Construction method and application of recombinant yarrowia lipolytica with high yield of tyrosol

Abstract

The invention provides a construction method and application of recombinant yarrowia lipolytica with high yield of tyrosol, and belongs to the field of bioengineering. The recombinant yarrowia lipolytica with high yield of tyrosol is named as a yarrowia lipolytica XJ-TL strain, and the recombinant yarrowia lipolytica with high yield of tyrosol is named as a yarrowia lipolytica XJ-TL strain. The recombinant yarrowia lipolytica disclosed by the invention expresses an ethanol dehydrogenase encoding gene, and simultaneously expresses any one or a combination of more of a DAHP synthase mutant encoding gene, a chorismate mutase mutant encoding gene, a glucose 6-phosphate dehydrogenase encoding gene, a transketolase encoding gene and a transaldolase encoding gene; meanwhile, a natural promoter of a pyruvate kinase coding gene is truncated. After the recombinant yarrowia lipolytica XJ-TL strain is subjected to fed-batch fermentation, tyrosol can be synthesized at a relatively high level. The construction method of the recombinant yarrowia lipolytica is easy to operate, and efficient synthesis of tyrosol can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a method for constructing a recombinant Yarrowia lipolytica with high tyrosol production and its application. Background Art

[0002] Tyrosol, scientific name: 4-(2-Hydroxyethyl)phenol, and its chemical structural formula is shown as Formula I:

[0003]

[0004] Tyrosol mainly exists in olive oil and is a powerful natural antioxidant. It has significant effects in anti-hypoxia, anti-fatigue, anti-radiation, anti-cancer, anti-aging, regulating body immunity, etc. It is mainly used as an intermediate in medicine and spice synthesis, such as synthesizing cardiovascular drugs metoprolol, betaxolol, etc. (D’Angelo C, Franceschelli S, Quiles JL, et al. Wide biological role of hydroxytyrosol: possible therapeutic and preventive properties in cardiovascular diseases[J]. Cells, 2020, 9(9):1932.). At the same time, tyrosol can also be used as a flavoring agent for alcoholic beverages and plays an important role in improving the taste of alcoholic beverages, especially in sake, beer, and wine (Chinese Patent CN110452865A). In addition, tyrosol also has antibacterial, anti-corrosion, etc. effects and can extend the shelf life of food to ensure the health and safety of consumers (Almanza-Aguilera E, Davila-Cordova E, -Fort D, et al. Correlation Analysis between Dietary Intake of Tyrosols and Their Food Sources and Urinary Excretion of Tyrosol and Hydroxytyrosol in a European Population[J]. Antioxidants, 2023, 12(3):715.). It is precisely because of the high value of tyrosol that it is highly favored in the market and has broad market prospects.

[0005] Traditionally, tyrosol is mainly obtained by direct extraction from olive leaves, but this method faces problems such as large planting area, long growth cycle and climate influence, and has low economic benefits. There are many existing chemical synthesis processes, such as phenol and its phenol derivative synthesis method, carboxylic acid synthesis method, phenylethanol synthesis method, p-nitrotoluene synthesis method and other processes. There are many synthesis methods for p-hydroxyphenylethanol, but at present, the only method that truly realizes industrial production in China is to use phenol and its derivatives and phenylethanol as raw materials for synthesis, and the two synthesis methods are relatively mature in process research. Taking the phenylethanol synthesis method as an example, most of them adopt the method of protecting the hydroxyl group first, then nitrifying, reducing, diazotizing, and hydrolyzing to obtain p-hydroxyphenylethanol, with a yield of 70%. In this approach, in the process of reducing the nitro group to the amino group, researchers can obtain amines with different yields using different catalysts. The main catalysts are platinum black and tin dichloride, and the yields obtained are 90% and 88% respectively. This process has a high yield, but its disadvantage is that the price of the raw material phenylethanol is expensive and the supply is tight. Other chemical synthesis processes, such as the p-nitrotoluene synthesis method, have relatively low raw material prices, more sources of goods, and lower costs, but the steps are long, the yield is low, and it is unfavorable for the protection of the environment; the p-hydroxystyrene synthesis method has a yield of 96% and a purity of 99%. Both the yield and the purity are very high, and have certain value, but the large amount of inputs in the cost of raw materials has also restricted large-scale industrial production (Chinese patent CN106754607A). In addition, since tyrosol is an important raw material for food, medicine, and health products, the product obtained by chemical method is not popular with people.

[0006] Compared with plant extraction and chemical synthesis, microbial fermentation has a short growth cycle and can be produced around the clock. It also has the advantages of low cost, mild conditions, and less environmental pollution, and is currently a hot topic of research. As an unconventional oil-producing yeast, Yarrowia lipolytica has a natural Ehrlich pathway and a high-throughput pentose phosphate pathway, and as a Crabtree-negative yeast, it is very suitable for industrial production. Therefore, using Yarrowia lipolytica to produce tyrosol is an economical and efficient way. In the prior art, there is a lack of microorganisms that can efficiently produce tyrosol. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a construction method and application of a recombinant Yarrowia lipolytica with high tyrosol production in view of the deficiencies in the prior art.

[0008] In order to solve the above technical problems, the present invention discloses a construction method and application of a recombinant Yarrowia lipolytica with high tyrosol production. The specific technical scheme is as follows:

[0009] A method for constructing a recombinant Yarrowia lipolytica with high tyrosol production, wherein the recombinant Yarrowia lipolytica expresses an ethanol dehydrogenase encoding gene and simultaneously expresses any one or a combination of a DAHP synthase mutant encoding gene, a chorismate mutase mutant encoding gene, a 6-phosphogluconate dehydrogenase encoding gene, a transketolase encoding gene, and a transaldolase encoding gene; preferably, the recombinant Yarrowia lipolytica simultaneously expresses an ethanol dehydrogenase encoding gene, a DAHP synthase mutant encoding gene, a chorismate mutase mutant encoding gene, a 6-phosphogluconate dehydrogenase encoding gene, a transketolase encoding gene, and a transaldolase encoding gene.

[0010] Among them, the ethanol dehydrogenase encoding gene is derived from Saccharomyces cerevisiae, namely ScADH, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0011] Among them, the recombinant Yarrowia lipolytica expresses 1 to 3 copies of the ethanol dehydrogenase encoding gene. Preferably, the recombinant Yarrowia lipolytica expresses 2 copies of the ethanol dehydrogenase encoding gene.

[0012] Among them, the DAHP synthase mutant encoding gene and the chorismate mutase mutant encoding gene are both derived from Saccharomyces cerevisiae; the 6-phosphogluconate dehydrogenase encoding gene, the transketolase encoding gene, and the transaldolase encoding gene are all derived from Yarrowia lipolytica.

[0013] Among them, the nucleotide sequence of the DAHP synthase mutant encoding gene (ScARO4 K229L ) is as shown in SEQ ID NO.2; the nucleotide sequence of the chorismate mutase mutant encoding gene (ScARO7 G141S ) is as shown in SEQ ID NO.3; the nucleotide sequence of the 6-phosphogluconate dehydrogenase encoding gene (YlZWF1) is as shown in SEQ ID NO.4; the nucleotide sequence of the transketolase encoding gene (YlTKT) is as shown in SEQ ID NO.5; the nucleotide sequence of the transaldolase encoding gene (YlTAL) is as shown in SEQ ID NO.6.

[0014] Among them, the recombinant Yarrowia lipolytica simultaneously truncates the natural promoter P of the pyruvate kinase encoding gene (YlPYK) from 500 bp to 300 bp, and the nucleotide sequence of the truncated promoter (P PYK ) is as shown in SEQ ID NO.7. Among them, the natural promoter P of endogenous YlPYK PYK-jd ) PYKThe nucleotide sequence of (the 500 bp of the ATG front end) is shown in SEQ ID NO. 8.

[0015] Preferably, the present invention provides a method for constructing a recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica expresses two copies of the ethanol dehydrogenase-encoding gene ScADH derived from Saccharomyces cerevisiae, and expresses the DAHP synthase mutant-encoding gene ScARO4 derived from Saccharomyces cerevisiae K229L and the chorismate mutase mutant-encoding gene ScARO7 G141S , and simultaneously expresses the 6-phosphogluconate dehydrogenase-encoding gene YlZWF1, the transketolase-encoding gene YlTKT, and the transaldolase-encoding gene YlTAL, and truncates the endogenous promoter P of the pyruvate kinase-encoding gene YlPYK PYK to a length of 300 bp of P PYK-jd ; wherein, the nucleotide sequence of the ScADH is shown in SEQ ID NO. 1, the ScARO4 K229L nucleotide sequence is shown in SEQ ID NO. 2, the ScARO7 G141S nucleotide sequence is shown in SEQ ID NO. 3, the nucleotide sequence of YlZWF1 is shown in SEQ ID NO. 4, the nucleotide sequence of YlTKT is shown in SEQ ID NO. 5, the nucleotide sequence of YlTAL is shown in SEQ ID NO. 6, and the nucleotide sequence of the P PYK-jd is shown in SEQ ID NO. 7.

[0016] Among them, the starting strain of the recombinant Yarrowia lipolytica is Yarrowia lipolytica Po1f in which the KU70 gene has been knocked out. Po1f was purchased from the American Type Culture Collection, with the number ATCC MYA-2613.

[0017] Among them, the encoding gene is integrated into the genome of Yarrowia lipolytica, and the integration site is selected from any one or more of the A08 site, 26s rDNA site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site, SCP2 site or YlSCD site. Preferably, the integration sites are the A08 site, IntA site, IntB site, IntC site, IntD site, IntE site, and IntF site of Yarrowia lipolytica.

[0018] Preferably, the method for constructing the recombinant Yarrowia lipolytica is specifically as follows: integrating the target gene into the yeast genome in the form of a gene expression cassette. The specific steps are as follows:

[0019] (1) Constructing the target gene expression cassette: Amplify the promoter, target gene, and terminator to obtain the promoter gene fragment, target gene fragment, and terminator gene fragment respectively, and simultaneously ligate them into a plasmid vector to obtain a recombinant plasmid vector containing the target gene expression cassette; Preferably, the promoter is any one of the promoters P TEF 、P hp4d 、P TEFin 、P FBA 、P FBAin 、P POX2 、P POT1 or P GPD of Yarrowia lipolytica; the terminator is any one of the terminators T xpr2t 、T mig1t 、T lip2t 、T cyc1t 、T pex3t 、T pex10t or T pex20t of Yarrowia lipolytica; the target gene includes any one of the ethanol dehydrogenase encoding gene, DAHP synthase mutant encoding gene, chorismate mutase mutant encoding gene, 6-phosphoglucose dehydrogenase encoding gene, transketolase encoding gene, or transaldolase encoding gene; Further preferably, the promoter is the promoter P TEFin of Yarrowia lipolytica, and the terminator is the terminator T xpr2t .

[0020] (2) Introduce the recombinant plasmid vector containing the target gene expression cassette into Yarrowia lipolytica, so that the gene expression cassette is integrated into the Yarrowia lipolytica genome;

[0021] Preferably, the construction method includes step (3): constructing a truncated promoter expression cassette, including the 1500 bp upstream homologous arm PPYKjd-up at the position 500 bp upstream of the start codon of the pyruvate kinase encoding gene YlPYK (i.e., the promoter position) and the 1500 bp downstream homologous arm PPYKjd-dm at the position 300 bp downstream of the start codon of YlPYK (i.e., the truncated promoter position), and integrating it into the Yarrowia lipolytica genome obtained in step (2).

[0022] Second, the present invention provides the recombinant Yarrowia lipolytica constructed by the construction method described in the first aspect.

[0023] Third aspect, the present invention provides the application of the recombinant Yarrowia lipolytica described in the second aspect in the fermentation production of tyrosol. Among them, the recombinant Yarrowia lipolytica is inoculated into a fermentation medium and cultured at 28-32 °C for 4-7 days to obtain tyrosol. Preferably, it is cultured at 28 °C for 6 days, and the fermentation medium includes 20 g / L of glucose, 20 g / L of yeast extract, and 20 g / L of peptone.

[0024] Beneficial effects:

[0025] The recombinant Yarrowia lipolytica XJ-TL strain constructed in the present invention is based on Yarrowia lipolytica with the non-homologous recombination gene KU70 knocked out, which enhances its homologous recombination ability. Through the homologous recombination function of Yarrowia lipolytica itself, gene integration is achieved, and the genetic stability of the introduced gene can be greatly improved. The construction method of this recombinant Yarrowia lipolytica is efficient and simple to operate.

[0026] Compared with the prior art, the recombinant Yarrowia lipolytica XJ-TL strain of the present invention overexpresses the genes encoding DAHP synthase mutant, chorismate mutase mutant, and two copies of alcohol dehydrogenase, as well as overexpresses the genes encoding 6-phosphogluconate dehydrogenase, transketolase, and transaldolase. At the same time, the natural promoter of the endogenous pyruvate kinase encoding gene YlPYK of Yarrowia lipolytica is truncated to 300 bp. Experiments prove that this recombinant Yarrowia lipolytica can produce 9262.8 mg / L of tyrosol after fed-batch fermentation, realizing the efficient synthesis of tyrosol in Yarrowia lipolytica. Brief description of the drawings

[0027] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0028] Figure 1 It is the tyrosol synthesis metabolism diagram provided by the embodiment of the present invention. YlZWF1: 6-phosphogluconate dehydrogenase; YlTKT: transketolase; YlTAL: transaldolase; ScARO4 K229L : DAHP synthase mutant; ScARO7 G141S : chorismate mutase mutant; ScADH: alcohol dehydrogenase.

[0029] Figure 2 It is the plasmid map of the recombinant plasmid pUC-A08-ScADH, where A08-up represents the upstream homologous arm of the A08 site, A08-dm represents the downstream homologous arm of the A08 site, TEFin represents the promoter P TEFin xpr2t represents the terminator Txpr2t , LEU represents the 3(β)-isopropylmalate dehydrogenase-encoding gene expression cassette (containing the promoter P endogenous to Yarrowia lipolytica TEFin , 3(β)-isopropylmalate dehydrogenase, and the terminator T xpr2t ), and ScADH6 is the ethanol dehydrogenase-encoding gene from Saccharomyces cerevisiae.

[0030] Figure 3 is the plasmid map of the recombinant plasmid pUC-HUH-IntB-ScARO4 K229L , where intB-up represents the homologous arm upstream of the IntB site, intB-dm represents the homologous arm downstream of the IntB site, TEFin represents the promoter P TEFin , xpr2t represents the terminator T xpr2t , URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (containing the promoter P endogenous to Yarrowia lipolytica TEFin , orotidine-5'-phosphate decarboxylase, and the terminator T xpr2t ), and ScARO4 K229L is the DAHP synthase mutant-encoding gene from Saccharomyces cerevisiae.

[0031] Figure 4 is a comparison of the tyrosol content in the fermentation of recombinant strains 1-9 (YL-1 to YL-9). TL: tyrosol. Specific implementation manners

[0032] The present invention will be further described below through specific examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0033] In the following examples, the YPD liquid medium has the following formula: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0034] The YPD plate containing 5-fluoroorotic acid has the following formula: Dissolve 1 g of 5-fluoroorotic acid powder in 10 mL of dimethyl sulfoxide (DMSO) to obtain a 5-fluoroorotic acid solution. Add 1 volume part of the 5-fluoroorotic acid solution to 99 volume parts of the YPD solid medium (23 g / L agar powder added to the YPD liquid medium) to obtain the YPD plate containing 5-fluoroorotic acid.

[0035] The formula of the screening medium SD-Leu is: glucose 20 g / L, YNB (yeast nitrogen base without amino acids, purchased from BBI Life Sciences) 6.7 g / L, CSM-Leu (complete supplement mixture minus leucine, purchased from MP Biomedicals) 0.67 g / L, agar powder 23 g / L, and the solvent is water.

[0036] The formula of the screening medium SD-Ura is: glucose 20 g / L, YNB (yeast nitrogen base without amino acids, purchased from BBI Life Sciences) 6.7 g / L, CSM-Ura (complete supplement mixture minus uracil, purchased from MP Biomedicals) 0.67 g / L, agar powder 23 g / L, and the solvent is water.

[0037] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection, with the accession number ATCC MYA-2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::HisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1fΔku70, was constructed by knocking out the coding gene KU70 responsible for non-homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2): 63-72).

[0038] The A08-site integration plasmid was obtained by inserting a 2521-bp sequence (upstream homologous arm) upstream of the start codon and a 2031-bp sequence (downstream homologous arm) downstream of the stop codon on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-Leu vector (the construction method is shown in Example 1). The Leu coding gene is between the upstream and downstream homologous arms of the A08 site.

[0039] The IntA site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntA site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG tag-encoding genes are between the upstream and downstream homologous arms of the IntA site.

[0040] The IntB site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntB site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG tag-encoding genes are between the upstream and downstream homologous arms of the IntB site.

[0041] The IntC site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntC site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG tag-encoding genes are between the upstream and downstream homologous arms of the IntC site.

[0042] The IntD site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntD site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG tag-encoding genes are between the upstream and downstream homologous arms of the IntD site.

[0043] The IntE site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntE site on the chromosome of the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG tag-encoding genes are between the upstream and downstream homologous arms of the IntE site.

[0044] The IntF-site integration plasmid was obtained by inserting a 1500-bp sequence (upstream homologous arm) upstream of the start codon and a 1500-bp sequence (downstream homologous arm) downstream of the stop codon of the IntF site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-HisG-Ura-HisG vector (the construction method is shown in Example 1). The two HisG-tag encoding genes are between the upstream and downstream homologous arms of the IntF site.

[0045] In the following examples, the promoter P TEFin and the terminator T xpr2t The nucleotide sequences of are shown in Chinese Patent CN202410521402X.

[0046] The synthetic metabolic map of tyrosol described in the present invention is as Figure 1 shown.

[0047] Example 1 Amplification of Gene Elements and Preparation of Target Plasmids

[0048] (I) Preparation of Target Genes

[0049] According to the nucleotide sequence of the ethanol dehydrogenase-encoding gene (GenBank accession number: NC_001145) from Saccharomyces cerevisiae provided on NCBI, after codon optimization, the optimized ethanol dehydrogenase-encoding gene ScADH (SEQ ID NO.1) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into the plasmid pUC57 (purchased from GenScript), obtaining the plasmid pUC57-ScADH.

[0050] According to the nucleotide sequence of the DAHP synthase mutant-encoding gene from Saccharomyces cerevisiae provided on NCBI, after codon optimization, the optimized DAHP synthase mutant-encoding gene ScARO4 K229L (SEQ ID NO.2) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into the plasmid pUC57 (purchased from GenScript), obtaining the plasmid pUC57-ScARO4 K229L .

[0051] According to the nucleotide sequence of the chorismate mutase mutant-encoding gene from Saccharomyces cerevisiae provided on NCBI, after codon optimization, the optimized chorismate mutase-encoding gene ScARO7G141S (SEQ ID NO.3) and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-ScARO7 G141S .

[0052] Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template and IntC::YlZWF1-F and IntC::YlZWF1-R shown in Table 2 as primers, the target gene encoding glucose-6-phosphate dehydrogenase gene (YlZWF1) was amplified, and its nucleotide sequence is as shown in SEQ ID NO.4.

[0053] Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template and IntE::YlTKT-F and IntE::YlTKT-R shown in Table 2 as primers, the target gene encoding transketolase gene (YlTKT) was amplified, and its nucleotide sequence is as shown in SEQ ID NO.5.

[0054] Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template and IntF::YlTAL-F and IntF::YlTAL-R shown in Table 2 as primers, the target gene encoding transaldolase gene (YlTAL) was amplified, and its nucleotide sequence is as shown in SEQ ID NO.6.

[0055] According to the nucleotide sequence of orotidine-5'-phosphate decarboxylase encoding gene URA of Yarrowia lipolytica (GenBank accession number: AJ306421.1) and HisG tag (GenBank accession number: AF324729.1) provided on NCBI, it was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The two HisG tag encoding gene sequences were inserted into plasmid pUC57, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (consisting of the promoter P TEFin endogenous to Yarrowia lipolytica, orotidine-5'-phosphate decarboxylase encoding gene URA and terminator T xpr2t ) was inserted between the two HisG tag encoding gene sequences to enable Ura marker recycling, obtaining plasmid pUC57-HisG-Ura-HisG.

[0056] According to the nucleotide sequence of the 3(β)-isopropylmalate dehydrogenase-encoding gene LEU of Yarrowia lipolytica provided on NCBI (GenBank accession number: CP061014.1), it was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. An expression cassette of the 3(β)-isopropylmalate dehydrogenase-encoding gene LEU (consisting of the promoter P TEFin endogenous to Yarrowia lipolytica, the 3(β)-isopropylmalate dehydrogenase-encoding gene Leu, and the terminator T xpr2t ) was inserted into the plasmid pUC57 to obtain the plasmid pUC57-Leu.

[0057] (II) Construction of recombinant plasmids

[0058] The structure of the recombinant plasmid is shown in Table 1; the primers used for constructing the recombinant plasmid are shown in Table 2.

[0059] 1. Construction of the recombinant plasmid pUC-A08-ScADH

[0060] The recombinant plasmid pUC-A08-ScADH uses pUC57-Leu as the backbone, inserts the upstream homologous arm A08-up upstream of the start codon and the downstream homologous arm A08-dm downstream of the stop codon at the A08 site in Yarrowia lipolytica Po1fΔku70, and also inserts the ScADH expression cassette (P TEFin -ScADH-T xpr2t ) between the upstream and downstream homologous arms. The LEU expression cassette (containing the promoter P TEFin endogenous to Yarrowia lipolytica, the 3(β)-isopropylmalate dehydrogenase-encoding gene LEU, and the terminator T xpr2t ) is also between the upstream and downstream homologous arms. The specific structure is shown in Figure 2 .

[0061] Using the genomic DNA of Yarrowia lipolytica Po1fΔku70 as a template, the promoter P TEFin and the terminator T xpr2t of the ScADH expression cassette were amplified respectively using the primers in Table 2.

[0062] Using the plasmid pUC57-ScADH as a template, the ScADH gene was amplified using the primers in Table 2.

[0063] The above PCR amplification system was: Primer STAR Max Premix 25 μL, template 1 μL, primer F 2 μL, primer R 2 μL, distilled water 20 μL.

[0064] Among them, PrimerSTAR Max Premix was purchased from Takara Biotechnology (Beijing) Co., Ltd.

[0065] The procedure of the above PCR was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C (extension time = target fragment length / 1 kb, unit: min), and repeating for 30 cycles.

[0066] Each fragment was purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Biosciences Co., Ltd.).

[0067] After digesting the A08-site integration plasmid with the restriction endonuclease SnaBⅠ from NEB, the linearized A08-site integration plasmid was recovered by agarose gel electrophoresis.

[0068] The linearized A08-site integration plasmid and each element (promoter P TEFin , gene ScADH, and terminator T xpr2t ) in the ScADH gene expression cassette constructed in this example were used to achieve one-step cloning using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novoprotein Scientific Inc. The ScADH gene expression cassette was inserted between the upstream and downstream homologous arms of the A08-site integration plasmid to obtain a circular recombinant vector, which was transformed into Escherichia coli DH5α competent cells. Positive recombinant plasmids were screened by LB plates with ampicillin resistance and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-A08-ScADH. After digesting the plasmid pUC-A08-ScADH with the restriction endonuclease NotⅠ from NEB, the linearized pUC-A08-ScADH plasmid was recovered by agarose gel electrophoresis for yeast transformation.

[0069] 2. Construction of recombinant plasmid pUC-HUH-IntA-ScADH

[0070] The recombinant plasmid pUC-HUH-IntA-ScADH was based on pUC57-HisG-Ura-HisG, inserted with the upstream homologous arm IntA-up of the start codon at the IntA site and the downstream homologous arm IntA-dm of the stop codon in Yarrowia lipolytica Po1fΔku70. The ScADH gene expression cassette (P TEFin -ScADH-T xpr2t ) was also inserted between the upstream and downstream homologous arms. The URA expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowia lipolytica TEFin, orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homologous arms. Its construction method is similar to that of recombinant plasmid pUC-A08-ScADH. Using the primers described in Table 2 and taking recombinant plasmid pUC-A08-ScADH as the template, the expression cassette P TEFin -ScADH-T xpr2t was amplified. After digesting the IntA site integration plasmid with the restriction endonuclease SnaBⅠ from NEB company, the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis gel extraction. The expression cassette P TEFin -ScADH-T xpr2t and the linearized IntA site integration plasmid were used to obtain the positive recombinant plasmid pUC-HUH-IntA-ScADH through one-step cloning and verification.

[0071] 3. Construction of recombinant plasmid pUC-HUH-IntA-2ScADH

[0072] Recombinant plasmid pUC-HUH-IntA-2ScADH uses pUC57-HisG-Ura-HisG as the backbone, inserts the upstream homologous arm IntA-up of the IntA site start codon and the downstream homologous arm IntA-dm of the stop codon in Yarrowia lipolytica Po1fΔku70, and also inserts two copies of the ScADH gene expression cassette (P TEFin -ScADH-T xpr2t ) between the upstream and downstream homologous arms. The URA expression cassette containing HisG fragments at both ends (including the promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homologous arms. Its construction method is similar to that of recombinant plasmid pUC-HUH-IntA-ScADH. Using the primers described in Table 2 (sequence numbers 9 - 12) and taking recombinant plasmid pUC-A08-ScADH as the template, the expression cassettes 1-P TEFin -ScADH-T xpr2t and 2-P TEFin -ScADH-T xpr2t were amplified. After digesting the IntA site integration plasmid with the restriction endonuclease EcoRⅠ from NEB company, the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis gel extraction. The expression cassettes 1-P TEFin -ScADH-T xpr2t , 2-P TEFin -ScADH-T xpr2tThe linearized IntA site integration plasmid was used to obtain the positive recombinant plasmid pUC-HUH-IntA-2ScADH containing two copies of ScADH through one-step cloning and verification.

[0073] 4. Construction of recombinant plasmid pUC-HUH-IntB-ScARO4 K229L Construction

[0074] The recombinant plasmid pUC-HUH-IntB-ScARO4 K229L was based on pUC57-HisG-Ura-HisG, into which the upstream homologous arm IntB-up of the start codon and the downstream homologous arm IntB-dm of the stop codon of the IntB site in Yarrowia lipolytica Po1fΔku70 were inserted. Between these upstream and downstream homologous arms, the ScARO4 K229L gene expression cassette (P TEFin -ScARO4 K229L -T xpr2t ) was also inserted. The URA expression cassette containing HisG fragments at both ends (including the promoter P TEFin , orotidine-5'-phosphate decarboxylase, and terminator T xpr2t ) was also between the upstream and downstream homologous arms. Its construction method was similar to that of the recombinant plasmid pUC-A08-ScADH, except that the ScADH expression cassette was replaced with the ScARO4 K229L expression cassette, that is, the primers and templates used for amplifying each element (promoter P TEFin , gene ScARO4 K229L , and terminator T xpr2t ) were different, as shown in Table 2. After digesting the IntB site integration plasmid with the restriction endonuclease SnaBⅠ from NEB, the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. The expression cassette P TEFin -ScARO4 K229L -T xpr2t and the linearized IntB site integration plasmid were used to obtain the positive recombinant plasmid pUC-HUH-IntB-ScARO4 K229L through one-step cloning and verification, as Figure 3 shown.

[0075] 5. Construction of recombinant plasmid pUC-HUH-IntD-ScARO7 G141S Construction

[0076] The recombinant plasmid pUC-HUH-IntD-ScARO7 G141SUsing pUC57-HisG-Ura-HisG as the backbone, the homologous arm IntD-up upstream of the start codon and the homologous arm IntD-dm downstream of the stop codon at the IntD locus of Yarrowia lipolytica Po1fΔku70 were inserted. Between these upstream and downstream homologous arms, the ScARO7 G141S gene expression cassette (P TEFin -ScARO7 G141S -T xpr2t ) was also inserted. The URA expression cassette containing HisG fragments at both ends (containing the endogenous promoter P TEFin of Yarrowia lipolytica, orotidine-5'-phosphate decarboxylase, and terminator T xpr2t ) was also between the upstream and downstream homologous arms. Its construction method was similar to that of the recombinant plasmid pUC-A08-ScADH, except that the ScADH expression cassette was replaced with the ScARO7 G141S expression cassette, that is, the primers and templates used for amplifying each element (promoter P TEFin , gene ScARO7 G141S , and terminator T xpr2t ) were different, as shown in Table 2 in detail. After digesting the IntD locus integration plasmid with the restriction endonuclease SnaBⅠ from NEB, the linearized IntD locus integration plasmid was recovered by agarose gel electrophoresis. The expression cassette P TEFin -ScARO7 G141S -T xpr2t and the linearized IntD locus integration plasmid were used to obtain the positive recombinant plasmid pUC-HUH-IntD-ScARO7 G141S through one-step cloning and verification.

[0077] 6. Construction of the recombinant plasmid pUC-HUH-IntC-YlZWF1

[0078] The recombinant plasmid pUC-HUH-IntC-YlZWF1 uses pUC57-HisG-Ura-HisG as the backbone, inserts the homologous arm IntC-up upstream of the start codon and the homologous arm IntC-dm downstream of the stop codon at the IntC locus of Yarrowia lipolytica Po1fΔku70. Between these upstream and downstream homologous arms, the YlZWF1 gene expression cassette (P TEFin -YlZWF1-T xpr2t ) was inserted. The URA expression cassette containing HisG fragments at both ends (containing the endogenous promoter P TEFin of Yarrowia lipolytica, orotidine-5'-phosphate decarboxylase, and terminator T xpr2t) is also between the upstream and downstream homologous arms. Its construction method is similar to that of recombinant plasmid pUC-A08-ScADH, except that the ScADH expression cassette is replaced with the YlZWF1 expression cassette, that is, the primers and templates used for amplifying each element (promoter P TEFin , gene YlZWF1 and terminator T xpr2t ) are different. See Table 2 for details. After digesting the IntC site integration plasmid with the restriction endonuclease SnaBⅠ from NEB company, the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis gel extraction. The elements in the expression cassette P TEFin -YlZWF1-T xpr2t and the linearized IntC site integration plasmid were used to obtain the positive recombinant plasmid pUC-HUH-IntC-YlZWF1 through one-step cloning and verification.

[0079] 7. Construction of recombinant plasmid pUC-HUH-IntE-YlTKT

[0080] The recombinant plasmid pUC-HUH-IntE-YlTKT uses pUC57-HisG-Ura-HisG as the backbone, inserts the upstream homologous arm IntE-up of the start codon and the downstream homologous arm IntE-dm of the stop codon of the IntE site in Yarrowia lipolytica Po1fΔku70, and also inserts the YlTKT gene expression cassette (P TEFin -YlTKT-T xpr2t ) between the upstream and downstream homologous arms. The URA expression cassette containing HisG fragments at both ends (including the promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homologous arms. Its construction method is similar to that of recombinant plasmid pUC-A08-ScADH, except that the ScADH expression cassette is replaced with the YlTKT expression cassette, that is, the primers and templates used for amplifying each element (promoter P TEFin , gene YlTKT and terminator T xpr2t ) are different. See Table 2 for details. After digesting the IntE site integration plasmid with the restriction endonuclease SnaBⅠ from NEB company, the linearized IntE site integration plasmid was recovered by agarose gel electrophoresis gel extraction. The elements in the expression cassette P TEFin -YlTKT-T xpr2t and the linearized IntE site integration plasmid were used to obtain the positive recombinant plasmid pUC-HUH-IntE-YlTKT through one-step cloning and verification.

[0081] 8. Construction of recombinant plasmid pUC-HUH-IntF-YlTAL

[0082] The recombinant plasmid pUC-HUH-IntF-YlTAL uses pUC57-HisG-Ura-HisG as the backbone, inserts the upstream homologous arm IntF-up of the start codon and the downstream homologous arm IntF-dm of the stop codon at the IntF locus in Yarrowia lipolytica Po1fΔku70. Between these upstream and downstream homologous arms, the YlTAL gene expression cassette (P TEFin -YlTAL-T xpr2t ) is also inserted. The URA expression cassette containing the HisG fragment at both ends (containing the promoter P TEFin of Yarrowia lipolytica, orotidine-5'-phosphate decarboxylase and the terminator T xpr2t ) is also between the upstream and downstream homologous arms. Its construction method is similar to that of the recombinant plasmid pUC-A08-ScADH, except that the ScADH expression cassette is replaced with the YlTAL expression cassette, that is, the primers and templates used for amplifying each element (the promoter P TEFin , the gene YlTAL and the terminator T xpr2t ) are different, as detailed in Table 2. After digesting the IntF locus integration plasmid with the restriction endonuclease SnaBⅠ from NEB, the linearized IntF locus integration plasmid is recovered by agarose gel electrophoresis and gel extraction. The positive recombinant plasmid pUC-HUH-IntF-YlTAL is obtained by one-step cloning and verification of each element in the expression cassette P TEFin -YlTAL-T xpr2t and the linearized IntF locus integration plasmid.

[0083] 9. Construction of the recombinant plasmid pUC-HUH-PPYK-PPYKjd

[0084] The recombinant plasmid pUC-HUH-PPYK-PPYKjd uses pUC57-HisG-Ura-HisG as the backbone, inserts the 1500 bp upstream homologous arm PPYKjd-up at the 500 bp position before the start codon of pyruvate kinase PYK (YALI1_F12842g) in Yarrowia lipolytica Po1fΔku70 (i.e., the promoter P PYK position) and the 1500 bp downstream homologous arm PPYKjd-dm at the 300 bp position before the start codon of PYK (i.e., the truncated promoter P PYK-jd position). Between these upstream and downstream homologous arms, the URA expression cassette containing the HisG fragment at both ends (containing the promoter P TEFin of Yarrowia lipolytica, orotidine-5'-phosphate decarboxylase and the terminator T xpr2t) Using the primers described in Table 2, the upstream and downstream homologous arms PPYKjd-up and PPYKjd-dm were amplified with the genome of Yarrowia lipolytica Po1fΔku70 as the template. After double-digesting the plasmid pUC57-HisG-Ura-HisG with the restriction enzymes EcoRⅠ and HindⅢ from NEB, the linearized pUC57-HisG-Ura-HisG plasmid was recovered by agarose gel electrophoresis. The PPYKjd-up, PPYKjd-dm and the linearized pUC57-HisG-Ura-HisG plasmid were used to obtain the positive recombinant plasmid pUC-HUH-PPYK-PPYKjd through one-step cloning and verification. The nucleotide sequence of the said P PYK-jd is shown in SEQ ID NO.7, and the nucleotide sequence of the endogenous promoter P PYK is shown in SEQ ID NO.8.

[0085] Table 1 Information on the inserted sequences in each recombinant plasmid

[0086]

[0087]

[0088] Table 2 Primer sequences

[0089]

[0090]

[0091]

[0092] Example 2 Construction of recombinant Yarrowia lipolytica (1) Construction of recombinant strain 1 (expressing ScADH)

[0093] The recombinant plasmid pUC-A08-ScADH was introduced into Yarrowia lipolytica Po1fΔku70, and the ScADH expression cassette was integrated into the genomic A08 locus to obtain recombinant strain 1.

[0094] The specific method for constructing recombinant strain 1 is as follows:

[0095] Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium. Competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation, and the linearized recombinant plasmid pUC-A08-ScADH was transformed into the competent cells of Yarrowia lipolytica Po1fΔku70 for homologous recombination. Positive clones were screened using the selection medium SD-Leu, and then PCR identification was performed. The strain with correct PCR identification was the recombinant strain 1.

[0096] (II) Construction of recombinant strains 2 - 3 (multi-copy expression of ScADH)

[0097] The recombinant plasmid pUC-HUH-IntA-ScADH was introduced into recombinant strain 1, and the ScADH expression cassette was integrated into the genomic IntA locus to obtain recombinant strain 2. Then, under the selection pressure of 5-fluoroorotic acid, one HisG tag and the Ura selection marker were lost for use in the next round of Ura selection.

[0098] The recombinant plasmid pUC-HUH-IntA-2ScADH was introduced into recombinant strain 1, and two copies of the ScADH expression cassette were integrated into the genomic IntA locus to obtain recombinant strain 3. Then, under the selection pressure of 5-fluoroorotic acid, one HisG tag and the Ura selection marker were lost for use in the next round of Ura selection.

[0099] The specific method for constructing recombinant strain 2 is as follows:

[0100] (1) Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium, and then competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation. The linearized recombinant plasmid pUC-HUH-IntA-ScADH was transformed into recombinant strain 1 for homologous recombination. Positive clones were screened using the selection medium SD-Ura, and then PCR identification was performed.

[0101] (2) Single colonies of positive clones with correct PCR identification were streaked simultaneously on YPD plates containing 5-fluoroorotic acid and SD-Ura plates. Single colonies that could grow on the YPD plates containing 5-fluoroorotic acid but not on the SD-Ura plates indicated successful loss of the Ura selection marker, and recombinant strain 2 was obtained.

[0102] The construction method of recombinant bacterium 3 is the same as that of recombinant bacterium 2, except that the plasmid used for homologous recombination is correspondingly changed. (III) Construction of recombinant bacteria 4-5 (expressing ScADH, ScARO4 K229L and ScARO7 G141S )

[0103] The recombinant plasmid pUC-HUH-IntB-ScARO4 K229L was introduced into recombinant bacterium 2 that had lost Ura, and the ScARO4 K229L expression cassette was integrated into the IntB site of the genome to obtain recombinant bacterium 4. Then, under the screening pressure of 5-fluoroorotic acid, one HisG tag and the Ura screening marker were lost for the next round of Ura screening. pUC-HUH-IntD-ScARO7 G141S was introduced into recombinant bacterium 4 that had lost Ura, and the ScARO7 G141S expression cassette was integrated into the IntD site of the genome to obtain recombinant bacterium 5. Then, under the screening pressure of 5-fluoroorotic acid, one HisG tag and the Ura screening marker were lost for the next round of Ura screening.

[0104] The construction method of recombinant bacteria 4-5 is the same as that of recombinant bacterium 2, except that the plasmid used for homologous recombination and the host used for transformation need to be correspondingly changed.

[0105] (IV) Construction of recombinant bacteria 6-8 (expressing ScADH, ScARO4 K229L , ScARO7 G141S , YlZWF1, YlTKT and YlTAL)

[0106] The recombinant plasmid pUC-HUH-IntC-YlZWF1 was introduced into recombinant bacterium 5 that had lost Ura, and the YlZWF1 expression cassette was integrated into the IntC site of the genome to obtain recombinant bacterium 6. Then, under the screening pressure of 5-fluoroorotic acid, one HisG tag and the Ura screening marker were lost for the next round of Ura screening. The recombinant plasmid pUC-HUH-IntE-YlTKT was introduced into recombinant bacterium 6 that had lost Ura, and the YlTKT expression cassette was integrated into the IntE site of the genome to obtain recombinant bacterium 7. Then, under the screening pressure of 5-fluoroorotic acid, one HisG tag and the Ura screening marker were lost for the next round of Ura screening. The recombinant plasmid pUC-HUH-IntF-YlTAL was introduced into recombinant bacterium 7 that had lost Ura, and the YlTAL expression cassette was integrated into the IntF site of the genome to obtain recombinant bacterium 8. Then, under the screening pressure of 5-fluoroorotic acid, one HisG tag and the Ura screening marker were lost for the next round of Ura screening.

[0107] (V) Construction of recombinant bacterium 9 (expressing ScADH, ScARO4 K229L, ScARO7 G141S , YlZWF1, YlTKT and YlTAL, truncated PYK promoter)

[0108] The recombinant plasmid pUC-HUH-PPYK-PPYKjd was introduced into the recombinant bacterium 8 that had lost Ura, and the endogenous promoter P of the pyruvate kinase-encoding gene YlPYK PYK was truncated to P with a length of 300 bp PYK-jd to obtain the recombinant bacterium 9.

[0109] The construction method of the recombinant bacterium 9 was the same as that of the recombinant bacterium 2, except that the plasmid used for homologous recombination and the host used for transformation needed to be changed accordingly.

[0110] Example 3 Application of recombinant Yarrowia lipolytica in the production of tyrosol (I) Cultivation of engineered bacteria and product extraction

[0111] Yarrowia lipolytica Po1fΔku70, the initial bacterium, and the recombinant bacteria 1-9 in Example 2 were respectively used to produce tyrosol. The specific method was as follows: The initial bacterium and the recombinant bacteria were activated and cultured in YPD liquid medium at 30 °C and 220 rpm for 16 h to obtain the seed liquid. The seed liquid was inoculated into 50 mL of fermentation medium at an inoculation amount of 1% v / v and shaken at 30 °C and 220 rpm for 5 days. After fermentation, the fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 5 min, and the supernatant was taken for liquid-phase detection of the tyrosol concentration. Subsequently, the remaining samples were decanted, placed in an oven (75 °C) and dried to a constant weight for measuring the biomass.

[0112] The fermentation medium formula was: 20 g / L peptone, 20 g / L yeast extract and 20 g / L glucose, and the solvent was water.

[0113] (II) Qualitative and quantitative analysis of tyrosol

[0114] 1. Detection of tyrosol

[0115] Detection conditions: Diode array detector, equipped with a C18 reverse chromatographic column, mobile phase A liquid was: pure acetonitrile, mobile phase B liquid was: 1 L ddH 2 O + 0.1% v / v formic acid, the mobile phase concentration gradient was: 0 - 5 min, 10% A liquid; 5 - 10 min, 10% - 20% A liquid; 10 - 20 min, A liquid linearly increased from 21% to 95%; 20 - 23 min, 95% A liquid; 23 - 30 min, 10% A liquid. The detection wavelength was 280 nm, the column oven temperature was 30 °C, and the mobile phase flow rate was 1 mL / min.

[0116] After 5 days of fermentation, a certain amount of tyrosol was detected in Yarrowia lipolytica Po1fΔku70, as Figure 4 shown. The tyrosol yields of recombinant strains 1-9 (YL-1 to YL-9) were 100.3, 273.5, 181.7, 627.8, 883.4, 1023.9, 1146.1, 1210.5, and 1452.2 mg / L, respectively. Among them, the recombinant strain 9 had the highest tyrosol yield, reaching 1452.2 mg / L, that is, 1452.2 mg of tyrosol was produced per liter of fermentation broth, which was significantly higher than that of the initial strain and recombinant strain 2. The recombinant strain 9 was named Yarrowia lipolytica XJ-TL strain.

[0117] Table 3 Tyrosol yields of the initial strain and recombinant strains

[0118] Fermentation method Strain Biomass (g / L) Tyrosol production (mg / L) Erlenmeyer flask Initial bacteria 4.52 5.6 Erlenmeyer flask Recombinant bacterium 2 4.96 273.5 Erlenmeyer flask Recombinant bacterium 8 4.81 1210.5 Erlenmeyer flask Recombinant bacterium 9 4.75 1452.2 Fermenter Recombinant bacterium 9 78.42 9262.8

[0119] The recombinant strain 9 was inoculated into 50 mL of seed culture medium (YPD medium) and cultured at 30 °C and 220 rpm for 24 h, and then the seed liquid was inoculated into a 5 L fermenter at an inoculation amount of 5% v / v. 2.5 L of fermentation medium was added to the 5 L fermenter. During the fermentation for 0-48 h, the dissolved oxygen was controlled at 20%; when the fermentation time was more than 48 h, the dissolved oxygen was controlled at 0-5%. During the fermentation process, the pH value was constantly controlled at 5.5 until the end of fermentation. Feedings were carried out at 40 h and 96 h of fermentation, and 800 mL of an aqueous solution containing 11.1 g of ammonium sulfate and 900 g / L of glucose was added each time per liter. The fermentation temperature was 28 °C and the culture time was 6 days. Among them, the components of the fermentation medium were 20 g / L of glucose, 20 g / L of yeast extract, and 20 g / L of peptone. After 5 days of fermentation, the highest tyrosol yield of the recombinant strain 9 reached 9262.8 mg / L, that is, 9262.8 mg of tyrosol was produced per liter of fermentation broth (Table 3), which was significantly higher than that of the initial strain and recombinant strain 2.

[0120] The present invention provides a method for constructing and applying a recombinant Yarrowia lipolytica with high tyrosol production. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica with high tyrosol production, characterized in that: The recombinant Yarrowia lipolytica expresses an alcohol dehydrogenase encoding gene, and simultaneously expresses any one or more combinations of a DAHP synthase mutant encoding gene, a chorismate mutase mutant encoding gene, a 6-phosphate glucose dehydrogenase encoding gene, a transketolase encoding gene, and a transaldolase encoding gene; The alcohol dehydrogenase encoding gene is derived from Saccharomyces cerevisiae, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The construction method according to claim 1, characterized in that: The recombinant Yarrowia lipolytica expresses 1 to 3 copies of alcohol dehydrogenase encoding gene.

3. The construction method according to claim 2, characterized in that: The recombinant Yarrowia lipolytica expresses two copies of alcohol dehydrogenase encoding genes.

4. The construction method according to claim 1, characterized in that: The DAHP synthase mutant encoding gene and the chorismate mutase mutant encoding gene are both derived from Saccharomyces cerevisiae; the 6-phosphate glucose dehydrogenase encoding gene, the transketolase encoding gene and the transaldolase encoding gene are all derived from Yarrowia lipolytica.

5. The construction method according to claim 4, characterized in that: The nucleotide sequence of the gene encoding the DAHP synthase mutant is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the chorismate mutase mutant is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding 6-phosphoglucose dehydrogenase is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the transketolase is shown in SEQ ID NO.5; and the nucleotide sequence of the gene encoding the transaldolase is shown in SEQ ID NO.

6.

6. The construction method according to claim 1, characterized in that: The recombinant Yarrowia lipolytica simultaneously expresses the natural promoter P of the pyruvate kinase encoding gene PYK The promoter was truncated from 500 bp to 300 bp, and the nucleotide sequence of the truncated promoter is shown in SEQ ID NO.

7.

7. The construction method according to claim 1, characterized in that: The recombinant Yarrowia lipolytica is derived from a strain of Yarrowia lipolytica Po1f in which the KU70 gene is knocked out.

8. The recombinant Yarrowia lipolytica constructed by the construction method according to any one of claims 1 to 7.

9. Use of the recombinant Yarrowia lipolytica according to claim 8 in fermentation production of tyrosol.

10. The use according to claim 9, characterized in that: The recombinant Yarrowia lipolytica is inoculated into a fermentation medium and cultured at 28-32° C. for 4-7 days to obtain tyrosol.

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