Construction process of high-yield vanillin chlamys farreri

Abstract

The invention discloses a construction process of high-yield vanillin chlamys farreri, and belongs to the technical field of vanillin production, which comprises the following steps: with ferulic acid as a substrate, vanillin is generated through conversion of a metabolic pathway, and the ferulic acid is an easily available natural compound and widely exists in plant cell walls. According to a definite vanillin synthesis route and a cofactor circulation route, amycolatopsis which can metabolize toxins and is low in vanillin degradation rate is selected as a chassis, and gene modification is performed on a strain by integrating vanillin synthetase of vanilla, so that efficient one-step synthesis of vanillin by the amycolatopsis is realized.

Description

Technical Field

[0001] The invention relates to the technical field of vanillin production, in particular to a construction process of a high-vanillin-yielding Ctenophora coli. Background Art

[0002] Vanillin, chemically known as 3-methoxy-4-hydroxybenzaldehyde, is a compound with a significant vanilla aroma. Due to its unique aroma characteristics, vanillin is widely used in the food, beverage, cosmetics and pharmaceutical industries. In the food industry, it is often used to bake cakes, candies and chocolates; in the beverage industry, it is used to prepare a variety of vanilla-flavored beverages; in the cosmetics industry, it is used to make products such as perfumes and creams; in the pharmaceutical industry, it is also used to prepare drugs. Vanillin is widely used in various industries, providing an important material basis for the development of these industries.

[0003] Currently, the sources of vanillin in the market are mainly divided into two types: natural extraction and artificial synthesis. Naturally extracted vanillin mainly comes from the tropical plant vanilla beans, whose pods are rich in vanillin. However, the output of naturally extracted vanillin is limited and it is difficult to meet the large-scale market demand, so artificially synthesized vanillin has become an important supplement. Artificially synthesized vanillin is made by converting raw materials through chemical reactions. The technology is mature and the cost is relatively low, so most of the vanillin on the market comes from this. Although naturally extracted vanillin may be better in quality, it is limited by production and cost and is mainly used in high-end markets or specific fields. Artificially synthesized vanillin is widely used in various industries such as food, beverages, cosmetics and medicine, bringing various wonderful experiences to people's lives.

[0004] At present, microorganisms from different sources such as Bacillus subtilis, Escherichia coli, Amycolatopsis sp., Pseudomonas putida, Saccharomyces cerevisiae, etc. have been modified to synthesize vanillin through substrates such as ferulic acid, eugenol, isoeugenol, glucose, etc. Among them, Amycolatopsis sp. and Streptomyces sp. can stably produce 20g / L vanillin with ferulic acid as substrate, but the conversion rate and efficiency are not high. There are also Escherichia coli integrated metabolic pathways to synthesize vanillin, but the yield is also low, only 5g / L. The research and application prospects of microbial synthesis of vanillin are broad, and a new approach is provided for the sustainable production of vanillin, but the above-mentioned synthesis method has low efficiency and yield, and it is difficult to meet the market demand for natural synthesis of vanillin by microbial method. Therefore, the present invention provides a high-yield vanillin pseudo-ctenoid bacteria construction process to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a process for constructing a high-vanillin-producing Ctenopharyngeal Acid Bacteria to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A process for constructing a high-vanillin-producing pseudo-ctenopharyngeal bacteria comprises: using ferulic acid as a substrate to generate vanillin through conversion of a metabolic pathway, wherein ferulic acid is a readily available natural compound that is widely present in plant cell walls and contains methoxy and hydroxyl groups in its structure, and is suitable as a precursor for the synthesis of vanillin; before the production operation is performed, pseudo-ctenopharyngeal bacteria and a recombinant vanillin synthase gene (VpVAN) are mixed and cultured to obtain a new colony;

[0008] Strain construction: The gene sequence of vpVAN (GenBank: KP278240.1) was first obtained from NCBI and synthesized;

[0009] Then, the upstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by specific primers vdhL-F and vdhL-R, and then the downstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by vdhR-F and vdhR-R primers;

[0010] Then, using seamless cloning technology, the three gene sequences were precisely connected to the plasmid pULcrRNA-km-Cas12, and the plasmid pULΔvdh:vpVAN was successfully constructed;

[0011] Then, the glycerol bacteria of Amycolatopsis sp. stored in cold storage were taken out;

[0012] Subsequently, the thawed Amycolatopsis sp. glycerol bacteria were inoculated onto a solid culture medium for activation culture at 30° C. for 3 to 4 days until obvious colony growth was observed;

[0013] The constructed plasmid pULΔvdh:vpVAN was thoroughly mixed with Amycolatopsis spp. and then electro-transformed;

[0014] After a single colony grew on the culture medium, colony PCR was performed using primers VDHcheck-F and VDHcheck-R to verify the correct insertion of the gene;

[0015] After colony verification, the correct strain was confirmed and glycerol was added for long-term storage and named AsvpVAN;

[0016] Fermentation Catalysis:

[0017] The AsvpVAN glycerol bacteria stored at -80°C were taken out and thawed naturally at room temperature. After thawing, the strain was inoculated on the surface of the activated solid culture medium and cultured at 30°C for 3 to 4 days until the colonies grew significantly.

[0018] Subsequently, the seed solution was transferred to the fermentation tank at a 10% inoculation rate;

[0019] A total of 90 g of ferulic acid solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 to 60 hours.

[0020] As a further solution of the present invention, the cultured strain was inoculated into an activation medium and cultured for 2 days at 30°C and 220 rpm. When the OD value reached 0.7-0.8, the bacteria were collected for subsequent use.

[0021] As a further embodiment of the present invention, the transformed cells are added to an activation medium and incubated at 30°C and 220 rpm. The incubated cells are spread on a solid medium containing 50 μg / ml apramycin and cultured at 30°C.

[0022] As a further solution of the present invention, a single colony is picked from the plate using a sterile tool, inoculated into a seed culture medium, and cultured at 30°C and 220rpm for 24 to 48 hours until the OD600 value reaches about 7 and microscopic examination shows that the bacteria have good morphology and are free of foreign bacteria.

[0023] As a further scheme of the present invention, taking a 5L fermenter as an example, 3L of fermentation medium is prepared, the pH is adjusted to 7.2, the temperature is set to 30°C, the stirring speed is set to 500rpm, the ventilation rate is controlled at 1-2vvm, and after fermentation for about 12 to 16 hours, the OD600 value should reach the range of 3-4.

[0024] As a further solution of the present invention, 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 5 min, diluted 10 times, filtered through a 0.22 μM water filter membrane, and analyzed by HPLC.

[0025] As a further solution of the present invention, C18 column, injection 10 μL, column temperature 35°C, mobile phase 30% methanol + 0.1% acetic acid, flow rate 1 mL / min, wavelength 280 nm, detection, then it was found that the vanillin yield was 66.34 g, the concentration was 22.11 g / L, and the molar conversion rate was 94.17%. In this way, the yield and conversion rate of vanillin can be greatly improved.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The invention adopts a clear vanillin synthesis pathway and a cofactor circulation pathway, selects Amycolatopsis which can metabolize toxins and has a low vanillin degradation rate as a base, and genetically modifies the strain by integrating vanilla vanillin synthase, thereby realizing efficient one-step synthesis of vanillin by Amycolatopsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The synthesis pathway of vanillin was constructed in the process of constructing a high-vanillin-producing Ctenopharyngeal Acid Bacteria.

[0029] Figure 2 This is the HPLC spectrum of vanillin detection in the process of constructing a high-vanillin-producing Ctenobacterium spp. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] See also Figures 1-2 In an embodiment of the present invention, a process for constructing a high-yield vanillin-producing pseudo-ctenopharyngeal bacteria comprises: using ferulic acid as a substrate to generate vanillin through conversion of a metabolic pathway, wherein ferulic acid is a readily available natural compound that is widely present in plant cell walls, and contains methoxy and hydroxyl groups in its structure, and is suitable as a precursor for the synthesis of vanillin; before the production operation is performed, pseudo-ctenopharyngeal bacteria are mixed and cultured with a recombinant vanillin synthase gene (VpVAN) to obtain a new colony;

[0032] Specific:

[0033] 1. Strain construction:

[0034] 1. First, the gene sequence of vpVAN (GenBank: KP278240.1) was obtained from NCBI and synthesized.

[0035] 2. The upstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by specific primers vdhL-F and vdhL-R, and the downstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by vdhR-F and vdhR-R primers;

[0036] 3. Then, using seamless cloning technology, the three gene sequences were precisely connected to the plasmid pULcrRNA-km-Cas12, and the plasmid pULΔvdh:vpVAN was successfully constructed;

[0037] Among them, the upstream and downstream sequences of the vpVAN gene and the vdh gene were connected to the plasmid pULcrRNA-km-Cas12 through seamless cloning technology, which can help further study the gene regulation and functional verification in the vanillin synthesis pathway;

[0038] 4. Then, take out the refrigerated Amycolatopsis sp. glycerol bacteria;

[0039] Among them, it is worth noting that: the vanillin synthase gene (VpVAN) is derived from vanilla beans, and its nucleotide sequence is as shown in No. 1; the vanillin dehydrogenase gene (vdh) is derived from Amycolatopsis spp., and its nucleotide sequence is as shown in No. 2;

[0040] Among them, the plasmid pULcrRNA-km-Cas12 containing Cas12a and crRNA co-expression was used as a template, and the spacer sequence of crRNA was replaced to obtain the recombinant plasmid pULΔvdh;

[0041] The genome of Amycolatopsis CCTCC NO:M2011265 was used as a template to amplify the upstream and downstream homologous arm fragments of the vdh gene, respectively. The amplified upstream and downstream products were recovered by a DNA recovery kit, and vpVAN was connected between the upstream and downstream homologous arms of the vdh gene by Overlap recombination. The obtained Fvdh+vpVAN+Rvdh gene fragment was then connected to the recombinant plasmid pULΔvdh by a one-step cloning method to obtain the plasmid pULΔvdh:vpVAN;

[0042] It is worth noting that Amycolatopsis should be stored in a -80℃ refrigerator and thawed at room temperature, and stored for a long time at -80℃ ultra-low temperature. In such a low temperature environment, the metabolic activity of the strain is almost stopped, which can maintain the activity and stability of the strain to the greatest extent, and the storage time can reach 2-5 years or even longer;

[0043] The Amycolatopsis sp. glycerol strain is a strain preservation form obtained by mixing a bacterial suspension of Amycolatopsis sp. with a certain proportion of glycerol;

[0044] Subsequently, the thawed Amycolatopsis sp. glycerol bacteria were inoculated onto a solid culture medium for activation culture at 30° C. for 3 to 4 days until obvious colony growth was observed.

[0045] Afterwards, the cultured strain was inoculated into the activation medium and cultured for 2 days at 30°C and 220 rpm. When the OD value reached 0.7-0.8, the bacteria were collected for subsequent use.

[0046] Then, the constructed plasmid pULΔvdh:vpVAN was thoroughly mixed with Amycolatopsis spp. and then electro-transformed;

[0047] Subsequently, the transformed cells were added to the activation medium and incubated at 30°C and 220 rpm;

[0048] The incubated cells were plated on solid medium containing 50 μg / ml apramycin and cultured at 30°C;

[0049] After a single colony grew on the culture medium, colony PCR was performed using primers VDHcheck-F and VDHcheck-R to verify the correct insertion of the gene;

[0050] After colony verification, the correct strain was confirmed and glycerol was added for long-term storage and named AsvpVAN;

[0051] Among them, colony PCR detection using VDHcheck-F and VDHcheck-R primers can quickly verify whether the gene is correctly inserted into the constructed plasmid. This is a common step to confirm whether seamless cloning is successful;

[0052] The specific steps are:

[0053] S1: Pick colonies:

[0054] Use a sterile toothpick or pipette tip to gently pick up individual colonies after transformation and add them to the PCR reaction system. To preserve the colonies, streak the bacterial liquid on the toothpick onto an LB medium plate containing antibiotics.

[0055] Prepare PCR system: Prepare PCR reaction system, usually the total reaction volume is 20-25μL:

[0056] VDHcheck-F primer: 0.1-0.5 μM

[0057] VDHcheck-R primer: 0.1-0.5 μM

[0058] PCR premix: Add appropriate amount according to product instructions

[0059] Bacterial template: picked colonies

[0060] Sterile water: make up to the total reaction volume

[0061] Set up PCR program: Set up PCR cycle conditions:

[0062] Pre-denaturation: 95°C, 5 minutes (to ensure bacterial lysis and release of DNA).

[0063] Cycling conditions (30-35 cycles):

[0064] Denaturation: 95°C, 30 sec.

[0065] Annealing: Depending on primer design, usually between 50-60°C, 30 seconds.

[0066] Extension: 72°C, the time is determined by the length of the amplified fragment, generally 1 minute per 1000 bp.

[0067] Final extension: 72°C, 5 min to ensure complete extension of all PCR products.

[0068] 3. Test results

[0069] Agarose gel electrophoresis: PCR products are electrophoresed, usually using 1%-1.5% agarose gel.

[0070] Band detection: Observe the electrophoresis results under ultraviolet light. If the primers VDHcheck-F and VDHcheck-R can amplify the expected bands, it means that the target gene has been successfully inserted into the plasmid.

[0071] 2. Fermentation catalysis:

[0072] Take out the AsvpVAN glycerol bacteria stored at -80°C and thaw naturally at room temperature;

[0073] After thawing, the strain was inoculated on the surface of the activated solid culture medium and cultured at 30°C for 3 to 4 days until the colonies grew significantly;

[0074] Use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30°C and 220 rpm for 24 to 48 hours until the OD600 value reaches about 7 and microscopic examination shows that the bacteria have good morphology and no foreign bacteria;

[0075] Subsequently, the seed solution was transferred to the fermentation tank at a 10% inoculation rate;

[0076] Taking a 5L fermenter as an example, prepare 3L of fermentation medium, adjust the pH to 7.2, set the temperature to 30°C, set the stirring speed to 500rpm, and control the aeration rate at 1-2vvm;

[0077] After about 12 to 16 hours of fermentation, the OD600 value should be in the range of 3-4;

[0078] At this time, a total of 90 g of ferulic acid solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 to 60 hours;

[0079] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 5 min, dilute 10 times, filter through 0.22 μM water filter membrane, and analyze by HPLC;

[0080] C18 column, injection 10μL, column temperature 35℃, mobile phase 30% methanol + 0.1% acetic acid, flow rate 1mL / min, wavelength 280nm, detection, then it was concluded that the vanillin yield was 66.34g, the concentration was 22.11g / L, and the molar conversion rate was 94.17%. This method can greatly improve the yield and conversion rate of vanillin.

[0081] Wherein, the formula of the activated fixed body culture medium is: glucose 10g / L, glycerol 10g / L, beef extract 1g / L, peptone 2g / L, yeast extract 1g / L, agar powder 20g / L, and the pH is adjusted to 7.2. The formula of the seed culture medium is: glucose 5g / L, yeast extract 10g / L, KH2PO41g / L, Na2HPO44g / L, MgSO4·7H2O0.2g / L, NaCl0.2g / L, CaCl20.05g / L, and the pH is adjusted to 7.2. The formula of the fermentation culture medium is: sucrose 103g / L, yeast extract 1.5g / L, Na2HPO44g / L, NaCl0.2g / L, KH2PO40.1g / L, MgSO4·7H2O0.2g / L, CaCl20.05g / L, and the pH is adjusted to 7.2;

[0082] 3. Sequences and primers involved in the present invention

[0083]

[0084]

[0085]

[0086] The PCR amplification system of the present invention

[0087]

[0088]

[0089] PCR program designed by the present invention

[0090]

[0091] 4. Nucleic acid sequence labeling:

[0092] No.1

[0093] MAAKLLFFLLFLVSALSVALAGFEEDNPIRSVTQRPDSIEPAILGVLGSCRHAFHFARFARRYGKSYGSE

[0094] EEIKKRFGIFVENLAFIRSTNRKDLSYTLGINQFADLTWEEFRTNRLGAAQNCSATAKGNHRFVDGVLPV

[0095] TRDWREQGIVSPVKDQGSCGSCWTFSTTGALEAAYTQLTGKSISLSEQQLVDCASAFNNFGCNGGLPSQA

[0096] FEYVKYNGGIDTEQTYPYLGVNGICNFKQENVGVKVIDSINITLGAEDELKHAVGLVRPVSVAFEVVKGF

[0097] NLYKKGVYSSDTCGRDPMDVNHAVLAVGYGVEDGIPYWLIKNSWGTNWGDNGYFKMELGKNMCGVATCAS

[0098] NO.2

[0099] 1msflddekwtgrvftgsweraaggdaaviepatgdelgrvgiaspqdlaasaakaaeaqr

[0100] 61awaatsfqeraavlrragdlwqqhaaelkdwliresgsipgkadfelhvaaqecyeaaal

[0101] 121pshptgevlpseaprlsmarrvpagvvgviapfnaplilsirsvapalalgnsvvlkpdp

[0102] 181rtavcggvalarvfeeaglpagvlhvlpggpdvgaalvedkhvrvisftgstaagravge

[0103] 241sagrhlkrahlelggnsalivlddadleqamsaaawgsffhqgqicmttgrhlvhaslyd

[0104] 301eyvdrladkashlpvgnpfteqvalgpiidakqrdkihglvtssvdagakvaaggtyedl

[0105] 361fyratvlagagpsvpaydqevfgpvapvakftsldeaaklaseseyglslgiitadvakg

[0106] 421laladriptgiahindqtvndealapfggvfdsgtgsrfggpaanieaftetrwvtmrgd

[0107] 481vagypf

[0108] NO.3

[0109] 1atggcagctaagctcctcttcttcctactcttcctggtctccgccctctccgtcgcgctc

[0110] 61gccggtttcgaagaagacaatccaatccggtccgttacacaaaggcctgactcgattgag

[0111] 121cctgccatcctcggcgtccttggcagttgccgccacgccttccacttcgcacggttcgcc

[0112] 181cgcaggtacgggaagagctacggatcggaggaggagatcaagaagaggttcgggatcttc

[0113] 241gtggagaatctagcgtttatccggtccactaatcggaaggatctgtcgtataccctagga

[0114] 301atcaaccaattcgccgacctgacctgggaggaattccggaccaatcgccttggtgcggcg

[0115] 361cagaactgctcggcgactgcgcatggaaaccaccggtttgtcgatggcgtgcttcctgta

[0116] 421acgagggattggagggagcaagggatagtgagccctgtaaaggaccaaggaagctgtgga

[0117] 481tcttgctggactttcagtactactggagcactagaggctgcatatacacagctaactgga

[0118] 541aagagcacatcattatctgaacagcaacttgtggactgtgcctcagcattcaataacttt

[0119] 601ggatgcaatggaggtttgccttcccaagcctttgaatacgttaagtacaatggaggcatc

[0120] 661gacacagaacagacttatccataccttggtgtcaatggtatctgcaacttcaagcaggag

[0121] 721aatgttggtgtcaaggtcattgattcgataaacatcaccctgggtgctgaggatgagttg

[0122] 781aagcatgcagtgggcttggtgcgtccagttagcgttgcatttgaggttgtgaaaggtttc

[0123] 841aatctgtacaagaaaggtgtatacagcagtgacacctgtggaagagatccaatggatgtg

[0124] 901aaccacgcagttcttgccgtcggttatggagtcgaggacgggattccttattggctcatc

[0125] 961aagaactcatggggtacaaattggggtgacaatggctactttaagatggaactcggcaag

[0126] 1021aacatgtgtggtgttgcaacttgcgcatcttatcccattgtggctgtgtag

[0127] NO.4

[0128] 1gtgagctttctcgacgacgagaagtggaccggacgcgtcttcaccggcagctgggagcgc

[0129] 61gcggcgggcggcgacgcggccgtcatcgagcccgcgaccggcgacgaactggggcgcgtc

[0130] 121ggcatcgcctcgccccaggacctggcggcctccgcggccaaggcggccgaggcgcagcgc

[0131] 181gcctgggcggcgacctccttccaagaacgcgccgcggtcctgcgccgcgccggcgacctg

[0132] 241tggcagcagcacgccgccgagctgaaggactggctgatccgcgagtcgggcagcatcccc

[0133] 301ggcaaggccgacttcgaactgcacgtcgccgcgcaggagtgctacgaggccgccgcgctg

[0134] 361ccctcccacccgacgggtgaggtcctgccgagcgaggcgccgcggctgagcatggcccgc

[0135] 421cgcgtgcccgccggcgtggtcggcgtgatcgcgccgttcaacgcgccgctgatcctgtcg

[0136] 481atccgctcggtcgcgccggcgctggcgctgggcaacagcgtcgtgctcaagccggacccc

[0137] 541cgcaccgcggtctgcggtggcgtggcgctggccagggtcttcgaggaggccgggctgccc

[0138] 601gccggggtcctgcacgtgctgccgggcggcccggacgtcggcgccgcgctggtcgaggac

[0139] 661aagcacgtccgcgtcatctcgttcaccggatcgaccgccgcgggccgcgcggtcggcgag

[0140] 721tccgcgggccgccacctcaagcgcgcccacctggaactgggcggcaactcggcgctgatc

[0141] 781gtgctcgacgacgccgacctggagcaggcgatgagcgccgccgcgtggggctcgttcttc

[0142] 841caccagggccagatctgcatgaccaccgggcggcacctggtgcacgcctcactctacgac

[0143] 901gaatacgtggaccgcctggcggacaaggccagccacctgccggtgggcaacccgttcacc

[0144] 961gagcaggtcgcgctcggcccgatcatcgacgccaagcagcgcgacaagatccacggcctg

[0145] 1021gtgacgtcca gtgtggacgc cggcgcgaag gtcgccgcgg gcggcaccta cgaggacctc

[0146] 1081ttctaccgcg ccaccgtgct cgccggcgcg ggcccctcgg tgcccgccta cgaccaggag

[0147] 1141gtgttcggcc cggtcgcccc ggtcgcgaag ttcaccagcc tggacgaggc cgcgaagctc

[0148] 1201gcgtcggaga gcgagtacgg gctgtcgctg ggcatcatca ccgcggacgt ggcgaaggga

[0149] 1261ctggcgctgg ccgaccgcat cccgaccggc atcgcgcaca tcaacgacca gacggtcaac

[0150] 1321gacgaggcgc tggccccgtt cggcggcgtg ttcgactccg gcaccggctc ccgcttcggc

[0151] 1381gggccggccg cgaacatcga ggcgttcacc gagacccgct gggtcacgat gcgcggcgac

[0152] 1441gtcgccggct acccgttctg a

[0153] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A process for constructing a high-yield vanillin-producing Ctenopharyngeal Acid Bacteria. comprising, characterized in that, Using ferulic acid as a substrate, vanillin is generated through conversion of metabolic pathways, wherein ferulic acid is a readily available natural compound widely present in plant cell walls, and contains methoxy and hydroxyl groups in its structure, which is suitable as a precursor for the synthesis of vanillin. Before the production operation, Amycolatopsis pseudomycoticus is mixed and cultured with a recombinant vanillin synthase gene (VpVAN) to obtain a new colony; Strain construction: The gene sequence of vpVAN (GenBank: KP278240.1) was first obtained from NCBI and synthesized; Then, the upstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by specific primers vdhL-F and vdhL-R, and then the downstream sequence of the vanillin dehydrogenase gene (vdh) was amplified by vdhR-F and vdhR-R primers; Then, using seamless cloning technology, the three gene sequences were precisely connected to the plasmid pULcrRNA-km-Cas12, and the plasmid pULΔvdh:vpVAN was successfully constructed; Then, the glycerol bacteria of Amycolatopsis sp. stored in cold storage were taken out; Subsequently, the thawed Amycolatopsis sp. glycerol bacteria were inoculated onto a solid culture medium for activation culture at 30° C. for 3 to 4 days until obvious colony growth was observed; The constructed plasmid pULΔvdh:vpVAN was thoroughly mixed with Amycolatopsis spp. and then electro-transformed; After a single colony grew on the culture medium, colony PCR was performed using primers VDHcheck-F and VDHcheck-R to verify the correct insertion of the gene; After colony verification, the correct strain was confirmed and glycerol was added for long-term storage and named AsvpVAN; Fermentation Catalysis: The AsvpVAN glycerol bacteria stored at -80°C were taken out and thawed naturally at room temperature. After thawing, the strain was inoculated on the surface of the activated solid culture medium and cultured at 30°C for 3 to 4 days until the colonies grew significantly. Subsequently, the seed solution was transferred to the fermentation tank at a 10% inoculation rate; A total of 90 g of ferulic acid solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 to 60 hours.

2. The process for constructing a high-vanillin-producing Ctenobacterium according to claim 1, characterized in that: The cultured strain was inoculated into the activation medium and cultured at 30°C and 220 rpm for 2 days. When the OD value reached 0.7-0.8, the bacteria were collected for subsequent use.

3. The process for constructing a high-vanillin-producing Ctenopharyngeal Acid Bacteria according to claim 1, characterized in that: The transformed cells were added to an activation medium and incubated at 30°C and 220 rpm. The incubated cells were spread on a solid medium containing 50 μg / ml apramycin and cultured at 30°C.

4. The process for constructing a high-vanillin-producing Ctenobacterium according to claim 1, characterized in that: Use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30°C and 220 rpm for 24 to 48 hours until the OD600 value reaches about 7 and microscopic examination shows that the bacteria have good morphology and are free of foreign bacteria.

5. The process for constructing a high-vanillin-producing Ctenobacterium according to claim 1, characterized in that: Taking a 5L fermenter as an example, prepare 3L of fermentation medium, adjust the pH to 7.2, set the temperature to 30°C, set the stirring speed to 500rpm, and control the aeration rate at 1-2vvm. After about 12 to 16 hours of fermentation, the OD600 value should reach the range of 3-4.

6. The process for constructing a high-vanillin-producing Ctenobacterium according to claim 1, characterized in that: Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 5 min, dilute 10 times, filter through 0.22 μM water filter membrane, and analyze by HPLC.

7. According to the construction process of a high-yield vanillin Pseudocanoic acid bacteria as described in claim 1, C18 column, injection 10 μL, column temperature 35°C, mobile phase 30% methanol + 0.1% acetic acid, flow rate 1 mL / min, wavelength 280 nm, detection, and then it was concluded that the vanillin yield was 66.34 g, the concentration was 22.11 g / L, and the molar conversion rate was 94.17%. This method can greatly improve the yield and conversion rate of vanillin.

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