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

Abstract

This invention belongs to the field of genetic engineering and microbial engineering technology, and discloses a recombinant *E. coli* strain capable of de novo synthesis of ethyl vanillin, its construction method, and its applications. This invention uses *E. coli* RARE as the starting strain, and achieves this by overexpressing 3-dehydroshikimate dehydrogenase, carboxyl reductase, 4′-phosphopantoylthioethylaminotransferase, and catechol- O α-methyltransferase, methionine adenosine transferase, O The encoding genes for α-acetylhomoserine thiosulfate and acylhomoserine transferase were identified, and a de novo biosynthetic pathway for ethyl vanillin using glucose as the sole carbon source and ethanethiol as the ethyl donor was constructed. The heterologous genes involved were codon-optimized and expressed in modules using different plasmids. This recombinant strain can produce ethyl vanillin via fermentation, with a maximum yield of 7.3 mg / L achieved through shake-flask fermentation, providing feasible technical support for the green industrial production of ethyl vanillin.

Description

Technical Field

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

[0002] Ethyl vanillin, also known as 3-ethoxy-4-hydroxybenzaldehyde (CAS No. 121-32-4), is an important fragrance compound. Its aroma intensity is about 3 to 4 times that of vanillin (3-methoxy-4-hydroxybenzaldehyde, CAS No. 121-33-5). It is characterized by low dosage and stable properties, and is widely used in food, daily chemical and pharmaceutical fields. It can also be used as an electroplating brightener and feed additive.

[0003] For a long time, the industrial production of ethyl vanillin has mainly relied on chemical synthesis methods. Traditional chemical synthesis processes suffer from problems such as lengthy process chains, numerous reaction steps, high costs for byproduct separation, limited yields, large consumption of organic solvents, and significant environmental pollution. Regarding biosynthesis, although studies suggest that naturally occurring ethyl vanillin may exist in plants such as strawberries, its biosynthetic pathway and metabolic mechanism remain unclear, making it difficult to directly apply to the construction of microbial biosynthetic pathways. Furthermore, compared to methylation modification, the donor forms for ethylation modification in vivo are more limited. Achieving a continuous supply of ethyl donors within microbial cells and the directed transfer of ethyl groups to the phenolic hydroxyl groups of precursor compounds is a major challenge in constructing a de novo biosynthetic pathway for ethyl vanillin. Therefore, establishing a mild and environmentally friendly microbial synthesis method for ethyl vanillin using inexpensive carbon sources is of significant technical importance. Summary of the Invention

[0004] The technical problem this invention aims to solve is to address the shortcomings of existing chemical synthesis processes for ethyl vanillin, such as lengthy process chains, high byproduct separation costs, limited yields, large consumption of organic solvents, and severe environmental pollution. This invention provides a recombinant *E. coli* strain for de novo synthesis of ethyl vanillin, its construction method, and its applications. This enables the microbial fermentation synthesis of ethyl vanillin using inexpensive glucose as the sole carbon source and ethanethiol as the ethyl donor, providing feasible technical support for the green industrial preparation of ethyl vanillin. To achieve this technical objective, this invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing recombinant Escherichia coli for de novo synthesis of ethyl vanillin, the method comprising the following steps: S1. Construct the first, second, third, and fourth recombinant expression vectors, respectively, wherein: The first recombinant expression vector carries the gene encoding 3-dehydroshikimate dehydrogenase; The second recombinant expression vector carries the gene encoding carboxylic acid reductase and the gene encoding 4′-phosphopanylthioethylamine transferase; The third recombinant expression vector carries catechol- O 1-methyltransferase encoding gene and methionine adenosine transferase encoding gene; The fourth recombinant expression vector carries O -Genes encoding acetylhomoserine thiolactone sulfatase and acylhomoserine transferase; S2. The first recombinant expression vector was introduced into Escherichia coli RARE competent cells, and positive strains containing the first recombinant expression vector were screened. Then, the second, third and fourth recombinant expression vectors were introduced into the positive strains in sequence. Positive transformants were screened after each round of introduction. S3. Obtain a recombinant Escherichia coli strain capable of de novo synthesis of ethyl vanillin using glucose as a carbon source and ethanethiol as an ethyl donor.

[0005] Furthermore, in the above construction method, in the first recombinant expression vector, the 3-dehydroshikimate dehydrogenase encoding gene is derived from Bacillus thuringiensis (Bt). Bacillus thuringiensis )of asbF The gene, whose nucleotide sequence is shown in SEQ ID NO:1.

[0006] Furthermore, in the above construction method, in the second recombinant expression vector, the carboxylate reductase encoding gene is derived from marine mycobacteria (…). Mycobacterium marinum )of car The gene, whose nucleotide sequence is shown in SEQ ID NO:2; the gene encoding the 4′-phosphopantoylthioethylamine transferase is derived from Bacillus subtilis (… Bacillus subtilis )of sfp The gene, whose nucleotide sequence is shown in SEQ ID NO:3.

[0007] Furthermore, in the above construction method, in the third recombinant expression vector, the catechol- O The gene encoding methyltransferase is derived from the ditch rat ( Rattus norvegicus )of COMT Y200L The gene, whose nucleotide sequence is shown in SEQ ID NO:4; the methionine adenosine transferase encoding gene is derived from Homo sapiens (…). Homo sapiens )of MAT2A I322V The gene, whose nucleotide sequence is shown in SEQ ID NO:5.

[0008] Furthermore, in the above construction method, in the fourth recombinant expression vector, the... O -The gene encoding acetylhomoserine thiosulfate is derived from Corynebacterium glutamicum ( Corynebacterium glutamicum )of metY The gene may have originated from brewer's yeast ( Saccharomyces cerevisiae )of MET17 Genes, the ones mentioned metY The nucleotide sequence of the gene is shown in SEQ ID NO:6. MET17 The nucleotide sequence of the gene is shown in SEQ ID NO:7; The gene encoding the acylhomoserine transferase is derived from Bacillus cereus ( Bacillus cereus )of metXbc The gene may have originated from Leptospira mesei ( Leptospira meyeri )of metXlm mut Genes, the ones mentioned metXbc The nucleotide sequence of the gene is shown in SEQ ID NO:8. metXlm mut The nucleotide sequence of the gene is shown in SEQ ID NO:9.

[0009] Furthermore, in the above construction method, each coding gene was optimized using E. coli codons.

[0010] Furthermore, in the above construction method, the first recombinant expression vector is based on pTac15K, the second recombinant expression vector is based on pBBR1Tac, the third recombinant expression vector is based on pTrc99A, and the fourth recombinant expression vector is based on pCDFTac.

[0011] Secondly, the present invention provides a recombinant Escherichia coli for de novo synthesis of ethyl vanillin, wherein the recombinant Escherichia coli is constructed by the construction method described above.

[0012] Furthermore, the recombinant Escherichia coli contains: carrying asbF pTac15K-derived plasmids carrying the gene car Genes and sfp pBBR1Tac-derived plasmids carrying the gene COMT Y200L Genes and MAT2A I322V pTrc99A-derived plasmids of the gene, and those carrying O - Acetylhomoserine thiolase encoding gene (selected from...) metY or MET17 ) and acylhomoserine transferase encoding genes (selected from metXbc ormetXlm mut pCDFTac-derived plasmids.

[0013] Thirdly, the present invention seeks protection for the use of the recombinant Escherichia coli described above in the fermentation preparation of ethyl vanillin.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a complete de novo biosynthesis pathway for ethyl vanillin by introducing heterologous synthetic genes into *E. coli* and combining them in a modular fashion, achieving de novo synthesis of ethyl vanillin using inexpensive glucose as the sole carbon source and ethanethiol as the ethyl donor. Shake-flask fermentation verification showed that the recombinant *E. coli* containing the complete synthetic pathway achieved a ethyl vanillin yield of 7.3 mg / L with the addition of 1 mM ethanethiol; in the control system using L-ethionine as the ethyl donor, the yield reached 8.5 mg / L with the addition of 5 mM L-ethionine, demonstrating the effectiveness of the constructed metabolic pathway.

[0015] At the functional module level, the upstream protocatechuic aldehyde synthesis module exhibited high catalytic efficiency. Specifically, the recombinant strain overexpressing 3-dehydroshikimate dehydrogenase accumulated 709.0 mg / L of protocatechuic acid. Further introduction of a carboxylic acid reductase system completely converted protocatechuic acid to protocatechuic aldehyde, yielding 310.7 mg / L with no protocatechuic acid residue, providing sufficient precursor substrate for the downstream ethylation reaction. The downstream ethyl transfer system module was also validated. Under conditions of L-ethionine as the ethyl donor and exogenous addition of protocatechuic aldehyde, the recombinant strain converted the substrate to 22.8 mg / L of ethyl vanillin. Under conditions of ethanethiol as the ethyl donor, recombinant strains with four different gene combinations could synthesize ethyl vanillin, with yields ranging from 4.7 mg / L to 6.9 mg / L, indicating that… O The combination of acetylhomoserine thiolate and acylhomoserine transferase can effectively convert ethanethiol into an intracellular ethyl donor.

[0016] Furthermore, this invention uses microbial fermentation as the core to replace the traditional chemical synthesis process. The production process does not require the consumption of large amounts of organic solvents, which can effectively alleviate the problems of long traditional process routes, high cost of by-product separation, and serious environmental pollution. It has the characteristics of low raw material cost, mild reaction conditions, and environmental friendliness, and has the potential for large-scale industrial production. At the same time, it provides a feasible technical basis and research direction for the subsequent construction of higher-yield ethyl vanillin engineered strains. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the biosynthetic pathway and genetic engineering modification of ethyl vanillin in recombinant Escherichia coli according to the present invention.

[0018] Figure 2 This is a plasmid map of plasmid pLEV-01 in an embodiment of the present invention.

[0019] Figure 3 This is a plasmid map of plasmid pLEV-02 in an embodiment of the present invention.

[0020] Figure 4 This is a plasmid map of plasmid pLEV-03 in an embodiment of the present invention.

[0021] Figure 5 This is a plasmid map of plasmid pLEV-S-01 in an embodiment of the present invention.

[0022] Figure 6 This is a plasmid map of plasmid pLEV-S-02 in an embodiment of the present invention.

[0023] Figure 7 This is a plasmid map of plasmid pLEV-S-03 in an embodiment of the present invention.

[0024] Figure 8 This is a plasmid map of plasmid pLEV-S-04 in an embodiment of the present invention.

[0025] Figure 9 This is a plasmid map of plasmid pLEV-S-05 in an embodiment of the present invention.

[0026] Figure 10 This is a plasmid map of plasmid pLEV-S-06 in an embodiment of the present invention.

[0027] Figure 11 The bar chart shows the fermentation results of strains LEV-01, LEV-02, and LEV-03 in the embodiments of the present invention.

[0028] Figure 12 The bar chart shows the fermentation results of strains LEV-04 and LEV-S-01 in the embodiments of the present invention.

[0029] Figure 13 This is a bar chart showing the fermentation results of strains LEV-S-02 to LEV-S-06 in the embodiments of the present invention.

[0030] Figure 14 The bar chart shows the fermentation results of strains LEV-S-07 and LEV-S-08 in the embodiments of the present invention.

[0031] Figure 15 These are high-performance liquid chromatography (HPLC) peak chromatograms of the fermentation products of strains LEV-01 and LEV-S-07 in the embodiments of this invention, detected in the 258nm detection channel. Wherein, A is the chromatogram of the mixed standard; B is the chromatogram of the control strain LEV-01; and C is the chromatogram of the fermentation sample of strain LEV-S-07.

[0032] Figure 16 These are high-performance liquid chromatography (HPLC) peak chromatograms of the fermentation products of strains LEV-01 and LEV-S-07 in the embodiments of this invention, detected in the 280nm detection channel. Wherein, A is the chromatogram of the mixed standard; B is the chromatogram of the control strain LEV-01; and C is the chromatogram of the fermentation sample of strain LEV-S-07. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] The genotypes of the strains and plasmids involved in the examples are shown in Table 1.

[0036] Table 1. Genotypes of strains and plasmids

[0037] The primers and their specific sequences involved in the examples are shown in Table 2.

[0038] Table 2. Primer sequence listing

[0039] The culture medium components involved in the examples are as follows: 1. LB medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, and deionized water to a final volume of 1L.

[0040] 2. SOC resuscitation medium: 2.0 g tryptone, 0.5 g yeast extract, 0.05 g sodium chloride, 2.5 mM potassium chloride, and deionized water to a final volume of 100 mL. Adjust the pH to 7.0. Simultaneously prepare a 2 M magnesium chloride stock solution and a 1 M glucose stock solution. All three components are separately autoclaved. Before use, add the stock solutions to the medium to achieve a final magnesium chloride concentration of 10 mM and a final glucose concentration of 20 mM.

[0041] 3. Fermentation medium: glucose 10g / L, MgSO4 The following concentrations were added: 7H₂O 0.8 g / L, KH₂PO₄ 6.67 g / L, (NH₄)₂HPO₄ 4 g / L, citric acid 0.8 g / L, and trace metal salt solution 5 mL / L, pH 7.0. The trace metal salt solution consisted 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 a separately sterilized mother liquor with concentrations of 500 g / L and 80 g / L, which should be added before use.

[0042] Example 1: Overall genetic engineering modification and synthetic pathway design of recombinant Escherichia coli.

[0043] This embodiment uses Escherichia coli RARE as the starting strain and employs a modular genetic engineering strategy to construct a complete artificial biosynthesis pathway for ethyl vanillin within the strain's cells, ultimately achieving de novo synthesis of ethyl vanillin using inexpensive glucose as a substrate and ethanethiol as the ethyl donor.

[0044] The RARE (Reduced Aromatic Aldehyde Reduction) strain was purchased from the Addgene Plasmid Depository (Addgene, Watertown, MA, USA). The depository's contact information is: Room 100, 490 Arsenal Road, Watertown, MA, 02472, USA; website: www.addgene.org; email: help@addgene.org; phone: +1 (617) 233-4363; strain number: Addgene #61440. The depositor is the laboratory of Kristala LJ Prather (Massachusetts Institute of Technology), located at 77 Massachusetts Avenue, Cambridge, MA, MIT; official email: kljp@mit.edu. The construction method of this strain can be found in the literature: Kunjapur AM, Tarasova Y, Prather KLJ. Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli . Journal of the American Chemical Society , 2014, 136(33): 11644-11654. Information about this strain is also disclosed in patent CN122706728A.

[0045] The overall modification and synthesis approach 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 the pentose phosphate pathway (PPP) within the cell. PEP and E4P are then processed by 3-deoxy-d-arabinohepenolate-7-phosphate (DAHP) synthase (from endogenous sources). aroG , aroF , aroH The gene encodes DAHP, which is then catalyzed to 3-dehydroshikimate (3-DHS) via the shikimate pathway. 3-DHS is then dehydroshikimate dehydrogenase (AsbF) by 3-dehydroshikimate dehydrogenase. asbF The gene encodes (carboxylic acid) to catalyze the conversion of protocatechuic acid (PCA), which is then catalyzed by a carboxylic acid reductase system to protocatechuic aldehyde (PCALD). This enzyme system includes carboxylic acid reductase (Car, which is produced by...) carThe gene encodes) and its activating enzyme, 4′-phosphopantoylthioethylaminotransferase (Sfp, by) sfp (Gene encoding); PCALD in catechol- O -Methyltransferase (COMT, by...) COMT Y200L Under the catalysis of gene encoding, receiving S The ethyl group on the adenosine-L-ethionine (SAE) molecule is transferred to the meta-phenolic hydroxyl group, converting it into ethyl vanillin, while SAE undergoes deethylation to convert into... S -Adenosine-L-homocysteine ​​(SAH). SAE can be produced by L-ethionine via methionine adenosine transferase (MAT). MAT2A I322V L-ethionine is obtained under the catalysis of gene encoding (and can be obtained from) O - Acetylhomoserine (OAH) in O - Acetylhomoserine thiolase (MetY / MET17, by...) metY or MET17 Under the catalysis of gene encoding, OAH reacts with ethanethiol to produce acetic acid, thereby achieving the transfer of ethyl from ethanethiol to ethyl vanillin. OAH can be obtained from L-homose (L-Hse) converted from glucose intracellularly via acylhomoserine transferase (MetX). metXbc or metXlm mut The reaction, catalyzed by the gene encoding acetyl-CoA, consumes acetyl-CoA to generate coenzyme A (CoA), which in turn reacts with endogenous acetyl-CoA synthase (Acs) to produce coenzyme A. acs Under the catalysis of gene encoding, acetyl-CoA is regenerated.

[0046] To achieve efficient operation of the above pathway, the starting strain underwent the following basic modifications in this embodiment to construct a complete artificial synthesis pathway. Each modification corresponds to... Figure 1 The annotations in the text are as follows: 1. 3-Dehydroshikimate dehydrogenase (AsbF) overexpression modification Overexpression of the gene encoding 3-dehydroshikimate dehydrogenase asbF This modification enables highly efficient catalysis of 3-DHS to PCA, providing a reaction substrate for the subsequent conversion of intermediate products. This gene can be selected from Bacillus thuringiensis (Bt). Bacillus thuringiensis )of asbF The gene, or other optional exogenous gene encoding 3-dehydroshikimate dehydrogenase. asbF The optimized nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0047] 2. Overexpression modification of carboxylic acid reductase system (Car+Sfp) Overexpression of the gene encoding carboxylic acid reductase car Simultaneously, overexpression of the gene encoding 4′-phosphoubiotylethylamine transferase sfp Car is phosphorylated to achieve its normal function. This modification enables the catalytic conversion of PCA to PCALD, opening the entry point from the upstream precursor to the downstream synthetic pathway. car Genes can be selected from marine mycobacteria ( Mycobacterium marinum )of car The gene, or other optional exogenous carboxylic acid reductase encoding gene. sfp Genes can be selected from Bacillus subtilis ( Bacillus subtilis )of sfp The gene, or other optional exogenous gene encoding 4′-phosphopantoylthioethylamine transferase. car The codon-optimized nucleotide sequence of the gene is shown in SEQ ID NO:2; sfp The optimized nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0048] 3. Catechins- O 2-methyltransferase (COMT) overexpression modification Overexpression of catechol- O α-methyltransferase encoding gene COMT Y200L This modification enables PCALD to accept ethyl groups from SAE molecules, ultimately catalyzing the conversion to the target product, ethyl vanillin. COMT Y200L Genes can be selectively derived from ditch rats ( Rattus norvegicus )of COMT Y200L Genes, or other optional exogenous catechols - O The gene encoding α-methyltransferase. COMT Y200L The optimized nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0049] 4. Methionine adenosine transferase (MAT) overexpression modification Overexpression of the gene encoding methionine adenosine transferase MAT2A I322V This modification enables the catalytic conversion of L-ethionine to SAE, providing a substrate for the modification in step 3. MAT2A I322V Genes can be selected from Homo sapiens ( Homo sapiens )of MAT2AI322V The gene, or other optional gene encoding methionine adenosine transferase. MAT2A I322V The optimized nucleotide sequence of the gene is shown in SEQ ID NO:5.

[0050] 5. O - Acetylhomoserine thiosulfate enzyme (MetY / MET17) overexpression modification overexpression O The gene encoding α-acetylhomoserine thiosulfate enzyme. This modification enables the reaction of OAH with ethanethiol to synthesize L-acetylthionine, and, combined with the modification in step 4, achieves the transfer of ethyl groups from ethanethiol to ethyl vanillin. The gene can be selected from Corynebacterium glutamicum (…). Corynebacterium glutamicum )of metY The gene may have originated from brewer's yeast ( Saccharomyces cerevisiae )of MET17 Genes, or other optional exogenous substances O The gene encoding acetylhomoserine thiosulfate. metY The codon-optimized nucleotide sequence of the gene is shown in SEQ ID NO:6. MET17 The optimized nucleotide sequence of the gene is shown in SEQ ID NO:7.

[0051] 6. Acylhomoserine transferase (MetX) overexpression modification Overexpression of the gene encoding acyl-homoserine transferase. This modification enables the catalytic conversion of endogenous L-homoserine to OAH, completing the construction of the entire artificial synthetic pathway. The gene can be selected from Bacillus cereus (…). Bacillus cereus )of metXbc The gene may originate from Leptospira mesei ( Leptospira meyeri )of metXlm mut The gene, or the gene encoding another optional exogenous acyl-homoserine transferase. metXbc The codon-optimized nucleotide sequence of the gene is shown in SEQ ID NO:8. metXlm mut The optimized nucleotide sequence of the gene is shown in SEQ ID NO:9.

[0052] It should be noted that all heterologous genes in this invention have been codon-optimized to fit the expression system of E. coli.

[0053] To achieve efficient expression of metabolic pathways, this embodiment uses different plasmid vectors to carry the above-mentioned functional genes in modules: among which, asbF The gene was expressed using the pTac15K plasmid;car and sfp The gene was expressed via the pBBR1Tac plasmid; COMT Y200L The gene was expressed via the pTrc99A plasmid; MAT2A I322V The gene was expressed using the pCDFTac plasmid. To avoid plasmid conflicts, subsequent steps will... COMT Y200L Genes and MAT2A I322V All genes were expressed using the pTrc99A plasmid. metY Gene, MET17 Gene, metXbc Genes and metXlm mut All genes were expressed using the pCDFTac plasmid.

[0054] Those skilled in the art can understand the strain modification strategy of the present invention based on the description of this embodiment. Through the above modification, a complete de novo synthesis pathway of ethyl vanillin with ethyl mercaptan as the ethyl donor can be constructed in Escherichia coli, providing a foundation for subsequent fermentation production.

[0055] Example 2: Construction of recombinant expression plasmid.

[0056] 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.

[0057] 1. Construction of pLEV-01 plasmid The plasmid uses pTac15K as a base vector to carry substances derived from Bacillus thuringiensis (Bt). Bacillus thuringiensis )of asbF Gene. The pTac15K plasmid was double-digested with restriction endonucleases EcoRI and SacI to obtain a linearized pTac15K plasmid vector fragment; simultaneously, using asbF_F and asbF_R as primers, a gene containing the fully synthesized gene was generated. asbF Using the gene plasmid as a template, amplification asbF The gene, whose nucleotide sequence is shown in SEQ ID NO:1, was used to synthesize the linearized pTac15K fragment after enzyme digestion and amplification. asbF The gene fragment was subjected to agarose gel electrophoresis, the correct band was excised, and purified using a gel extraction kit. The purified... asbF The gene fragment was double-digested with restriction endonucleases EcoRI and SacI, and then purified using a gel extraction kit. The purified fragment was then... asbFThe gene fragment and the linearized pTac15K fragment were ligated using T4 DNA ligase. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing kanamycin. Colony PCR was used to verify single clones; positive clones were sent to a sequencing company for sequencing to confirm the successfully ligated plasmid pLEV-01. Its plasmid map is shown below. Figure 2 As shown.

[0058] 2. Construction of pLEV-02 plasmid The plasmid is based on pBBR1Tac and is used to carry bacteria derived from marine mycobacteria (PbBR1Tac). Mycobacterium marinum )of car Genes and derived from Bacillus subtilis ( Bacillus subtilis )of sfp Genes. Using pBBR1Tac plasmid as a template and pBBR1Tac_F and pBBR1Tac_R as primers, linearized plasmid fragments were amplified by PCR; simultaneously, using car-1_F and car-2_R, and sfp_F and sfp_R as primers, respectively, gene sequences containing fully synthesized genes were amplified. car Genes and sfp Using the gene plasmid as a template, amplification car Genes and sfp The gene, whose nucleotide sequences are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The linearized pBBR1Tac fragment, car Gene fragments and sfp 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. Colony PCR was used to verify single clones, and positive clones were sent to a sequencing company for sequencing to confirm the successfully ligated plasmid pLEV-02. Its plasmid map is shown below. Figure 3 As shown.

[0059] 3. Construction of pLEV-03 plasmid The essential granule uses pTrc99A as the base carrier to carry granulation material derived from ditch rats ( Rattus norvegicus )of COMT Y200L Gene. The pTrc99A plasmid was double-digested with restriction endonucleases EcoR I and Kpn I to obtain a linearized pTrc99A plasmid vector fragment; simultaneously, using COMT_F and COMT_R as primers, a gene containing the fully synthesized gene was generated. COMT Y200L Using the gene plasmid as a template, amplification COMT Y200LThe gene, whose nucleotide sequence is shown in SEQ ID NO:4, was used. The linearized pTrc99A fragment after enzyme digestion was compared with the amplified... COMT Y200L The gene fragment was subjected to agarose gel electrophoresis, the correct band was excised, and purified using a gel extraction kit. The purified... COMT Y200L The gene fragment was double-digested with restriction endonucleases EcoRI and KpnI, and then purified using a gel extraction kit. The purified fragment was then... COMT Y200L The gene fragment and the linearized pTrc99A fragment were ligated using T4 DNA ligase. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing ampicillin. Colony PCR was used to verify single clones; positive clones were sent to a sequencing company for sequencing to confirm the successfully ligated plasmid pLEV-03. Its plasmid map is shown below. Figure 4 As shown.

[0060] 4. Construction of pLEV-S-01 plasmid The essential particles are based on pCDFTac as a carrier, used to carry information derived from Homo sapiens (…). Homo sapiens )of MAT2A I322V Gene. The pCDFTac plasmid was double-digested with restriction endonucleases EcoRI and SacRI to obtain a linearized pCDFTac plasmid vector fragment; simultaneously, using MAT2A_F and MAT2A_R as primers, a gene containing the fully synthesized gene was extracted. MAT2A I322V Using the gene plasmid as a template, amplification MAT2A I322V The gene, whose nucleotide sequence is shown in SEQ ID NO:5, was used. The linearized pCDFTac fragment after enzyme digestion was compared with the amplified... MAT2A I322V The gene fragment was subjected to agarose gel electrophoresis, the correct band was excised, and purified using a gel extraction kit. The purified... MAT2A I322V The gene fragment was double-digested with restriction endonucleases EcoRI and SacI, followed by purification using a gel extraction kit. The purified fragment was then... MAT2A I322V The gene fragment and the linearized pCDFTac fragment were ligated using T4 DNA ligase. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin sulfate. Colony PCR was used to verify single clones; positive clones were sent to a sequencing company for sequencing to confirm the successfully ligated plasmid pLEV-S-01. Its plasmid map is shown below. Figure 5 As shown.

[0061] 5. Construction of pLEV-S-02 plasmid The essential granule uses pTrc99A as the base carrier to carry granulation material derived from ditch rats ( Rattus norvegicus )of COMT Y200L Genes and originating from Homo sapiens ( Homo sapiens )of MAT2A I322V Genes. The pLEV-03 plasmid was digested with restriction endonuclease Xba I to obtain a linearized pLEV-03 plasmid fragment; simultaneously, using MAT2A_F2 and MAT2A_R2 as primers, a gene containing the fully synthesized gene was extracted. MAT2A I322V Using the gene plasmid as a template, amplification MAT2A I322V The gene, whose nucleotide sequence is shown in SEQ ID NO:5, was used. The linearized pLEV-03 fragment after enzyme digestion was compared with the amplified... MAT2A I322V The gene fragment was subjected to agarose gel electrophoresis, the correct band was excised, and purified using a gel extraction kit. The purified... MAT2A I322V The gene fragment and the linearized pLEV-03 fragment were recombinated using a seamless cloning kit. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing ampicillin. Colony PCR was used to verify single clones, and positive clones were sent to a sequencing company for sequencing to confirm the successfully ligated plasmid pLEV-S-02. Its plasmid map is shown below. Figure 6 As shown.

[0062] 6. Construction of plasmids pLEV-S-03 to pLEV-S-06 All of the above plasmids are based on pCDFTac as a vector for carrying... O - Different combinations of genes encoding acetylhomoserine thiosulfate and acylhomoserine transferase. The pCDFTac plasmid was double-digested with restriction endonucleases EcoRI and BamHRI to obtain a linearized pCDFTac plasmid vector fragment; simultaneously, amplification of *Corynebacterium glutamicum* was performed using metY_F and metY_R primers, respectively. Corynebacterium glutamicum )of metY Gene (nucleotide sequence as shown in SEQ ID NO:6); amplified from Saccharomyces cerevisiae using MET17_F and MET17_R primers ( Saccharomyces cerevisiae )of MET17 Gene (nucleotide sequence as shown in SEQ ID NO:7); amplified from Bacillus cereus using metXbc_F and metXbc_R primers ( Bacillus cereus )of metXbc Gene (nucleotide sequence as shown in SEQ ID NO:8); amplification of Leptospira mesoni using metXlm_F and metXlm_R primers ( Leptospira meyeri )of metXlm mut Gene (nucleotide sequence as shown in SEQ ID NO:9). The above fragments were subjected to agarose gel electrophoresis, the correct bands were excised, and purified using a gel extraction kit.

[0063] Purified metY Gene fragments, metXbc The gene fragment and the linearized pCDFTac fragment were recombined using a seamless cloning kit. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin sulfate. Colony PCR and sequencing confirmed the recombinant result, yielding plasmid pLEV-S-03. Its plasmid map is shown below. Figure 7 As shown.

[0064] Purified metY Gene fragments, metXlm mut The gene fragment and the linearized pCDFTac fragment were recombined using a seamless cloning kit. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin sulfate. Colony PCR and sequencing confirmed the recombinant result, yielding plasmid pLEV-S-04, the plasmid map of which is shown below. Figure 8 As shown.

[0065] Purified MET17 Gene fragments, metXbc The gene fragment and the linearized pCDFTac fragment were recombined using a seamless cloning kit. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin sulfate. Colony PCR and sequencing confirmed the recombinant result, yielding plasmid pLEV-S-05, the plasmid map of which is shown below. Figure 9 As shown.

[0066] Purified MET17 Gene fragments, metXlm mut The gene fragment and the linearized pCDFTac fragment were recombined using a seamless cloning kit. The reaction product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing streptomycin sulfate. Colony PCR and sequencing confirmed the recombinant result, yielding plasmid pLEV-S-06, the plasmid map of which is shown below. Figure 10 As shown.

[0067] This embodiment constructed a series of modular functional expression plasmids, providing expression vectors for the construction of the recombinant strain in Example 3. To clarify the vector descriptions, the plasmids are explained as follows: plasmid pLEV-01 is the first recombinant expression vector, plasmid pLEV-02 is the second recombinant expression vector, and plasmid pLEV-S-02 is the third recombinant expression vector; plasmids pLEV-S-03, pLEV-S-04, pLEV-S-05, and pLEV-S-06 are parallel implementations of the fourth recombinant expression vector, and the four are respectively... O - Acetylhomoserine thiolase encoding gene ( metY or MET17 ) and the gene encoding acylhomoserine transferase ( metXbc or metXlm mut Different gene combinations. Plasmid pLEV-03 carries only the gene. COMT Y200L Plasmid pLEV-S-01 carries only the gene MAT2A I322V The first, second, third, and fourth recombinant expression vectors are intermediate plasmids obtained during the construction of the third recombinant expression vector pLEV-S-02, and are used for segmental functional verification of the downstream ethyl transfer system module. The empty plasmids pTac15K, pTrc99A, and pCDFTac do not carry functional genes and are used only as blank controls. The replicons of the first, second, third, and fourth recombinant expression vectors are p15A, pBBR1, pBR322, and CloDF13, respectively, and the resistance selection markers are kanamycin, chloramphenicol, ampicillin, and streptomycin, respectively. These four belong to different incompatible groups and can coexist stably in the same E. coli cell.

[0068] Example 3: Construction of the target strain.

[0069] 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 protocatechuic aldehyde synthesis module and the downstream ethyl transfer system module, as well as the construction of the final complete ethyl vanillin synthesis pathway strain.

[0070] 1. Preparation and electroporation transformation of electrocompetent cells The strain to be transformed was inoculated into a test tube containing 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 to 16 hours. Then, 2% of the inoculum was transferred to 15 mL of fresh LB medium and cultured at 37°C and 200 rpm for 1.5 hours, until the bacterial culture reached OD500. 600 Reach a concentration of 0.4-0.6. Centrifuge and discard the supernatant. Wash the cells twice with 10% glycerol solution. Finally, resuspend the cells in an appropriate amount of 10% glycerol solution to a final volume of 100 μL to obtain electrocompetent cells.

[0071] Take the above competent cells and mix thoroughly with approximately 50 ng of the plasmid to be transformed. Transfer the mixture to a pre-chilled 1 mm electroporation cuvette and incubate on ice for 20 min. Wipe the outside of the electroporation cuvette dry with paper and place it in an electroporator. Electroporate once using the Ec1 setting. Immediately after electroporation, add 600 μL of pre-chilled SOC resuscitation medium to the cuvette, mix gently, and tilt the cuvette to transfer the entire mixture to a sterile 1.5 mL centrifuge tube. Incubate at 37°C and 200 rpm for 45 min. Spread 80 μL of the bacterial culture onto an LB agar plate containing the appropriate antibiotic and incubate overnight at 37°C to obtain the transformants.

[0072] 2. Construction of the upstream protocatechuic aldehyde synthesis module validation strain 2.1 Electrocompetent cells of strain RARE were prepared using the method described in step 1. The empty plasmid pTac15K was introduced into RARE and spread onto LB plates containing kanamycin sulfate. The plates were cultured overnight at 37°C to obtain strain LEV-01, which served as the blank control strain for the upstream module.

[0073] 2.2 Electrocompetent cells of strain RARE were prepared using the method described in step 1. Plasmid pLEV-01 was introduced into RARE, plated onto LB agar plates containing kanamycin sulfate, and cultured overnight at 37°C to obtain strain LEV-02. This strain can express… asbF The gene, which is an intermediate strain in the upstream module, can synthesize protocatechuic acid.

[0074] 2.3 Using the method described in step 1, electrocompetent cells of strain LEV-02 were prepared. Plasmid pLEV-02 was introduced into LEV-02 and plated onto LB agar plates containing kanamycin sulfate and chloramphenicol. The plates were incubated overnight at 37°C to obtain strain LEV-03. This strain can express… asbF Gene, car Genes and sfp The gene, which is the final upstream module strain, can synthesize protocatechuic aldehyde.

[0075] 3. Construction of the downstream ethyl transfer system module validation strain 3.1 Validation strains using L-ethionine as the ethyl donor Electrocompetent cells of strain RARE were prepared using the method described in step 1. The empty plasmid pTrc99A was introduced into RARE and plated onto LB agar plates containing ampicillin sodium. The plates were then incubated overnight at 37°C to obtain strain RARE / pTrc99A. Electrocompetent cells of strain RARE / pTrc99A were then prepared using the method described in step 1. The empty plasmid pCDFTac was introduced into RARE and plated onto LB agar plates containing ampicillin sodium and streptomycin sulfate. The plates were then incubated overnight at 37°C to obtain strain LEV-04, which served as a blank control strain for the downstream module using L-ethionine as the ethyl donor.

[0076] Electrocompetent cells of strain RARE were prepared using the method described in step 1. Plasmid pLEV-03 was introduced into RARE, and the cells were plated onto LB agar plates containing ampicillin sodium and incubated overnight at 37°C to obtain strain RARE / pLEV-03. Electrocompetent cells of strain RARE / pLEV-03 were then prepared using the method described in step 1. Plasmid pLEV-S-01 was introduced into RARE, and the cells were plated onto LB agar plates containing ampicillin sodium and streptomycin sulfate and incubated overnight at 37°C to obtain strain LEV-S-01. This strain can express… COMT Y200L Genes and MAT2A I322V The gene, as an intermediate strain in the downstream module, can utilize protocatechuic aldehyde and L-ethionine to produce ethyl vanillin.

[0077] 3.2 Validation strains using ethanethiol as the ethyl donor Electrocompetent cells of strain RARE were prepared using the method described in step 1. The plasmid pLEV-S-02 was introduced into RARE and plated onto LB plates containing ampicillin sodium. The plates were then incubated overnight at 37°C to obtain strain RARE / pLEV-S-02.

[0078] Electrocompetent cells of strain RARE / pLEV-S-02 were prepared using the method described in step 1. Empty plasmid pCDFTac, plasmid pLEV-S-03, plasmid pLEV-S-04, plasmid pLEV-S-05, and plasmid pLEV-S-06 were introduced into them, respectively, and then plated onto LB plates containing ampicillin sodium and streptomycin sulfate. The plates were cultured overnight at 37°C to obtain strains LEV-S-02, LEV-S-03, LEV-S-04, LEV-S-05, and LEV-S-06, respectively.

[0079] Among them, LEV-S-02 can only express COMT Y200L Genes and MAT2A I322V The gene serves as a blank control strain for the downstream module, using ethanethiol as the ethyl donor. LEV-S-03 can express... COMT Y200L Gene, MAT2A I322V Gene, metY Genes and metXbc Gene; LEV-S-04 can be expressed COMT Y200L Gene, MAT2A I322V Gene, metY Genes and metXlm mut Gene; LEV-S-05 can be expressed COMT Y200L Gene, MAT2A I322V Gene, MET17 Genes and metXbc Gene; LEV-S-06 can be expressed COMT Y200L Gene, MAT2A I322V Gene, MET17 Genes and metXlm mut Genes. Strains LEV-S-03, LEV-S-04, LEV-S-05, and LEV-S-06 are all final downstream module strains that can utilize protocatechuic aldehyde and ethanethiol to produce ethyl vanillin.

[0080] 4. Construction of recombinant strains with complete synthetic pathway 4.1 Electrocompetent cells of strain LEV-03 were prepared using the method described in step 1. Plasmid pLEV-S-02 was introduced into the cells and plated onto LB agar plates containing kanamycin sulfate, chloramphenicol, and ampicillin sodium. The plates were incubated overnight at 37°C to obtain strain LEV-S-07. This strain incorporates all the modifications described in Examples 1-4 and enables the de novo synthesis of ethyl vanillin using glucose as a substrate and L-ethionine as the ethyl donor.

[0081] 4.2 Electrocompetent cells of strain LEV-S-07 were prepared using the method described in step 1. Plasmid pLEV-S-05 was introduced into the cells and plated onto LB agar plates containing kanamycin sulfate, chloramphenicol, ampicillin sodium, and streptomycin sulfate. The plates were incubated overnight at 37°C to obtain strain LEV-S-08. This strain incorporates all the modifications described in Examples 1-6 of Example 1 and enables the de novo synthesis of ethyl vanillin using glucose as a substrate and ethanethiol as the ethyl donor.

[0082] This embodiment yielded a series of gradient-modified validation strains, providing a foundation for subsequent fermentation function validation experiments.

[0083] Example 4: Fermentation culture and product detection of recombinant Escherichia coli.

[0084] This embodiment is used to ferment and culture the recombinant strains constructed in Example 3 to verify the catalytic effect of each functional module and the final de novo synthesis capability of ethyl vanillin.

[0085] 1. Culture medium preparation 1.1 LB seed culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, and deionized water to a final volume of 1L.

[0086] 1.2 Fermentation medium: glucose 10 g / L, MgSO4 The concentrations of the following substances are present: 7H₂O 0.8 g / L, KH₂PO₄ 6.67 g / L, (NH₄)₂HPO₄ 4 g / L, citric acid 0.8 g / L, and trace metal salt solution 5 mL / L, pH 7.0. 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 was used to prepare separately sterilized stock solutions with concentrations of 500 g / L and 80 g / L, which were added to the culture medium before use.

[0087] 2. Preparation of Seed Liquid Select a single clone from the corresponding strain plate and inoculate it into a test tube containing 5 mL of LB seed medium and the corresponding antibiotic. Incubate at 37℃ and 200 rpm for 12-16 hours to obtain activated seed liquid.

[0088] 3. 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 and the corresponding antibiotics. Incubate at 30℃ and 200rpm with shaking for a total fermentation period of 60 hours. After 8 hours of post-inoculation culture, observe the OD of the bacterial culture. 600 When the concentration reached 0.5–0.8, IPTG was added to a final concentration of 1.0 mM to induce the expression of functional genes. Three biological replicates were set up for fermentation of all strains to ensure the reliability of the results.

[0089] For strains used for different validation purposes, additional substrates or ethyl donors were added during fermentation: For strains LEV-04 and LEV-S-01, used for validation of downstream modules using L-ethionine as an ethyl donor, an additional 200 mg / L (approximately 1.45 mM) of protocatechuic aldehyde and 236 mg / L (approximately 1.45 mM) of L-ethionine were added 12 h after inoculation to supplement the reaction substrates for the downstream modules and validate their catalytic function; for strains LEV-S-02, LEV-S-03, LEV-S-04, LEV-S-05, and LEV-S-06, used for validation of downstream modules using ethanethiol as an ethyl donor... S-06, after 12 hours of inoculation, was supplemented with a final concentration of 276 mg / L (2 mM) of protocatechuic aldehyde and 10 mM of ethanethiol to replenish the reaction substrate of the downstream module and verify the catalytic function of the downstream module; strain LEV-S-07, which has a de novo synthesis pathway with L-ethionine as the ethyl donor, was supplemented with a final concentration of 1 mM, 3 mM and 5 mM of L-ethionine as the ethyl donor after 12 hours of inoculation; strain LEV-S-08, which has a de novo synthesis pathway with ethanethiol as the ethyl donor, was supplemented with a final concentration of 1 mM, 10 mM and 100 mM of ethanethiol as the ethyl donor after 12 hours of inoculation.

[0090] 4. Processing and detection of fermentation samples 4.1 Sample Pretreatment: After fermentation, take 1 mL of fermentation broth, and then take 100 μL of bacterial culture, dilute it 10 times, and measure 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 particulate impurities such as bacteria, and the sample to be tested is obtained.

[0091] 4.2 HPLC Detection Conditions: High-performance liquid chromatography (HPLC) was used for sample detection. The chromatographic system used was a Shimadzu LC-16 series with a UV-Vis spectrophotometric detection module. The chromatographic column was an Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm). The mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and phase B was methanol. The flow rate was 0.65 mL / min. The elution program was as follows: 0 min–1 min, 10% B; 1 min–10 min, phase B linearly increased from 10% to 70%; 10 min–12 min, maintained at 70% B; 12 min–14 min, phase B linearly decreased from 70% to 10%; 14 min–23 min, maintained at 10% B. The sample injection volume was 5 μL, and the column oven temperature was 30℃. The detection wavelength is set to two channels: Channel 1 has a detection wavelength of 280 nm and is used for the quantitative detection of protocatechuic aldehyde, byproduct vanillin and ethyl vanillin; Channel 2 has a detection wavelength of 258 nm and is used for the quantitative detection of protocatechuic acid.

[0092] Product identification was performed using the peak elution time of the standards. The chromatographic results of the mixed standards at a 258 nm detection channel are as follows: Figure 15 As shown in Figure A, peak 1 is the protocatechuic acid standard, with an elution time of 10.350 min. The chromatographic results of the mixed standards under a 280 nm detection channel are shown below. Figure 16 As shown in Figure A, peak 2 is protocatechuic aldehyde standard with an elution time of 11.733 min, peak 3 is vanillin standard with an elution time of 13.883 min, and peak 4 is ethyl vanillin standard with an elution time of 15.225 min. The qualitative analysis of the product can be achieved by comparing the elution times of the fermented sample with those of the standards, while the quantitative analysis can be achieved based on the standard curve.

[0093] 5. Fermentation Results and Functional Verification 5.1 Functional Verification of the Upstream Protocatechuic Aldehyde Synthesis Module For strains LEV-01 to LEV-03 used to verify the function of the upstream module, the fermentation results of each strain are as follows: Figure 11 As shown in the figure. The results showed that LEV-02 produced 709.0 mg / L of protocatechuic acid, LEV-03 produced 310.7 mg / L of protocatechuic aldehyde with no protocatechuic acid residue, while the control strain LEV-01 produced neither protocatechuic acid nor protocatechuic aldehyde, demonstrating the effectiveness of the upstream protocatechuic aldehyde synthesis module.

[0094] 5.2 Functional Verification of the Downstream Ethyl Transfer System Module For strains LEV-04 and LEV-S-01, used to validate the downstream module using L-ethionine as the ethyl donor, the fermentation results of the two strains are as follows: Figure 12 As shown in the figure. The results showed that strain LEV-S-01 could convert added protocatechuic aldehyde and an equimolar amount of L-ethionine into ethyl vanillin at a concentration of 22.8 mg / L, while simultaneously generating 26.0 mg / L of vanillin as a byproduct. In contrast, only protocatechuic aldehyde could be detected in the culture medium in the control strain LEV-04, demonstrating the effectiveness of the downstream ethyl transfer system module with L-ethionine as the ethyl donor.

[0095] For strains LEV-S-02 to LEV-S-06 used to validate the downstream module using ethanethiol as the ethyl donor, the fermentation results of each strain are as follows: Figure 13 As shown in the figure. The results showed that the control strain LEV-S-02 produced no ethyl vanillin, while strains LEV-S-03, LEV-S-04, LEV-S-05 and LEV-S-06 produced ethyl vanillin at concentrations of 4.8 mg / L, 4.7 mg / L, 6.9 mg / L and 6.2 mg / L, respectively, demonstrating the effectiveness of the downstream ethyl transfer system module with ethanethiol as the ethyl donor.

[0096] 5.3 Functional Verification of the Complete Synthetic Pathway For the complete pathway strains LEV-S-07 and LEV-S-08, the fermentation results of the two strains are as follows: Figure 14 As shown in the figure, the results indicated that strain LEV-S-07 could achieve de novo synthesis of ethyl vanillin using glucose as a substrate under different concentrations of L-ethionine: the yield was 5.8 mg / L with a final concentration of 1 mM L-ethionine, 7.0 mg / L with a final concentration of 3 mM L-ethionine, and 8.5 mg / L with a final concentration of 5 mM L-ethionine. Under different concentrations of ethanethiol, strain LEV-S-08 could also achieve de novo synthesis of ethyl vanillin using glucose as a substrate: the yield was 7.3 mg / L with a final concentration of 1 mM ethanethiol, 7.0 mg / L with a final concentration of 10 mM ethanethiol, and 5.4 mg / L with a final concentration of 100 mM ethanethiol. These experimental results demonstrate the effectiveness of the complete ethyl vanillin synthesis pathway.

[0097] 5.4 Qualitative verification of products Chromatographic results of one group of fermentation samples of strain LEV-S-07 and the control strain LEV-01 are as follows: Figure 15 B, C (258nm detection channel) and Figure 16 The chromatographic results of the control strain LEV-01 under the 258nm detection channel are shown in Figures B and C. Figure 15As shown in B, the characteristic peak of protocatechuic acid (elution time 10.350 min) was not detected; the chromatographic results of the LEV-S-07 fermentation sample are as follows. Figure 15 As shown in C, peak 5 was identified as protocatechuic acid, and its retention time was consistent with that of peak 1 in A.

[0098] Under the 280nm detection channel, the chromatographic results of the control strain LEV-01 are as follows: Figure 16 As shown in B, the characteristic peaks of protocatechuic aldehyde (peak time 11.733 min), vanillin (peak time 13.883 min), and ethyl vanillin (peak time 15.225 min) were not detected; the chromatographic results of the LEV-S-07 fermentation sample are as follows. Figure 16 As shown in Figure C, peak 6 was identified as protocatechuic aldehyde, peak 7 as vanillin, and peak 8 as ethyl vanillin. The retention times of these three peaks were consistent with those of peaks 2, 3, and 4 in Figure A, respectively. These results indicate that the substances detected in the fermentation sample correspond one-to-one with the standards, while the control strain LEV-01 did not show peaks for these four substances, further verifying the successful synthesis of the target product, ethyl vanillin.

[0099] This embodiment clarifies the method of fermentation culture and product detection, verifies the effectiveness of the gene modification of the present invention, and realizes the de novo synthesis of ethyl vanillin using inexpensive glucose as a substrate.

[0100] 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 for de novo synthesis of ethyl vanillin, characterized in that, Includes the following steps: S1. Construct the first, second, third, and fourth recombinant expression vectors, respectively, wherein: The first recombinant expression vector carries the gene encoding 3-dehydroshikimate dehydrogenase; The second recombinant expression vector carries the gene encoding carboxylic acid reductase and the gene encoding 4′-phosphopanylthioethylamine transferase; The third recombinant expression vector carries catechol- O 1-methyltransferase encoding gene and methionine adenosine transferase encoding gene; The fourth recombinant expression vector carries O -Genes encoding acetylhomoserine thiolactone sulfatase and acylhomoserine transferase; S2. The first recombinant expression vector was introduced into Escherichia coli RARE competent cells, and positive strains containing the first recombinant expression vector were screened. Then, the second, third and fourth recombinant expression vectors were introduced into the positive strains in sequence. Positive transformants were screened after each round of introduction. S3. Obtain a recombinant Escherichia coli strain capable of de novo synthesis of ethyl vanillin using glucose as a carbon source and ethanethiol as an ethyl donor.

2. The construction method according to claim 1, characterized in that, In the first recombinant expression vector, the gene encoding 3-dehydroshikimate dehydrogenase is: asbF The gene, whose nucleotide sequence is shown in SEQ ID NO:

1.

3. The construction method according to claim 1, characterized in that, In the second recombinant expression vector, the carboxylic acid reductase encoding gene is: car The gene, whose nucleotide sequence is shown in SEQ ID NO:2; the gene encoding the 4′-phosphopantoylthioethylamine transferase is... sfp The gene, whose nucleotide sequence is shown in SEQ ID NO:

3.

4. The construction method according to claim 1, characterized in that, In the third recombinant expression vector, the catechol- O The gene encoding α-methyltransferase is: COMT Y200L The gene, whose nucleotide sequence is shown in SEQ ID NO:4; the methionine adenosine transferase encoding gene is MAT2A I322V The gene, whose nucleotide sequence is shown in SEQ ID NO:

5.

5. The construction method according to claim 1, characterized in that, In the fourth recombinant expression vector, the O The gene encoding acetylhomoserine thiolase is: metY Gene or MET17 Genes, the ones mentioned metY The nucleotide sequence of the gene is shown in SEQ ID NO:

6. MET17 The nucleotide sequence of the gene is shown in SEQ ID NO:7; The gene encoding the acylhomoserine transferase is: metXbc Gene or metXlm mut Genes, the ones mentioned metXbc The nucleotide sequence of the gene is shown in SEQ ID NO:

8. metXlm mut The nucleotide sequence of the gene is shown in SEQ ID NO:

9.

6. The construction method according to claim 1, characterized in that, Each coding gene was optimized using E. coli codons.

7. The construction method according to claim 1, characterized in that, The first recombinant expression vector is based on pTac15K, the second recombinant expression vector is based on pBBR1Tac, the third recombinant expression vector is based on pTrc99A, and the fourth recombinant expression vector is based on pCDFTac.

8. A recombinant *Escherichia coli* strain capable of de novo synthesis of ethyl vanillin, characterized in that, The recombinant Escherichia coli was constructed using the construction method described in any one of claims 1 to 7.

9. The recombinant Escherichia coli according to claim 8, characterized in that, The recombinant E. coli respectively contain: carrying asbF pTac15K-derived plasmids carrying the gene car Genes and sfp pBBR1Tac-derived plasmids carrying the gene COMT Y200L Genes and MAT2A I322V pTrc99A-derived plasmids of the gene, and those carrying O pCDFTac-derived plasmids encoding acetylhomoserine thiosulfate and acylhomoserine transferase genes.

10. The use of the recombinant Escherichia coli according to claim 8 or 9 in the fermentation preparation of ethyl vanillin.

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