ERG20_ Linker obtained in the above, the DNA fragment tERG20 obtained in the step S2 and the DNA fragment Linker _ SmVS obtained in the step S3 are connected together by performing overlap extension PCR reaction with primers ERG20-F and tERG20-R to obtain a fusion expression ERG20-Linker-SmVS module, namely ERG20_ Linker _ SmVS _ tERG 20.

The Linker comprises Linker1, Linker2 and Linker3, the corresponding amino acid sequences are GSG, GGGS and GSGGSG, the G corresponds to the nucleic acid sequence GGT, and the S corresponds to the nucleic acid sequence TCT.

Step S3, construction of knockout galactose regulatory protein GAL80 Gene expression Module

1) PCR was carried out using Saccharomyces cerevisiae 3000B genomic DNA as a template and GAL80left-F, GAL80left-R, GAL80right-F and GAL80right-R primers, respectively, to obtain the left and right homologous arms GAL80left and GAL80right of DNA fragment GAL 80.

2) And carrying out PCR reaction by using the plasmid vector pFZ201 as a template and primers Hyg-F and Hyg-R to obtain the hygromycin expression cassette Hyg.

3) The over-expressed tHMG1 module and the ERG20-Linker-SMVS module, GAL80left, GAL80right and Hyg five DNA fragments are connected together by performing overlap extension PCR reaction with primers GAL80left-F and GAL80right-R to obtain the DNA fragment which is the knockout galactose regulatory protein GAL80 gene expression module.

The Linker3 is selected for connection, and a module can be obtained:

GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_SmVS_tERG20_GAL80righ；。

4) connecting the obtained module with a pMD19-T vector, transferring into a large intestine for amplification, and obtaining a recombinant plasmid vector pWZ 300:

pWZ300ΔGAL80：：Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_SmVS_tERG20；

the structure of plasmid vector pWZ300 is shown in FIG. 2.

5) The recombinant plasmid vector pWZ300 constructed above is linearized respectively with restriction enzyme PmeI, a fragment with a target gene is recovered, the fragment is transformed to Saccharomyces cerevisiae GS-A3 by a yeast lithium acetate transformation method, the YPD plate containing 500 mug/mL hygromycin is coated with the fragment, a transformant is picked, genome PCR verification is carried out, and a corresponding correct transformation strain GS-A3-W3 is obtained.

Step S4 construction of a Squalene synthase pathway Down-regulated expression plasmid

pCZ101ΔpEGR9：：G418_pCUP1

1) Taking the 3000B genome DNA of the saccharomyces cerevisiae as a template, and respectively using pERG9-left-

F, pERG9-left-R, pCUP1-F, pCUP1-R, pERG9-right-F and pERG9-right-R primers were subjected to PCR reaction, and DNA fragments pERG9-left, pCUP1 and pERG9-right were obtained by amplification.

2) The G418 expression cassette G418 was obtained by PCR reaction using the plasmid vector pFZ202 as a template and the primers G418-F and G418-R.

3) The DNA fragments pERG9-left, pCUP1 and pERG9-right obtained above and the expression cassette G418 obtained in step S1 were ligated together by overlap extension PCR reaction using the primers pERG9-left-F and pERG9-right-R to obtain the DNA fragment G418_ pCUP 1/. DELTA.pEGR 9.

4) The DNA fragment G418_ pCUP 1/delta pEGR9 obtained above was ligated with the plasmid pMD19-T to obtain a squalene synthase pathway down-regulated expression plasmid vector, designated pCZ101, whose structure is shown in FIG. 3.

Step S5, linearizing plasmid pCZ101 with restriction enzyme PmeI, recovering a fragment with a target gene, transforming the fragment into the saccharomyces cerevisiae GS-A3-W3 strain by a yeast lithium acetate transformation method, coating the strain on a YPD plate containing 500 mu G/mL G418 and 200 mu mol/L copper ions, picking up a transformant and carrying out genome PCR verification to obtain a corresponding correct high-yield transforming strain GS-A3-W4 of the valencene.

Example 2

Construction of genetically engineered bacterium GS-A3-W5:

the construction method is the same as that in example 1, wherein the Linker selected in the step S3 is connected to Linker1, and the constructed knockout galactose regulatory protein GAL80 gene expression DNA fragment is as follows:

GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_SmVS_tERG20_GAL80right；

the constructed recombinant plasmid vector pWZ200 was:

pWZ200ΔGAL80：：Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_SmVS_tERG20；

the constructed engineering bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene are GS-A3-W1.

Example 3

Construction of genetically engineered bacterium GS-A3-W6:

the construction method is the same as that in example 1, wherein the Linker selected in the step S3 is connected to Linker2, and the constructed knockout galactose regulatory protein GAL80 gene expression DNA fragment is as follows:

GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_SmVS_tERG20_GAL80right；

the constructed recombinant plasmid vector pWZ100 was:

pWZ100ΔGAL80：：Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_SmVS_tERG20；

the constructed engineering bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene are GS-A3-W2.

Comparative example 1

Construction of genetically engineered bacterium 30000B-W2

The construction method is the same as that of example 1, the plasmid vector pWZ300 obtained in the step S3 and the plasmid vector pCZ101 obtained in the step S4 are linearized by restriction enzyme PmeI, the fragment with the target gene is recovered, the fragment is transformed into a saccharomyces cerevisiae chassis strain 30000B by a yeast lithium acetate transformation method in two steps, a transformant is selected and genome PCR verification is carried out, and the corresponding correct high-yield transformed strain 30000B-W2 of the valencene is obtained.

Comparative example 2

Construction of genetically engineered bacterium CEN. PK2-1D-W2

The construction method is the same as that of example 1, the plasmid vector pWZ300 obtained in the step S3 and the plasmid vector pCZ101 obtained in the step S4 are linearized by restriction endonuclease PmeI, the fragment with the target gene is recovered, the fragment is transformed into a saccharomyces cerevisiae chassis strain S.cerevisiae CEN.PK2-1D by a yeast lithium acetate transformation method in two steps, a transformant is selected and subjected to genome PCR verification, and the transformed strain CEN.PK2-1D-W2 with high yield of the corresponding correct valencene is obtained.

Comparative example 3

Construction of genetically engineered bacterium BY474-W2

The construction method is the same as that of example 1, the plasmid vector pWZ300 obtained in the step S3 and the plasmid vector pCZ101 obtained in the step S4 are linearized BY restriction enzyme PmeI, the fragment with the target gene is recovered, the fragment is transformed into a saccharomyces cerevisiae chassis strain BY474 BY a yeast lithium acetate transformation method in two steps, a transformant is picked up and genome PCR verification is carried out, and the corresponding correct high-yield transformed strain BY474-W2 of the valencene is obtained.

In order to illustrate the mechanism and the application capability of the high-yield valencene engineering bacteria, the influence of different saccharomyces cerevisiae chassis strains on the yield of valencene is modified by the influence of different linkers on the yield of valencene, and comparative research is carried out.

The method for measuring the concentration of the valencene in the fermentation liquor comprises the following steps:

a detection process;

putting 45mL of fermentation liquor into a 50mL centrifuge tube, centrifuging for 10min at 10000r/min, carefully absorbing the upper isopropyl myristate liquid, and diluting with chromatographic grade n-hexane to a proper concentration for detection.

The instrument uses an Agilent 7890A gas chromatograph, a chromatographic column is Agilent HP-5(30mx0.32 mmx0.25 mu m), the temperature of a column box is 100 ℃, the initial temperature is kept for 2min, the temperature is increased to 280 ℃ at 10 ℃/min, the temperature is kept for 3min, the temperature of a sample inlet is 280 ℃, the sample injection amount is 1 mu L, the flow rate of the column is 1mL/min, the injection split ratio is 1:50, the temperature of a detector (FID) is 280 ℃, nitrogen is used as carrier gas, the inlet pressure is 12-18psi, the mode is a constant current mode, and a standard substance: valencene (SIGMA).

1. Comparing the Effect of different Linke junctions on Warrene production

Respectively taking 10 mu L of strain GS-A3-W1, strain GS-A3-W2 and strain GS-A3-W3 stored in glycerinum tubing to a PA bottle containing 5mLYPD (final concentration of copper-containing ions is 200 mu mol/L) culture medium, and culturing for 16h at 30 ℃ by a shaking table at 220rpm to obtain first-grade seeds; the first seed was transferred to a shake flask containing 200mL YPD medium at 1% transfer amount, and cultured on a shaker at 220rpm at 30 ℃ for five days to determine the content of valencene.

As shown in FIG. 4, the results showed that the content of valencene in GS-A3-W3 cells was 73.64. + -. 3.53mg/L higher than that of GS-A3-W1 and GS-A3-W2. Therefore, the Llinker-GSGGSG is more beneficial to fusion expression of ERG20 and SmVS, and the reaction efficiency from IPP to FPP to valencene is improved, so that the yield of valencene is improved.

2. Comparison of the Effect of pERG9 replacement with the promoter pCUP1 on Warrene production

Collecting 10 μ L of strain GS-A3-W4 stored in Glycine max (L.) Gaertn, placing in PA bottle containing 5mLYPD (final concentration of copper ion is 200 μmol/L) culture medium, and culturing at 30 deg.C with shaking table at 220rpm for 16h to obtain primary seed; the first seed was transferred to a shake flask containing 200mL YPD medium at 1% transfer amount, and cultured on a shaker at 220rpm at 30 ℃ for five days to determine the content of valencene.

The results show that the yield of GS-A3-W3-H is increased to 117.52 +/-7.22 mg/L compared with 73.64 +/-3.53 mg/L of GS-A3-W3. Therefore, the promoter pCUP1 is used for replacing pERG9, so that the flow of FPP to ergosterol is reduced, more flow to a valencene pathway is increased, and the yield of the valencene is increased.

3. Comparing the yields of valencene of different Saccharomyces cerevisiae chassis strains

Respectively taking 10 mu L of strain GS-A3-W4, strain 30000B-W2, strain CEN.PK2-1D-W2 and strain BY474-W2 which are stored in glycerinum tubing to a PA bottle containing 5mLYPD (the final concentration of copper ions is 200 mu mol/L) culture medium, and culturing for 16h at 30 ℃ BY shaking at 220rpm to obtain primary seeds; the first seed was transferred to a shake flask containing 200mL YPD medium at 1% transfer amount, and cultured on a shaker at 220rpm at 30 ℃ for five days to determine the content of valencene.

As shown in FIG. 5, the results showed that the yield of valencene was significantly higher for strain GS-A3-W4 than for strain G30000B-W2 and strain CEN. PK2-1D-W2 but was comparable to BY 474-W2. It can be seen that GS-A3 is a high-yield squalene strain screened and evolved by the company, and can provide more sufficient precursor substance, so that the yield of the valencene is improved, but the catalytic efficiency of the valencene synthetase SmVS is low, only part of FPP can be converted into the valencene, and under the condition, the yield cannot be obviously improved by improving the supply of the precursor substance.

3. Fermentation production of valencene by using strain GS-A3-W4

Taking 10 μ L of the strain preserved in Glycine max (L.) Gaertn, placing into PA bottle containing 5mLYPD (final concentration of copper ion is 200 μmol/L) culture medium, and culturing at 30 deg.C with shaking table at 220rpm for 16h to obtain first-class seed; transferring the primary seeds into a shake flask containing 200mL YPD (final concentration of copper-containing ions is 200 mu mol/L) culture medium by 1 percent of transfer amount, and culturing for 12h at 30 ℃ by a shaking table at 220rpm to obtain secondary seeds; 2.5L of fermentation medium was added to a 5L fermenter and the activated secondary seed liquid was inoculated at 10% of the transfer amount. In the fermentation process, the temperature is controlled at 30 ℃, the pH is controlled at 5.0 by using concentrated ammonia water in the whole process, the initial rotating speed of a fermentation tank is 200rpm, the ventilation capacity is 1L/min, the dissolved oxygen is 100 percent, the OD is continuously increased along with the fermentation, the dissolved oxygen can be continuously reduced, and the dissolved oxygen is controlled at about 20 percent by gradually increasing the rotating speed and the ventilation capacity until the maximum rotating speed and the ventilation capacity are reached; the glucose concentration in the fermentation tank is controlled below 1g/L by adjusting the feeding materials in the early stage; when the fermentation time is 30 hours, adding 15% isopropyl myristate of fermentation volume as an organic phase, and using a second feeding material to maintain the ethanol concentration in the fermentation tank below 1g/L for fermentation for 5 days.

The fermentation medium comprises the following components: 40g/L glucose, 5g/L yeast extract, 12g/L ammonium sulfate, 8g/L potassium dihydrogen phosphate, 6.2g/L magnesium sulfate heptahydrate, 200mg/L thiamine, 200mg/L pyridoxine, 500mg/L inositol, 50mg/L biotin and 200mg/L calcium pantothenate.

The supplementary material component comprises: 800g/L glucose and 50g/L yeast extract.

The supplementary material comprises the following components: 800g/L of sucrose and 50g/L of yeast extract.

The yield of the valencene produced by the final fermentation tank reaches 8g/L, and the industrial production capacity is high.

The features of the embodiments and embodiments described herein above may be combined with each other without conflict.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Hubei Guanzhongtong technology Co., Ltd

<120> high-yield valencene genetic engineering bacterium and preparation method and application thereof

<141> 2021-10-22

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 668

<212> DNA

<213> Saccharomyces cerevisiae pGAL1-10

<400> 1

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60

atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120

tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180

taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240

acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300

aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360

tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420

gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480

taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540

cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600

tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660

aaactata 668

<210> 2

<211> 1509

<212> DNA

<213> Saccharomyces cerevisiae tHMG1

<400> 2

ttaggattta atgcaggtga cggacccatc tttcaaacga tttatatcag tggcgtccaa 60

attgttaggt tttgttggtt cagcaggttt cctgttgtgg gtcatatgac tttgaaccaa 120

atggccggct gctagggcag cacataagga taattcacct gccaagacgg cacaggcaac 180

tattcttgct aattgacgtg cgttggtacc aggagcggta gcatgcgggc ctcttacacc 240

taataagtcc aacatggcac cttgtggttc tagaacagta ccaccaccga tggtacctac 300

ttcgatggat ggcatggata cggaaattct caaatcaccg tccacttctt tcatcaatgt 360

tatacagttg gaactttcaa cattttgtgc aggatcttgt cctaatgcca agaaaacagc 420

tgtcactaaa ttagctgcat gtgcgttaaa tccaccaaca gacccagcca ttgcagatcc 480

aaccaaattc ttagcaatgt tcaactcaac caatgcggaa acatcacttt ttaacacttt 540

tctgacaaca tcaccaggaa tagtagcttc tgcgacgaca ctcttaccac gaccttcgat 600

ccagttgatg gcagctggtt ttttgtcggt acagtagtta ccagaaacgg agacaacctc 660

catatcttcc cagccatact cttctaccat ttgctttaat gagtattcga cacctttaga 720

aatcatattc atacccattg cgtcaccagt agttgttcta aatctcatga agagtaaatc 780

tcctgctaga caagtttgaa tatgttgcag acgtgcaaat cttgatgtag agttaaaagc 840

ttttttaatt gcgttttgtc cctcttctga gtctaaccat atcttacagg caccagatct 900

tttcaaagtt gggaaacgga ctactgggcc tcttgtcata ccatccttag ttaaaacagt 960

tgttgcacca ccgccagcat tgattgcctt acagccacgc atggcagaag ctaccaaaca 1020

accctctgta gttgccattg gtatatgata agatgtacca tcgataacca aggggcctat 1080

aacaccaacg ggcaaaggca tgtaacctat aacattttca caacaagcgc caaatacgcg 1140

gtcgtagtca taatttttat atggtaaacg atcagatgct aatacaggag cttctgccaa 1200

aattgaaaga gccttcctac gtaccgcaac cgctctcgta gtatcaccta attttttctc 1260

caaagcgtac aaaggtaact taccgtgaat aaccaaggca gcgacctctt tgttcttcaa 1320

ttgttttgta tttccactac ttaataatgc ttctaattct tctaaaggac gtattttctt 1380

atccaagctt tcaatatcgc gggaatcatc ttcctcacta gatgatgaag gtcctgatga 1440

gctcgattgc gcagatgata aacttttgac tttcgatcca gaaatgactg ttttattggt 1500

taaaaccat 1509

<210> 3

<211> 1059

<212> DNA

<213> Saccharomyces cerevisiae ERG20

<400> 3

atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60

gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120

gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180

gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240

aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600

tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660

gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720

gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780

gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840

aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900

attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960

gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020

actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059

<210> 4

<211> 1641

<212> DNA

<213> Salvia miltiorrhiza SmVS

<400> 4

atggcaacca cccaagttga aattcaaaga cccattgcta attttagtcc ctctttgtgg 60

ggagaccaat ttataaagaa cgacagcggt gctaaggctg ctgaaaaaca ctgtaaagct 120

gttgaagaat taaagaaaga ggtaatgaac atgatcacag ccgcagaatc taatttggta 180

gaagcaatga acttgataga tacattggaa agactaggta taagctatca tttcgaaaaa 240

gagatagacc agaaattgaa tcacttcttt tcactaaaca ccgattatag cgatgaaagt 300

tatgatttgt acacagtgag cttgcatttt agattgttta gacaacatgg tcatagaata 360

tcatctgata ttttcggcag atggattgat gaaagtggaa aatttaagga gggtttgaaa 420

actgatggca aaggtttgtt gtctttgtat gaagcttcat atctaaggac tagaggtgaa 480

actattttgg atgatgcatt ggagtttgct acagctacat tgaacagtat tgctccacac 540

ttagagtctc cattgtctaa acaggttgtg catgctttga ttcaaccatt gcattatgga 600

aaccctagga ttgaagctca taattttata tctatctacg aggaaaacca ggacaaaaat 660

gaatttttac tgaagttcgc caagttggat tataatttac tacaaatgtt gcataaggaa 720

gagttgcacg aagtttctag gtggtggaaa gaattggatc tagtctcaaa attgccctat 780

gctagggata gagttgttga atgttttttt tgggccatgg gtgtttatca tgagcctcag 840

tattctaggg ctaggattat gttgactaag actattacta tgacctcaat tatcgacgat 900

acatatgacg cttatggtgt tattgaggag ttggatatat ttactgaagc aattgaaagg 960

tggaatattg aagagatgga tagattgccc gaatatgtta aaccatttta taaggctctg 1020

ttggaattgt atgagcagtt tgaagaggag ctggcagaag aaggaagatc ttatgcagct 1080

cattatgcaa tagaaagttt gaaagaactg gttaggagct atcatgtgga agctaaatgg 1140

tttattcagg gttacctgcc accatttgaa gaatatttga aaaatgctct gatcacttgt 1200

acttattgct atcatactac aacttctttg ttgggtgtgg aatctgctgt tgaagaggat 1260

ttccaatggt tggctaaaaa gcctaaaatg ttggtggcag gtttgttgat ttgtagggtt 1320

attgatgata tcgctactta tgaagttgag aaggaaagag gtcaaagtgc tactggtatt 1380

gaatcttata tgagagataa caacgctact attgaagaag ctgttgcaaa atttttcgag 1440

attgctacag atgcttggaa agatattaat gaagaatgtc taatgccctc tccatactct 1500

agggatgttt tgatgagaat acttaacttg gaaagaatca tcgatgtcac ttataaagga 1560

aatgaagatg gttataccca accagaaaaa gtattgaaac cccacattat tgccttgttt 1620

gttgatccta ttaagatgta a 1641

<210> 5

<211> 459

<212> DNA

<213> Saccharomyces cerevisiae pCUP1

<400> 5

cgatcccatt accgacattt gggcgctata cgtgcatatg ttcatgtatg tatctgtatt 60

taaaacactt ttgtattatt tttcctcata tatgtgtata ggtttatacg gatgatttaa 120

ttattacttc accacccttt atttcaggct gatatcttag ccttgttact agttagaaaa 180

agacattttt gctgtcagtc actgtcaaga gattcttttg ctggcatttc ttctagaagc 240

aaaaagagcg atgcgtcttt tccgctgaac cgttccagca aaaaagacta ccaacgcaat 300

atggattgtc agaatcatat aaaagagaag caaataactc cttgtcttgt atcaattgca 360

ttataatatc ttcttgttag tgcaatatca tatagaagtc atcgaaatag atattaagaa 420

aaacaaactg tacaatcaat caatcaatca tcacataaa 459

<210> 6

<211> 450

<212> DNA

<213> Saccharomyces cerevisiae pERG9

<400> 6

tgcgaagcct gctaaaatgc agtggaggcc gtgtaccctt tgccaaattg gctattggaa 60

tcggcagaga acctgggtcc cgttctagag accctgcgag cgtgtcccgg tgggttctgg 120

gagctctaac tccgcaggaa ctacaaacct tgcttacaca gagtgaacct gctgcctggc 180

gtgctctgac tcagtacatt tcatagccca tcttcaacaa caataccgac ttaccatcct 240

atttgctttg ccctttttct tttccactgc actttgcatc ggaaggcgtt atcggttttg 300

ggtttagtgc ctaaacgagc agcgagaaca cgaccacggg ctatataaat ggaaagttag 360

gacaggggca aagaataaga gcacagaaga agagaaaaga cgaagagcag aagcggaaaa 420

cgtatacacg tcacatatca cacacacaca 450

<210> 7

<211> 273

<212> DNA

<213> Saccharomyces cerevisiae tCYC1

<400> 7

gcaaattaaa gccttcgagc gtcccaaaac cttctcaagc aaggttttca gtataatgtt 60

acatgcgtac acgcgtttgt acagaaaaaa aagaaaaatt tgaaatataa ataacgttct 120

taatactaac ataactataa aaaaataaat agggacctag acttcaggtt gtctaactcc 180

ttccttttcg gttagagcgg atgtgggggg agggcgtgaa tgtaagcgtg acataactaa 240

ttacatgata tcgacaaagg aaaaggggcc tgt 273

<210> 8

<211> 149

<212> DNA

<213> Saccharomyces cerevisiae tERG20

<400> 8

aactaacgct aatcgataaa acattagatt tcaaactaga taaggaccat gtataagaac 60

tatatacttc caatataata tagtataagc tttaagatag tatctctcga tctaccgttc 120

cacgtgacta gtccaaggat tttttttaa 149

Claims

1. A construction method of gene engineering bacteria for high yield of valencene is characterized in that: the method comprises the following steps:

s1, constructing a saccharomyces cerevisiae MVA pathway related gene expression module: the over-expression tHMG1 module comprises an inducible bidirectional strong promoter pGAL1-10 and an MVA pathway rate-limiting enzyme coding gene tHMG1, wherein the nucleotide sequence of pGAL1-10 is shown as SEQ NO.1, and the nucleotide sequence of tHMG1 is shown as SEQ NO. 2;

s2, constructing a valencene production related gene expression module: the ERG20-Linker-SmVS module comprises an encoding gene ERG20 of FPP synthase and an encoding gene SmVS of valencene synthetase, wherein the nucleotide sequence of the ERG20 is shown as SEQ NO.3, and the nucleotide sequence of the SmVS is shown as SEQ NO. 4;

s3, constructing engineering bacteria for knocking out galactose regulatory protein GAL80 gene expression: integrating the saccharomyces cerevisiae MVA pathway related gene expression module in the step S1 and the valencene production related gene expression module in the step S2 to a galactose regulatory protein GAL80 gene locus, simultaneously knocking out a galactose regulatory protein GAL80 gene to obtain a knocked-out galactose regulatory protein GAL80 gene expression module, transforming the knocked-out galactose regulatory protein GAL80 gene expression module into the constructed saccharomyces cerevisiae genetically engineered bacterium GS-A3, and obtaining the genetically engineered bacterium with the galactose regulatory protein GAL80 gene expression knocked out through multiple genetic engineering operations;

s4, constructing a squalene synthase pathway down-regulation expression plasmid, wherein a copper ion-induced promoter pCUP1 is replaced by a squalene synthase gene ERG9 promoter, and the nucleotide sequence of pCUP1 is shown as SEQ NO. 5; the squalene synthase gene ERG9 promoter is obtained 450bp before the initiation codon of ERG9 gene, and the nucleotide sequence is shown as SEQ NO. 6;

s5, transforming the squalene synthase pathway down-regulated expression plasmid in the step S5 into the genetic engineering bacteria expressed by the knockout galactose regulatory protein GAL80 gene in the step S3, sequentially screening antibiotic resistance, and then obtaining the high-yield valencene genetic engineering bacteria through colony PCR verification;

2. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 1, wherein the method comprises the following steps: in step S1, the method for constructing the tmgb 1 module includes the steps of:

s1, using Saccharomyces cerevisiae 3000B genome DNA as a template, and respectively using primers of tCYC1-F, tCYC1-R, tHMG1-F, tHMG1-R, pGAL1pGAL10-F and pGAL1pGAL10-R to carry out PCR reaction to obtain DNA fragments tCYC1, tHMG1 and pGAL10pGAL 1;

s2, the three DNA fragments tCYC1, tHMG1 and pGAL10pGAL1 obtained in step S1 were ligated together by overlap extension PCR reaction using primers tCYC1-F and pGAL1pGAL10-R to obtain an overexpressed tHMG1 module, i.e., tCYC1_ tHMG1_ pGAL10pGAL 1.

3. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 1, wherein the method comprises the following steps: in step S2, the method for constructing the ERG20-Linker-SmVS module includes the steps of:

s2, carrying out PCR reaction by using primers tERG20-W-F and tERG20-R, and amplifying to obtain a DNA fragment tERG 20;

s3, carrying out PCR reaction by using a plasmid pGZ221 as a template and primers Linker-SmVS-F and SmVS-R, and amplifying to obtain a DNA fragment Linker-SmVS;

s4, connecting the DNA fragment ERG20_ Linker obtained in the step S1, the DNA fragment tERG20 obtained in the step S2 and the DNA fragment Linker _ SmVS obtained in the step S3 together by performing overlap extension PCR reaction by using primers ERG20-F and tERG20-R to obtain a fusion expression ERG20-Linker-SmVS module, namely ERG20_ Linker _ SmVS _ tERG 20.

4. The method for constructing the high-yield Valencene gene engineering bacterium as claimed in claim 3, wherein the method comprises the following steps: linker comprises glycine and serine, and its combined structure comprises any one of GSG, GGGS and GSGGSG, wherein G corresponds to nucleic acid sequence GGT, and S corresponds to nucleic acid sequence TCT.

5. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 4, wherein: in step S2, the Linker in the ERG20-Linker-SmVS module is GSGGSG.

6. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 1, wherein the method comprises the following steps: in step S3, the method for constructing a module expressing a knockout galactose regulatory protein GAL80 gene includes the steps of:

s1, taking Saccharomyces cerevisiae 3000B genomic DNA as a template, respectively carrying out PCR reaction by using GAL80left-F, GAL80left-R, GAL80right-F and GAL80right-R primers, and amplifying to obtain left and right homologous arms GAL80left and GAL80right of a DNA fragment GAL 80;

s2, carrying out PCR reaction by using a plasmid vector pFZ201 as a template and primers Hyg-F and Hyg-R to obtain a hygromycin expression cassette Hyg;

s3, connecting the over-expressed tHMG1 module and the ERG20-Linker-SmVS module, GAL80left, GAL80right and Hyg five DNA fragments together by performing overlap extension PCR reaction by using primers GAL80left-F and GAL80right-R to obtain a DNA fragment of the knockout galactose regulatory protein GAL80 gene expression module;

s4, connecting the DNA fragment obtained in the step S3 with a plasmid pMD19-T to obtain a recombinant plasmid vector, linearizing the recombinant plasmid by using a restriction enzyme PmeI, recovering the fragment with a target gene, transforming the fragment into a host bacterium Saccharomyces cerevisiae by using a yeast lithium acetate transformation method, sequentially screening antibiotic resistance, and then obtaining a positive bacterial colony by colony PCR to obtain an engineering bacterium with the expression of the galactose regulatory protein GAL80 knocked out.

7. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 6, wherein: in step S3, the host bacteria saccharomyces cerevisiae comprises any one of saccharomyces cerevisiae 30000B, saccharomyces cerevisiae s.cerevisiae cen.pk2-1D, saccharomyces cerevisiae BY4741 and saccharomyces cerevisiae GS-A3, wherein the saccharomyces cerevisiae GS-A3 is deposited in the chinese type culture collection at 2021, 9 and 17 days, with the deposition numbers: CCTCC NO: M20211191.

8. The method for constructing the high-yield valencene gene engineering bacteria as claimed in claim 1, wherein the method comprises the following steps: in step S4, the construction method of the squalene synthase pathway down-regulation expression plasmid comprises the following steps:

s2, carrying out PCR reaction by using a plasmid vector pFZ202 as a template and using primers G418-F and G418-R to obtain a G418 expression cassette G418;

s3, connecting five DNA fragments of the DNA fragments pERG9-left, pCUP1 and pERG9-right obtained in the step S1 and the expression cassette G418 obtained in the step S1 together by performing overlap extension PCR reaction with primers pERG9-left-F and pERG9-right-R to obtain a DNA fragment G418_ pCUP 1/delta pEGR 9;

s4, connecting the DNA fragment G418_ pCUP 1/delta pEGR9 obtained in the step S3 with a plasmid pMD19-T to obtain a squalene synthase pathway down-regulated expression plasmid vector which is marked as pCZ 101.

9. A high-yield valencene genetic engineering bacterium is characterized in that: obtained by the method of construction according to any one of claims 1 to 8.

10. The application of the high-yield valencene genetic engineering bacteria as claimed in claim 9, wherein: the liquid fermentation medium components of the gene engineering bacteria for producing the valencene in a fermentation way comprise 20-50g/L of glucose, 5-10g/L of yeast extract, 6-15g/L of ammonium sulfate, 3-8g/L of potassium dihydrogen phosphate, 5-10g/L of magnesium sulfate heptahydrate, 500mg/L of thiamine 100-containing materials, 500mg/L of pyridoxine 100-containing materials, 800mg/L of inositol 400-containing materials, 20-100mg/L of biotin and 500mg/L of calcium pantothenate 100-containing materials.