A recombinant Escherichia coli producing 2,5-dimethylpyrazine and its application

Abstract

This invention discloses a recombinant 2,5-dimethylpyrazine-producing *Escherichia coli* and its applications, belonging to the field of genetic engineering technology. Utilizing the CRISPR / Cas9 gene editing system, and starting with *E. coli* TWF001, this invention obtained a recombinant 2,5-dimethylpyrazine-producing *E. coli* strain that can be stably expressed in industrial fermentation by knocking out or overexpressing genes related to the glucose-based 2,5-dimethylpyrazine metabolic pathway. The recombinant *E. coli* provided by this invention is genetically stable, requires mild fermentation conditions, and is low in cost, making it applicable to the food and pharmaceutical industries and possessing broad application prospects in industrial production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a recombinant Escherichia coli that produces 2,5-dimethylpyrazine and its applications. Background Technology

[0002] Alkylpyrazines are a class of flavoring compounds widely found in traditionally fermented and heat-treated foods, and their importance to the food industry is expected to continue to grow in the coming years. Chemical synthesis is currently the main method for synthesizing alkylpyrazines; however, with increasing awareness of food sourcing, consumers are no longer satisfied with chemically synthesized products and prefer "natural" products synthesized through biotechnology. Therefore, the process of synthesizing alkylpyrazines through biotechnology has received increasing attention. 2,5-Dimethylpyrazine (2,5-DMP) is an important alkylpyrazine with a strong roasted peanut aroma and chocolate / cream flavor, and can be used as a food flavoring. In addition, it also has important applications in the pharmaceutical field; for example, 2,5-DMP is a substrate for synthesizing the hypoglycemic drug glipizide and the lipid-lowering drug acipimox. In recent years, the biosynthesis of 2,5-DMP has received increasing attention.

[0003] In fact, researchers obtained 2,5-DMP through microbial fermentation as early as 1997 and proved that 2,5-DMP is derived from L-threonine. However, it wasn't until 2019 that the complete biosynthetic pathway from L-threonine to 2,5-DMP was successfully elucidated, including one enzymatic reaction and three spontaneous reactions. Currently, optimization of recombinant engineered bacteria producing 2,5-dimethylpyrazine focuses on overexpressing key enzymes in the metabolic pathway or constructing novel, highly efficient pathways. However, the impact of the balance between the supply and conversion rate of the precursor L-threonine on 2,5-dimethylpyrazine fermentation remains unresolved, and problems such as low substrate utilization and difficulty in increasing product yield during metabolism persist. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention employs the CRISPR / Cas9 gene editing system to knock out or overexpress certain genes related to the 2,5-dimethylpyrazine metabolic pathway in L-threonine-producing *E. coli* TWF001. This reduces L-threonine efflux and enhances L-threonine uptake, balancing the supply and conversion rate of L-threonine. This results in extremely low extracellular L-threonine accumulation and significantly promotes 2,5-dimethylpyrazine accumulation, yielding a high-yield recombinant *E. coli* strain producing 2,5-dimethylpyrazine. This recombinant strain exhibits increased fermentation yield, stable inheritance during fermentation, and eliminates the need for antibiotics or inducers, thus saving fermentation and production costs.

[0005] The first objective of this invention is to provide a recombinant *Escherichia coli* that produces 2,5-dimethylpyrazine, and overexpresses the threonine dehydrogenase gene *tdh* and the aminoacetone oxidase gene *aao* in the *Escherichia coli* genome. So cofactor NAD + Synthetic enzyme genes pncB and nadE, NADH oxidase gene Smnox, and antifeedback inhibition gene lysC fbr and thrA fbr BC and the threonine uptake protein gene sstT, knock out the DNA-binding transcriptional repressor gene lacI, the 2-amino-3-ketobutyrate CoA ligase gene kbl, the glyoxylate pathway repressor protein gene iclR, and the threonine efflux system gene rhtA.

[0006] Among them, the TRC starter is used to control AAO. So pncB, nadE, Smnox, lysC fbr ,thrA fbr The expression of BC and sstT genes was achieved by using the trc promoter to express two copies of the tdh gene, and also by using the lpp promoter to express two copies of the tdh gene.

[0007] Furthermore, the nucleotide sequence of the tdh gene is shown in SEQ ID NO.1, aao So The nucleotide sequences of the genes are shown in SEQ ID NO.2, the pncB gene in SEQ ID NO.3, the nadE gene in SEQ ID NO.4, and the Smnox gene in SEQ ID NO.5. lysC fbr The nucleotide sequence of the gene is shown in SEQ ID NO.6, thrA fbr The nucleotide sequence of the BC gene is shown in SEQ ID NO.7, and the nucleotide sequence of the sstT gene is shown in SEQ ID NO.8.

[0008] Furthermore, the nucleotide sequence of the lacI gene is shown in SEQ ID NO.9, the nucleotide sequence of the kbl gene is shown in SEQ ID NO.10, the nucleotide sequence of the iclR gene is shown in SEQ ID NO.11, and the nucleotide sequence of the rhtA gene is shown in SEQ ID NO.12.

[0009] Furthermore, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.13, and the nucleotide sequence of the lpp promoter is shown in SEQ ID NO.14.

[0010] Furthermore, gene editing of E. coli was performed using the CRISPR / Cas9 system.

[0011] Furthermore, the recombinant Escherichia coli used Escherichia coli TWF001 as its host.

[0012] A second objective of this invention is to provide a microbial preparation containing recombinant Escherichia coli.

[0013] A third objective of this invention is to provide a method for producing 2,5-dimethylpyrazine using glucose as a substrate and fermentation with the aforementioned recombinant Escherichia coli or the aforementioned microbial preparation.

[0014] Furthermore, the above-mentioned Escherichia coli or the above-mentioned microbial preparation is added to the reaction system, and the dissolved oxygen is controlled at 25-35% and the glucose concentration is controlled at 1-3g / L.

[0015] A fourth objective of this invention is to provide the application of the above-mentioned recombinant Escherichia coli or the above-mentioned microbial preparation in the food industry or pharmaceutical synthesis.

[0016] The beneficial effects of this invention are:

[0017] This invention utilizes the CRISPR / Cas9 gene editing system to perform a series of metabolic engineering modifications on the genome of *E. coli* TWF001, obtaining a genetically engineered strain capable of fermenting glucose and producing high levels of 2,5-DMP. This strain enhances L-threonine uptake and balances the relationship between L-threonine supply and conversion rate by optimizing the copy number and expression intensity of genes such as tdh on the chromosome, significantly promoting the accumulation of 2,5-dimethylpyrazine. No antibiotics or inducers are required during cultivation, saving fermentation costs and facilitating industrial applications. The recombinant strain also maintains genetic stability during fermentation. Attached Figure Description

[0018] Figure 1 It is a biological pathway for the synthesis of 2,5-dimethylpyrazine using glucose as a substrate;

[0019] Figure 2 These are the results of shake-flask fermentation of recombinant Escherichia coli in Examples 1-4;

[0020] Figure 3 These are the results of shake-flask fermentation of recombinant Escherichia coli in Examples 4-7;

[0021] Figure 4 The recombinant Escherichia coli NADH and NAD in Examples 4-7 + Content and NADH / NAD + ratio;

[0022] Figure 5 These are the results of shake-flask fermentation of recombinant Escherichia coli in Examples 7-10;

[0023] Figure 6 These are the results of shake-flask fermentation of recombinant Escherichia coli in Examples 11-14;

[0024] Figure 7 This is the result of the fed-batch fermentation of recombinant Escherichia coli in a 5L fermenter in Example 16;

[0025] Figure 8 In Comparative Example 1, P fliC The effect of promoters on the fermentation yield of recombinant Escherichia coli. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] Escherichia coli TWF001 was published in "Combined metabolic analyses for the biosynthesis pathway of L-threonine in Escherichia coli" (Frontiers in bioengineering and biotechnology, 2022).

[0028] Detection of 2,5-DMP: 2,5-DMP was detected using high-performance liquid chromatography (HPLC) 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: 0-10 min, 100% A, run time 10 min, flow rate 0.8 mL / min, UV detection wavelength 275 nm, column temperature 25 °C, and sample loading volume 10 μL.

[0029] NAD+ / NADH detection: CoenzymeⅠNAD(H)Content Assay Kit (Solarbio, Beijing, China).

[0030] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract; solid medium supplemented with 2% agar powder; sterilize at 121°C for 20 min. Add resistant kanamycin (final concentration 50 μg / mL) and spectinomycin (final concentration 50 μg / mL) as needed.

[0031] Example 1: Construction of a strain with lacI gene deletion

[0032] (1) Preparation of electrocompetent cells: First, the plasmid pEcCas was chemically transformed into E. coli TWF001, and positive transformants were screened on LB plates containing kanamycin (final concentration 50 μg / mL). The positive transformants were then inoculated into LB liquid medium containing 10 mM arabinose and cultured at 37°C until OD. 600 After the concentration was set to 0.6-0.8, electrocompetent cells were prepared.

[0033] (2) Construction of plasmid pEcgRNA-lacI: Using plasmid pEcgRNA as a template, linearized plasmid pEcgRNA-lacI was obtained by amplification using primers lacI-xx-S / lacI-xx-A. Then, the linearized plasmid pEcgRNA-lacI was transformed into E. coli JM109 competent cells to obtain plasmid pEcgRNA-lacI with a supercoiled structure.

[0034] (3) Constructing donor DNA: Using the Escherichia coli genome as a template, the upstream and downstream homologous arms were amplified using primers UP-lacI-S / UP-lacI-A and DN-lacI-S / DN-lacI-A, and the donor DNA was obtained by fusion PCR.

[0035] (4) Electroporation: The plasmid pEcgRNA-lacI and donor DNA were co-transformed into E. coli TWF001 electrocompetent cells containing plasmid pEcCas. The cells were then recovered in LB medium at 37°C for 2 h. The cells were then plated on LB plates containing kanamycin (final concentration 50 μg / mL) and spectinomycin (final concentration 50 μg / mL). Positive transformants were identified and sequenced using primers lacI-JD-S / lacI-JD-A.

[0036] (5) Elimination of plasmid pEcgRNA-lacI: The correct transformants were cultured at 37°C for 12 h in LB medium containing rhamnose (10 mM) and kanamycin (50 μg / mL), and then plated on LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight to obtain E. coli recombinants without plasmid pEcgRNA-lacI.

[0037] (6) Elimination of plasmid pEcCas: The correct transformant was cultured in LB medium containing glucose (5 g / L) at 37°C for 12 h, and then spread on LB plates containing glucose (5 g / L) and sucrose (10 g / L) and cultured at 37°C for 12 h. Finally, the plasmid-free Escherichia coli recombinant strain was obtained and named TWF001ΔlacI.

[0038] Table 1 Primer sequences

[0039]

[0040]

[0041] Example 2: Construction of a strain overexpressing the tdh gene

[0042] (1) Preparation of electrocompetent cells: First, the plasmid pEcCas was chemically transformed into TWF001ΔlacI, and positive transformants were screened on LB plates containing kanamycin (final concentration 50 μg / mL). The positive transformants were then inoculated into LB liquid medium containing 10 mM arabinose and cultured at 37°C until OD. 600 After the concentration was set to 0.6-0.8, electrocompetent cells were prepared.

[0043] (2) Construction of plasmid pEcgRNA-yjiV: Using plasmid pEcgRNA as a template, linearized plasmid pEcgRNA-yjiV was obtained by amplification using primers yjiV-xx-S / yjiV-xx-A. Then, the linearized plasmid pEcgRNA-yjiV was transformed into E. coli JM109 competent cells to obtain plasmid pEcgRNA-yjiV with a supercoiled structure.

[0044] (3) Constructing donor DNA: Using the E. coli genome as a template, the upstream and downstream homologous arms were amplified using primers UP-yjiV-S / UP-yjiV-A and DN-yjiV-S / DN-yjiV-A, and the trc-tdh expression cassette was amplified using primers tdh-S and tdh-A. Finally, the upstream and downstream homologous arms and the trc-tdh expression cassette were used as templates to amplify the donor DNA using primers UP-yjiV-S and DN-yjiV-A.

[0045] (4) Electroporation: The plasmid pEcgRNA-yjiV and donor DNA were co-transformed into TWF001ΔlacI electrocompetent cells containing plasmid pEcCas. The cells were then recovered in LB medium at 37°C for 2 h. The cells were then plated on LB plates containing kanamycin (final concentration 50 μg / mL) and spectinomycin (final concentration 50 μg / mL). Positive transformants were identified and sequenced using primers yjiV-in-S / yjiV-in-A.

[0046] (5) Elimination of plasmid pEcgRNA-yjiV: The correct transformants were cultured at 37°C for 12 h in LB medium containing rhamnose (10 mM) and kanamycin (50 μg / mL), and then plated on LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight to obtain E. coli recombinants without plasmid pEcgRNA-yjiV.

[0047] (6) Elimination of plasmid pEcCas: The correct transformant was cultured in LB medium containing glucose (5 g / L) at 37°C for 12 h, and then spread on LB plates containing glucose (5 g / L) and sucrose (10 g / L) and cultured at 37°C for 12 h. Finally, the plasmid-free Escherichia coli recombinant strain was obtained and named D1.

[0048] Table 2 Primer sequences

[0049]

[0050] Example 3: Construction of strains with double copies of the tdh gene and kbl deletion

[0051] Using the method described in Example 2, the trc-tdh expression cassette was integrated into the kbl site of the genome of strain D1. The resulting strain was named D2.

[0052] Table 3 Primer sequences

[0053]

[0054]

[0055] Example 4: Constructing aao So Gene overexpression strains

[0056] Using the method in Example 2, with D2 as the starting strain, trc-aao was integrated into the yafU site on its genome. So Expression box. The obtained strain was named D3.

[0057] Table 4 Primer sequences

[0058]

[0059] Example 5: Construction of a strain overexpressing the pncB gene

[0060] Using the method of Example 2, with strain D3 as the starting strain, the trc-pncB expression cassette was integrated into the yeeP site on its genome. The resulting strain was named D4.

[0061] Table 5 Primer sequences

[0062]

[0063]

[0064] Example 6: Construction of a nadE gene overexpressing strain

[0065] Using the method described in Example 2, with strain D4 as the starting strain, the trc-nadE expression cassette was integrated into the mazG site on its genome. The resulting strain was named D5.

[0066] Table 6 Primer sequences

[0067]

[0068] Example 7: Construction of Smnox gene overexpression strain

[0069] Using the method described in Example 2, with strain D5 as the starting strain, the trc-Smnox expression cassette was integrated into the yjhV site on its genome. The resulting strain was named D6.

[0070] Table 7 Primer sequences

[0071]

[0072] Example 8: Constructing lysC fbr Gene overexpression strains

[0073] Using the method of Example 2, with D6 as the starting strain, trc-lysC was integrated into the yncI site on its genome. fbr Expression box, in which lysC is an anti-feedback inhibition mutant lysC fbr It was obtained by mutating the 1055th base C of lysC to T. The resulting strain was named D7.

[0074] Table 8 Primer sequences

[0075]

[0076]

[0077] Example 9: Constructing thrA fbr BC gene overexpression strains

[0078] Using the method of Example 2, with D7 as the starting strain, trc-thrA was integrated into the ltaE site on its genome. fbr BC expression cassette, in which the anti-feedback inhibition mutant thrA of thrA fbr It was obtained by mutating the 1034th base C of thrA to T. The resulting strain was named D8.

[0079] Table 9 Primer sequences

[0080]

[0081] Example 10: Construction of iclR gene deletion strain

[0082] Using the method described in Example 1, the iclR gene was knocked out of strain D8. The resulting strain was named D9.

[0083] Table 10 Primer Sequences

[0084]

[0085] Example 11: Construction of lpp-tdh gene overexpression strain

[0086] Using the method described in Example 2, with strain D9 as the starting strain, the lpp-tdh expression cassette was integrated into the ykgP site on its genome. The resulting strain was named D13.

[0087] Table 11 Primer sequences

[0088]

[0089] Example 12: Construction of a strain overexpressing the double-copy lpp-tdh gene

[0090] Using the method of Example 2, with D13 as the starting strain, the lpp-tdh expression cassette was integrated into the lfhA site on its genome, and the resulting strain was named D14.

[0091] Table 12 Primer sequences

[0092]

[0093] Example 13: Construction of a strain overexpressing the three-copy lpp-tdh gene

[0094] Using the method of Example 2, with D14 as the starting strain, the lpp-tdh expression cassette was integrated into the ykiA site on its genome, and the resulting strain was named D15.

[0095] Table 13 Primer sequences

[0096]

[0097]

[0098] Example 14: Construction of strains with sstT gene overexpression and rhtA gene deletion

[0099] Using the method described in Example 2, with strain D14 as the starting strain, the trc-sstT expression cassette was integrated into the rhtA site on its genome. The resulting strain was named D19.

[0100] Table 14 Primer Sequences

[0101]

[0102] Example 15: Production of 2,5-DMP by shake-flask fermentation

[0103] The culture medium formula is as follows:

[0104] Seed culture medium (LB): 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0105] Fermentation medium: 20 g / L glucose, 3 g / L (NH4)2SO4, 0.9 g / L KCl, 2 g / L citric acid, 0.5 g / L betaine, 0.8 g / L MgSO4·7H2O, 1.8 g / L phosphoric acid, 20 mg / L FeSO4·7H2O, 20 mg / L MnSO4·H2O, 2% phenol red, adjusted to pH 7.

[0106] The recombinant strains prepared in Examples 1-13 were taken from a -80℃ freezer and inoculated onto LB agar plates for overnight incubation at 37℃. Then, a loopful of the strain was taken from the plate and inoculated into a test tube containing 5 mL of seed culture medium, and incubated at 37℃ for 8 h. Finally, a 10% inoculum was inoculated into a 500 mL Erlenmeyer flask containing 45 mL of fermentation medium and incubated at 37℃ for 48 h. During the incubation process, 25% ammonia (v / v) was added to maintain the pH value according to the color of the fermentation broth.

[0107] like Figure 2 As shown, D1 accumulated 52.89 mg / L of 2,5-DMP, and its growth was improved compared to TWF001ΔlacI, indicating that the presence of the tdh gene enables *E. coli* to synthesize 2,5-DMP. Simultaneously, knocking out the kbl gene and overexpressing the tdh gene (D2) increased the yield to 173.35 mg / L. Overexpression of aao... So The gene further increased the yield by 28.8%, reaching 223.29 mg / L.

[0108] like Figure 3 As shown, overexpression of pncB alone (D4) increased the yield of 2,5-DMP to 347.7 mg / L, a 55.72% increase compared to D3. Simultaneous overexpression of pncB and nadE genes increased the yield of 2,5-DMP to 423.31 mg / L, an 89.58% increase compared to D3. This indicates that overexpression of pncB and nadE genes can increase NAD+ yield. + The production of 2,5-DMP was improved by increasing its content. In addition, further overexpression of the NADH oxidase encoding gene Smnox(D6) increased the yield of 2,5-DMP to 477.7 mg / L.

[0109] like Figure 4As shown, overexpression of pncB(D4) alone reduces NAD + The intracellular content of NAD+ increased by 60.67%, while simultaneous overexpression of pncB and nadE genes (D5) reduced NAD+ levels. + The intracellular content of NAD+ further increased by 105.8%, indicating that overexpression of both pncB and nadE genes increased intracellular NAD+ levels. + In addition, further overexpression of the NADH oxidase encoding gene Smnox(D6) significantly increased intracellular NAD content. + The level has increased by 37% compared to D5.

[0110] like Figure 5 As shown, overexpression of lysC fbr The gene (D7) did not have a significant effect on the production of 2,5-DMP. Overexpression of thrA fbr Both the BC gene cluster (D8) and the knockout of the iclR gene (D9) promoted the accumulation of 2,5-DMP. Strain D9 accumulated 560.5 mg / L of 2,5-DMP, which was 17.3% higher than that of strain D6.

[0111] like Figure 6 As shown, single copy P lpp The yield of strain D13 of -tdh reached 929.8 mg / L, and the double-copy P lpp The -tdh strain D14 further increased the yield to 1064.3 mg / L; however, the three-copy P strain... lpp The -tdh strain D15 did not continue to increase yield, indicating that the double-copy P lpp -tdh already met the TDH requirements for 2,5-DMP production. Further, by knocking out the rhtA gene and overexpressing the sstT gene (D19) on the basis of D14, the yield of 2,5-DMP increased to 1589.1 mg / L, while the extracellular accumulation of L-threonine was reduced to a very low level. This indicates that by optimizing tdh gene expression and modifying the L-threonine transport system, combined with the relationship between L-threonine supply and L-threonine conversion rate, the 2,5-DMP production performance of the strain was significantly improved.

[0112] Example 16: Production of 2,5-DMP by fed-batch fermentation in a 5L fermenter

[0113] The culture medium formula is as follows:

[0114] Fermentation tank culture medium: 80 g / L glucose, 3 g / L yeast extract, 0.5 g / L betaine, 2 g / L KH2PO4, 10 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·H2O, adjusted to pH 7.

[0115] First, strain D19 was removed from a -80℃ freezer and inoculated onto LB agar plates for overnight incubation at 37℃. Then, a loopful of the strain was taken from the plate and inoculated into a test tube containing 5 mL of LB medium, incubated overnight at 37℃. Next, the entire 5 mL of bacterial culture was added to a 500 mL Erlenmeyer flask containing 45 mL of seed culture medium and incubated at 37℃ for 12 h. Finally, a 10% inoculum was added to a 5 L bioreactor containing 2 L of fermentation medium and incubated at 37℃ for 48 h. During fermentation, the pH was maintained at 7 by adding 25% ammonia (v / v), and dissolved oxygen was maintained at 25-35% by adjusting the stirring speed and aeration rate. When the initial glucose in the fermentation medium was depleted, a 60% glucose solution (w / v) was automatically added to maintain the glucose concentration at 0-3 g / L.

[0116] like Figure 7 As shown, the biomass of D19 reached its maximum value of 54.6 (OD) at 24 h. 600 The titer of 2,5-DMP then gradually decreased, while the titer of 2,5-DMP reached a peak of 3.1 g / L at 36 h, with a production intensity of 2.1 g / (L·d), which is the highest reported yield of 2,5-DMP from fermented glucose to date.

[0117] Comparative Example 1: Using promoter P fliC Regulating the supply of L-threonine

[0118] To further improve the supply of L-threonine, the synthetic pathways of L-lysine, L-methionine, and L-isoleucine were modified. Multiple studies have confirmed that P... fliC A promoter is a self-regulating promoter that automatically downregulates gene expression during the late exponential phase and the stationary phase, and has been used extensively in metabolic engineering. Here, P... fliC Promoters were used to dynamically regulate key genes lysA, metA, and ilvA in the synthesis pathways of L-lysine, L-methionine, and L-isoleucine, aiming to ensure a greater carbon flux to L-threonine during the stationary phase. Compared to directly knocking out these genes, this approach avoids the generation of auxotrophic strains and saves economic costs. The natural promoters of the lysA, metA, and ilvA genes in D9 were replaced one by one with P... fliCThe promoter was activated, producing D12. The results showed that although D12 could grow without the addition of the corresponding amino acids, it severely impacted cell growth; compared to D9, the OD of D12 was significantly lower. 600 The yield decreased by 155%, and the production of 2,5-DMP and L-threonine also decreased significantly. Figure 8 This suggests that the lysA, metA, and ilvA genes may require more refined regulation.

[0119] Table 15 Primer Sequences

[0120]

[0121]

[0122] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A recombinant *Escherichia coli* producing 2,5-dimethylpyrazine, characterized in that: The recombinant Escherichia coli overexpressed the threonine dehydrogenase gene tdh and the aminoacetone oxidase gene aao. So cofactor NAD + Synthetic enzyme genes pncB and nadE, NADH oxidase gene Smnox, and antifeedback inhibition gene lysC fbr and thrA fbr BC and L-threonine transporter gene sstT, knock out DNA-binding transcriptional repressor gene lacI, 2-amino-3-ketobutyrate CoA ligase gene kbl, glyoxylate pathway repressor gene iclR, and threonine efflux system gene rhtA. wherein the expression of the aao So , pncB, nadE, Smnox, lysC fbr , thrA fbr BC and sstT genes, double copy expression of the tdh gene using the trc promoter, and double copy expression of the tdh gene using the lpp promoter.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the tdh gene is shown in SEQ ID NO.1, aao So The nucleotide sequences of the genes are shown in SEQ ID NO.2, the pncB gene in SEQ ID NO.3, the nadE gene in SEQ ID NO.4, the Smnox gene in SEQ ID NO.5, and lysC... fbr The nucleotide sequence of the gene is shown in SEQ ID NO.6, thrA fbr The nucleotide sequence of the BC gene is shown in SEQ ID NO.7, and the nucleotide sequence of the sstT gene is shown in SEQ ID NO.

8.

3. The recombinant Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the lacI gene is shown in SEQ ID NO. 9, the nucleotide sequence of the kbl gene is shown in SEQ ID NO. 10, the nucleotide sequence of the iclR gene is shown in SEQ ID NO. 11, and the nucleotide sequence of the rhtA gene is shown in SEQ ID NO.

12.

4. The recombinant Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the trc promoter is shown in SEQ ID NO.13, and the nucleotide sequence of the lpp promoter is shown in SEQ ID NO.

14.

5. The recombinant Escherichia coli according to claim 1, characterized in that: The recombinant Escherichia coli was gene-edited using the CRISPR / Cas9 system.

6. The recombinant Escherichia coli according to claim 1, characterized in that: The recombinant Escherichia coli uses Escherichia coli TWF001 as the host.

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

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

9. The method according to claim 8, characterized in that: Add the *Escherichia coli* of any one of claims 1-6 or the microbial preparation of claim 7 to the reaction system, and control the dissolved oxygen at 25-35% and the glucose concentration at 1-3 g / L.

10. The use of the recombinant Escherichia coli according to any one of claims 1-6 or the microbial preparation according to claim 7 in the food industry or pharmaceutical synthesis.

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