A method for de novo synthesis of vanillin based on co-culture of recombinant bacteria and application thereof

Abstract

The application belongs to the field of genetic engineering and bioengineering technology, and discloses a vanillin de novo synthesis method based on recombinant bacteria co-culture and application. The method designs a de novo biosynthesis pathway containing tyrosine deaminase, ferulic acid decarboxylase and other key enzymes, constructs an Escherichia coli co-culture system composed of an upstream recombinant strain producing ferulic acid from glucose and a downstream recombinant strain converting ferulic acid into vanillin, and after optimizing the inoculation ratio of the strains, the highest yield of 7.8 mg / L is obtained under specific fermentation conditions with glucose as the substrate. The method is environmentally friendly and efficient, establishes a microbial platform for industrial production of vanillin, and provides technical support for large-scale production of vanillin and its derivatives.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and bioengineering technology, and relates to a method for de novo synthesis of vanillin based on co-culture of recombinant bacteria and its application. Background Technology

[0002] Vanillin ethyl ketone (CAS Registry No.: 498-02-2), chemically known as 4-hydroxy-3-methoxyacetophenone, is also called Apocynum venetum, Oleander ethyl ketone, and Vanillin ethyl ketone. As the main active ingredient in the traditional Chinese medicine Coptis chinensis, vanillin ethyl ketone holds an important position in traditional medicine, used to treat various diseases and possessing multiple biological functions such as anti-inflammatory, anti-tumor, and immunomodulatory effects. Furthermore, it is a key organic synthesis raw material, widely used in the synthesis of the psychotropic drug ipraristone and various novel antimalarial drugs. It is also commonly used as an antioxidant and in the medical field as a cardiotonic and diuretic, its applications spanning multiple important areas including pharmaceuticals and chemicals.

[0003] Currently, vanillin is mainly obtained through two methods: plant extraction and chemical synthesis. Plant extraction is limited by the plant's growth cycle, requires a long cultivation process, and involves cumbersome extraction steps, resulting in low efficiency and making it difficult to meet the needs of large-scale production. Chemical synthesis, on the other hand, has high requirements for reaction conditions and is prone to generating pollutants during production, which not only does not conform to the trend of environmental protection but may also have potential impacts on product purity and safety.

[0004] With the deepening of environmental protection concepts and the increasing demand for sustainable development, developing an efficient, environmentally friendly method for producing vanillin based on renewable raw materials has become a focus of industry attention. The biosynthetic method of producing vanillin from renewable biomass using microbial cell factories demonstrates broad application prospects due to its mild reaction conditions and environmental friendliness. More importantly, biosynthesized vanillin is considered natural, a characteristic that makes it more competitive in fields with stringent requirements for raw material safety, such as food, cosmetics, and pharmaceuticals. Therefore, overcoming the shortcomings of existing production technologies and constructing a de novo vanillin synthesis system based on microbial cell factories to achieve efficient, environmentally friendly, and large-scale production not only fills a technological gap in the industry but also effectively meets the diversified market demand for high-quality vanillin, possessing significant practical significance and industrial value. Summary of the Invention

[0005] This invention aims to address the technical problems of low efficiency, environmental pollution, insufficient product safety, and difficulty in large-scale production of vanillin in existing technologies. Specifically, existing plant extraction methods are limited by plant growth cycles and resource reserves, resulting in cumbersome extraction processes and low yields, failing to meet industrial demands. Chemical synthesis methods require harsh reaction conditions, easily generate toxic byproducts, cause severe environmental pollution, and struggle to guarantee product purity and safety, limiting their application in high-end fields such as food and pharmaceuticals. Existing microbial synthesis technologies mostly employ single-strain fermentation, which suffers from excessive metabolic burden, insufficient accumulation of precursor substances, or incomplete conversion, leading to low vanillin yields and hindering industrial-scale conversion. To fill these technological gaps, this invention provides a de novo synthesis method for vanillin based on recombinant bacterial co-culture and its application, achieving efficient, environmentally friendly, and large-scale production of vanillin.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] First, this invention provides a method for de novo synthesis of vanillin based on co-culture of recombinant bacteria, comprising:

[0008] include:

[0009] The design includes tyrosine deaminase TAL, 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC, and caffeic acid 3- O -Methyltransferase COMT, ferulic acid decarboxylase FDC, ferulic acid decarboxylase mutant FDC V46E The de novo biosynthetic pathway of (S)1-phenylethanol dehydrogenase ped;

[0010] A co-culture system was constructed consisting of an upstream recombinant strain that produces ferulic acid from glucose and a downstream recombinant strain that produces vanillin from ferulic acid;

[0011] The upstream and downstream recombinant strains were inoculated into a glucose-containing fermentation medium for fermentation and induction to obtain vanillin.

[0012] Furthermore, in the above method, the upstream recombinant strain is based on Escherichia coli MG1655 as the starting strain, and is modified in at least one of the following ways:

[0013] A. Overexpression originates from Rhodotorula glutinis. Rhodotorula glutinis Tyrosine deaminase RgTAL, derived from Escherichia coli Escherichia coli 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC and derived from Arabidopsis thaliana Arabidopsis thaliana caffeic acid 3- O The gene encoding the methyltransferase AtCOMT;

[0014] B. Knock out the tyrosine metabolism repression gene based on A. tyrR ;

[0015] C. Overexpression of Pseudomonas motilityis based on B. Zymomonas mobilis The gene encoding the prebenzoic acid dehydrogenase TyrC;

[0016] D. Overexpression of Escherichia coli based on C. Escherichia coli AroG, a feedback inhibition mutant of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase D146N The gene that encodes it.

[0017] Furthermore, in the above method, in step A, the codon of the tyrosine deaminase RgTAL is optimized, and its nucleotide sequence is shown in SEQ ID NO:1;

[0018] In A, the 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC comprises: 4-hydroxyphenylacetic acid-3-monooxygenase EcHpaB and flavin reductase EcHpaC; the nucleotide sequence of the encoding gene of the 4-hydroxyphenylacetic acid-3-monooxygenase EcHpaB is shown in SEQ ID NO:2, and the nucleotide sequence of the encoding gene of the flavin reductase EcHpaC is shown in SEQ ID NO:3;

[0019] In A, the caffeic acid 3- O The gene encoding the methyltransferase AtCOMT ( Atcomt The nucleotide sequence of the gene is shown in SEQ ID NO:4;

[0020] In B, the tyrosine metabolism repression gene tyrR The nucleotide sequence is shown in SEQ ID NO:5;

[0021] In C, the gene encoding the prephenylacetic acid dehydrogenase TyrC ( tyrC The nucleotide sequence of the gene is shown in SEQ ID NO:6;

[0022] In D, the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase... D146N The coding gene ( aroG D146N The nucleotide sequence of the gene is shown in SEQ ID NO:7.

[0023] Furthermore, in A, using pCDFTac as a vector, the encoding genes for the tyrosine deaminase RgTAL and the 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC were overexpressed; and using pTrc99A as a vector, the encoding genes for caffeic acid 3-... O The gene encoding the methyltransferase AtCOMT.

[0024] Furthermore, the downstream recombinant strain is based on Escherichia coli MG1655 as the starting strain and is modified in at least one of the following ways:

[0025] E. Overexpression originates from Enterobacteriaceae. Enterobacter sp. Px6-4 The gene encoding ferulic acid decarboxylase FDC ( FDC Gene);

[0026] F. Overexpression originates from Enterobacteriaceae. Enterobacter sp. Px6-4 The ferulic acid decarboxylase mutant FDC V46E The coding gene ( FDC V46E Gene);

[0027] G. Overexpression originates from Aspergillus aromatica Aromatoleum aromaticum The gene encoding (S)1-phenylethanol dehydrogenase ped ( ped Gene).

[0028] Furthermore, in E, the nucleotide sequence of the gene encoding the ferulic acid decarboxylase FDC is shown in SEQ ID NO:8;

[0029] In F, the ferulic acid decarboxylase mutant FDC V46E The nucleotide sequence of the encoding gene is shown in SEQ ID NO:9;

[0030] In G, the nucleotide sequence of the gene encoding the (S)1-phenylethanol dehydrogenase ped is shown in SEQ ID NO:10.

[0031] Furthermore, in E~G, pTrc99A was used as the vector for overexpression.

[0032] Furthermore, in the above method, the inoculation ratio of the upstream recombinant strain to the downstream recombinant strain is 1:3 to 3:1.

[0033] Furthermore, in the above method, the fermentation medium further includes: MgSO4. A solution of 7H2O, KH2PO4, (NH4)2HPO4, citric acid, and trace metal salts, with a pH of 6.5–7.5.

[0034] Furthermore, the composition of the trace metal salt solution is: 10 g / L FeSO4 7H2O, 2.65 g / L CaCl2 2H₂O, 2.2 g / L ZnSO₄ 7H2O, 0.58 g / L MnSO4 5H2O, 1g / L CuSO4 5H₂O, 0.1 g / L (NH₄)₆Mo₇O 24 4H₂O, 0.02 g / L Na₂B₄O₇ 10H2O and 10 mL / L of 35% HCl.

[0035] Secondly, the present invention seeks protection for the application of the above-described de novo method for the synthesis of vanillin in the preparation of products containing vanillin by microbial fermentation.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] Firstly, production efficiency is significantly improved. This invention uses a co-culture system to separate the metabolic burden, with upstream strains efficiently supplying ferulic acid and downstream strains efficiently converting it into vanillin. The shake flask yield is stable at 7.5~7.8 mg / L, and it overcomes the resource and cycle limitations of plant extraction methods, meeting the needs of large-scale production.

[0038] Secondly, it is environmentally friendly and uses renewable raw materials. It uses glucose as the sole carbon source, and the reaction conditions are mild (normal temperature and pressure, neutral pH). It does not require harsh chemical conditions, and the production process produces no toxic byproducts. Compared with chemical synthesis methods, it significantly reduces environmental pollution and is in line with the trend of green chemical development.

[0039] Third, the product is highly safe. The microbial fermentation products can be identified as natural sources with no chemical reagent residues. It can be directly applied to fields such as food and medicine where the safety requirements for raw materials are stringent, thus breaking through the application limitations of chemically synthesized products.

[0040] Fourth, the technology has wide applicability. The recombinant plasmids and co-culture schemes constructed are applicable to common engineered strains such as Escherichia coli MG1655 and DH5α. The host can be flexibly selected according to different production scenarios. At the same time, large-scale fermentation verification shows that the process has strong stability and is easy to scale up industrially.

[0041] Fifth, the technology chain is complete, forming a closed loop from strain construction, fermentation optimization, product purification to application verification. It not only provides an efficient method for producing vanillin, but also expands its application scenarios in multiple fields, significantly enhancing the industrialization value of the technology and providing reliable technical support for the green industrial production of vanillin. Attached Figure Description

[0042] Figure 1 This diagram illustrates the biosynthetic pathway and genetic engineering modifications for the de novo synthesis of vanillin. Solid arrows represent single metabolic reactions, dashed arrows represent multiple consecutive metabolic reactions, black dashed lines with T-termini represent feedback inhibition, red X's represent the elimination of feedback inhibition, and blue labels represent overexpression of related genes.

[0043] Figure 2 This is a map of plasmid P01; the plasmid is 5903 base pairs in length, and the key elements marked include the CloDF13 origin of replication (CloDF13ori) and the streptomycin resistance gene (…). SmR ), regulatory gene lacI and target gene RgTAL (Encodes tyrosine deaminase).

[0044] Figure 3 This is a map of plasmid P02; this plasmid was constructed based on plasmid P01, with the target gene annotated in addition to the functional elements of P01. HpaBC (Include EchpaB and EchpaC They encode 4-hydroxyphenylacetic acid-3-monooxygenase and flavin reductase, respectively.

[0045] Figure 4 This is a map of plasmid P03; the plasmid is 5265 base pairs long, and the key elements marked include the pBR322 origin of replication (ori) and the ampicillin resistance gene (…). ApR ), regulatory gene lacI and target gene Atcomt (Code for caffeic acid 3-) O α-methyltransferase).

[0046] Figure 5 This is a map of plasmid P04; the full-length plasmid is 4680 base pairs and contains the pBR322 origin of replication (ori) and the ampicillin resistance gene (…). ApR ), regulatory gene lacI and target gene FDC (Encodes ferulic acid decarboxylase).

[0047] Figure 6 This is a map of plasmid P05; the full-length plasmid is 4680 base pairs, with the target gene marked at the core. FDC V46E (Encoding a ferulic acid decarboxylase mutant), the remaining functional elements (ori, ApR, lacI) are identical to those of PO4.

[0048] Figure 7 This is a map of plasmid P06; the full-length plasmid is 5205 base pairs long and is labeled with genes containing both target genes. FDC (wild-type ferulic acid decarboxylase gene) and FDC V46E (Mutant gene), while retaining ori, ApR Basic functional components such as lacI.

[0049] Figure 8 This is a map of plasmid P07; the full-length plasmid is 5975 base pairs and is labeled with three target genes. FDC , FDCV46E and ped (Encoding (S)1-phenylethanol dehydrogenase), which works in conjunction with the functional elements of ori, ApR, and lacI.

[0050] Figure 9 A bar chart showing the yield of ferulic acid (FA) produced by strain Up02 during fermentation; the vertical axis represents product concentration, and the horizontal axis represents fermentation-related groups.

[0051] Figure 10 The figures show the high-performance liquid chromatography (HPLC) chromatograms of caffeic acid, ferulic acid (FA) standards, and Up02 fermentation samples; the horizontal axis represents retention time (min), and the vertical axis represents absorbance; peak 1 is labeled as caffeic acid standard, with a peak time of 17.446 min; peak 2 is labeled as caffeic acid in the Up02 fermentation sample; peak 3 is labeled as ferulic acid standard, with a peak time of 22.046 min; and peak 4 is labeled as ferulic acid in the Up02 fermentation sample.

[0052] Figure 11 This is a bar chart showing the yield of vanillin produced by fermentation of strain Down02; the vertical axis represents the vanillin concentration, and the horizontal axis represents the fermentation-related groups.

[0053] Figure 12 The HPLC peak diagrams are of vanillin ethyl ketone standard and Down02 fermentation sample; the horizontal axis represents retention time (min) and the vertical axis represents absorbance; peak 5 is labeled as vanillin ethyl ketone standard, with an elution time of 20.355 min, and peak 6 is labeled as vanillin ethyl ketone in Down02 fermentation sample.

[0054] Figure 13 The bar chart shows the yield of vanillin produced by fermentation of strains Up02 and Down03 at different inoculation ratios; the horizontal axis represents the inoculation ratio (Up02:Down03), and the vertical axis represents the vanillin concentration.

[0055] Figure 14 The HPLC peak diagrams are of vanillin ethyl ketone standard and fermentation samples with different inoculation ratios; the horizontal axis represents retention time (min) and the vertical axis represents absorbance; peak 7 is labeled as vanillin ethyl ketone standard, with an elution time of 21.315 min; peaks 8, 9, and 10 are labeled as fermentation sample peaks with inoculation ratios of 1:3, 1:1, and 3:1, respectively. Detailed Implementation

[0056] 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.

[0057] 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.

[0058] The genotypes of the strains and plasmids involved in the examples are shown in Table 1.

[0059] Table 1. Genotypes of strains and plasmids

[0060]

[0061] The primers and their specific sequences involved in the examples are shown in Table 2.

[0062] Table 2. Primer sequence listing

[0063]

[0064] The culture medium components involved in the examples are as follows:

[0065] LB medium;

[0066] 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; The trace metal salt solution contains 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 The mother liquor concentrations of 7H2O were 500 g / L and 80 g / L, respectively, and were sterilized independently by moist heat.

[0067] Seed culture medium: LB medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L);

[0068] SOC medium: 2.0 g tryptone, 0.5 g yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM glucose, dissolved in deionized water, pH 7.0, autoclaved.

[0069] Information on the main experimental instruments involved in the examples:

[0070] High-performance liquid chromatography (HPLC) system (Shimadzu LC-20 series, including analytical and preparative types, with Shim-pack GIST C18-AQ columns 4.6×250mm and 20×250mm), PCR instrument, agarose gel electrophoresis system, high-speed refrigerated centrifuge, rotary evaporator under reduced pressure, constant temperature water bath, nuclear magnetic resonance spectrometer (NMR spectrometer). 1 H-NMR, 400MHz), constant temperature shaker, ultraviolet spectrophotometer (for detecting OD) 600 Equipment includes: (value), electronic balance, sterile operating table, 0.22μm aqueous / organic phase filter membrane, etc.

[0071] A schematic diagram illustrating the biosynthetic pathway and genetic engineering modification for the de novo synthesis of vanillin is shown below. Figure 1 As shown in the figure, this diagram illustrates the complete metabolic pathway of glucose to vanillin via intermediates such as L-tyrosine, p-coumaric acid, caffeic acid, and ferulic acid, as well as the genetic modification targets.

[0072] Example 1

[0073] This example describes the construction of the core expression plasmid.

[0074] I. Experimental Methods

[0075] (a) Test materials

[0076] 1. Host strain: Escherichia coli DH5α, used for plasmid construction and amplification.

[0077] 2. Vector backbone: pCDFTac (SmR, tac promoter, CDF replication origin, lacI regulatory gene) and pTrc99A (ApR, trc promoter, pBR322 replication origin, lacI regulatory gene) were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0078] 3. Target gene template: Contains a tyrosine deaminase gene optimized by E. coli codons. Rgtal Gene, SEQ ID NO:1), 4-hydroxyphenylacetic acid-3-hydroxylase gene HpaBC (include EchpaB and EchpaC , EchpaBThe nucleotide sequence is shown in SEQ ID NO:2. EchpaC The nucleotide sequence is shown in SEQ ID NO:3), caffeic acid 3- O -Methyltransferase gene ( Atcomt Gene, SEQ ID NO:4), ferulic acid decarboxylase gene ( FDC Gene, SEQ ID NO:8), ferulic acid decarboxylase mutant gene ( FDC V46E (S)1-phenylethanol dehydrogenase gene (SEQ ID NO:9) ped The gene (SEQ ID NO:10) was synthesized by Sangon Biotech.

[0079] 4. Primers: The primer sequences required for construction are shown in Table 2 and were synthesized by Qingke Company.

[0080] 5. Tools, enzymes, and kits: EcoRI, SacI, NcoI, KpnI, SalI restriction endonucleases, and DpnI methyltransferase were purchased from TAKARA; the seamless cloning kit was purchased from Ibotek; and the gel recovery kit was purchased from Junuode.

[0081] 6. Culture medium and resistance: LB medium; ampicillin (Ap) final concentration 100 mg / L, streptomycin (Sm) final concentration 50 mg / L, used for positive clone screening.

[0082] (II) Core Expression Plasmid Construction Steps

[0083] 1. Construction of P01 plasmid: The pCDFTac vector was digested with EcoRI restriction endonuclease to obtain a linearized vector fragment; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Rgtal Using the fully synthesized template of the gene (sequence shown in SEQ ID NO:1) as a template, PCR amplification was performed using primers RgTAL_TY_F2 and RgTAL_TY_R to obtain the target gene fragment. The linearized vector and the target gene fragment were separated by agarose gel electrophoresis, and the correct band was excised and purified using a gel extraction kit. Recombinant reactions were performed using a seamless cloning kit, and the reaction product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing Sm, and incubated overnight at 37°C. Single clones were picked for colony PCR verification; positive clones were sent to a sequencing company for sequencing to confirm correct insertion of the target gene, yielding the P01 plasmid.

[0084] 2. Construction of P02 plasmid: The P01 plasmid was digested with SacI restriction endonuclease to obtain a linearized vector fragment; using the *E. coli* genome as a template, PCR amplification was performed using primers SacI_Rg_EC_F and SacI_Rg_Ec_R to obtain... hpaBC Gene fragments (including) EchpaB and EchpaC , EchpaB The nucleotide sequence is shown in SEQ ID NO:2. EchpaC The nucleotide sequence is shown in SEQ ID NO:3. Subsequent agarose gel electrophoresis, gel recovery, seamless cloning, transformation screening, and sequencing verification followed the same procedure as for P01 plasmid construction, yielding plasmids containing... Rgtal and hpaBC P02 plasmid containing two genes.

[0085] 3. Construction of P03 plasmid: The pTrc99A vector was digested with EcoRI restriction endonuclease to obtain a linearized vector fragment; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Atcomt Using the fully synthesized template of the gene (sequence shown in SEQ ID NO:4) as a template, PCR amplification was performed using primers 99A_optAtCOMT_F and 99A_optAtCOMT_R to obtain the target gene fragment. Subsequent processes included gel recovery, seamless cloning, transformation (plating onto LB plates containing Ap), screening, and sequencing verification, following the same procedure as for plasmid P01 construction, to obtain plasmid P03.

[0086] 4. Construction of P04 plasmid: The pTrc99A vector was digested with NcoI restriction endonuclease to obtain a linearized vector fragment; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] FDC Using the fully synthesized template of the gene (sequence shown in SEQ ID NO:8) as a template, PCR amplification was performed using primers 99A_FDC_F and 99A_FDC_R to obtain the target gene fragment. Subsequent processes included gel recovery, seamless cloning, transformation (plating onto LB plates containing Ap), screening, and sequencing verification, following the same procedure as for P01 plasmid construction, to obtain plasmid P04.

[0087] 5. Construction of P05 plasmid: Using the P04 plasmid, which was verified by sequencing, as a template, PCR amplification was performed using primers V46E_F and V46E_R to obtain plasmid containing P05. FDC V46E The linear plasmid fragment of the gene (sequence shown in SEQ ID NO:9) was obtained; template plasmid P04 was digested with Dpn I enzyme to remove wild-type plasmid interference. After gel purification, self-circularization recombination was performed using a seamless cloning kit, and the cells were transformed into *E. coli* DH5α competent cells (plated on LB agar plates containing Ap). Colony PCR and sequencing verification were performed to obtain the gene containing... FDC V46E The P05 plasmid of the gene.

[0088] 6. Construction of P06 plasmid: The P04 plasmid was digested with KpnI restriction endonuclease to obtain a linearized vector fragment; using the P05 plasmid as a template, PCR amplification was performed using primers KpnI_FDC_F and 46_FDC_R to obtain... FDC V46E The target gene fragment was obtained. Subsequent processes included gel extraction, seamless cloning, transformation screening, and sequencing verification, following the same procedure as the P01 plasmid construction. FDC and FDC V46E P06 plasmid containing two genes.

[0089] 7. Construction of P07 plasmid: The P06 plasmid was digested with SalI restriction endonuclease to obtain a linearized vector fragment; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] ped Using the fully synthesized template of the gene (sequence shown in SEQ ID NO:10) as a template, PCR amplification was performed using primers SalI_ped_F and ped_R2 to obtain the target gene fragment. Subsequent gel extraction, seamless cloning, transformation screening, and sequencing verification followed the same procedure as for P01 plasmid construction, yielding a fragment containing... FDC , FDC V46E and ped P07 plasmid containing three genes.

[0090] (III) Plasmid Validation Methods

[0091] Colony PCR verification: Specific primers were designed for the target gene of each plasmid, and the selected single clones were amplified by PCR. The size of the amplified product was detected by agarose gel electrophoresis. The clones that were consistent with the expected target gene fragment size were considered positive clones.

[0092] Sequencing verification: Colony PCR-positive clones were sent to a sequencing company for bidirectional sequencing. The sequencing results were compared with the theoretical sequence of the target gene (SEQ ID NO: 1~4, 8~10) to verify the accuracy of the gene sequence.

[0093] Plasmid mapping verification: Plasmids with correct sequencing are identified by enzyme digestion, and the size of the enzyme digested fragments is detected by agarose gel electrophoresis. Combined with the sequencing results, the correct assembly of the plasmid backbone, target gene and regulatory elements is confirmed.

[0094] II. Test Results

[0095] 1. Colony PCR results showed that the positive clones corresponding to each plasmid P01 to P07 amplified specific bands consistent with the expected size of the target gene, without any interference from other bands, indicating that the target gene has been initially inserted into the vector backbone.

[0096] 2. Sequencing results showed that the inserted plasmid P01 contained... RgtalThe gene sequence is completely identical to SEQ ID NO:1; the inserted gene in plasmid P02 is... hpaBC Gene( EchpaB , EchpaC The sequences are completely identical to SEQ ID NO:2 and 3, respectively; the inserted sequence in plasmid P03 is... Atcomt The gene sequence is completely identical to SEQ ID NO:4; the inserted gene in plasmid P04 is... FDC The gene sequence is completely identical to SEQ ID NO:8; the inserted gene in plasmids P05 and P06 is... FDC V46E The gene sequence is completely identical to SEQ ID NO:9; the inserted gene in plasmid P07 is... ped The gene sequence is completely identical to SEQ ID NO:10, and there are no base mutations, deletions or frameshifts in any of the target genes.

[0097] 3. Plasmid mapping validation results

[0098] P01 plasmid map as follows Figure 2 As shown, it includes the CloDF13 origin of replication (CloDF13 ori), the streptomycin resistance gene (SmR), the lacI regulatory gene, and Rgtal The target gene, with a total length of 5903 base pairs, meets the design expectations.

[0099] P02 plasmid map as follows Figure 3 As shown, insertion was successfully performed on the P01 plasmid. hpaBC Genes, with a complete structure.

[0100] P03 plasmid map as follows Figure 4 As shown, it includes the pBR322 origin of replication, the ampicillin resistance gene (ApR), the lacI regulatory gene, and... Atcomt The target gene, with a total length of 5265 base pairs, meets the design expectations.

[0101] P04 plasmid map as follows Figure 5 As shown, it includes the pBR322 replication origin and the ApR resistance gene. 、 lacI-regulated genes and FDC The target gene, with a total length of 4680 base pairs, meets the design expectations.

[0102] P05 plasmid map as follows Figure 6 As shown, its insertion FDC V46E The mutated gene sequence is correct, with a total length of 4680 base pairs, which meets the design expectations.

[0103] P06 plasmid map as follows Figure 7 As shown, insertion was successfully performed on the P04 plasmid. FDCV46E The gene, with a total length of 5205 base pairs, meets the design expectations.

[0104] P07 plasmid map as follows Figure 8 As shown, insertion was successfully performed on the P06 plasmid. ped The gene, with a total length of 5975 base pairs, meets the design expectations.

[0105] In summary, the core expression plasmids P01 to P07 were successfully constructed, with accurate gene sequences and correct vector assembly, and can be used for the construction of subsequent recombinant strains.

[0106] Example 2

[0107] This embodiment describes the stepwise modification and validation of the upstream recombinant strain.

[0108] I. Experimental Methods

[0109] (a) Test materials

[0110] 1. Starting strain: Escherichia coli MG1655, which served as the starting point for the modification of the upstream recombinant strain.

[0111] 2. Helper plasmids: pKD46 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; pJW168 and pUTrc were purchased from Nanjing Genscript Biotech Co., Ltd.

[0112] 3. Target gene template: Escherichia coli BL21(DE3) genomic DNA (for amplification) hpaBC (gene), pBBR1Tac-aroG D146N Plasmid.

[0113] 4. Primers: The primer sequences used for modification are shown in Table 2, including... ΔtyrR Gene knockout primers, tyrC Gene integration primers, aroG D146N The gene insertion primers were synthesized by Qingke Company.

[0114] 5. Tools, enzymes, and kits: EcoRI, SacI, NcoI, KpnI, SalI restriction endonucleases, and DpnI methyltransferase were purchased from TAKARA; the seamless cloning kit was purchased from Ibotek; and the gel extraction kit and genomic DNA extraction kit were purchased from Junuode.

[0115] 6. Culture medium and resistance: LB medium; ampicillin (Ap) final concentration 100 mg / L, chloramphenicol (Cm) final concentration 34 mg / L, streptomycin (Sm) final concentration 50 mg / L, used for strain screening and verification.

[0116] 7. Fermentation-related reagents: glucose, MgSO4 The fermentation medium components, including 7H2O, KH2PO4, (NH4)2HPO4, and citric acid, were all of analytical grade. Caffeic acid standard was purchased from ACMEC, ferulic acid standard was purchased from Maclean's, and isopropyl-β-D-thiogalactoside (IPTG) was purchased from Maclean's.

[0117] (II) Step-by-step modification of upstream recombinant strains

[0118] The upstream recombinant strains were modified sequentially by knocking out the repressor gene, integrating the functional gene, inserting the mutant gene, and introducing the expression plasmid, ultimately obtaining strains Up01 and Up02. The specific steps are as follows:

[0119] 1. Construction of M01 strain (knockout) tyrR Gene)

[0120] 1) Linearized fragment preparation

[0121] Using pUTrc plasmid as a template, primers ΔtyrR_F1 and ΔtyrR_R1 were used to amplify plasmids containing pUTrc plasmid. tyrR The lox71-CmR-lox66 fragment (KO-ΔtyrR1) containing 50bp homologous arms upstream and downstream of the gene (SEQ ID NO:5) was amplified using KO-ΔtyrR1 as a template with primers ΔtyrR_F2 and ΔtyrR_R2 to obtain the linearized fragment KO-ΔtyrR containing 100bp homologous arms upstream and downstream.

[0122] 2) Assisted plasmid introduction

[0123] (1) Preparation of Escherichia coli MG1655 electrocompetent cells: Laboratory-preserved Escherichia coli MG1655 was inoculated into test tubes containing 5 mL LB medium and cultured at 30℃ and 200 rpm for 12-16 h; 2% of the cells were inoculated into 100 mL of fresh LB medium and cultured at 30℃ and 200 rpm for 1.5 h. When the 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 store at -80℃. (2) Electroporation: Take the prepared Escherichia coli MG1655 electroporation competent cells, thaw them on ice, mix them thoroughly with 50 ng pKD46 helper plasmid, transfer them to a pre-cooled 1 mm electroporation cup and incubate on ice for 30 min; wipe the water mist off the outside and bottom of the electroporation cup, place it in an electroporator and electroporate once using the Ec1 setting; immediately add 600 μL of pre-cooled SOC medium, mix gently and tilt the electroporation cup, transfer the entire mixture to a sterile 1.5 mL centrifuge tube, and incubate at 30℃ and 200 rpm for 60-80 min; take 80 μL and spread it on an LB plate containing Ap, incubate overnight at 30℃ to obtain the MG1655 strain containing pKD46.

[0124] 3) Fragment insertion and filtering

[0125] (1) Preparation of electrocompetent cells: Pick the above single clones and inoculate them into 5 mL LB tubes containing ampicillin sodium. Culture them at 30℃ and 200 rpm for 16-20 h. Inoculate them into 15 mL of fresh LB medium at a volume of 2% and add L-arabinose to a final concentration of 10 mM. Culture them at 30℃ and 200 rpm until 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 the final volume 100μL to obtain electrocompetent cells. (2) Electroporation: transfer the obtained competent cells to a sterile 1.5mL centrifuge tube, mix thoroughly with 500ngKO-ΔtyrR fragment, perform electroporation in the same way as in step 2), and spread the mixture after recovery culture onto LB plates containing Ap and Cm, and incubate overnight at 30℃; verify positive clones by colony PCR to confirm fragment insertion.

[0126] 4) Elimination of helper plasmids and selection markers

[0127] (1) Elimination of pKD46 helper plasmid: The positive clone from step 3) was streaked onto an LB agar plate containing Cm and cultured overnight at 42°C to eliminate pKD46. (2) Elimination of CmR selection marker: The strain with pKD46 helper plasmid eliminated was inoculated into an LB agar plate containing Cm and cultured at 37°C and 200 rpm for 12-16 h; 2% of the culture was inoculated into 15 mL of fresh LB medium and cultured at 37°C and 200 rpm until 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 the final volume 100μL; transfer to a sterile 1.5mL centrifuge tube, mix thoroughly with 50ng of pJW168 helper plasmid, perform electroporation transformation in the same way, and plate onto LB plates containing Ap and 1mM IPTG, incubate overnight at 30℃ to induce Cre recombinase expression and eliminate CmR selection marker; verify positive clones by colony PCR to confirm the elimination of selection marker. (3) Elimination of pJW168 helper plasmid: streak the positive clone from step (2) onto LB plates, incubate overnight at 42℃ to eliminate pJW168, and obtain tyrR Gene knockout strain M01 (MG1655) ΔtyrR ).

[0128] 2. Construction (integration) of strain M02 tyrC Gene)

[0129] 1) Linearized fragment preparation

[0130] Using pUTrc plasmid as a template, INS tyrC _F1 and INS tyrC Amplification of the lox71-CmR-lox66 selectable marker fragment (LTL) using primer R1; using the prephenyl acid dehydrogenase TyrC synthesis gene optimized by E. coli ( tyrC Using the gene as a template, amplification was performed using primers tyrC_F1 and tyrC_R1. tyrC Gene fragment (SEQ ID NO:6); in LTL and tyrC Using gene fragments as templates, overlap extension PCR was used to obtain samples containing... yjjP-yjjR Edited fragment INS-tyrC with 100bp homologous arms upstream and downstream of the site (structure: upstream homologous arm - lox71-CmR-lox66-tyrC-downstream homologous arm).

[0131] 2) Assisted plasmid import and fragment insertion

[0132] Following the M01 strain construction procedure, the pKD46 plasmid was introduced into the M01 strain, followed by the INS-tyrC fragment. The mixture was then plated on LB plates containing Ap and Cm and incubated overnight at 30°C. Positive clones were verified by colony PCR.

[0133] 3) Labeling and helper plasmid elimination

[0134] Following the elimination method for strain M01, pKD46, CmR markers, and pJW168 were eliminated sequentially to obtain... tyrC Gene-integrated strain M02 (MG1655) ΔtyrR yjjP-yjjR::tyrC ).

[0135] 3. Construction of strain M03 (insertion) aroG D146N Gene)

[0136] 1) Linearized fragment preparation

[0137] Using pUTrc plasmid as a template, the lox71-CmR-lox66 selectable marker fragment (LCL) was amplified using INS_F1 and INS_R1 primers; pBBR1Tac-aroG was used as a template. D146N Using plasmids as templates, aroG D146N _F1 and aroG D146N _R1 primer amplification aroG D146N Gene fragment (SEQ ID NO:7); then LCL fragment and aroG D146N Using gene fragments as templates, INS_F1 and aroG were used. D146N The R1 primer was used for overlap extension PCR to obtain the intermediate product INS-aroG. D146N 1; Finally, use INS-aroG D146N 1 is the template, using INS_F2 and aroG D146N The final PCR amplification was performed using primers R2 to obtain the final edited fragment INS-aroG containing 100bp homologous arms upstream and downstream of the rsmG-atpI site. D146N (Structure: upstream homologous arm - lox71-CmR-) aroG D146N -Downstream homologous arm).

[0138] 2) Assisted plasmid import and fragment insertion

[0139] pKD46 plasmid was introduced into strain M02, followed by INS-aroG. D146N The fragments were plated on LB plates containing Ap and Cm, incubated overnight at 30°C, and positive clones were verified by colony PCR.

[0140] 3) Labeling and helper plasmid elimination

[0141] Following the method described above, pKD46, CmR markers, and pJW168 are eliminated sequentially to obtain... aroG D146N Gene-inserted strain M03 (MG1655) ΔtyrRyjjP-yjjR::tyrCrsmG-atpI::aroG D146N ).

[0142] 4. Construction of Up01 and Up02 strains (importing expression plasmids)

[0143] 1) Preparation of electrocompetent cells for strain M03

[0144] Inoculate strain M03 into 5 mL of LB medium and incubate at 37°C and 200 rpm for 12–16 h; then inoculate 2% of the culture medium into 100 mL of fresh LB medium and incubate at 37°C and 200 rpm until OD (dose-free survival). 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 the final volume 500μL. Aliquot 100μL into a 1.5mL centrifuge tube and freeze at -80℃.

[0145] 2) Plasmid import and screening

[0146] One electrotransformation competent cell was mixed with 50 ng of pTrc99A plasmid and 50 ng of pCDFTac plasmid. After electrotransformation using the same method, the mixture was plated on LB plates containing Ap and Sm and cultured overnight at 37°C to obtain strain Up01 (M03 harboring pTrc99A and pCDFTac). Another electrotransformation competent cell was mixed with 50 ng of P02 plasmid and 50 ng of P03 plasmid. After electrotransformation, the mixture was plated on LB plates containing Ap and Sm and cultured overnight at 37°C to obtain strain Up02 (M03 harboring P02 and P03).

[0147] (III) Strain Validation Methods

[0148] 1. Genotype verification: Colony PCR was performed on strains M01, M02, M03, Up01, and Up02. The target fragments were amplified using specific primers corresponding to each modification step, and the fragment size was verified by agarose gel electrophoresis. Positive clones were sent to a sequencing company for sequencing to confirm the accuracy of gene knockout, integration, insertion, and plasmid introduction.

[0149] 2. Fermentation performance verification of Up01 and Up02

[0150] Seed culture: Select single clones of Up01 and Up02 strains and inoculate them into 5 mL of LB medium containing the corresponding resistance, and culture overnight at 37°C and 200 rpm.

[0151] Shake-flask fermentation: Inoculate the seed culture at a rate of 2% into a 250mL baffled shake flask containing 50mL of fermentation medium, and incubate at 30℃ and 200rpm for 60h with shaking; after 8h of inoculation (OD 600 =0.5~0.8) Add IPTG to a final concentration of 0.5mM to induce expression.

[0152] Product detection: After fermentation, 1 mL of fermentation broth was centrifuged at 16000 × g for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane, and the contents of caffeic acid and ferulic acid were determined by high performance liquid chromatography (HPLC). The HPLC system was a Shimadzu LC-20 series, and the column was a Shim-pack GIST C18-AQ (4.6 × 250 mm). Mobile phase A was ddH2O containing 0.1% trifluoroacetic acid, and mobile phase B was methanol. The flow rate was 0.65 mL / min, and the gradient program was as follows: 0–1 min, 30% B; 1–16 min, B linearly increased to 60%; 16–17 min, maintained at 60% B; 17–25 min, B linearly decreased to 30%; 25–50 min, maintained at 30% B; column temperature was 30℃, injection volume was 5 μL, and detection wavelength was 300 nm.

[0153] II. Test Results

[0154] 1. Genotype verification results: Colony PCR and sequencing results showed that strain M01 contained... tyrR The gene has been successfully knocked out with no residual fragments; strain M02 has accurately integrated at the yjjP-yjjR site. tyrC No gene or sequence mutations were found; strain M03 successfully inserted the gene into the rsmG-atpI site. aroG D146N The genes were successfully integrated in the correct direction; strain Up01 was successfully introduced into the pTrc99A and pCDFTac empty vectors, and strain Up02 was successfully introduced into the P02 and P03 expression plasmids, and the target gene was successfully integrated. Rgtal (SEQ ID NO:1) hpaBC (SEQ IDNO:2~3) Atcomt The sequence (SEQ ID NO:4) is consistent with the design, with no base deletions or mutations. These results indicate that the stepwise modification of the upstream recombinant strain achieved the expected goals, and the genotype is correct.

[0155] 2. Fermentation performance verification results: The shake-flask fermentation results of strains Up01 and Up02 are as follows: Figure 9 As shown, Figure 9 The study demonstrated the accumulation of ferulic acid during fermentation by strain Up02, along with the accompanying accumulation of caffeic acid. No caffeic acid or ferulic acid accumulation was detected in strain Up01 (including the empty vector). Strain Up02 (including the functional expression plasmid) successfully synthesized the target product, with a ferulic acid yield of 57.3 mg / L and an accumulation of 59.3 mg / L of caffeic acid. HPLC chromatograms showed (…). Figure 10The elution time of caffeic acid standard (peak 1) was 17.446 min, and the corresponding peak (peak 2) in sample Up02 was identified as caffeic acid upon comparison. The elution time of ferulic acid standard (peak 3) was 22.046 min, and the corresponding peak (peak 4) in sample Up02 was identified as ferulic acid upon comparison. Furthermore, each peak was uniform in shape and free from significant interference from other peaks, indicating that the detection results were accurate and reliable. The accumulation of caffeic acid may stem from two main reasons: one is caffeic acid 3- O The low catalytic efficiency of methyltransferase (AtCOMT) and the insufficient methyl donors in the fermentation system affect the conversion of caffeic acid to ferulic acid.

[0156] In summary, by gradually knocking out the repressor gene, integrating the functional gene, inserting the mutant gene, and introducing the expression plasmid, the upstream recombinant strain Up02 with ferulic acid synthesis capability was successfully constructed, providing a stable precursor supply strain for the subsequent de novo synthesis of vanillin in the co-culture system.

[0157] Example 3

[0158] This embodiment describes the functional verification of key enzymes in downstream recombinant strains.

[0159] I. Experimental Methods

[0160] (a) Test materials

[0161] 1. Host strain: Escherichia coli MG1655, used as the construction host for downstream recombinant strains.

[0162] 2. Core plasmid: Core expression plasmid P04 (containing FDC Genes), P05 (containing FDC V46E Gene), P06 (containing) FDC + FDC V46E Gene), P07 (containing) FDC + FDC V46E + ped All genes were plasmids constructed and verified correctly in Example 1; control plasmid: empty vector pTrc99A, used to construct the control strain, purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0163] 3. Reagents and Kits: Ampicillin (Ap), final concentration 100 mg / L; ferulic acid standard, purchased from Maclean's; 4-hydroxy-3-methoxy-α-methylbenzyl alcohol standard, purchased from Aladdin; 4-hydroxy-3-methoxystyrene standard and vanillin ethyl ketone standard, purchased from Maclean's; IPTG, purchased from Maclean's; restriction endonuclease and DpnI methyltransferase, purchased from TAKARA; seamless cloning kit, purchased from Ibotek; plasmid extraction kit and gel extraction kit, purchased from Junuode; all reagents used in high performance liquid chromatography (HPLC) were of analytical grade.

[0164] 4. Culture media: SOC medium; LB medium; fermentation medium, in which ferulic acid with a final concentration of 500 mg / L is added as a reaction substrate.

[0165] (II) Construction of downstream recombinant strains

[0166] The construction process for downstream recombinant strains containing single or combined target enzyme genes based on different plasmids is as follows (unless otherwise specified):

[0167] Competent cell preparation: MG1655 cells were inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12–14 h; then, 2% of the cells were inoculated into 100 mL of fresh LB medium and cultured at 37°C and 200 rpm until OD200 was reached. 600 =0.4~0.6; collect bacterial cells by centrifugation at 4℃ and 4000×g for 10 min, wash 3 times with pre-cooled 10% glycerol solution, and finally resuspend with an appropriate amount of 10% glycerol. Aliquot into 100μL and store at -80℃ for later use.

[0168] Electroporation transformation: Take 100 μL of electrocompetent cells, thaw them in an ice bath, add the corresponding plasmid, mix gently, and incubate in an ice bath for 30 min; transfer to a pre-cooled 1 mm electroporation cuvette, and electroporate once using the Ec1 setting; immediately add 600 μL of pre-cooled SOC medium, and revive and incubate at 37℃ and 200 rpm for 60-80 min.

[0169] Screening and validation: Spread the revived bacterial culture onto LB agar plates containing the corresponding resistance and incubate overnight at 37°C; select single clones for colony PCR validation, and send positive clones to a sequencing company for sequencing to confirm the correct target gene sequence.

[0170] The specific construction parameters for each strain are as follows:

[0171] 1. Control strain Down01: Mix 50 ng of pTrc99A empty vector with MG1655 competent cells, transform according to the above procedure, plate on LB plates containing Ap, and verify the introduction of the empty vector by colony PCR to obtain a control strain without the target enzyme gene.

[0172] 2. FDC-expressing strain Down04: 50 ng of P04 plasmid was mixed with MG1655 competent cells, transformed, and plated on LB agar plates containing Ap. Colony PCR and sequencing were then used for verification. FDC The gene (SEQ ID NO:8) sequence is correct, and a strain expressing only FDC is obtained.

[0173] 3. Only express FDC V46E strain Down05: 50 ng of P05 plasmid was mixed with MG1655 competent cells, transformed, and plated on LB agar plates containing Ap. Colony PCR and sequencing were then performed for verification. FDC V46E The gene (SEQ ID NO:9) sequence is correct, yielding a result expressing only FDC. V46E strains.

[0174] 4. Co-expression FDC+FDC V46E The strain Down06 was obtained by mixing 50 ng of P06 plasmid with MG1655 competent cells, transforming them, and then plating them on LB plates containing Ap. The correct dual gene sequences were verified by colony PCR and sequencing, and a strain co-expressing the two enzymes was obtained.

[0175] 5. Co-expression FDC+FDC V46E For strain Down02 with +ped plasmid: 50 ng of P07 plasmid was mixed with MG1655 competent cells, transformed, and plated on LB agar plates containing Ap. Colony PCR and sequencing were then performed for verification. FDC , FDC V46E , ped The three gene sequences (SEQ ID NO: 8~10) were correct, and a strain co-expressing the three enzymes was obtained.

[0176] 6. Strains co-expressing FDC+FDCV46E+ped and containing pCDFTac: Down03 was obtained by mixing 50 ng of P07 plasmid and 50 ng of pCDFTac plasmid and introducing them into MG1655 competent cells. After transformation, the cells were plated on LB plates containing Ap+Sm double resistance. Colony PCR and sequencing were used to verify that the target genes and plasmid backbone sequences were correct, and Down03 strain was obtained.

[0177] (III) Fermentation Validation and Product Detection

[0178] Seed culture: Single clones of strains Down01, Down04, Down05, Down06, Down02, and Down03 were picked and inoculated into 5 mL of LB medium containing the corresponding resistance. They were cultured overnight at 37°C and 200 rpm for 12-14 h to obtain seed culture.

[0179] Shake-flask fermentation: Seed culture of each strain was inoculated at a 2% inoculum into 250 mL baffled shake flasks containing 50 mL of fermentation medium (containing 500 mg / L ferulic acid), and cultured at 30 °C and 200 rpm for 60 h with shaking; after 8 h of inoculation (OD... 600 =0.5~0.8) Add IPTG to a final concentration of 0.5mM to induce enzyme expression, and set up three biological replicates for each group.

[0180] Product detection: After fermentation, 1 mL of fermentation broth was centrifuged at 16000 × g for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane and the target product was detected by HPLC using the same method as above. By comparing the peak time and peak area of ​​each standard, the product type was qualitatively analyzed and the product concentration was quantitatively calculated.

[0181] II. Test Results

[0182] 1. Results of strain construction and validation

[0183] Colony PCR and sequencing results showed that strains Down01, Down04, Down05, Down06, Down02, and Down03 were all successfully introduced into their corresponding plasmids, with Down04 showing the best results. FDC Gene (SEQ ID NO:8), Down05 FDC V46E The sequences of the gene (SEQ ID NO: 9), the double gene of Down06, and the triple gene (SEQ ID NO: 8~10) of Down02 and Down03 are consistent with the design, with no base mutations, deletions or frameshifts; the sequences of key elements such as the resistance gene and replication origin of the pCDFTac plasmid in the Down03 strain are correct, and the construction of each strain meets expectations.

[0184] 2. Results of key enzyme function verification

[0185] Down01 strain (empty vector control): as follows Figure 11 As shown, only the unconverted ferulic acid substrate peak was detected in the fermentation broth, and the target products such as 4-hydroxy-3-methoxystyrene, 4-hydroxy-3-methoxy-α-methylbenzyl alcohol and vanillin were not detected, indicating that MG1655 itself does not have the relevant enzyme activity for catalyzing the conversion of ferulic acid.

[0186] Down04 strain (expressing FDC only): 4-hydroxy-3-methoxystyrene (FDC-specific catalytic product) was successfully detected at a concentration of 323.23 mg / L. No subsequent intermediates or vanillin were detected, verifying the first step function of FDC in catalyzing the decarboxylation of ferulic acid.

[0187] Down05 strain (expresses FDC only) V46E4-Hydroxy-3-methoxy-α-methylbenzyl alcohol was successfully detected at a concentration of 61.06 mg / L, validating the FDC assay. V46E Its catalytic function.

[0188] Down06 strain (co-expressing FDC+FDC) V46E 4-Hydroxy-3-methoxystyrene (286.81 mg / L) and 4-hydroxy-3-methoxy-α-methylbenzyl alcohol (41.62 mg / L) were detected, but vanillin was not detected, indicating that FDC is different from FDC. V46E It can synergistically complete two-step continuous catalysis.

[0189] Down02 strain (co-expressing FDC+FDC) V46E +ped): Vanillin, the final product, was successfully detected, with a yield of 52.9 mg / L (e.g., vanillin). Figure 11 As shown); HPLC chromatograms show ( Figure 12 The peak time of vanillin ethyl ketone standard was 20.355 min (peak 5). The corresponding peak (peak 6) in the Down02 sample was identified as vanillin ethyl ketone. The peak shape was single and there were no impurity peaks, which verified the function of PED catalytic terminal dehydrogenation.

[0190] Down03 strain (co-expressing three enzymes + containing pCDFTac): Vanillin was successfully detected in the fermentation broth, and the yield was not significantly different from that of Down02 strain, and the peak time was consistent with that of vanillin standard, indicating that the introduction of pCDFTac plasmid did not affect the catalytic function of key enzymes, and the strain can be used for subsequent co-culture system construction.

[0191] In summary, this embodiment successfully constructed a series of downstream recombinant strains and clarified the synergistic catalytic mechanism of key enzymes: FDC initiates ferulic acid decarboxylation, FDC... V46E The catalytic intermediate conversion, ped, completes the terminal dehydrogenation to produce vanillin, providing a functionally validated downstream strain for the de novo synthesis of vanillin in the co-culture system.

[0192] Example 4

[0193] This embodiment describes the co-culture of upstream and downstream recombinant strains and the optimization of the inoculation ratio.

[0194] I. Experimental Methods

[0195] (a) Test materials

[0196] 1. Test strains: Up02, the upstream recombinant strain, and Down03, the downstream recombinant strain, were constructed and verified to be correct in the aforementioned examples. They were used to synthesize ferulic acid precursor and convert ferulic acid into vanillin, respectively.

[0197] 2. Culture media: Seed culture medium: LB medium, Up02 strain culture medium supplemented with ampicillin (Ap, final concentration 100mg / L) and streptomycin (Sm, final concentration 50mg / L), Down03 strain culture medium supplemented with the same concentration of Ap and Sm to maintain plasmid stability; fermentation culture medium.

[0198] 3. Reagents and Instruments: Isopropyl-β-D-thiogalactoside (IPTG) was purchased from Maclean's; vanillin ethyl ketone standard and ferulic acid standard were purchased from Maclean's; high performance liquid chromatography (HPLC) system; high-speed refrigerated centrifuge was purchased from HERMLE; constant temperature shaker was purchased from Shanghai Zhichu Instrument Co., Ltd.; ultraviolet spectrophotometer was purchased from Shanghai Meipuda Instrument Co., Ltd.

[0199] 4. Resistance reagents: Ampicillin (Ap) final concentration 100 mg / L, streptomycin (Sm) final concentration 50 mg / L, used to maintain plasmid stability during fermentation.

[0200] (II) Experimental Design

[0201] The core evaluation index is the yield of vanillin in the co-culture system, and the specific design is as follows:

[0202] 1. Inoculation ratio setting: The total inoculation amount was fixed at 2% (volume fraction), and the inoculation ratio gradient between Up02 and Down03 was set to 1:3, 1:1, and 3:1, for a total of 3 experimental groups, with three biological replicates in each group.

[0203] 2. Fermentation Condition Control: Uniformly optimized fermentation parameters were used: fermentation temperature 30℃, shaking speed 200 rpm, initial culture medium pH 7.0, and fermentation cycle 60 h; OD... 600 When the concentration is 0.5~0.8, add IPTG at a final concentration of 0.5mM to induce the expression of the target enzyme.

[0204] (III) Fermentation and Detection Methods

[0205] 1. Seed culture: Select Up02 and Down03 single clones respectively, inoculate them into 5 mL of LB medium containing the corresponding resistance, and culture overnight at 37℃ and 200 rpm for 12-14 h to obtain seed culture.

[0206] 2. Co-culture fermentation: The OD of the seed culture of each strain was measured using a UV spectrophotometer. 600 Calculate the inoculation volume of each seed culture according to the preset ratio, ensuring that the total inoculation volume is 1 mL (corresponding to 2% inoculation volume of 50 mL fermentation medium); inoculate the seed culture mixed in the ratio into a 250 mL baffle shaker flask containing 50 mL fermentation medium, and shake and culture according to the set conditions.

[0207] 3. Product Detection: After fermentation, 1 mL of fermentation broth was centrifuged at 16000 × g for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane, and the concentrations of vanillin and ferulic acid were determined by HPLC. The HPLC detection conditions were the same as in the previous examples. The product concentration was quantitatively calculated by comparing the peak time and peak area of ​​the standards.

[0208] II. Test Results

[0209] 1. Vanillin yield at different inoculation ratios

[0210] like Figure 13 As shown, Up02 and Down03 successfully synthesized vanillin at three inoculation ratios, but the yields differed significantly. The highest vanillin yield (7.8 mg / L) was observed at an inoculation ratio of 1:1; 3.1 mg / L at an inoculation ratio of 1:3 (upstream:downstream); and 4.4 mg / L at an inoculation ratio of 3:1. These results indicate that an imbalance in the inoculation ratio between upstream and downstream strains leads to a mismatch between precursor supply and product transformation, thus affecting the final yield.

[0211] 2. HPLC validation results

[0212] HPLC detection chromatogram as follows Figure 14 As shown, the elution time of vanillin standard was 21.315 min (peak 7); in the fermentation sample with Up02:Down03=1:3, peak 8 was identified as vanillin after comparison, and the peak shape showed no obvious impurities; in the fermentation sample with Up02:Down03=1:1, peak 9 was vanillin, with the largest peak area, consistent with the yield data; in the fermentation sample with Up02:Down03=3:1, peak 10 was vanillin, with a smaller peak area than peak 9. These results confirm that the fermentation product under different inoculation ratios was the target product vanillin, and the detection results are accurate and reliable.

[0213] In summary, the optimal inoculation ratio of upstream recombinant strain Up02 to downstream recombinant strain Down03 in the co-culture system is 1:1. At this ratio, the two strains exhibit coordinated growth, achieving the best match between precursor supply and product conversion efficiency, with the highest vanillin ethyl ketone yield reaching 7.8 mg / L in shake flasks. This provides key process parameter support for the large-scale application of the co-culture system.

[0214] 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 de novo synthesis of vanillin based on co-culture of recombinant bacteria, characterized in that, include: The design includes tyrosine deaminase TAL, 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC, and caffeic acid 3- O -Methyltransferase COMT, ferulic acid decarboxylase FDC, ferulic acid decarboxylase mutant FDC V46E The de novo biosynthetic pathway of (S)1-phenylethanol dehydrogenase ped; A co-culture system was constructed consisting of an upstream recombinant strain producing ferulic acid from glucose and a downstream recombinant strain producing vanillin from ferulic acid, wherein the upstream recombinant strain was based on *Escherichia coli* MG1655 and modified in at least one of the following ways: A. Overexpression originates from Rhodotorula genus. Rhodotorula glutinis Tyrosine deaminase RgTAL, derived from Escherichia coli Escherichia coli 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC and derived from Arabidopsis thaliana Arabidopsis thaliana caffeic acid 3- O The gene encoding the methyltransferase AtCOMT, and the optimized codon of the tyrosine deaminase RgTAL, have the nucleotide sequence shown in SEQ ID NO:1; the 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC comprises: 4-hydroxyphenylacetic acid-3-monooxygenase EcHpaB and flavin reductase EcHpaC, the nucleotide sequence of the gene encoding the 4-hydroxyphenylacetic acid-3-monooxygenase EcHpaB is shown in SEQ ID NO:2, and the nucleotide sequence of the gene encoding the flavin reductase EcHpaC is shown in SEQ ID NO:3; the caffeic acid 3- O The nucleotide sequence of the gene encoding the methyltransferase AtCOMT is shown in SEQ ID NO:

4. The genes encoding the tyrosine deaminase RgTAL and the 4-hydroxyphenylacetic acid-3-hydroxylase EcHpaBC are overexpressed using pCDFTac as a vector, and the genes encoding the caffeic acid 3-hydroxylase are overexpressed using pTrc99A as a vector. O The gene encoding the methyltransferase AtCOMT; B. Knock out the tyrosine metabolism repression gene based on A. tyrR The tyrosine metabolism repression gene tyrR The nucleotide sequence is shown in SEQ ID NO:5; C. Overexpression of Pseudomonas motilityis based on B. Zymomonas mobilis The gene encoding prephenyl acid dehydrogenase TyrC, the nucleotide sequence of which is shown in SEQ ID NO:6; D. Overexpression of Escherichia coli based on C. Escherichia coli AroG, a feedback inhibition mutant of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase D146N The gene encoding the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase. D146N The nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:7; The downstream recombinant strain is based on Escherichia coli MG1655 as the starting strain, and is modified in at least one of the following ways: E. Overexpression originates from Enterobacteriaceae. Enterobactersp. Px6-4 The gene encoding ferulic acid decarboxylase FDC, the nucleotide sequence of which is shown in SEQ ID NO:8; F. Overexpression originates from Enterobacteriaceae. Enterobactersp. Px6-4 The ferulic acid decarboxylase mutant FDC V46E The gene encoding the ferulic acid decarboxylase mutant FDC V46E The nucleotide sequence of the encoding gene is shown in SEQ ID NO:9; G. Overexpression originates from Aspergillus aromatica Aromatoleumaromaticum The gene encoding (S)1-phenylethanol dehydrogenase ped, the nucleotide sequence of which is shown in SEQ ID NO:

10. The upstream and downstream recombinant strains were inoculated into a glucose-containing fermentation medium at a 1:1 ratio and co-cultured at 30°C, 200 rpm on a shaker, and an initial pH of 7.0 for 60 h. The OD values ​​were then calculated. 600 When the concentration of the target enzyme is 0.5-0.8, IPTG with a final concentration of 0.5 mM is added to induce expression of the target enzyme, and vanillin is obtained.

2. The de novo synthesis method of vanillin ethyl ketone according to claim 1, characterized in that, In E~G, pTrc99A was used as the vector for overexpression.

3. The de novo synthesis method of vanillin ethyl ketone according to claim 1, characterized in that, The fermentation medium also includes: MgSO4 A solution of 7H2O, KH2PO4, (NH4)2HPO4, citric acid, and trace metal salts, with a pH of 6.5–7.

5.

4. The de novo synthesis method of vanillin ethyl ketone according to claim 3, characterized in that, The composition of the trace metal salt solution is: 10 g / L FeSO4 7H2O, 2.65 g / L CaCl2 2H₂O, 2.2 g / L ZnSO₄ 7H2O, 0.58 g / L MnSO4 5H2O, 1g / L CuSO4 5H₂O, 0.1 g / L (NH₄)₆Mo₇O 24 4H₂O, 0.02 g / L Na₂B₄O₇ 10H2O and 10 mL / L of 35% HCl.

5. The application of the de novo synthesis method of vanillin according to any one of claims 1 to 4 in the preparation of products containing vanillin by microbial fermentation.

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