A genetically engineered bacterium with high β-bisabolene yield, a construction method and application thereof

Abstract

This invention discloses a genetically engineered bacterium producing high levels of bisabolene, its construction method, and its applications, belonging to the field of genetic engineering technology. Using a *Saccharomyces cerevisiae* strain overexpressing the mevalonic acid pathway as the starting strain, this invention integrates a fusion protein expression cassette of farnesyl pyrophosphate synthase and bisabolene synthase, overexpresses endogenous transcription factors and transport proteins of *Saccharomyces cerevisiae*, and replaces the original squalene synthase promoter with an inducible promoter, thus constructing a genetically engineered bacterium producing high levels of bisabolene. This invention achieves heterologous bisabolene synthesis in *Saccharomyces cerevisiae* by introducing the bisabolene synthase gene, while simultaneously integrating rate-limiting enzyme fusion expression and transport protein overexpression to alleviate metabolic stress in *Saccharomyces cerevisiae* cells, downregulating competitive pathways to divert the substrate farnesyl pyrophosphate to the bisabolene metabolic pathway, thereby enhancing the strain's ability to produce bisabolene. This invention provides a method for achieving high bisabolene production, superior to existing technologies, and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a genetically engineered bacterium that produces high levels of bisabolene, its construction method, and its applications. Background Technology

[0002] Bisabolene, also known as bisabolene or sweet bisabolene, is a common natural sesquiterpene product found in plants. In plants, bisabolene acts as a chemical defense against insects, herbivores, and fungal pathogens. It has significant market potential as an important precursor in the production of green biofuels, food, flavorings, and pharmaceuticals.

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

[0004] Compared to traditional chemical and plant extraction methods, the microbial synthesis of bisabolene offers several advantages. First, bisabolene synthases are highly specific, enabling the production of high-purity, single-configuration bisabolene. Second, using microorganisms as a substrate in a mild reaction system makes it a more environmentally friendly method. Finally, many microbial substrates themselves generate bisabolene precursors through their metabolic pathways, making them highly advantageous for direct use as recombinant microorganisms in bisabolene production. Saccharomyces cerevisiae is a commonly used substrate organism, possessing advantages such as a clear genetic background, recognized safety, and mature high-density fermentation technology. Therefore, utilizing Saccharomyces cerevisiae as a host for bisabolene production holds great potential and promise.

[0005] Currently, the highest shake-flask yield of α-bisabolene from engineered strains of *Saccharomyces cerevisiae* is 866.7 mg / L (Enhancing glycerol metabolism to improve bisabolene yield in engineered yeast strains. *Chinese 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 using *Yarrowia lipolytica* is only 68.2 mg / L (High-efficiency production of bisabolene from waste cooking oil by metabolically engineered *Yarrowia lipolytica*. *Microb Biotechnol*, 2021, 14(6):2497-2513). These bisabolene yields are still far from industrial application.

[0006] Factors affecting the yield of bisabolene in engineered strains of *Saccharomyces cerevisiae* include the soluble expression of plant-derived bisabolene synthase, the supply of precursors, competitive consumption and efficient conversion of metabolic intermediates, and extracellular secretion of the product. 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 purpose of this invention is to provide a genetically engineered bacterium capable of producing high yields of bisabolene, which efficiently prepares α- and β-bisabolene separately or simultaneously through microbial heterologous synthesis.

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

[0009] This invention provides a genetically engineered bacterium that produces high levels of bisabolene. The bacterium originates from a *Saccharomyces cerevisiae* strain overexpressing the mevalonic acid pathway, with the squalene synthase ERG9 promoter in the genome replaced by an inducible promoter. Furthermore, the genome integrates coding sequences for the transcription factor PDR1 and the transporter SNQ2, which are multi-directional resistance proteins, as well as α-bisabolene synthase fusion protein expression cassettes and / or β-bisabolene synthase fusion protein expression cassettes. Alternatively, a recombinant expression plasmid containing PDR1 and SNQ2, as well as α-bisabolene synthase fusion proteins and / or β-bisabolene synthase fusion proteins, is introduced. The α-bisabolene synthase fusion protein is formed by the fusion of farnesyl pyrophosphate synthase and α-bisabolene synthase via a linker peptide, and the β-bisabolene synthase fusion protein is formed by the fusion of β-bisabolene synthase and farnesyl pyrophosphate synthase via a linker peptide.

[0010] This invention uses an engineered *Saccharomyces cerevisiae* strain overexpressing the mevalonate pathway as the starting strain. This strain can synthesize abundant isoprene pyrophosphate and dimethylpropenyl pyrophosphate through the mevalonate pathway, and synthesize the precursor of bisabolene, farnesyl pyrophosphate, via the endogenous farnesyl pyrophosphate synthase ERG20. Based on this, this invention introduces the gene encoding an α-bisabolene synthase fusion protein to express the fusion protein of ERG20 and α-bisabolene synthase, achieving heterologous synthesis of α-bisabolene. By replacing the α-bisabolene synthase expression cassette with a β-bisabolene synthase expression cassette, heterologous synthesis of β-bisabolene can be achieved. Combining the α-bisabolene and β-bisabolene synthase expression cassettes allows for the simultaneous synthesis of α- and β-bisabolene. Furthermore, to facilitate the metabolic pathway of converting the substrate farnesyl pyrophosphate to bisabolene, this invention replaces the original promoter of squalene synthase with an inducible promoter, thereby downregulating the squalene synthesis pathway. To alleviate the metabolic stress on yeast cells, this invention overexpresses the transcription factor PDR1 and the transporter protein SNQ2, which are endogenous multi-directional drug resistance proteins in Saccharomyces cerevisiae, thereby increasing the yield of bisabolene.

[0011] Preferably, the starting strains are BY4742-C04 and BY4741-C04, which overexpress the mevalonate pathway in their cytoplasm and are complementary crossbreeding engineered strains. These strains are publicly available materials, and their construction method is described in the reference (Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nat Commun, 2016, 7:12851).

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

[0013] This invention explores the effect of protein fusion expression sequence on bisabolene yield. The study found that fusion expression of farnesene pyrophosphate synthase ERG20 with two bisabolene synthases effectively promotes bisabolene synthesis; however, due to differences in protein structure, the preferred expression sequences differ. For α-bisabolene synthase AgBIS derived from fir trees, ERG20 fusion at its N-terminus is more effective; while for β-bisabolene synthase SaBIS derived from sandalwood, ERG20 fusion at its C-terminus is more effective.

[0014] Farnesyl pyrophosphate synthase and bisabolene synthase are linked by a flexible linker peptide, preferably having the amino acid sequence GGGGS.

[0015] The present invention can optimize codons and synthesize the coding sequence of the fusion protein according to the host cell's preferences.

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

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

[0018] To downregulate the competitive pathway of bisabolene, this invention replaces the original promoter of squalene synthase with an inducible promoter. In the absence of inducing factors, the squalene synthesis pathway cannot be initiated, allowing the substrate farnesyl pyrophosphate to flow into the bisabolene metabolic pathway. Preferably, the inducible promoter is a glucose-inducible promoter. The glucose-inducible promoter can be, but is not limited to, P... HXT1 .

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

[0020] To further increase bisabolene production, this invention overexpresses endogenous alcohol dehydrogenase ADH2, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase ACS2 in Saccharomyces cerevisiae, thereby enhancing the supply of acetyl-CoA in the cytoplasm and increasing 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 transport protein 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 sequences of the above-mentioned endogenous proteins of *Saccharomyces cerevisiae* through gene cloning.

[0022] Preferably, the genetically engineered bacteria are diploid strains obtained through hybridization. This invention can obtain diploid strains that produce high yields of α-bisabolene and β-bisabolene, or simultaneously high yields of both, through hybridization-complementary mating genetically engineered bacteria.

[0023] The present invention also provides a method for constructing the high-bisabolene-producing genetically engineered strain, the method comprising: using a Saccharomyces cerevisiae strain overexpressing the mevalonic acid pathway as the starting strain, replacing the promoter of squalene synthase with an inducible promoter by means of an integration plasmid or CRISPR / Cas9 technology, integrating the gene sequences encoding transcription factor PDR1 and transport protein SNQ2 and α-bisabolene synthase fusion protein expression cassette and / or β-bisabolene synthase fusion protein expression cassette into the genome of the engineered strain to obtain a high-bisabolene-producing genetically engineered strain;

[0024] Alternatively, using an engineered strain of *Saccharomyces cerevisiae* that overexpresses the mevalonic acid pathway as the starting strain, the promoter of squalene synthase can be replaced with an inducible promoter using an integrative plasmid or CRISPR / Cas9 technology. Recombinant expression plasmids for expressing transcription factor PDR1, transport protein SNQ2, α-bisabolene synthase fusion protein, and / or β-bisabolene synthase fusion protein can be introduced to obtain a genetically engineered strain that produces high levels of bisabolene.

[0025] This invention achieves the construction of a high-yield bisabolene genetically engineered strain by integrating strategies such as rate-limiting enzyme fusion expression, competitive pathway downregulation, and overexpression of endogenous transcription factors and transport proteins.

[0026] Preferably, the integrative plasmid is a pUMRI series assembly tool plasmid.

[0027] Furthermore, the present invention can further increase the yield of α-bisabolene by strategies such as enhancing the supply of the precursor acetyl-CoA, expressing multi-copy fusion protein expression cassettes, and strain hybridization.

[0028] Preferably, the method for constructing the high-bisabolene-producing genetically engineered bacterium includes the following steps:

[0029] (1) The encoding gene of the α-bisabolene synthase fusion protein ERG20-AgBIS was cloned into the pUMRI-GAL1-7HA P GAL1 With T CYC1 Between these steps, the recombinant plasmid pUMRI-GAL1-7HA-EA was obtained, and the gene encoding the lysis tag was then 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 endogenous multi-directional drug resistance proteins in Saccharomyces cerevisiae were cloned into the pUMRI-ROX1 P gene. GAL1 With T CYC1 Between these steps, 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 pUMRI-GAL1-7HA, respectively. GAL1 With T CYC1 and P GAL10 With T ADH1 Between these steps, the recombinant plasmid pUMRI-GAL1-7HA-ADH2-ALD6 was obtained;

[0032] (4) Cloning the acetyl-CoA synthase encoding gene into pUMRI-DPP1 P GAL1 With T CYC1 Between these steps, 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, which overexpresses the mevalonate pathway. The SNQ2 coding sequence and the TRX1-ERG20-AgBIS expression cassette were integrated into the genome using CRISPR / Cas9 technology, and then P… HXT1 By replacing the original promoter of squalene synthase ERG9, a genetically engineered bacterium producing high levels of α-bisabolene was obtained.

[0034] Alternatively, recombinant plasmids pUMRI-GAL1-7HA-TRX1-EA and pUMRI-ROX1-SNQ2 can be transformed into the engineered strain BY4741-C04, which overexpresses the mevalonate pathway. Using CRISPR / Cas9 technology, the coding sequences for ADH2-ALD6, ACS2, and PDR1, along with the TRX1-ERG20-AgBIS expression cassette, can be integrated into the genome, and then P... HXT1 By replacing the original promoter of squalene synthase ERG9, a genetically engineered bacterium producing high levels of α-bisabolene was obtained.

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

[0036] Preferably, the recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 is transformed into engineered bacteria, thereby replacing the original promoter of squalene synthase ERG9 with 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 diploid strains that produce high levels of α-bisabolene or β-bisabolene, or simultaneously high levels of both, through hybridization of complementary genetically engineered bacteria. By hybridizing different haploid strains, diploid strains producing α-bisabolene and β-bisabolene in different copy number ratios can be obtained.

[0039] Preferably, using the defect-free whole-genome Saccharomyces cerevisiae genome as a template, after obtaining the target band by PCR, the nutritional deficiencies of two hybrid strains, URA3, HIS3, and LEU2, are supplemented respectively. Through hybridization technology, the retained Δmet of BY4741 and Δlys of BY4742 can be mutually supplemented to obtain a nutritionally deficient fermentation strain. No additional amino acids need to be added to the fermentation medium, which can save fermentation costs.

[0040] This invention also provides the application of the genetically engineered strain that produces high levels of bisabolene in the preparation of bisabolene. The bisabolene is isolated from the fermentation product of the genetically engineered strain constructed according to this invention. The bisabolene is α-bisabolene, β-bisabolene, or a mixture of both.

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

[0042] 1) After the genetically engineered bacteria producing high bisabolene were cultured on a large scale, they were inoculated into YPD fermentation medium with an in-situ extractant added for two-phase culture. The extractant was dodecane, and the amount of extractant added was 5%-20%. The culture was carried out at 24-30℃ for 80-120h to obtain fermentation broth.

[0043] 2) Collect the organic phase in the fermentation broth and separate bisabolene.

[0044] This invention also provides a method for fed-batch high-density fermentation of genetically engineered bacteria, comprising: expanding the culture of the genetically engineered bacteria that produce high levels of bisabolene, then transferring it to a secondary YPD seed culture at a 1% inoculum, and culturing until OD... 600 The inoculum was 8-10, and then transferred to YPD fermentation medium at a 10% inoculum rate. The fermentation tank pressure was controlled at 0.04 MPa, the dissolved oxygen (DO) in the fermentation broth was 40% ± 15%, and the pH was 5.0 ± 0.5. When the ethanol content in the fermentation broth was lower than 3 g / L, the fermentation feed concentrate was added. After 24 hours of fermentation, 10% (v / v) of dodecane was added for product extraction. When the cells entered the stationary phase (about 48 hours of fermentation), the glucose in the feed concentrate was replaced with a 75% (w / v) ethanol solution.

[0045] The beneficial effects of this invention are as follows:

[0046] (1) This invention uses an engineered strain of *Saccharomyces cerevisiae* overexpressing the mevalonate pathway as the starting strain, integrates a fusion protein expression cassette of farnesyl pyrophosphate synthase and α-bisabolene synthase, overexpresses endogenous transcription factors and transport proteins of *Saccharomyces cerevisiae*, and replaces the original promoter of squalene synthase with an inducible promoter to construct a genetically engineered strain that produces high levels of bisabolene. This invention achieves heterologous synthesis of α-bisabolene in *Saccharomyces cerevisiae* by introducing the α-bisabolene synthase gene, while simultaneously integrating rate-limiting enzyme fusion expression and transport protein overexpression to alleviate metabolic stress in *Saccharomyces cerevisiae* cells, downregulating competitive pathways to divert the substrate farnesyl pyrophosphate to the bisabolene metabolic pathway, and enhancing the strain's ability to produce bisabolene.

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

[0048] (3) This invention provides a method for achieving high yield of bisabolene. The yield of bisabolene synthesized by the engineered strain constructed by this invention after two-phase fermentation in shake flask is better than that reported in the prior art and has good application prospects. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the pUMRI-GAL1-7HA-TRX1-EA plasmid structure.

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

[0051] Figure 3 This is a schematic diagram of the pUMRI-GAL1-7HA-SE plasmid structure.

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

[0053] Figure 5 The effect of fusion-enhancing labels on bisabolene production.

[0054] Figure 6 A schematic diagram illustrating the construction process of a diploid strain that produces high levels of bisabolene.

[0055] Figure 7 This is a schematic diagram of the replacement of the β-bisabolene synthesis expression cassette.

[0056] Figure 8 A schematic diagram illustrating the construction of a diploid strain capable of simultaneously producing α-bisabolene and β-bisabolene.

[0057] Figure 9 The GC spectra of β-bisabolene standard and α-bisabolene and β-bisabolene are shown.

[0058] Figure 10 The diagram shows the production output of YD02-11, YDZ07, and YDZ-C-α.

[0059] Figure 11 The yield of α-bisabolene from YDZ01 and the original YD01 strain.

[0060] Figure 12 The results of shake-flask culture of the high-yield β-bisabolene strain YDZSE.

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

[0062] Figure 14The results of fed-batch fermentation of a hybrid strain that simultaneously produces α- and β-bisabolene. Detailed Implementation

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

[0064] The strains BY4741-C04 and BY4742-C04, which overexpress the mevalonate pathway in the cytoplasm, and the plasmid pUMRI-GAL1-7HA were all previously constructed by our research group. The construction method is referenced in (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 also previously constructed by our research group. The construction method is referenced in (Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae, Metab Eng, 2015, 28:8-18).

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

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

[0067] In this embodiment, farnesene pyrophosphate synthase (ERG20) is fused to the N-terminus of α-bisabolene synthase (AgBIS) via a linker peptide (GGGGS). 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 aforementioned fusion protein were synthesized artificially. The gene sequence of farnesene pyrophosphate synthase ERG20 is available at accession number NM_001181600. The exogenous gene α-bisabolene synthase (AgBIS) is derived from Abiesgrandis, accession number AF006195.1. The coding sequence of the linker peptide is 5'-ggaggcggtgggtcc-3'.

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

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

[0071] The gene fragments of the above-mentioned fusion protein were artificially synthesized. The exogenous gene β-bisabolene synthase (SaBIS) was derived from Santalum album, gene accession number: KJ665778.1. Codon optimization of Saccharomyces cerevisiae was performed during synthesis, 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 h and centrifuge at 12000 rpm for 10 min at room temperature.

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

[0076] (3) Centrifuge at 12000 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 precipitate, and aspirate the supernatant into a new 1.5 mL centrifuge tube.

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

[0078] (5) Add 5 μL of RNase A and mix well. 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 aspirate 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 of Buffer HB, centrifuge at 10,000 rpm for 30 seconds, and discard the filtrate.

[0082] (9) Place the adsorption column back into the collection tube, add 700 μL DNA Wash Buffer, centrifuge at 10000 rpm for 30 s, and discard the filtrate.

[0083] (10) Repeat the previous step, run empty, and remove the residual DNA Wash Buffer.

[0084] (11) Place the adsorption column into a new 1.5 mL centrifuge tube, add 50-100 μL of Elution Buffer, let stand at 65 °C for 3-5 min, centrifuge at 12000 rpm at room temperature for 1 min, and collect the DNA solution and store at -20 °C.

[0085] 4. Cloning of transcription factor encoding genes (PDR1), transport protein encoding genes (SNQ2), alcohol dehydrogenase encoding genes (ADH2), aldehyde dehydrogenase encoding genes (ALD6), and acetyl-CoA synthase encoding genes (ACS2). Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed using the high-fidelity DNA polymerase KOD One™ PCR Master Mix.

[0086] The sequences of the transcription factor encoding gene PDR1 are shown in SEQ ID NO. 6 (Gen Registry No. NM_001180878.1); the sequence of the transporter protein encoding gene SNQ2 is shown in SEQ ID NO. 7 (Gen Registry No. NM_001180319.1); the sequence of the alcohol dehydrogenase encoding gene ADH2 is shown in SEQ ID NO. 8 (Gen Registry No. NM_001182812.1); the sequence of the acetaldehyde dehydrogenase encoding gene ALD6 is shown in SEQ ID NO. 9 (Gen Registry No. NM_001183875.1); and the sequence of the acetyl-CoA synthase encoding gene is shown in SEQ ID NO. 10 (Gen Registry No. NM_001182040.1). Primer designs are shown in Table 1 below.

[0087] Table 1. Primers used for the gene required for the biosynthesis of clonal bisabolene

[0088]

[0089]

[0090]

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

[0092] The PCR reaction system (50 μL) is 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 is as follows: (1) Pre-denaturation at 98℃ for 3 min; (2) Denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 68℃ for 1 kb / 10 s for 39 cycles; (3) Extension at 68℃ for 5 min; (4) Storage at 4℃ for 5 min.

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

[0095] 1. Enzyme digestion and gel recovery

[0096] The pUMRI series of integrative plasmids (pUMRI-LPP1, pUMRI-DPP1, pUMRI-GAL80, pUMRI-HO, pUMRI-GAL1-7HA, pUMRI-ROX1) were all preserved in our laboratory. Both the plasmids and the target fragments from the PCR products were double-digested with Takara restriction endonucleases according to the Takara restriction endonuclease instructions. After digestion, the DNA was recovered via DNA gel extraction, following the specific procedures outlined in the Axygen kit instructions.

[0097] 2. Enzyme-linked

[0098] The ligation was performed using T4 DNA ligase. 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] Connect in a 22℃ metal bath for 30-50 minutes.

[0100] 3. Transformation

[0101] Add 10 μL of the ligation product to the competent E. coli solution, place on ice for 15-25 min, heat shock at 42°C in a metal bath for 90 s, and then quickly place on ice for 3 min. Add 1 mL of LB liquid medium, and revive at 37°C in a shaker for 45 min. Then centrifuge at 12000 rpm for 1 min, discard 1 mL of supernatant, mix well, and take an appropriate amount to spread on the corresponding resistant LB plate. Incubate at 37°C for 12-16 h.

[0102] Specifically, the recombinant plasmid is constructed by enzyme digestion and ligation as follows:

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

[0104] The lysis-promoting 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 [link to relevant documentation]). Figure 1 and Figure 2 The sequence of the lysosomal 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-directional drug resistance proteins (PDR1, SNQ2) in Saccharomyces cerevisiae were cloned into the multiple cloning site after PGAL1 of pUMRI-ROX1 to obtain recombinant plasmids pUMRI-ROX1-PDR1 and pUMRI-ROX1-SNQ2.

[0106] The target fragments of alcohol dehydrogenase (ADH2) and aldehyde 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 synthase (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. (See [link to relevant documentation]) Figure 3 and Figure 4 .

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

[0110] 1. Gene integration via pUMRI system

[0111] Our research group developed the pUMRI system for gene integration in *Saccharomyces cerevisiae*. The pUMRI plasmid was linearized by Sfi I restriction enzyme digestion and transformed into competent *Saccharomyces cerevisiae* cells. Homologous arms on the plasmid underwent homologous recombination with the region of the genome to be replaced, achieving the integration of the target gene. Because the plasmid carries the KanMX (G418 resistance gene) and URA3 selection markers, positive clones can be screened using plates containing G418 or SD-URA plates.

[0112] SfiⅠ restriction enzyme system (20 μL): 17 μL plasmid, 2 μL 10×QuickCut Buffer, 1 μL SfiⅠ restriction enzyme. Restriction conditions: 50℃ water bath for 2 h.

[0113] 2. Use CRISPR / Cas9 integration tools

[0114] Using plasmid p426-SpSgH as a template, a 20bp gRNA was designed into primers. PCR was performed to obtain the full-length plasmid containing the gRNA. After template removal by digestion with DpnI at 37℃ for 2 hours, the plasmid was transformed into competent E. coli cells for transformation. The 40bp ends of the genome to be integrated were used as homologous arms of the donor to ensure integration efficiency. The expression cassette containing the homologous arms obtained by PCR was the donor required for chemical transformation.

[0115] Specifically, the construction steps of recombinant 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 at 30℃ and 220 rpm for 12-16 h in a constant temperature shaker. Then streak the YPD plate and incubate at 30℃ for 2 days.

[0117] (2) Pick a single colony from the plate and inoculate it into a 5mL YPD test tube. Incubate at 30℃ and 220rpm for 12-16h, then detect the OD. 600 The initial value was set to 0.05 and added to 50 mL of YPD liquid medium. The mixture was then incubated on a shaker at 220 rpm until the OD value reached [value missing]. 600 It is 1.5-2.0.

[0118] (3) Heat the pre-prepared ssDNA at 100℃ for 5 minutes and then quickly place it on ice to cool for later use.

[0119] (4) Centrifuge the bacterial culture 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 once.

[0120] (5) Centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend in sterile water, mix thoroughly, and then, according to the required amount of chemical conversion, take 100 μL and dispense into 1.5 mL EP tubes, centrifuge at 12000 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) After thoroughly shaking and mixing the above system, place it in a water bath at 42°C for 45 minutes.

[0125] (8) Centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL of YPD liquid to resuspend and mix well, wrap the EP tube with sealing film, and incubate in a shaker at 30℃ and 220 rpm for 2 h.

[0126] (9) After removing the sealing film in the ultra-clean workbench, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL of sterile water to resuspend and wash, and repeat twice.

[0127] (10) Centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL of sterile water to suspend and mix well, take 100 μL of bacterial solution and spread it on a plate containing resistance or auxotrophic type with resistance, and place it in a 30℃ 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 mating-complementary engineered yeast strains BY4741-C04 and BY4742-C04, which integrated the TRX1-ERG20-AgBIS expression cassette into the chromosomes of BY4742-C04 and BY4741-C04, resulting in YD01 and YDZ01.

[0133] The YD01 strain introduced an additional TRX1 solubilizing tag compared to the CEA strain, and its effect on bisabolene yield is shown in [reference needed]. Figure 5 .

[0134] 5. Construction of strain YDZ07

[0135] YDZ02 was constructed by introducing ADH2 (controlled by the GAL1 promoter) and ALD6 (controlled by the GAL10 promoter) at the Int10 site of YDZ01 using CRISPR / Cas9. YDZ03 was constructed by introducing ACS2 (controlled by the GAL10 promoter) at the Int7 site of YDZ02 using CRISPR / Cas9. The recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 was transformed into strain YDZ03, allowing PHXT1 to integrate into the YD03 chromosome, thus obtaining YDZ02. YDZ04; YDZ05 was constructed by introducing a TRX1-ERG20-AgBIS expression cassette controlled by the GAL1 promoter at the Int16 site of YDZ04 using CRISPR / Cas9; YDZ06 was constructed by introducing a PDR1 controlled by the GAL1 promoter at the Int17 site of YDZ05 using CRISPR / Cas9; and YDZ07 was constructed by transforming the recombinant plasmid PUMRI-ROX1-SNQ2 into the YDZ06 strain, which integrated SNQ2 into the YDZ04 chromosome.

[0136] 6. Construction of strain YD02-11

[0137] The recombinant plasmid PUMRI-ROX1-PDR1 was transformed into strain YD01, allowing PDR1 to integrate into the YD01 chromosome. SNQ2, controlled by the GAL1 promoter, was introduced at the Int17 site of YD01 using CRISPR / Cas9 to construct YD01-11. The recombinant plasmid PUMRI-12-HXT1-ERG9-TEF1 was then transformed into strain YD01-11. A TRX1-ERG20-AgBIS expression cassette, controlled by the GAL1 promoter, was introduced at the Int16 site of YD01-11 using CRISPR / Cas9, allowing it to integrate into the YD01-11 chromosome, resulting in YD02-11.

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

[0139] The auxotrophic types URA3, HIS3, and LEU2 of YDZ07 and YD02-11 were supplemented to obtain YDZ08 and YD02-12. The method for supplementing the auxotrophic types was as follows: using a non-auxotrophic whole-genome of *Saccharomyces cerevisiae* as a template, PCR was performed to obtain a defective marker with approximately 1000 bp homologous arms at the beginning and end, followed by alcohol precipitation to obtain a high-concentration marker fragment, which was then chemically transformed. In this experiment, the auxotrophic types URA3, HIS3, and LEU2 of BY4741 and BY4742 were supplemented, while the Δmet of BY4741 and the Δlys of BY4742 were retained to facilitate subsequent screening of successful hybrid strains through double-auxotrophic selection.

[0140] YDZ08 and YD02-12 were hybridized to obtain YDZ-C-α, the construction method of which is described in [link to documentation]. Figure 6 The specific procedure is as follows: haploid strains are cultured overnight in 5 mL of YPD medium. 100 μL of each strain is taken and mixed and cultured in 5 mL of YPD medium for 12-16 hours. 1 mL of bacterial solution is taken into a 1.5 mL sterile EP tube, centrifuged at 12000 rpm for 1 min, washed 3 times with 1 mL of sterile water, and then resuspended in 1 mL of sterile water. The solution is then streaked onto an SD selection plate without additional amino acids.

[0141] 8. Construction of strain YDZSE

[0142] YDZSE was constructed by introducing SABIS-ERG20, controlled by the GAL1 promoter, into the TRX1-ERG20-AgBIS site of YDZ07 using the CRISPR / Cas9 method. The construction approach is described in [link to documentation]. Figure 7 .

[0143] 9. Construction of strains Ydαβ and Ydα3β

[0144] Ydαβ was constructed by introducing a GAL1 promoter-controlled SABIS-ERG20 at a TRX1-ERG20-AgBIS site (GAL1-7HA) on YD02-11 using CRISPR / Cas9. YDαβ and YDZSE were then hybridized to obtain Ydα3β. (See [link to documentation]). Figure 8 .

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

[0146] 1. Pick a single colony from the streak plate and inoculate it into a 5mL YPD tube. Incubate at 30℃ and 220rpm in a shaker for 16-24 hours to allow its initial OD to develop. 600The culture medium was 0.05% transferred to a shake flask containing 50 mL YPD and cultured for 24 h. After 24 h, 10% dodecane was added for two-phase culture and cultured 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 the internal standard dibutyl phthalate and mix thoroughly. Filter the mixed system through a 0.22 μm organic filter into a new 1.5 mL centrifuge tube for GC detection.

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

[0149] The Fuli gas chromatograph is equipped with a flame ionization detector (FID) and an HP-5 capillary column. It uses nitrogen at 0.1 MPa as the carrier gas, with hydrogen and air adjusted to 0.1 MPa. The injection port temperature is 250℃, and the detector temperature is 320℃. The temperature program is shown in Table 2 below, and the peak times and gas chromatograph detection parameters are shown in [Table 2]. Figure 9 .

[0150] Table 2

[0151]

[0152] The results are as follows Figure 10 As shown, the α-bisabolene yields of strains YDZ07, YD02-11, and YDZ-C-α were 923.97 mg / L, 1400.96 mg / L, and 1433.33 mg / L, respectively. Among them, strains YD02-11 and YDZ01 showed differences in their α-bisabolene production capacity due to their different auxotrophic forms, as shown in the results. Figure 11 As shown.

[0153] The YDZSE strain produced β-bisabolene at a concentration of 1222.95 mg / L. (See [reference needed]) Figure 12 .

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

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

[0156] Pick a single colony of diploid strain from an SD plate (with added amino acid-free YNB) and incubate it in approximately 5 mL YPD tubes. Incubate at 30°C and 220 rpm for 12-16 h. Then, streak the colony onto a YPD solid plate for activation. Again, pick a single colony and inoculate it into a 5 mL YPD tube. Incubate at 30°C and 220 rpm for 12 h. Transfer 1% of the inoculum from the 5 mL tubes to a secondary YPD seed culture (425 mL YPD) and incubate overnight at 30°C and 220 rpm until OD (dose elapsed). 600 The initial inoculum was 8-10, and then 10% of the secondary seed culture was added to 1.8L of fermentation medium. The fermenter pressure was controlled at 0.04MPa. During fermentation, dissolved oxygen (DO) and pH electrodes were used to monitor the dissolved oxygen and pH in the fermentation broth in real time and linked to the fermentation controller. The pH was set to 5.0 after inoculation, and ammonia was added by gravity to adjust the pH and stabilize it at around 5.0. Before feeding, dissolved oxygen was linked to the rotation speed, with DO set at 40% and the rotation speed range at 200-600 rpm. The initial aeration rate was set to 1 vvm, and then to 2 vvm after feeding began. After feeding, if DO < 20%, the rotation speed was increased by 50 rpm, with a maximum speed of 600 rpm. When the ethanol content in the fermentation broth was below 3 g / L, the fermentation feed concentrate was added, and the feeding was dynamically linked to its DO content. After approximately 24 hours, 10% v / v dodecane was added for extraction. When the cells entered the stationary phase, glucose was replaced with 75% w / w ethanol as the feed solution, and the upper limit of dynamic feed-related DO was appropriately lowered. Dodecane was added according to the concentration of intracellular products. During the later stages of fermentation, dissolved oxygen continuously increased, so the feed rate was adjusted to constant and the feed rate was reduced. Each time, 9-10 mL of fermentation broth was taken, allowed to stand, and then 200 μL of the oil phase and 50 μL of the internal standard dibutyl phthalate were thoroughly mixed. The mixed system was filtered through a 0.22 μm organic filter into a new 1.5 mL centrifuge tube for GC analysis. 1 mL of the non-oil phase fermentation broth was centrifuged, appropriately diluted, and glucose and ethanol were analyzed using a Silman Technology glucose analyzer according to the instructions. After high-density fermentation of the YDZ-C-α strain, the yield of α-bisabolene reached 58.64 g / L. (See [link to relevant documentation]). Figure 13 As can be seen from the figure, when the cells enter the stationary phase after 48 hours of fermentation, replacing glucose with 75% w / w ethanol feed solution downregulates the squalene synthesis pathway, causing the substrate to flow into the bisabolene metabolic pathway, which is beneficial to the formation of bisabolene.

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

[0158] To test whether a one-pot synthesis of bisabolene with mixed configurations could be achieved by simultaneously expressing α-bisabolene synthase and β-bisabolene synthase, the inventors attempted to replace one copy of AgBIS in YD02-11 with SaBIS, naming the strain YDαβ. It was cultured at 24℃ and 220rpm for 5 days, using the same method as in Example 4. The α-bisabolene yield was measured to be 742.94 mg / L, and the β-bisabolene yield was 134.67 mg / L, with a β / α ratio of 18.13%. YDαβ was hybridized with YDZSE to obtain strain YDα3β, containing one copy of the α-bisabolene synthase gene and three copies of the β-bisabolene synthase gene. Cultured at 24℃ and 220rpm for 5 days, its α-bisabolene yield was measured to be 311.32 mg / L, and the β-bisabolene yield was 240.18 mg / L, with a β / α ratio of 77.14%.

[0159] Considering the different optimal temperatures of the two enzymes (α-bisabolene synthase at 30℃ and β-bisabolene synthase at 24℃), this example adjusts the ratio of the two enzymes by adjusting the temperature during fed-batch high-density fermentation of YDα3β. After 112 hours of fermentation, the yield of α-bisabolene was 32.61 g / L, with an intracellular content of 0.86 g / L; the yield of β-bisabolene was 12.54 g / L, with an intracellular content of 1.89 g / L, and the β / α-bisabolene ratio was 38.45%. (See [link to relevant documentation]). Figure 14 .

Claims

1. A genetically engineered bacterium that produces high levels of bisabolene, characterized in that, The genetically engineered bacteria used a Saccharomyces cerevisiae strain that overexpressed the mevalonic acid pathway as the starting strain, and the promoter of squalene synthase in the genome was replaced with an inducible promoter. Furthermore, the genome integrates the coding sequences of transcription factor PDR1 and transporter SNQ2, which are multi-directional drug resistance proteins, as well as ≥1 copy of α-bisabolene synthase fusion protein expression cassette and / or ≥1 copy of β-bisabolene synthase fusion protein expression cassette, or introduces a recombinant expression plasmid containing the transcription factor PDR1 and transporter SNQ2, as well as α-bisabolene synthase fusion protein and / or β-bisabolene synthase fusion protein; The amino acid sequence of the transcription factor PDR1 is shown in SEQ ID NO.6, and the amino acid sequence of the transport protein SNQ2 is shown in SEQ ID NO.

7. The α-bisabolene synthase fusion protein is formed by fusing the N-terminus of α-bisabolene synthase with farnesyl pyrophosphate synthase via a linker peptide, and the amino acid sequence of α-bisabolene synthase is shown in SEQ ID NO.1; the β-bisabolene synthase fusion protein is formed by fusing the C-terminus of β-bisabolene synthase with farnesyl pyrophosphate synthase via a linker peptide, and the amino acid sequence of β-bisabolene synthase is shown in SEQ ID NO.2; The fusion protein has a solubilizing tag TRX1 introduced at its N-terminus, and the amino acid sequence of the solubilizing tag TRX1 is shown in SEQ ID NO.

5.

2. The genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, The amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID NO.3, and the amino acid sequence of the linker peptide is GGGGS.

3. The genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, The genetically engineered bacteria contain 1-4 copies of the α-bisabolene synthase fusion protein expression cassette and / or 1-4 copies of the β-bisabolene synthase fusion protein expression cassette.

4. The genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, The inducible promoter is a glucose-inducible promoter.

5. The genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, The genome of the genetically engineered bacteria integrates gene sequences encoding alcohol dehydrogenase, aldehyde dehydrogenase, and acetyl-CoA synthase. The amino acid sequence of alcohol dehydrogenase is shown in SEQ ID NO.8, the amino acid sequence of aldehyde dehydrogenase is shown in SEQ ID NO.9, and the amino acid sequence of acetyl-CoA synthase is shown in SEQ ID NO.

10.

6. The genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, The genetically engineered bacteria are diploid strains obtained through hybridization.

7. The method for constructing a genetically engineered bacterium producing high levels of bisabolene as described in claim 1, characterized in that, include: Using an engineered strain of Saccharomyces cerevisiae overexpressing the mevalonic acid pathway as the starting strain, the promoter of squalene synthase was replaced with an inducible promoter by using an integrative plasmid or CRISPR / Cas9 technology. The gene sequences encoding transcription factor PDR1 and transport protein SNQ2, as well as α-bisabolene synthase fusion protein expression cassette and / or β-bisabolene synthase fusion protein expression cassette, were integrated into the genome of the engineered strain to obtain a genetically engineered strain that produces high levels of bisabolene. Alternatively, using an engineered strain of Saccharomyces cerevisiae overexpressing the mevalonic acid pathway as the starting strain, the promoter of squalene synthase can be replaced with an inducible promoter using an integrative plasmid or CRISPR / Cas9 technology, and a recombinant expression plasmid for expressing transcription factor PDR1, transport protein SNQ2, and α-bisabolene synthase fusion protein and / or β-bisabolene synthase fusion protein can be introduced.

8. The application of the genetically engineered bacterium that produces high levels of bisabolene as described in any one of claims 1-6 in the preparation of bisabolene.

9. The application as described in claim 8, characterized in that, include: After expanding the culture of the genetically engineered bacteria that produce high levels of bisabolene, it was transferred to a secondary YPD seed culture at a 1% inoculum and cultured until OD reached. 600 The inoculum was 8-10, and then transferred to YPD fermentation medium at a 10% inoculum rate. The fermentation tank pressure was controlled at 0.04 MPa, the dissolved oxygen (DO) in the fermentation broth was 40%±15%, and the pH was 5.0±0.

5. When the ethanol content in the fermentation broth was lower than 3 g / L, the fermentation feed concentrate was added. After 24 h of fermentation, 10% (v / v) of dodecane was added for extraction. When the cells entered the stationary phase, the glucose in the feed concentrate was replaced with a 75% (w / v) ethanol solution.

Images