Yarrowia lipolytica engineering bacterium with high yield of p-coumaric acid by taking glucose as substrate as well as construction method and application of Yarrowia lipolytica engineering bacterium

Abstract

The invention relates to the technical field of biology, and discloses yarrowia lipolytica engineering bacteria with high yield of p-coumaric acid by taking glucose as a substrate as well as a construction method and an application of the yarrowia lipolytica engineering bacteria. The construction method comprises the following steps: integrating a tyrosine ammonolysis enzyme gene TAL into a Yarrowia lipolytica strain by adopting a CRISPR / Cas9 positioning integration method, strengthening DAHP synthetase genes ARO4, DHS1 and AROG, overexpressing a tyrosine synthetase TYR gene and a histidine phosphate aminotransferase HIS5 protease gene, and carrying out high-throughput screening to obtain the Yarrowia lipolytica strain. And further integrating exogenous phenylalanine deamination hydroxylation pathway genes for synthesizing p-coumaric acid, wherein the exogenous phenylalanine deamination hydroxylation pathway genes comprise a phenylalanine ammonialyase gene, a cinnamate hydroxylase gene and a P450 reductase gene. The yield of p-coumaric acid produced by the Yarrowia lipolytica engineering strain is the highest yield of known Yarrowia lipolytica, the highest yield in a shake flask can reach 1.7 g / L, the fed-batch fermentation yield in a 5L fermentation tank can reach 30 g / L, and the Yarrowia lipolytica engineering strain has remarkable industrial application value.

Description

Field of the Invention

[0001] The invention relates to the field of biotechnology, and in particular to a recombinant Yarrowia lipolytica producing p-coumaric acid, a construction method and an application thereof. Background Art

[0002] p-Coumaric acid (p-CA), as an upstream metabolite of the plant phenylpropanoid pathway, is a common precursor of phenylpropanoids, lignans, flavonoids and stilbenes. Various in vivo and in vitro studies have revealed its scavenging and antioxidant properties in reducing oxidative stress and inflammatory responses. Therefore, p-coumaric acid has many important applications in nutrition, pharmaceuticals, materials and chemical industries. At present, the main sources of p-coumaric acid are plant extraction, chemical synthesis and biosynthesis. Plant extraction faces problems such as long growth cycle, low yield and great environmental impact, while chemical synthesis faces problems such as high energy consumption, low yield and environmental pollution. Biosynthesis is considered to be the most promising production method in the 21st century because of its advantages such as low cost, high yield and green environmental protection.

[0003] Yarrowia lipolytica is an important industrial microbial species that is recognized as a food-grade safe microorganism. Lipid yeasts naturally have high fluxes of malonyl-CoA and the pentose phosphate pentose pathway (PPP), and these metabolic traits may be particularly relevant to the production of shikimate pathway-derived compounds and plant natural products that require aromatic amino acids and malonyl-CoA-derived building blocks. Therefore, Yarrowia lipolytica yeast is an excellent host for p-coumaric acid biosynthesis. Summary of the invention

[0004] The first object of the present invention is to provide a recombinant Yarrowia lipolytica which can stably and highly produce p-coumaric acid using glucose as a substrate.

[0005] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Yarrowia lipolytica with high p-coumaric acid production.

[0006] The third object of the present invention is to provide an application of the above-mentioned recombinant Yarrowia lipolytica with high p-coumaric acid production.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides an engineered Yarrowia lipolytica strain that uses glucose as a substrate to produce high p-coumaric acid, wherein the engineered strain is integrated with L-tyrosine ammonia lyase gene, DAHP synthase ARO4, DHS1, AROG gene, tyrosine synthase TYR gene, histidine phosphate aminotransferase HIS5 gene, and exogenous phenylalanine deamination hydroxylation pathway phenylalanine ammonia lyase gene, cinnamate hydroxylase gene and P450 reductase gene.

[0009] According to the above scheme, the L-tyrosine ammonia lyase gene is the L-tyrosine ammonia lyase gene FjTAL derived from Flavobacterium; among the DAHP synthase ARO4, DHS1, and AROG genes, the enzyme ARO4 and DHS1 genes are endogenous enzyme genes, and the AROG gene is the EcAROG gene derived from Escherichia coli; the tyrosine synthase TYR gene and the histidine phosphate aminotransferase HIS5 gene are endogenous genes; the exogenous phenylalanine deamination hydroxylation pathway genes are the phenylalanine ammonia lyase gene AtPAL2, the cinnamate hydroxylase gene AtC4H, and the P450 reductase gene AtATR2 derived from Arabidopsis thaliana.

[0010] According to the above scheme, the nucleotide sequence of the gene FjTAL is shown as SEQ ID No.1.

[0011] According to the above scheme, the nucleotide sequence of the gene ARO4 is shown as SEQ ID No.2.

[0012] According to the above scheme, the nucleotide sequence of the gene DHS1 is shown as SEQ ID No.3.

[0013] According to the above scheme, the nucleotide sequence of the gene AROG is shown as SEQ ID No.4.

[0014] According to the above scheme, the tyrosine synthesis TYR gene and HIS5 gene are endogenous genes, the nucleotide sequence of the TYR gene is shown as SEQ ID No.5, and the nucleotide sequence of the HIS5 gene is shown as SEQ ID No.6.

[0015] According to the above scheme, the phenylalanine ammonia lyase is the AtPAL2 gene from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.7.

[0016] According to the above scheme, the cinnamate hydroxylase gene is the AtC4H gene from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.8.

[0017] According to the above scheme, the P450 reductase gene is AtPAL2 from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.9.

[0018] The second aspect of the present invention provides a method for constructing the above-mentioned Yarrowia lipolytica engineered bacteria that uses glucose as a substrate to produce high-yield p-coumaric acid. The CRISPR / Cas9 targeted integration method is used to integrate the tyrosine ammonia lyase TAL gene into the Yarrowia lipolytica strain, strengthen the DAHP synthase genes ARO4, DHS1 and AROG, overexpress the tyrosine synthase TYR gene and the histidine phosphate aminotransferase HIS5 protease gene, and further integrate exogenous phenylalanine deamination hydroxylation pathway genes for synthesizing p-coumaric acid, including phenylalanine ammonia lyase gene, cinnamate hydroxylase gene and P450 reductase gene.

[0019] According to the above scheme, the tyrosine ammonia lyase gene is integrated at two different gene sites to integrate two copies of the tyrosine ammonia lyase gene.

[0020] In the present invention, the above-mentioned genes can be expressed separately at the genomic locus, or multiple genes can be expressed at the same genomic locus. Preferably, the recombinant genetic engineering bacteria of the present invention express L-tyrosine ammonia lyase gene, DAHP synthase gene (ARO4, DHS1, AROG) separately at different gene editing sites such as intE1, intC3, intE14, and DGA1, tyrosine synthase TYR gene and histidine phosphate aminotransferase HIS5 gene and exogenous phenylalanine deamination hydroxylation pathway genes including phenylalanine ammonia lyase gene, cinnamate hydroxylase gene and P450 reductase gene, and finally obtain the Yarrowia lipolytica engineering bacteria with high yield of p-coumaric acid using glucose as substrate. Specifically, pylcas9-sgRNA plasmids and plasmids expressing target genes can be constructed for the gene locus to be integrated based on the Crispr / Cas9 gene editing system, and the construction of Yarrowia lipolytica engineering bacteria can be carried out by co-transferring to Yarrowia lipolytica. More specifically, Yarrowia lipolytica, such as the po1fΔKu70 strain, is used as a starting strain, the L-tyrosine ammonia lyase TAL gene is integrated at the intE1 site of the genome, the DAHP synthase ARO4, DHS1, EcAROG and TAL genes are overexpressed at the intC3 site, the upstream tyrosine synthesis TYR gene and the histidine phosphate aminotransferase HIS5 gene are enhanced at the intE14 site, and the exogenous phenylalanine deamination hydroxylation pathway genes including the phenylalanine ammonia lyase gene, the cinnamate hydroxylase gene and the P450 reductase gene are overexpressed at the DGA1 site.

[0021] According to the above scheme, the method for constructing the Yarrowia lipolytica engineered bacteria that produces high p-coumaric acid using glucose as a substrate comprises the following steps:

[0022] 1) Integrate the L-tyrosine ammonia lyase gene TAL into the intE1 site of Yarrowia lipolytica to obtain a recombinant Yarrowia lipolytica that synthesizes p-coumaric acid from glucose;

[0023] 2) Based on the strain obtained in step 1), DAHP synthase ARO4, DHS1, AROG and L-tyrosine ammonia lyase gene TAL are further overexpressed at the intC3 site to remove the upstream DAHP synthesis restriction, increase the TAL copy number, and enhance the synthesis of p-coumaric acid;

[0024] 3) Based on the strain obtained in step 2), the upstream tyrosine synthesis TYR gene and the histidine phosphate aminotransferase HIS5 gene are strengthened at the intE14 site to further improve the p-coumaric acid production of the engineered strain;

[0025] 4) Overexpressing exogenous phenylalanine deamination hydroxylation pathway genes including phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, and P450 reductase gene at the diacylglycerol transferase DGA1 site of the strain obtained in step 3) to significantly increase the production of p-coumaric acid.

[0026] Specifically, step (1) is:

[0027] An FjTAL gene expression cassette was constructed and the FjTAL gene expression cassette fragment was amplified. The upstream homology arm and downstream homology arm fragments of the intE1 site were amplified using the yeast genome as a template, and the upstream homology arm and downstream homology arm fragments of the intE1 site were cloned and connected with the FjTAL gene expression cassette fragment in one step using a plasmid to construct a plasmid.

[0028] Based on the sgRNA sequence targeting the intE1 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, the pYlCas9-sgRNA-intE1 fragment containing Cas9 protein and targeting the intE1 site was amplified. The vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intE1 plasmid.

[0029] The above two plasmids were co-transformed into the starting strain PO1f, and colony PCR screening was used to obtain the recombinant strain jz101.

[0030] Step (2) is: constructing ARO4, DHS1, EcAROG, and FjTAL gene expression cassettes respectively, and using plasmids to clone the amplified ARO4, DHS1, EcAROG, and FjTAL gene expression cassette fragments and the intC3 upstream homology arm and intC3 upstream homology arm fragments in one step to construct a plasmid;

[0031] Based on the sgRNA sequence targeting the intC3 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, the pYlCas9-sgRNA-intC3 fragment containing Cas9 protein and targeting the intC3 site was amplified. The vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intC3 plasmid.

[0032] The recombinant strain jz102 was obtained by co-transformation into the jz101 strain and screening by colony PCR.

[0033] Step (3) is: construct the TYR gene expression cassette and the HIS5 gene expression cassette respectively, clone the amplified TYR and HIS5 gene expression cassette fragments and the intE14 upstream homology arm and the intE14 upstream homology arm fragment in one step using a plasmid to construct a plasmid.

[0034] Based on the sgRNA sequence targeting the intE14 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, a pYlCas9-sgRNA-intE14 fragment containing Cas9 protein and targeting the intE14 site was amplified. The vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intE14 plasmid;

[0035] It was co-transformed into jz102 and screened by colony PCR to obtain the recombinant strain jz104.

[0036] Step (4) is: constructing gene expression cassettes for phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, and P450 reductase gene, respectively,

[0037] The upstream homology arm fragment and the downstream homology arm fragment DGA1Up and DGA1down of the amplified diacylglycerol acyltransferase DGA1 are used to construct a DGA1 site integration vector, and the amplified phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, P450 reductase gene gene expression cassette fragment and the DGA1 site integration vector amplified fragment are cloned in one step to construct a first plasmid;

[0038] Based on the sgRNA targeting the DGA1 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, the pYlCas9-sgRNA-DGA1 fragment containing Cas9 protein and localized to the DGA1 site was amplified. The vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-DGA1 plasmid.

[0039] Using the first plasmid as a template, primers were used to amplify and recover the homology arms and the gene expression cassette. The recovered fragments and the pYlCas9-sgRNA-DGA1 plasmid were co-transformed into the recombinant strain jz104, and colony PCR screening was used to obtain the recombinant strain jz106.

[0040] The third aspect of the present invention provides the use of the above-mentioned Yarrowia lipolytica engineered bacteria in high production of p-coumaric acid using glucose as a substrate.

[0041] The present invention integrates the tyrosine ammonia lyase gene TAL, strengthens the DAHP synthase genes ARO4, DHS1 and AROG, overexpresses the tyrosine synthesis upstream TYR and HIS5 protease genes, and further integrates the exogenous phenylalanine deamination hydroxylation pathway genes for synthesizing p-coumaric acid, including the phenylalanine ammonia lyase gene, the cinnamate hydroxylase gene and the P450 reductase gene to obtain the lipolytic yeast strain, which can achieve high production of coumaric acid using glucose as a substrate.

[0042] Beneficial effects of the present invention:

[0043] The p-coumaric acid producing yeast strain constructed by the present invention has a p-coumaric acid yield of 1.7 g / L in a shake flask and a p-coumaric acid yield of 30 g / L in a 5L fermenter by batch feeding fermentation, and has significant industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Metabolic pathways for high p-coumaric acid-producing Yarrowia lipolytica strains;

[0045] Figure 2 is the p-coumaric acid production of the key gene overexpression strain;

[0046] Figure 3 Batch fermentation of p-coumaric acid by engineered Yarrowia lipolytica strain jz106. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with examples. The following examples are intended to enable those skilled in the art to better understand the present invention, but do not impose any limitation on the present invention.

[0048] The DNA ligase and other molecular biological reagents used were purchased from TaKaRa (https: / / www.takara.com.cn / ); the Uniclone One Step Seamless Cloning Kit was purchased from Beijing Jinsha Biotechnology Co., Ltd. (https: / / 400-688-7890.company.lookchem.cn / ); and other biochemical reagents were purchased from China Reagent Network (https: / / www.labgogo.com / ).

[0049] The formula of LB liquid culture medium is: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, sterilized at 121℃ for 30min.

[0050] The formula of LB solid culture medium is: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar powder, sterilized at 121℃ for 30min.

[0051] YPD liquid medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, sterilized at 115°C for 30 min;

[0052] YPD solid medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, 20 g / L agar, sterilized at 115°C for 30 min;

[0053] YPD-40 liquid medium: 10 g / L yeast extract, 20 g / L tryptone, 40 g / L glucose, sterilized at 115°C for 30 min;

[0054] YNB-Leu liquid culture medium: 6.7g / L YNB, 1.5g / L (NH4)2SO4, 20g / L glucose, 0.1g / L Ura, sterilized at 115℃ for 30min

[0055] YNB-Leu solid culture medium: 6.7 g / L YNB, 1.5 g / L (NH4)2SO4, 20 g / L glucose, 0.1 g / L Ura, 20 g / L agar, sterilized at 115°C for 30 min.

[0056] Preparation of Yarrowia lipolytica competent cells:

[0057] (1) Pick a fresh single colony of Saccharomyces lipolytica from the YPD plate and add it to 5 ml of YPD liquid medium. Culture it at 30°C and 250 rpm overnight.

[0058] (2) Determine the OD600 value of the overnight culture;

[0059] (3) Inoculate the overnight culture into fresh medium to obtain an initial OD600 value of 0.2 to 0.4;

[0060] (4) Continue culturing in a shaking incubator at 28-30°C for 3-6 hours until the OD600 value reaches 0.6-1.0;

[0061] (5) Take 50 mL of culture medium and centrifuge at 3000 g for 3 min at room temperature to collect yeast cells and discard the supernatant;

[0062] (6) Resuspend the cells in 50 mL of sterile water, then centrifuge at 3000 g for 3 min at room temperature to collect the cells and discard the supernatant;

[0063] (7) Resuspend the yeast cells in 1.5 mL of 1×LiAc / 0.5×TE, then centrifuge at 3000 g for 3 min at room temperature to collect the cells and discard the supernatant;

[0064] (8) Finally, resuspend the yeast cells in 200 μL 1×LiAc / 0.5×TE and dispense into 50 μL tubes.

[0065] Transformation of Yeast lipolytica cells:

[0066] (1) Take 50 μL of competent cells, add 2-5 μL of each plasmid to be transfected, and then add 5 μL (10 mg / L) denatured salmon protamine, and mix well;

[0067] (2) Add 300 μL of transformation solution 1×LiAc / 40% PEG-3350 / 1×TE and tap the tube wall to mix;

[0068] (3) Bath in 30°C water for 0.5-2 h, tapping the tube wall every 15 min to mix;

[0069] (4) Transfer to a 42°C water bath and heat shock for 15-18 min;

[0070] (5) Centrifuge at 3000 g for 3 min, retain the precipitate, and discard the supernatant;

[0071] (6) Resuspend the precipitate in 50-150 μL of distilled water, apply to the corresponding screening plate, and incubate the plate upside down at 30°C for 2-3 days.

[0072] Reagents:

[0073] 10×TE (100 mL): 100 mM Tris (1.2114 g), 10 mM EDTA (0.29224 g), adjust pH to 7.5, filter sterilize and store at 4°C;

[0074] 10×LiAc: Dissolve 10.2 g LiAc in 90 mL distilled water, adjust pH to 7.5, and dilute to 100 mL with distilled water. Filter sterilize and store at 4°C;

[0075] 50% PEG-3350: Weigh 25 g PEG-3350, add a small amount of distilled water and slowly heat to dissolve it, dilute to 50 mL with distilled water, filter and sterilize, and store at 4°C;

[0076] 1×LiAc / 0.5×TE: Mix 100 μL 10×LiAc and 50 μL 10×TE and dilute to 1 mL with distilled water.

[0077] 1×LiAc / 50% PEG-3350 / 1×TE: Mix 100μL 10×LiAc, 100μL 10×TE and 800μL 50% PEG-3350;

[0078] Genome extraction of Yarrowia lipolytica transformants

[0079] (1) Add 100 μL of bacterial solution to a 1.5 mL centrifuge tube, centrifuge at 5000 rpm for 3 min, and discard the supernatant;

[0080] (2) Add 100 μL of 0.2 M LiAc 1% SDS solution (m / v) to resuspend the cells; place in a metal bath at 75°C for 10 min;

[0081] (3) Add 300 μL of anhydrous ethanol, vortex, centrifuge at 15,000 rpm for 3 min, and remove the supernatant with a pipette;

[0082] (4) Add 150 μL of 70% ethanol, centrifuge at 15,000 rpm for 3 min, and remove the supernatant with a pipette;

[0083] (5) Drying in a 37°C incubator;

[0084] (6) Add 50 μL of sterile water, resuspend, and centrifuge at 15,000 rpm for 1 min. The supernatant is the genome template.

[0085] Example 1. Construction of p-coumaric acid-producing Yarrowia lipolytica

[0086] The L-tyrosine ammonia lyase FjTAL from Flavobacterium was integrated into the po1fΔKu70 strain of Yarrowia lipolytica to construct a recombinant Yarrowia lipolytica that synthesizes p-coumaric acid from scratch using glucose as a substrate. The specific operation is as follows:

[0087] The FjTAL gene sequence is shown in SEQ ID No. 1. The FjTAL gene fragment was amplified using primers (SEQ ID No. 10-11) to amplify the L-tyrosine ammonia lyase FjTAL gene fragment. The FjTAL gene expression cassette P was constructed in Escherichia coli DH5α using plasmid pUC19. TEF1 -FjTAL-T lip2t, using primers (SEQ ID No.12-15) for amplifying upstream and downstream homology arms of the intE1 site of the yeast genome, respectively amplifying the fragments of the upstream homology arms and downstream homology arms of the intE1 site using the yeast genome as a template, using primers (SEQ ID No.16-17) for amplifying the FjTAL gene expression cassette to amplify the FjTAL gene expression cassette to obtain the FjTAL gene expression cassette fragment, and using plasmid pUC19 to clone and connect the upstream homology arms and downstream homology arms of the intE1 site with the FjTAL gene expression cassette fragment in one step to construct a plasmid pUC19-intE1-P TEF1 -FjTAL-T lip2t .

[0088] Based on the sgRNA sequence targeting the intE1 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, primers (SEQ ID No. 18-19) were used to amplify the pYlCas9-sgRNA-intE1 fragment containing Cas9 protein and positioned at the intE1 site to amplify the pYlCas9-sgRNA-intE1 fragment containing Cas9 protein and positioned at the intE1 site. The PCR product was recovered after overnight digestion with Dpn I, and the vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intE1 plasmid.

[0089] SEQ ID No.1 Flavobacterium johnsoniae derived gene

[0090] DNA sequence of FjTAL

[0091]

[0092] ATCTCTTCTGTGTCTAAGAAGTGGTACGACGAGATTCGAAACATCATCCCCACCTTTAAGGAAGATCAGGTGATGTACCCCTTCGTGCAGAAGGTCAAGGACCATCTGATTAACAACTAG

[0093] Primer sequences for amplifying FjTAL gene

[0094] FjTAL-F: TTGCAGTACTAACCGCAGAACACCATCAACGAGTACCTG, SEQ ID No.10

[0095] FjTAL-R: GGTTGATTCCGAACAGAAGCTAGTTGTTAATCAGATG, SEQ ID No.11

[0096] Amplify the upstream and downstream homology arm primer sequences of the yeast genome intE1 site

[0097] puc19-intE1-F1:TTGTAAAACGACGGCCAGTGCACTGTTGATTGCTTCAGTTAC, SEQ IDNo.12

[0098] intE1-pTEF1-R1: GCCGCCAACCCGGTCTCTAAGCACTATCCTCTGCTGCG, SEQ ID No. 13

[0099] Lip2t-intE1-F2: ACCGCCCAAGAATGCATCTGTTGTGTCGAAATACAACAGCC, SEQ ID No. 14

[0100] intE1-puc19-R2: CCATGATTACGCCAAGCTTGCTTAAAGCGATGTGGCGCAG, SEQ ID No.15

[0101] Primer sequences for amplifying the FjTAL gene expression cassette

[0102] pTEF1-UP: AGAGACCGGGTTGCGGC, SEQ ID No. 16

[0103] Lip2t-R: CAGATGCATTCTTGGGCGGT, SEQ ID No.17

[0104] Amplify the primer sequence of the pYlCas9-sgRNA-intE1 fragment containing Cas9 protein and located at the intE1 site

[0105] sgRNA-intE1-F:GGGCTATCTGAGCTGGATCGGTTTTAGAGCTAGAAAT, SEQ ID No.18

[0106] sgRNA-intE1-R: CGATCCAGCTCAGATAGCCCTAACCAACCTGCGCCGA, SEQ ID No.19

[0107] The pUC19-intE1-PTEF1-FjTAL-Tlip2t and pYlCas9-sgRNA-intE1 plasmids were co-transfected into Yarrowia lipolytica po1fΔKu70 by the "lithium acetate / ssDNA / PEG3350" thermal transfer method and plated on the leucine-deficient screening plate YNB-leu. After 2-3 days, several transformants were randomly selected in the corresponding liquid YNB-leu medium, cultured at 30°C and 250rpm for 48 hours, and the genome was extracted for PCR verification, and positive transformants were selected to obtain the recombinant strain jz101.

[0108] A single colony of the recombinant strain jz101 was picked and inoculated into a test tube containing 2 mL of YPD medium, and cultured in a shaker at 250 rpm and 30°C for 24 h; the first-level seed liquid was inoculated into a 50 mL conical flask containing 10 mL of YPD medium and cultured for 24 h with an inoculation amount of 1% (v / v) to obtain a second-level seed liquid; the second-level seed liquid was inoculated into a 250 mL conical flask containing 30 mL of YPD-40 medium with an inoculation amount of 5% (v / v). After fermentation under the same conditions for 96 h, the yield of p-coumaric acid was 100.8 mg / L.

[0109] Example 2. Strengthening upstream DAHP synthesis and increasing FjTAL copy number

[0110] The sequences of yeast endogenous DAHP synthase genes ARO4, DHS1 and Escherichia coli-derived DAHP synthase gene EcAROG are shown in SEQ ID No. 2-4, and the endogenous DAHP synthase genes ARO4, DHS1 and Escherichia coli-derived DAHP synthase gene EcAROG of Yarrowia lipolytica are further overexpressed to remove the upstream DAHP synthesis restriction and increase the copy number of L-tyrosine ammonia lyase gene FjTAL. The specific operation is as follows:

[0111] The ARO4, DHS1, and EcAROG gene fragments were amplified using primers (SEQ ID No. 20-25) for amplifying ARO4, DHS1, and EcAROG gene fragments, respectively, and the expression cassette P was constructed in Escherichia coli DH5α using plasmid pUC19. FAB1in -ARO4-T Pex20 , P hp4d -ECAROG-T xpr2t , P GPD1 -DHS1-T xpr2t Primers for amplifying the intC3 upstream homology arm and intC3 downstream homology arm fragments (SEQ ID No.26-29) were used to amplify the intC3 upstream homology arm and intC3 downstream homology arm fragments using the Yarrowia lipolytica genome as a template, and primers for amplifying the ARO4, DHS1, EcAROG, and FjTAL gene expression cassettes (SEQ ID No.34-35, SEQ ID No.36-37, SEQ ID No.32-33, and SEQ ID No.30-31, respectively) were used to amplify the ARO4, DHS1, EcAROG, and FjTAL gene expression cassette fragments. The amplified ARO4, DHS1, EcAROG, and FjTAL gene expression cassette fragments were cloned in one step with the intC3 upstream homology arm and intC3 upstream homology arm fragments using plasmid pUC19 to construct plasmid pUC19-intC3-FjTAL-ARO4-EcAROG-DHS1. Based on the sgRNA sequence targeting the intC3 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, primers (SEQ ID No.38-39) were used to obtain a pYlCas9-sgRNA-intC3 fragment containing Cas9 protein and positioned at the intC3 site. The pYlCas9-sgRNA-intC3 fragment containing Cas9 protein and positioned at the intC3 site was amplified. The PCR product was recovered after overnight digestion with Dpn I, and the vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intC3 plasmid.

[0112] SEQ ID No.2 Endogenous Yarrowia lipolytica

[0113] DNA sequence of ARO4

[0114]

[0115] SEQ ID No.3 Endogenous Yarrowia lipolytica

[0116] DNA sequence of DHS1

[0117]

[0118] SEQ ID No.4 Gene derived from Escherichia coli

[0119] DNA sequence of EcAROG

[0120]

[0121] Primer sequences for amplifying ARO4 gene fragment

[0122] ARO4-F: CAAACTAACCCAGCTCTCCATGTCCCGTTCCTCCT, SEQ ID No.20

[0123] ARO4-R:ACTTCCCCCATCCACACTTTTAGTTCTTGTTTC, SEQ ID No.21

[0124] Primer sequences for amplifying DHS1 gene fragments

[0125] DHS1-F: CCCGAAACTAAGGATCATGCCCGCTATGCACAA, SEQ ID No. 22

[0126] DHS1-R: ACACAAGTTCCGTAGTTGCTAACCTCGTCGAGTC, SEQ ID No.23

[0127] Primer sequences for amplifying AROG gene fragment

[0128] EcAROG-F: GTACTAACACAGGTCATGAATTATCAGAACGA, SEQ ID No. 24

[0129] EcAROG-R: AGGCCATGGAGGTACCTTACCCGCGACGCGCTTTT, SEQ ID No. 25

[0130] Amplify the upstream and downstream homology arm primer sequences of the yeast genome intC3 site

[0131] puc19-intC3-F1: TAGCTTATCGATACGCGTAAAAAACTGTAGTAGTGTGGTGAT, SEQ IDNo.26

[0132] IntC3-R1:TTTCAGTCTCCTCTTCACCATACAGTGTCTATCAACGGGGC, SEQ ID No. 27

[0133] IntC3-F2: TAAATTTAGTCTGCAGCCCAGCCATAGCACTATTGTAGAGTGG, SEQ ID No. 28

[0134] intC3-puc19-R2: ACCATGATTACGCCAAGCTTGAATTATTGCACAGGACACAC, SEQ ID No. 29

[0135] Primer sequences for amplifying the FjTAL gene expression cassette

[0136] IntC3-FjTAL-F: TGGTGAAGAGGAGACTGAAATAGAGACCGGGTTGGCGG, SEQ ID No. 30 FjTAL-EcAROG-R: GCAGGACATCCTACTGCGTCCAGATGCATTCTTGGGCGG, SEQ ID No. 31

[0137] Primer sequences for amplifying the EcAROG gene expression cassette

[0138] FjTAL-EcAROG-F: CCGCCCAAGAATGCATCTGGACGCAGTAGGATGTCCTGC, SEQ ID No. 32

[0139] EcAROG-ARO4-R: AGTTAGCTCACTCATTAGGCACCCATCTCACTTGCGTAT, SEQ ID No. 33

[0140] Primer sequences for amplifying the ARO4 gene expression cassette

[0141] EcAROG-ARO4-F: ATACGCAAGTGAGATGGGTGCCTAATGAGTGAGCTAACT, SEQ ID No. 34 ARO4-DHS1-R: CACGGCTCCGCCAGAGACGCAACTAACATGAATGA, SEQ ID No. 35 Primer sequence for amplifying DHS1 gene expression cassette

[0142] ARO4-DHS1-F: TCATTCATGTTAGTTGCGTCTCTGGCGGAGCCGTG, SEQ ID No. 36

[0143] DHS1-IntC3--R: AGTTAGCTCACTCATTAGGCACCCATCTCACTTGCGTAT, SEQ ID No. 37

[0144] Amplify the primer sequence of the pYlCas9-sgRNA-intC3 fragment containing Cas9 protein and located at the intC3 site

[0145] sgRNA-intC3-F: CTCGCTCCATGATCGTCGTAGTTTTAGAGCTAGAAAT, SEQ ID No. 38

[0146] sgRNA-intC3-R: TACGACGATCATGGAGCGAGTAACCAACCTGCGCCGA, SEQ ID No. 39

[0147] pUC19-intC3-FjTAL-ARO4-EcAROG-DHS1 and pYlCas9-sgRNA-intC3 were co-transfected into Yarrowia lipolytica jz101 by the "lithium acetate / ssDNA / PEG3350" thermal transfer method and spread on the leucine-deficient screening plate YNB-leu. After 2-3 days, several transformants were randomly selected in the corresponding liquid YNB-leu medium, cultured at 30°C and 250rpm for 48 hours, and the genome was extracted for PCR verification, and positive transformants were selected to obtain the recombinant strain jz102.

[0148] A single colony of the recombinant strain jz102 was picked and inoculated into a test tube containing 2 mL of YPD medium, and cultured in a shaker at 250 rpm and 30°C for 24 h; the first-level seed liquid was inoculated into a 50 mL conical flask containing 10 mL of YPD medium and cultured for 24 h with an inoculation amount of 1% (v / v) to obtain a second-level seed liquid; the second-level seed liquid was inoculated into a 250 mL conical flask containing 30 mL of YPD-40 medium with an inoculation amount of 5% (v / v). After fermentation under the same conditions for 96 h, the yield of p-coumaric acid was 355.9 mg / L.

[0149] Example 3. Strengthening the tyrosine synthesis pathway and increasing tyrosine flux

[0150] Strengthen the endogenous upstream tyrosine synthesis TYR gene and HIS5 gene of Yarrowia lipolytica to further increase the p-coumaric acid production of the engineered strain. The specific operations are as follows:

[0151] The sequences of the endogenous upstream tyrosine synthesis TYR gene and HIS5 gene of Yarrowia lipolytica are shown in SEQ ID No. 5 and 6. The TYR gene fragment was amplified using primers (SEQ ID No. 40-41) and primers (SEQ ID No. 42-43) for amplifying the HIS5 gene fragment using the Yarrowia lipolytica genome as a template to amplify the TYR gene and HIS5 gene fragments. The expression cassette P was constructed in Escherichia coli DH5α using plasmid pUC19. GPD1 -TYR-T lip2 , P TEFin -HIS5-T lip2tAs a template, primers for amplifying upstream and downstream homology arms of the yeast genome intE14 site (SEQ ID No.44-47) were used to amplify the intE14 upstream homology arm and the intE14 upstream homology arm fragment using the Yarrowia lipolytica genome as a template, and primers for amplifying the TYR gene expression cassette (SEQ ID No.48-49) and primers for amplifying the HIS5 gene expression cassette (SEQ ID No.50-51) were used to amplify the TYR and HIS5 gene expression cassette fragments, and the TYR and HIS5 gene expression cassette fragments and the intE14 upstream homology arm and the intE14 upstream homology arm fragment were cloned in one step using plasmid pUC19 to construct the plasmid pUC19-intE14-TYR-HIS5 plasmid. Based on the sgRNA sequence targeting the intE14 site, a tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, primers (SEQ ID No.52-53) were used to amplify the pYlCas9-sgRNA-intE14 fragment containing Cas9 protein and positioned at the intE14 site. The pYlCas9-sgRNA-intE14 fragment containing Cas9 protein and positioned at the intE14 site was amplified. The PCR product was recovered after overnight digestion with Dpn I, and the vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-intE14 plasmid.

[0152] SEQ ID No.5 Endogenous Yarrowia lipolytica

[0153] DNA sequence of TYR

[0154]

[0155] SEQ ID No.6 Endogenous Yarrowia lipolytica

[0156] DNA sequence of HIS5

[0157]

[0158] Primer sequences for amplifying TYR gene fragment

[0159] TYR-F: CAATGTACTAACACAGGTCTCTATTGAGGAATGGAAG, SEQ ID No.40

[0160] TYR-R: GTTGTAAAGAGTGATAAATAGCTTAAGTGGTTGACAGAATGGTG, SEQ ID No. 41

[0161] Primer sequences for amplifying HIS5 gene fragment

[0162] HIS5-F: CTGAGTATAAGAATCATTCAAAATGCCCAACACGAAATTCAACCTC, SEQ ID No. 42

[0163] HIS5-R: GTTGTAAAGAGTGATAAATAGCCTACTGTCTCAGGGCAGCAATTG, SEQ ID No. 43

[0164] Amplify the upstream and downstream homology arm primer sequences of the yeast genome intE14 site

[0165] puc19-intE14-F:GTAAACGACGGCCAGTGCCCCATCTGTAGAGCTAGTCAGT, SEQ ID No.44

[0166] IntE14-R1:TTTCAGTCTCCTCTTCACCAGAGTACAACGCCGACATCCAC, SEQ ID No.45

[0167] IntE14-F2: TAAATTTAGTCTGCAGCCCATTTGCCCTATGCTCAGTCAAC, SEQ ID No. 46

[0168] intE14-puc19-R2: CCATGATTACGCCAAGCTTGAGCCTTACATAGACTGGTAC, SEQ ID No. 47

[0169] Primer sequences for amplifying TYR gene expression cassette

[0170] IntE14-TYR-F: TGGTGAAGAGGAGACTGAAATGACGCAGTAGGATGTCCTG, SEQ ID No. 48

[0171] TYR-HIS5-R: CTGACCCTAGTTACGGTACACAGATGCATTCTTGGGCGGT, SEQ ID No. 49 Primer sequence for amplifying HIS5 gene expression cassette

[0172] TYR-HIS5-F: GTACCGTAACTAGGGTCAGAGAGACCGGGTTGCGGCGCATT, SEQ ID No.50

[0173] HIS5-IntE14-R: TGGGCTGCAGACTAAATTTACAGATGCATTCTTGGGCGGT, SEQ ID No.51, amplified the primer sequence of the pYlCas9-sgRNA-intE14 fragment containing Cas9 protein and located at the intE14 site

[0174] sgRNA-intE14-F:GCTTCGGTGGACACACACTGGTTTTAGAGCTAGAAAT, SEQ ID No.52

[0175] sgRNA-intE14-R: CAGTGTGTGTCCACCGAAGCTAACCAACCTGCGCCGA, SEQ ID No. 53

[0176] pUC19-intE14-TYR-HIS5 and pYlCas9-sgRNA-intE14 were co-transfected into Yarrowia lipolytica jz102 by the "lithium acetate / ssDNA / PEG3350" thermal transfer method and plated on the leucine-deficient screening plate YNB-leu. After 2-3 days, several transformants were randomly selected in the corresponding liquid YNB-leu medium, cultured at 30°C and 250rpm for 48 hours, and the genome was extracted for PCR verification, and positive transformants were selected to obtain the recombinant strain jz104.

[0177] A single colony of the engineered strain jz104 was picked and inoculated into a test tube containing 2 mL of YPD culture medium, and cultured in a shaker at 250 rpm and 30° C. for 24 hours; the first-level seed liquid was inoculated into a 50 mL conical flask containing 10 mL of YPD culture medium, and cultured for 24 hours with an inoculation amount of 1% (v / v) to obtain a second-level seed liquid; the second-level seed liquid was inoculated into a 250 mL conical flask containing 30 mL of YPD-40 culture medium with an inoculation amount of 5% (v / v). After fermentation under the same conditions for 96 hours, the yield of p-coumaric acid was 463.6 mg / L.

[0178] Example 4. Synthesis of p-coumaric acid by integrating exogenous phenylalanine deamination and hydroxylation pathway

[0179] The exogenous phenylalanine deamination hydroxylation pathway AtPAL2, AtC4H, AtATR2 were integrated into the diacylglycerol transferase DGA1 site. The specific operation is as follows:

[0180] The gene sequences of AtPAL2, AtC4H, and AtATR2 are shown in SEQ ID No.7, 8, and 9, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (https: / / store.sangon.com / ). The AtPAL2, AtC4H, and AtATR2 genes were amplified using the synthetic sequences as templates using primers for amplifying the AtPAL2 gene, primers for amplifying the AtC4H gene, and primers for amplifying the AtATR2 gene (shown in SEQ ID No.54-55, SEQ ID No.56-57, and SEQ ID No.58-59, respectively), and then the expression cassette P was constructed in Escherichia coli DH5α using plasmid pUC19. TEF1 -AtPAL2-T xpr2t / P TEF1 -AtC4H-T xpr2t / P TEF1 -AtATR2-T xpr2t Primers (SEQ ID No. 60-63) for amplifying the upstream homology arm fragment and downstream homology arm fragment of diacylglycerol acyltransferase DGA1 were used with the Yarrowia lipolytica genome as a template to amplify the upstream homology arm fragment and downstream homology arm fragment DGA1Up and DGA1down of diacylglycerol acyltransferase DGA1, and plasmid pUC19 was used to construct the DGA1 site integration vector pUC19-DGA1Up-DGA1down in Escherichia coli DH5α. The gene expression cassettes of AtPAL2, AtC4H and AtATR2 were amplified using primers (SEQ ID No.64-65, SEQ ID No.66-67 and SEQ ID No.68-69, respectively) respectively, and the upstream homology arm fragment and downstream homology arm primers (SEQ ID No.70-71) for amplifying diacylglycerol acyltransferase DGA1 were amplified using pUC19-DGA1Up-DGA1down plasmid with homology arms, and the gene expression cassette fragments of AtPAL2, AtC4H and AtATR2 and the vector pUC19-DGA1Up-DGA1down fragment were cloned in one step to construct plasmid pUC19-ΔDGA1-AtPAL2-AtC4H-AtATR2;

[0181] The gRNA design website chopchop (https: / / chopchop.cbu.uib.no / ) was used to design sgRNA targeting the DGA1 site, and the tool plasmid pylcas9 expressing Cas9 protein and sgRNA was constructed. Using it as a template, primers (SEQ ID No.72-73) were used to amplify the pYlCas9-sgRNA-DGA1 fragment containing Cas9 protein and targeting the DGA1 site. The pYlCas9-sgRNA-DGA1 fragment containing Cas9 protein and targeting the DGA1 site was amplified. The PCR product was recovered after overnight digestion with Dpn I, and the vector fragment was constructed by T5 self-ligation to obtain the pYlCas9-sgRNA-DGA1 plasmid.

[0182] Using plasmid pUC19-ΔDGA1-AtPAL2-AtC4H-AtATR2 as a template, primers (sequences shown in SEQ ID No. 60 and 63) were used to amplify and recover the homology arms and gene expression cassette. The recovered fragments and pYlCas9-sgRNA-DGA1 plasmid were transformed into yeast jz104 using the "lithium acetate / single-stranded DNA / PEG3350" thermal transfer method and coated on the leucine-deficient screening plate YNB-leu. After 2-3 days, several transformants were randomly selected in the corresponding liquid YNB-leu medium, cultured at 30°C and 250rpm for 48 hours, and the genome was extracted for PCR verification, and positive transformants were selected to obtain the recombinant strain jz106.

[0183] SEQ ID No.7 Arabidopsis thaliana derived gene

[0184] DNA sequence of AtPAL2

[0185]

[0186] SEQ ID No.8 Arabidopsis thaliana derived gene

[0187] DNA sequence of AtC4H

[0188]

[0189] SEQ ID No.9 Arabidopsis thaliana derived gene

[0190] DNA sequence of AtATR2

[0191]

[0192] Primer sequences for amplifying the AtPAL2 gene

[0193] AtPAL2-F: TAAGAATCATTCAAAATGGACCAGATCGAGGCCATG, SEQ ID No. 54

[0194] AtPAL2-R: ACAGGCCATGGAGGTACTTAACAGATGGGGATGGGGGCA, SEQ ID No. 55

[0195] Primer sequences for amplifying AtC4H gene

[0196] AtC4H-F: TAAGAATCATTCAAAATGGACCTGCTGCTGCTGGA, SEQ ID No. 56

[0197] AtC4H-R: ACAGGCCATGGAGGTACTTAGCAGTTTCGGGGCTTCA, SEQ ID No. 57

[0198] Primer sequences for amplifying AtATR2 gene

[0199] AtATR2-F: TAAGAATCATTCAAAATGTCTTCTTCTTCCTCTTC, SEQ ID No.58

[0200] AtATR2-R: ACAGGCCATGGAGGTACTTACCAGACGTCTCGCAGGT, SEQ ID No. 59

[0201] Primer sequences for amplifying the upstream homology arm fragment and downstream homology arm of diacylglycerol acyltransferase DGA1

[0202] puc19-dga1-F: GTAAAACGACGGCCAGTGTATGTAGATGGCTTCTCTTCTCCA, SEQ ID No.60

[0203] DGA1-R1: AGCTTTTGTTTTGTGTGACTTG, SEQ ID No.61

[0204] DGA1-F2: AGTCACACAAAACAAAAGCTGAAAACTGCCTGGGTTAGGC, SEQ ID No. 62

[0205] dga1-puc19-R2: CCATGATTACGCCAAGCTTGGCAGTTCTCGTTCTCTTCATGC, SEQ ID No. 63

[0206] Primer sequences for amplifying the AtPAL2 gene expression cassette

[0207] dga1up-AtPAL2-F: CAAGTCACACAAAACAAAAGCTAGAGACCGGGTTTGGCGGC, SEQ ID No. 64

[0208] Feature13-AtPAL2-R: GCTATCTAATTTCGTGGCCCGCATCTCACTTGCGTATGTATGGA, SEQ IDNo.65

[0209] Primer sequences for amplifying the AtC4H gene expression cassette

[0210] Feature13-AtC4H-F: GGCCACGAAATTAGATAGCCAGAGACCGGGTTGGCGGC, SEQ ID No.66

[0211] Feature14-AtC4H-R: TGGACAGGACTAGTTTTAGTCATCTCACTTGCGTATGTATGGA, SEQ IDNo.67

[0212] Primer sequences for amplifying the AtATR2 gene expression cassette

[0213] Feature14-AtATR2-F: ACTAAAACTAGTCCTGTCCACGGAGAGACCGGGTTTGGCGGC, SEQ IDNo.68

[0214] dga1down-AtATR2-R: GCCTAACCCAGGCAGTTTTCCATCTCACTTGCGTATGTATGGA, SEQ IDNo.69

[0215] Amplify the pUC19-DGA1Up-DGA1down primer sequence with homology arms

[0216] dga1down-F: GAAAACTGCCTGGGTTAGGC, SEQ ID No.70

[0217] dga1up-R:AGCTTTTGTTTTGTGTGACTTG, SEQ ID No.71

[0218] Amplify the primer sequence of the pYlCas9-sgRNA-DGA1 fragment containing Cas9 protein and located at the DGA1 site

[0219] sgRNA-dga-F: GTTAGGCTCCAACGGCCACGCCTCGTTTTAGAGCTAGAAAT, SEQ ID No. 72

[0220] sgRNA-dga-R: AACGAGGCGTGGCCGTTGGAGCCTAACCAACCTGGCCCGAC, SEQ ID No. 73

[0221] A single colony of the engineered strain jz106 was picked and inoculated into a test tube containing 2 mL of YPD medium, and cultured in a shaker at 250 rpm and 30°C for 24 h; the first-level seed liquid was inoculated into a 50 mL conical flask containing 10 mL of YPD medium, and cultured for 24 h with an inoculation amount of 1% (v / v) to obtain a second-level seed liquid; the second-level seed liquid was inoculated into a 250 mL conical flask containing 30 mL of YPD-40 medium with an inoculation amount of 5% (v / v). After fermentation under the same conditions for 96 h, the yield of p-coumaric acid was 1719.0 mg / L.

[0222] Example 5. Production of p-coumaric acid by fed-batch fermentation of strain jz106

[0223] The production of p-coumaric acid by fed-batch fermentation in a 5L fermenter is as follows:

[0224] Pick jz106 monoclone from the plate, inoculate into a test tube containing 2 ml of YPD medium, shake culture at 30°C and 220 rpm for 24 hours, transfer to a 500 ml Erlenmeyer flask containing 100 ml of YPD medium, shake culture for 24 hours, and obtain the seed solution required for batch fermentation. All 100 ml of seed solution is transferred to 2 L of fermentation medium, where the components of the fermentation medium are: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 12 ml / L vitamins, and 10 ml / L trace metal salts. The components of the trace metal salt solution are: 5.75g / L ZnSO4*7H2O, 0.32g / L MnCI2, 0.32g / L CuSO4, 0.47g / L CoCl2, 0.48g / L Na2MoO4, 2.9g / L CaCl2·2H2O, 2.8g / L FeSO4*7H2O, 0.5M EDTA. The components of the vitamin solution are: 0.05g / L biotin, 1g / L calcium pantothenate, 1g / L niacin, 25g / L inositol, 1g / L thiamine hydrochloride, 1g / L pyridoxal phosphate, 0.2g / L p-aminobenzoic acid.

[0225] The batch fermentation temperature was 30°C, and ammonia was used to control the pH to 5.5. The feed medium contained 800 g / L glucose. The feed medium was added during the fermentation process to control the glucose concentration of the fermentation broth to be less than 0.1 g / L. The production of products was detected during the fermentation process. Finally, the jz106 strain fermented and produced 29.4 g / L of p-coumaric acid. Figure 3 As shown, it is the highest level reported for the de novo synthesis of p-coumaric acid.

[0226] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. An engineered strain of Yarrowia lipolytica that produces high p-coumaric acid using glucose as a substrate, characterized in that: The engineered bacteria integrate L-tyrosine ammonia lyase gene, DAHP synthase ARO4, DHS1, AROG gene, tyrosine synthase TYR gene, histidine phosphate aminotransferase HIS5 gene, and exogenous phenylalanine deamination hydroxylation pathway phenylalanine ammonia lyase gene, cinnamate hydroxylase gene, and P450 reductase gene.

2. The engineered Yarrowia lipolytica according to claim 1, characterized in that: The L-tyrosine ammonia lyase gene is the L-tyrosine ammonia lyase gene FjTAL from Flavobacterium; among the DAHP synthase ARO4, DHS1, and AROG genes, ARO4 and DHS1 genes are endogenous enzyme genes, and the AROG gene is the EcAROG gene from Escherichia coli; the tyrosine synthase TYR gene and the histidine phosphate aminotransferase HIS5 gene are endogenous genes; the exogenous phenylalanine deamination hydroxylation pathway genes are the phenylalanine ammonia lyase gene AtPAL2, the cinnamate hydroxylase gene AtC4H, and the P450 reductase gene AtATR2 from Arabidopsis thaliana.

3. The engineered Yarrowia lipolytica according to claim 1, characterized in that: The nucleotide sequence of the TAL gene is shown in SEQ ID No.

1.

4. The engineered Yarrowia lipolytica according to claim 1, characterized in that: The nucleotide sequence of the ARO4 gene is shown in SEQ ID No.2; the nucleotide sequence of the DHS1 gene is shown in SEQ ID No.3; and the nucleotide sequence of the AROG gene is shown in SEQ ID No.

4.

5. The engineered Yarrowia lipolytica according to claim 1, characterized in that: The tyrosine synthase TYR gene and HIS5 gene are endogenous genes. The nucleotide sequence of the TYR gene is shown in SEQ ID No.5, and the nucleotide sequence of the HIS5 gene is shown in SEQ ID No.

6.

6. The engineered Yarrowia lipolytica according to claim 1, characterized in that: The phenylalanine ammonia lyase gene is the AtPAL2 gene from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.7; the cinnamate hydroxylase gene is the AtC4H gene from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.8; the P450 reductase gene is the AtATR2 gene from Arabidopsis thaliana, and the nucleotide sequence is shown in SEQ ID No.

9.

7. The method for constructing the Yarrowia lipolytica engineered bacteria that uses glucose as a substrate to produce high p-coumaric acid according to claim 1, characterized in that: The CRISPR / Cas9 targeted integration method was used to integrate the tyrosine ammonia lyase gene TAL into the Yarrowia lipolytica strain, strengthen the DAHP synthase genes ARO4, DHS1 and AROG, overexpress the tyrosine synthase TYR gene and the histidine phosphate aminotransferase HIS5 protease gene, and further integrate the exogenous phenylalanine deamination hydroxylation pathway genes for synthesizing p-coumaric acid, including the phenylalanine ammonia lyase gene, the cinnamate hydroxylase gene and the P450 reductase gene.

8. The construction method according to claim 7, characterized in that: The tyrosine ammonia lyase gene is integrated at two different gene loci to integrate two copies of the tyrosine ammonia lyase gene.

9. The construction method according to claim 7, characterized in that: The steps include: 1) Integrate the L-tyrosine ammonia lyase gene TAL into the intE1 locus of Yarrowia lipolytica; 2) Based on the strain obtained in step 1), overexpressing DAHP synthase ARO4, DHS1, AROG and L-tyrosine ammonia lyase gene TAL at the intC3 site to relieve the upstream DAHP synthesis restriction and increase the TAL copy number; 3) Based on the strain obtained in step 2), the upstream tyrosine synthesis TYR gene and the histidine phosphate aminotransferase HIS5 gene are strengthened at the intE14 site; 4) overexpressing exogenous phenylalanine deamination hydroxylation pathway genes including phenylalanine ammonia lyase gene, cinnamate hydroxylase gene and P450 reductase gene at the diacylglycerol transferase DGA1 site of the strain obtained in step 3).

10. Use of the engineered Yarrowia lipolytica according to any one of claims 1 to 6 in high production of p-coumaric acid using glucose as a substrate.

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