2, 5-dimethylpyrazine production strain as well as construction method and application thereof

Abstract

The invention provides a 2, 5-dimethylpyrazine production strain and a construction method and application thereof.According to the strain, key feedback inhibition is relieved through a perfect metabolic engineering strategy and strategies of knocking out a competitive pathway, introducing heterologous aminoacetone oxidase, deleting L-threonine efflux protein and the like, competitive enzyme conversion of aminoacetone is reduced through a system, and the 2, 5-dimethylpyrazine production strain is obtained. Spontaneous transformation of aminoacetone in Escherichia coli is enhanced, metabolism of L-threonine is expanded to synthesis of 2, 5-dimethylpyrazine, the yield of 2, 5-dimethylpyrazine is further increased, the constructed strain does not contain plasmids and has the advantages of clear genetic background, sustainable transformation and the like, and the 2, 5-dimethylpyrazine is produced through fermentation. The method is simple to operate, can be used for directly synthesizing 2, 5-dimethylpyrazine from the beginning by using a cheap carbon source, and has a very good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a 2,5-dimethylpyrazine-producing strain, its construction method, and its application. Background Technology

[0002] Pyrazines are volatile nitrogen-containing heterocyclic compounds widely found in plants, insects, fungi, and bacteria, and are used in numerous fields such as pesticides, dyes, food, and pharmaceuticals. As consumer preferences shift towards natural products, the demand for "natural" pyrazines is continuously growing. However, due to the low content of naturally derived pyrazines—for example, beet molasses contains only 0.01 wt%—extraction and concentration techniques based solely on natural sources cannot meet market demand.

[0003] Microbial fermentation has emerged as a promising method for pyrazine production. Among pyrazines, 2,5-dimethylpyrazine (2,5-DMP) is a key contributor to the distinctive flavor of cocoa and soybean fermentation products, as well as oolong tea. Besides its role as a natural flavor compound, 2,5-DMP also serves as an important intermediate in pharmaceutical synthesis, particularly in the production of 5-methylpyrazine-2-carboxylic acid (a precursor to commercial drugs such as acilimex and glipizide). The biosynthesis of 2,5-DMP has attracted attention, especially in Bacillus subtilis, where its derivatization from L-threonine has been confirmed. This biosynthetic pathway involves one enzymatic reaction and three spontaneous reactions. In this pathway, threonine dehydrogenase catalyzes the conversion of L-threonine to L-2-aminoacetoacetic acid, followed by decarboxylation to aminoacetone. Aminoacetone then condenses to form 3,6-dihydro-2,5-dimethylpyrazine, which is further dehydrogenated to yield 2,5-DMP. Recombinant Escherichia coli strains have been engineered to produce 2,5-DMP de novo from L-threonine or via glucose. However, metabolic engineering studies on 2,5-DMP production remain limited, and the production efficiency achieved to date is insufficient for industrial applications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a 2,5-dimethylpyrazine producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned 2,5-dimethylpyrazine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned 2,5-dimethylpyrazine producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A 2,5-dimethylpyrazine-producing strain, named strain MP14, is derived from wild-type Escherichia coli. E.coliW3110 knockout of lac repressor gene lacI The starting strain was obtained through genetic modification: knockout of the aspartate kinase I gene. thrA Isocitrate lyase inhibitor gene iclR threonine aldolase gene ltaE L-2-aminoacetoacetate CoA ligase gene kbl Tyrosine oxidase gene tynA glycerol dehydrogenase gene gldA Genes encoding threonine export proteins rhtA and rhtBC The phosphoenolpyruvate carboxylase gene ppc The original promoter was replaced with P trc Promoter, integrating threonine-operating genes thrA C1034T BC And by P trc Promoter activation integrates the gene encoding membrane-binding transhydrogenase. pntAB And by P trc Promoter activation, introducing streptococci from oligofermentation ( Streptococcus oligofermentan aminoacetone oxidase gene aao And by P trc Promoter activation, integration of catalase gene katE By P trc The T7 RNAP gene is integrated using the promoter, and the threonine dehydrogenase gene is integrated using the lacUV5 promoter. tdh It is then started by the T7 bootloader.

[0008] The above-mentioned 2,5-dimethylpyrazine-producing strain is based on wild-type Escherichia coli. E.coli W3110 Knockout of lac repressor gene lacI As the starting strain, the aspartate kinase I gene was knocked out to eliminate transcriptional repression by the thrABC operon. thrA and integrate at that site thrA C1034T BC By P trc Promoter activation; to improve the availability of oxaloacetate, the gene encoding phosphoenolpyruvate carboxylase will be activated. ppc The original promoter uses P trc Promoter substitution; to improve L-threonine formation, the isocitrate lyase inhibitor gene was knocked out. iclR To alleviate NADPH restriction, in the genome gapC Site overexpression of the gene encoding membrane-binding transhydrogenase pntAB Knock out the gene encoding threonine aldolase, which is mainly responsible for L-threonine degradation. ltaEand the gene encoding L-2-aminoacetoacetate CoA ligase kbl To reduce the competitive enzymatic conversion of aminoacetone and thus increase its availability in the biosynthesis of 2,5-DMP, the gene encoding tyrosine oxidase was knocked out. tynA and the gene encoding glycerol dehydrogenase gldA Knock out the gene encoding the threonine export protein rhtA , rhtB and rhtC This inhibits the export of L-threonine to the extracellular space; to enhance the spontaneous conversion of aminoacetone in Escherichia coli, streptococci from oligofermentative bacteria (Streptococcus) were introduced. Streptococcus oligofermentan Gene encoding aminoacetone oxidase aao The H2O2 produced by overexpression of this gene can generate highly reactive hydroxyl radicals and singlet oxygen to cellular macromolecules, thereby introducing... aao This resulted in a very weak impact on the growth of the strain, leading to a 18% increase in yield; subsequent introduction katE To continue mitigating the impact, introduce katE The strain showed a 10% increase in growth and yield, and overexpression of the catalase-encoding gene. katE Reduce DNA damage aao and katE Both genes are produced by P trc Startup sub-boot; to improve tdh The expression level in the genome yeeP The site integrates the T7 RNAP gene, which is initiated by the lacUV5 promoter, and the corresponding genome... ycgH Site integration encoding threonine dehydrogenase gene tdh It is started by the T7 bootloader.

[0009] Preferably, the genetic modification method of the above-mentioned 2,5-dimethylpyrazine producing strain is CRISPR / Cas9-mediated gene editing technology.

[0010] Preferably, the above-mentioned 2,5-dimethylpyrazine-producing strain, wherein P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.1, the nucleotide sequence of the Lacuv5 promoter is shown in SEQ ID NO.3, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.4.

[0011] Preferably, the above-mentioned 2,5-dimethylpyrazine-producing strain, thrA C1034T BC The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing. iclRThe nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing. pntAB The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing. ltaE The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing. kbl The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing. tynA The nucleotide sequence is shown in SEQ ID NO. 10 of the sequence listing. gldA The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing. aao The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing. katE The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing. rhtA The nucleotide sequence is shown in SEQ ID NO.14 of the sequence listing. rhtBC The nucleotide sequence is shown in SEQ ID NO. 15 of the sequence listing. tdh The nucleotide sequence of the T7 RNAP is shown in SEQ ID NO.16, and the nucleotide sequence of the T7 RNAP is shown in SEQ ID NO.17. thrA The nucleotide sequence is shown in SEQ ID NO.18 of the sequence listing. lacI The nucleotide sequence is shown in SEQ ID NO.19 of the sequence listing.

[0012] The above-mentioned method for constructing the 2,5-dimethylpyrazine-producing strain involves modifying the starting strain, wherein the starting strain is a strain with the lac repressor protein gene knocked out. lacI wild-type Escherichia coli E.coli For W3110, the specific steps are as follows: (1) Eliminate transcriptional repression of the thrABC operon: knock out the aspartate kinase I gene on the genome. thrA and introduce at this site thrA C1034T BC , by P trc Startup sub-boot; (2) Increase the carbon flux of oxaloacetate: [This refers to the gene for phosphoenolpyruvate carboxylase.] ppc The promoter uses P trc Promoter replacement; (3) Knock out the isocitrate lyase inhibitor gene in the genome iclR ; (4) Reduce NADPH restriction: Overexpress membrane-binding transhydrogenase gene pntAB ; (5) Reduce L-threonine degradation: Knock out threonine aldolase gene ltaE and L-2-aminoacetoacetate CoA ligase gene kbl ; (6) Enhance the spontaneous conversion of aminoacetone in Escherichia coli and reduce the effects of hydrogen peroxide produced on the strain: overexpression of aminoacetone oxidase gene aao and catalase gene katE ; (7) Reduce the competitive enzymatic conversion of aminoacetone: knock out the tyramine oxidase gene tynA and glycerol dehydrogenase gene gldA ; (8) Blocking L-threonine export, reducing byproduct accumulation, and improving its availability: Knockout of genes encoding threonine export proteins rhtA and rhtBC ; (9) Improve tdh Expression levels: The T7 RNAP gene is integrated and activated by the lacUV5 promoter, correspondingly integrating the threonine dehydrogenase gene. tdh It is started by the T7 bootloader.

[0013] Application of the above-mentioned 2,5-dimethylpyrazine producing strains in the fermentation production of 2,5-dimethylpyrazine (2,5-DMP).

[0014] Preferably, in the above application, 2,5-dimethylpyrazine is produced by fermentation: the 2,5-dimethylpyrazine producing strain is contacted with a fermentation medium and fermented to obtain 2,5-dimethylpyrazine.

[0015] Preferably, in the above applications, the fermentation culture includes shake flask fermentation or fermenter fermentation.

[0016] Preferably, in the above application, the steps of shake-flask fermentation are as follows: inoculum amount of 10%-15%, temperature maintained at 37℃±0.2, shaking culture at 220r / min, pH maintained at 7.0 by adding 25% ammonia water during fermentation; and adding 60% glucose solution to supplement the carbon source required by the cells.

[0017] Preferably, in the above application, the specific steps of fermentation using a fermenter are as follows: (1) Activation of strain: The strain was transferred from the glycerol tube to the slant medium and activated for 12-14 h; the strain was then transferred from the slant medium to the eggplant flask medium and activated and expanded for 10-12 h at a temperature of 37℃. (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained at 30±10%. When the OD was 15, the culture was transferred to a fermenter for fermentation culture. (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained at 30±10%. During the culture, 80% glucose solution was added to maintain the carbon source required by the fermentation cells. During this period, 25% ammonia water was added to adjust the pH to maintain it at 7.0±0.1.

[0018] Preferably, in the above application, a tail gas recovery bottle is used to collect the volatile 2,5-dimethylpyrazine, and the tail gas recovery bottle contains 1000ml of water.

[0019] Preferably, in the above applications, the slant culture medium and the flask culture medium used are: beef extract 10 g / L, glucose 5 g / L, sodium chloride 5 g / L, peptone 10 g / L, yeast powder 5 g / L, agar powder 25 g / L, and pH adjusted to 7.0-7.2.

[0020] Preferably, in the above application, the seed culture medium used is: glucose 30g / L, yeast extract 5g / L, peptone 5g / L, corn steep liquor 4g / L, potassium dihydrogen phosphate 3g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate heptahydrate 20mg / L, manganese sulfate 10mg / L, vitamins B1, B3, B5, and B12 2mg / L each, with the remainder being water.

[0021] Preferably, in the above application, the fermentation medium used is: glucose 20g / L, peptone 5g / L, yeast extract 5g / L, corn steep liquor 6g / L, potassium dihydrogen phosphate 3g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 2g / L, citrate 0.5g / L, L-lysine 0.2g / L, L-methionine 0.2g / L, ferrous sulfate heptahydrate 20mg / L, manganese sulfate 10mg / L, vitamins B1, B3, B5, and B12 2mg / L each, with the remainder being water.

[0022] All of the above-mentioned culture media can be prepared using standard methods.

[0023] Beneficial effects: The aforementioned 2,5-dimethylpyrazine-producing strain, through a sophisticated metabolic engineering strategy, including knocking out competing pathways, introducing heterologous aminoacetone oxidase, and deleting L-threonine efflux proteins, relieved key feedback inhibition. By systematically reducing the competitive enzymatic conversion of aminoacetone, the spontaneous conversion of aminoacetone in *E. coli* was enhanced, extending L-threonine metabolism to 2,5-dimethylpyrazine synthesis, thus further increasing the yield of 2,5-dimethylpyrazine. The constructed strain is plasmid-free, possesses a clear genetic background, and is suitable for sustainable modification. The fermentation production of 2,5-dimethylpyrazine is simple to operate and can directly synthesize 2,5-DMP de novo using inexpensive carbon sources, showing great application potential. Attached Figure Description

[0024] Figure 1 This is a diagram illustrating the gene modification process of the 2,5-dimethylpyrazine strain.

[0025] Figure 2 Comparison of shake-flask fermentation yields of 2,5-dimethylpyrazine-producing strains.

[0026] Figure 3 Figure showing the fermentation yield of the MP14 strain for producing 2,5-dimethylpyrazine in a fermenter. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0028] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0029] The starting strain used in the examples was wild-type Escherichia coli. E.coli W3110 ATCC 27325 Knockout of lac repressor gene lacI This serves as the starting strain. The corresponding promoter, terminator, and genes are listed in the sequence listing. Primers used in the construction of the involved strains are listed in Table 1.

[0030] Table 1 Primers used in strain construction

[0031]

[0032]

[0033]

[0034] The gene editing methods used in the above gene manipulations are referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in that article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after ligation with a promoter and terminator.

[0035] Example 1 like Figure 1 As shown, this embodiment uses wild-type Escherichia coli. E.coli W3110 ATCC 27325 Knockout of lac repressor gene lacI This example serves as the starting strain, aiming to illustrate the specific construction steps of strain MP14. Specifically, if a similar gene manipulation method is used in the examples, it will only be provided once and annotated, without further elaboration.

[0036] 1.1 Gene knockout thrA and introduce thrA C1034T BC : wild-type Escherichia coli E.coli Using the genome of W3110 as a template, respectively with thrA -UF、 thrA -UR and thrA -DF、 thrA -DR is the primer, and upstream and downstream homologous arms are obtained by PCR amplification; since the PAM sequence of plasmid 1 was ligated to the 5' and 3' ends of the primer during primer design, the overlapping fragment consists of "upstream homologous arm - PAM sequence of plasmid 1 - downstream homologous arm". thrA -pGRB-UP, thrA Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... thrA -pGRB; Prepare MP0 electrotransformation competent cells, and combine overlapping fragments with... thrA- pGRB was electroporated into competent cells, and positive transformants were selected to obtain strain MP-1-1; wild-type Escherichia coli was used. E.coli Using the W3110 genome as a template, Ptrc- thrA -F、Ptrc- thrA- R and thrAC1034T -UF、 thrA C1034T -UR and thrA C1034T -DF、 thrA C1034T -DR refers to the homologous arms obtained by PCR amplification using primers. Since P was already included in the primer design... trc promoters and thrA C1034T The primers were attached to the 5' and 3' ends, so the overlapping fragments carried P. trc promoters and thrA C1034T That is, the overlapping segment is composed of the "upstream homologous arm - P trc promoter- thrA C1034T The gRNA fragment was obtained by annealing with 1-pGRB-UP and 1-pGRB-DN as primers and then ligating it with the pGRB vector to obtain 1-pGRB. The overlapping fragment and 1-pGRB were electroporated into competent cells using the above-mentioned MP-1-1 competent cells, and positive transformants were screened to obtain strain MP1.

[0037] 1.2 Genes ppc The promoter uses P trc Starter replacement: wild-type Escherichia coli E.coli Using the genome of W3110 as a template, respectively with ppc -UF、 ppc -UR and ppc -DF、 ppc -DR, upstream and downstream homologous arms were obtained by PCR amplification. Then, using the upstream and downstream homologous arms as templates, ppc -UF、 ppc -DR is the primer, and overlapping fragments are obtained by overlapping PCR amplification. Since P was already included in the primer design... trc The promoter is attached to the 5' and 3' ends of the primer, so the overlapping fragment carries P trc The promoter, i.e., the overlapping segment, is generated by the "upstream homologous arm - P trc Composed of "promoter-target gene-downstream homologous arm"; with Pppc -pGRB-UP, ​ Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... ​ -pGRB; Prepare MP1 electrocompetent cells, and combine overlapping fragments with... ​ -pGRB was electroporated into competent cells, and positive transformants were obtained through screening to obtain strain MP2.

[0038] 1.3 Gene Knockout​ : wild-type Escherichia coli ​ Using the genome of W3110 as a template, respectively with ​ -UF、 ​ -UR and ​ -DF、 ​ -DR is used as a primer. Upstream and downstream homologous arms are obtained by PCR amplification. Then, using the upstream and downstream homologous arms as templates, ... ​ -UF、 ​ -DR is the primer, and overlapping PCR amplification yields an overlapping fragment, which consists of an "upstream homologous arm - downstream homologous arm"; ​ -pGRB-UP, ​ Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... ​ -pGRB; Prepare MP2 electrocompetent cells, and combine overlapping fragments with... ​ -pGRB was electroporated into competent cells, and positive transformants were obtained through screening to obtain strain MP3.

[0039] 1.4 Overexpression ​ : wild-type Escherichia coli ​ Using the genome of W3110 as a template, respectively with ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR was used as a primer, and upstream and downstream homologous arms were obtained by PCR amplification; ​ -F、 ​ -R is a primer, obtained through PCR amplification. ​ Gene fragments; finally, using the upstream homologous arm, downstream homologous arm, and target gene fragment as templates, ​ -UF、 ​ -DR is the primer, and overlapping fragments are obtained by overlapping PCR amplification. Since P was already included in the primer design... trc promoters and P trc Termination (P) trc The nucleotide sequence of the terminator (as shown in SEQ ID NO.2) was attached to the 5' and 3' ends of the primer, respectively, thus the overlapping fragment carried P. trc promoters and P trc Terminator, i.e., the overlapping segment is formed by the upstream homologous arm - P trc Promoter-target gene-P trc Composed of "terminator-downstream homologous arm"; with ​ -pGRB-UP, ​Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... ​ -pGRB; Prepare MP4 electrotransformation competent cells, and combine overlapping fragments with... ​ -pGRB was electroporated into competent cells, and positive transformants were obtained through screening to obtain strain MP4.

[0040] 1.5 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​ -pGRB-DN, using MP4 competent cells, to obtain strain MP5.

[0041] 1.6 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​ -pGRB-DN, using MP5 competent cells, strain MP6 was obtained.

[0042] 1.7 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​ -pGRB-DN, using MP6 competent cells, strain MP7 was obtained.

[0043] 1.8 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​-pGRB-DN, using MP7 competent cells, strain MP8 was obtained.

[0044] 1.9 Overexpression ​ : From the oligofermentant Streptococcus oligofermentans ​ The gene (its unoptimized nucleotide sequence is shown in SEQ ID NO. 20) was synthesized by Genewiz (Suzhou, China) and codon optimization was performed for *E. coli* (nucleotide sequence shown in SEQ ID NO. 12). [The gene was then used in conjunction with other gene sequences.] ​ So -F、 ​ So -R is a primer, obtained through PCR amplification. ​ Target fragment; wild-type Escherichia coli ( ​ Using the genome of W3110 as a template, respectively with ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR was used as primers to obtain upstream and downstream homologous arms via PCR amplification; finally, the upstream and downstream homologous arms and the target gene fragment were used as templates to... ​ -UF、 ​ -DR is the primer, and overlapping fragments are obtained by overlapping PCR amplification. Since P was already included in the primer design... trc promoters and P trc The terminators were attached to the 5' and 3' ends of the primers, respectively, so the overlapping fragments carried P. trc promoters and P trc Terminator, i.e., the overlapping segment is formed by the upstream homologous arm - P trc Promoter-target gene-P trc It consists of a terminator and a downstream homologous arm. ​ -pGRB-UP, ​ Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... ​ -pGRB; Prepare MP9 electrocompetent cells, and combine overlapping fragments with... ​ -pGRB was electroporated into competent cells, and positive transformants were obtained through screening to obtain strain MP9.

[0045] 1.10 Overexpression ​ : The procedure is the same as step 1.4, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​-DR、 ​ -F、 ​ -R、 ​ -pGRB-UP, ​ -pGRB-DN, competent cells were MP10, electroporation was performed to enter competent cells, and positive transformants were obtained through screening to obtain strain MP10.

[0046] 1.11 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​ -pGRB-DN, using MP10 competent cells, strain MP11 was obtained.

[0047] 1.12 Knockout ​ : The procedure is the same as in step 1.1, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -pGRB-UP, ​ -pGRB-DN, using MP11 competent cells, strain MP12 was obtained.

[0048] 1.13 Integration of T7 RNAP: Using the genome of Escherichia coli BL21(DE3) as a template, T7RNAP-F and T7RNAP-R were used. The primers were used to amplify the T7 RNAP gene, which was then promoted using the lacUV5 promoter and terminated by P. trc Terminator; using the genome of Escherichia coli W3110 as a template ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR, to amplify the upper and lower homologous arms in the primers; with ​ -UF、 ​ -DR is used as a primer; overlapping fragments are obtained through overlapping PCR amplification. ​ -pGRB-UP, ​ Using pGRB-DN as a primer, annealing yields a gRNA fragment, which is then ligated into the pGRB vector to obtain... ​ -pGRB; This will merge overlapping fragments with... ​-pGRB was electroporated into competent cells MP12, and positive transformants were obtained through screening to obtain strain MP13.

[0049] 1.14 Using the T7 promoter ​ : The procedure is the same as step 1.4, except that the primer is... ​ -UF、 ​ -UR、 ​ -DF、 ​ -DR、 ​ -F、 ​ -R、 ​ -pGRB-UP, ​ -pGRB-DN, T7 promoter and T7 terminator (the nucleotide sequence of the T7 terminator is shown in SEQ ID NO.123 in the sequence listing), competent cells were MP14, electroporated into competent cells, and positive transformants were obtained by screening to obtain strain MP14.

[0050] The strain information for the above modification process is shown in Table 2.

[0051] Table 2. Strain Information

[0052] Example 2 Fermentation was carried out for 24 hours in 500mL Erlenmeyer flasks using strains MP0, MP7, MP8, MP9, and MP14 from Example 1, respectively. The specific steps are as follows: 2.1 Activation of bacterial strain: The bacterial strain was transferred from the glycerol tube to an LB test tube and activated for 13 hours at 37°C and 220 rpm. 2.2 Seed culture: Under aseptic conditions, the bacterial strain on the slant culture medium in the test tube in step 2.1 was inoculated into 30 mL of seed culture medium and cultured at 37℃ and 220 rpm for 12 h; 2.3 Fermentation Culture: Under aseptic conditions, the seed culture solution from step 2.2 was inoculated into the fermentation medium at a 13% inoculum volume using a sterile pipette, bringing the total volume of the fermentation medium to 30 mL. The fermentation was carried out at 37℃ and 220 rpm. During fermentation, the pH value and glucose content were determined by the color change of the phenol red indicator. The pH value was maintained at 7.0-7.2 by adding small amounts of 25% ammonia water multiple times. The growth of the strain was maintained by adding 60% (m / v) glucose. The fermentation cycle was 24 h.

[0053] In the above shake-flask fermentation process, the LB test tube culture medium used was: sodium chloride 10 g / L, peptone 10 g / L, yeast powder 5 g / L, sterilized at 121℃ for 20 min; after sterilization, the test tube culture medium was prepared.

[0054] The seed culture medium used was: glucose 30 g / L, yeast extract 5 g / L, peptone 5 g / L, corn steep liquor 4 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamins B1, B3, B5, and B12 2 mg / L each, with the remainder being water.

[0055] The fermentation medium used was: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, corn steep liquor 6 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, citrate 0.5 g / L, L-lysine 0.2 g / L, L-methionine 0.2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamins B1, B3, B5, and B12 2 mg / L each, with the remainder being water.

[0056] like ​ As shown, the OD of strain MP0 after 24 h of fermentation in a 500 mL Erlenmeyer flask... 600 47.1, no 2,5-DMP was generated in the fermentation broth; shake flask data showed that the introduction of... ​ The MP8 strain showed an 18% increase in yield compared to MP7; overexpression ​ The yield of strain MP9 was increased by 10% compared to MP8; the OD of strain MP14 was... 600 16. The yield of 2,5-DMP in the fermentation broth can reach 1.56 g / L.

[0057] Example 3 The strain MP14 from Example 1 was cultured in a 5L fermenter. The volatilized 2,5-DMP was collected using a tail gas recovery bottle, which contained 1000ml of water. The specific steps are as follows: 3.1 Strain activation: The strain was transferred from the glycerol tube to the slant medium and activated for 13 hours; the strain was then transferred from the slant medium to the eggplant flask medium for further activation and expansion culture for 12 hours at a temperature of 37℃. 3.2 Seed culture: During the culture process, the pH was maintained at 7.0±0.1, the temperature at 37±0.2℃, and the dissolved oxygen at 30±10%. When the OD reached 15, the culture was transferred to a fermenter for fermentation culture. (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained at 30±10%. During the culture, 80% glucose solution was added to maintain the carbon source required by the fermentation cells. During this period, 25% ammonia water was added to adjust the pH to maintain it at 7.0±0.1.

[0058] The slant culture medium and flask culture medium used in the above fermentation process were: beef extract 10g / L, glucose 5g / L, sodium chloride 5g / L, peptone 10g / L, yeast powder 5g / L, agar powder 25g / L, pH adjusted to 7.0-7.2, sterilized at 121℃ for 20min; after sterilization, test tube slant culture medium and flask culture medium were prepared respectively.

[0059] The seed culture medium used was: glucose 30 g / L, yeast extract 5 g / L, peptone 5 g / L, corn steep liquor 4 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamins B1, B3, B5, and B12 2 mg / L each, with the remainder being water.

[0060] The fermentation medium used was: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, corn steep liquor 6 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, citrate 0.5 g / L, L-lysine 0.2 g / L, L-methionine 0.2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamins B1, B3, B5, and B12 2 mg / L each, with the remainder being water.

[0061] like ​ As shown, 2,5-DMP was produced by fermentation in a 5L fermenter, and the OD of the strain was [value missing] after 32 hours. 600 The yield of 17.5,2,5-DMP reached 3.44 g / L, and 1.8 g / L was detected in the tail gas recovery bottle. The total yield in the overall fermentation tank was 4.04 g / L, which laid the foundation for subsequent fermentation production of 2,5-DMP.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A strain for producing 2,5-dimethylpyrazine, characterized in that: wild-type Escherichia coli E. coli W3110 Knockout of lac repressor gene lacI The starting strain was obtained through genetic modification: knockout of the aspartate kinase I gene. thrA Isocitrate lyase inhibitor gene iclR threonine aldolase gene ltaE L-2-aminoacetoacetate CoA ligase gene kbl Tyrosine oxidase gene tynA glycerol dehydrogenase gene gldA Genes encoding threonine export proteins rhtA and rhtBC The phosphoenolpyruvate carboxylase gene PPC The original promoter was replaced with P trc Promoter, integrating threonine operator gene thrA C1034T BC And by P trc Promoter activation integrates the gene encoding membrane-binding transhydrogenase. pntAB And by P trc Promoter activation introduces the aminoacetone oxidase gene from oligofermentative streptococci. aao And by P trc Promoter activation, integration of catalase gene katE By P trc The T7 RNAP gene is integrated using the promoter, and the threonine dehydrogenase gene is integrated using the lacUV5 promoter. tdh It is then started by the T7 bootloader.

2. The 2,5-dimethylpyrazine-producing strain according to claim 1, characterized in that: The P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.1, the nucleotide sequence of the Lacuv5 promoter is shown in SEQ ID NO.3, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.

4.

3. The 2,5-dimethylpyrazine-producing strain according to claim 1, characterized in that: The thrA C1034T BC The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing. iclR The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing. pntAB The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing. ltaE The nucleotide sequence is shown in SEQ ID NO. 8 of the sequence listing. kbl The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing. tynA The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing. gldA The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing. aao The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing. katE The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing. rhtA The nucleotide sequence is shown in SEQ ID NO.14 of the sequence listing. rhtBC The nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing. tdh The nucleotide sequence of the T7 RNAP is shown in SEQ ID NO.16, and the nucleotide sequence of the T7 RNAP is shown in SEQ ID NO.

17. thrA The nucleotide sequence is shown in SEQ ID NO.18 of the sequence listing. lacI The nucleotide sequence is shown in SEQ ID NO.19 of the sequence listing.

4. The method for constructing the 2,5-dimethylpyrazine-producing strain according to any one of claims 1-3, characterized in that: The strain was modified based on the starting strain, which was a strain with the lac repressor protein gene knocked out. lacI wild-type Escherichia coli E. coli For W3110, the specific steps are as follows: (1) Eliminate transcriptional repression of the thrABC operon: knock out the aspartate kinase I gene on the genome. thrA and introduce at this site thrA C1034T BC , by P trc Startup sub-boot; (2) Increase the carbon flux of oxaloacetate: [This refers to the gene for phosphoenolpyruvate carboxylase.] PPC The promoter uses P trc Promoter replacement; (3) Knock out the isocitrate lyase inhibitor gene in the genome. iclR ; (4) Reduce NADPH restriction: Overexpress membrane-binding transhydrogenase gene pntAB ; (5) Reduce L-threonine degradation: Knock out threonine aldolase gene ltaE and L-2-aminoacetoacetate CoA ligase gene kbl ; (6) Enhance the spontaneous conversion of aminoacetone in Escherichia coli and reduce the effects of hydrogen peroxide produced on the strain: overexpression of aminoacetone oxidase gene aao and catalase gene katE ; (7) Reduce the competitive enzymatic conversion of aminoacetone: knock out the tyramine oxidase gene tynA and glycerol dehydrogenase gene gldA ; (8) Blocking L-threonine export, reducing byproduct accumulation, and improving its availability: Knockout of genes encoding threonine export proteins rhtA and rhtBC ; (9) Improve tdh Expression levels: The T7 RNAP gene is integrated and activated by the lacUV5 promoter, correspondingly integrating the threonine dehydrogenase gene. tdh It is started by the T7 bootloader.

5. The use of the 2,5-dimethylpyrazine-producing strain according to any one of claims 1-3 in the fermentation production of 2,5-dimethylpyrazine.

6. The application according to claim 5, characterized in that: 2,5-Dimethylpyrazine was produced by fermentation: the 2,5-dimethylpyrazine-producing strain was contacted with a fermentation medium and fermented to obtain 2,5-dimethylpyrazine.

7. The application according to claim 5 or 6, characterized in that: The steps for shake-flask fermentation are as follows: inoculate 10%-15% of the culture, maintain the temperature at 37℃±0.2, and culture with shaking. During the fermentation process, ammonia water is added to maintain the pH at 7.0; glucose solution is added to supplement the carbon source required by the cells.

8. The application according to claim 5 or 6, characterized in that: The specific steps for fermentation using a fermentation tank are as follows: (1) Activation of strain: The strain was transferred from the glycerol tube to the slant medium and activated for 12-14 h; the strain was then transferred from the slant medium to the eggplant flask medium and activated and expanded for 10-12 h at a temperature of 37℃. (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained at 30±10%. When the OD was 15, the culture was transferred to a fermenter for fermentation culture. (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained at 30±10%. During the culture, glucose solution was added to maintain the carbon source required by the fermentation cells. During this period, ammonia water was added to adjust the pH to maintain it at 7.0±0.

1.

9. The application according to claim 8, characterized in that: The volatile 2,5-dimethylpyrazine was collected using an exhaust gas recovery bottle containing 1000 ml of water.

10. The application according to claim 8, characterized in that: The slant and flask culture media used were: beef extract 10 g / L, glucose 5 g / L, sodium chloride 5 g / L, peptone 10 g / L, yeast extract 5 g / L, agar powder 25 g / L, pH adjusted to 7.0-7.2; the seed culture medium used was: glucose 30 g / L, yeast extract 5 g / L, peptone 5 g / L, corn steep liquor 4 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamin B1. The following were added: B3, B5, and B12, each 2 mg / L, with the remainder being water; the fermentation medium used was: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, corn steep liquor 6 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 2 g / L, citrate 0.5 g / L, L-lysine 0.2 g / L, L-methionine 0.2 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate 10 mg / L, vitamins B1, B3, B5, and B12 each 2 mg / L, with the remainder being water.

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