Microorganism capable of improving production level of p-coumaric acid or p-coumaric acid derivative as well as construction method and application of microorganism

Abstract

The invention discloses a microorganism capable of improving the production level of p-coumaric acid or p-coumaric acid derivatives as well as a construction method and application of the microorganism. The construction method comprises the following steps: transforming genetic targets related to metabolic pathways of p-coumaric acid of the microorganism; the target spot is one or a combination of two or more of 4-coumaroyl coenzyme A ligase, hydroxycinnamyl coenzyme A hydratase lyase, hydroxyacyl coenzyme A dehydrogenase, beta-keto-acyl coenzyme A thiolated enzyme and 3-(aryl) acrylate reductase, and is transformed to remove part or all of the activity of genetic target spots related to a coumaric acid metabolic pathway. Genetic targets related to metabolic pathways of p-coumaric acid are modified, so that the degree of degrading p-coumaric acid by microorganisms is reduced, and the yield and / or production efficiency of p-coumaric acid and p-coumaric acid derivatives produced by the microorganisms are / is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives and a construction method and application thereof. Background Art

[0002] p-Coumaric acid ( p -Coumaric acid, p -CA) is a compound with multiple pharmacological effects such as antibacterial, anti-inflammatory, and antioxidant effects. It is also an important precursor metabolite of many compounds including phenolic acids (representative substances include caffeic acid, ferulic acid, etc.), flavonoids (representative substances include naringenin, catechins, etc.), stilbenes (representative substances include resveratrol, silver pinot nouril, etc.) and coumarins (representative substances include umbelliferone, scopolamine, etc.). At present, the production of p-coumaric acid and p-coumaric acid derivatives mainly relies on plant extraction. However, the planting area of ​​economic crops used as extraction raw materials is limited, the cultivation cycle is long, and they are easily affected by climate and seasonal changes. The sufficient and stable supply of p-coumaric acid and its derivatives cannot be guaranteed. Compared with the production method of plant extraction, the method of constructing a microbial cell factory to produce coumaric acid and its derivatives is more environmentally friendly and has a more stable yield. Synthetic biology has achieved the construction of artificial biological systems through bioengineering design, promoted the revolutionary development of natural product biosynthesis, and provided a new strategy for the stable production of natural products. It has been a research hotspot in recent years.

[0003] The key to producing p-coumaric acid and its derivatives through microbial cell factories is to construct high-yield strains. By introducing exogenous synthesis pathways into strains through genetic modification tools, microorganisms can heterologously synthesize p-coumaric acid and its derivatives, but it is still necessary to iteratively design and optimize the metabolic network of the strain to achieve high yields. In the past nearly 20 years, researchers have obtained a series of microbial cell factories for efficient heterologous synthesis of p-coumaric acid and its derivatives by optimizing the source and expression of exogenous pathway genes and increasing the supply of precursor metabolites.

[0004] p-Coumaric acid biodegradation is an important factor restricting the efficient production of p-coumaric acid and its derivatives by host microorganisms. Taking Yarrowia lipolytica, an important host for the production of polyphenolic compounds, as an example, Sáez-Sáez et al. (Sáez-Sáez, J., Wang, G., Marella, E. R., Sudarsan, S., Cernuda Pastor, M., & Borodina, I. (2020). Engineering the oleaginous yeast Yarrowia lipolyticafor 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 Yarrowia lipolytica As a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose. Journal of agricultural and food chemistry, 72(11), 5867–5877.) In the studies on the construction of cell factories for the synthesis of p-coumaric acid and p-coumaric acid derivatives, it was reported that Yarrowia lipolytica has the ability to degrade a large amount of p-coumaric acid, which in turn limits the increase in the production of p-coumaric acid and its derivatives. However, only a few p-coumaric acid degrading enzymes in other species have been reported. The process of p-coumaric acid metabolism in various microorganisms, including Yarrowia lipolytica, and the genes involved in this process are still unclear. Identifying the metabolic pathways and related genes of p-coumaric acid and carrying out corresponding genetic engineering transformation of the strains to reduce and eliminate the degradation and consumption of p-coumaric acid by the strains are the key to improving the level of microbial cells in the synthesis of p-coumaric acid and p-coumaric acid derivatives. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, and a construction method and application thereof, by modifying genetic targets related to the p-coumaric acid metabolic pathway to reduce the degree of microbial degradation of p-coumaric acid, thereby improving 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] To this end, the technical solution provided by the present invention is: A microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, wherein genetic targets related to the p-coumaric acid metabolic pathway of the microorganism are modified, wherein the modification is to remove part or all of the activity of the genetic targets related to the p-coumaric acid metabolic pathway, and the modification method comprises knocking out the corresponding gene, mutating the corresponding gene, RNA interference, gene downregulation or removing the enzyme activity of the genetic target. In the construction method of the microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, the genetic targets related to the p-coumaric acid metabolic pathway have the catalytic ability of one or a combination of two or more of 4-coumaroyl-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).

[0009] Preferably, in the method for constructing a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, the p-coumaric acid metabolic pathway-related genetic targets are one or a combination of two or more of 4-coumaroyl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, and 3-(aryl)acrylate reductase.

[0010] Preferably, in the method for constructing a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, the 4-coumaroyl-CoA ligase comprises an amino acid sequence as shown in SEQ ID NO: 1 or an analog thereof; The gene of hydroxycinnamoyl-CoA hydratase lyase comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or an analog thereof; The hydroxyacyl-CoA dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 4 or an analog thereof; The β-ketoacyl-CoA thiolase comprises the amino acid sequence shown in SEQ ID NO: 5 or an analog thereof; The 3-(aryl)acrylate reductase includes the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7 or an analog thereof.

[0011] Preferably, the analogue of the p-coumaric acid metabolic pathway-related genetic target 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.

[0012] In the above method, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by 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 searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0013] Preferably, in the microorganism with improved 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.

[0014] Preferably, in the microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, the transformation method is to knock out 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.

[0015] Preferably, in the microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, the construction method is to knock out the coding genes of the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5.

[0016] Preferably, the microorganism is a fungus or a bacterium; Preferably, the fungus is selected from the genus Yarrowia, Kluyveromyces, Saccharomyces, Candida, Pichia, Aspergillus or Fusarium; The bacterial cell is selected from the genus Lactobacillus or Bacillus; Preferably, the Yarrowia spp. is Yarrowia lipolytica ( Yarrowia lipolytica )、 Yarrowia alimentaria , Yarrowia Bubula , Yarrowia hollandica, Yarrowia deformans or Yarrowia divulgata Any one of; Preferably, the Kluyveromyces genus is any one of Kluyveromyces marxianus, Kluyveromyces marxianus variant, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickheimii or Kluyveromyces thermotolerans; Preferably, the Bacillus is any one of Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis; Preferably, the Lactobacillus is any one of Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum or Lactobacillus salivarius; More preferably, the microorganism is Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Rhodosporidium toruloides, Pichia fermentans, Kluyveromyces marxianus, Hansenula, Debaryomyces hansenii, Zygosaccharomyces bailii, Wickhamia anomala or Pichia kudria, Trichoderma reesei or Aspergillus niger.

[0017] More preferably, the microorganism is Yarrowia lipolytica ( Yarrowia lipolytica ).

[0018] A method for constructing a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, which is constructed according to any of the contents described above.

[0019] Application of the microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, Specifically, the p-coumaric acid derivatives include compounds with p-coumaric acid and / or p-coumaryl-CoA as metabolic precursors.

[0020] Preferably, the p-coumaric acid derivative is a flavonoid, a phenolic acid, a lignan, or a stilbene; Further, the p-coumaric acid derivative is a stilbene, flavonoid, or pigment platform compound; Further, the phenolic acid compound is selected from p-coumaric acid, caffeic acid, and ferulic acid; the stilbene compound is selected from resveratrol, pinus erythrin, picetol, polydatin, pterostilbene, morin and other resveratrol derivatives; the flavonoid compound is selected from naringenin and its derivatives, preferably, the naringenin and its derivatives are selected from naringenin, baicalein, quercetin, tangerine, luteolin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, 7-O -(2-naphthylmethyl)naringenin, 5-O-methylnaringenin, 7,4′-2-O-methylnaringenin, 7-O-methylnaringenin (sakurain) and isosakurain (4′-O-methylnaringenin); the pigment compound is selected from curcumin, anthocyanin and its derivatives, preferably, the curcumin, anthocyanin and its derivatives are selected from demethoxycurcumin, bisdemethoxycurcumin, cyanidin, pelargonidin, peony pigment, delphinidin, morning glory pigment and malva pigment.

[0021] Preferably, the application comprises the following steps: First, the genetic target sites related to the p-coumaric acid metabolic pathway of Yarrowia lipolytica are modified, wherein the modification is to remove part or all of the activities of the genetic target sites related to the p-coumaric acid metabolic pathway. Then the modified Yarrowia lipolytica is cultured in a culture medium for 2 to 8 days, and p-coumaric acid and p-coumaric acid derivatives are harvested from the fermentation broth; The culture medium includes a carbon source and a nitrogen source, which may be a combination of one or more of the following: (i) the carbon source is selected from a fermentable carbon source or a non-fermentable carbon source. Preferably, the fermentable carbon source includes but is not limited to glucose, fructose, galactose, xylose, and sucrose, and the non-fermentable carbon source includes but is not limited to ethanol, acetic acid and acetate, glycerol, lactate, methanol, and formic acid; (ii) The nitrogen source is selected from an organic nitrogen source or an inorganic nitrogen source. Preferably, the organic nitrogen source includes but is not limited to peptone, yeast powder, and corn steep liquor, and the inorganic nitrogen source includes but is not limited to ammonium sulfate, ammonium chloride, nitrate, and ammonia water.

[0022] (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues and wastes.

[0023] Use of the microorganism or the enzyme 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; The present invention has at least the following beneficial effects: The present invention identifies targets related to the metabolic pathway for degradation of p-coumaric acid by Yarrowia lipolytica: 4-coumaroyl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase and 3-(aryl)acrylate reductase. By combining and modifying the above genes in Yarrowia lipolytica, p-coumaric acid degradation can be reduced by 81.23%. The method is applied to a p-coumaric acid production strain, and the p-coumaric acid yield can be increased by 2.51 times compared with before the modification. The method is applied to a production strain of resveratrol, a derivative of p-coumaric acid, and the efficiency of resveratrol production by microbial monomer cells can be increased to 1.34 times (in mg / L / OD) compared with before the modification.

[0024] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is a graph showing the p-coumaric acid production of Yarrowia lipolytica ST015 and ST198 cultured in MMG and YPD medium in an example of the present invention.

[0026] Figure 2 Graph showing the content of resveratrol synthesized in the microbial monomer cells of Yarrowia lipolytica ST655 and ST200 in the examples of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for illustration: The present invention is based on Yarrowia lipolytica ( Yarrowia lipolytica) as an example, the targets related to the metabolic pathway of p-coumaric acid degradation by Yarrowia lipolytica were identified for the first time: 4-coumaroyl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase and 3-(aryl)acrylate reductase. By combining and modifying the encoding genes of the above enzymes in Yarrowia lipolytica, the degradation of p-coumaric acid can be reduced by 81.23%. The method was applied to the p-coumaric acid production strain, which increased the p-coumaric acid yield by 2.51 times compared with the original strain. The method was applied to the production strain of resveratrol, a derivative of p-coumaric acid, which increased the efficiency of resveratrol production by microbial monomer cells to 1.34 times (in mg / L / OD) compared with the original strain.

[0030] In the content analysis of the following examples, standard products of p-coumaric acid and resveratrol were used respectively, and standard curves were drawn by high performance liquid chromatography to quantitatively analyze the contents of p-coumaric acid and resveratrol in the test samples.

[0031] The Yarrowia lipolytica ST015 in the embodiment is obtained by transforming the Yarrowia lipolytica W29, and the KU70 gene of W29 is replaced by the Cas9 protein encoding gene and the hygromycin resistance gene HygR to facilitate genome editing. The strains involved in the embodiments of the present invention are all obtained by subsequent transformation of this strain.

[0032] Genome editing method for Yarrowia lipolytica strains: Use CRISPR / Cas9 technology to integrate exogenous gene fragments into the target location to achieve genome editing of Yarrowia lipolytica strains. Through the synergistic effect of the SpCas9 protein integrated and expressed in the genome of Yarrowia lipolytica strain ST015 and the guide RNA (gRNA) expressed by the plasmid, DNA breaks are introduced at the target site of the genome, and the repair fragments are integrated at the corresponding site of the genome through homologous recombination. Gene knockout can be achieved using the repair fragment composed of the upstream and downstream fragments of the open reading frame.

[0033] The experimental methods without specific conditions in the examples were carried out according to conventional conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001).

[0034] Example 1: Single gene knockout of genes related to p-coumaric acid degradation in Yarrowia lipolytica Since the p-coumaric acid degradation pathway and genes in Yarrowia lipolytica are unclear, we used bioinformatics prediction to mine genes that may be involved in the p-coumaric acid degradation process in Yarrowia lipolytica. The specific information is shown in Table 1. YALI1_B10231g(SEQ ID NO: 1) There is an annotation of 4-coumaryl-CoA ligase that has not been experimentally verified in the database, and other genes have not been annotated with enzyme names consistent with the present invention. Single gene knockout of the above genes was performed in the ST015 strain to verify whether these genes are involved in the process of degradation of p-coumaric acid by Yarrowia lipolytica. The knockout method is: amplify the upstream and downstream homologous regions of the gene to be knocked out by PCR, and connect the upstream and downstream homologous regions of the gene by overlap PCR to obtain the donor fragment of the gene to be knocked out. Select a suitable gRNA sequence located in the gene to be knocked out to construct a plasmid expressing the gRNA, transform it into ST015 together with the donor fragment, and obtain a single gene knockout strain by colony PCR identification. The knockout strain was cultured in a basal salt medium (MMG) supplemented with 300 mg / L p-coumaric acid, and cultured in a shaker at 30°C and 700 rpm. Take the culture that has been cultured for 48 hours, and use high performance liquid chromatography to determine the remaining amount of p-coumaric acid in the sample. The results are shown in Table 2. At 48 h, ST015 had degraded 96.66% of p-coumaric acid. YALI1_B14044g , YALI1_F29301g , YALI1_C12024g , YALI1_E22238g , YALI1_D19252g , YALI1_E15555g The knockout of all genes significantly slowed down the degradation of p-coumaric acid, indicating that these genes are involved in the degradation of p-coumaric acid in Yarrowia lipolytica.

[0035] Table 1 Degradation targets of p-coumaric acid Table 2 Results of single gene knockout of p-coumaric acid degradation-related genes in Yarrowia lipolytica Example 2: Combinatorial modification of p-coumaric acid degradation-related targets The above-mentioned 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 stacked manner, and the knockout strain was cultured in a basic salt medium (MMG) supplemented with 300 mg / L p-coumaric acid. The culture was taken after 48 hours of cultivation, and 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_C12024gThe superposition knockout of can further reduce the degradation of p-coumaric acid. YALI1_B14044g +△ YALI1_E22238g The degradation of p-coumaric acid can be reduced by 81.23%. This modification method is relatively simple and can reduce the degradation of p-coumaric acid to a large extent.

[0036] Table 3 Combination modification of p-coumaric acid degradation-related targets Example 3: Knocking out p-coumaric acid degradation genes to increase the level of p-coumaric acid synthesis in microbial cells The p-coumaric acid production strain ST115 without knockout of p-coumaric acid degradation gene was compared with the p-coumaric acid production strain ST198 with knockout of p-coumaric acid degradation gene as a demonstration of improving the production of p-coumaric acid by microorganisms. The p-coumaric acid production strain ST115 was obtained by integrating the p-coumaric acid synthesis gene expression cassette into the chromosome E_3 site of Yarrowia lipolytica ST015. The expression cassette consists of: promoter PrTEF1in, derived from Flavobacterium johnsoniae The tyrosine ammonia lyase FjTAL encoding gene (sequence shown in SEQ ID NO: 8), terminator Tpex20. The p-coumaric acid production strain ST198 obtained by the method of the present invention is obtained by knocking out the following genes from Yarrowia lipolytica ST015: YALI1_B14044g , YALI1_D19252g , YALI1_E15556g , YALI1_F29302g and YALI1_E22238g The p-coumaric acid synthesis gene expression cassette was integrated into the chromosome E_3 site. The expression cassette consisted of: promoter PrTEF1in, derived from Flavobacterium johnsoniae The tyrosine ammonia lyase FjTAL encoding gene and the terminator Tpex20. The above strains were cultured in a shaker at 30°C and 700 rpm using MMG and YPD culture medium for 72 hours, and the content of p-coumaric acid in the samples was determined by high performance liquid chromatography. The results are shown in Figure 1 As shown, the p-coumaric acid production strain ST115 without p-coumaric acid degradation gene knockout in MMG medium did not produce p-coumaric acid, while the p-coumaric acid production strain ST198 with p-coumaric acid degradation gene knockout could detect 18.95±0.43 mg / L p-coumaric acid. The p-coumaric acid gene knockout strain had a 2.51-fold increase in production in YPD medium compared to the non-knockout strain.

[0037] Example 4: Knocking out p-coumaric acid degradation genes to improve the efficiency of synthesizing p-coumaric acid derivatives by microbial monomer cells The resveratrol production strain ST655 without knockout of p-coumaric acid degradation gene was compared with the resveratrol production strain ST200 with knockout of p-coumaric acid degradation gene as a demonstration of improving the yield of p-coumaric acid derivatives produced by microorganisms. The resveratrol production strain ST655 was derived from Yarrowia lipolytica and ST015 was integrated with a resveratrol synthesis gene expression cassette at the chromosome C_2 site. The expression cassette consisted of: promoter PrTEF1in, derived from Flavobacterium johnsoniae The tyrosine ammonia lyase FjTAL encoding gene, terminator Tlip2; promoter PrPGD, derived from Arabidopsis thaliana The 4-coumaroyl-CoA ligase At4CL encoding gene (sequence see 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 production strain obtained by the method of the present invention is ST200, which is obtained by knocking out the following genes from Yarrowia lipolytica ST015: YALI1_B14044g , YALI1_D19252g , YALI1_E15556g , YALI1_F29302g and YALI1_E22238g The resveratrol synthesis gene expression cassette was integrated into the chromosome C_2 site. The expression cassette consisted of: promoter PrTEF1in, derived from Flavobacterium johnsoniae The tyrosine ammonia lyase FjTAL encoding gene, terminator Tlip2; promoter PrPGD, derived from Arabidopsis thaliana The 4-coumaryl-CoA ligase At4CL encoding gene, terminator Tcyc; promoter PrTEF1in, derived from Vitis vinifera The resveratrol synthase VvVST encoding gene was cultured in a YPD medium at 30°C and 700 rpm in a shaker for 72 hours, and the contents of p-coumaric acid and resveratrol in the sample were determined by high performance liquid chromatography. The efficiency of resveratrol synthesis by microbial monomer cells is measured in mg / L / OD. Figure 2 As shown, the production efficiency of resveratrol synthesized by microbial monomer cells increased by 1.34 times (ST655 VS. ST200). It can be seen that the method of the present invention can improve the efficiency of synthesizing p-coumaric acid and p-coumaric acid derivatives by microbial monomer cells.

[0038] In summary, the present invention identifies and characterizes the metabolic pathway and related genes for the degradation of p-coumaric acid by Yarrowia lipolytica, and the extent of p-coumaric acid degradation by Yarrowia lipolytica can be reduced by knocking out the p-coumaric acid degradation gene. The p-coumaric acid degradation-related targets are modified in combination to achieve a reduction of 81.23% of p-coumaric acid degradation by wild-type Yarrowia lipolytica. The method is applied to strains producing p-coumaric acid and p-coumaric acid derivatives, and the yield and / or production efficiency of p-coumaric acid and p-coumaric acid derivatives produced by the strains can be increased.

[0039] YALI1_B10231g (SEQ ID NO: 1): MSIIHKSPVPDVQLFYGSWPDLMRTSPHAHNDSKPVVFDFDTKQQLTWKQVWQLSARLRAQLYHKYGIGKPGALAPFHNDPSLGDVVIFYTPNTYSSLPYHLALHDLGATISPASTSYDVKDICHQIVTTDAVVVVAAAEKSEIAREAV QLSGRDVRVVVMEDLINNAPTVAQNDIDSAPHVSLSRDQARAKIAYLGMSSGTSGGLPKAVRLTHFNVTSNCLQVSAAAPNLAQNVVASAVIPTTHIYGLTMFLSVLPYNGSVVIHHKQFNLRDLLEAQKTYKVSLWILVPPVIVQLAKN PMVDEYLDSIRAHVRCIVSGAAPLGGNVVDQVSVRLTGNKEGILPNGDKLVIHQAYGLTESSPIVGMLDPLSDHIDVMTVGCLMPNTEARIVDEEGNDQPAVHVTDTRGIGAAVKRGEKIPSGELWIRGPQIMDGYHKNPESSRESLEP STETYGLQHFQDRWLRTGDVAVIDTFGRVMVVDRTKELIKSMSRQVAPAELEALLLNHPSVNDVAVVGVHNDDNGTESARAFVVLQPGDACDPTTIKHWMDQQVPSYKRLYGGIVVIDTVPKNASGKILRRLLRQRRDDRVWGLAKVAKL YALI1_B14044g (SEQ ID NO: 2): MRSLYINVPGLFPSTSLARETVHHRTEMLRTIRSSSRLGVRAMSTAATRRAAQIGFHTRVPTVVTKAPTLRMQTTPFSSSAPAQTFGDKKYEHILTSTPVPKVALVTLNRPKALNALCTPLIKELNEALQAADADPTIGAIVLTGSEKSFAAGADIKEMKDKTVTSVLNENFIEEWGNMANIKKPIIAAVNGFALGGGCELAMMADIIYAGAKAKFGQPEIKLGVIPGAGGTQRLTRAIGLYRANHYILTGEMFTAQQAADWGLAAKVYEPAQLVDESVKAAAQIASYGQLAVQAAKASVHQSAEVGLRAGLEFERVRFHGLFGTHDQKEGMAAFAEKREPNFKNE YALI1_F29301g (SEQ ID NO:3): MLRTISRTRTMVPSRHLISYRFFSDVSTTKGETFTLTKHFLDASNTAHIAVYSLNRPEAMNSISKKLLEEFETYINSLAAEGRHQNVTNTRALILSSELPKVFCAGADLKERKTFTDADTAAFLNKLNGTLDTIQSLHMPTITAIQGFALGGGAEISLATDFRVLSDVAQFGLPETRLAILPGAGGTKRLPKLIGYSRALDLVLTGRRVKADEALHLGIANRTGENALETALEMAKLICEGGPIAINAAKMAVRGQSKEWEIAAYNKVVNSEDKFEALSAFKEKRKPIFKGR YALI1_C12024g (SEQ ID NO:4): MFRLTTARIASVRGFSTSASLSKKVDSLSVIGAGQMGLGIALVAANKAGLQVNLIDANQGALDKGLKFMDKLLEKDVGKGRLTSDEAQAVRGRVTGHTNLQSAVADVDMIIEAVPEIPKLKFDIFRDLNEWTQKDTILATNTSSISITKIAAA AGAGAPRVISAHFMNPVPVQKGVEIITGLQTSPETLATTLEVVKRMGKIPSIKDSPGFLANRILMPYINEAITILLETGVGEKEDIDNILKNGCAMPMGPLALADFIGLDTCLAIMRVLYEDTGDSKYRPSVLLNKYVDAGWLGKKSKGGFYDY YALI1_E22238g (SEQ ID NO: 5): MDRLNNLATQLEQNPAKGLDAITSKNPDDVVITAAYRTAHTKGGKGLFKDTSSSELLASLLEGLVKESKIDPKLIGDVVCGNVLAAGAGATEHRAACLVAGIPETVPFVALNRQCSSGLMAVNDVANKIRAGQIDIGIGCGVESMSNQYGPNSVTPFSNKFQNNEEAKKCLIPMGITSENVAAKYNVSRKAQDAFAAKSYEKAAAAQAAGKFDQEILPIKTTTVLDDDNEKEVTVNKDDGIRPGVTAEKLGKLKPAFSAEGTTHAGNASQISDGAGAVLLMRRSVAEKLGQPILAKFVHCKTVGVPPELMGIGPAYAIPAVLEDLGLTVNDVDVFEINEAFASQALFSIQHCGIDESKVNPRGGAIAIGHPLGATGARQFATLLSELKESGKKVGVTSMCIGTGMGAASLVVAE YALI1_D19252g (SEQ ID NO: 6): MLSIRSITRSLPIGSRICQQSAMKASTVRPLALRAYSTRPPVTHFSEEEEMFRDMVSKFADEVIAPKVREMDEAEQMDKTIIQDMFDNGLMGIETPEEFGGAGANFTSAIIVVEELAKVDPSVSVMNDVHNTLVNTCIRSWGSDALRNKYLPQLAAQKVGSFALSEPSSGSDAFAMKSRATKTDDGYILNGSKMWITNAAEAELFIVFANLDPSKGYKGITAFVVEKDMGVQIAKKEQKLGIRASSTCVLNFEDVFIPKENLLGEEGKGYKIAIECLNEGRIGIAAQMLGLAGGAFKKATGYAFNDRKQFGQYIGEFQGMQHQIGQAATEIEAARLLVYNAARLKEAGVPFTKEAAMAKLYASQVAGNVASKAVEWMGGVGFTREETLEKFFRDSKIGAIYEGTSNIQLQTIAKIIQKESA YALI1_E15555g (SEQ ID NO:7): MLRSTLSHMQKSVRVASMARQSMAAARLYSTDTNGSGSGIEEIEELRQAAIDFCAAEITPELQEKTDVDNAFPNHLWEKFGEAGFLGMTAPEKYGGLNLGYLAHSVVMEELSRASGSIALSYSAHSQLCINQLSLHTTPEQGEKYLPDLIAGKKIGALAMSEAGAGSDVVSMKTTAKKVDGGYVLNGTKMWITNGPDADYMVVYAKTDPAAGARGITAFIVEKGMDGFSCARKLDKLGMRGSNTGELIFEDVFVPEGNVLGKINGGVYVLMSGLDLERLVLAAGPIGLMHRALDEALPYVHTRTQFGKPIAHNQLLQGKLADMYTAFASTRAFQYQTAKAADLGQHTSKECASVILLAAEQATQVALDAIQCMGGMGYMMETVPQRLLRDAKLYEIGAGTSEVRRMLIGRAFNKDFA FjTAL(SEQ ID NO:8): NTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPFVQKVKDHLINN At4CL(SEQ ID NO:9): MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIFQNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFVLSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIVCIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMPKFEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL VvVST(SEQ ID NO:10): ASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADYYFRVTKSEHMTELKKKFNRICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPRLGRDAALKALKEWGQPKSKITHLVFCTTSGVEMPGADYKLANLLGLETSVRRVMLYHQGCYAGGTVLRTAKDLAENNAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSSAVIVGSDPDVSIERPLFQLVSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWNSLFWIAHPGGPAILDAVEAKLNLEKKKLEATRHVLSEYGNMSSACVLFILDEMRKKSLKGEKATTGEGLDWGVLFGFGPGLTIETVVLHSVPTVTN Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, characterized in that: The genetic target sites related to the p-coumaric acid metabolic pathway of the microorganism are modified. The construction method of the microorganism is to remove part or all of the activity of the genetic target sites related to the p-coumaric acid metabolic pathway. The modification method includes knocking out the corresponding gene, mutating the corresponding gene, RNA interference, gene downregulation or removing the enzyme activity of the genetic target. The genetic targets related to the p-coumaric acid metabolic pathway have the catalytic ability of one or a combination of two or more of 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).

2. The microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives according to claim 1, characterized in that The genetic targets related to the p-coumaric acid metabolic pathway are one or a combination of two or more of 4-coumaryl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, and 3-(aryl)acrylate reductase.

3. The microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives according to claims 1-2, characterized in that The 4-coumaroyl-CoA ligase comprises the amino acid sequence shown in SEQ ID NO: 1 or an analog thereof; The gene of hydroxycinnamoyl-CoA hydratase lyase comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or an analog thereof; The hydroxyacyl-CoA dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 4 or an analog thereof; The β-ketoacyl-CoA thiolase comprises the amino acid sequence shown in SEQ ID NO: 5 or an analog thereof; The 3-(aryl)acrylate reductase includes the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7 or an analog thereof.

4. The p-coumaric acid metabolic pathway-related genetic target according to any one of claims 1 to 3, characterized in that: The analogs are mutants 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.

5. The microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives according to claims 1-4, characterized in that The p-coumaric acid metabolic pathway-related genetic target 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.

6. The microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives according to claims 1-5, characterized in that The modification method is to knock out 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; Preferably, the modification method is to knock out the genes encoding the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO:

5.

7. The microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives according to claims 1-6, characterized in that The microorganisms are fungi and bacteria; Preferably, the fungus is selected from the genus Yarrowia, Kluyveromyces, Saccharomyces, Candida, Pichia, Aspergillus or Fusarium; The bacterial cell is selected from the genus Lactobacillus or Bacillus; Preferably, the Yarrowia spp. is Yarrowia lipolytica ( Yarrowia lipolytica )、 Yarrowia.alimentaria , Yarrowia Bubula , Yarrowia hollandica, Yarrowia deformans or Yarrowia divulgata Any one of; Preferably, the Kluyveromyces genus is any one of Kluyveromyces marxianus, Kluyveromyces marxianus variant, Kluyveromyces lactis, Kluyveromyces hubeiensis, Kluyveromyces wickheimii or Kluyveromyces thermotolerans; Preferably, the Bacillus is any one of Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis; Preferably, the Lactobacillus is any one of Lactobacillus delbrueckii bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum or Lactobacillus salivarius; More preferably, the microorganism is Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Rhodosporidium toruloides, Pichia fermentans, Kluyveromyces marxianus, Hansenula, Debaryomyces hansenii, Zygosaccharomyces bailii, Wickhamia anomala or Pichia kudria, Trichoderma reesei or Aspergillus niger; More preferably, the microorganism is Yarrowia lipolytica ( Yarrowia lipolytica ).

8. A method for constructing a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, characterized in that: Constructed according to any one of claims 1 to 7.

9. Use of the microorganism and construction method according to claims 1 to 8 in increasing the yield of p-coumaric acid and p-coumaric acid derivatives, characterized in that: synthesizing the polyphenolic compound by fermenting the microorganism in a suitable culture medium; Preferably, the p-coumaric acid derivatives include compounds with p-coumaric acid and / or p-coumaryl-CoA as metabolic precursors; More preferably, the p-coumaric acid derivative is a flavonoid, a phenolic acid, a lignan, or a stilbene; Further, the p-coumaric acid derivative is a stilbene, flavonoid, or pigment platform compound; Further, the phenolic acid compound is selected from p-coumaric acid, caffeic acid, and ferulic acid; the stilbene compound is selected from resveratrol, pinus erythrin, picetol, polydatin, pterostilbene, morin and other resveratrol derivatives; the flavonoid compound is selected from naringenin and its derivatives, preferably, the naringenin and its derivatives are selected from naringenin, baicalein, quercetin, tangerine, luteolin, myricetin, catechin, epicatechin, isoliquiritigenin, xanthohumol, icariin, 7-O -(2-naphthylmethyl)naringenin, 5-O-methylnaringenin, 7,4′-2-O-methylnaringenin, 7-O-methylnaringenin (sakurain) and isosakurain (4′-O-methylnaringenin); the pigment compound is selected from curcumin, anthocyanin and its derivatives, preferably, the curcumin, anthocyanin and its derivatives are selected from demethoxycurcumin, bisdemethoxycurcumin, cyanidin, pelargonidin, peony pigment, delphinidin, morning glory pigment and malva pigment.

10. The use according to claim 9, characterized in that: The steps include: First, the genetic target sites related to the p-coumaric acid metabolic pathway of the microorganism are modified, characterized in that they are constructed according to any one of claims 1 to 9, Then the modified strain is cultured in a culture medium for 2 to 8 days, and p-coumaric acid and / or p-coumaric acid derivatives are harvested from the culture; The culture medium includes a carbon source and a nitrogen source, which may be a combination of one or more of the following: (i) the carbon source is selected from a fermentable carbon source or a non-fermentable carbon source. Preferably, the fermentable carbon source includes but is not limited to glucose, fructose, galactose, xylose, and sucrose, and the non-fermentable carbon source includes but is not limited to ethanol, acetic acid and acetate, glycerol, lactate, methanol, and formic acid; (ii) the nitrogen source is selected from an organic nitrogen source or an inorganic nitrogen source. Preferably, the organic nitrogen source includes but is not limited to peptone, yeast powder, corn steep liquor, and the inorganic nitrogen source includes but is not limited to ammonium sulfate, ammonium chloride, nitrate and ammonia water; (iii) Biomass containing carbon and nitrogen sources, including but not limited to agricultural, industrial and forestry residues and wastes.

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