CN113061560A - Genetically engineered bacterium of amycolatopsis as well as construction method and application thereof - Google Patents

Abstract

The invention discloses a genetically engineered bacterium of amycolatopsis as well as a construction method and application thereof, belonging to the technical field of genetic engineering. The invention utilizes CRISPR/Cas9 editing system to knock out genes in Amycolatopsis producing vanillin, the method introduces CRISPR/Cas9 editing plasmid pLYZYP01 containing Vdh gene homologous arm into Amycolatopsis CCTCCM 2011265 to realize Vdh gene knock out, the yield of vanillin reaches 9.19g/L and the molar conversion rate is 97.7 under the condition of adding 12g/L substrate ferulic acid to the obtained genetic engineering strain Amycolatopsis sp. Showing good industrial application value.

Description

Genetically engineered bacterium of amycolatopsis as well as construction method and application thereof

Technical Field

The invention relates to genetically engineered bacteria of amycolatopsis as well as a construction method and application thereof, in particular to genetically engineered bacteria obtained by knocking out a vanillin dehydrogenase Vdh gene in amycolatopsis by using a CRISPR/Cas9 editing system, and belongs to the technical field of genetic engineering.

Background

Vanillin is the main component of vanilla, also called vanillin, with the chemical name 3-methoxy-4-hydroxybenzaldehyde, and has the reputation of "perfume queen". Vanillin is one of the most widely used spices in industry, and is widely used in food, chemical industry, medicine, agriculture and other aspects. In the aspect of food, the product can be widely used as a food additive in high-grade foods such as chocolate, cream, cocoa, ice cream, famous wine and the like; in chemical engineering, it can be used for toothpaste, perfumed soap, perfume and various cosmetics, and is a raw material for preparing various perfumes and cosmetics for sanitary articles; in the aspect of medicine, the derivative can be used as a medicine intermediate for synthesizing L-dopa, L-methyldopa and other medicines; in agriculture, the compound can be used as a yield increasing agent and a ripener, and can also be used for synthesizing herbicides; in addition, it can be used as a bactericide, a defoaming agent, a plating additive, etc.

The worldwide annual demand for vanillin exceeds 16000 t. There are three types of vanillin currently available on the market, namely: (1) natural vanillin extracted from vanilla pods; (2) vanillin produced by chemical synthesis (such as guaiacol method, lignin method, safrole method, eugenol method, p-hydroxybenzaldehyde method, 4-methyl guaiacol method, p-cresol method, etc.); (3) vanillin is produced by a bioconversion method, wherein vanillin produced by microbial conversion using a natural raw material as a substrate by using biotechnology is recognized as natural vanillin by food regulations in Europe and the United states. Because the vanillin extracted from plants is expensive and about 300 times of the synthetic vanillin, the vanillin production by transforming natural substrates with microorganisms becomes a new channel for producing natural vanillin and attracts attention. According to research, only a plurality of strains of Amycolatopsis and Streptomyces can convert a substrate ferulic acid into natural vanillin with higher yield (more than 10g/L) at present, and the method is basically applicable to industrial production. For example, Streptomyces setonii ATCC39116 and Amycolatopsis sp HR167 can convert ferulic acid to produce vanillin, the concentration of vanillin can reach 12g/L and 11.5g/L respectively, the molar conversion rate of substrate ferulic acid is 50-70%, but when two strains of bacteria produce vanillin, byproducts of vanillin, guaiacol and the like are more, and the separation difficulty of products is enlarged.

The CRISPR/Cas9 system is a novel technology developed at present for quickly and simply carrying out site-directed editing on a genome, and can recognize and cut a specific DNA sequence to complete efficient gene editing only by combining a single Cas9 protein with sgRNA. By utilizing the characteristic, the Cas9 system can be successfully combined with an in vitro recombination system to accurately clone the fragment DNA directly from a chromosome. Since the application of the CRISPR/Cas9 technology to gene knockout in 2013, the molecular tool with the advantages of quick construction, simple structure and high efficiency is quickly accepted by people, and is successfully applied to eukaryotes such as rice, wheat, plasmodium, fruit flies, zebra fish, mammals and the like. In eukaryotes, the system is superior to other prior art such as traditional homologous recombination in the aspects of in vivo gene function research of prokaryotes, such as escherichia coli, streptomyces which are important sources of microbial drugs and the like. However, the application of CRISPR/Cas9 in amycolatopsis is currently in the beginning.

M2011265(CN102321563A) is a strain capable of producing vanillin by fermentation with ferulic acid as precursor, but the vanillin dehydrogenase Vdh of the strain can further degrade vanillin to generate vanillic acid. Knocking out the gene coding for vanillin dehydrogenase may improve the production of vanillin, but has many uncertain factors, and researches show that more than one pathway for metabolizing vanillin into vanillic acid in amycolatopsis is provided. Therefore, even if vanillin dehydrogenase is knocked out, vanillic acid may continue to accumulate. Atmospheric room temperature plasma mutagenesis (ARTP) was performed on the strains, and although the molar yield of the strains to the precursor was increased to 72.1%, other byproducts such as vanillic acid were still accumulated. In addition, M2011265 (Amycolatopsis ramosum CCTCC NO) is a wild strain for industrial production of vanillin, and the gene operation research is less, and a set of mature gene editing tools are not developed, so that the gene editing of the Amycolatopsis ramosus is a challenge.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is that when M2011265 takes ferulic acid as a precursor to ferment and produce vanillin, the vanillin dehydrogenase Vdh of the amycolatopsis can further degrade vanillin to generate vanillic acid, thereby reducing the yield of vanillin; the problem of lacking a mature gene editing method is faced when the genetic engineering transformation is carried out on the amycolatopsis CCTCC NO: M2011265.

[ solution ]

The invention provides a method for knocking out a vdh gene of M2011265 of amycolatopsis CCTCC NO based on a CRISPR/Cas9 editing system, which comprises the following steps:

step one, constructing a backbone plasmid pKCHMCs 9Vdh containing Cas9 and sgRNA;

the construction method of the framework plasmid pKCHMCs 9Vdh containing Cas9 and sgRNA comprises the following steps: carrying out double digestion on pKCcas9dO by using Nde I/Hind III to obtain a cas9sco vector; then, taking pKCcas9dO as a template, and carrying out PCR amplification by using primer sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2 to obtain an sg RNA recombinant fragment; then, connecting the Cas9sco vector, the sgRNA recombinant fragment, the Km promoter fragment and the PermE promoter fragment to obtain a CRISPR/Cas9 skeleton plasmid pKCmCas 9 Vdh;

step two, constructing a Vdh gene homology arm;

the construction method of the Vdh gene homology arm comprises the following steps: taking amycolatopsis zhp06 genome as template, respectively amplifying the upstream and downstream homologous arm fragments by using the primer sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 and the primer sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8; recovering the amplified upstream and downstream products by a DNA recovery kit, performing Overlap recombination by using primer sequences shown as SEQ ID NO. 5 and SEQ ID NO. 8, and recovering the products by the DNA recovery kit to obtain a Vdh homologous arm fragment;

step three, editing plasmid pLYZYP01 containing Vdh gene homology arm;

the construction method of the editing plasmid pLYZYP01 containing the Vdh gene homology arm comprises the following steps: linearizing the backbone plasmid pKCHMCs 9Vdh obtained in the first step by using a restriction enzyme; then, integrating the Vdh gene homology arm of the second step into the linearized pCKmCas 9Vdh by using a homologous recombination kit to obtain an editing plasmid pLYZYP01 containing the Vdh gene homology arm;

and step four, electrically converting the editing plasmid pLYZYP01 into amycolatopsis to knock out the Vdh gene.

The invention provides a CRISPR/Cas9 editing plasmid for knocking out a Vdh gene of M2011265 (amycolatopsis CCTCC NO: M2011265), which is a CRISPR/Cas9 editing plasmid pLYZYP01 containing a Vdh gene homology arm, wherein the CRISPR/Cas9 editing plasmid pLYZYP01 is obtained by performing homologous recombination on a backbone plasmid pCKmCas 9Vdh and the Vdh gene homology arm.

The CRISPR/Cas9 editing plasmid pLYZYP01 containing the Vdh gene homologous arm can be used for preparing vanillin high-yield bacteria.

The CRISPR/Cas9 editing plasmid pLYZYP01 containing the Vdh gene homologous arm can be used for knocking genes into the genome of amycolatopsis CCTCC NO: M2011265 or preparing site-directed integration amycolatopsis mutant strains. When used for knocking in a gene, the gene to be knocked in is ligated between the upstream and downstream homology arms of the Vdh gene.

The invention provides a primer pair sequence of sgRNA of a vanillin dehydrogenase Vdh gene of amycolatopsis CCTCC M2011265, which can be effectively edited, and the sequences are shown as SEQ ID NO 1 and SEQ ID NO 2.

The invention provides Amycolatopsis sp.DELTA.vdh of Amycolatopsis gene engineering bacteria with a Vdh gene knocked out, and the bacterial strain reduces the concentration of side product vanillic acid and improves the conversion rate of the vanillic acid to the vanillic aldehyde through the deletion of the vanillic aldehyde dehydrogenase Vdh gene.

[ advantageous effects ]

The CRISPR/Cas9 gene editing system is constructed in the amycolatopsis CCTCC M2011265 for the first time, has the advantages of high integration efficiency and relatively simple genetic operation, and can be used for performing other genetic operations on the amycolatopsis CCTCC M2011265.

The yield of vanillin reaches 9.19g/L and the molar conversion rate is increased from 88.6% to 97.7% under the condition that 12g/L substrate ferulic acid is added to the Amycolatopsis sp.

Drawings

FIG. 1 is a schematic structural diagram of pKCcas9dO plasmid.

FIG. 2 is a schematic diagram of the structure of pKCHMCs 9Vdh backbone plasmid.

FIG. 3 is a schematic structural diagram of CRISPR/Cas9 editing plasmid pLYZYP 01.

FIG. 4 is a PCR-verified plot of the knockout strain of example 5; wherein, lanes 1-12 of panel A are pKCcas9dO plasmid; lanes 13-24 are positive knock-out strains; b lanes 1-12 of the drawings are original strains of Amycolatopsis zhp 06; lane M is 5kb DNA Ladder marker (5kb DNA molecular weight gradient scale).

FIG. 5 production amounts of vanillin (A), vanillic acid (B) and consumption amount of substrate ferulic acid (C) of the original strain and the mutant strain.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Reagents and plasmid sources used in the following examples:

1. the amycolatopsis zhp06 is preserved in China center for type culture Collection of microorganisms, with the preservation number being CCTCC NO: m2011265;

2. coli JM110 and pKCcas9dO are commercially available products. The structure of pKCas 9dO plasmid is shown in FIG. 1.

Example 1 knocking out Vanillin dehydrogenase Vdh Gene in Amycolatopsis by Using CRISPR/Cas9 editing System

1. Construction and verification of plasmid pKCHMCs 9Vdh

The first step,

The cas9sco vector is obtained by Nde I/Hind III double digestion of pKCcas9dO, and the digestion system is as follows: mu.L of plasmid, 1. mu.L of each of Nde I and Hind III endonuclease, 1. mu.L of Buffer, and 10. mu.L of water supplemented, and reacted in a water bath at 37 ℃ for 30 min.

Step two, obtaining of sgRNA recombinant fragment

The sgRNA was designed using the vanillin dehydrogenase Vdh gene of amycolatopsis zhp06 as the editing target, pKCcas9dO as the template, and primers P1 and P2 were used to perform PCR amplification to obtain the sg RNA recombinant fragment (SEQ ID NO:11) with a fragment size of 127bp, which contained the target gene (Vdh) sequence of 20 bp.

P1:CGCCTCGCCCCAGGACCTGGCGTTTTAGAGCTAGAAATAGCAAGT

(SEQ ID NO:1)

P2:CGACGGCCAGTGCCAAGCTTCTCAAAAAAAGCACCGACTC

(SEQ ID NO:2)

The PCR reaction system is as follows: PrimeSTAR Max DNA Polymerase 25. mu.l, P1 and P2 each 1. mu.L, pKCcas9dO 1. mu.L, dd H₂O was added to 50. mu.L.

The PCR reaction program is: hot starting at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 s; the annealing temperature is 55 ℃ and 5 s; extension at 72 ℃ the efficiency of PrimeSTAR Max DNA Polymerase enzyme system was: 5s/kb, extension 5 s. The three steps of denaturation, annealing and extension are 30 cycles. Finally, the extension is carried out at 72 ℃ for 10min and the mixture is placed at 4 ℃.

Step three, obtaining Km promoter

Synthesizing a Km promoter, wherein the fragment size is 177bp, and the sequence is shown as SEQ ID NO 3;

step four, obtaining PermE promoter

Synthesizing a PermE promoter, wherein the size of a fragment is 310bp, and the sequence is shown as SEQ ID NO. 4;

step five, construction of CRISPR/Cas9 skeleton plasmid pKCHMCs 9Vdh

The sgRNA recombinant fragment and the PermE promoter fragment were subjected to Overlap (Overlap extension PCR) recombination, and the obtained fragment and the Km promoter fragment were subjected to Overlap recombination again. Then, the obtained fusion fragment is integrated into a Cas9sco vector through one-step cloning to obtain a CRISPR/Cas9 backbone plasmid pKCKmCas9Vdh, and the structural schematic diagram is shown in fig. 2. And transforming the ligation product, introducing a competent cell JM109, selecting a monoclonal antibody in an LB liquid culture medium, carrying out overnight culture at 30 ℃, and extracting a plasmid for later use after verification.

Overlap recombinant PCR program: the primer uses the F primer of the upstream fragment and the R primer of the downstream fragment, and the extension time t is the time required by connecting the two fragments into a large fragment. The template is a mixture of upstream and downstream fragments. The rest is the same as the ordinary PCR procedure.

One-step cloning method the fragment (0.06pmol) and vector (0.03pmol) were mixed in the same PCR tube and 2-4. mu.L of the mixture was addedHistone buffer and 1-2. mu.L of recombinase, plus ddH₂The reaction was carried out at 37 ℃ for 30min while adding 10 to 20. mu.L of O.

2. Construction of homologous arm of CRISPR/Cas9 gene editing system

Step one, amplifying upstream and downstream homologous arm fragments by using a genome of amycolatopsis zhp06 as a template and respectively using P5, P6, P7 and P8;

P7:GAGTCGGTGCTTTTTTTGAGAGCTCGTCCCACGACG(SEQ ID NO:5)

P8:GGTCGTTGATGTCCACTTCTCGTCGTCGAG(SEQ ID NO:6)

P9:AGAAGTGGACATCAACGACCAGACGGTCAAC(SEQ ID NO:7)

P10:CGACGGCCAGTGCCAAGCTTTGCGAGCACCTGGAGAAGG(SEQ ID NO:8)

and step two, recovering the amplified upstream and downstream products by a DNA recovery kit, and performing Overlap recombination by using P7 and P10 and then recovering the products.

3. Construction of CRISPR/Cas9 editing plasmid containing Vdh gene homology arm

The Vdh gene homology arm obtained in the previous step is integrated into pKCHMCs 9Vdh by one-step cloning to obtain an editing plasmid containing the Vdh gene homology arm. The specific operation is as follows:

step one, carrying out enzyme digestion linearization on pKCHMCs 9Vdh framework plasmid by using Hind III;

step two, connecting the linearized pKCHMCs 9Vdh framework plasmid obtained in the step one with the Vdh gene homology arm obtained in the example 2 through one-step cloning

And step three, after the ligation reaction is finished, uniformly mixing all the products with 100 mu l of JM109 competent cells by blowing, converting by a conventional method, coating on an LB solid plate containing corresponding resistance, and carrying out inversion overnight culture at 30 ℃. The correct plasmid is the CRISPR/Cas9 editing plasmid pLYZYP01, and the structural schematic diagram is shown in figure 3.

4. Electrotransformation of CRISPR/Cas9 editing plasmid into Amycolatopsis zhp06

Step one, converting a CRISPR/Cas9 editing plasmid pLYZYP01 into E.coli JM110 for demethylation;

and step two, taking 2-5 mu g of demethylated plasmid into an EP tube which is precooled by ice and is 1.5ml, adding 60-100 mu L of amycolatopsis zhp06 competent cells, blowing, sucking and mixing uniformly, and immediately putting into a precooled electric rotor cup. And (3) electrotransformation conditions are as follows: 7.5-1.25kV/cm, and the time length is 5 milliseconds.

And step three, after electrotransformation, resuspending the thalli by using 1mL of TSB, transferring the thalli into a 15mL glass test tube, and carrying out shake culture at 28 ℃ for 5-8 hours.

And step four, coating 100 mu L of bacterial liquid on a bennet plate culture medium containing corresponding antibiotics, performing centrifugal concentration on the residual bacterial liquid, coating the residual bacterial liquid on a corresponding plate, and culturing for 5-8 days at 28 ℃.

5. Elimination and verification of CRISPR/Cas9 editing plasmid in positive strain

A plurality of transformants were selected and cultured in 3mL of TSB liquid medium containing Apr resistance at 28 ℃ for 2-3 days. After the transformant bacterial liquid is subjected to streaking of single colonies on a nonreactive Bunsen cell culture medium at 37 ℃ for overnight culture, selecting a single colony control and coating the single colony control on a nonreactive Bunsen cell culture medium containing Apr, and culturing the single colony control at 28 ℃ for 2-3 days, wherein the single colony which normally grows on the nonreactive plate and does not grow on the resistant plate is the positive bacterial strain which successfully eliminates the plasmid. The results are shown in FIG. 4, and the following verification primers are used:

P11:AGCTCGTCCCACGACGGTAGCAAC(SEQ ID NO:9)

P12:TGCGAGCACCTGGAGAAGGCACG(SEQ ID NO:10)

the experimental results are as follows: PCR was performed with primers P11 and P12, and a band of 1000bp was obtained for positive transformants and a band of 2500bp was obtained for negative controls. The sequencing verification proves that the 1000bp band is the upstream and downstream homologous arms of the Vdh gene.

And (4) conclusion: a correctly edited positive strain Amycolatopsis sp.

Example 2 analysis of fermentation product of Amycolatopsis sp. Δ vdh, an engineered Amycolatopsis pseudoamycolatopsis gene

The seed culture medium formula comprises: 5g of glucose, 10g of yeast extract and Na₂HPO₄ 4g，KH₂PO₄ 1g，NaCl 0.2g，MgSO₄·7H₂O 0.2g，CaCl₂·2H₂0.05g of O and 1000mL of distilled water, and the pH is adjusted to 7.2;

the formula of the culture medium for transforming the growing cells comprises the following components: grapeSugar 50g, yeast extract 1g, Na₂HPO₄ 4g，KH₂PO₄ 0.1g，NaCl 0.2g，MgSO₄ 0.2g，CaCl₂0.05g, 1000mL of distilled water, pH adjusted to 7.2.

The Amycolatopsis sp. delta. vdh mutant strain and the original strain are respectively inoculated in a seed culture medium and cultured in a constant temperature shaking table at 30 ℃ and 180r/min for 36 h. Inoculating a growth cell transformation medium with an inoculation amount of 8%, culturing in a constant-temperature shaking table at 30 ℃ and 180r/min, adding a substrate ferulic acid with a final concentration of 12g/L after 20h, simultaneously increasing the culture temperature to 35 ℃, sampling every 6h after 12h, and carrying out quantitative analysis (HPLC), wherein the fermentation process is shown in figure 5, the substrate ferulic acid with the concentration of 12g/L is added for conversion for 72h, the ferulic acid is basically consumed completely, and the vanillin yield of the original strain and the mutant strain Amycolatopsis sp.delta vdh is 8.32g/L and 9.02g/L respectively; when the vanillic acid is concentrated for 84 hours, 222mg/L and 200mg/L are respectively obtained. The vanillin concentration of the mutant strain product is increased by 10.3%, the byproduct concentration is reduced by 9.4%, and the molar conversion rate is increased from 88.6% to 97.7%.

Comparative example 1 sgRNA without target gene sequence failed to knock down vdh gene

In the second construction and verification step of plasmid pKCcAs 9Vdh of example 1, PCR amplification was performed using pKCcas9dO as a template and primers P2(SEQ ID NO:2) and P13(SEQ ID NO:14) to obtain a recombinant sgRNA fragment without the target gene sequence, and the rest was performed as in example 1. Finally, it was found by genomic PCR verification that a strain in which the vdh gene was knocked out could not be obtained.

P2:CGACGGCCAGTGCCAAGCTTCTCAAAAAAAGCACCGACTC

(SEQ ID NO:2)

P13:TGGTAGGATCCGTTTTAGAGCTAGAAATAGCAAGT

(SEQ ID NO:12)

Comparative example 2 recombinant plasmid not containing homology arm of vdh Gene failed to knock out the vdh Gene

In example 1, the backbone plasmid pKCKmCas9Vdh without homology arms was directly transformed into amycolatopsis, and the rest of the procedure was performed as in example 1. Finally, it was found by genomic PCR verification that a strain in which the vdh gene was knocked out could not be obtained.

SEQ ID NO.1：cgcctcgccc caggacctgg cgttttagag ctagaaatag caagt 45

SEQ ID NO.2：cgacggccag tgccaagctt ctcaaaaaaa gcaccgactc 40

SEQ ID NO.3：

GCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCCCCGGGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGC

SEQ ID NO.4：

CCAGCCCGACCCGAGCACGCGCCGGCACGCCTGGTCGATGTCGGACCGGAGTTCGAGGTACGCGGCTTGCAGGTCCAGGAAGGGGACGTCCATGCGAGTGTCCGTTCGAGTGGCGGCTTGCGCCCGATGCTAGTCGCGGTTGATCGGCGATCGCAGGTGCACGCGGTCGATCTTGACGGCTGGCGAGAGGTGCGGGGAGGATCTGACCGACGCGGTCCACACGTGGCACCGCGATGCTGTTGTGGGCACAATCGTGCCGGTTGGTAGGATC

SEQ ID NO.5：gagtcggtgc tttttttgag agctcgtccc acgacg 36

SEQ ID NO.6：ggtcgttgat gtccacttct cgtcgtcgag 30

SEQ ID NO.7：agaagtggac atcaacgacc agacggtcaa c 31

SEQ ID NO.8：cgacggccag tgccaagctt tgcgagcacc tggagaagg 39

SEQ ID NO.9：agctcgtccc acgacggtag caac 24

SEQ ID NO.10：tgcgagcacc tggagaaggc acg 23

SEQ ID NO.11：cgcctcgccc caggacctgg cgttttagag ctagaaatag caagttaaaa taaggctagt 60

ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgagaag cttggcactg 120

gccgtcg127

SEQ ID NO:12：tggtaggatc cgttttagag ctagaaatag caagt 35

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims (10)

1. An editing plasmid based on a CRISPR/Cas9 editing system, which is prepared by the following steps:

carrying out double digestion on pKCcas9dO by using Nde I/Hind III to obtain a cas9sco vector;

connecting a Cas9sco vector, an sgRNA recombinant fragment, a Km promoter fragment and a PermE promoter fragment to obtain a CRISPR/Cas9 skeleton plasmid pKCmCas 9 Vdh;

the Vdh gene homology arm was integrated into the linearized pKCKmCas9Vdh to obtain an editing plasmid containing the Vdh gene homology arm.

2. The editing plasmid based on the CRISPR/Cas9 editing system according to claim 1, wherein the sgRNA recombinant fragment is obtained by performing PCR amplification by using primer sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 and pKCcas9dO as a template.

3. The editing plasmid based on CRISPR/Cas9 editing system according to claim 1, wherein the sequence of Km promoter fragment is shown as SEQ ID NO. 3.

4. The editing plasmid based on CRISPR/Cas9 editing system according to claim 1, wherein the sequence of the PermE promoter fragment is shown in SEQ ID NO. 4.

5. The editing plasmid based on CRISPR/Cas9 editing system according to any one of claims 1-4, wherein the vdh gene is derived from Amycolatopsis CCTCC NO: M2011265.

6. The editing plasmid based on CRISPR/Cas9 editing system according to claim 5, wherein the vdh gene homology arm uses Amycolatopsis CCTCC NO: M2011265 genome as a template, and the primer sequences shown as SEQ ID NO:5 and SEQ ID NO:6, respectively, are used for amplifying the upstream and downstream homology arm fragments.

7. The method for knocking out the vdh gene of amycolatopsis CCTCC NO: M2011265 as claimed in claim 5, wherein the method comprises electrically transforming the editing plasmid into amycolatopsis to knock out the vdh gene and then eliminating the editing plasmid.

8. Use of a plasmid according to any one of claims 1 to 6 for the knock-out or knock-in of an amycolatopsis species.

9. The amycolatopsis gene engineering bacterium is characterized in that the vdh gene in the amycolatopsis CCTCC M2011265 genome is knocked out.

10. Use of the plasmid according to any one of claims 1 to 6 for the preparation of a vanillin-producing bacterium.

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