Yarrowia lipolytica strain for producing tyrosol as well as construction method and application of Yarrowia lipolytica strain

Abstract

The invention relates to a yarrowia lipolytica strain for producing tyrosol as well as a construction method and application of the yarrowia lipolytica strain, and belongs to the technical field of genetic engineering. According to the present invention, ScADH6, ScARO10F138L and D218G genes are expressed in the yarrowia lipolytica W29 [delta] KU70; the shikimic acid feedback inhibition is relieved by expressing the Y1ARO3K225L gene, the Y1ARO4K2214 gene and the Y1AR07G139S gene; pmLAAD, EcTyrAM53I and A354V genes are introduced, so that the conversion from FAD to FADH2 is promoted, and the supply of NADH (Nicotinamide Adenine Dinucleotide Hormone) in cytoplasm is enhanced; a 4-hydroxyphenylpyruvate dioxygenase (HPD) gene is knocked out; and overexpressing Y1DHS1, Y1DHS2 and Y1DHS3 genes of a shikimic acid pathway to obtain the yarrowia lipolytica strain HY5 for producing tyrosol. The invention lays a foundation for industrial production of tyrosol, and has important social significance and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a Yarrowia lipolytica strain for producing tyrosol, a construction method and an application thereof. Background Art

[0002] Tyrosol is widely found in plants such as Rhodiola rosea, Ligustrum lucidum, and olives. Its mild properties and virtually no toxic side effects make it a powerful therapeutic food and dietary supplement, exhibiting diverse pharmacological activities such as antioxidant, anti-inflammatory, and neuroprotective properties. It is widely used in the pharmaceutical and food industries. It is also a key precursor for the synthesis of high-value chemicals such as hydroxytyrosol and salidroside.

[0003] Tyrosol production methods include plant extraction, chemical synthesis, and microbial fermentation. However, the low abundance of tyrosol from plant sources and the complex extraction process result in low production capacity and high costs. The chemical synthesis of tyrosol, typically using phenol and its derivatives, has low yields and is environmentally friendly. In contrast, microbial cell factories exhibit many qualities that favor the low-cost, eco-friendly, and sustainable production of hydroxytyrosol.

[0004] De novo tyrosol synthesis has been achieved in Escherichia coli and Saccharomyces cerevisiae, but yields are low, making industrial-scale production difficult. Yarrowia lipolytica, a typical non-conventional oleaginous yeast, possesses comprehensive genetic manipulation tools and holds great promise for industrial fermentation. Currently, there are no reports of Yarrowia lipolytica producing tyrosol using simple carbon sources such as glucose. Therefore, establishing de novo tyrosol synthesis in Yarrowia lipolytica has significant scientific and social benefits. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and to provide a Yarrowia lipolytica strain for producing tyrosol and a construction method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A first aspect of the present invention provides a method for constructing a Yarrowia lipolytica strain for producing tyrosol, comprising the following steps:

[0008] Step (1), expressing ScADH6 and ScARO10 genes in Yarrowia lipolytica to obtain engineered bacteria HY1;

[0009] The nucleotide sequence of ScADH6 is SEQ ID NO.1; the nucleotide sequence of ScARO10 is SEQ ID NO.2;

[0010] Step (2): for the engineered bacteria HY1, by expressing YlARO3 K225L、Y1ARO4 K2214 and Y1AR07 G139S Genetically relieve the feedback inhibition of shikimic acid to obtain the engineered bacterium HY2;

[0011] Step (3): Introduce PmLAAD and EcTyrA into engineered bacteria HY2 M53I,A354V Gene, and obtained the engineered bacteria HY3;

[0012] The nucleotide sequence of the PmLAAD is SEQ ID NO. 3; EcTyrA M53I,A354V The nucleotide sequence is SEQ ID NO.4;

[0013] Step (4), knocking out the 4-hydroxyphenylpyruvate dioxygenase HPD gene in the engineered bacteria HY3 to obtain the engineered bacteria HY4;

[0014] In step (5), the engineered bacteria HY4 is overexpressed with genes YlDHS1, YlDHS2 and YlDHS3 of the shikimate pathway to obtain the engineered bacteria HY5; the engineered bacteria HT1 to HT5 are all Yarrowia lipolytica strains that produce tyrosol.

[0015] Furthermore, the specific method of step (1) is:

[0016] Using the DNA fragment shown in SEQ ID No. 1 as a template, the primer pair YL-ScADH6-F and YL-Sc ADH6R were used to amplify the ScADH6 gene fragment;

[0017] The DNA fragment shown in SEQ ID No. 2 was used as a template and primers YL-ScARO10-F and YL-Sc ARO10-R were used to amplify the gene ScARO10. F138L,D218G fragment.

[0018] The linearized p-IntD1 vector backbone, fused bidirectional promoter fragment, gene ScADH6 fragment, and gene ScARO10 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P2-ScADH6-ScARO10;

[0019] The recombinant plasmid P2-ScADH6-ScARO10 was amplified using primer pair P2-F and P2-R to obtain the linearized fragment P2-ScADH6-ScARO10. F138L,D218G ; Linearized fragment P2-ScADH6-ScARO10 F138L,D218 G was integrated into Yarrowia lipolytica W29ΔKU70 to obtain the engineered strain HT1.

[0020] Furthermore, the specific method of step (2) is:

[0021] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO3-F and YL-ARO3-R were used to amplify the YLARO3 gene fragment;

[0022] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO4-F and YL-ARO4-R were used to amplify the YLARO4 gene fragment;

[0023] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO7-F and YL-ARO7-R were used to amplify the YLARO7 gene fragment.

[0024] The linearized p-IntC2 vector backbone, fused bidirectional promoter fragment, gene YLARO4 fragment, and gene YLARO7 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P3-YLARO4-YLARO7;

[0025] The recombinant plasmid P3-YLARO4-YLARO7 was amplified by two rounds of mutation using primer pair AR04-K221 LF, AR04-K221 LR and primer pair AR07-G 139S-F, AR07-G139S-R to obtain the recombinant plasmid P3-YLARO4 K221 L -YLARO7 G139S ;

[0026] The recombinant plasmid P3-YLARO4 was amplified using primer pair P3-F and P3-R. K221 L -YLARO7 G139S , and obtain the linearized fragment P3-YLARO4 K221L -YLARO7 G139S ;

[0027] The linearized p-IntE 1 vector backbone, fused unidirectional promoter fragment, and gene YLARO3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P4-YLARO3;

[0028] The recombinant plasmid P4-YLARO3 was mutated by primer pair ARO3-K225L-F and ARO3-K225L-R to obtain the recombinant plasmid P4-YLARO3. K225L ;

[0029] The recombinant plasmid P4-YLARO3 was amplified using primer pair P4-F and P4-R. K225L, and obtain the linearized fragment P4-YLARO3 K225L ;

[0030] Linearized fragment P3-YLARO4 K221 L -YLARO7 G139S and P4-YLARO3 K225L Integrate into engineered bacteria HT1 to obtain engineered bacteria HT2.

[0031] Furthermore, the specific method of step (3) is:

[0032] Using the DNA fragment shown in SEQ ID No. 3 as a template, the primer pair YL-LAAD-F and YL-LAAD-R were used to amplify the gene PmLAAD fragment;

[0033] The DNA fragment shown in SEQ ID No. 4 was used as a template and primers YL-EcTyrA-F and YL-EcT yrA-R were used to amplify the gene EcTyrA. M53I,A354V fragment.

[0034] The linearized p-IntC3 vector backbone, fusion bidirectional promoter fragment, gene PmLAAD fragment, gene EcTyrA M53I,A354V The fragments were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P5-LAAD-EcTyrA M53I,A354V ;

[0035] The recombinant plasmid P5-LAAD-EcTyrA was amplified using primer pair P5-F and P5-R M53I,A354V， Obtained linearized fragment P5-LAAD-EcTyrA M53I,A354V ;

[0036] Linearized fragment P5-LAAD-EcTyrA M53I,A354V Integrate into engineered bacteria HT2 to obtain engineered bacteria HT3.

[0037] Furthermore, the specific method of step (4) is:

[0038] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primers HPD-UP-F and HPD-UP-R were used to amplify the upstream homology arm fragment HPD-UP of the HPD gene.

[0039] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primers HPD-DN-F and HPD-DN-R were used to amplify the downstream homology arm fragment HPD-DN of the HPD gene.

[0040] Using the upstream homology arm fragment HPD-UP and the downstream homology arm fragment HPD-DN as templates, PCR amplification was performed with primer pair HPD-UP-F and HPD-DN-R to obtain the repaired fragment HPD-UP-DN.

[0041] The repaired fragment HPD-UP-DN was integrated into the engineered bacteria HT3 to obtain the engineered bacteria HT4.

[0042] Furthermore, the specific method of step (5) is:

[0043] The YL-DHS1 gene fragment was obtained by amplification using the Yarrowia lipolytica W29ΔKU70 genome as a template and primer pair YL-DHS1-F and YL-DHS1-R;

[0044] The YL-DHS2 gene fragment was obtained by amplification using the Yarrowia lipolytica W29ΔKU70 genome as a template and primer pair YL-DHS2-F and YL-DHS2-R;

[0045] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-DHS3-F and YL-DHS3-R were used to amplify the YLDHS3 gene fragment.

[0046] The linearized p-D17 backbone fragment, the fused bidirectional promoter fragment, the YLDHS1 gene fragment, and the YLDHS2 gene fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P6-YLDHS1-YLDHS2;

[0047] The recombinant plasmid P6-YLDHS1-YLDHS2 was amplified using primer pair P6-F and P6-R to obtain the linearized fragment P6-YLDHS1-YLDHS2.

[0048] The linearized p-IntE4 vector backbone, fused unidirectional promoter fragment, and gene YLDHS3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P7-YLDHS3;

[0049] The recombinant plasmid P7-YLDHS3 was amplified using primer pair P7-F and P7-R to obtain the linearized fragment P7-YL DHS3;

[0050] The linearized fragments P6-YLDHS1-YLDHS2 and the linearized fragment P7-YLDHS3 were integrated into the engineered bacteria HT4 to obtain the engineered bacteria HT5.

[0051] The second aspect of the present invention provides a method for constructing the above-mentioned Yarrowia lipolytica strain that produces tyrosol, to obtain the Yarrowia lipolytica strain that produces tyrosol.

[0052] The third aspect of the present invention provides the use of the tyrosol-producing Yarrowia lipolytica strain in the production of tyrosol.

[0053] A fourth aspect of the present invention provides a method for producing tyrosol, comprising fermenting the tyrosol-producing Yarrowia lipolytica strain according to claim 8 in YNB medium to obtain tyrosol.

[0054] When producing tyrosol, the tyrosol-producing Yarrowia lipolytica strain is preferably inoculated into YPD medium and cultured for 12 hours, then transferred to YNB medium, and fermented at 30° C. and 220 rpm in a shaker for 96 hours to produce tyrosol.

[0055] The present invention provides an engineered strain of Yarrowia lipolytica that produces high hydroxytyrosol production, as well as its construction method and application. The strain is constructed based on the Yarrowia lipolytica strain W29ΔKU70, a wild-type W29 strain with the gene encoding the Cas9 protein integrated into its KU70 locus. This strain is described in the paper "Production of the antidepressant orcinolglucoside in Yarr owia Lipolytica with yields over 6,400-fold higher than plant extraction."

[0056] First, pyruvate decarboxylase (ScARO10) and alcohol dehydrogenase (ScADH6) from Saccharomyces cerevisiae were integrated into the Yarrowia lipolytica genome for expression. The nucleotide sequence of ScADH6 is SEQ ID NO. 1; the nucleotide sequence of ScARO10 is SEQ ID NO. 2.

[0057] Secondly, the mutants of Yarrowia lipolytica endogenous Y1ARO3, Y1ARO4 and Y1AR07 genes that relieve shikimate feedback inhibition were integrated and expressed. K225L 、Y1ARO4 K2214 and Y1AR07 G139S The GenBank number of the Y1ARO3 gene is XM_501119.3; the GenBank number of the Y1ARO4 gene is XM_501538.3; and the GenBank number of the Y1AR07 gene is XM_504065.2.

[0058] Next, the L-amino acid deaminase (PmLA AD) from Proteus mirabilis was integrated to promote the conversion of FAD to FADH2 and EcTyrA from Escherichia coli was expressed. M53I,A354 V enhances the supply of NADH in the cytoplasm. The nucleotide sequence of the PmLAAD is SEQ ID NO.3; EcTyrA M53I,A354V The nucleotide sequence is SEQ ID NO.4.

[0059] Thirdly, the endogenous 4-hydroxyphenylpyruvate dioxygenase encoding HPD gene of Yarrowia lipolytica was knocked out. The GenBank number of the HPD gene is XM_501197.3.

[0060] Finally, overexpression of the YIDHS1, YIDHS2, and YIDHS3 genes involved in the shikimate pathway increased precursor flux. The YIDHS1 gene (GenBank ID: YALI0B20020g), the YIDHS2 gene (GenBank ID: YALI0B22440g), and the YIDHS3 gene (GenBank ID: YALI0C06952g) further boosted tyrosol production.

[0061] In this study, we established an efficient artificial pathway for tyrosol synthesis in Yarrowia lipolytica. First, we integrated pyruvate decarboxylase (ScARO10) and alcohol dehydrogenase (ScADH6) from Saccharomyces cerevisiae into the Yarrowia lipolytica genome to construct a hydroxytyrosol biosynthesis pathway. Second, we integrated and expressed the endogenous YlARO3 enzyme, which relieves shikimate feedback inhibition. K225L 、Y1ARO4 K2214 and Y1AR07 G139S Then, the L-amino acid deaminase (PmLAAD) from Proteus mirabilis was integrated to promote the conversion of FAD to FADH2 and EcTyrA from Escherichia coli was expressed. M53I,A354V Enhance the supply of NADH in the cytoplasm. Again, the endogenous 4-hydroxyphenylpyruvate dioxygenase encoding HPD gene of Yarrowia lipolytica was knocked out to weaken the branching pathway. Then, the YlDHS1, YlDHS2 and YlDHS3 genes of the shikimate pathway were overexpressed to increase the precursor flux and further improve the production of hydroxytyrosol.Figure 1 shown.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention uses Yarrowia lipolytica W29 as a host and realizes the de novo synthesis of tyrosol by expressing pyruvate decarboxylase (ScARO10) and alcohol dehydrogenase (ScADH6) from Saccharomyces cerevisiae. M53I,A354V By enhancing cofactor supply and knocking out the HPD bypass pathway, Yarrowia lipolytica achieved efficient tyrosol synthesis, ultimately achieving a shake flask yield of 1.5g / L. This invention integrates plant-derived metabolic pathways into Yarrowia lipolytica to achieve high tyrosol production, laying the foundation for subsequent industrial expansion of fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the tyrosol biosynthesis pathway;

[0065] Figure 2 This is the YNB medium shake flask fermentation yield of engineered bacteria HT1-HT7;

[0066] Figure 3 This is a graph showing the tyrosol production by fed-batch fermentation of engineered bacteria HY5 in a 1.3 L fermenter. DETAILED DESCRIPTION

[0067] The present invention is described in further detail below with reference to the embodiments.

[0068] Those skilled in the art will understand that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Materials or equipment used without manufacturer identification are commercially available conventional products.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, generally according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0070] (1) Culture medium

[0071] LB medium: 5 g / L yeast extract powder, 10 g / L peptone, 10 g / L sodium chloride, solvent is deionized water; preparation: dissolve each component in deionized water, stir to dissolve, and sterilize.

[0072] LB solid medium (LB solid plate): Add 15 g / L agar powder to LB medium and sterilize.

[0073] YNB culture medium: 20 g / L glucose, 6.7 g / L YNB, 5 g / L ammonium sulfate, solvent is deionized water; preparation: dissolve each component in deionized water, stir to dissolve, and sterilize.

[0074] YPD culture medium: 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, solvent is deionized water; preparation: dissolve each component in deionized water, stir to dissolve, and sterilize.

[0075] YPD solid medium (YPD solid plate): Add 15 g / L agar powder to YPD medium and sterilize it.

[0076] MM medium: 400 mL of initial medium [containing ammonium sulfate (5 g / L), potassium dihydrogen phosphate (3 g / L), magnesium sulfate (0.5 g / L), and glucose (40 g / L)], 2 mL of trace metal solution, and 1 mL of vitamin solution.

[0077] Trace metal solution (1 L): 4.5 g calcium chloride dihydrate, 4.5 g zinc sulfate heptahydrate, 3 g ferric sulfate heptahydrate, 1 g boric acid, 1 g manganese chloride tetrahydrate, 0.4 g sodium molybdate dihydrate, 0.3 g cobalt chloride hexahydrate, 0.1 g copper sulfate pentahydrate, 0.1 g potassium iodide, 15 g ethylenediaminetetraacetic acid. Prepare the trace metal solution by dissolving all ingredients except ethylenediaminetetraacetic acid in 900 mL ultrapure water (pH = 6). Gently heat the solution to approximately 50°C and add ethylenediaminetetraacetic acid. Finally, adjust the pH to 4 with hydrochloric acid, bring the solution volume to 1 L with ultrapure water, and autoclave (121°C, 20 min). Store the solution at 4°C until ready for use.

[0078] Vitamin solution (1 L): 50 mg biotin, 200 mg para-aminophenylpropionic acid, 1 g niacin, 1 g calcium pantothenate, 1 g vitamin B6, 1 g vitamin B1, 25 g inositol. Dissolve the biotin in 20 mL 0.1 M NaOH, then dissolve all components in 900 mL ultrapure water (pH 6). Adjust the pH to 6.5 with hydrochloric acid, and bring the final volume to 1 L with ultrapure water. Sterile filter and store at 4°C until ready for use.

[0079] MM feed medium (1 L): glucose (600 g / L), ammonium sulfate (50 g / L), potassium dihydrogen phosphate (30 g / L), magnesium sulfate (5 g / L), 20 mL vitamin solution, 10 mL trace metal solution.

[0080] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0081] (2) To construct the integrated strain, a single gRNA vector and homologous donor plasmid for gene knock-in were constructed using the CRISPR / Cas9 system.

[0082] The original gRNA knockout plasmid vector, pCfB6627, was purchased from Addgene (https: / / www.addgene.org / ) with catalog number #106159. Knockout vectors were constructed using the original gRNA plasmid pCfB6627 targeting different integration sites (IntC-2, IntC-3, IntD-1, IntE-1, IntE-3, IntE-4, D17, and HPD). For detailed construction of the knockout vector, see Step 1 of Example 1.

[0083] The homologous donor plasmid, i.e., the integration vector, was constructed with pMD-19T as the backbone to form integration vectors with different integration sites (P-IntC2, P-IntC3, P-IntD1, P-IntE1, P-IntE3, P-IntE4, P-D17). The integration site sequences were referenced to the literature (Holkenbrink C, Dam MI, Kildegaard KR, et al. EasyCloneYALI: CRISPR / Cas9-based synthetic toolbox for engineering of the yellow Yarrowia lipolytica[J]. Biotechnology Journal, 2018, 13(9): 1700543.). The specific construction method of the integration vector is shown in step 2 of Example 1.

[0084] (3) Yarrowia lipolytica genome integration method

[0085] 1. Prepare a YPD solid plate (i.e., YPD solid medium) in advance, streak the Yarrowia lipolytica glycerol strain on the YPD solid plate, and place it in a 30°C constant temperature incubator for 16-24 hours to allow the strain to recover.

[0086] 2. For each strain to be transformed, prepare a 1.5 mL centrifuge tube containing 1 mL of sterile water. Use an inoculation needle to scrape the cells from the plate and transfer them to a 1.5 mL centrifuge tube containing 1 mL of sterile water. Slowly pipette up and down to resuspend the cells to obtain a suspension. One transformation reaction uses 5*10 7 cells (equivalent to OD600 of 5).

[0087] 3. Centrifuge the suspension at 3000 rpm for 5 min and remove the supernatant.

[0088] 4. Resuspend the cells in 1 mL of sterile water to wash the cells, centrifuge the suspension at 3000 rpm for 5 min, and remove the supernatant; repeat the wash twice.

[0089] 5. Discard the supernatant and use the cells for competent yeast transformation.

[0090] 6. Transfer 600 ng of each linearized fragment and the corresponding knockout plasmid gRNA into the yeast competent cells. The transformation system is shown in Table 1. After addition, gently pipette to mix.

[0091] Table 1 Yarrowia lipolytica transformation system

[0092] Required solution For 1X conversion buffer PEG (initial concentration 50%; sterile filtered; final concentration 43.8%) 87.5μL LiAc (initial concentration 2 M; pH 6.0; sterile filtration; final concentration 0.1 M) 5.0μL ssDNA (initial concentration 10 mg / mL; final concentration 0.25 g / L) 5.0μL DTT (initial concentration 2 M; sterile filtered; final concentration 100 mM) 5.0μL Linearized fragment 600ng Knockout plasmid gRNA 600ng

[0093] 7. Place the mixed transformation system in a 39°C metal bath and incubate for 60 minutes.

[0094] 8. After taking out, centrifuge at 3000 rpm for 5 minutes to centrifuge the cells.

[0095] 9. Remove the supernatant and add 500 μL of YPD medium. Gently pipette to resuspend the yeast cells in the YPD medium and incubate at 30°C and 220 rpm for 2 h.

[0096] 10. Take out the tube, centrifuge at 3000 rpm for 5 min, and remove the supernatant.

[0097] 11. Add 100 μL of sterile water and spread the cells on YPD solid plates containing hygromycin B (400 mg / L) and nourseothricin (250 mg / L) to screen for positive transformants.

[0098] 12. Re-inoculate the positive transformants into 3 mL of new YPD medium and culture for 1 day. Spread them on YPD solid plates to obtain more successful gRNA marker deletion for subsequent strain transformation.

[0099] (3) HPLC detection conditions

[0100] Quantification was performed using a Shimadzu high-performance liquid chromatography (HPLC). HPLC conditions included a SHIMSEN SuperbIIC18 column (5 μm, 4.6 mm × 250 mm), a column temperature of 30°C, a flow rate of 1 mL / min, a detection wavelength of 274 nm, and an injection volume of 10 μL. Hydroxytyrosol was analyzed and quantified. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile. The HPLC elution program was: 10% to 40% B (0-5 min), 40% to 60% B (5-8 min), 60% to 100% B (8-13 min), 100% to 100% B (13-15 min), 100% to 10% B (15-20 min), and 10% to 10% B (20-25 min). During gradient elution, linear gradient transformation was used.

[0101] The primer sequences involved in the following examples are shown in Table 2. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0102] Table 2 Primer sequences of the present invention

[0103]

[0104]

[0105]

[0106]

[0107] In this invention, all the Gibson assembly methods involved are assembled using US EVERBRIG HT INC.'s seamless cloning kit was used for Gibson assembly, and the corresponding system preparation and assembly were performed according to the instructions in the manual.

[0108] In the present invention, all involved amplifications, the amplification system is Phanta Max Master Mix polymerase 25 μL; upstream primer (10 μM) 2 μL; downstream primer (10 μM) 2 μL; template 2 μL; dd H2O 19 μL.

[0109] The amplification procedures are shown in Table 3.

[0110] Example 1: Construction of integrated plasmid vector

[0111] 1. Construction of knockout plasmid

[0112] (I) Construction of linearized fragments

[0113] Using the gRNA plasmid pCfB6627 as a template, perform the following operations to obtain the corresponding linearized fragments:

[0114] (1) Amplify the linearized fragment of G-IntE3 using the primer pair IntE3-F and IntE3-R;

[0115] (2) Amplification of the linearized fragment of G-IntD1 was performed using primer pair IntD1-F and IntD1-R;

[0116] (3) Amplify the linearized fragment of G-IntC2 using primer pair IntC2-F and IntC2-R;

[0117] (4) Amplify the linearized fragment of G-IntE1 using primer pair IntE1-F and IntE1-R;

[0118] (5) Amplify the linearized fragment of G-IntC3 using primer pair IntC3-F and IntC3-R;

[0119] (6) Amplify the linearized fragment of G-HPD using primers G-HPD-F and G-HPD-R;

[0120] (7) Amplify the linearized fragment of G-D17 using primer pair D17-F and D17-R;

[0121] (8) Amplify the linearized fragment of G-IntE4 using primers IntE4-F and IntE4-FR

[0122] The PCR enzyme used in the amplification was 2× Phanta Max Master Mix polymerase from Nanjing Novozymes Biotechnology Co., Ltd. The PCR amplification system was as follows: 25 μL of Phanta Max Master Mix polymerase; 2 μL of upstream primer (10 μM); 2 μL of downstream primer (10 μM); 2 μL of template; and 19 μL of dd H2O.

[0123] The PCR amplification program is shown in Table 3.

[0124] Table 3

[0125]

[0126] (II) Construction of knockout plasmid gRNA

[0127] (1) Construction of knockout plasmid gRNA G-IntE3: The linearized fragment PCR product of G-IntE3 amplified was treated with SpeedyCut DpnI enzyme, and then transformed into DH5α competent medium. The plasmid was screened on LB solid plates containing 100 mg / L ampicillin resistance (, and the plasmid was extracted and sequenced using primers PgRN A-JC-F to obtain the correct knockout plasmid gRNA G-IntE3;

[0128] Similarly, for the construction of knockout plasmid gRNA G-IntC2, knockout plasmid gRNA G-IntC3, knockout plasmid gRNA G-IntD1, knockout plasmid gRNA G-IntE1, knockout plasmid gRNA G-HPD, knockout plasmid gRNA G-IntE4, and knockout plasmid gRNA G-D17: the corresponding linearized fragment PCR product was used, treated with SpeedyCut DpnI quick digester, and transformed into DH5α competent state, screened by LB solid plate containing 100 mg / L ampicillin resistance, extracted plasmids, and sequenced and verified using primers PgRNA-JC-F to obtain the correct knockout plasmid gRNA G-IntC2, knockout plasmid gRNA G-IntC3, knockout plasmid gRNA G-IntD1, knockout plasmid gRNA G-IntE1, knockout plasmid gRNA G-HPD, knockout plasmid gRNA G-IntE4, knockout plasmid gRNA G-D17.

[0129] The SpeedyCut DpnI enzyme reaction system is shown in Table 4.

[0130] Table 4

[0131]

[0132] 2. Construction of homologous donor vector

[0133] (1) First, the vector pMD-19T was linearized and amplified using the primer pair p-T1-F and p-T1-R to obtain the vector backbone fragment;

[0134] (2) Then, using the Yarrowia lipolytica W29ΔKU70 genome as a template, the following operations were performed to obtain the corresponding upstream and downstream homology arms or fragments:

[0135] (1) The upstream and downstream homology arms of the integration site IntE3 were amplified using primer pairs IntE3-UP-F and IntE3-UP-R, and primer pairs IntE3-DN-F and IntE3-DN-R, respectively;

[0136] (2) The upstream and downstream homology arms of the integration site IntD1 were amplified using primer pairs IntD1-UP-F and IntD1-UP-R and primer pairs IntD1-DN-F and IntD1-DN-R, respectively;

[0137] (3) The upstream and downstream homology arms of the integration site IntC2 were amplified using primer pairs IntC2-UP-F and IntC2-UP-R and primer pairs IntC2-DN-F and IntC2-DN-R, respectively;

[0138] (4) Using primer pairs IntE1-UP-F and IntE1-UP-R and primer pairs IntE1-DN-F and IntE1-DN-R, respectively, the upstream and downstream homology arms of the integration site IntE1 were amplified;

[0139] (5) Using primer pairs IntC3-UP-F, IntC3-UP-R and primer pairs IntC3-DN-F, IntC3-DN-R, respectively, the upstream and downstream homology arms of the integration site IntC3 were amplified;

[0140] (6) Use primer pairs D17-UP-F and D17-UP-R and primer pairs D17-DN-F and D17-DN-R to amplify the upstream and downstream homology arms of integration site D17 respectively;

[0141] (7) Using primer pairs IntE4-UP-F, IntE4-UP-R and primer pairs IntE4-DN-F, IntE4-DN-R, respectively, the upstream and downstream homology arms of the integration site IntE4 were amplified;

[0142] (8) Amplify the terminator tPEX20 fragment with homology arms using primer pair p1-tPEX20-F and p1-tPEX20-R;

[0143] (9) The terminator tLIP2 fragment with homology arms was amplified using primer pair p1-tlip2-F and p1-tlip2-R;

[0144] (3) Vector construction

[0145] (1) Construction of vector:

[0146] The vector pMD-19T was linearized to obtain the vector backbone fragment, and the upstream and downstream homology arms of the integration site IntE3, the terminator tPEX20 fragment with homology arms, and the terminator tLIP2 fragment with homology arms were assembled by the Gibson assembly method to obtain the P-IntE3 vector.

[0147] Similarly, for the construction of P-IntC2 vector, P-IntC3 vector, P-IntD1 vector, P-IntE1 vector, P-IntE4 vector, and P-D17 vector: the vector pMD-19T was linearized to obtain the vector backbone fragment, and the upstream and downstream homology arms of the integration corresponding sites obtained above, the terminator tPEX20 fragment with homology arms, and the terminator tLIP2 fragment with homology arms were assembled by the Gibson assembly method to obtain P-IntC2 vector, P-IntC3 vector, P-IntD1 vector, P-IntE1 vector, P-IntE4 vector, and P-D17 vector.

[0148] When assembling by Gibson assembly, use US EVERBRIGHT INC.'s seamless cloning kit was used for Gibson assembly, and the corresponding system preparation and assembly were performed according to the instructions in the manual.

[0149] Example 2: Construction of a high-yielding Yarrowia lipolytica strain

[0150] First, the Yarrowia lipolytica W29ΔKU70 genome was used as a template, and the primer pairs pGPD-F and pGPD-R and pTEFIN-F and pTEFIN-R were used to amplify the strong endogenous promoters GPD and TEFIN of Yarrowia lipolytica, respectively;

[0151] At the same time, using promoters GPD and TEFIN as templates, the primer pair pGPD-F and pTEFIN-R were used to amplify the fusion bidirectional promoter fragment common to both genes;

[0152] At the same time, using promoters GPD and TEFIN as templates, the fusion unidirectional promoter fragment was amplified using primer pair p2-tpex20-pGPD and pTEFI NR.

[0153] The p-IntE3 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntE3 vector backbone;

[0154] The p-IntC2 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntC2 vector backbone;

[0155] The p-IntC3 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntC3 vector backbone;

[0156] The p-IntD1 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntD1 vector backbone;

[0157] The p-IntE1 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntE1 vector backbone;

[0158] The p-IntE4 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntE4 vector backbone;

[0159] The p-IntD17 vector was PCR amplified using primer pairs p-T1-tPEX20-F and p-T1-tLIP2-F to obtain the linearized p-IntD17 vector backbone;

[0160] 2. Using the DNA fragment shown in SEQ ID No. 1 as a template, amplify with primer pair YL-ScADH6-F and YL-ScADH6R to obtain the ScADH6 gene fragment;

[0161] The DNA fragment shown in SEQ ID No. 2 was used as a template and primers YL-ScARO10-F and YL-Sc ARO10-R were used to amplify the gene ScARO10. F138L,D218G fragment.

[0162] The linearized p-IntD1 vector backbone, fused bidirectional promoter fragment, gene ScADH6 fragment, and gene ScARO10 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells. The positive recombinant plasmid P2-ScADH6-ScARO10 was obtained by screening on LB solid plates containing 100 mg / L ampicillin and sequencing to verify its correctness.

[0163] The recombinant plasmid P2-ScADH6-ScARO10 was amplified using primer pair P2-F and P2-R to obtain the linearized fragment P2-ScADH6-ScARO10. F138L,D218G ; Linearized fragment P2-ScADH6-ScARO10 F138L,D218 G was integrated into Yarrowia lipolytica W29ΔKU70 (for specific methods, see the Yarrowia lipolytica genome integration method above) to obtain the engineered strain HT1.

[0164] Third, the Yarrowia lipolytica W29ΔKU70 genome was used as a template and primers YL-ARO3-F and YL-ARO3-R were used to amplify the YLARO3 gene fragment;

[0165] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO4-F and YL-AR O4-R were used to amplify the YLARO4 gene fragment;

[0166] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO7-F and YL-AR O7-R were used to amplify the YLARO7 gene fragment.

[0167] The linearized p-IntC2 vector backbone, fused bidirectional promoter fragment, gene YLARO4 fragment, and gene YLARO7 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells. The positive recombinant plasmid P3-YLARO4-YLARO7 was obtained after screening on LB solid plates containing 100 mg / L ampicillin and sequencing verification.

[0168] The recombinant plasmid P3-YLARO4-YLARO7 was amplified by two rounds of mutation using primer pair AR04-K221L-F, AR04-K221L-R and primer pair AR07-G139S-F, AR 07-G139S-R to obtain the recombinant plasmid P3-YLARO4 K221L -YLARO7 G139S ;

[0169] The recombinant plasmid P3-YLARO4 was amplified using primer pair P3-F and P3-R. K221L -YLARO7 G139S , and obtain the linearized fragment P3-YLARO4 K221L -YLARO7 G139S ;

[0170] The linearized p-IntE1 vector backbone, fused unidirectional promoter fragment, and gene YLARO3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells. The positive recombinant plasmid P4-YLARO3 was obtained by screening on LB solid plates containing 100 mg / L ampicillin and sequencing to verify the correctness.

[0171] The recombinant plasmid P4-YLARO3 was mutated by primer pair ARO3-K225L-F and ARO3-K225L-R to obtain the recombinant plasmid P4-YLARO3. K225L ;

[0172] The recombinant plasmid P4-YLARO3 was amplified using primer pair P4-F and P4-R. K225L， Obtained linearized fragment P4-YLARO3 K225L .

[0173] Linearized fragment P3-YLARO4K221L -YLARO7 G139S and P4-YLARO3 K225L The engineered bacteria HT2 was obtained by integrating the yeast into the engineered bacteria HT1 (for the specific method, see the above Yarrowia lipolytica genome integration method, the difference being that during rejuvenation, the Yarrowia lipolytica was replaced by the engineered bacteria HT1).

[0174] 4. Using the DNA fragment shown in SEQ ID No. 3 as a template, amplify with primer pair YL-LAAD-F and YL-LAAD-R to obtain the gene PmLAAD fragment;

[0175] The DNA fragment shown in SEQ ID No. 4 was used as a template and primers YL-EcTyrA-F and YL-EcT yrA-R were used to amplify the gene EcTyrA. M53I,A354V fragment.

[0176] The linearized p-IntC3 vector backbone, fusion bidirectional promoter fragment, gene PmLAAD fragment, gene EcTyrA M53I,A354V The fragments were assembled using the Gibson assembly method, transformed into DH5α competent cells, screened on LB solid plates containing 100 mg / L ampicillin resistance and sequenced to obtain the positive recombinant plasmid P5-LAAD-EcTyrA M53I,A354V ;

[0177] The recombinant plasmid P5-LAAD-EcTyrA was amplified using primer pair P5-F and P5-R M53I,A354V， Obtained linearized fragment P5-LAAD-EcTyrA M53I,A354V ;

[0178] Linearized fragment P5-LAAD-EcTyrA M53I,A354V The engineered bacteria HT2 was integrated (for the specific method, see the above Yarrowia lipolytica genome integration method, the difference being that during rejuvenation, Yarrowia lipolytica was replaced by the engineered bacteria HT2) to obtain the engineered bacteria HT3.

[0179] Fifth, using the genome of Yarrowia lipolytica W29ΔKU70 as a template, the primer pair HPD-UP-F and HPD-UP-R were used to amplify the upstream homology arm fragment HPD-UP of the HPD gene;

[0180] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pairs HPD-DN-F and HPD-DN-R were used to amplify the downstream homology arm fragment HPD-DN of the HPD gene.

[0181] Using the upstream homology arm fragment HPD-UP and the downstream homology arm fragment HPD-DN as templates, PCR amplification was performed with primer pair HPD-UP-F and HPD-DN-R to obtain the repaired fragment HPD-UP-DN.

[0182] The repaired fragment HPD-UP-DN was integrated into the engineered bacterium HT3 (for the specific method, see the Yarrowia lipolytica genome integration method above, the difference being that during rejuvenation, Yarrowia lipolytica was replaced by the engineered bacterium HT3) to obtain the engineered bacterium HT4.

[0183] 6. Using the genome of Yarrowia lipolytica W29ΔKU70 as a template, primer pair YL-DHS1-F and YL-DHS1-R were used to amplify the YLDHS1 gene fragment;

[0184] The YL-DHS2 gene fragment was obtained by amplification using the Yarrowia lipolytica W29ΔKU70 genome as a template and primer pair YL-DHS2-F and YL-DH S2-R;

[0185] The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-DHS3-F and YL-DH S3-R were used to amplify the YLDHS3 gene fragment.

[0186] The linearized p-D17 backbone fragment, the fused bidirectional promoter fragment, the YLDHS1 gene fragment, and the YLDHS2 gene fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells. The positive recombinant plasmid P6-YLDHS1-YLDHS2 was obtained after screening on LB solid plates containing 100 mg / L ampicillin and sequencing verification.

[0187] The recombinant plasmid P6-YLDHS1-YLDHS2 was amplified using primer pair P6-F and P6-R to obtain the linearized fragment P6-YLDHS1-YLDHS2.

[0188] The linearized p-IntE4 vector backbone, fused unidirectional promoter fragment, and gene YLDHS3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells. The positive recombinant plasmid P7-YLDHS3 was obtained by screening on LB solid plates containing 100 mg / L ampicillin resistance and sequencing verification.

[0189] The recombinant plasmid P7-YLDHS3 was amplified using primer pair P7-F and P7-R to obtain the linearized fragment P7-YLD HS3.

[0190] The linearized fragments P6-YLDHS1-YLDHS2 and P7-YLDHS3 were integrated into the engineered bacterium HT4 (for the specific method, see the Yarrowia lipolytica genome integration method above, the difference being that during rejuvenation, Yarrowia lipolytica was replaced with the engineered bacterium HT4) to obtain the engineered bacterium HT5.

[0191] Example 3: Production of Tyrosol by Shake Flask Fermentation of Engineered Yarrowia lipolytica

[0192] The engineered bacteria HT1, HT2, HT3, HT4, and HT5 were activated in 24-well plates containing 3 mL of YPD medium for 48 h to obtain the corresponding seed solutions.

[0193] The seed liquid obtained in step (1) was inoculated into 250 mL of sterilized YNB medium at a ratio of 1:30 (volume ratio). After 4 days of fermentation at 30°C and 220 rpm, HPLC-MS analysis was performed to quantify tyrosol. The yield of engineered strains HT1 to HT5 in shake flasks was as follows: Figure 2 As shown, the tyrosol production of the engineered bacteria HT5 reached the highest 1547.65 mg / L.

[0194] Example 4: Application of engineered bacteria HY5 in fed-batch fermentation in a bioreactor

[0195] Taking the engineered bacteria HY5 as an example, the engineered bacteria HY5 was streaked on a YPD solid plate and cultured at 30°C for 48 hours. A single colony was picked from the plate and inoculated into 4 mL of MM medium, and cultured at 30°C and 220 rpm for 48 hours. During the logarithmic growth phase, the colony was transferred to a 250 mL shake flask containing fresh 40 mL of MM medium at a ratio of 1:30 (volume ratio) and cultured at 30°C and 220 rpm for 24 hours.

[0196] The bacteria in the shake flask were centrifuged at 5000 rpm for 5 minutes to remove the supernatant, and washed twice with double distilled water; each time the cells were centrifuged at 5000 rpm for 5 minutes to remove the supernatant; the bacteria were resuspended in 20 mL of double distilled water to obtain a cell suspension; the cell suspension was inoculated into a 1.3 L Dipil bioreactor (initially containing 400 mL of MM culture medium) for culture. The fermentation temperature was 30 ° C, and potassium hydroxide with a concentration of 4 M was automatically added. The pH value was maintained at 5.0. During the fed-batch stage, the DO was maintained at 30% by a two-stage series connection of stirring (600-1200 rpm) and airflow (0.5 SLPM-1.5 SLPM). After the glucose was exhausted, the MM fed-batch culture (constant rate feeding) medium was added at a rate of 3 mL / h until the end of the fermentation. During this period, samples were taken every 12 hours for testing. The fermentation results showed that the hydroxytyrosol production of the engineered bacteria HT5 reached 7516.37 mg / L ( Figure 3 ).

[0197] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a Yarrowia lipolytica strain for producing tyrosol, characterized in that: The steps include: Step (1), expressing ScADH6 and ScARO10 genes in Yarrowia lipolytica to obtain engineered bacteria HY1; The nucleotide sequence of ScADH6 is SEQ ID NO.1; the nucleotide sequence of ScARO10 is SEQ ID NO.2; Step (2): for the engineered bacteria HY1, by expressing YlARO3 K225L 、Y1ARO4 K2214 and Y1AR07 G139S Genetically relieve the feedback inhibition of shikimic acid to obtain the engineered bacterium HY2; Step (3): Introduce PmLAAD and EcTyrA into engineered bacteria HY2 M53I,A354V Gene, and obtained the engineered bacteria HY3; The nucleotide sequence of the PmLAAD is SEQ ID NO. 3; EcTyrA M53I,A354V The nucleotide sequence is SEQ ID NO.4; Step (4), knocking out the 4-hydroxyphenylpyruvate dioxygenase HPD gene in the engineered bacteria HY3 to obtain the engineered bacteria HY4; In step (5), the engineered bacteria HY4 is overexpressed with genes YlDHS1, YlDHS2 and YlDHS3 of the shikimate pathway to obtain the engineered bacteria HY5; the engineered bacteria HT1 to HT5 are all Yarrowia lipolytica strains that produce tyrosol.

2. The method for constructing the Yarrowia lipolytica strain for producing tyrosol according to claim 1, wherein The specific method of step (1) is: Using the DNA fragment shown in SEQ ID No. 1 as a template, the primer pair YL-ScADH6-F and YL-Sc ADH6R were used to amplify the ScADH6 gene fragment; The DNA fragment shown in SEQ ID No. 2 was used as a template and primers YL-ScARO10-F and YL-Sc ARO10-R were used to amplify the gene ScARO10. F138L,D218G fragment. The linearized p-IntD1 vector backbone, fused bidirectional promoter fragment, gene ScADH6 fragment, and gene ScARO10 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P2-ScADH6-ScARO10; The recombinant plasmid P2-ScADH6-ScARO10 was amplified using primer pair P2-F and P2-R to obtain the linearized fragment P2-ScADH6-ScARO10. F138L,D218G ; Linearized fragment P2-ScADH6-ScARO10 F138L,D218 G was integrated into Yarrowia lipolytica W29ΔKU70 to obtain the engineered strain HT1.

3. The method for constructing the Yarrowia lipolytica strain for producing tyrosol according to claim 1, wherein The specific method of step (2) is: The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO3-F and YL-ARO3-R were used to amplify the YLARO3 gene fragment; The Yarrowia lipolytica W29ΔKU70 genome was used as a template and the primer pair YL-ARO4-F and YL-ARO4-R were used to amplify the YLARO4 gene fragment; The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-ARO7-F and YL-ARO7-R were used to amplify the YLARO7 gene fragment. The linearized p-IntC2 vector backbone, fused bidirectional promoter fragment, gene YLARO4 fragment, and gene YLARO7 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P3-YLARO4-YLARO7; The recombinant plasmid P3-YLARO4-YLARO7 was amplified by two rounds of mutation using primer pairs AR04-K221L-F, AR04-K221L-R and primer pairs AR07-G139S-F, AR07-G139S-R to obtain the recombinant plasmid P3-YLARO4 K221L -YLARO7 G139S ; The recombinant plasmid P3-YLARO4 was amplified using primer pair P3-F and P3-R. K221L -YLARO7 G139S , and obtain the linearized fragment P3-YLARO4 K221L -YLARO7 G139S ; The linearized p-IntE1 vector backbone, fused unidirectional promoter fragment, and gene YLARO3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P4-YLARO3; The recombinant plasmid P4-YLARO3 was mutated by primer pair ARO3-K225L-F and ARO3-K225L-R to obtain the recombinant plasmid P4-YLARO3. K225L ; The recombinant plasmid P4-YLARO3 was amplified using primer pair P4-F and P4-R. K225L , and obtain the linearized fragment P4-YLARO3 K225L ; Linearized fragment P3-YLARO4 K221L -YLARO7 G139S and P4-YLARO3 K225L Integrate into engineered bacteria HT1 to obtain engineered bacteria HT2.

4. The method for constructing the Yarrowia lipolytica strain for producing tyrosol according to claim 1, wherein The specific method of step (3) is: Using the DNA fragment shown in SEQ ID No. 3 as a template, the primer pair YL-LAAD-F and YL-LAAD-R were used to amplify the gene PmLAAD fragment; The DNA fragment shown in SEQ ID No. 4 was used as a template and primers YL-EcTyrA-F and YL-EcTyrA-R were used to amplify the gene EcTyrA. M53I,A354V fragment. The linearized p-IntC3 vector backbone, fusion bidirectional promoter fragment, gene PmLAAD fragment, gene EcTyrA M53I ,A354V The fragments were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P5-LAAD-EcTyrA M53I,A354V ; The recombinant plasmid P5-LAAD-EcTyrA was amplified using primer pair P5-F and P5-R M53I,A354V， Obtained linearized fragment P5-LAAD-EcTyrA M53I,A354V ; Linearized fragment P5-LAAD-EcTyrA M53I,A354V Integrate into engineered bacteria HT2 to obtain engineered bacteria HT3.

5. The method for constructing the Yarrowia lipolytica strain for producing tyrosol according to claim 1, wherein The specific method of step (4) is: The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primers HPD-UP-F and HPD-UP-R were used to amplify the upstream homology arm fragment HPD-UP of the HPD gene. The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primers HPD-DN-F and HPD-DN-R were used to amplify the downstream homology arm fragment HPD-DN of the HPD gene. Using the upstream homology arm fragment HPD-UP and the downstream homology arm fragment HPD-DN as templates, PCR amplification was performed with primer pair HPD-UP-F and HPD-DN-R to obtain the repaired fragment HPD-UP-DN. The repaired fragment HPD-UP-DN was integrated into the engineered bacteria HT3 to obtain the engineered bacteria HT4.

6. The method for constructing the Yarrowia lipolytica strain for producing tyrosol according to claim 1, wherein The specific method of step (5) is: The YL-DHS1 gene fragment was obtained by amplification using the Yarrowia lipolytica W29ΔKU70 genome as a template and primer pair YL-DHS1-F and YL-DHS1-R; The YL-DHS2 gene fragment was obtained by amplification using the Yarrowia lipolytica W29ΔKU70 genome as a template and primer pair YL-DHS2-F and YL-DHS2-R; The Yarrowia lipolytica W29ΔKU70 genome was used as a template and primer pair YL-DHS3-F and YL-DHS3-R were used to amplify the YLDHS3 gene fragment. The linearized p-D17 backbone fragment, the fused bidirectional promoter fragment, the YLDHS1 gene fragment, and the YLDHS2 gene fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P6-YLDHS1-YLDHS2; The recombinant plasmid P6-YLDHS1-YLDHS2 was amplified using primer pair P6-F and P6-R to obtain the linearized fragment P6-YLDHS1-YLDHS2. The linearized p-IntE4 vector backbone, fused unidirectional promoter fragment, and gene YLDHS3 fragment were assembled using the Gibson assembly method and transformed into DH5α competent cells to obtain the positive recombinant plasmid P7-YLDHS3; The recombinant plasmid P7-YLDHS3 was amplified using primer pair P7-F and P7-R to obtain the linearized fragment P7-YLDHS3; The linearized fragments P6-YLDHS1-YLDHS2 and the linearized fragment P7-YLDHS3 were integrated into the engineered bacteria HT4 to obtain the engineered bacteria HT5.

7. A tyrosol-producing Yarrowia lipolytica strain obtained by the method for constructing a tyrosol-producing Yarrowia lipolytica strain according to any one of claims 1 to 6.

8. Use of the tyrosol-producing Yarrowia lipolytica strain according to any one of claim 7 in the production of tyrosol.

9. A method for producing tyrosol, characterized in that: The tyrosol-producing Yarrowia lipolytica strain according to claim 8 is used for fermentation in YNB culture medium to obtain tyrosol.

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