Genetically engineered bacterium for high-yield production of bisabolol as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for high yield of bisabolol as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. Saccharomyces cerevisiae engineering bacteria in an overexpression mevalonic acid pathway are used as starting bacteria, a fusion protein expression cassette of farnesyl pyrophosphate synthetase and bisabolol synthetase is integrated, endogenous transcription factors and transporter of the saccharomyces cerevisiae are overexpressed, an original promoter of squalene synthetase is replaced by an inducible promoter, and the recombinant squalene synthetase is obtained. And the genetically engineered bacterium for high-yield production of bisabolol is constructed. According to the present invention, the bisabolol synthase gene is introduced to achieve the heterologous synthesis of the bisabolol in the Saccharomyces cerevisiae, the rate-limiting enzyme fusion expression and the transport protein overexpression are integrated so as to alleviate the metabolism pressure of the Saccharomyces cerevisiae cells, and the competition pathway is down-regulated so as to enable the substrate farnesyl pyrophosphate to flow to the metabolism pathway of the bisabolol; the bisabolol production capability of the strain is improved. The invention provides a method for realizing high yield of bisabolol, which is superior to reports in the prior art and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a genetic engineering bacterium with high production of bisabolene, a construction method and application thereof. Background Art

[0002] Bisabolene, also known as bisabolene and sweet bisabolene, is a common natural sesquiterpene product in nature. In plants, bisabolene is a chemical defense against insects, herbivores and fungal pathogens. Bisabolene can be used as an important precursor in the production of green biofuels, food, flavors and fragrances, and medicines, and has broad market prospects.

[0003] At present, the synthesis methods of bisabolene mainly include plant extraction and chemical synthesis. Chemical synthesis mainly includes semi-synthesis method using limonene as raw material and total synthesis method using isoprene as raw material. These two methods have the following problems: limonene as raw material needs to be extracted from plants, and the obtained bisabolene has low purity; isoprene and methyl acrylate are used as raw materials to generate the main ring structure through the catalysis of anhydrous aluminum chloride, and then after a series of reactions, β-bisabolene is synthesized by Witting reaction. The reaction steps are numerous and toxic and harmful catalysts are required, and the reaction conversion rate is low. Although there are literature reports on the optimization of the synthesis conditions by the three-step synthesis method of isoprene, the yield is only 40% (New Synthesis of (±)-β-Bisabolene. Journal of East China University of Chemical Technology, 1985, (04): 447-450.). The chemical synthesis of α-bisabolene using the Witting method requires more than 10 steps of reaction to complete, and the last reaction step requires flammable and explosive n-butyl lithium as a catalyst, resulting in a low reaction conversion rate (The wittig-horner route to tri-substituted alkanes: synthesis of Z-α-bisabolene. Tetrahedron Lett, 1983, 24(1): 111-114.).

[0004] Compared with traditional chemical and plant extraction methods, the advantages of synthesizing bisabolene by microbial methods are that, first, the specificity of bisabolene synthase is strong, and it can produce single-configuration bisabolene with high purity. Secondly, using microorganisms as a chassis to produce in a mild reaction system is a relatively green and environmentally friendly method. Finally, many metabolic pathways of the microbial chassis themselves will generate precursors of bisabolene, and it is very advantageous to directly use recombinant bacteria to produce bisabolene. Saccharomyces cerevisiae is a commonly used chassis organism with the advantages of clear genetic background, recognized safety, and mature high-density fermentation technology. Therefore, using Saccharomyces cerevisiae as a host to produce bisabolene has great potential and prospects.

[0005] At present, the highest shake flask yield of α-bisabolene of Saccharomyces cerevisiae engineered strains is 866.7 mg / L (Enhancing glycerol metabolism to increase the bisabolene yield of yeast engineered strains. Journal of Biotechnology, 2024, 40(3):847-857.). However, there are no reports on the synthesis of β-bisabolene using Saccharomyces cerevisiae, and the shake flask yield of β-bisabolene synthesized by Yarrowia lipolytica is only 68.2 mg / L (High-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowialipolytica. Microb Biotechnol, 2021, 14(6):2497-2513). These bisabolene yields are still far from industrial application.

[0006] Possible factors affecting the production of bisabolene in engineered strains of Saccharomyces cerevisiae include the soluble expression of plant-derived bisabolene synthase, the supply of precursors, competitive consumption and effective conversion of metabolic intermediates, and extracellular secretion of products. Therefore, how to efficiently prepare bisabolene using Saccharomyces cerevisiae is a technical problem that needs to be solved by those skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a genetically engineered bacterium capable of producing a high yield of bisabolene, and to prepare α- and β-bisabolene separately or simultaneously in an efficient manner by means of microbial heterologous synthesis.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The invention provides a genetically engineered bacterium with high yield of bisabolene. The genetically engineered bacterium uses an engineered strain of saccharomyces cerevisiae overexpressing a mevalonate pathway as a starting bacterium, and a promoter of squalene synthase ERG9 in the genome is replaced with an inducible promoter; and the genome is integrated with coding sequences of a transcription factor PDR1 of a pleiotropic drug resistance protein and a transporter SNQ2, as well as an α-bisabolene synthase fusion protein expression cassette and / or a β-bisabolene synthase fusion protein expression cassette, or a recombinant expression plasmid containing a transcription factor PDR1, a transporter SNQ2, an α-bisabolene synthase fusion protein and / or a β-bisabolene synthase fusion protein is introduced, wherein the α-bisabolene synthase fusion protein is formed by fusion of farnesyl pyrophosphate synthase and α-bisabolene synthase via a connecting peptide, and the β-bisabolene synthase fusion protein is formed by fusion of β-bisabolene synthase and farnesyl pyrophosphate synthase via a connecting peptide.

[0010] The present invention uses an engineered strain of Saccharomyces cerevisiae that overexpresses a mevalonate pathway as a starting strain. The strain overexpresses a mevalonate pathway to synthesize abundant isoprenyl pyrophosphate and dimethylallyl pyrophosphate, and synthesizes farnesyl pyrophosphate, a precursor of bisabolene, through endogenous farnesyl pyrophosphate synthase ERG20. On this basis, the present invention introduces a coding gene for an α-bisabolene synthase fusion protein to express a fusion protein of ERG20 and α-bisabolene synthase to achieve heterologous synthesis of α-bisabolene; by replacing an α-bisabolene synthase expression cassette with a β-bisabolene synthase expression cassette, heterologous synthesis of β-bisabolene can be achieved; and by introducing a combination of an α-bisabolene synthase expression cassette and a β-bisabolene synthase expression cassette, simultaneous synthesis of α and β-bisabolene can be achieved. Furthermore, in order to direct the substrate farnesyl pyrophosphate to the metabolic pathway of bisabolene, the present invention replaces the original promoter of squalene synthase with an inducible promoter to downregulate the squalene synthesis pathway; in order to alleviate the metabolic pressure of yeast cells, the present invention overexpresses the transcription factor PDR1 and the transporter SNQ2 of the endogenous pleiotropic drug resistance protein of Saccharomyces cerevisiae, thereby increasing the production of bisabolene.

[0011] Preferably, the starting bacteria are strains BY4742-C04 and BY4741-C04 that overexpress the mevalonate pathway in the cytoplasm, both of which are complementary mating type engineered strains. These strains are publicly available materials, and their construction methods refer to the literature (Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nat Commun, 2016, 7: 12851).

[0012] Preferably, α-bisabolene synthase is derived from Abies grandis, and the amino acid sequence is shown in SEQ ID NO.1, and the N-terminus of α-bisabolene synthase is fused to farnesyl pyrophosphate synthase through a connecting peptide; β-bisabolene synthase is derived from Santalum album, and the amino acid sequence is shown in SEQ ID NO.2, and the C-terminus of β-bisabolene synthase is fused to farnesyl pyrophosphate synthase through a connecting peptide; the amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID NO.3.

[0013] The present invention explores the effect of protein fusion expression order on the production of bisabolene. The study found that the fusion expression of farnesene pyrophosphate synthase ERG20 and two bisabolene synthases can effectively promote the synthesis of bisabolene, but due to the difference in protein structure, the preferred expression order of the two is different. For the α-bisabolene synthase AgBIS from Abies grandis, ERG20 fusion at its N-terminus has a better effect; while for the β-bisabolene synthase SaBIS from sandalwood, ERG20 fusion at its C-terminus has a better effect.

[0014] Farnesyl pyrophosphate synthase and bisabolene synthase are connected via a flexible connecting peptide, and preferably, the amino acid sequence of the connecting peptide is GGGGS.

[0015] The present invention can perform codon optimization and synthesize the coding sequence of the fusion protein according to the preference of the host cell.

[0016] Further, in order to increase the expression solubility of the fusion protein and promote the correct folding of the protein, the present invention introduces a soluble expression tag at the N-terminus of the fusion protein, which may be, but not limited to, thioredoxin TRX1, small ubiquitin-like protein SMT3, glutathione S-transferase GST or maltose binding protein MBP. Preferably, the N-terminus of the fusion protein introduces a soluble tag TRX1, and the amino acid sequence of the soluble tag TRX1 is shown in SEQ ID NO.5. Specifically, the present invention obtains the optimal expression cassette TRX1-ERG20-GGGGS-AgBIS by exploring the effect of protein fusion expression order and soluble tags on yield.

[0017] The genetically engineered bacteria provided by the present invention contain ≥1 copy of an α-bisabolene synthase fusion protein expression cassette and / or ≥1 copy of a β-bisabolene synthase fusion protein expression cassette. In order to increase the yield of bisabolene, the present invention increases the copy number of the bisabolene expression cassette. Preferably, the genetically engineered bacteria contain 1-4 copies of an α-bisabolene synthase fusion protein expression cassette and / or 1-4 copies of a β-bisabolene synthase fusion protein expression cassette; more preferably, the copy number of the fusion protein expression cassette is 2-3.

[0018] In order to down-regulate the competitive pathway of bisabolene, the present invention replaces the original promoter of squalene synthase with an inducible promoter. In the absence of an inducing factor, the squalene synthesis pathway cannot be started, so that the substrate farnesyl pyrophosphate flows to the metabolic pathway of bisabolene. Preferably, the inducible promoter is a glucose-inducible promoter. The glucose-inducible promoter can be, but is not limited to, P HXT1 .

[0019] Preferably, gene sequences encoding alcohol dehydrogenase, acetaldehyde dehydrogenase and acetyl-CoA synthase are integrated into the genome of the genetically engineered bacteria.

[0020] In order to further increase the yield of bisabolene, the present invention overexpresses endogenous alcohol dehydrogenase ADH2, acetaldehyde dehydrogenase ALD6 and acetyl-CoA synthase ACS2 of Saccharomyces cerevisiae, thereby enhancing the supply of acetyl-CoA in the cytoplasm and the production capacity of downstream products of the MVA pathway.

[0021] Preferably, the amino acid sequence of the transcription factor PDR1 is shown in SEQ ID NO.6, the amino acid sequence of the transporter SNQ2 is shown in SEQ ID NO.7, the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.8, the amino acid sequence of the acetaldehyde dehydrogenase is shown in SEQ ID NO.9, and the amino acid sequence of the acetyl-CoA synthase is shown in SEQ ID NO.10. The present invention can obtain the coding gene sequence of the above-mentioned endogenous protein of Saccharomyces cerevisiae by gene cloning.

[0022] Preferably, the genetically engineered bacteria are diploid strains obtained by hybridization. The present invention can obtain diploid strains that can produce high yields of α-bisabolene and β-bisabolene respectively or simultaneously by hybridizing genetically engineered bacteria with complementary mating types.

[0023] The present invention also provides a method for constructing the high-yield bisabolene genetic engineering bacteria, the construction method comprising: using a saccharomyces cerevisiae engineering strain that overexpresses the mevalonate pathway as a starting strain, replacing the promoter of squalene synthase with an inducible promoter by integrating a plasmid or CRISPR / Cas9 technology, integrating the gene sequence encoding the transcription factor PDR1 and the transporter protein SNQ2 and the α-bisabolene synthase fusion protein expression cassette and / or the β-bisabolene synthase fusion protein expression cassette into the engineering strain genome, and obtaining a high-yield bisabolene genetic engineering bacteria;

[0024] Alternatively, an engineered strain of Saccharomyces cerevisiae that overexpresses the mevalonate pathway is used as the starting bacterium, the promoter of squalene synthase is replaced with an inducible promoter through integration of plasmids or CRISPR / Cas9 technology, and a recombinant expression plasmid for expressing the transcription factor PDR1 and the transporter protein SNQ2 as well as α-bisabolene synthase fusion protein and / or β-bisabolene synthase fusion protein is introduced to obtain a genetically engineered bacterium with high bisabolene production.

[0025] The invention realizes the construction of a high-yield genetic engineering strain of bisabolene by integrating strategies such as rate-limiting enzyme fusion expression, competitive pathway down-regulation, endogenous transcription factor and transporter protein overexpression.

[0026] Preferably, the integration plasmid uses the pUMRI series assembly tool plasmid.

[0027] Furthermore, the present invention can further improve the yield of α-bisabolene by strategies such as enhancing the supply of the precursor substance acetyl-CoA, expressing a multi-copy fusion protein expression cassette, and hybridizing strains.

[0028] Preferably, the method for constructing the high-yield bisabolene genetic engineering bacteria comprises the following steps:

[0029] (1) The coding gene of the α-bisabolene synthase fusion protein ERG20-AgBIS was cloned into the pUMRI-GAL1-7HA GAL1 With T CYC1 In between, the recombinant plasmid pUMRI-GAL1-7HA-EA was obtained, and then the lytic tag encoding gene was integrated into the N-terminus of the fusion protein to obtain the recombinant plasmid pUMRI-GAL1-7HA-TRX1-EA;

[0030] (2) The genes encoding the transcription factor PDR1 and the transporter SNQ2 of the endogenous multi-drug resistance protein of Saccharomyces cerevisiae were cloned into the P GAL1 With T CYC1 Between, the recombinant plasmids pUMRI-ROX1-PDR1 and pUMRI-ROX1-SNQ2 were obtained;

[0031] (3) The alcohol dehydrogenase encoding gene and the acetaldehyde dehydrogenase encoding gene were cloned into the P GAL1 With T CYC1 and P GAL10 With T ADH1 The recombinant plasmid pUMRI-GAL1-7HA-ADH2-ALD6 was obtained;

[0032] (4) Clone the gene encoding acetyl-CoA synthetase into the P GAL1 With T CYC1 The recombinant plasmid pUMRI-DPP1-ACS2 was obtained;

[0033] (5) The recombinant plasmids pUMRI-GAL1-7HA-TRX1-EA and pUMRI-ROX1-PDR1 were transformed into the engineered strain BY4742-C04 overexpressing the mevalonate pathway, and the SNQ2 coding sequence and TRX1-ERG20-AgBIS expression cassette were integrated into the genome using CRISPR / Cas9 technology. HXT1 Replace the original promoter of squalene synthase ERG9 to obtain a genetically engineered bacterium with high production of α-bisabolene;

[0034] Alternatively, the recombinant plasmids pUMRI-GAL1-7HA-TRX1-EA and pUMRI-ROX1-SNQ2 were transformed into the engineered strain BY4741-C04 overexpressing the mevalonate pathway, and the ADH2-ALD6 coding sequence, ACS2 coding sequence, PDR1 coding sequence, and TRX1-ERG20-AgBIS expression cassette were integrated into the genome using CRISPR / Cas9 technology, and the expression cassette was expressed using P HXT1 Replace the original promoter of squalene synthase ERG9 to obtain a genetically engineered bacterium with high production of α-bisabolene;

[0035] (6) Using CRISPR / Cas9 technology, one or all of the TRX1-ERG20-AgBIS expression cassettes in the genetically engineered bacteria that produce high α-bisabolene production in step (5) are replaced with the β-bisabolene synthase fusion protein SABIS-ERG20 expression cassette to obtain a genetically engineered bacterium that simultaneously produces α and β-bisabolene or a genetically engineered bacterium that produces β-bisabolene.

[0036] Preferably, the recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 is transformed into the engineered bacteria so that the original promoter of squalene synthase ERG9 is replaced by P HXT1 Promoter.

[0037] The plasmids PUMRI-12-HXT1-ERG9-TEF1, pUMRI-GAL1-7HA, pUMRI-ROX1, and pUMRI-DPP1 are all publicly available materials.

[0038] Preferably, the present invention obtains a diploid strain that produces high α-bisabolene or β-bisabolene or both α-bisabolene and β-bisabolene by hybridizing genetically engineered bacteria with complementary mating types. By hybridizing different haploid strains, a diploid strain that produces α-bisabolene and β-bisabolene at different copy number ratios can be obtained.

[0039] Preferably, the whole genome of Saccharomyces cerevisiae without defects is used as a template, and after the target band is obtained by PCR, the nutritional deficiencies of URA3, HIS3, and LEU2 of the two hybrid strains are complemented respectively. The retained △met of BY4741 and △lys of BY4742 can be complemented with each other through hybridization technology to obtain a fermentation strain without nutritional deficiency. No additional amino acids need to be added to the fermentation medium, which can save fermentation costs.

[0040] The present invention also provides the use of the genetically engineered bacteria with high bisabolene production in the preparation of bisabolene. The bisabolene is separated from the fermentation product of the genetically engineered bacteria constructed by the present invention. The bisabolene is α-bisabolene or β-bisabolene or a mixture of the two.

[0041] Preferably, the application comprises the following steps:

[0042] 1) After the genetically engineered bacteria with high production of bisabolene are expanded and cultured, they are inoculated into a YPD fermentation medium with an in-situ extractant added thereto for two-phase culture, wherein the extractant is dodecane and the amount of the extractant added is 5%-20%, and the culture is carried out at 24-30° C. for 80-120 hours to obtain a fermentation liquid;

[0043] 2) Collecting the organic phase in the fermentation broth and separating and obtaining bisabolene.

[0044] The present invention also provides a method for high-density fermentation of genetically engineered bacteria by batch feeding, comprising: expanding the culture of the genetically engineered bacteria with high yield of bisabolene, transferring it to a secondary YPD seed solution at a 1% inoculation amount, and culturing it until OD 600 The inoculum volume is 8-10, and then transferred to the YPD fermentation medium according to 10%, the pressure of the fermentation tank is controlled to be 0.04Mpa, the dissolved oxygen DO of the fermentation liquid is 40%±15%, and the pH value is 5.0±0.5; when the ethanol content in the fermentation liquid is lower than 3g / L, the fermentation feed concentrate is started; when the fermentation is 24h, 10% by volume of dodecane is added for product extraction; when the cells enter the stable period (about 48h of fermentation), the glucose in the feed solution is replaced by 75% by mass ethanol solution.

[0045] The present invention has the following beneficial effects:

[0046] (1) The present invention uses an engineered yeast strain of Saccharomyces cerevisiae that overexpresses the mevalonate pathway as a starting strain, integrates a fusion protein expression cassette of farnesyl pyrophosphate synthase and α-bisabolene synthase, overexpresses endogenous transcription factors and transporters of Saccharomyces cerevisiae, and replaces the original promoter of squalene synthase with an inducible promoter, thereby constructing a genetically engineered strain that produces high levels of bisabolene. The present invention achieves heterologous synthesis of α-bisabolene in Saccharomyces cerevisiae by introducing the α-bisabolene synthase gene, while integrating the rate-limiting enzyme fusion expression and overexpression of transporters to alleviate the metabolic pressure of Saccharomyces cerevisiae cells, downregulating the competitive pathway so that the substrate farnesyl pyrophosphate flows to the metabolic pathway of bisabolene, and improving the ability of the strain to produce bisabolene.

[0047] (2) The present invention constructs a genetically engineered bacterium that produces high β-bisabolene by replacing the α-bisabolene synthase fusion protein expression cassette with a β-bisabolene fusion protein expression cassette; and obtains a diploid strain that produces high α and / or β-bisabolene by hybridizing genetically engineered bacteria with complementary mating types.

[0048] (3) The present invention provides a method for achieving high production of bisabolene. The production of bisabolene synthesized by the engineered strain constructed by the present invention after two-phase fermentation in shake flasks is better than that reported in the prior art and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the pUMRI-GAL1-7HA-TRX1-EA plasmid structure.

[0050] Figure 2 This is a diagram for constructing the expression cassette of the genetically engineered strain TRX1-ERG20-AgBIS that produces high levels of bisabolene.

[0051] Figure 3 Schematic diagram of the pUMRI-GAL1-7HA-SE plasmid structure.

[0052] Figure 4 This is a diagram for constructing the expression cassette of the genetically engineered strain SaBIS-ERG20 that produces high levels of bisabolene.

[0053] Figure 5 Effects of fusion-promoting tags on bisabolene production.

[0054] Figure 6 A roadmap for constructing a diploid strain with high bisabolene production.

[0055] Figure 7 Schematic diagram of the replacement of the expression cassette for β-bisabolene synthesis.

[0056] Figure 8 A roadmap for constructing a diploid strain for the simultaneous production of α-bisabolene and β-bisabolene.

[0057] Fig. 9 This is the GC detection spectrum of β-bisabolene standard and α-bisabolene and β-bisabolene.

[0058] Fig.10 Schematic diagram of the production of YD02-11, YDZ07, and YDZ-C-α.

[0059] Fig.11 The yield of α-bisabolene in YDZ01 and YD01 original strains.

[0060] Fig.12 These are the results of shake flask culture of the high-yield β-bisabolene strain YDZSE.

[0061] Fig.13 These are the results of fed-batch fermentation of the high-yield α-bisabolene hybrid strain YDZ-C-α.

[0062] Fig.14These are the results of fed-batch fermentation of a hybrid strain that simultaneously produces α- and β-bisabolene. DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the examples are conventional technical means in the art, and the raw materials and reagents are all commercially available products.

[0064] The strains BY4741-C04 and BY4742-C04 and plasmid pUMRI-GAL1-7HA that overexpress the mevalonate pathway in the cytoplasm were constructed by our research group in the early stage. The construction method is based on the reference (Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nat Commun, 2016, 7: 12851); the plasmid PUMRI-12-HXT1-ERG9-TEF1 was constructed by our research group in the early stage. The construction method is based on the reference (Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae, Metab Eng, 2015, 28: 8-18).

[0065] Example 1: Cloning of genes required for the production of bisabolene

[0066] 1. Fusion of farnesene pyrophosphate synthase ERG20 and α-bisabolene synthase AgBIS

[0067] In this example, α-bisabolene synthase (AgBIS) is fused to farnesene pyrophosphate synthase ERG20 via a linker peptide (GGGGS) at the N-terminus of the AgBIS. The amino acid sequence of AgBIS is shown in SEQ ID NO.1, and the amino acid sequence of ERG20 is shown in SEQ ID NO.3.

[0068] The gene fragments of the above fusion protein are artificially synthesized. The gene sequence of farnesene pyrophosphate synthase ERG20 is shown in the gene accession number: NM_001181600. The exogenous gene α-bisabolene synthase (AgBIS) is derived from Abiesgrandis, and the gene accession number is: AF006195.1. The coding sequence of the connecting peptide is 5'-ggaggcggtgggtcc-3'.

[0069] 2. Fusion of β-bisabolene synthase SaBIS and farnesene pyrophosphate synthase ERG20

[0070] In this example, the C-terminus of β-bisabolene synthase (SaBIS) is fused to farnesene pyrophosphate synthase ERG20 via a connecting peptide (GGGGS). The amino acid sequence of SaBIS is shown in SEQ ID NO.2.

[0071] The gene fragment of the above fusion protein is artificially synthesized. The exogenous gene β-bisabolene synthase (SaBIS) is derived from Santalum album, and the gene accession number is KJ665778.1. During synthesis, the codon of Saccharomyces cerevisiae is optimized, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; the gene sequence of ERG20 is shown in gene accession number NM_001181600.

[0072] 3. Extraction of Saccharomyces cerevisiae genomic DNA

[0073] The specific steps for extracting genomic DNA from Saccharomyces cerevisiae BY4741-C04 are as follows:

[0074] (1) Take 2-4 mL of yeast culture medium that has been cultured for 16-24 hours and centrifuge at 12000 rpm for 10 minutes at room temperature.

[0075] (2) Discard the supernatant, add 480 μL Buffer SE, 10 μL mercaptoethanol, and 20 μL lyase, resuspend the precipitate, and incubate at 30°C for 30 min.

[0076] (3) Centrifuge at 12,000 rpm for 5 min at room temperature, discard the supernatant, add 200 μL Buffer YL and 50 mg glass beads (0.4-0.6 mm), vortex for 3-5 min, let stand to allow the glass beads to settle, and aspirate the supernatant into a new 1.5 mL centrifuge tube.

[0077] (4) Add 25 μL of Proteinase K, mix thoroughly, and shake at 65°C for 30 min.

[0078] (5) Add 5 μL RNase A, mix thoroughly, and let stand at room temperature for 10 min.

[0079] (6) Add 220 μL of Buffer YDL and 220 μL of anhydrous ethanol and vortex for 20 s.

[0080] (7) Place the adsorption column into the collection tube, use a pipette to draw all the supernatant into the adsorption column, centrifuge at 10,000 rpm for 1 min, and discard the supernatant.

[0081] (8) Place the adsorption column back into the collection tube, add 500 μL Buffer HB, centrifuge at 10,000 rpm for 30 s, and discard the filtrate.

[0082] (9) Place the adsorption column back into the collection tube, add 700 μL DNA Wash Buffer, centrifuge at 10,000 rpm for 30 seconds, and discard the filtrate.

[0083] (10) Repeat the previous step, spin, and remove the remaining DNA Wash Buffer.

[0084] (11) Place the adsorption column in a new 1.5 mL centrifuge tube, add 50-100 μL Elution Buffer, incubate at 65°C for 3-5 min, centrifuge at 12,000 rpm for 1 min at room temperature, and collect the resulting DNA solution and store it at -20°C.

[0085] 4. Clone the transcription factor encoding gene (PDR1), transporter encoding gene (SNQ2), alcohol dehydrogenase encoding gene (ADH2), acetaldehyde dehydrogenase encoding gene (ALD6) and acetyl-CoA synthase encoding gene (ACS2). Use the Saccharomyces cerevisiae genome as template and high-fidelity DNA polymerase KOD OneTM PCR Master Mix for PCR amplification.

[0086] Among them, the sequence of the transcription factor encoding gene PDR1 is shown in gene accession number NM_001180878.1, and the amino acid sequence is shown in SEQ ID NO.6; the sequence of the transporter encoding gene SNQ2 is shown in gene accession number NM_001180319.1, and the amino acid sequence is shown in SEQ ID NO.7; the sequence of the alcohol dehydrogenase encoding gene ADH2 is shown in gene accession number NM_001182812.1, and the amino acid sequence is shown in SEQ ID NO.8; the sequence of the acetaldehyde dehydrogenase encoding gene ALD6 is shown in gene accession number NM_001183875.1, and the amino acid sequence is shown in SEQ ID NO.9; the sequence of the acetyl-CoA synthase encoding gene is shown in gene accession number NM_001182040.1, and the amino acid sequence is shown in SEQ ID NO.10. The primer design is shown in Table 1 below.

[0087] Table 1. Primers used to clone genes required for bisabolene biosynthesis

[0088]

[0089]

[0090]

[0091] Note: N in the sequence represents any base A / T / C / G.

[0092] The PCR reaction system (50 μL) was as follows: KODONE polymerase: 25 μL, primer F: 1.5 μL, primer R: 1.5 μL, genomic DNA / template: 1 μL, water: 17 μL.

[0093] The PCR program was as follows: (1) pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 68°C, 1 kb / 10 s, for 39 cycles; (3) extension at 68°C for 5 min; (4) storage at 4°C for 5 min.

[0094] Example 2: Construction of plasmids required for bisabolene synthesis

[0095] 1. Enzyme digestion and gel recovery

[0096] The pUMRI series integration plasmids (pUMRI-LPP1, pUMRI-DPP1, pUMRI-GAL80, pUMRI-HO, pUMRI-GAL1-7HA, pUMRI-ROX1) were all preserved in the laboratory. The plasmids and PCR product target fragments were double-digested with Takara restriction endonucleases. The double-digestion system was in accordance with the Takara restriction endonuclease instructions. After digestion, the system was subjected to DNA gel recovery treatment. The specific steps were carried out in accordance with the Axygen kit instructions.

[0097] 2. Enzyme Link

[0098] T4 DNA ligase was used for ligation, and the ligation system (10 μL) was as follows: 10×T4 DNA Buffer: 1 μL, gene fragment: 3.5 μL, vector fragment: 5.5 μL, T4 DNA ligase: 0.5 μL.

[0099] The connection was carried out in a metal bath at 22°C for 30-50 min.

[0100] 3. Conversion

[0101] Add 10 μL of the ligation product to the competent E. coli solution, place it on ice for 15-25 minutes, heat shock it in a metal bath at 42°C for 90 seconds, and quickly place it in an ice bath for 3 minutes, add 1 mL of LB liquid culture medium, resuscitate it in a shaker at 37°C for 45 minutes, then centrifuge it at 12000 rpm for 1 minute, discard 1 mL of supernatant, mix it well, take an appropriate amount and spread it on the corresponding resistance LB plate, and place it in a 37°C incubator for 12-16 hours.

[0102] Specifically, enzyme digestion and ligation are performed in the following manner to construct a recombinant plasmid:

[0103] The gene encoding the fusion protein of farnesyl pyrophosphate synthase and α-bisabolene synthase was cloned into the multiple cloning site behind PGAL1 of pUMRI-GAL1-7HA to obtain the recombinant plasmid pUMRI-GAL1-7HA-EA.

[0104] The lytic tag encoding gene TRX1 was integrated into the N-terminus of the ERG20-AgBIS expression cassette to obtain the recombinant plasmid pUMRI-GAL1-7HA-TRX1-EA, see Figure 1 and Figure 2 The sequence of the lytic tag encoding gene TRX1 is shown in NM_001181930, and the amino acid sequence is shown in SEQ ID NO.5.

[0105] The genes encoding endogenous multi-drug resistance proteins (PDR1, SNQ2) of Saccharomyces cerevisiae were cloned into the multiple cloning site behind PGAL1 of pUMRI-ROX1 to obtain recombinant plasmids pUMRI-ROX1-PDR1 and pUMRI-ROX1-SNQ2.

[0106] The target fragments of alcohol dehydrogenase (ADH2) and acetaldehyde dehydrogenase (ALD6) were cloned into the multiple cloning sites after PGAL1 and PGAL10 of pUMRI-GAL1-7HA, respectively, to obtain the recombinant plasmid pUMRI-GAL1-7HA-ADH2-ALD6.

[0107] The gene encoding acetyl-CoA synthetase (ACS2) was cloned into the multiple cloning site after PGAL1 of pUMRI-DPP1 to obtain the recombinant plasmid pUMRI-DPP1-ACS2.

[0108] The gene encoding the fusion protein SABIS-ERG20 of β-bisabolene synthase and farnesyl pyrophosphate synthase was cloned into the multiple cloning site after PGAL1 of pUMRI-GAL1-7HA to obtain the recombinant plasmid pUMRI-GAL1-7HA-SE. Figure 3 and Figure 4 .

[0109] Example 3: Construction of a Bisabolene-producing Saccharomyces cerevisiae strain

[0110] 1. Gene integration through the pUMRI system

[0111] Our group used the pUMRI system for gene integration in Saccharomyces cerevisiae. The pUMRI plasmid was linearized by Sfi I digestion and transformed into competent cells of Saccharomyces cerevisiae. The homologous arms on the plasmid and the part to be replaced on the genome underwent homologous recombination to achieve the integration of the target gene. Since the plasmid carries KanMX (G418 resistance gene) and URA3 screening markers, positive clones can be screened using plates containing G418 or SD-URA plates.

[0112] SfiⅠ digestion system (20μL): 17μL plasmid, 2μL 10×QuickCut Buffer, 1μL SfiⅠ endonuclease. Digestion conditions: in a water bath at 50℃ for 2h.

[0113] 2. Use CRISPR / Cas9 integration tools

[0114] Using plasmid p426-SpSgH as a template, 20 bp gRNA was designed into the primers, and the full-length plasmid containing gRNA was obtained by PCR. After being digested with DpnⅠ at 37℃ for 2h to remove the template, it was transferred into competent E. coli cells for transformation. The 40 bp at both ends of the genome to be integrated serve as the homology arms of the donor to ensure the integration efficiency. The expression cassette containing the homology arms obtained by PCR is the donor required for chemical transformation.

[0115] Specifically, the steps for constructing the recombinant yeast Saccharomyces cerevisiae are as follows:

[0116] (1) Take an appropriate amount of Saccharomyces cerevisiae from the glycerol tube and inoculate it into 5 mL of YPD medium. Incubate it in a constant temperature shaker at 30°C and 220 rpm for 12-16 h. Then streak it on a YPD plate and place it in an incubator at 30°C for 2 days.

[0117] (2) Pick a single colony from the plate and place it in a 5 mL YPD tube. Incubate at 30°C, 220 rpm in a constant temperature shaker for 12-16 hours and measure the OD 600 , so that its initial value is 0.05, inoculated into 50mL YPD liquid medium, cultured in a constant temperature shaker at 220rpm, and cultured until OD 600 It is 1.5-2.0.

[0118] (3) Heat the pre-made ssDNA at 100°C for 5 min and immediately cool it on ice for later use.

[0119] (4) Centrifuge the bacterial solution at 4000 rpm for 5 min, discard the supernatant, wash with 25 mL of sterile water, centrifuge at 4000 rpm for 5 min, discard the supernatant, and repeat.

[0120] (5) Centrifuge at 12,000 rpm for 1 min, discard the supernatant, resuspend in sterile water, mix thoroughly, and dispense 100 μL of each into 1.5 mL EP tubes according to the required number of transformations. Centrifuge at 12,000 rpm for 1 min, and discard the supernatant.

[0121] (6) Configure according to the following conversion system:

[0122] 1) Linearized plasmid transformation system: PEG3350 (50% w / V) 240 μL, LiAC (1.0 M) 36 μL, ssDNA (2.0 mg / mL) 50 μL, plasmid (linear) 5 μL.

[0123] 2) CRISPR / Cas9 transformation system: PEG3350 (50% w / V) 240 μL, LiAC (1.0 M) 36 μL, ssDNA (2.0 mg / mL) 50 μL, Cas9 plasmid (300 ng / μL) 10 μL, donor (500 ng / μL) 10 μL, gRNA (300 ng / μL) 10 μL.

[0124] (7) The above system was thoroughly shaken and mixed, and then placed in a 42°C water bath for 45 min.

[0125] (8) Centrifuge at 12,000 rpm for 1 min, discard the supernatant, add 1 mL of YPD liquid to resuspend and mix, wrap the EP tube with sealing film, and incubate in a shaker at 30°C and 220 rpm for 2 h.

[0126] (9) Remove the sealing film in a clean bench and centrifuge at 12,000 rpm for 1 min. Discard the supernatant and add 1 mL of sterile water to resuspend and wash. Repeat this process twice.

[0127] (10) Centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL of sterile water to suspend and mix, take 100 μL of the bacterial solution and apply it to the resistance or nutritional deficiency type plus resistance plate, and place it in a 30°C incubator for 2-3 days.

[0128] 3. Construction of strains CEA and CSE

[0129] The recombinant plasmid pUMRI-GAL1-7HA-EA was transformed into BY4742-C04 to obtain CEA.

[0130] The recombinant plasmid pUMRI-GAL1-7HA-SaBIS-ERG20 was transformed into BY4742-C04 to obtain CSE.

[0131] 4. Construction of strains YD01 and YDZ01

[0132] The recombinant plasmid pUMRI-GAL1-7HA-TRX1-EA was transformed into the mating type-complemented engineered yeast strains BY4741-C04 and BY4742-C04, so that the TRX1-ERG20-AgBIS expression cassette was integrated into the chromosomes of BY4742-C04 and BY4741-C04 to obtain YD01 and YDZ01;

[0133] The YD01 strain introduced an additional TRX1 solubility-promoting tag compared to the CEA strain. Its effect on the production of bisabolene is shown in Figure 5 .

[0134] 5. Construction of strain YDZ07

[0135] The CRISPR / Cas9 method was used to introduce ADH2 controlled by the GAL1 promoter and ALD6 controlled by the GAL10 promoter into the Int10 site of YDZ01 to construct YDZ02; the CRISPR / Cas9 method was used to introduce ACS2 controlled by the GAL10 promoter into the Int7 site of YDZ02 to construct YDZ03; the recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 was transformed into strain YDZ03, so that PHXT1 was integrated into the YD03 chromosome, and YDZ03 was obtained. DZ04; the TRX1-ERG20-AgBIS expression cassette controlled by the GAL1 promoter was introduced at the Int16 site of YDZ04 by the CRISPR / Cas9 method to construct YDZ05; the PDR1 controlled by the GAL1 promoter was introduced at the Int17 site of YDZ05 by the CRISPR / Cas9 method to construct YDZ06; the recombinant plasmid PUMRI-ROX1-SNQ2 was transformed into the strain YDZ06, so that SNQ2 was integrated into the YDZ04 chromosome to obtain YDZ07.

[0136] 6. Construction of strain YD02-11

[0137] The recombinant plasmid PUMRI-ROX1-PDR1 was transformed into strain YD01, so that PDR1 was integrated into the YD01 chromosome, and SNQ2 controlled by the GAL1 promoter was introduced at the Int17 site of YD01 by the CRISPR / Cas9 method to construct YD01-11; the recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 was transformed into strain YD01-11, and the TRX1-ERG20-AgBIS expression cassette controlled by the GAL1 promoter was introduced at the Int16 site of YD01-11 by the CRISPR / Cas9 method, so that it was integrated into the YD01-11 chromosome to obtain YD02-11.

[0138] 7. Construction of hybrid strain YDZ-C-α

[0139] The nutritional deficiency of URA3, HIS3, and LEU2 of YDZ07 and YD02-11 was supplemented to obtain YDZ08 and YD02-12. The method for supplementing the nutritional deficiency is: using the whole genome of Saccharomyces cerevisiae without defects as a template, PCR obtains a defective marker with homology arms of about 1000 bp before and after, and then ethanol precipitation is performed to obtain a marker fragment with a high concentration, and then chemical transformation is performed. In this experiment, the nutritional deficiency of URA3, HIS3, and LEU2 of BY4741 and BY4742 was supplemented, and the △met of BY4741 and the △lys of BY4742 were retained to facilitate the subsequent screening of successful hybrid strains through double deficiency.

[0140] YDZ08 and YD02-12 were hybridized to obtain YDZ-C-α. The construction idea is shown in Figure 6 The specific operation is to culture the haploid strains in 5 mL of YPD medium overnight, take 100 μL of each, mix them and culture them in 5 mL of YPD medium for 12-16 hours, take 1 mL of bacterial solution in a 1.5 mL sterile EP tube, centrifuge at 12000 rpm for 1 min, wash 3 times with 1 mL of sterile water, resuspend with 1 mL of sterile water, and streak on SD screening plates without additional amino acids.

[0141] 8. Construction of strain YDZSE

[0142] The SABIS-ERG20 gene controlled by the GAL1 promoter was introduced into the TRX1-ERG20-AgBIS locus of YDZ07 by CRISPR / Cas9 to construct YDZSE. The construction strategy is shown in Figure 7 .

[0143] 9. Construction of strains Ydαβ and Ydα3β

[0144] SABIS-ERG20 controlled by the GAL1 promoter was introduced into a TRX1-ERG20-AgBIS site (GAL1-7HA) of YD02-11 by CRISPR / Cas9 to construct Ydαβ. YDαβ and YDZSE were hybridized to obtain Ydα3β, see Figure 8 .

[0145] Example 4: Fermentation and cultivation of genetically engineered bacteria and extraction and gas phase analysis of bisabolene

[0146] 1. Pick a single colony from the streaked plate and place it in a 5 mL YPD test tube and culture it in a shaker at 30°C and 220 rpm for 16-24 hours. 600The concentration of 0.05 was transferred to a 50 mL YPD shake flask for culture. After 24 h, 10% dodecane was added for two-phase culture. The culture was maintained at 30°C or 24°C for 120 h.

[0147] 2. Transfer 50 mL of fermentation broth to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min. Transfer the upper oil phase to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 1 min. Take 200 μL of the upper oil phase and 50 μL of internal standard dibutyl phthalate and mix thoroughly. Filter the mixed system with a 0.22 μm organic filter into a new 1.5 mL centrifuge tube for GC detection.

[0148] 3. The conditions for detecting bisabolene in Saccharomyces cerevisiae by GC are as follows:

[0149] Fuli gas chromatograph, the equipment is equipped with hydrogen flame detector (FID) and HP-5 capillary column, using 0.1Mpa nitrogen as carrier gas, adjusting hydrogen to 0.1Mpa, air to 0.1Mpa. The injection port temperature is 250℃, and the detector temperature is 320℃. The heating program is shown in Table 2 below, and its peak time and gas phase detection are shown in Fig. 9 .

[0150] Table 2

[0151]

[0152] The results are as follows Fig.10 As shown in the figure, the α-bisabolene production of strains YDZ07, YD02-11, and YDZ-C-α were measured to be 923.97 mg / L, 1400.96 mg / L, and 1433.33 mg / L, respectively. Among them, strains YD02-11 and YDZ01 have different abilities to produce α-bisabolene due to their different auxotrophic types. The results are shown in the figure. Fig.11 shown.

[0153] YDZSE strain produces 1222.95 mg / L of β-bisabolene, see Fig.12 .

[0154] Example 5: High-density fed-batch fermentation of genetically engineered bacteria

[0155] 1. Fed-batch high-density fermentation of engineered strain YDZ-C-α

[0156] Pick a single colony of the diploid strain from the SD plate (add YNB without amino acids) and place it in a 5mL YPD tube. Culture it at 30℃, 220rpm for 12-16h, then streak it on a YPD solid plate to activate it. Pick a single colony again and inoculate it in a 5mL YPD tube and culture it at 30℃, 220rpm for 12h. Take 1% of the inoculum from the 5mL tube and transfer it to the secondary YPD seed solution (425mL YPD), and culture it at 30℃, 220rpm overnight until the OD 600 The inoculation volume is 8-10, and then the secondary seed liquid is connected to 1.8L fermentation medium with an inoculation volume of 10%. The fermentation tank pressure is controlled to 0.04MPa. During the fermentation process, the dissolved oxygen (DO) electrode and pH electrode are used to perform real-time online detection of dissolved oxygen and pH in the fermentation liquid, and are associated with the fermentation controller. The pH value after inoculation is set to 5.0, and ammonia water is added by gravity to adjust the pH and stabilize it at around 5.0. From inoculation to feeding, the dissolved oxygen is associated with the speed, and the DO is set to 40%, and the speed range is 200-600rpm. The initial ventilation volume is set to 1vvm, and it is set to 2vvm after the start of feeding. After feeding, if DO <20%, increase the speed by 50rpm, and the maximum speed is 600rpm. When the ethanol content in the fermentation broth is lower than 3g / L, start to supplement the fermentation feed concentrate, and dynamically feed it in conjunction with its DO. Add 10% v / v dodecane for extraction after about 24h. When the cells enter the stable phase, replace glucose with 75% w / w ethanol feed solution, and appropriately lower the upper limit of dynamic feeding-associated DO. Add dodecane according to the concentration of intracellular products. In the later stage of fermentation, the dissolved oxygen continues to rise, and the feed is adjusted to a constant rate feed and the feed rate is reduced. Take 9-10mL of fermentation broth each time, let it stand, take 200μL of oil phase and 50μL of internal standard dibutyl phthalate and mix them thoroughly. Filter the mixed system with a 0.22μm organic filter into a new 1.5mL centrifuge tube for GC detection. Take 1mL of non-oil phase fermentation broth and centrifuge it, then dilute it appropriately. Use the glucose analyzer from Silman Technology to detect glucose and ethanol according to the instructions. After high-density fermentation of the YDZ-C-α strain, the production of α-bisabolene reached 58.64g / L, see Fig.13 As can be seen from the figure, when the cells entered the stable phase at 48h of fermentation, glucose was replaced with 75% w / w ethanol feed solution, which downregulated the squalene synthesis pathway and made the substrate flow to the metabolic pathway of bisabolene, which was beneficial to the production of bisabolene.

[0157] 2. Shake flask fermentation and fed-batch high-density fermentation of engineered strain YDα3β

[0158] In order to test whether the one-pot synthesis of mixed configuration bisabolene can be achieved by simultaneously expressing α-bisabolene synthase and β-bisabolene synthase, the inventors tried to replace one copy of AgBIS in YD02-11 with SaBIS, and the strain was named YDαβ. It was cultured at 24°C and 220rpm for 5 days. The method was the same as in Example 4. The α-bisabolene production was 742.94 mg / L, the β-bisabolene production was 134.67 mg / L, and the β / α ratio was 18.13%. YDαβ and YDZSE were hybridized to obtain strain YDα3β containing 1 copy of α-bisabolene synthase gene and 3 copies of β-bisabolene synthase gene. It was cultured at 24°C and 220rpm for 5 days. The α-bisabolene production was 311.32 mg / L, the β-bisabolene production was 240.18 mg / L, and the β / α ratio was 77.14%.

[0159] Considering that the two enzymes have different optimal temperatures, α-bisabolene synthase is 30°C, and β-bisabolene synthase is 24°C. In this embodiment, the ratio of the two enzymes is adjusted by adjusting the temperature during the batch feeding high-density fermentation of YDα3β. After 112 hours of fermentation, the α-bisabolene production is 32.61 g / L, and the intracellular content is 0.86 g / L; the β-bisabolene production is 12.54 g / L, and the intracellular content is 1.89 g / L. The β / α-bisabolene ratio is 38.45%, see Fig.14 .

Claims

1. A genetically engineered bacterium with high yield of bisabolene, characterized in that: The genetically engineered bacteria uses a saccharomyces cerevisiae engineered strain that overexpresses the mevalonate pathway as a starting strain, and the promoter of squalene synthase in the genome is replaced with an inducible promoter; In addition, the genome is integrated with the coding sequences of the transcription factor PDR1 and the transporter SNQ2 of the pleiotropic drug resistance protein, as well as an α-bisabolene synthase fusion protein expression cassette and / or a β-bisabolene synthase fusion protein expression cassette, or a recombinant expression plasmid containing a protein for expressing the transcription factor PDR1 and the transporter SNQ2 and an α-bisabolene synthase fusion protein and / or a β-bisabolene synthase fusion protein is introduced, wherein the α-bisabolene synthase fusion protein is formed by fusion of farnesyl pyrophosphate synthase and α-bisabolene synthase via a connecting peptide, and the β-bisabolene synthase fusion protein is formed by fusion of β-bisabolene synthase and farnesyl pyrophosphate synthase via a connecting peptide.

2. The genetically engineered bacterium with high production of bisabolene according to claim 1, characterized in that: The amino acid sequence of α-bisabolene synthase is shown in SEQ ID NO.1, and the N-terminus of α-bisabolene synthase is fused to farnesyl pyrophosphate synthase via a connecting peptide; the amino acid sequence of β-bisabolene synthase is shown in SEQ ID NO.2, and the C-terminus of β-bisabolene synthase is fused to farnesyl pyrophosphate synthase via a connecting peptide; the amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID NO.3, and the amino acid sequence of the connecting peptide is GGGGS.

3. The genetically engineered bacterium with high production of bisabolene according to claim 1 or 2, characterized in that: A lytic tag TRX1 is introduced into the N-terminus of the fusion protein, and the amino acid sequence of the lytic tag TRX1 is shown in SEQ ID NO.

5.

4. The genetically engineered bacterium with high production of bisabolene according to claim 1, characterized in that: The genetically engineered bacteria contain 1-4 copies of an α-bisabolene synthase fusion protein expression cassette and / or 1-4 copies of a β-bisabolene synthase fusion protein expression cassette.

5. The genetically engineered bacterium with high production of bisabolene according to claim 1, characterized in that: The inducible promoter is a glucose-inducible promoter.

6. The genetically engineered bacterium for high production of bisabolene according to claim 1, characterized in that: The amino acid sequence of the transcription factor PDR1 is shown in SEQ ID NO.6, and the amino acid sequence of the transporter SNQ2 is shown in SEQ ID NO.7; the gene sequences encoding alcohol dehydrogenase, acetaldehyde dehydrogenase, and acetyl-CoA synthase are integrated into the genome of the genetically engineered bacteria, the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.8, the amino acid sequence of the acetaldehyde dehydrogenase is shown in SEQ ID NO.9, and the amino acid sequence of the acetyl-CoA synthase is shown in SEQ ID NO.

10.

7. The genetically engineered bacterium for high production of bisabolene according to claim 1, characterized in that: The genetically engineered bacteria are diploid strains obtained through hybridization.

8. The method for constructing a genetically engineered bacterium with high bisabolene production as claimed in claim 1, characterized in that: include: Using an engineered strain of Saccharomyces cerevisiae that overexpresses the mevalonate pathway as a starting strain, the promoter of squalene synthase is replaced with an inducible promoter by integrating a plasmid or CRISPR / Cas9 technology, and the gene sequences encoding the transcription factor PDR1 and the transporter protein SNQ2 and the α-bisabolene synthase fusion protein expression cassette and / or the β-bisabolene synthase fusion protein expression cassette are integrated into the genome of the engineered strain to obtain a genetically engineered strain with high production of bisabolene; Alternatively, an engineered strain of Saccharomyces cerevisiae that overexpresses the mevalonate pathway is used as the starting strain, and the promoter of squalene synthase is replaced with an inducible promoter through integration of plasmids or CRISPR / Cas9 technology, and a recombinant expression plasmid for expressing the transcription factor PDR1 and the transporter protein SNQ2 as well as α-bisabolene synthase fusion protein and / or β-bisabolene synthase fusion protein is introduced.

9. Use of the genetically engineered bacterium with high bisabolene production as claimed in any one of claims 1 to 7 in the preparation of bisabolene.

10. The use according to claim 9, characterized in that include: After the high-yield bisabolene genetically engineered bacteria were expanded and cultured, they were transferred to the secondary YPD seed solution at a 1% inoculum and cultured until OD 600 The fermentation medium is 8-10, and then transferred to the YPD fermentation medium according to a 10% inoculation amount, the fermentation tank pressure is controlled to be 0.04Mpa, the fermentation liquid dissolved oxygen DO is 40%±15%, and the pH value is 5.0±0.5; when the ethanol content in the fermentation liquid is lower than 3g / L, the fermentation feed concentrate is started; when the fermentation is 24h, 10% by volume of dodecane is added for extraction; when the cells enter the stable period, the glucose in the feed solution is replaced by 75% by mass ethanol solution.

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