A yarrowia lipolytica engineering strain with improved p-coumaric acid accumulation level, construction method and application

Abstract

This invention relates to the fields of synthetic biology and biochemical engineering, and provides an engineered *Yersinia lipolytica* strain that enhances coumaric acid accumulation, its construction method, and its applications. The engineered *Yersinia lipolytica* strain of this invention uses *Yersinia lipolytica* as the substrate strain, expresses the tyrosine ammonia-lyase encoding gene RgTAL derived from *Rhodotorula glutinis*, and completely knocks out the aminotransferase encoding gene containing the nucleotide sequence shown in SEQ ID NO 1 (database number YALI0A08734g). RgTAL converts endogenous L-tyrosine to coumaric acid. This invention reduces dependence on complex metabolic regulation and increases the coumaric acid accumulation level in *Yersinia lipolytica*.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and biochemical engineering, and in particular to an engineered strain of Yersinia lipolytica that enhances the accumulation level of coumaric acid, its construction method, and its application. Background Technology

[0002] p-Coumaric acid is a derivative of cinnamic acid with a monohydroxylated benzene ring. Its phenolic hydroxyl structure endows it with various biological activities, including anti-melanogenesis, anti-inflammation, antibacterial, anticancer, and antioxidant effects, which are beneficial to human health. In the pharmaceutical field, p-coumaric acid has wide applications, showing protective effects against carcinogenesis, atherosclerosis, oxidative stress-induced cardiac damage, and neuronal damage, while also exhibiting good anti-inflammatory activity. Furthermore, p-coumaric acid is an important precursor in the synthesis of plant secondary metabolites, participating in the biosynthesis of various compounds such as naringenin, kaempferol, quercetin, resveratrol, and pterostilbene. Therefore, p-coumaric acid is considered a fundamental compound in the pharmaceutical and nutritional health fields, possessing significant application value.

[0003] The preparation methods of p-coumaric acid mainly fall into three categories: natural extraction, chemical synthesis, and biosynthesis. Each method has its advantages and disadvantages, and process optimization, efficiency improvement, and cost control are the core research areas for coumaric acid preparation technology. Currently, natural extraction is mainly used to extract p-coumaric acid from agricultural by-products, achieving comprehensive resource utilization; chemical synthesis is a traditional preparation method with mature technology but environmental problems; biosynthesis, as a green synthesis technology, has become a research hotspot in recent years due to its environmental friendliness and high product purity, and is also the core development direction for future large-scale production.

[0004] In recent years, extensive research has been conducted on the construction of microbial cell factories for the de novo synthesis of coumaric acid, with positive progress achieved. These studies mainly focus on enzyme design and modification to improve catalytic efficiency; (dynamic) metabolic regulation of the cell factory to achieve metabolic flux balance; and deregulation of metabolic flux, such as knocking out regulatory factors and mutating regulated enzymes. However, increasing coumaric acid accumulation in *Yarrowia lipolytica* by knocking out aminotransferase-encoding genes is an unexplored method, and the underlying regulatory mechanisms remain unclear. Summary of the Invention

[0005] The purpose of this invention is to provide an engineered *Yersinia lipolytica* strain that enhances the accumulation of coumaric acid, its construction method, and its application. This invention addresses the problem that existing microbial synthesis technologies for coumaric acid mainly rely on complex implementation through enzyme engineering, dynamic metabolic flux regulation, or multi-target metabolic deregulation. It achieves the effect of enhancing the coumaric acid accumulation level of *Yersinia lipolytica* by knocking out the aminotransferase encoding gene with database number YALI0A08734g.

[0006] To achieve the above objectives, the present invention provides the following solution: A Yersinia lipolytica engineered strain that enhances the accumulation level of coumaric acid, wherein the Yersinia lipolytica engineered strain uses Yersinia lipolytica as the chassis strain, expresses the tyrosine ammonia-lyase encoding gene RgTAL derived from Rhodotorula glutinis, and the aminotransferase encoding gene in the Yersinia lipolytica engineered strain is knocked out in its entirety. The tyrosine ammonia-lyase encoding gene RgTAL is used to convert endogenous L-tyrosine in the engineered Yersinia lipolytica into p-coumaric acid. The database number of the aminotransferase encoding gene is YALI0A08734g, and the aminotransferase encoding gene contains the nucleotide sequence shown in SEQ ID NO 1.

[0007] Optionally, the *Yersinia lipolytica* strain is a *Yersinia lipolytica* strain Po1f that is deficient in both uracil and leucine, and the genotype of the *Yersinia lipolytica* strain Po1f is MATA ura3-302 leu2-270 xpr2-322 axp2-deltaNU49 XPR2::SUC2.

[0008] Optionally, the tyrosine ammonia-lyase encoding gene RgTAL comprises the nucleotide sequence shown in SEQ ID NO 2.

[0009] Optionally, the tyrosine ammonia-lyase encoding gene RgTAL is integrated into the ADH3 site of the engineered Yersinia lipophila genome.

[0010] Optionally, the expression of the tyrosine ammonia-lyase encoding gene RgTAL is controlled by the TEF1 promoter and the XPR2 terminator.

[0011] Optionally, the aminotransferase-encoding gene is knocked out entirely via homologous recombination.

[0012] Optionally, the homologous recombination is performed using a knockout fragment provided by the tool plasmid pUrloxP3-AT, which contains the nucleotide sequence shown in SEQ ID NO 3.

[0013] Optionally, under the same culture conditions, the p-coumaric acid accumulation level of the engineered *Yersinia lipolytica* strain is 8.05-9.14 times that of the *Yersinia lipolytica* strain that has not knocked out the aminotransferase encoding gene and expresses the tyrosine ammonia-lyase encoding gene RgTAL.

[0014] A method for constructing an engineered *Yarrowia lipolytica* strain that enhances coumaric acid accumulation levels includes the following steps: The tyrosine ammonia-lyase encoding gene RgTAL from Rhodotorula glutinis was integrated into the ADH3 site of the Yersinia lipolytica genome to obtain a Yersinia lipolytica strain expressing the tyrosine ammonia-lyase encoding gene RgTAL. The aminotransferase encoding gene with database number YALI0A08734g in the *Yersinia lipolytica* strain expressing the tyrosine ammonia-lyase encoding gene RgTAL was knocked out in its entirety to obtain an engineered *Yersinia lipolytica* strain with increased coumaric acid accumulation. The aminotransferase encoding gene contains the nucleotide sequence shown in SEQ ID NO 1.

[0015] The above-mentioned engineered *Yersinia lipolytica* strain or the engineered *Yersinia lipolytica* strain obtained by the above construction method is used in the production of p-coumaric acid.

[0016] The present invention discloses the following beneficial effects: Existing microbial synthesis technologies for p-coumaric acid mainly revolve around enzyme engineering, dynamic metabolic flux regulation, and metabolic deregulation. These typically require the design and optimization of catalytic enzyme structures, regulatory elements, or multi-gene metabolic networks, involving numerous implementation steps. This invention expresses the tyrosine ammonia-lyase encoding gene RgTAL from *Rhodotorula glutinis* in *Yersinia lipolytica*, enabling the conversion of endogenous L-tyrosine in *Yersinia lipolytica* into p-coumaric acid. Building upon this p-coumaric acid synthesis capacity, the invention further knocks out the entire frame of the aminotransferase encoding gene (database number YALI0A08734g) containing the nucleotide sequence shown in SEQ ID NO 1, thereby reducing the adverse effects of this endogenous aminotransferase encoding gene on p-coumaric acid accumulation levels. Compared to modification methods relying on multi-target metabolic regulation or rational enzymatic design, this invention, by knocking out only one aminotransferase encoding gene, can increase the accumulation level of p-coumaric acid in *Yersinia lipolytica* by 8.05-9.14 times. The genetic modification target is clearly defined, and the increased accumulation level can be directly characterized by the concentration of p-coumaric acid in the fermentation broth. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1The diagram shows the integration verification of the tyrosine ammonia-lyase encoding gene RgTAL and the verification of coumaric acid synthesis. In diagram A, the colony PCR verification result shows the integration of the tyrosine ammonia-lyase encoding gene RgTAL at the ADH3 site; the 1702-bp band indicates a positive clone. C1 and C2 are negative controls using wild-type Po1f as a template. In diagram B, the high-performance liquid chromatography (HPLC) verification result shows the coumaric acid synthesis result; the retention time for coumaric acid is 7.55 min, with wild-type Po1f serving as a negative control.

[0019] Figure 2 This figure shows the validation of the entire AT-encoding gene knockout and the comparison of coumaric acid accumulation levels. In the figure, A represents the validation results of URA3 tag recovery; B represents the results of colony PCR validation of the AT-encoding gene knockout, with a 1223-bp specific band indicating AT-encoding gene knockout; C represents the comparison of coumaric acid accumulation levels between the AT-encoding gene knockout strain ΔADH3::TAL-ΔAT::URA3 and the strain without AT-encoding gene knockout. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The following examples are used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods described below shall be performed in accordance with conventional molecular biology experimental methods in the art; unless otherwise specified, the reagents, culture medium components, enzyme preparations and materials described below may be obtained commercially.

[0022] This invention provides an engineered *Yarrowia lipolytica* strain that enhances coumaric acid accumulation, its construction method, and its application. The engineered *Yarrowia lipolytica* strain uses *Yarrowia lipolytica* as the substrate strain and expresses the expression derived from *Rhodotorula glutinis* (…). Rhodotorula glutinis The RgTAL gene, which encodes tyrosine ammonia-lyase (TAL), was knocked out entirely in the engineered *Yarrowia lipolytica* strain, and the aminotransferase-encoding gene was also knocked out entirely. For ease of description, the aminotransferase-encoding gene will be referred to as the AT-encoding gene below.

[0023] The tyrosine ammonia-lyase encoding gene RgTAL is used to convert endogenous L-tyrosine in engineered *Yarrowia lipolytica* strains into p-coumaric acid. The AT encoding gene, with database ID YALI0A08734g, is labeled as capable of synthesizing an aminotransferase similar to *Schizosaccharomyces cerevisiae*. The AT encoding gene contains the nucleotide sequence shown in SEQ ID NO 1.

[0024] In one embodiment, the *Yersinia lipolytica* strain is Po1f, with ATCC serial number MYA-2613. *Yersinia lipolytica* Po1f is a uracil- and leucine-deficient strain with the genotype MATA ura3-302 leu2-270xpr2-322 axp2-deltaNU49 XPR2::SUC2.

[0025] In one embodiment, the tyrosine ammonia-lyase encoding gene RgTAL is derived from *Rhodotorula glutinis*, and the tyrosine ammonia-lyase encoding gene RgTAL contains the nucleotide sequence shown in SEQ ID NO 2. The tyrosine ammonia-lyase encoding gene RgTAL is integrated into the ADH3 site of the *Yarrowia lipolytica* genome and its expression is controlled by the TEF1 promoter and the XPR2 terminator.

[0026] In one implementation, the entire frame knockout of the AT-encoding gene is achieved via homologous recombination. Homologous recombination is performed using a knockout fragment provided by the tool plasmid pUrloxP3-AT, which contains the nucleotide sequence shown in SEQ ID NO 3. The knockout fragment contains an AT site homologous arm and a URA3 selectable marker gene used for screening positive transformants.

[0027] The genes, plasmids, and strains used in the embodiments of this invention are as follows.

[0028] The tyrosine ammonia-lyase encoding gene RgTAL is derived from Rhodotorula glutinis. The sequence of the tyrosine ammonia-lyase encoding gene RgTAL is shown in SEQ ID NO 2.

[0029] Plasmid pYLXP' contains the TEF1 promoter and the XPR2 terminator. The sequence of plasmid pYLXP' is shown in SEQ ID NO 4.

[0030] The plasmid pUrloxP3-ADH3 is a genome-specific integration tool used for integration at the ADH3 site. The sequence of plasmid pUrloxP3-ADH3 is shown in SEQ ID NO 5.

[0031] The plasmid pUrloxP3-AT is an AT site manipulation tool used for whole-frame knockout of AT-encoding genes. The sequence of plasmid pUrloxP3-AT is shown in SEQ ID NO 3.

[0032] The plasmid pYLXP'-Cre contains the gene encoding the recombinase Cre, which is used to recover the screening marker gene URA3. The sequence of plasmid pYLXP'-Cre is shown in SEQ ID NO 6.

[0033] The *Yarrowia lipolyticis* Po1f strain was a commercially available strain with the ATCC serial number MYA-2613.

[0034] The culture medium used in this embodiment of the invention is as follows.

[0035] SD-Ura medium is a uracil-deficient synthetic medium with the following components: glucose 20 g / L, YNB (amino acid-free and ammonium sulfate) 1.7 g / L, ammonium sulfate 5 g / L, and DO Supplement-Ura 1.29 g / L; agar powder 20 g / L is added when preparing the solid medium.

[0036] SD-Leu medium is a leucine-deficient synthetic medium with the following components: glucose 20 g / L, YNB (amino acid-free and ammonium sulfate) 1.7 g / L, ammonium sulfate 5 g / L, and DO Supplement-Leu 1.29 g / L; agar powder 20 g / L is added when preparing the solid medium.

[0037] The components of YPD medium are: 20 g / L tryptone, 10 g / L yeast extract, and 20 g / L glucose; 20 g / L agar powder is added when preparing solid medium.

[0038] In this embodiment of the invention, high-performance liquid chromatography (HPLC) was used to analyze p-coumaric acid. The HPLC system was an Agilent 1220 system, and the column was an Eclipse Plus C18 column with dimensions of 4.6 mm × 250 mm × 5 μm. A UV detector was used to monitor the synthesis and accumulation levels of p-coumaric acid at a detection wavelength of 290 nm. The column oven temperature was controlled at 40℃. Mobile phase A was an aqueous solution containing 0.1% (v / v) glacial acetic acid, and mobile phase B was a methanol solution containing 0.1% (v / v) glacial acetic acid. The mobile phase flow rate was 1 mL / min, and the injection volume was 5 μL. The gradient elution parameters of the mobile phase are shown in Table 1.

[0039] Table 1. Gradient elution parameters of mobile phase in high performance liquid chromatography The primer sequences used in the embodiments of this invention are shown in Table 2.

[0040] Table 2 Primer sequences used in this invention Example 1: Construction of a *Yarrowia lipolytica* strain expressing the tyrosine ammonia-lyase encoding gene RgTAL This embodiment constructs a Yersinia lipolytica strain capable of synthesizing p-coumaric acid.

[0041] The tyrosine ammonia-lyase encoding gene RgTAL was amplified using primer pairs RgTAL-pYLXP'F and RgTAL-pYLXP'R. The amplified tyrosine ammonia-lyase encoding gene RgTAL was then cloned into plasmid pYLXP' using a one-step cloning kit from Nanjing Novizan Biotechnology Co., Ltd. (C115). Sna Between the BI sites, the recombinant plasmid pYLXP'-RgTAL was obtained. The recombinant plasmid pYLXP'-RgTAL was used... TEF1 promoters and XPR2 Terminator controls the gene encoding tyrosine ammonia-lyase RgTAL The expression.

[0042] use AvrII and SalI The recombinant plasmid pYLXP'-RgTAL was digested with two enzymes, and the fragment containing the TEF1-RgTAL-XPR2 gene cassette was recovered by gel extraction. NheI and SalI The plasmid pUrloxP3-ADH3 was digested with two enzymes, and the plasmid backbone was recovered by gel electrophoresis. The recovered TEF1-RgTAL-XPR2 gene cassette fragment and the plasmid backbone were ligated using T4 ligase to obtain the recombinant plasmid pUrloxP3-ADH3-RgTAL.

[0043] use BamHI and HindIII Double digestion of recombinant plasmid pUrloxP3-ADH3-RgTAL, gel recovery contains ADH3 site homology arm, URA3 Screening for marker genes and fragments containing the TEF1-RgTAL-XPR2 gene cassette. The fragment was 6428 bp in length. The recovered fragment contained... ADH3 Upper and lower homologous arms of the site and URA3 By selecting marker genes, the tyrosine ammonia-lyase encoding gene RgTAL can be integrated into the genome of *Yarrowia lipolytica* through homologous recombination. ADH3 The site was identified, and positive transformants were screened using uracil-deficient plates.

[0044] The recovered 6428-bp fragment was transformed into *Yarrowia lipolyticis* Po1f using the lithium acetate method, and positive transformants were screened on SD-Ura plates. Colony PCR was performed using primer pairs RgTAL-pYLXP'F and RgTAL-pYLXP'R to further screen and confirm positive clones. Results are as follows: Figure 1As shown in Figure A, the 1702-bp band confirms that the strain is a positive clone. The resulting recombinant strain is labeled ΔADH3::TAL-URA3.

[0045] The recombinant strain ΔADH3::TAL-URA3 was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of YPD liquid medium. The incubation was performed at 220 rpm and 30 °C. The wild-type strain Po1f was used as a blank control under the same conditions. High-performance liquid chromatography (HPLC) was used to analyze p-coumaric acid and its accumulation levels.

[0046] The results are as follows Figure 1 As shown in Figure B, strain ΔADH3::TAL-URA3 exhibited a detection peak at the retention time of 7.55 min corresponding to coumaric acid, while the wild-type strain Po1f did not show a corresponding detection peak. This result demonstrates that Yersinia lipolytica can synthesize coumaric acid by integrating the exogenous tyrosine ammonia-lyase encoding gene RgTAL at the ADH3 site. The calculated concentration of coumaric acid synthesized by strain ΔADH3::TAL-URA3 was 12.86 mg / L.

[0047] Example 2: Whole-frame knockout of the AT-encoding gene and detection of coumaric acid accumulation levels In this embodiment, based on the strain ΔADH3::TAL-URA3 obtained in Example 1, the AT coding gene was knocked out in its entirety to increase the accumulation level of coumaric acid in Yersinia lipolytica.

[0048] In the genome of *Yarrowia lipolytica*, the gene numbered YALI0A08734g (https: / / gryc.inrae.fr / db / yarrowia-lipolytica / clib-122 / yali0a / yali0a08734g) is labeled as capable of synthesizing an aminotransferase similar to that of *Schizosaccharomyces cerevisiae*. For ease of description, this invention refers to this gene as the AT-encoding gene, the sequence of which is shown in SEQ ID NO 1.

[0049] To knock out the AT-coding gene, the AT gene was first recovered from strain ΔADH3::TAL-URA3. URA3 Tagging. The plasmid pYLXP'-Cre was transformed into strain ΔADH3::TAL-URA3 using the lithium acetate method, and positive transformants were screened on SD-Leu plates. Clones that grew normally on SD-Leu plates but not on SD-Ura plates were considered clones with the URA3 tag removed. URA3 tag recovery verification results are as follows... Figure 2 As shown in A. Eliminated. URA3 The tagged strain was named ΔADH3::TAL and used in the next round of cloning.

[0050] use Swa I and Hind III. Double digestion of the recombinant plasmid pUrloxP3-AT, gel recovery of homologous arms containing the AT site and... URA3 Fragments containing marker genes were selected. These fragments were 3332 bp in length. The recovered fragments contained upper and lower homologous arms at the AT site. URA3 The marker gene can be integrated into the AT site through homologous recombination, while the AT-encoding gene is knocked out, and positive transformants are screened using uracil-deficient plates.

[0051] The recovered 3332-bp fragment was transformed into the chassis strain ΔADH3::TAL (with the URA3 tag removed) using the lithium acetate method. Positive transformants were screened on SD-Ura plates. Colony PCR was performed using primer pairs URA3T F and AT-Locus-Check R to further screen and confirm positive clones. Results are as follows: Figure 2 As shown in Figure B, the 1223-bp specific band confirms that strains #1 and #5 are positive clones. The resulting recombinant strain is labeled ΔADH3::TAL-ΔAT::URA3.

[0052] The recombinant strain ΔADH3::TAL-ΔAT::URA3 and the strain ΔADH3::TAL-URA3 without the AT coding gene knocked out were inoculated into 250 mL Erlenmeyer flasks containing 25 mL of YPD liquid medium, respectively. The incubator speed was 220 rpm and the temperature was 30 °C. The p-coumaric acid content and accumulation level in the cultures were analyzed by high performance liquid chromatography.

[0053] The results are as follows Figure 2 As shown in Figure C, the coumaric acid accumulation levels of the AT gene-knockout strain ΔADH3::TAL-ΔAT::URA3 ranged from 79.06 to 103.57 mg / L, while those of the strain without AT gene knockout ranged from 8.65 to 12.85 mg / L. The coumaric acid accumulation level of the AT gene-knockout strain ΔADH3::TAL-ΔAT::URA3 was 8.05-9.14 times that of the strain without AT gene knockout. At 192 h, the coumaric acid accumulation level of strain ΔADH3::TAL-ΔAT::URA3 reached 103.57 mg / L, while that of strain ΔADH3::TAL-URA3 was 12.85 mg / L, with the former being 8.05 times higher than the latter.

[0054] The above results indicate that expressing the tyrosine ammonia-lyase encoding gene RgTAL in Yersinia lipolytica can establish a synthetic pathway for the conversion of endogenous L-tyrosine into p-coumaric acid. Based on this synthetic pathway, knocking out the AT encoding gene with the database number YALI0A08734g in its entirety can increase the accumulation level of p-coumaric acid in Yersinia lipolytica.

[0055] The above embodiments are merely specific implementations of the present invention. For those skilled in the art, conventional adjustments can be made to the specific operating conditions without departing from the technical concept of the present invention. Such adjustments do not affect the technical essence of the present invention, which involves expressing the tyrosine ammonia-lyase encoding gene RgTAL and knocking out the entire AT encoding gene to increase the coumaric acid accumulation level in *Yersinia lipolytica*.

[0056] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0057] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A *Yarrowia lipolyticis* engineered strain that enhances the accumulation level of coumaric acid, characterized in that, The engineered *Yersinia lipolytica* strain uses *Yersinia lipolytica* as the chassis strain and expresses the tyrosine ammonia-lyase encoding gene RgTAL derived from *Rhodotorula glutinis*. Furthermore, the aminotransferase encoding gene in the engineered *Yersinia lipolytica* strain is knocked out entirely. The tyrosine ammonia-lyase encoding gene RgTAL is used to convert endogenous L-tyrosine in the engineered Yersinia lipolytica into p-coumaric acid. The database number of the aminotransferase encoding gene is YALI0A08734g, and the aminotransferase encoding gene contains the nucleotide sequence shown in SEQ ID NO 1.

2. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The *Yersinia lipolytica* strain is a *Yersinia lipolytica* strain Po1f that is deficient in both uracil and leucine. The genotype of the *Yersinia lipolytica* strain Po1f is MATA ura3-302 leu2-270 xpr2-322 axp2-deltaNU49 XPR2::SUC2.

3. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The tyrosine ammonia-lyase encoding gene RgTAL contains the nucleotide sequence shown in SEQ ID NO 2.

4. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The tyrosine ammonia-lyase encoding gene RgTAL is integrated into the ADH3 site of the genome of the engineered Yersinia lipophila.

5. The engineered *Yarrowia lipophila* strain according to claim 4, characterized in that, The tyrosine ammonia-lyase encoding gene RgTAL is expressed under the control of the TEF1 promoter and the XPR2 terminator.

6. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, The aminotransferase-encoding gene was knocked out entirely via homologous recombination.

7. The engineered *Yarrowia lipophila* strain according to claim 6, characterized in that, The homologous recombination was performed using a knockout fragment provided by the tool plasmid pUrloxP3-AT, which contains the nucleotide sequence shown in SEQ ID NO 3.

8. The engineered *Yarrowia lipophila* strain according to claim 1, characterized in that, Under the same culture conditions, the p-coumaric acid accumulation level of the engineered *Yersinia lipolytica* strain was 8.05-9.14 times that of the *Yersinia lipolytica* strain that did not knock out the aminotransferase encoding gene and expressed the tyrosine ammonia-lyase encoding gene RgTAL.

9. A method for constructing an engineered strain of *Yarrowia lipolytica* that enhances the accumulation level of coumaric acid, characterized in that, Includes the following steps: The tyrosine ammonia-lyase encoding gene RgTAL from Rhodotorula glutinis was integrated into the ADH3 site of the Yersinia lipolytica genome to obtain a Yersinia lipolytica strain expressing the tyrosine ammonia-lyase encoding gene RgTAL. The aminotransferase encoding gene with database number YALI0A08734g in the *Yersinia lipolytica* strain expressing the tyrosine ammonia-lyase encoding gene RgTAL was knocked out in its entirety to obtain an engineered *Yersinia lipolytica* strain with increased coumaric acid accumulation. The aminotransferase encoding gene contains the nucleotide sequence shown in SEQ ID NO 1.

10. The engineered *Yersinia lipolytica* strain according to any one of claims 1 to 8, or the engineered *Yersinia lipolytica* strain obtained by the construction method according to claim 9, in the production of p-coumaric acid.

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