Microorganism with improved production level of p-coumaric acid or p-coumaric acid derivative, and construction method and application thereof

Abstract

The application discloses a kind of p-coumaric acid or p-coumaric acid derivative production level improved microorganism and its construction method and application, comprising: the genetic target point related to p-coumaric acid metabolic pathway of microorganism is reformed, target point is 4-coumaroyl coenzyme A ligase, hydroxycinnamoyl coenzyme A hydratase lyase, hydroxyacyl coenzyme A dehydrogenase, beta-ketoacyl-coa thiolase, 3- (aryl) propenoate reductase one or more than two combinations, reformation is removed the partial activity or all activity of p-coumaric acid metabolic pathway related genetic target point.The application reduces the degree of degradation of p-coumaric acid by microorganism by reformation p-coumaric acid metabolic pathway related genetic target point, to improve the yield and / or production efficiency of p-coumaric acid and p-coumaric acid derivative produced by microorganism.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a microorganism that improves the production level of p-coumaric acid or p-coumaric acid derivatives, its construction method, and its application. Background Technology

[0002] p-coumaric acid ( p -Coumaric acid p Coumaric acid (CA) is a compound with various pharmacological effects, including antibacterial, anti-inflammatory, and antioxidant properties. It is also an important precursor metabolite for many compounds, including phenolic acids (such as caffeic acid and ferulic acid), flavonoids (such as naringenin and catechins), stilbenes (such as resveratrol and pinocembrin), and coumarins (such as umbelliferone and scopolamine). Currently, the production of coumaric acid and its derivatives mainly relies on plant extraction. However, the planting area of ​​the economic crops used as raw materials is limited, the cultivation cycle is long, and they are easily affected by climate and seasonal changes, making it impossible to guarantee a sufficient and stable supply of coumaric acid and its derivatives. Compared to plant extraction, constructing microbial cell factories to produce coumaric acid and its derivatives is more environmentally friendly and yields more stable output. Synthetic biology, through bioengineering design, has enabled the construction of artificial biological systems, driving a revolutionary development in the biosynthesis of natural products and providing new strategies for the stable production of natural products. It has become a research hotspot in recent years.

[0003] The key to producing coumaric acid and its derivatives through microbial cell factories lies in the construction of high-yield strains. Introducing exogenous synthetic pathways into strains using genetic engineering tools enables heterologous synthesis of coumaric acid and its derivatives, but iterative design and optimization of the strain's metabolic network are still necessary to achieve high yields. Over the past 20 years, researchers have obtained a series of highly efficient microbial cell factories for heterologous synthesis of coumaric acid and its derivatives by optimizing the source and expression of exogenous pathway genes and increasing the supply of precursor metabolites.

[0004] Biodegradation of p-coumaric acid is a significant factor limiting the efficient production of p-coumaric acid and its derivatives by host microorganisms. Taking *Yarrowia lipolytica*, an important host for polyphenol production, as an example, Sáez-Sáez et al. (Sáez-Sáez, J., Wang, G., Marella, ER, Sudarsan, S., Cernuda Pastor, M., & Borodina, I. (2020). Engineering the oleaginous yeast) Yarrowialipolyticafor high-levelresveratrol production. Metabolic engineering, 62, 51–61. ) and Zhu et al. (Zhu, J., Yang, S., Cao, Q., Li, X., Jiao, L., Shi, Y., Yan, Y., Xu, L., Yang, M., Xie, X., Madzak, C., & Yan, J. (2024). Engineering Yarrowialipolytica (From *Journal of Agricultural and Food Chemistry*, 72(11), 5867–5877.) Studies on constructing cell factories for the synthesis of p-coumaric acid and its derivatives have reported that *Yersinia lipolytica* possesses a high capacity to degrade p-coumaric acid, thus limiting the production of p-coumaric acid and its derivatives. However, only a few p-coumaric acid-degrading enzymes from other species have been reported so far, and the processes by which various microorganisms, including *Yersinia lipolytica*, metabolize p-coumaric acid and the genes involved in these processes remain unclear. Identifying and characterizing the metabolic pathways and related genes of p-coumaric acid, and then genetically engineering the strains accordingly to reduce or eliminate the degradation and consumption of p-coumaric acid by the strains, is key to improving the synthesis of p-coumaric acid and its derivatives by microbial cells. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0006] Another objective of this invention is to provide a microorganism for improving the production level of p-coumaric acid or p-coumaric acid derivatives, its construction method, and its application. By modifying the genetic targets related to the p-coumaric acid metabolic pathway, the degree of microbial degradation of p-coumaric acid is reduced, thereby increasing the yield and / or production efficiency of p-coumaric acid and p-coumaric acid derivatives produced by the microorganism.

[0007] Another object of the present invention is to provide enzymes related to the p-coumaric acid metabolic pathway and methods for producing p-coumaric acid and p-coumaric acid derivatives.

[0008] Therefore, the technical solution provided by this invention is as follows:

[0009] A microorganism for improving the production level of coumaric acid or coumaric acid derivatives is disclosed. The microorganism is modified to remove some or all of the activity of the genetic target sites related to the coumaric acid metabolic pathway by means of gene knockout, gene mutation, RNA interference, gene downregulation, or removal of the enzyme activity of the genetic target sites. In the method for constructing the microorganism for improving the production level of coumaric acid or coumaric acid derivatives, the genetic target sites related to the coumaric acid metabolic pathway possess catalytic activity of one or more of the following: 4-coumaryl-CoA ligase (EC 6.2.1.12), hydroxycinnamoyl-CoA hydratase lyase (EC 4.1.2.61), hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), β-ketoacyl-CoA thiolase (EC 2.3.1.16), and 3-(aryl)acrylate reductase (EC 1.3.8.15).

[0010] Preferably, in the method for constructing microorganisms that improve the production level of p-coumaric acid or p-coumaric acid derivatives, the genetic target related to the p-coumaric acid metabolic pathway is one or a combination of two or more of the following: 4-coumaryl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, and 3-(aryl)acrylate reductase.

[0011] Preferably, in the method for constructing microorganisms that improve the production level of p-coumaric acid or p-coumaric acid derivatives, the 4-coumaryl-CoA ligase includes the amino acid sequence shown in SEQ ID NO: 1 or an analogue thereof;

[0012] The gene for hydroxycinnamoyl-CoA hydratase lyase includes the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 or their analogues;

[0013] Hydroxyacyl-CoA dehydrogenases include the amino acid sequence shown in SEQ ID NO: 4 or analogues thereof;

[0014] β-ketoacyl-CoA thiolase includes the amino acid sequence shown in SEQ ID NO: 5 or its analogues;

[0015] 3-(aryl)acrylate reductase includes the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7 or their analogues.

[0016] Preferably, the analogue is a mutant of the corresponding genetic target, and the amino acid sequence of the mutant has at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% sequence identity with the amino acid sequence of the corresponding genetic target.

[0017] In the above methods, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using the blastp tool, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0018] Preferably, in the microorganisms that improve the production level of p-coumaric acid or p-coumaric acid derivatives, the genetic target related to the p-coumaric acid metabolic pathway is one or a combination of two or more of the genes encoding the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0019] Preferably, in the microorganisms that improve the production level of p-coumaric acid or p-coumaric acid derivatives, the modification method is to knock out one or more combinations of the coding genes of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0020] Preferably, in the microorganism that improves the production level of p-coumaric acid or p-coumaric acid derivatives, the construction method is to knock out the coding gene of the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 5.

[0021] Preferably, the microorganism is a fungus or bacteria;

[0022] Preferably, the fungus is selected from the genera *Yersinia*, *Kluyveromyces*, *Saccharomyces*, *Candida*, *Pichia*, *Aspergillus*, or *Fusarium*.

[0023] The bacterial cells are selected from Lactobacillus or Bacillus.

[0024] Preferably, the *Yersinia* species is *Yersinia lipolytica*. Yarrowia lipolytica ), Yarrowia alimentaria , Yarrowia Bubula , Yarrowia hollandica, Yarrowia deformans or Yarrowia divulgata Any one of them;

[0025] Preferably, the Kluyveromyces genus is any one of Kluyveromyces marx, Kluyveromyces marx, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickheimensis, or Kluyveromyces thermostableis.

[0026] Preferably, the Bacillus genus is any one of Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus thermophilus, or Bacillus subtilis;

[0027] Preferably, the Lactobacillus genus is any one of Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus curvatureii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, or Lactobacillus salivarius.

[0028] More preferably, the microorganism is *Yersinia lipophila*, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Pichia cylindrica*, *Pichia fermentata*, *Kluyveromyces marx*, *Hansenula polymorpha*, *Hansenula baliche*, *Bayer zygosacchari*, *Wickham aberrantia*, or *Pichia kudria*, *Trichoderma reesei*, or *Aspergillus niger*.

[0029] More preferably, the microorganism is *Yarrowia lipolyticis* (Yarrowia lipolyticis). Yarrowia lipolytica ).

[0030] A method for constructing microorganisms to improve the production level of p-coumaric acid or p-coumaric acid derivatives, wherein the microorganisms are constructed in accordance with any of the contents described herein.

[0031] The application of microorganisms that improve the production level of p-coumaric acid or p-coumaric acid derivatives.

[0032] Specifically, the p-coumaric acid derivatives include compounds with p-coumaric acid and / or p-coumaryl-CoA as metabolic precursors.

[0033] Preferably, the p-coumaric acid derivative is a flavonoid, phenolic acid, lignan, or stilbene.

[0034] Furthermore, the p-coumaric acid derivative is a stilbene, flavonoid, or pigment platform compound;

[0035] Further, the phenolic acid compounds are selected from p-coumaric acid, caffeic acid, and ferulic acid; the stilbene compounds are selected from resveratrol, pinocembrin, sprucetin, polygalactoside, pterostilbene, morin, and other resveratrol derivatives; the flavonoid compounds are selected from naringenin and its derivatives, preferably, naringenin and its derivatives are selected from naringenin, baicalein, quercetin, hesperidin, luteolin, myricetin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, and 7-O. -(2-naphthylmethyl)naringenin, 5-O-methylnaringenin, 7,4′-2-O-methylnaringenin, 7-O-methylnaringenin (cherry blossom pigment) and isochorin (4′-O-methylnaringenin); the pigment compounds are selected from curcumin, anthocyanins and their derivatives, preferably, the curcumin, anthocyanins and their derivatives are selected from demethoxycurcumin, bisdemethoxycurcumin, cyanidin, pelargonidin, peony pigment, delphinidin, morning glory pigment, and mallow pigment.

[0036] Preferably, the application includes the following steps:

[0037] First, the genetic targets related to the p-coumaric acid metabolic pathway in *Yarrowia lipolytica* were modified by removing some or all of the activity of the genetic targets related to the p-coumaric acid metabolic pathway.

[0038] Then, the yeast was cultured in the modified Yersinia lipolytica medium for 2-8 days, and p-coumaric acid and p-coumaric acid derivatives were harvested from the fermentation broth.

[0039] The culture medium includes a carbon source and a nitrogen source, specifically one or more of the following combinations:

[0040] (i) The carbon source is selected from fermentable carbon sources or non-fermentable carbon sources. Preferably, fermentable carbon sources include, but are not limited to, glucose, fructose, galactose, xylose, and sucrose, while non-fermentable carbon sources include, but are not limited to, ethanol, acetic acid and acetates, glycerol, lactate, methanol, and formic acid.

[0041] (ii) The nitrogen source is selected from organic nitrogen sources or inorganic nitrogen sources. Preferably, organic nitrogen sources include, but are not limited to, peptone, yeast powder, and corn steep liquor, while inorganic nitrogen sources include, but are not limited to, ammonium sulfate, ammonium chloride, nitrate, and ammonia.

[0042] (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues and wastes.

[0043] The application of the microorganisms or the enzymes related to the p-coumaric acid metabolic pathway in increasing the yield of p-coumaric acid and p-coumaric acid derivatives, preferably, the p-coumaric acid derivatives include compounds with p-coumaric acid and / or p-coumaryl-CoA as metabolic precursors;

[0044] The present invention has at least the following beneficial effects:

[0045] This invention identifies the following metabolic targets related to the degradation of p-coumaric acid in *Yersinia lipolytica*: 4-coumaryl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, and 3-(aryl)acrylate reductase. By combining and modifying these genes in *Yersinia lipolytica*, p-coumaric acid degradation can be reduced by 81.23%. Applying this method to p-coumaric acid-producing strains increases p-coumaric acid yield by 2.51 times compared to before modification. Applying this method to resveratrol-producing strains, a derivative of p-coumaric acid, increases the efficiency of resveratrol production by microbial monomer cells by 1.34 times (in mg / L / OD) compared to before modification.

[0046] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0047] Figure 1 shows the coumaric acid production of Yersinia lipophila ST015 and ST198 in MMG and YPD media in the embodiments of the present invention.

[0048] Figure 2 This is a graph showing the content of resveratrol synthesized by microbial monomer cells of Yeast Extract ST655 and ST200 in embodiments of the present invention. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0050] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for illustration:

[0052] This invention uses Yersinia lipophila ( Yarrowia lipolyticaFor example, the following targets were identified for the first time in *Yersinia lipolytica* for the metabolic pathway of p-coumaric acid degradation: 4-coumaryl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, and 3-(aryl)acrylate reductase. By modifying the encoding genes of these enzymes in *Yersinia lipolytica*, p-coumaric acid degradation was reduced by 81.23%. Applying this method to p-coumaric acid-producing strains increased p-coumaric acid production by 2.51 times compared to before modification. Applying this method to resveratrol-producing strains, a derivative of p-coumaric acid, increased the efficiency of resveratrol production by microbial monomer cells by 1.34 times (in mg / L / OD) compared to before modification.

[0053] In the following examples, standards for coumaric acid and resveratrol were used for content analysis. Standard curves were plotted using high performance liquid chromatography (HPLC) to quantitatively analyze the content of coumaric acid and resveratrol in the test samples.

[0054] The *Yersinia lipolytica* ST015 in the examples is a strain obtained by modifying *Yersinia lipolytica* W29. The KU70 gene of W29 was replaced with the Cas9 protein-coding gene and the hygromycin resistance gene HygR to facilitate genome editing. All strains involved in the examples of this invention were obtained by further modification of this strain.

[0055] Genome editing method for *Yersinia lipolytica* strain: Genome editing of *Yersinia lipolytica* strain was achieved by integrating exogenous gene fragments into target sites using CRISPR / Cas9 technology. Through the synergistic effect of the SpCas9 protein integrated and expressed in the genome of *Yersinia lipolytica* strain ST015 and the guide RNA (gRNA) expressed on the plasmid, DNA breaks were introduced at the target site in the genome, and the repair fragments were integrated into the corresponding sites through homologous recombination. Gene knockout could be achieved using repair fragments composed of upstream and downstream fragments connected in an open reading frame.

[0056] Experimental methods not specified in the examples were performed under standard conditions as described in Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 2001).

[0057] Example 1: Single gene knockout of coumaric acid degradation-related genes in Yersinia lipolytica

[0058] Since the degradation pathway and genes of coumaric acid in *Yarrowia lipolytica* are unknown, we used bioinformatics prediction to identify genes that may be involved in the degradation process of coumaric acid in *Yarrowia lipolytica*. Specific information is shown in Table 1. Besides... YALI1_B10231g(SEQ ID NO: 1) A 4-coumaryl-CoA ligase annotation exists in the database but has not been experimentally verified. Other genes are not annotated with enzyme names consistent with those used in this invention. Single-gene knockout of the above genes was performed in the ST015 strain to verify whether these genes are involved in the degradation of p-coumaric acid by *Yarrowia lipolytica*. The knockout method was as follows: homologous regions upstream and downstream of the gene to be knocked out were amplified by PCR, and the upstream and downstream homologous regions were ligated using overlap PCR to obtain the donor fragment of the gene to be knocked out. A plasmid expressing the gRNA was constructed using a suitable gRNA sequence located in the gene to be knocked out, and transformed into ST015 along with the donor fragment. Single-gene knockout strains were identified by colony PCR. The knockout strain was cultured in basal salt medium (MMG) supplemented with 300 mg / L p-coumaric acid at 30°C and 700 rpm in a shaker. After 48 hours of culture, the remaining p-coumaric acid in the sample was determined by high-performance liquid chromatography (HPLC). The results are shown in Table 2. After 48 hours, ST015 had degraded 96.66% of p-coumaric acid. YALI1_B14044g , YALI1_F29301g , YALI1_C12024g , YALI1_E22238g , YALI1_D19252g , YALI1_E15555g Knockout of all genes significantly slowed down the degradation of coumaric acid, indicating that these genes are involved in the degradation of coumaric acid by Yeastra lipolytica.

[0059] Table 1. Target sites for coumaric acid degradation

[0060]

[0061] Table 2. Results of single-gene knockout of genes related to coumaric acid degradation in Yersinia lipolytica.

[0062]

[0063] Example 2: Combinatorial modification of targets related to coumaric acid degradation

[0064] The above-verified p-coumaric acid degradation gene YALI1_B14044g (SEQ ID NO: 2), YALI1_ F29301g (SEQ ID NO: 3), YALI1_C12024g (SEQ ID NO: 4), YALI1_E22238g (SEQ ID NO: 5), YALI1_D19252g (SEQ ID NO: 6), YALI1_E15555g(SEQ ID NO: 7) was knocked out in a superimposed manner. The knockout strain was cultured in basal salt medium (MMG) supplemented with 300 mg / L p-coumaric acid. After 48 hours of culture, the p-coumaric acid content in the sample was determined by high performance liquid chromatography. The results are shown in Figure 3. YALI1_B14044g, YALI1_ D19252g, YALI1_E15555g, YALI1_F29301g, YALI1_E22238g, YALI1_C12024g The superposition and knockout can further reduce the degradation of coumaric acid. Among them, △ YALI1_B14044g +△ YALI1_E22238g It can reduce the degradation of coumaric acid by 81.23%. This modification method is relatively simple and can significantly reduce the degradation of coumaric acid.

[0065] Table 3. Targets related to coumaric acid degradation through combined modification

[0066]

[0067] Example 3: Knocking out the p-coumaric acid degradation gene to increase the level of p-coumaric acid synthesis in microbial cells.

[0068] The p-coumaric acid producing strain ST115 (without knockout of the p-coumaric acid degradation gene) was compared with the p-coumaric acid producing strain ST198 (with the p-coumaric acid degradation gene knocked out) to demonstrate improved microbial p-coumaric acid production. The p-coumaric acid producing strain ST115 was obtained from *Yarrowia lipolytica* by integrating a p-coumaric acid synthesis gene expression cassette from ST015 at chromosome E_3. This expression cassette consisted of the promoter PrTEF1in, derived from... Flavobacterium johnsoniae The gene encoding the tyrosine ammonia-lyase FjTAL (sequence shown in SEQ ID NO: 8) is terminated by Tpex20. Using the method described in this invention, a p-coumaric acid-producing strain, ST198, was obtained. This strain was derived from *Yarrowia lipolytica* by knocking out the following gene in ST015: YALI1_B14044g , YALI1_D19252g , YALI1_E15556g , YALI1_F29302g and YALI1_E22238g And a coumaric acid synthesis gene expression cassette is integrated at the E3 site on chromosome 1. This expression cassette consists of a promoter PrTEF1in, derived from... Flavobacterium johnsoniae The gene encoding the tyrosine ammonia-lyase FjTAL was identified, with the terminator Tpex20. The above strains were cultured using MMG and YPD in a shaker at 30°C and 700 rpm for 72 hours. The content of p-coumaric acid in the samples was determined using high-performance liquid chromatography (HPLC). Results are as follows... Figure 1As shown, in MMG medium, no p-coumaric acid was detected in the p-coumaric acid producing strain ST115 without knockout of the p-coumaric acid degradation gene, while 18.95 ± 0.43 mg / L of p-coumaric acid was detectable in the p-coumaric acid producing strain ST198 with knockout of the p-coumaric acid degradation gene. In YPD medium, the strain with knockout of the p-coumaric acid gene produced 2.51 times more p-coumaric acid than the strain without knockout.

[0069] Example 4: Knocking out the p-coumaric acid degradation gene to improve the efficiency of microbial monomer cells in synthesizing p-coumaric acid derivatives.

[0070] The resveratrol-producing strain ST655, which did not have the p-coumaric acid degradation gene knocked out, was compared with the resveratrol-producing strain ST200, which had the p-coumaric acid degradation gene knocked out, to demonstrate the improvement in microbial production of p-coumaric acid derivatives. The resveratrol-producing strain ST655 was developed by lipase inhibitors who integrated a resveratrol synthesis gene expression cassette at the C2 site on chromosome C015. This expression cassette consists of the promoter PrTEF1in, derived from... Flavobacterium johnsoniae The gene encoding the tyrosine ammonia-lyase FjTAL has the terminator Tip2 and the promoter PrPGD, derived from... Arabidopsis thaliana The gene encoding the 4-coumaroyl-CoA ligase At4CL (sequence shown in SEQ ID NO: 9), terminator Tcyc; promoter PrTEF1in, derived from Vitis vinifera The resveratrol synthase VvVST encoding gene (sequence shown in SEQ ID NO: 10). The resveratrol-producing strain ST200 was obtained using the method described in this invention. This strain was obtained by knocking out the following gene in *Yarrowia lipolytica* ST015: YALI1_B14044g , YALI1_D19252g , YALI1_E15556g , YALI1_F29302g and YALI1_E22238g Furthermore, a resveratrol synthesis gene expression cassette is integrated at the C2 site on chromosome 2. This cassette consists of a promoter, PrTEF1in, derived from... Flavobacterium johnsoniae The gene encoding the tyrosine ammonia-lyase FjTAL has the terminator Tip2 and the promoter PrPGD, derived from... Arabidopsis thaliana The gene encoding the 4-coumaroyl-CoA ligase At4CL, terminator Tcyc; promoter PrTEF1in, derived from... Vitis vinifera The resveratrol synthase VvVST encoding gene was determined. The above-mentioned strains were cultured in a YPD culture medium at 30℃ and 700 rpm for 72 hours. The contents of p-coumaric acid and resveratrol in the samples were determined by high-performance liquid chromatography (HPLC). The efficiency of resveratrol synthesis by microbial monomer cells was expressed as mg / L / OD. The results are as follows: Figure 2As shown, the production efficiency of resveratrol synthesized by microbial monomer cells was increased by 1.34 times (ST655 vs. ST200). This demonstrates that the method described in this invention can improve the efficiency of microbial monomer cell synthesis of p-coumaric acid and its derivatives.

[0071] In summary, this invention identifies and determines the metabolic pathway and related genes involved in the degradation of p-coumaric acid by *Yersinia lipolytica*. Knocking out p-coumaric acid degradation genes reduces the degree of p-coumaric acid degradation in *Yersinia lipolytica*. By combining and modifying p-coumaric acid degradation-related targets, an 81.23% reduction in p-coumaric acid degradation was achieved in wild-type *Yersinia lipolytica*. Applying this method to p-coumaric acid and p-coumaric acid derivative-producing strains can improve the yield and / or efficiency of p-coumaric acid and p-coumaric acid derivative production.

[0072] YALI1_B10231g (SEQ ID NO: 1):

[0073] MSIIHKSPVPDVQLFYGSWPDLMRTSPHAHNDSKPVVFDFDTKQQLTWKQVWQLSARLRAQLYHKYGIGKPGALAPFHNDPSLGDVVIFYTPNTYSSLPYHLALHDLGATISPASTSYDVKDICHQIVTTDAVVVVAAAEKSEIAREAV QLSGRDVRVVVMEDLINNAPTVAQNDIDSAPHVSLSRDQARAKIAYLGMSSGTSGGLPKAVRLTHFNVTSNCLQVSAAAPNLAQNVVASAVIPTTHIYGLTMFLSVLPYNGSVVIHHKQFNLRDLLEAQKTYKVSLWILVPPVIVQLAKN PMVDEYLDSIRAHVRCIVSGAAPLGGNVVDQVSVRLTGNKEGILPNGDKLVIHQAYGLTESSPIVGMLDPLSDHIDVMTVGCLMPNTEARIVDEEGNDQPAVHVTDTRGIGAAVKRGEKIPSGELWIRGPQIMDGYHKNPESSRESLEP STETYGLQHFQDRWLRTGDVAVIDTFGRVMVVDRTKELIKSMSRQVAPAELEALLLNHPSVNDVAVVGVHNDDNGTESARAFVVLQPGDACDPTTIKHWMDQQVPSYKRLYGGIVVIDTVPKNASGKILRRLLRQRRDDRVWGLAKVAKL

[0074] YALI1_B14044g(SEQ ID NO:2):

[0075] MRSLYINVPGLFPSTSLARETVHHRTEMLRTIRSSSRLGVRAMSTAATRRAAQIGFHTRVPTVVTKAPTLRMQTTPFSSSAPAQTFGDKKYEHILTSTPVPKVALVTLNRPKALNALCTPLIKELNEALQAADADPTIGAIVLTGSEKSFAAGADIKEMKDKTVTSVLNENFIEEWGNMANIKKPIIAAVNGFALGGGCELAMMADIIYAGAKAKFGQPEIKLGVIPGAGGTQRLTRAIGLYRANHYILTGEMFTAQQAADWGLAAKVYEPAQLVDESVKAAAQIASYGQLAVQAAKASVHQSAEVGLRAGLEFERVRFHGLFGTHDQKEGMAAFAEKREPNFKNE

[0076] YALI1_F29301g (SEQ ID NO:3):

[0077] MLRTISRTRTMVPSRHLISYRFFSDVSTTKGETFTLTKHFLDASNTAHIAVYSLNRPEAMNSISKKLLEEFETYINSLAAEGRHQNVTNTRALILSSELPKVFCAGADLKERKTFTDADTAAFLNKLNGTLDTIQSLHMPTITAIQGFALGGGAEISLATDFRVLSDVAQFGLPETRLAILPGAGGTKRLPKLIGYSRALDLVLTGRRVKADEALHLGIANRTGENALETALEMAKLICEGGPIAINAAKMAVRGQSKEWEIAAYNKVVNSEDKFEALSAFKEKRKPIFKGR

[0078] YALI1_C12024g (SEQ ID NO:4):

[0079] MFRLTTARIASVRGFSTSASLSKKVDSLSVIGAGQMGLGIALVAANKAGLQVNLIDANQGALDKGLKFMDKLLEKDVGKGRLTSDEAQAVRGRVTGHTNLQSAVADVDMIIEAVPEIPKLKFDIFRDLNEWTQKDTILATNTSSISITKIAAA AGAGAPRVISAHFMNPVPVQKGVEIITGLQTSPETLATTLEVVKRMGKIPSIKDSPGFLANRILMPYINEAITILLETGVGEKEDIDNILKNGCAMPMGPLALADFIGLDTCLAIMRVLYEDTGDSKYRPSVLLNKYVDAGWLGKKSKGGFYDY

[0080] YALI1_E22238g (SEQ ID NO: 5):

[0081] MDRLNNLATQLEQNPAKGLDAITSKNPDDVVITAAYRTAHTKGGKGLFKDTSSSELLASLLEGLVKESKIDPKLIGDVVCGNVLAAGAGATEHRAACLVAGIPETVPFVALNRQCSSGLMAVNDVANKIRAGQIDIGIGCGVESMSNQYGPNSVTPFSNKFQNNEEAKKCLIPMGITSENVAAKYNVSRKAQDAFAAKSYEKAAAAQAAGKFDQEILPIKTTTVLDDDNEKEVTVNKDDGIRPGVTAEKLGKLKPAFSAEGTTHAGNASQISDGAGAVLLMRRSVAEKLGQPILAKFVHCKTVGVPPELMGIGPAYAIPAVLEDLGLTVNDVDVFEINEAFASQALFSIQHCGIDESKVNPRGGAIAIGHPLGATGARQFATLLSELKESGKKVGVTSMCIGTGMGAASLVVAE

[0082] YALI1_D19252g (SEQ ID NO: 6):

[0083] MLSIRSITRSLPIGSRICQQSAMKASTVRPLALRAYSTRPPVTHFSEEEEMFRDMVSKFADEVIAPKVREMDEAEQMDKTIIQDMFDNGLMGIETPEEFGGAGANFTSAIIVVEELAKVDPSVSVMNDVHNTLVNTCIRSWGSDALRNKYLPQLAAQKVGSFALSEPSSGSDAFAMKSRATKTDDGYILNGSKMWITNAAEAELFIVFANLDPSKGYKGITAFVVEKDMGVQIAKKEQKLGIRASSTCVLNFEDVFIPKENLLGEEGKGYKIAIECLNEGRIGIAAQMLGLAGGAFKKATGYAFNDRKQFGQYIGEFQGMQHQIGQAATEIEAARLLVYNAARLKEAGVPFTKEAAMAKLYASQVAGNVASKAVEWMGGVGFTREETLEKFFRDSKIGAIYEGTSNIQLQTIAKIIQKESA

[0084] YALI1_E15555g (SEQ ID NO:7):

[0085] MLRSTLSHMQKSVRVASMARQSMAAARLYSTDTNGSGSGIEEIEELRQAAIDFCAAEITPELQEKTDVDNAFPNHLWEKFGEAGFLGMTAPEKYGGLNLGYLAHSVVMEELSRASGSIALSYSAHSQLCINQLSLHTTPEQGEKYLPDLIAGKKIGALAMSEAGAGSDVVSMKTTAKKVDGGYVLNGTKMWITNGPDADYMVVYAKTDPAAGARGITAFIVEKGMDGFSCARKLDKLGMRGSNTGELIFEDVFVPEGNVLGKINGGVYVLMSGLDLERLVLAAGPIGLMHRALDEALPYVHTRTQFGKPIAHNQLLQGKLADMYTAFASTRAFQYQTAKAADLGQHTSKECASVILLAAEQATQVALDAIQCMGGMGYMMETVPQRLLRDAKLYEIGAGTSEVRRMLIGRAFNKDFA

[0086] FjTAL(SEQ ID NO:8):

[0087] NTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPFVQKVKDHLINN

[0088] At4CL(SEQ ID NO:9):

[0089] MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIFQNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFVLSFLAASFRGATATAANPFFTPAEIAKQAKASNTKL IITEARYVDKIKPLQNDDGVVIVCIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMP KFEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL

[0090] VvVST (SEQ ID NO: 10):

[0091] ASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADYYFRVTKSEHMTELKKKFNRICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPRLGRDAALKALKEWGQPKSKITHLVFCTTSGVEMPGADYKLANLLGLETSVRRVMLYHQGCYAGGTVLRTAKDLAENNAGARVLVVCSEITVV TFRGPSEDALDSLVGQALFGDGSSAVIVGSDPDVSIERPLFQLVSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWNSLFWIAHPGGPAILDAVEAKLNLEKKKLEATRHVLSEYGNMSSACVLFILDEMRKKSLKGEKATTGEGLDWGVLFGFGPGLTIETVVLHSVPTVTN

[0092] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A microorganism for improving the production level of p-coumaric acid or p-coumaric acid derivatives, characterized in that: Genetic targets related to the p-coumaric acid metabolic pathway in microorganisms were modified, wherein the microorganism is Yersinia lipophila ( Yarrowia lipolytica The modification method is to knock out the coding gene of the amino acid sequence shown in SEQ ID NO:2 and SEQ ID NO:

5. For example, the amino acid sequence shown in SEQ ID NO:2 is hydroxycinnamoyl-CoA hydratase lyase, and the amino acid sequence shown in SEQ ID NO:5 is β-ketoyl-CoA thiolase.

2. A method for constructing microorganisms to improve the production level of p-coumaric acid or p-coumaric acid derivatives, characterized in that, Construct according to the content described in claim 1.

3. The application of the microorganism as described in claim 1 or the construction method as described in claim 2 in improving the yield of coumaric acid and resveratrol, characterized in that, Includes the following steps: The modified Yersinia lipolytica strain was cultured in a culture medium for 2-8 days, and p-coumaric acid and / or resveratrol were harvested from the culture. The culture medium includes a carbon source and a nitrogen source, specifically one or more of the following combinations: (i) The carbon source is selected from fermentable carbon sources or non-fermentable carbon sources. Fermentable carbon sources include glucose, fructose, galactose, xylose, and sucrose. Non-fermentable carbon sources include ethanol, acetic acid and acetates, glycerol, lactate, methanol, and formic acid. (ii) The nitrogen source is selected from organic or inorganic nitrogen sources. Organic nitrogen sources include peptone, yeast powder, and corn steep liquor. Inorganic nitrogen sources include ammonium sulfate, ammonium chloride, nitrates, and ammonia. (iii) Biomass containing carbon and nitrogen sources, including agricultural, industrial and forestry residues and wastes.

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