Method for constructing vanillin-producing engineering bacteria and application thereof

Abstract

The application provides a construction method of vanillin engineering bacteria and application thereof. Based on a SAM supply, ATP synthesis reinforcement and intracellular oxidation regulation coordination strategy, the vanillin engineering bacteria are constructed. The vanillin engineering bacteria take E. coli as a starting strain, and overexpress DSD, OMT, CAR, VaoA, Mtn, LuxS and gltA R119L genes. Mtn and LuxS reinforce intracellular SAM regeneration cycle, and remove the feedback inhibition of SAH on methyltransferase; gltA R119L optimizes central carbon metabolism and oxidative phosphorylation pathway, improves tricarboxylic acid cycle flux, and provides sufficient ATP energy guarantee for vanillin synthesis; VaoA effectively solves the problem that vanillin is degraded into vanillyl alcohol by endogenous enzymes by regulating intracellular oxidation level. The yield of the vanillin engineering bacteria constructed by the application in fermentation production reaches 729.81 mg / L, and can meet the needs of industrialized low-cost and high-efficiency production of vanillin.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and microbial biomanufacturing technology, specifically relating to a method for constructing vanillin-producing engineered bacteria and its application. Background Technology

[0002] Vanillin, chemically known as 3-methoxy-4-hydroxybenzaldehyde, is the world's most widely used flavoring monomer, with applications in food, daily chemicals, pharmaceuticals, and fine chemicals. Currently, vanillin is mainly produced through three methods: plant extraction, chemical synthesis, and biotransformation. Traditional plant extraction uses vanilla beans as raw material, but its large-scale application is significantly limited due to the low yield and dependence on specific growing conditions. Chemical synthesis suffers from complex reaction steps, requires precise control of process conditions, and demands high standards for equipment and operation. Furthermore, it generates wastewater and waste residue, resulting in high subsequent treatment costs. In contrast, biotransformation offers advantages such as being environmentally friendly, having mild reaction conditions, producing high-purity products, and eliminating chemical residues, making it the mainstream development direction for green vanillin manufacturing.

[0003] Escherichia coli (E. coli) is one of the optimal chassis strains for constructing the vanillin synthesis pathway due to its clear genetic background, rapid growth rate, and low culture cost. However, current E. coli vanillin synthesis technologies mostly focus on overexpression of key enzymes in the vanillin synthesis pathway, optimization of precursor synthesis, and knockout modification of genes involved in side reactions. These technologies generally suffer from core bottlenecks such as insufficient intracellular cofactor supply, energy metabolism imbalance, and poor vanillin tolerance. These problems result in low vanillin synthesis throughput, high metabolic stress on the strain, and limited final product yield, making it difficult to meet the requirements of industrial fermentation production. Summary of the Invention

[0004] Technical Problem to be Solved: Addressing the shortcomings of existing Escherichia coli vanillin-producing strains, such as insufficient cofactors, energy depletion, product degradation, and low yield, this invention provides a method for constructing a vanillin-producing engineered bacterium and its application. Through multi-dimensional synergistic modification of Escherichia coli, simultaneously optimizing the intracellular SAM regeneration pathway, ATP energy metabolism pathway, and product protection, the metabolic bottleneck in vanillin synthesis is systematically resolved, significantly increasing vanillin biosynthesis yield.

[0005] Technical Solution: This invention provides a vanillin-producing engineered bacterium, using *Escherichia coli* as the starting strain, which overexpresses the following genes: dehydroshikimate dehydratase (DSD), 3-O-methyltransferase (OMT), carboxylic acid reductase (CAR), vanillyl alcohol oxidase (VaoA), 5'-methylthioadenosine nucleosidase (Mtn), S-ribose homocysteine ​​lyase (LuxS), and citrate synthase mutant gene (gltA). R119L .

[0006] This invention also provides the above-mentioned dehydroshikimate dehydratase gene DSD, 3-O-methyltransferase gene OMT, carboxylic acid reductase gene CAR, vanillyl alcohol oxidase gene VaoA, 5'-methionine adenosine nucleosidase gene Mtn, S-ribose homocysteine ​​lyase gene LuxS, and citrate synthase mutant gene gltA. R119L The nucleotide sequence.

[0007] The aforementioned dehydroshikimate dehydratase gene DSD is derived from Propionibacterium, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The 3-O-methyltransferase gene OMT mentioned above is derived from Leptospira, and its nucleotide sequence is shown in SEQ ID NO.2.

[0009] The carboxylic acid reductase gene CAR mentioned above is derived from Nocardia iowanensis, and its nucleotide sequence is shown in SEQ ID NO.3.

[0010] The vanillyl oxidase gene VaoA mentioned above is derived from Penicillium spp., and its nucleotide sequence is shown in SEQ ID NO.4.

[0011] The 5'-methionine nucleosidase gene Mtn mentioned above is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.5.

[0012] The S-ribosomal homocysteine ​​lyase gene LuxS mentioned above is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.6.

[0013] The aforementioned citrate synthase mutant was obtained by mutating arginine at position 119 of the wild-type citrate synthase amino acid sequence to leucine; the citrate synthase mutant gene gltA R119L The nucleotide sequence is shown in SEQ ID NO.7, gltA R119L The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.8; the amino acid sequence of the wild-type citrate synthase is shown in SEQ ID NO.9.

[0014] The present invention also provides a method for constructing the above-mentioned vanillin-producing engineered bacteria, comprising the following steps:

[0015] S1. The pRSFDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the CAR gene was inserted into the MCS1 site of pRSFDuet-1 using homologous recombination; the pRSFDuet-1 expression vector was digested with restriction endonucleases NdeI and XhoI, and the VaoA gene was inserted into the MCS2 site of pRSFDuet-1 using homologous recombination to obtain the pRSFDuet-CAR-VaoA expression vector;

[0016] S2. The pETDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the OMT gene was inserted into the MCS1 site of pETDuet-1 using homologous recombination; the pETDuet-1 expression vector was digested with restriction endonucleases EcoRV and XhoI, and the DSD gene was inserted into the MCS2 site of pETDuet-1 using homologous recombination to obtain the pETDuet-OMT-DSD expression vector;

[0017] S3. The pCDFDuet-1 expression vector was double-digested with restriction endonucleases BamHI and HindIII, and the Mtn gene was inserted into the MCS1 site of pCDFDuet-1 using homologous recombination. The pCDFDuet-1 expression vector was double-digested with restriction endonucleases NdeI and XhoI, and the LuxS gene was inserted into the MCS2 site of pCDFDuet-1 using homologous recombination to obtain the pCDFDuet-Mtn-LuxS expression vector. The pCDFDuet-Mtn-LuxS expression vector was used as a template for linearization, and gltA was ligated via homologous recombination. R119L Obtain pCDFDuet-Mtn-LuxS-gltA R119L Expressive vehicle;

[0018] S4. Co-transform Escherichia coli with pRSFDuet-CAR-VaoA expression vector and pETDuet-OMT-DSD expression vector to obtain engineered strains pRSFDuet-CAR-VaoA and pETDuet-OMT-DSD;

[0019] S5. pCDFDuet-Mtn-LuxS-gltA R119L The expression vectors were transformed into engineered strains pRSFDuet-CAR-VaoA and pETDuet-OMT-DSD to obtain vanillin-producing engineered strains.

[0020] The present invention also provides the application of the above-mentioned vanillin-producing engineered bacteria or the vanillin-producing engineered bacteria constructed by the above-mentioned construction method in the fermentation production of vanillin.

[0021] This invention also provides a method for producing vanillin by fermentation, comprising the following steps:

[0022] (1) The vanillin-producing engineered bacteria according to any one of claims 1 to 3 are streaked on LB solid medium containing ampicillin, streptomycin and kanamycin resistance, and a single colony is picked and inoculated into LB liquid medium containing ampicillin, streptomycin and kanamycin resistance, and cultured at 35 to 38°C and 180 to 220 rpm for 8 to 16 h to obtain seed culture;

[0023] (2) Inoculate the seed culture into M9Y fermentation medium containing ampicillin, streptomycin and kanamycin resistance, and culture at 35-38℃ with shaking at 180-220 rpm until the bacterial cell OD 600 When the value reaches 0.6~0.9, IPTG is added, and the mixture is induced and cultured at 14~18℃ and 180~220 rpm for 16~20 h, and then fermented at 28~32℃ and 180~220 rpm for 40~60 h to synthesize vanillin.

[0024] Preferably, the inoculation amount of seed liquid in step (2) above is 1~5%.

[0025] Preferably, the final concentration of IPTG in step (2) above is 0.1~2 mM.

[0026] Beneficial effects:

[0027] 1. This invention breaks through the technical limitations of traditional single-modification synthetic pathways. It adopts a synergistic modification strategy of methyl donor supply, energy metabolism support and intracellular oxidation regulation, systematically breaking through the bottleneck of vanillin synthesis metabolism from three core dimensions: substrate cofactor, energy support and cell homeostasis, with significant synergistic effects.

[0028] 2. Through multi-pathway synergistic optimization, the engineering method constructed in this invention, under conventional shake-flask fermentation conditions and using a simple carbon source as raw material, achieved a stable vanillin fermentation yield of 729.81 mg / L, which is at a relatively high level compared to similar E. coli biosynthesis vanillin technologies, demonstrating a significant yield increase effect.

[0029] 3. This invention achieves in-situ anti-degradation protection of vanillin by regulating intracellular oxidation levels, effectively avoiding the product loss problem of vanillin being degraded into vanillyl alcohol by endogenous enzymes, and ensuring the efficient accumulation of synthetic products.

[0030] 4. The modification method of this invention is simple to operate, requiring no expensive substrates, complex equipment, or special culture conditions. The strain cultivation cost is low, the fermentation process is mature, and it can be directly adapted to existing industrial fermenter scale-up production systems. The entire process is green and low-carbon, possessing good economic feasibility and promising prospects for promotion, thus driving the industrialization of vanillin green biomanufacturing.

[0031] 5. The multi-dimensional synergistic modification strategy of cofactor supply, energy metabolism enhancement and cellular redox regulation established in this invention is not only highly applicable to the biosynthesis of vanillin, but also provides important technical reference and general platform strategy for the microbial synthesis of other natural products that rely on methylation reactions, are energy-intensive and have easily degradable products. Attached Figure Description

[0032] Figure 1 This is the HPLC chromatogram of vanillin fermented in shake flask in Example 2. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0034] Materials sourced from: Escherichia coli BL21(DE3) was commercially available; pRSFDuet-1, pETDuet-1, and pCDFDuet-1 were storage vectors from the applicant's Zhongyuan Food Laboratory; KOD one high-fidelity DNA polymerase was purchased from TOYOBO; 2×TransStart® FastPfu Fly PCR high-fidelity DNA polymerase was purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases, plasmid extraction kits, DNA purification kits, and gel recovery kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; vanillin and vanillin were purchased from Maclean's.

[0035] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl were dissolved in distilled water and the pH was adjusted to 7.0. The mixture was then autoclaved at 121°C for 20 min.

[0036] M9Y medium: 6.8 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 10.0 g / L glycerol, and 5.0 g / L yeast extract were dissolved in distilled water and autoclaved at 121℃ for 20 min. Then, MgSO4, CaCl2, and glucose were added. The final concentrations of MgSO4, CaCl2, and glucose were 2.5 g / L.

[0037] Example 1

[0038] This embodiment describes a method for constructing vanillin-producing engineered bacteria, including the following steps:

[0039] S1. Preparation of target gene fragments: PodDSD, NiCAR, VaoA, and LiOMT genes were synthesized by BGI Genomics and processed using KOD One. TM PCR Master Mix was used to amplify PodDSD containing EcoRV and XhoI restriction sites, NiCAR containing BamHI and HindIII restriction sites, VaoA containing NdeI and XhoI restriction sites, and LiOMT containing BamHI and HindIII restriction sites; using E. coli BL21(DE3) genome as a template, KOD One was used. TM PCR Master Mix amplification yielded Mtn, LuxS, and gltA R119LGene (first amplify to obtain gltA, then use gltA) R119L -F / R mutation); PCR primers, amplification reaction system and amplification conditions are shown in Tables 1-3. PCR products were detected by agarose gel electrophoresis. After the band size was found to be correct, the gel was recovered for subsequent operations.

[0040] Table 1 PCR Primers

[0041]

[0042] Table 2 PCR reaction system

[0043]

[0044] Table 3 PCR amplification conditions

[0045]

[0046] S2. The pRSFDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII. The NiCAR gene was inserted into the MCS1 site of pRSFDuet-1 using the ClonExpress Ultra One Step Cloning Kit V2 homologous recombination kit. The pRSFDuet-1 expression vector was digested with restriction endonucleases NdeI and XhoI. The VaoA gene was inserted into the MCS2 site of pRSFDuet-1 using homologous recombination to obtain the pRSFDuet-NiCAR-VaoA expression vector.

[0047] S3. The pETDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the LiOMT gene was inserted into the MCS1 site of pETDuet-1 using homologous recombination. The pETDuet-1 expression vector was digested with restriction endonucleases EcoRV and XhoI, and the PodDSD gene was inserted into the MCS2 site of pETDuet-1 using homologous recombination to obtain the pETDuet-LiOMT-PodDSD expression vector.

[0048] S4. The pCDFDuet-1 expression vector was double-digested with restriction endonucleases BamHI and HindIII. Homologous recombination was used to insert the Mtn gene into the MCS1 site of pCDFDuet-1. The pCDFDuet-1 expression vector was then double-digested with restriction endonucleases NdeI and XhoI. Homologous recombination was used to insert the LuxS gene into the MCS2 site of pCDFDuet-1, yielding the pCDFDuet-Mtn-LuxS expression vector. Linearization was performed using the pCDFDuet-Mtn-LuxS expression vector as a template, and gltA was ligated via homologous recombination.R119L Obtain pCDFDuet-Mtn-LuxS-gltA R119L Expressive vehicle;

[0049] S5. Construction of engineered bacteria: pRSFDuet-NiCAR-VaoA expression vector and pETDuet-LiOMT-PodDSD expression vector were co-transformed into *E. coli* BL21(DE3). BL21(DE3) competent cells were immediately thawed on ice after being removed from -80℃. The expression vectors were added and gently mixed, then incubated on ice for 30 min. The cells were then heat-shocked at 42℃ for 45 s, and immediately incubated on ice for 3 min. 900 μL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37℃ and 200 rpm for 1 h. The bacterial culture was spread on LB solid medium containing ampicillin and kanamycin resistance and cultured overnight at 37℃. Colonies growing on the antibiotic-resistant medium were identified as BL21-pRSFDuet-NiCAR-VaoA, pETDuet-LiOMT-PodDSD. Single colonies were picked and inoculated into LB liquid medium containing ampicillin and kanamycin resistance and cultured overnight at 37℃. A 1% transfer ratio was then performed to 50... Incubate in LB liquid medium containing ampicillin and kanamycin resistance at 37°C and 200 rpm until OD500. 600 Reach 0.5; incubate on ice for 10 min, centrifuge at 4000 rpm, 4℃ for 8 min and discard the supernatant; resuspend the bacterial cells in ice water, centrifuge at 4000 rpm, 4℃ for 8 min and discard the supernatant; add 30 mL of pre-cooled 0.1 mM calcium chloride solution, let stand for 30 min, centrifuge at 4000 rpm, 4℃ for 5 min and discard the supernatant; add 500 μL of 0.1 mM calcium chloride-20% glycerol solution to obtain BL21-pRSFDuet-NiCAR-VaoA, pETDuet-LiOMT-PodDSD competent cells; use chemical transformation to convert pCDFDuet-Mtn-LuxS-gltA... R119L The expression vector was transformed into the above competent cells to obtain cells carrying pRSFDuet-NiCAR-VaoA, pETDuet-LiOMT-PodDSD, and pCDFDuet-Mtn-LuxS-gltA. R119L Engineered bacteria for expression vectors.

[0050] Example 2

[0051] This embodiment describes a method for synthesizing vanillin using the engineered bacteria constructed in Example 1, including the following steps:

[0052] S1. Take the recombinant engineered bacteria constructed in Example 1, streak it onto LB solid medium containing ampicillin, streptomycin and kanamycin resistance, activate it, pick a single colony, and inoculate it into LB liquid medium containing ampicillin, streptomycin and kanamycin resistance. Culture at 37°C and 200 rpm for 12 h to prepare seed culture;

[0053] S2. Transfer the seed culture to M9Y fermentation medium containing ampicillin, streptomycin, and kanamycin resistance at a 1% volume inoculation rate, and incubate at 37°C with shaking at 200 rpm until the bacterial cell OD... 600 When the value reached 0.8, IPTG at a final concentration of 0.5 mM was added to induce protein expression, and the mixture was cultured at 16℃ and 200 rpm for 20 h; then the culture temperature was adjusted to 30℃ and fermented at 200 rpm for 48 h to synthesize vanillin.

[0054] S3. After fermentation, samples were taken, and the cell reaction was terminated by adding 2 volumes of ice-cold methanol. The vanillin content was determined by high-performance liquid chromatography (HPLC). The detection equipment was a Shimadzu LC-2050 HPLC system, and the chromatographic column was a Shimadzu ShimNex CS C18 column (5 μm, 4.6 × 150 mm). The detection parameters were: flow rate 1 mL / min, column temperature 40℃, injection volume 10 μL; mobile phase was 0.1% formic acid aqueous solution (phase A) - acetonitrile (phase B); HPLC detection conditions were 5-30% B, 25 min, and detection wavelength 280 nm.

[0055] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 2 is that the engineered bacteria in this comparative example do not contain the Mtn-LuxS gene, but contain the pRSFDuet-NiCAR-VaoA expression vector, the pETDuet-LiOMT-PodDSD expression vector, and the pACYCDuet-gltA expression vector. R119L Expression vector, pACYCDuet-gltA R119L The expression vector was constructed as follows: the pACYCDuet-1 expression vector was double-digested with BamHI and HindIII, the gltA gene was amplified by PCR, and homologous recombination was performed to obtain the pACYCDuet-gltA expression vector. The gltA gene was then used... R119L -F / R performs site-directed mutagenesis to obtain pACYCDuet-gltA R119LThe expression vectors, pRSFDuet-NiCAR-VaoA and pETDuet-LiOMT-PodDSD, were constructed using the same methods as in Example 1, and the engineered bacteria were constructed using the same methods as in Example 2.

[0058] Comparative Example 2

[0059] The difference between this comparative example and Example 2 is that the engineered bacteria in this comparative example do not contain gltA. R119L The construction methods of the gene, pRSFDuet-NiCAR-VaoA expression vector, pETDuet-LiOMT-PodDSD expression vector, and pCDFDuet-Mtn-LuxS expression vector are the same as in Example 1, and the construction method of the engineered bacteria is the same as in Example 2.

[0060] Comparative Example 3

[0061] The difference between this comparative example and Example 2 is that the engineered bacteria in this comparative example contain only the pRSFDuet-NiCAR expression vector and the pETDuet-LiOMT-PodDSD expression vector, and do not contain VaoA, Mtn-LuxS, or gltA. R119L The pRSFDuet-NiCAR expression vector was constructed as follows: the pRSFDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the NiCAR gene was inserted into the MCS1 site of pRSFDuet-1 using the ClonExpress Ultra One Step Cloning Kit V2 homologous recombination kit to obtain the pRSFDuet-NiCAR expression vector; the pETDuet-LiOMT-PodDSD expression vector was constructed in the same way as in Example 1, and the engineered bacteria were constructed in the same way as in Example 2.

[0062] Table 4 Vanillin Production

[0063]

[0064] SAM (S-adenosylmethionine) is a core limiting cofactor in the methylation step of the vanillin synthesis pathway. Synthesized via the methionine cycle, its intracellular supply directly determines the efficiency of the methylation reaction. This invention overexpresses the genes encoding the key rate-limiting enzymes of the methionine cycle, Mtn and LuxS, to achieve dual enhancement: Mtn accelerates the degradation of S-adenosyl-L-homocysteine ​​(SAH), and LuxS accelerates the synthesis of SAM precursors, thereby strengthening the intracellular SAM regeneration cycle, increasing the effective supply of SAM, and avoiding feedback inhibition of methyltransferases by SAH.

[0065] The synthesis of vanillin and the cofactor SAM both require a large amount of ATP, and the intracellular ATP level directly determines the overall efficiency of the synthetic pathway. Simultaneously, vanillin accumulation triggers a cellular stress response, with more ATP being used to combat vanillin-induced damage, leading to a severe shortage of energy available for the synthetic pathway. This invention modifies the central carbon metabolism and oxidative phosphorylation pathway in *E. coli* by overexpressing the citrate synthase mutant gene *gltA*. R119L It enhances the flux of the tricarboxylic acid cycle, strengthens the efficient synthesis of ATP, provides continuous and sufficient energy support for vanillin and SAM synthesis, and ensures the catalytic activity of key enzymes in the synthesis pathway.

[0066] Vanillin is readily degraded into vanillin in Escherichia coli, limiting its accumulation. This invention introduces the vanillin oxidase gene VaoA to oxidize vanillin in situ, effectively increasing vanillin accumulation and reducing non-target conversion losses.

[0067] Through the synergistic modification of the three strategies mentioned above, this invention constructs a high-vanillin-producing recombinant engineered Escherichia coli strain. As shown in Table 4, under conventional shake-flask fermentation conditions, the vanillin fermentation yield of this strain consistently reaches 729.81 mg / L, a significant increase compared to strains that only undergo key enzyme overexpression (Comparative Example 3) and single cofactor optimization (Comparative Examples 1-2). This invention effectively solves common industry pain points such as insufficient cofactor supply, energy metabolism imbalance, severe vanillin degradation, and low final product yield commonly found in traditional strains, providing a feasible technical path for the green biomanufacturing of vanillin.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A vanillin-producing engineered bacterium, characterized in that, The vanillin-producing engineered bacteria used *Escherichia coli* as the starting strain, overexpressing the following genes: dehydroshikimate dehydratase (DSD), 3-O-methyltransferase (OMT), carboxylic acid reductase (CAR), vanillyl alcohol oxidase (VaoA), 5'-methylthioadenosine nucleosidase (Mtn), S-ribose homocysteine ​​lyase (LuxS), and citrate synthase mutant gene (gltA). R119L .

2. The vanillin-producing engineered bacteria according to claim 1, characterized in that: The dehydroshikimate dehydratase gene DSD is derived from Propionibacterium, and its nucleotide sequence is shown in SEQ ID NO.1; the 3-O-methyltransferase gene OMT is derived from Leptospira, and its nucleotide sequence is shown in SEQ ID NO.2; the carboxylic acid reductase gene CAR is derived from Nocardia iowanensis, and its nucleotide sequence is shown in SEQ ID NO.3; the vanillyl alcohol oxidase gene VaoA is derived from Penicillium jaundice, and its nucleotide sequence is shown in SEQ ID NO.4; the 5'-methylthioadenosine nucleosidase gene Mtn is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.5; the S-ribosomalosomal homocysteine ​​lyase gene LuxS is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.

6.

3. The vanillin-producing engineered bacteria according to claim 1, characterized in that: The citrate synthase mutant was obtained by mutating arginine at position 119 of the amino acid sequence of wild-type citrate synthase to leucine. citrate synthase mutant gene gltA R119L The nucleotide sequence is shown in SEQ ID NO.7, gltA R119L The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.8; the amino acid sequence of the wild-type citrate synthase is shown in SEQ ID NO.

9.

4. The method for constructing vanillin-producing engineered bacteria according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. The pRSFDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the CAR gene was inserted into the MCS1 site of pRSFDuet-1 using homologous recombination; the pRSFDuet-1 expression vector was digested with restriction endonucleases NdeI and XhoI, and the VaoA gene was inserted into the MCS2 site of pRSFDuet-1 using homologous recombination to obtain the pRSFDuet-CAR-VaoA expression vector; S2. The pETDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the OMT gene was inserted into the MCS1 site of pETDuet-1 using homologous recombination; the pETDuet-1 expression vector was digested with restriction endonucleases EcoRV and XhoI, and the DSD gene was inserted into the MCS2 site of pETDuet-1 using homologous recombination to obtain the pETDuet-OMT-DSD expression vector; S3. The pCDFDuet-1 expression vector was digested with restriction endonucleases BamHI and HindIII, and the Mtn gene was inserted into the MCS1 site of pCDFDuet-1 using homologous recombination. The pCDFDuet-1 expression vector was then digested with restriction endonucleases NdeI and XhoI, and the LuxS gene was inserted into the MCS2 site of pCDFDuet-1 using homologous recombination to obtain the pCDFDuet-Mtn-LuxS expression vector. The pCDFDuet-Mtn-LuxS expression vector was used as a template for linearization, and gltA was ligated via homologous recombination. R119L Obtain pCDFDuet-Mtn-LuxS-gltA R119L Expressive vehicle; S4. Co-transform Escherichia coli with pRSFDuet-CAR-VaoA expression vector and pETDuet-OMT-DSD expression vector to obtain engineered strains pRSFDuet-CAR-VaoA and pETDuet-OMT-DSD; S5. pCDFDuet-Mtn-LuxS-gltA R119L The expression vectors were transformed into engineered strains pRSFDuet-CAR-VaoA and pETDuet-OMT-DSD to obtain vanillin-producing engineered strains.

5. The application of the vanillin-producing engineered bacteria according to any one of claims 1 to 3 or the vanillin-producing engineered bacteria constructed by the construction method according to claim 4 in the fermentation production of vanillin.

6. The application according to claim 5, characterized in that, The method for producing vanillin by fermentation includes the following steps: (1) The vanillin-producing engineered bacteria according to any one of claims 1 to 3 are streaked on LB solid medium containing ampicillin, streptomycin and kanamycin resistance, and a single colony is picked and inoculated into LB liquid medium containing ampicillin, streptomycin and kanamycin resistance, and cultured at 35 to 38°C and 180 to 220 rpm for 8 to 16 h to obtain seed culture; (2) Inoculate the seed culture into M9Y fermentation medium containing ampicillin, streptomycin and kanamycin resistance, and culture at 35-38℃ with shaking at 180-220 rpm until the bacterial cell OD 600 When the value reaches 0.6~0.9, IPTG is added, and the mixture is induced and cultured at 14~18℃ and 180~220 rpm for 16~20 h, and then fermented at 28~32℃ and 180~220 rpm for 40~60 h to synthesize vanillin.

7. The application according to claim 6, characterized in that: The inoculation amount of seed liquid in step (2) is 1-5%.

8. The application according to claim 6, characterized in that: The final concentration of IPTG in step (2) is 0.1~2 mM.

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