Recombinant escherichia coli for synthesizing piperonyl from scratch, and construction method and application thereof

Abstract

This invention belongs to the field of genetic engineering and bioengineering technology, and discloses a recombinant *E. coli* strain capable of de novo synthesis of piperine, its construction method, and its applications. Using *E. coli* MG1655 as the starting strain, this invention constructs a piperine biosynthetic pathway by overexpressing tyrosine ammonia-lyase, carboxylic acid reductase, 4′-phosphopanylthioethylamine transferase, shinol acyltransferase, and eugenol synthase. Further overexpression of DAHP synthase feedback inhibition mutants and prephenylate dehydrogenase can be used to knock out tyrosine metabolism repression genes. tyrR To enhance precursor supply, each heterologous gene was codon-optimized and expressed in modules using different plasmids. This recombinant strain produced piperine by fermentation using glucose as a substrate, achieving a maximum yield of 47.9 mg / L in shake flasks, and can be applied to the microbial fermentation preparation of piperine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and bioengineering technology, and relates to a recombinant Escherichia coli that synthesizes piperonol de novo, its construction method and application. Background Technology

[0002] Chavicol, also known as p-allylphenol, with CAS Registry Number 501-92-8, is a colorless to pale yellow volatile oily liquid. It is an aromatic phenolic compound used in the food and beverage industry as a flavoring agent, and in the pharmaceutical industry as an antifungal and anti-inflammatory active ingredient in dental antibacterial materials and cancer adjuvant therapy products. Furthermore, it can be used as a monomer in the synthesis of high-performance thermosetting plastics and environmental remediation materials, finding wide application in multiple industries including food, pharmaceuticals, and materials.

[0003] Currently, the industrial production of piperine mainly relies on two processes: plant extraction and chemical synthesis. Plant extraction uses naturally occurring piperine-containing plants as raw materials, obtaining the target product through extraction and separation. However, this process is limited by the plant's growth characteristics; the cultivation cycle of the raw materials is long, and the natural accumulation of piperine in plant tissues is low, resulting in low raw material utilization, large fluctuations in extraction rates, and limited overall production capacity, making it difficult to meet the continuously growing industry demand. Chemical synthesis uses chemical raw materials as substrates, synthesizing piperine through multi-step catalytic reactions. This process produces many byproducts, has high costs for separating and purifying the target product, and consumes large amounts of organic solvents, easily causing environmental pollution. This does not meet the requirements of green production development and also limits the large-scale industrial application of this process.

[0004] To address these issues, the industry has attempted to develop microbial synthesis methods for preparing piperine, using low-cost sugars as substrates and synthesizing the target product through microbial metabolism. However, microorganisms themselves do not possess a natural biosynthetic pathway for piperine, requiring the introduction of heterologous synthetic genes to construct artificial metabolic pathways. Furthermore, heterologous genes exhibit poor expression adaptability in host strains, and the host strains themselves suffer from insufficient precursor supply. Additionally, the presence of natural metabolic feedback regulation mechanisms limits the synthesis throughput of the target product, resulting in low piperine yields in existing microbial synthesis attempts, failing to meet the requirements of industrial production. This has become a pressing technical problem in this field. Summary of the Invention

[0005] The technical problem this invention aims to solve is that existing piperine preparation processes suffer from limited plant extraction capacity, low raw material utilization, significant chemical synthesis pollution, and high byproduct separation costs. Furthermore, existing microbial synthesis technologies suffer from poor heterologous synthesis pathway compatibility, insufficient precursor supply, and low piperine yield due to natural metabolic feedback inhibition, failing to meet the demands of industrial production. This invention provides a recombinant Escherichia coli strain for de novo piperine synthesis, its construction method, and its application. This enables efficient synthesis of piperine using inexpensive glucose as a substrate, providing feasible technical support for the green industrial preparation of piperine.

[0006] To address the aforementioned technical problems, the technical solutions provided by this invention cover recombinant strains, strain construction methods, product production methods, and corresponding applications, as detailed below:

[0007] In a first aspect, the present invention provides a method for constructing a recombinant Escherichia coli strain for de novo synthesis of piperonol, using Escherichia coli MG1655 as the starting strain and performing the following basic modifications: A. Overexpression of the gene encoding tyrosine ammonia-lyase catalyzes the conversion of tyrosine to p-coumaric acid; B. Overexpression of the gene encoding carboxylic acid reductase and the gene encoding 4′-phosphopanylthioethylamine transferase catalyzes the conversion of p-coumaric acid to p-coumaraldehyde; C. Overexpression of the gene encoding needle-leaf acyltransferase converts p-coumarol to p-coumaroyl acetate (the conversion of p-coumarol to p-coumarol is catalyzed by endogenous aldehyde reductase in Escherichia coli). D. Overexpression of the gene encoding eugenol synthase will ultimately catalyze p-coumaryl acetate to piperine.

[0008] Furthermore, in step A of the above construction method, the gene encoding the tyrosine ammonia-lyase includes: a gene derived from Rhodococcus. Rpctal The gene may have originated from red yeast. RgTAL Gene; The Rpctal The nucleotide sequence of the gene is shown in SEQ ID NO:1; RgTAL The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0009] Furthermore, in step B of the above construction method, the carboxylic acid reductase gene is derived from marine mycobacteria. Mmcar The 4′-phosphopantoylthioethylamine transferase gene is derived from Bacillus subtilis. Bssfp Genes; the stated Mmcar The nucleotide sequence of the gene is shown in SEQ ID NO:3; Bssfp The nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0010] Furthermore, in step C of the above construction method, the needle-leaf acyltransferase gene is derived from petunia. PhCFAT Genes; the stated PhCFAT The nucleotide sequence of the gene is shown in SEQ ID NO:5.

[0011] Furthermore, in step D of the above construction method, the eugenol synthase gene is derived from basil. ObEGS Genes; the stated ObEGS The nucleotide sequence of the gene is shown in SEQ ID NO:6.

[0012] Furthermore, in addition to the basic renovations, the following optimizations and upgrades are also included: E. Overexpression of 3-deoxy-D-arabino-heptanuronate-7-phosphate synthase aroG D146N Gene; F. Overexpression of prephenylate dehydrogenase ZmtyrC Gene; G. Knocking out tyrosine metabolism repressor genes tyrR .

[0013] Furthermore, in the optimization and transformation, the aforementioned aroG D146N The nucleotide sequence of the gene is shown in SEQ ID NO:7; ZmtyrC The nucleotide sequence of the gene is shown in SEQ ID NO:8; tyrR The nucleotide sequence of the gene is shown in SEQ ID NO:9.

[0014] Secondly, the present invention provides a recombinant Escherichia coli that synthesizes piperonol de novo, wherein the recombinant Escherichia coli is constructed by the above-described construction method.

[0015] Thirdly, the present invention provides the application of the above-mentioned recombinant Escherichia coli in the fermentation preparation of piperine.

[0016] Furthermore, in the above application, the recombinant Escherichia coli was used as the fermentation strain and inoculated into the fermentation medium, with glucose as the substrate for fermentation culture.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes a complete de novo biosynthesis pathway of piperine by introducing a heterologous synthetic gene into Escherichia coli. By combining codon optimization of the heterologous gene with modular vector expression design, it effectively solves the problem of poor expression adaptability of the heterologous gene in the host strain, realizes the efficient operation of the artificial metabolic pathway, and achieves the first de novo synthesis of piperine using inexpensive glucose as a substrate.

[0018] Based on this, the present invention modifies the upstream precursor synthesis pathway, overexpresses the key enzyme that relieves feedback inhibition, and knocks out the metabolic repression gene, effectively relieving the natural metabolic feedback regulation of the host strain, significantly increasing the synthesis flux of precursor substances such as tyrosine and p-coumaric acid, and effectively improving the synthesis efficiency of piperine. Experimental verification shows that the modified recombinant Escherichia coli can produce up to 47.9 mg / L of piperine in shake-flask fermentation, which is far higher than the yield level of existing microbial synthesis technologies.

[0019] Meanwhile, the preparation process of this invention takes microbial fermentation as the core, replacing the traditional plant extraction and chemical synthesis process. It does not rely on natural plant raw materials with long growth cycles, nor does it consume a large amount of organic solvents. It effectively solves the defects of limited production capacity and large environmental pollution of traditional processes. The production process is green and environmentally friendly, and the raw material cost is low. It has the potential for large-scale industrial production. At the same time, it provides a feasible technical basis and research direction for the construction of higher-yield piperine engineered bacteria. Attached Figure Description

[0020] Figure 1 This diagram illustrates the constructed biosynthetic pathway of piperine in *E. coli* and its genetic engineering modification. It labels the intermediate metabolites in the pathway, including tyrosine, p-coumaric acid, p-coumaraldehyde, p-coumarol, and p-coumaroyl acetate. It also labels the genes encoding related enzymes in the pathway, including: aroG D146N , tyrA , tyrB , tyrC , tyrR , TAL , sfp , car , CAD , CFAT , EGS .

[0021] Figure 2 This is a plasmid map of plasmid pLML01 in this embodiment of the invention, which marks the repressor protein gene lacI, the origin of replication CloDF13 ori, the streptomycin resistance selection marker gene SmR, and three independent functional expression units: a tyrosine ammonia-lyase encoding gene driven by the Tac promoter. Rpctal Tac promoter-driven prephenyl acid dehydrogenase encoding gene ZmtyrC Tac promoter-driven feedback inhibition-relief DAHP synthase encoding gene aroG D146N .

[0022] Figure 3The image shown is an agarose gel electrophoresis diagram of the successful construction of plasmid pLML01 in this embodiment of the invention. Lane M is the marker, lane 1 is the pCDFTac empty vector, lane 2 is the pLML01-1 plasmid, lane 3 is the pLML01-2 plasmid, and lane 4 is the pLML01 plasmid.

[0023] Figure 4 This is a plasmid map of plasmid pLML02 in this embodiment of the invention, which marks the repressor protein gene lacI, the origin of replication CloDF13 ori, the streptomycin resistance selection marker gene SmR, and three independent functional expression units: a tyrosine ammonia-lyase encoding gene driven by the Tac promoter. RgTAL Tac promoter-driven prephenyl acid dehydrogenase encoding gene ZmtyrC Tac promoter-driven feedback inhibition-relief DAHP synthase encoding gene aroG D146N .

[0024] Figure 5 The image shown is an agarose gel electrophoresis diagram of the successful construction of plasmid pLML02 in this embodiment of the invention. Lane M is the marker, lane 1 is the pCDFTac empty vector, lane 2 is the pLML02-1 plasmid, lane 3 is the pLML02-2 plasmid, and lane 4 is the pLML02 plasmid.

[0025] Figure 6 This is a plasmid map of plasmid pLML03 in this embodiment of the invention, which marks the plasmid's origin of replication pBBR1oriV, the replication functional protein pBBR1 Rep, the chloramphenicol resistance selection marker gene CAT / CamR, and two independent functional expression units: a carboxylic acid reductase encoding gene driven by the Tac promoter. Mmcar The gene encoding 4′-phosphopantoylthioethylamine transferase driven by the Tac promoter. Bssfp .

[0026] Figure 7 This is an agarose gel electrophoresis image of plasmid pLML03 successfully constructed in an embodiment of the present invention, where lane M is the marker, lane 1 is the pBBR1Tac empty vector, and lane 2 is the pLML03 plasmid.

[0027] Figure 8 This is a plasmid map of plasmid pLML04 in this embodiment of the invention, which marks the plasmid's origin of replication p15Aori, the kanamycin resistance selection marker gene KanR, the terminator rrnBT1T2, and two independent functional expression units: a neemyl alcohol transferase encoding gene driven by the Tac promoter. PhCFAT The eugenol synthase encoding gene driven by the Tac promoter ObEGS .

[0028] Figure 9 This is an agarose gel electrophoresis image of the successful construction of plasmid pLML04 in an embodiment of the present invention, where lane M is the marker, lane 1 is the pTac15K empty vector, and lane 2 is the pLML04 plasmid.

[0029] Figure 10 As described in the embodiments of the present invention E. coli MG1655 tyrR Agarose gel electrophoresis image of gene knockout, where lane M is the marker and lane 1 is the gene knockout. E. coli MG1655, lane 2 is L01 strain (MG1655 Δ tyrR ).

[0030] Figure 11 This is a schematic diagram of the fermentation results of the upstream module of strains L03 and L04 in this embodiment of the invention, used to show the yield of p-coumaric acid synthesized de novo by the strains under the action of tyrosine ammonia-lyase from different sources.

[0031] Figure 12 This is a schematic diagram of the fermentation results of the downstream module of strain L07 in an embodiment of the present invention, used to demonstrate the synthesis of p-coumaric acid into piperine by this strain.

[0032] Figure 13 This is a schematic diagram of the de novo fermentation results of piperine synthesis by strains L08 and L09 in this embodiment of the invention, used to demonstrate the yield of piperine synthesized by the modified strains.

[0033] Figure 14 The figure shows the liquid chromatogram of the product detection in this embodiment of the invention. The peak positions of the standards for p-coumaric acid, p-coumaraldehyde, p-coumarol, and piperine, as well as the peak position of the fermentation sample, are marked. The detection wavelengths are 308 nm and 279 nm, respectively, to verify the synthesis of the target product. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0036] The genotypes of the strains and plasmids involved in the examples are shown in Table 1.

[0037] Table 1. Genotypes of strains and plasmids

[0038] The primers and their specific sequences involved in the examples are shown in Table 2.

[0039] Table 2. Primer sequence listing

[0040] The culture medium components involved in the examples are as follows: 1. LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, and deionized water to a final volume of 1L.

[0041] 2. SOC resuscitation medium: 2.0 g tryptone, 0.5 g yeast extract, 0.05 g sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 20 mM glucose, diluted to 100 mL with deionized water, and adjusted to pH 7.0. Preparation method: Dissolve the above ingredients in deionized water, adjust the pH to 7.0, autoclave, cool to room temperature, and finally add filtered and sterilized glucose solution.

[0042] 3. Fermentation medium: glucose 10g / L, MgSO4 7H₂O 0.8 g / L, KH₂PO₄ 6.67 g / L, (NH₄)₂HPO₄ 4 g / L, citric acid 0.8 g / L, trace metal salt solution 5 mL / L, pH 7.0; wherein the trace metal salt solution consists of: 10 g / L FeSO₄ 7H2O, 2.65g / L CaCl2 2H₂O, 2.2 g / L ZnSO₄ 7H2O, 0.58g / L MnSO4 5H2O, 1g / L CuSO4 5H₂O, 0.1 g / L (NH₄)₆Mo₇O 24 4H₂O, 0.02 g / L Na₂B₄O₇ 10H₂O, 10 mL / L 35% HCl, deionized water as solvent; glucose and MgSO₄ 7H2O is the mother liquor for independent sterilization, with concentrations of 500 g / L and 80 g / L, respectively.

[0043] The main reagents involved in the examples are: 1. Antibiotics: Ampicillin sodium (Ap, final concentration 100 mg / L), chloramphenicol (Cm, final concentration 34 mg / L), streptomycin (Sm, final concentration 50 mg / L), kanamycin (Km, final concentration 50 mg / L), used for screening positive clones after transformation and maintaining plasmid stability; 2. Inducers: IPTG (isopropyl-β-D-thiogalactoside, final concentration 0.1~1.0mM) and L-arabinose (final concentration 10mM) are used to induce the expression of exogenous proteins and recombinases, respectively. 3. Reagents for preparing competent cells: 10% glycerol solution, used for washing and resuspending electroporated competent cells to prepare competent cells; 4. Molecular biology reagents: *E. coli* DH5α competent cells were used for transformation and verification of recombinant plasmids. Restriction endonucleases, purchased from TAKARA, were used for plasmid digestion construction to achieve plasmid linearization. A seamless cloning kit, purchased from Ibotek, was used for recombination ligation of the target gene fragment with the vector. A gel extraction kit was used for gel extraction and purification of nucleic acid fragments.

[0044] 5. Reagents for product detection: Standards for p-coumaric acid, p-coumaraldehyde, p-coumarol, and piperonol are used for qualitative verification and quantitative analysis of fermentation products. Mobile phase reagents for high-performance liquid chromatography (HPLC) include ultrapure water containing 0.1% trifluoroacetic acid and methanol for chromatographic separation of the target product; a 0.22 μm aqueous filter membrane is used for pretreatment of the fermentation supernatant to remove cell impurities.

[0045] Information on the main experimental instruments involved in the examples: 1. Electroconversion instrument: used for electroconversion of Escherichia coli to introduce plasmids and homologous recombination linear fragments. In this example, the Ec1 setting is used to complete the electroconversion.

[0046] 2. PCR amplification instrument: used for amplification of target genes and homologous recombination screening fragments, as well as verification of positive clones by colony PCR.

[0047] 3. Agarose gel electrophoresis system: used for the separation and detection of nucleic acid fragments, to separate enzyme digestion products and PCR amplification products, and to provide pretreatment for gel recovery and purification.

[0048] 4. Clean bench: Provides a sterile operating environment for aseptic operations such as competent cell preparation, transformation, and strain inoculation.

[0049] 5. Constant temperature shaker: Used for seed culture and fermentation culture of strains. The culture temperature and shaking speed can be adjusted to meet the different needs of conventional strain culture at 37℃ and fermentation culture at 30℃.

[0050] 6. Constant temperature incubator: Used for culture on solid plates, screening of positive clones after transformation, and streak culture for eliminating auxiliary plasmids.

[0051] 7. Autoclave: Used for sterilization of culture media and laboratory equipment, and can be adapted to the independent sterilization requirements of fermentation culture media components.

[0052] 8. High-speed centrifuge: Used for cleaning competent cells and centrifuging fermentation samples. It can achieve high-speed centrifugation of 16,000 × g to separate the cells from the supernatant.

[0053] 9. Ultraviolet spectrophotometer: used for bacterial culture OD 600 The value is detected to monitor the growth status of the strain and determine the timing of adding the inducer.

[0054] 10. High Performance Liquid Chromatograph: Shimadzu LC-16 series, equipped with a UV-Vis spectrophotometric detection module, used for quantitative detection and qualitative verification of fermentation products, and can simultaneously detect p-coumaric acid, p-coumaraldehyde, p-coumarol, and piperonol.

[0055] 11. Liquid chromatography column: Shim-pack GIST C18-AQ (4.6×250mm), a dedicated chromatographic separation column for HPLC detection, used for the separation of target products.

[0056] Example 1: Overall genetic engineering modification and synthetic pathway design of recombinant Escherichia coli.

[0057] This embodiment uses Escherichia coli MG1655 as the starting strain. Through a modular genetic engineering modification strategy, a complete artificial biosynthetic pathway for piperine is constructed within the strain's cells. At the same time, the upstream precursor synthesis pathway is optimized to remove feedback inhibition from natural metabolism, ultimately achieving de novo synthesis of piperine using inexpensive glucose as a substrate.

[0058] The overall modification and synthesis pathway in this embodiment is as follows: Figure 1 As shown, this diagram labels the names of the metabolic substrates, intermediate products, and corresponding catalytic enzymes in the modified *E. coli*, and also indicates different types of gene modifications. The complete metabolic process is as follows: In E. coli, glucose is first converted into phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) via glycolysis and pentose phosphate pathways, respectively. PEP and E4P are then catalyzed by DAHP synthase to generate DAHP, which is subsequently catalyzed by shikimic acid pathway to branched acid (CHA). Branched acid is further converted into tyrosine. Tyrosine is catalyzed by tyrosine ammonia-lyase to generate p-coumaric acid, which is then catalyzed by carboxylic acid reductase system to generate p-coumaraldehyde. P-coumaraldehyde is then converted into p-coumarol by endogenous aldehyde reductase CAD in E. coli. P-coumarol is then catalyzed by needle-leaf acyltransferase to generate p-coumaroyl acetate, which is finally catalyzed by eugenol synthase to generate the target product, piperine.

[0059] To achieve efficient operation of the aforementioned pathway, this embodiment performed the following basic modifications on the starting strain to construct a complete artificial synthesis pathway. Each modification corresponds to... Figure 1 The annotations in the text are as follows: A. Tyrosine ammonia-lyase overexpression modification: Overexpression of the gene encoding tyrosine ammonia-lyase enables highly efficient catalysis of tyrosine to p-coumaric acid, opening the entry point for the upstream precursor to downstream synthetic pathway. This gene can be selected from Rhodococcus bacteria (…). Rhodococcus sp .)of Rpctal The gene may have originated from red yeast ( Rhodotorula glutinis )of RgTAL The gene, or other optional exogenous gene encoding tyrosine ammonia-lysine hydrolase. Rpctal The codon-optimized nucleotide sequence of the gene is shown in SEQ ID NO:1; RgTAL The optimized nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0060] B. Carboxylic acid reductase overexpression modification: Overexpression of the coding gene for carboxylic acid reductase and the coding gene for 4′-phosphopanylthioethylamine transferase. This modification enables the catalytic conversion of coumaric acid to p-coumaraldehyde, providing a reaction substrate for the subsequent conversion of intermediate products. The coding gene for carboxylic acid reductase can be selected from marine mycobacteria (…). Mycobacterium marinum )of Mmcar The gene encoding the 4′-phosphopantoylthioethylamine transferase, or other optional exogenous carboxylic acid reductase, may be selected from: Bacillus subtilis (…). Bacillus subtilis )of Bssfp The gene, or other optional exogenous gene encoding 4′-phosphopantoylthioethylamine transferase. Mmcar The codon-optimized nucleotide sequence of the gene is shown in SEQ ID NO:3; BssfpThe optimized nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0061] C. Overexpression of needle-leaf acyltransferase: Overexpression of the gene encoding needle-leaf acyltransferase enables the catalytic conversion of coumarin to p-coumaryl acetate, completing the intermediate step in downstream synthesis. The needle-leaf acyltransferase gene can be selected from: those derived from petunia (… Petunia hybrida )of PhCFAT The gene, or the gene encoding another optional exogenous needle-leaf acyltransferase. PhCFAT The optimized nucleotide sequence of the gene is shown in SEQ ID NO:5.

[0062] D. Eugenol synthase overexpression modification: Overexpression of the eugenol synthase encoding gene enables the final catalysis of coumaroyl acetate to the target product piperine, thus completing the construction of the entire artificial synthesis pathway. The eugenol synthase gene can be selected from basil (…). Ocimum basilicum )of ObEGS The gene, or other optional exogenous gene encoding eugenol synthase. ObEGS The optimized nucleotide sequence of the gene is shown in SEQ ID NO:6.

[0063] Building upon the aforementioned basic modifications, this embodiment further optimizes the upstream tyrosine synthesis pathway to enhance the supply of precursor substances and alleviate the natural metabolic feedback inhibition in *E. coli*. Each optimization corresponds to... Figure 1 The annotations in the text are as follows: E. Overexpression modification of feedback inhibition-relieved DAHP synthase: Overexpression of the gene encoding feedback inhibition-relieved DAHP synthase. This optimization modification can relieve the feedback inhibition of DAHP synthase by L-phenylalanine, significantly increase the carbon flux of the shikimic acid pathway, promote DAHP synthesis, and provide a sufficient carbon source for subsequent tyrosine synthesis. The feedback inhibition-relieved DAHP synthase gene can be selected from Escherichia coli. aroG D146N The gene, or other optional gene encoding exogenous feedback inhibition-relief DAHP synthase. aroG D146N The nucleotide sequence of the gene is shown in SEQ ID NO:7.

[0064] F. Prebenzoic acid dehydrogenase overexpression modification: Overexpression of the prebenzoic acid dehydrogenase encoding gene, through this optimization modification, can enhance the metabolic flux of the tyrosine branch pathway, increase the synthesis flux of tyrosine, and provide sufficient precursors for the downstream synthesis of p-coumaric acid. The prebenzoic acid dehydrogenase encoding gene can be selected from *Pseudomonas motilityans* (…). Zymomonas mobilis)of ZmtyrC Gene, or other optional exogenous prephenylacetic acid dehydrogenase gene. ZmtyrC The optimized nucleotide sequence of the gene is shown in SEQ ID NO:8.

[0065] G. Tyrosine metabolism repression gene knockout modification: Knocking out tyrosine metabolism repression genes tyrR This optimization and modification can remove the metabolic repression of the tyrosine synthesis pathway, further unblock natural metabolic regulation, increase the amount of tyrosine synthesized, and ensure the supply of precursors for downstream synthesis pathways. tyrR The gene is an endogenous gene of E. coli. tyrR The nucleotide sequence of the gene is shown in SEQ ID NO:9.

[0066] It should be noted that, in this invention, apart from endogenous... aroG D146N Gene (SEQ ID NO:7) and tyrR Apart from gene (SEQ ID NO:9), all heterologous genes were codon-optimized to fit the expression system of E. coli.

[0067] To achieve phased regulation of metabolic pathways, this embodiment uses different plasmid vectors to carry the above-mentioned functional genes in modules: among which, tyrosine ammonia-lyase gene, aroG D146N Genes and ZmtyrC All genes were expressed using the pCDFTac plasmid; Mmcar and Bssfp The gene was expressed via the pBBR1Tac plasmid; PhCFAT and ObEGS The gene is expressed via the pTac15K plasmid; and tyrR Gene knockout is a traceless knockout modification of the genome of the starting strain, which can achieve stable inheritance of the strain.

[0068] Those skilled in the art can understand the specific implementation of this modification strategy based on the description in this embodiment. Through the above modification, a complete de novo synthesis pathway of piperonol can be built in Escherichia coli, while significantly improving the supply efficiency of precursors, thus providing a foundation for subsequent fermentation production.

[0069] Example 2: Construction of plasmids pLML01~pLML04.

[0070] This embodiment is used to construct modular functional expression plasmids, each carrying one of the modified genes described in Example 1, to provide expression vectors for the subsequent construction of recombinant strains.

[0071] 1. Construction of pLML01 plasmid The plasmid uses pCDFTac as its base vector to carry substances derived from Rhodococcus. Rpctal Gene, ZmtyrC Genes and aroG D146N The specific steps for constructing the gene are as follows: The pCDFTac plasmid was used with restriction endonuclease EcoR I. Single enzyme digestion was performed to obtain linearized plasmid vector fragments; using RPCTAL_F and RPCTAL_R (SEQ ID NO: 10~11) as primers, a fully synthesized plasmid encoding tyrosine ammonia-lyase was generated. Rpctal Using the gene plasmid as a template, amplification Rpctal Gene fragment (sequence shown in SEQ ID NO:1). The linearized vector fragment after enzyme digestion and the amplified... Rpctal Gene fragments were subjected to agarose gel electrophoresis, and the target band of the correct size was excised and purified using a gel extraction kit. Then, recombination ligation reaction was performed using a seamless cloning kit. The reaction product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing streptomycin and incubated overnight at 37°C. Single clones were picked for colony PCR (primer sequences are shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After verification, the intermediate plasmid pLML01-1 was obtained.

[0072] The pLML01-1 plasmid was used with restriction endonuclease Kpn I. Single enzyme digestion was performed to obtain a linearized plasmid vector fragment; using tyrC_F1 (SEQ ID NO:14) and tyrC_R (SEQ ID NO:16) as primers, a fully synthesized plasmid encoding prephenyl acid dehydrogenase was used. ZmtyrC Using the gene plasmid as a template, PCR amplification was performed to obtain... ZmtyrC Gene fragment (sequence shown in SEQ ID NO:8). The linearized pLML01-1 fragment and the amplified... ZmtyrC Gene fragments were subjected to agarose gel electrophoresis, the correct bands were excised and purified using a gel extraction kit, and then recombinant reactions were performed using a seamless cloning kit. The reaction products were transformed into E. coli DH5α competent cells, plated on LB agar plates containing streptomycin, and single clones were picked after culture. Colony PCR was performed (primer sequences are shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After verification, the intermediate plasmid pLML01-2 was obtained.

[0073] The pLML01-2 plasmid was used with restriction endonuclease Bam H Ⅰ was used for single enzyme digestion to obtain linearized plasmid vector fragments; simultaneously, aroG was used...D146N _F1 (SEQ ID NO:17) and aroG D146N Using primer _R (SEQ ID NO:19) and plasmid pLMLPAA05 as a template, PCR amplification was performed to obtain aroG D146N Gene fragment (sequence shown in SEQ ID NO:7). The linearized pLML01-2 fragment and the amplified... aroG D146N Gene fragments were subjected to agarose gel electrophoresis, the correct bands were excised and purified using a gel extraction kit, and then recombinant reactions were performed using a seamless cloning kit. The reaction products were transformed into E. coli DH5α competent cells and plated on LB agar plates containing streptomycin. Single clones were picked for colony PCR (primer sequences are shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After verification, the pLML01 plasmid was obtained.

[0074] The pLML01 plasmid map is as follows: Figure 2 As shown, this is the expression vector for the upstream module, which can achieve overexpression of the corresponding functional genes; the electrophoresis diagram of colony PCR verification is shown below. Figure 3 As shown, lane M is the marker, lane 1 is the pCDFTac empty vector, lane 2 is the pLML01-1 plasmid, lane 3 is the pLML01-2 plasmid, and lane 4 is the pLML01 plasmid.

[0075] 2. Construction of pLML02 plasmid The plasmid also uses pCDFTac as its base carrier to carry substances derived from Rhodotorula glutinis. RgTAL Gene, ZmtyrC Genes and aroG D146N The specific steps for constructing the gene are as follows: The pCDFTac plasmid was used with restriction endonuclease Eco RⅠ was used for single enzyme digestion to obtain a linearized plasmid vector fragment; using RgTAL_F and RgTAL_R (SEQ ID NO:12~13) as primers, a fully synthesized plasmid encoding a tyrosine ammonia-lyase was generated. RgTAL Using the gene plasmid as a template, amplification RgTAL Gene fragment (sequence shown in SEQ ID NO:2). The linearized vector fragment after enzyme digestion and the amplified... RgTALGene fragments were subjected to agarose gel electrophoresis, and the correct bands were excised and purified using a gel extraction kit. Recombinant reactions were then performed using a seamless cloning kit. The reaction products were transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin. Single clones were picked for colony PCR verification (primer sequences shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After successful verification, the intermediate plasmid pLML02-1 was obtained.

[0076] The pLML02-1 plasmid was used with restriction endonuclease Kpn I. Single enzyme digestion was performed to obtain a linearized plasmid vector fragment; using tyrC_F2 and tyrC_R (SEQ ID NO:15~16) as primers, a fully synthesized plasmid encoding prephenylalanine dehydrogenase was used. ZmtyrC Using the gene plasmid as a template, amplification was performed to obtain... ZmtyrC Gene fragment (sequence shown in SEQ ID NO:8); linearized pLML02-1 fragment and amplified ZmtyrC Gene fragments were subjected to agarose gel electrophoresis, and the correct bands were excised and purified using a gel extraction kit. Recombinant reactions were then performed using a seamless cloning kit. The reaction products were transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin. Single clones were picked for colony PCR verification (primer sequences are shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After successful verification, the intermediate plasmid pLML02-2 was obtained.

[0077] The pLML02-2 plasmid was used with restriction endonuclease Bam H I was used for single enzyme digestion to obtain linearized plasmid vector fragments, which were then used with aroG D146N _F2 and aroG D146N Using primers _R (SEQ ID NO:18~19) and plasmid pLMLPAA05 as a template, amplification was performed to obtain... aroG D146N Gene fragment (sequence shown in SEQ ID NO:7). The linearized pLML02-2 fragment and the amplified... aroG D146N Gene fragments were subjected to agarose gel electrophoresis, and the correct bands were excised and purified using a gel extraction kit. Recombinant reactions were then performed using a seamless cloning kit. The reaction products were transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin. Single clones were picked for colony PCR (primer sequences shown in SEQ ID NO:34 and SEQ ID NO:37). Positive clones were sent to a sequencing company for verification. After verification, the pLML02 plasmid was obtained.

[0078] The spectrum of the pLML02 plasmid is as follows: Figure 4 As shown, this is another alternative expression vector for the upstream module, used to verify the expression effects of tyrosine ammonia-lyases from different sources; the electrophoresis image of the colony PCR verification is shown below. Figure 5 As shown, lane 1 is the pCDFTac empty vector, lane 2 is the pLML02-1 plasmid, lane 3 is the pLML02-2 plasmid, and lane 4 is the pLML02 plasmid.

[0079] 3. Construction of pLML03 plasmid The essential particles are based on pBBR1Tac as a carrier and are used to carry out... Mmcar Genes and Bssfp The specific steps for constructing the gene are as follows: Using pBBR1Tac plasmid as a template and pBBR1Tac_F and pBBR1Tac_R (SEQ ID NO:20~21) as primers, the linearized plasmid fragment was amplified by PCR. Simultaneously, using MmCAR-1_F and MmCAR-2_R (SEQ ID NO:22~23) and sfp_F and sfp_R (SEQ ID NO:24~25) as primers, a plasmid containing a fully synthetically encoded carboxyl reductase was amplified. Mmcar Genes and encoding 4′-phosphopantoylthioethylamine transferase Bssfp Using the gene plasmid as a template, amplification Mmcar Gene (sequence shown in SEQ ID NO:3) and Bssfp Gene (sequence shown in SEQ ID NO:4). Linearized pBBR1Tac fragment, Mmcar Gene fragments and Bssfp Gene fragments were subjected to agarose gel electrophoresis, the correct bands were excised and purified using a gel extraction kit, and then recombinant reactions were performed using a seamless cloning kit. The reaction products were transformed into E. coli DH5α competent cells and plated on LB agar plates containing chloramphenicol. Single clones were picked for colony PCR (primer sequences are shown in SEQ ID NO:35 and SEQ ID NO:37). Positive clones were sent to a sequencing company for sequencing verification. After verification, the pLML03 plasmid was obtained.

[0080] The pLML03 plasmid map is as follows: Figure 6 The image shown is the expression vector for the downstream carboxylic acid reductase module; the electrophoresis diagram for colony PCR verification is shown below. Figure 7 As shown, lane M is the marker, lane 1 is the pBBR1Tac empty vector, and lane 2 is the pLML03 plasmid.

[0081] 4. Construction of pLML04 plasmid The essential particles are based on pTac15K as a carrier and are used to support... PhCFAT Genes and ObEGS The specific steps for constructing the gene are as follows: The pTac15K plasmid was used with restriction endonuclease Eco R Ⅰ and Xba I. Double digestion was performed to obtain linearized plasmid vector fragments; simultaneously, using PhCFAT_F and PhCFAT_R (SEQ ID NO:26~27) and ObEGS_F and ObEGS_R (SEQ ID NO:28~29) as primers, respectively, a plasmid containing a fully synthetic encoding needle-leaf acyltransferase was developed. PhCFAT Genes and encoding eugenol synthase ObEGS Using the gene plasmid as a template, amplification PhCFAT Gene fragment (sequence shown in SEQ ID NO:5) and ObEGS Gene fragment (sequence shown in SEQ ID NO:6). The linearized vector fragment and the amplified... PhCFAT Gene fragments, ObEGS Gene fragments were subjected to agarose gel electrophoresis, the correct bands were excised and purified using a gel extraction kit, and then a recombination reaction was performed using a seamless cloning kit. The reaction product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing kanamycin. Single clones were picked for colony PCR (primer sequences are shown in SEQ ID NO:36~37). Positive clones were sent to a sequencing company for sequencing verification. After verification, the pLML04 plasmid was obtained.

[0082] The pLML04 plasmid map is as follows: Figure 8 The image shows the expression vector for the downstream acyl transfer and final product synthesis module; the electrophoresis diagram for colony PCR verification is shown below. Figure 9 As shown, lane M is the marker, lane 1 is the pTac15K empty vector, and lane 2 is the pLML04 plasmid.

[0083] Example 3: E. coli MG1655 tyrR Gene knockout.

[0084] 1. Preparation of linearized fragment KO-ΔtyrR Using pUTrc plasmid as a template, primers ΔtyrR_F1 and ΔtyrR_R1 (SEQ ID NO:30~31) were used to amplify the plasmid containing pUTrc plasmid. tyrRThe lox71-CmR-lox66 gene fragment with 50bp homologous arms upstream and downstream of the gene was designated KO-ΔtyrR1. Then, using primer pairs ΔtyrR_F2 and ΔtyrR_R2 (SEQ ID NO:32~33) as a template, the homologous arms at both ends of KO-ΔtyrR1 were amplified to obtain the gene containing... tyrR The linearized fragment KO-ΔtyrR consists of 100bp homologous arms upstream and downstream of the gene.

[0085] 2. Introduction of helper plasmid pKD46 E. coli Preparation of MG1655 electrocompetent states: Laboratory-preserved... E. coli MG1655 was inoculated into test tubes containing 5 mL of LB medium and cultured at 37°C and 200 rpm for 12–16 h. Then, 2% of the culture medium was inoculated into 100 mL of fresh LB medium and cultured at 37°C and 200 rpm for 1.5 h. When OD... 600 =0.4~0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, and finally add an appropriate amount of 10% glycerol to make the final volume 600~800μL. Aliquot 100μL into a 1.5mL centrifuge tube and freeze at -80℃ for later use.

[0086] Introduction of helper plasmid pKD46: Take one sample from a -80℃ freezer. E. coli MG1655 electroporation competent cells were thawed on ice and thoroughly mixed with approximately 50 ng of plasmid pKD46. The mixture was then transferred to a pre-chilled 1 mm electroporation cuvette and incubated on ice for 30 min. The cuvette was dried by wiping the outside and bottom with paper, placed in an electroporator, and electroporated once using the Ec1 setting. Immediately afterward, 600 μL of pre-chilled SOC medium was added, gently mixed, and the cuvette was tilted to transfer the entire mixture to a sterile 1.5 mL centrifuge tube. The mixture was incubated at 30°C and 200 rpm for 60–80 min. 80 μL of the incubated mixture was then spread onto LB agar plates containing ampicillin sodium and incubated overnight at 30°C.

[0087] 3. Insertion of linearized fragment KO-ΔtyrR Pick single colonies from the plate in step 2 and inoculate them into test tubes containing 5 mL of LB medium and ampicillin sodium. Incubate at 30°C and 200 rpm for 16–20 h. Inoculate 2% of the culture medium into 15 mL of fresh LB medium containing ampicillin sodium, and add L-arabinose to a final concentration of 10 mM. Incubate at 30°C and 200 rpm for 2 h. OD 600=0.4~0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, and finally add a certain amount of 10% glycerol to make a final volume of 100 μL. Transfer to a sterile 1.5 mL centrifuge tube, mix thoroughly with about 500 ng of the KO-ΔtyrR fragment, and perform electroporation transformation in the same manner as in step 2. Spread the entire mixture after recovery culture onto LB agar plates containing ampicillin sodium and chloramphenicol, and incubate overnight at 30°C. Pick single clones for colony PCR verification and screen for positive clones.

[0088] 4. Elimination of helper plasmid pKD46 The positive clones from step 3 were streaked onto LB agar plates containing chloramphenicol and incubated overnight at 42°C. Single clones from the plates were then inoculated into 5 mL LB tubes containing both ampicillin sodium and chloramphenicol, and into tubes containing only chloramphenicol, and incubated overnight to verify whether plasmid pKD46 had been eliminated.

[0089] 5. Elimination of the CmR screening marker The strain with pKD46 eliminated was inoculated into test tubes containing 5 mL of LB broth and chloramphenicol, and incubated at 37°C, 200 rpm, with shaking for 12–16 h. 2% of the culture was then inoculated into 15 mL of fresh LB broth containing chloramphenicol, and incubated at 37°C, 200 rpm, with shaking for 1.5 h. OD 600 =0.4~0.6, centrifuge and discard the supernatant, wash twice with 10% glycerol, and finally add a certain amount of 10% glycerol to make a final volume of 100 μL. Transfer to a sterile 1.5 mL centrifuge tube, mix thoroughly with about 50 ng of plasmid pjw168, and perform electroporation transformation in the same manner as in step 2. Spread the mixture onto LB agar plates containing ampicillin sodium and 1 mM IPTG, and incubate overnight at 30°C. Pick single clones for colony PCR and screen for positive clones.

[0090] 6. Elimination of helper plasmid pjw168 Streak the positive clones from step 5 onto LB agar plates and incubate overnight at 42°C. Inoculate single clones from the plates into 5 mL LB tubes containing or without ampicillin sodium to verify whether plasmid pjw168 has been eliminated. The genotype of the successfully eliminated strain is MG1655 Δ. tyrR It is designated as strain L01.

[0091] 7. Strain identification Genomic PCR verification was performed using identification primers Seq-ΔtyrR_F (SEQ ID NO:38) and Seq-ΔtyrR_R (SEQ ID NO:39). The electrophoresis results are as follows: Figure 10 As shown, lane M is the marker, and lane 1 is... E. coliMG1655, lane 2 is L01 strain (MG1655 Δ tyrR ).

[0092] Example 4: Construction of strains L02~L09.

[0093] This embodiment is used to introduce the modular expression plasmid constructed in Example 2 into the corresponding host strain to construct a series of gradient-modified recombinant strains, which are used for functional verification of the upstream precursor synthesis module and the downstream product synthesis module, as well as the construction of the final complete piperine synthesis pathway strain. The specific steps are as follows: 1. Upstream precursor synthesis module validates strain construction This step is used to construct a recombinant strain that only imports the upstream module plasmid, to verify the function of the upstream module for coumaric acid synthesis. The specific steps are as follows: Preparation of electrocompetent cells of strain L01: Inoculate strain L01 into test tubes containing 5 mL LB medium and incubate at 37°C, 200 rpm for 12–16 h. Inoculate 2% of the culture medium into 100 mL of fresh LB medium and incubate at 37°C, 200 rpm for 1.5 h, OD600 = 0.4–0.6. Centrifuge and discard the supernatant. Wash twice with 10% glycerol. Finally, add a certain amount of 10% glycerol to make a final volume of 300 μL. Aliquot 100 μL into 1.5 mL centrifuge tubes.

[0094] Each competent cell was thoroughly mixed with approximately 50 ng of plasmids pCDFTac, pLML01, and pLML02, and electroporated using the same method as step 2 in Example 3. The mixture was then plated onto LB agar plates containing streptomycin and incubated overnight at 37°C to obtain three strains: L02: A blank control strain in which an empty pCDFTac plasmid was introduced; L03: An upstream module strain that has had the pLML01 plasmid introduced, capable of expressing... Rpctal , ZmtyrC , aroG D146N Gene; L04: An upstream module strain that has had the pLML02 plasmid introduced, capable of expressing... RgTAL , ZmtyrC , aroG D146N Gene.

[0095] 2. Downstream product synthesis module to validate strain construction This step is used to construct a recombinant strain that only imports plasmids from the downstream module, to verify the function of the downstream coumaric acid to piperine synthesis module. The specific steps are as follows: First, prepare E. coliThe electrocompetent cells of MG1655 were prepared using a method that was completely consistent with the competent cell preparation method in step 1 of this embodiment.

[0096] Competent cells were thoroughly mixed with approximately 50 ng of empty plasmid pBBR1Tac and plasmid pLML03, respectively, and transformed by electroporation. The transformed cells were then plated on LB agar plates containing chloramphenicol and incubated overnight at 37°C to obtain two bacterial strains. L05: Blank control strain with an empty pBBR1Tac plasmid introduced; L06: A strain that has been inoculated with the pLML03 plasmid and can express... Mmcar , Bssfp Gene.

[0097] Subsequently, single clones of strain L06 were selected, and electrotransformation competent cells were prepared again. These cells were thoroughly mixed with approximately 50 ng of plasmid pLML04 and subjected to electrotransformation. The transformed bacterial culture was plated on LB agar plates containing chloramphenicol and kanamycin and incubated overnight at 37°C to obtain strain L07. This strain simultaneously introduced pLML03 and pLML04 plasmids and could express all functional genes of the downstream module, which was used for functional verification of the downstream module.

[0098] 3. Construction of recombinant strains with complete synthetic pathways This step is used to construct a recombinant strain that simultaneously introduces upstream and downstream module plasmids, thereby realizing a complete de novo synthesis pathway of piperine. The specific steps are as follows: Single clones of strains L03 and L04 obtained in step 1 were selected and electroporated into competent cells. Then, the pLML03 and pLML04 plasmids were sequentially introduced into the corresponding competent cells. The transformation procedure was the same as described above. After each transformation, selection was performed on LB agar plates supplemented with the corresponding selection antibiotics. Chloramphenicol was used for selection after pLML03 introduction, while chloramphenicol and kanamycin were used simultaneously after pLML04 introduction. Finally, recombinant strains with two complete pathways were obtained. L08: Using L03 as the host, and simultaneously introducing pLML03 and pLML04 plasmids, this strain contains all the modifications from A to G, enabling de novo synthesis of piperine using glucose as a substrate. L09: Using L04 as the host, pLML03 and pLML04 plasmids were introduced. This strain also included all the modifications to verify the effects of tyrosine ammonia-lyases from different sources on the yield of the final product.

[0099] This embodiment yielded a series of gradient-modified validation strains, providing a foundation for subsequent fermentation function validation experiments.

[0100] Example 5: Fermentation of strains L01~L09.

[0101] This embodiment is used to ferment and culture the L01~L09 series strains constructed in Example 4 to verify the catalytic effect of each functional module and the final de novo synthesis ability of piperine. The specific steps are as follows: 1. Preparation of seed liquid Pick a single clone from the corresponding strain plate and inoculate it into a test tube containing 5 mL of LB seed medium. Incubate overnight at 37°C and 200 rpm to obtain activated seed liquid.

[0102] 2. Shake-flask fermentation culture Inoculate the above seed culture at a volumetric inoculation rate of 2% into a 250mL baffled shake flask containing 50mL of fermentation medium. Incubate at 30℃ and 200rpm with shaking for a total fermentation period of 60 hours. After 8 hours of incubation following inoculation, the OD of the bacterial culture... 600 When the concentration reaches 0.5-0.8, IPTG with a final concentration of 1.0 mM is added to induce the expression of functional genes.

[0103] In this embodiment, fermentation of all strains was performed in triplicate to ensure the reliability of the results. Among them, strains L05, L06, and L07 used for downstream module verification were supplemented with a final concentration of 500 mg / L of p-coumaric acid 12 h after inoculation to supplement the reaction substrate of the downstream module and verify the catalytic function of the downstream module.

[0104] 3. Processing and detection of fermentation samples After fermentation, take 1 mL of fermentation broth, then take 100 μL of the bacterial culture, dilute it 10 times, and test the OD of the bacterial culture. 600 The value is used to monitor the growth of the bacteria; the remaining 900 μL of fermentation broth is centrifuged at 16000×g for 10 min, the supernatant is collected, and then filtered through a 0.22 μm aqueous filter membrane to remove bacterial impurities, thus obtaining the sample to be tested.

[0105] High-performance liquid chromatography (HPLC) was used to detect the samples. The HPLC system used was a Shimadzu LC-16 series, equipped with a UV-Vis spectrophotometric detection module, and the chromatographic column was a Shim-pack GIST C18-AQ (4.6×250 mm). The mobile phase settings were as follows: Phase A was ultrapure water containing 0.1% trifluoroacetic acid, and Phase B was methanol. The flow rate was 0.65 mL / min, and the elution program was as follows: 0–5 min, 10% B; 5–10 min, Phase B linearly increased from 10% to 90%; 10–25 min, maintained at 90% B; 25–30 min, Phase B linearly decreased from 90% to 10%; 30–34 min, maintained at 10% B. The sample injection volume was 5 μL, and the column oven temperature was 30℃. The detection wavelength was set to two channels: channel 1 was used for the quantitative detection of coumaric acid at a detection wavelength of 308 nm; channel 2 was used for the quantitative detection of coumarin, p-coumarol, and piperonol at a detection wavelength of 279 nm.

[0106] Product identification was performed using the peak elution time of the standard. The peak elution results of the standard are as follows: Figure 14 As shown: Peak 1 is p-coumaric acid standard, with a peak elution time of 16.242 min; Peak 2 is p-coumaraldehyde standard, with a peak elution time of 16.642 min; Peak 3 is p-coumarol standard, with a peak elution time of 14.947 min; Peak 4 is piperonol standard, with a peak elution time of 21.038 min. By comparing the peak elution times of the fermented sample with those of the standards, the product can be qualitatively identified, and the product can be quantitatively identified based on the standard curve.

[0107] 4. Fermentation Results and Functional Verification This step summarizes and analyzes the test results to verify the functionality of each module. The specific results are as follows: Functional validation of the upstream precursor synthesis module: For strains L03 and L04 of the upstream module, the test results showed that both strains achieved de novo synthesis of p-coumaric acid using glucose as a substrate. The corresponding validation results are as follows: Figure 11 As shown, the effectiveness of the upstream precursor synthesis module is demonstrated.

[0108] Functional validation of the downstream product synthesis module: For strain L07 in the downstream module, the test results showed that this strain could convert added p-coumaric acid into piperine, with a piperine yield of 27.3 mg / L. However, a large accumulation of p-coumaric acid was also detected, reaching 239.7 mg / L. It is speculated that this phenomenon is related to the insufficient expression of endogenous aldehyde reductase CAD in E. coli. The corresponding validation results are as follows: Figure 12 As shown, the effectiveness of the downstream product synthesis module is demonstrated.

[0109] Functional validation of the complete synthetic pathway: For strains L08 and L09 with the complete pathway, the results showed that both strains achieved de novo synthesis of piperine using glucose as a substrate. Strain L08 achieved a piperine yield of 40.8 mg / L, and strain L09 achieved a piperine yield of 47.9 mg / L. This result indicates that the catalytic effect of RgTAL derived from Rhodotorula glutinis is superior to that of Rpctal derived from Rhodococcus, and the accumulation of intermediate products in both strains was relatively low. The corresponding validation results are as follows: Figure 13 As shown, the effectiveness of the complete piperine synthesis pathway is demonstrated.

[0110] Meanwhile, the fermentation samples of strain L07 were tested, and the peak results of the samples were as follows: Figure 14 As shown, peak 5 was identified as p-coumaric acid, peak 6 as p-coumaraldehyde, peak 7 as p-coumarol, and peak 8 as piperine, further verifying the successful synthesis of the target product.

[0111] This embodiment clarifies the fermentation culture and product detection methods, verifies the effectiveness of the gene modification of the present invention, and realizes the efficient de novo synthesis of piperine using inexpensive glucose as a substrate.

[0112] In summary, this invention, through constructing a non-natural synthetic pathway for piperine in *E. coli* and performing genetic engineering, achieved a piperine yield of 47.9 mg / L by shake-flask fermentation of the engineered strain L09. Therefore, the piperine-producing genetically engineered strain constructed in this invention can accumulate piperine in the fermentation broth during fermentation, laying the foundation for constructing high-yield piperine-producing genetically engineered strains.

[0113] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for constructing recombinant Escherichia coli that synthesizes piperonol de novo, characterized in that, Using Escherichia coli MG1655 as the starting strain, the following basic modifications were made to it: A. Overexpression of the gene encoding tyrosine ammonia-lyase catalyzes the conversion of tyrosine to p-coumaric acid; B. Overexpression of the gene encoding carboxylic acid reductase and the gene encoding 4′-phosphopanylthioethylamine transferase catalyzes the conversion of p-coumaric acid to p-coumaraldehyde; C. Overexpression of the gene encoding needle-leaf acyltransferase converts p-coumarol to p-coumaroyl acetate; D. Overexpression of the gene encoding eugenol synthase will ultimately catalyze p-coumaryl acetate to piperine.

2. The construction method according to claim 1, characterized in that, In A, the gene encoding the tyrosine ammonia-lyase includes: derived from Rhodococcus. Rpctal The gene may have originated from red yeast. RgTAL Gene; The Rpctal The nucleotide sequence of the gene is shown in SEQ ID NO:1; RgTAL The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. The construction method according to claim 1, characterized in that, In B, the carboxylic acid reductase gene is derived from marine mycobacteria. Mmcar The 4′-phosphopantoylthioethylamine transferase gene is derived from Bacillus subtilis. Bssfp Genes; the stated Mmcar The nucleotide sequence of the gene is shown in SEQ ID NO:3; Bssfp The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. The construction method according to claim 1, characterized in that, In C, the needle-leaf acyltransferase gene is derived from petunia. PhCFAT Genes; the stated PhCFAT The nucleotide sequence of the gene is shown in SEQ ID NO:

5.

5. The construction method according to claim 1, characterized in that, In D, the eugenol synthase gene is derived from basil. ObEGS Genes; the stated ObEGS The nucleotide sequence of the gene is shown in SEQ ID NO:

6.

6. The construction method according to claim 1, characterized in that, In addition to the basic renovations, the following optimizations are also included: E. Overexpression of 3-deoxy-D-arabino-heptanuronate-7-phosphate synthase aroG D146N Gene; F. Overexpression of prephenylate dehydrogenase ZmtyrC Gene; G. Knocking out tyrosine metabolism repressor genes tyrR .

7. The construction method according to claim 6, characterized in that, In the optimization and transformation, the aforementioned aroG D146N The nucleotide sequence of the gene is shown in SEQ ID NO:7; ZmtyrC The nucleotide sequence of the gene is shown in SEQ ID NO:8; tyrR The nucleotide sequence of the gene is shown in SEQ ID NO:

9.

8. A recombinant Escherichia coli that synthesizes piperonol de novo, characterized in that, The recombinant Escherichia coli was constructed using the construction method described in any one of claims 1 to 7.

9. The application of the recombinant Escherichia coli according to claim 8 in the fermentation preparation of piperine.

10. The application according to claim 9, characterized in that, Using the recombinant Escherichia coli as described in claim 8 as the fermentation strain, it was inoculated into a fermentation medium and fermented using glucose as the substrate.

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