CN117417947A

CN117417947A - Recombinant escherichia coli for producing 2, 5-dimethylpyrazine and application thereof

Info

Publication number: CN117417947A
Application number: CN202311365400.8A
Authority: CN
Inventors: 罗玮; 曾明曦
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-10-20
Filing date: 2023-10-20
Publication date: 2024-01-19

Abstract

The invention discloses recombinant escherichia coli for producing 2, 5-dimethyl pyrazine and application thereof, and belongs to the technical field of genetic engineering. The invention uses CRISPR/Cas9 gene editing system, uses escherichia coli TWF001 as an initial strain, and obtains 2, 5-dimethyl pyrazine recombinant escherichia coli capable of stably expressing in industrial fermentation by knocking out or over-expressing related genes in a 2, 5-dimethyl pyrazine metabolic pathway which uses glucose as a substrate. The recombinant escherichia coli provided by the invention is stable in heredity, mild in fermentation condition and low in cost, can be applied to the food and medicine industries, and has a wide application prospect in industrial production.

Description

Recombinant escherichia coli for producing 2, 5-dimethylpyrazine and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to recombinant escherichia coli for producing 2, 5-dimethyl pyrazine and application thereof.

Background

Alkylpyrazines are a class of flavor compounds that are widely found in traditional fermented and heat treated foods, and the importance of alkylpyrazines to the food industry is expected to continue to increase in the coming years. Chemical synthesis is currently the primary means of synthesizing alkylpyrazine, however with increasing awareness of food sources, consumers are no longer satisfied with chemically synthesized products, but prefer "natural" products synthesized by biotechnology. Accordingly, a process for synthesizing alkylpyrazine by biotechnology has received increasing attention. 2, 5-dimethyl pyrazine (2, 5-DMP) is an important alkyl pyrazine with strong peanut flavor and chocolate, creamy flavor, and can be used as food flavor. In addition, the 2,5-DMP has important application value in the field of medicine, for example, the 2,5-DMP is a substrate for synthesizing the hypoglycemic glipizide and the lipid-lowering drug acipimox. In recent years, biosynthesis of 2,5-DMP has been increasingly focused.

In fact, researchers have obtained 2,5-DMP by microbial fermentation as early as 1997 and demonstrated that 2,5-DMP is derived from L-threonine. However, until 2019, the complete biosynthetic pathway from L-threonine to 2,5-DMP was not successfully resolved, involving a one-step enzymatic reaction and a three-step spontaneous reaction. At present, the optimization of 2, 5-dimethyl pyrazine recombinant engineering bacteria is expressed as over-expression of key enzymes in metabolic pathways, or a brand-new efficient pathway is constructed, the influence of the balance between the supply of precursor L-threonine and the conversion rate of L-threonine on 2, 5-dimethyl pyrazine fermentation is not focused, the substrate utilization rate in the metabolic process is low, the product yield is difficult to improve, and the like are difficult to solve all the time.

Disclosure of Invention

In order to solve the technical problems, a CRISPR/Cas9 gene editing system is adopted, partial genes related to a 2, 5-dimethylpyrazine metabolic pathway in the escherichia coli TWF001 for producing the L-threonine are knocked out or overexpressed, the outward emission of the L-threonine is weakened, the intake of the L-threonine is enhanced, and the like, so that the relation between the supply of the L-threonine and the conversion rate of the L-threonine is balanced by the strategy, the extracellular accumulation amount of the L-threonine is reduced to a very trace level, the accumulation of the 2, 5-dimethylpyrazine is greatly promoted, and the recombinant escherichia coli for producing the 2, 5-dimethylpyrazine in high yield is obtained. The fermentation quantity of the recombinant bacteria is improved, the genetically modified bacteria can be stably inherited in the fermentation production process, antibiotics or inducers are not required to be added, and the fermentation and production cost is saved.

It is a first object of the present invention to provideRecombinant escherichia coli for producing 2, 5-dimethyl pyrazine and overexpression of threonine dehydrogenase gene tdh and amino acetone oxidase gene aao in escherichia coli genome _So Cofactor NAD ⁺ Synthase gene pncB and nadE, NADH oxidase gene Smnox, feedback inhibition resistance gene lysC ^fbr And thrA ^fbr BC and threonine uptake protein gene sstT, knockout DNA binding transcription repressor gene lacI, 2-amino-3-ketobutyrate CoA ligase gene kbl, glyoxylate pathway repressor gene iclR and threonine exosystem gene rhtA.

Wherein the trc promoter is used to control aao _So 、pncB、nadE、Smnox、lysC ^fbr 、thrA ^fbr Expression of BC and sstT genes, double copy expression of tdh gene was performed using trc promoter, and double copy expression of tdh gene was performed using lpp promoter.

Further, the nucleotide sequence of the tdh gene is shown as SEQ ID NO.1, aao _So The nucleotide sequence of the gene is shown as SEQ ID NO.2, the nucleotide sequence of the pncB gene is shown as SEQ ID NO.3, the nucleotide sequence of the nadE gene is shown as SEQ ID NO.4, the nucleotide sequence of the Smnox gene is shown as SEQ ID NO.5, lysC ^fbr The nucleotide sequence of the gene is shown as SEQ ID NO.6, thrA ^fbr The nucleotide sequence of the BC gene is shown as SEQ ID NO.7, and the nucleotide sequence of the sstT gene is shown as SEQ ID NO. 8.

Further, the nucleotide sequence of the lacI gene is shown as SEQ ID NO.9, the nucleotide sequence of the kbl gene is shown as SEQ ID NO.10, the nucleotide sequence of the iclR gene is shown as SEQ ID NO.11, and the nucleotide sequence of the rhtA gene is shown as SEQ ID NO. 12.

Further, the nucleotide sequence of the trc promoter is shown as SEQ ID NO.13, and the nucleotide sequence of the lpp promoter is shown as SEQ ID NO. 14.

Further, gene editing was performed on E.coli using the CRISPR/Cas9 system.

Further, the recombinant E.coli is a host of E.coli TWF 001.

It is a second object of the present invention to provide a microbial preparation comprising recombinant E.coli.

The third object of the present invention is to provide a method for producing 2, 5-dimethylpyrazine using glucose as a substrate, and using the recombinant E.coli or the microbial agent as described above for fermentation production.

Further, the escherichia coli or the microbial preparation is added into a reaction system, dissolved oxygen is controlled to be 25-35%, and glucose concentration is controlled to be 1-3g/L.

A fourth object of the present invention is to provide the use of the recombinant E.coli or the microbial preparation described above in the food industry or pharmaceutical synthesis.

The invention has the beneficial effects that:

the invention utilizes a CRISPR/Cas9 gene editing system to carry out a series of metabolic engineering transformation on the genome of escherichia coli TWF001, thus obtaining a genetic engineering strain capable of fermenting glucose to produce 2,5-DMP with high yield, and the strain strengthens the uptake of L-threonine by optimizing the copy number and the expression strength of tdh isogenic genes on chromosomes, balances the relation between the supply of the L-threonine and the conversion rate of the L-threonine and greatly promotes the accumulation of 2, 5-dimethyl pyrazine. Antibiotics and inducers are not needed to be added in the culture process, so that the fermentation cost is saved, the industrial practical application is facilitated, and the genetic stability of the recombinant bacteria in the fermentation process is maintained.

Drawings

FIG. 1 is a biological pathway for the synthesis of 2, 5-dimethylpyrazine using glucose as a substrate;

FIG. 2 shows the results of shake flask fermentation of recombinant E.coli in examples 1-4;

FIG. 3 shows the results of shake flask fermentation of recombinant E.coli in examples 4-7;

FIG. 4 is the recombinant E.coli NADH and NAD in examples 4-7 ⁺ Content and NADH/NAD ⁺ Ratio of;

FIG. 5 shows the results of shake flask fermentation of recombinant E.coli of examples 7-10;

FIG. 6 shows the results of shake flask fermentation of recombinant E.coli of examples 11-14;

FIG. 7 is the result of fed-batch fermentation in a 5L fermenter of recombinant E.coli in example 16;

FIG. 8 is P in comparative example 1 _fliC Effect of promoter on fermentation yield of recombinant E.coli.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Coli TWF001 is published in Combined metabolic analyses for the biosynthesis pathway of L-threonine in Escherichia coli (Frontiers in bioengineering and biotechnology, 2022).

Detection of 2, 5-DMP: 2,5-DMP was detected by High Performance Liquid Chromatography (HPLC) equipped with an Agilent 5HC-C18 column (Agilent, CA, USA). Mobile phase a (acetonitrile: water=28:72) containing 0.1% trifluoroacetic acid was used, elution gradient: 0-10min,100% A, running time of 10min, flow rate of 0.8mL/min, ultraviolet detection wavelength of 275nm, column temperature of 25deg.C, and loading amount of 10 μl.

Detection of NAD+/NADH: coenzymeI NAD (H) Content Assay Kit (Solarbio, beijing, china) kit.

LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, 2% agar powder added to the solid medium, and sterilized at 121℃for 20min. Resistant kanamycin (final concentration 50. Mu.g/mL) and spectinomycin (final concentration 50. Mu.g/mL) were added as needed.

Example 1: construction of lacI Gene-deleted Strain

(1) Preparation of electrotransformation competence: first, plasmid pEcCas was chemically transformed into E.coli TWF001, plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) and positive transformants were selected, inoculated in LB liquid medium containing 10mM arabinose, and cultured at 37℃to OD ₆₀₀ After 0.6-0.8, the preparation of electrotransformation competent cells was started.

(2) Construction of plasmid pEcgRNA-lacI: the plasmid pEcgRNA is used as a template, a primer lacI-xx-S/lacI-xx-A is used for amplification to obtain a linearized plasmid pEcgRNA-lacI, and the linearized plasmid pEcgRNA-lacI is transformed into E.coli JM109 competent cells to obtain the plasmid pEcgRNA-lacI with a supercoiled structure.

(3) Construction of donor DNA: the upstream and downstream homology arms were amplified using E.coli genome as template and primers UP-lacI-S/UP-lacI-A and DN-lacI-S/DN-lacI-A and donor DNA was obtained by fusion PCR.

(4) Electric conversion: plasmid pEcgRNA-lacI and donor DNA were co-transformed into E.coli TWF001 electrotransformation competent containing plasmid pEcCas, resuscitated in LB medium at 37℃for 2h, and cells were plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) and spectinomycin (final concentration 50. Mu.g/mL), and positive transformants were identified using primers lacI-JD-S/lacI-JD-A for sequencing.

(5) Elimination of plasmid pEcgRNA-lacI: the correct transformants were cultured in LB medium containing rhamnose (10 mM) and kanamycin (50. Mu.g/mL) at 37℃for 12 hours, and then plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) for overnight culture to give E.coli recombinants without plasmid pEcgRNA-lacI.

(6) Elimination of plasmid pEcCas: the correct transformant was cultured in LB medium containing glucose (5 g/L) at 37℃for 12 hours, then spread on LB plate containing glucose (5 g/L) and sucrose (10 g/L) at 37℃for 12 hours, and finally a plasmid-free E.coli recombinant strain designated TWF001.DELTA.lacI was obtained.

TABLE 1 primer sequences

Example 2: construction of tdh Gene overexpression Strain

(1) Preparation of electrotransformation competence: first, plasmid pEcCas was chemically transformed into TWF001.DELTA.lacI, plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) and positive transformants were selected and inoculated in LB liquid medium containing 10mM arabinose and cultured at 37℃to OD ₆₀₀ After 0.6-0.8, starting to prepare electrotransformation competenceAnd (3) cells.

(2) Construction of plasmid pEcgRNA-yjiV: the plasmid pEcgRNA is used as a template, and a primer yjiV-xx-S/yjiV-xx-A is used for amplification to obtain a linearized plasmid pEcgRNA-yjiV, and then the linearized plasmid pEcgRNA-yjiV is transformed into E.coli JM109 competent cells to obtain the plasmid pEcgRNA-yjiV with a supercoiled structure.

(3) Construction of donor DNA: the E.coli genome was used as Sup>A template, the primers UP-yjiV-S/UP-yjiV-A and DN-yjiV-S/DN-yjiV-A were used to amplify the upstream and downstream homology arms, primers tdh-S and tdh-A were used to amplify the trc-tdh expression cassette, and finally the upstream and downstream homology arms and trc-tdh expression cassette were used as templates, and primers UP-yjiV-S and DN-yjiV-A were used to amplify the donor DNA.

(4) Electric conversion: plasmid pEcgRNA-yjiV and donor DNA were co-transformed into TWF 001. DELTA. LacI electrotransformation competent containing plasmid pEcCas, resuscitated in LB medium at 37℃for 2h, and cells were plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) and spectinomycin (final concentration 50. Mu.g/mL), and positive transformants were identified for sequencing using primers yjiV-in-S/yjiV-in-A.

(5) Elimination of plasmid pEcgRNA-yjiV: the correct transformants were cultured in LB medium containing rhamnose (10 mM) and kanamycin (50. Mu.g/mL) at 37℃for 12 hours, and then plated on LB plates containing kanamycin (final concentration 50. Mu.g/mL) for overnight culture to give E.coli recombinants without plasmid pEcgRNA-yjiV.

(6) Elimination of plasmid pEcCas: the correct transformant was cultured in LB medium containing glucose (5 g/L) at 37℃for 12 hours, then spread on LB plate containing glucose (5 g/L) and sucrose (10 g/L) at 37℃for 12 hours, and finally a recombinant E.coli strain containing no plasmid was obtained and designated as D1.

TABLE 2 primer sequences

Example 3: construction of tdh Gene double copy and kbl deletion Strain

The trc-tdh expression cassette was integrated at position kbl on its genome using the method of example 2, starting with D1. The obtained strain was designated as D2.

TABLE 3 primer sequences

Example 4: construction aao _So Gene overexpression strain

Using the method of example 2, D2 was the starting strain, and trc-aao was integrated at yafU site on its genome _So Expression cassette. The obtained strain was designated as D3.

TABLE 4 primer sequences

Example 5: construction of pncB Gene overexpression Strain

Using the method of example 2, D3 was the starting strain and the trc-pncB expression cassette was integrated at the yeeP site on its genome. The obtained strain was designated as D4.

TABLE 5 primer sequences

Example 6: construction of nadE Gene overexpression Strain

Using the procedure of example 2, D4 was the starting strain, integrating the trc-nadE expression cassette at the mazG site on its genome. The obtained strain was designated as D5.

TABLE 6 primer sequences

Example 7: construction of Smnox Gene overexpression Strain

Using the method of example 2, D5 was the starting strain, and the trc-Smnox expression cassette was integrated at the yjhV site on its genome. The obtained strain was designated as D6.

TABLE 7 primer sequences

Example 8: construction of lysC ^fbr Gene overexpression strain

By the method of example 2, D6 was the starting strain, and trc-lysC was integrated at the yncI site on its genome ^fbr Expression cassette in which lysC is an anti-feedback-inhibition mutant of lysC ^fbr Is obtained by mutating the 1055 th base C of lysC to T. The obtained strain was designated as D7.

TABLE 8 primer sequences

Example 9: construction of thrA ^fbr BC gene over-expression strain

By the method of example 2, D7 is the starting strain, with trc-thrA integrated at the ltaE site on its genome ^fbr BC expression cassette wherein the anti-feedback inhibition mutant thrA of thrA ^fbr Is obtained by mutating the 1034 th base C of thrA to T. The obtained strain was designated as D8.

TABLE 9 primer sequences

Example 10: construction of iclR Gene deletion Strain

Using the procedure of example 1, D8 was the starting strain, and the iclR gene was knocked out on its genome. The obtained strain was designated as D9.

TABLE 10 primer sequences

Example 11: construction of lpp-tdh Gene overexpression Strain

Using the method of example 2, D9 was the starting strain, and the lpp-tdh expression cassette was integrated at the ykgP site on its genome. The obtained strain was designated as D13.

TABLE 11 primer sequences

Example 12: construction of double-copy lpp-tdh Gene overexpression Strain

Using the method of example 2, D13 was the starting strain, and the lfhA site on its genome was integrated with the lpp-tdh expression cassette, and the resulting strain was designated as D14.

TABLE 12 primer sequences

Example 13: construction of three-copy lpp-tdh Gene overexpression Strain

Using the method of example 2, D14 was the starting strain, and the lpp-tdh expression cassette was integrated at the ykiA site on its genome, and the obtained strain was designated as D15.

TABLE 13 primer sequences

Example 14: construction of sstT Gene overexpression and rhtA Gene deletion Strain

Using the procedure of example 2, D14 was the starting strain, integrating the trc-sstT expression cassette at the rhtA site on its genome. The obtained strain was designated as D19.

TABLE 14 primer sequences

Example 15: 2,5-DMP produced by shake flask fermentation

The formula of the culture medium is as follows:

seed medium (LB): 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract.

Fermentation medium: 20g/L glucose, 3g/L (NH) ₄ ) ₂ SO ₄ 0.9g/L KCl,2g/L citric acid, 0.5g/L betaine, 0.8g/L MgSO ₄ ·7H ₂ O,1.8g/L phosphoric acid, 20mg/L FeSO ₄ ·7H ₂ O，20mg/L MnSO ₄ ·H ₂ O,2% phenol red, and the pH value is adjusted to 7.

The recombinant strains prepared in examples 1 to 13 were taken out from the-80℃refrigerator and inoculated into LB plates, respectively, for overnight culture at 37 ℃. A loop of strain was then removed from the plate and inoculated into a tube containing 5mL of seed medium and incubated at 37℃for 8h. Finally, the culture was further carried out in 500mL Erlenmeyer flasks containing 45mL of fermentation medium at 37℃for 48h at an inoculum size of 10%. During the incubation, 25% ammonia (v/v) was added to maintain the pH depending on the color of the broth.

As shown in FIG. 2, D1 accumulated 52.89mg/L of 2,5-DMP, and its growth was also improved compared to TWF001.DELTA.lacI, indicating that the presence of the tdh gene confers the ability to synthesize 2,5-DMP to E.coli. The simultaneous knockout of kbl gene and overexpression of tdh gene (D2) increased the yield to 173.35mg/L. Overexpression aao _So The gene further improves the yield by 28.8%, up to 223.29mg/L.

As shown in FIG. 3, overexpression of pncB (D4) alone increased 2,5-DMP production to 347.7mg/L compared to D3 by 55.72%, while overexpression of both pncB and nadE genes increased 2,5-DMP production to 423.31mg/L compared to D3 by 89.58%. Indicating that overexpression of the pncB and nadE genes can be achieved by increasing NAD ⁺ To improve 2,5-DMP production. In addition, further overexpression of the NADH oxidase-encoding gene Smnox (D6) increased 2,5-DMP production to 477.7mg/L.

As shown in FIG. 4, overexpression of pncB (D4) alone caused NAD ⁺ 60.67% increase in intracellular content while simultaneously overexpressing the pncB and nadE genes (D5) to NAD ⁺ Further increased by 105.8%, indicating that overexpression of both the pncB and nadE genes increases intracellular NAD ⁺ The content is as follows. In addition, further overexpression of NADH oxidase encoding gene Smnox (D6) significantly increases intracellular NAD ⁺ The level increased by 37% compared to D5.

As shown in FIG. 5, lysC was overexpressed ^fbr Gene (D7) did not have a significant effect on the yield of 2,5-DMP. Overexpression of thrA ^fbr Both the BC gene cluster (D8) and the knockout iclR gene (D9) promote 2,5-DMP accumulation. Strain D9 accumulated 560.5mg/L of 2,5-DMP, which was 17.3% higher than strain D6.

As shown in FIG. 6, a single copy P _lpp The yield of tdh strain D13 reaches 929.8mg/L, double copy P _lpp Strain D14 of tdh further increased the yield to 1064.3mg/L, however with three copies of P _lpp Strain D15 of tdh did not continue to increase yield, indicating double copy P _lpp TDH has met the TDH requirement for 2,5-DMP production. Further, the rhtA gene is knocked out and the sstT gene (D19) is overexpressed on the basis of D14, so that the yield of 2,5-DMP is increased to 1589.1mg/L, and the extracellular accumulation of L-threonine is reduced to a very trace level, which shows that the production performance of 2,5-DMP of the strain is greatly improved by optimizing tdh gene expression and modifying the relation between L-threonine supply and L-threonine conversion rate in combination with an L-threonine transport system strategy.

Example 16: fed-batch fermentation production of 2,5-DMP in 5L fermenter

The formula of the culture medium is as follows:

fermentation tank medium: 80g/L glucose, 3g/L yeast extract, 0.5g/L betaine, 2g/LKH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ ，0.5g/L MgSO ₄ ·7H ₂ O，5mg/L FeSO ₄ ·7H ₂ O，5mg/LMnSO ₄ ·H ₂ O, pH was adjusted to 7.

Strain D19 was first removed from the-80℃refrigerator and inoculated into LB plates for overnight culture at 37 ℃. A loop of strain was then removed from the plate and inoculated into a tube containing 5mL of LB medium for overnight culture at 37 ℃. Then, 5mL of the whole bacterial liquid was added to a 500mL Erlenmeyer flask containing 45mL of the seed medium, and the whole bacterial liquid was cultured at 37℃for 12 hours. Finally, the culture is carried out for 48 hours at 37 ℃ in a 5L bioreactor containing 2L of fermentation culture medium with 10 percent of inoculation amount. In the fermentation process, the pH value is maintained at 7 by feeding 25% ammonia water (v/v), dissolved oxygen is maintained at 25-35% by adjusting stirring speed and ventilation, and after the initial glucose in the fermentation medium is exhausted, 60% glucose solution (w/v) is automatically fed, so that the glucose concentration is maintained at 0-3g/L.

As shown in FIG. 7, the biomass of D19 reached a maximum of 54.6 (OD at 24h ₆₀₀ ) The gradual decrease starts thereafter, and the titer of 2,5-DMP reaches a peak value of 3.1g/L at 36h, with a production intensity of 2.1 g/(L.d), which is the highest yield of 2,5-DMP produced by fermenting glucose that has been reported.

Comparative example 1: by means of the promoter P _fliC Regulation of L-threonine supply

To further improve the supply of L-threonine, the synthetic pathway of L-lysine, L-methionine, L-isoleucine is modified. Multiple studies have demonstrated that P _fliC The promoter is a self-regulated promoter capable of automatically down-regulating the expression of genes in the late exponential phase and in the stationary phase, and has been used in metabolic engineering many times. Here, P _fliC The promoters are used to dynamically regulate the key genes lysA, metA, ilvA of the synthesis pathway of L-lysine, L-methionine, L-isoleucine, aiming at allowing more carbon flow during stabilizationL-threonine, compared with direct knockout of these genes, can avoid the generation of auxotrophic strains, and save economic cost. The natural promoters of lysA, metA, ilvA genes of D9 are replaced one by P _fliC Promoter, yielding D12. The results show that D12 can grow without adding the corresponding amino acid, but has a serious effect on cell growth, compared with D9, OD of D12 ₆₀₀ 155% decrease in the yield of 2,5-DMP and L-threonine was followed by a dramatic decrease (FIG. 8), suggesting that lysA, metA, ilvA genes may need to be more finely regulated.

TABLE 15 primer sequences

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A recombinant escherichia coli for producing 2, 5-dimethylpyrazine is characterized in that: overexpression of threonine dehydrogenase Gene tdh, amino acetone oxidase Gene aao in the recombinant E.coli _So Cofactor NAD ⁺ Synthase gene pncB and nadE, NADH oxidase gene Smnox, feedback inhibition resistance gene lysC ^fbr And thrA ^fbr BC and L-threonine transporter gene sstT, knockout of DNA binding transcription repressor gene lacI, 2-amino-3-ketobutyrate CoA ligase gene kbl, glyoxylate pathway repressor gene iclR and threonine exosystem gene rhtA;

2. The recombinant escherichia coli of claim 1, wherein: the nucleotide sequence of tdh gene is shown as SEQ ID NO.1, aao _So The nucleotide sequence of the gene is shown as SEQ ID NO.2, the nucleotide sequence of the pncB gene is shown as SEQ ID NO.3, the nucleotide sequence of the nadE gene is shown as SEQ ID NO.4, the nucleotide sequence of the Smnox gene is shown as SEQ ID NO.5, lysC ^fbr The nucleotide sequence of the gene is shown as SEQ ID NO.6, thrA ^fbr The nucleotide sequence of the BC gene is shown as SEQ ID NO.7, and the nucleotide sequence of the sstT gene is shown as SEQ ID NO. 8.

3. The recombinant escherichia coli of claim 1, wherein: the nucleotide sequence of the lacI gene is shown as SEQ ID NO.9, the nucleotide sequence of the kbl gene is shown as SEQ ID NO.10, the nucleotide sequence of the iclR gene is shown as SEQ ID NO.11, and the nucleotide sequence of the rhtA gene is shown as SEQ ID NO. 12.

4. The recombinant escherichia coli of claim 1, wherein: the nucleotide sequence of the trc promoter is shown as SEQ ID NO.13, and the nucleotide sequence of the lpp promoter is shown as SEQ ID NO. 14.

5. The recombinant escherichia coli of claim 1, wherein: and adopting a CRISPR/Cas9 system to carry out gene editing on the recombinant escherichia coli.

6. The recombinant escherichia coli of claim 1, wherein: the recombinant escherichia coli takes escherichia coli TWF001 as a host.

7. A microbial preparation comprising the recombinant escherichia coli of any one of claims 1-6.

8. A method for producing 2, 5-dimethylpyrazine, characterized in that: fermentation production using glucose as substrate, recombinant E.coli according to any one of claims 1 to 6 or a microbial preparation according to claim 7.

9. The method according to claim 8, wherein: adding the escherichia coli of any one of claims 1-6 or the microbial preparation of claim 7 into a reaction system, controlling dissolved oxygen to be 25-35%, and controlling glucose concentration to be 1-3g/L.

10. Use of the recombinant escherichia coli of any one of claims 1-6 or the microbial preparation of claim 7 in the food industry or pharmaceutical synthesis.