Construction method and application of engineering bacteria for improving yield of alpha-ketoglutaric acid

Abstract

The invention discloses a construction method and application of an engineering bacterium for increasing the yield of alpha-ketoglutaric acid. Wild E.coli.W3110 serves as a chassis strain, lactic dehydrogenase ldhA, pyruvate dehydrogenase poxB and formic acid C-acetyltransferase pflB are knocked out firstly, and the flow direction of pyruvic acid to lactic acid, acetic acid and formic acid is blocked; meanwhile, the promoter PBBaJ23109 is used for replacing a natural promoter of the sucA gene, so that alpha-ketoglutaric acid dehydrogenase is weakened, and the decomposition way of alpha-ketoglutaric acid is weakened; the phosphoenolpyruvate carboxylase ppc is overexpressed, and the flow direction of phosphoenolpyruvate to oxaloacetic acid is enhanced; isocitrate dehydrogenase icd from escherichia coli is overexpressed, so that synthesis of a product is promoted; besides, pyruvate dehydrogenase derived from saccharomyces cerevisiae is introduced and coded by genes PDB1, PDA1 and LAT1, so that an important precursor, namely pyruvic acid, is promoted to flow to acetyl coenzyme A; the engineering strain with high yield of alpha-ketoglutaric acid is finally obtained, the yield of 75.1 g / L can be achieved in a 5L fermentation tank through a fed-batch fermentation method for 40 hours, and the conversion rate is 39.6%.

Description

Technical Field

[0001] This invention relates to the field of compound biotechnology production, and in particular to a method and application for constructing engineered bacteria to increase the yield of α-ketoglutarate. Background Technology

[0002] The English scientific name of α-ketoglutaric acid is 2-Ketoglutaric acid. Its common molecular formula is C5H6O5, and its molecular weight is 146. It is easily soluble in water and alcohol, but extremely difficult to dissolve in ether. It will turn pale grayish-yellow after being left for a long time and is hygroscopic.

[0003] Alpha-ketoglutarate (AKG) is an intermediate in the tricarboxylic acid (TCA) cycle. As an important metabolic intermediate, it participates in cellular energy metabolism and amino acid synthesis, and is a key metabolite in the synthesis of glutamate family proteases and ammonia homeostasis. AKG can delay the aging process by regulating cellular metabolism and antioxidant pathways, showing potential application prospects in the treatment of metabolic syndrome. It can improve obesity, hyperglycemia, and cardiovascular disease risk by regulating macrophage metabolism and inflammatory responses, thus improving the pathological mechanisms of metabolic syndrome. In addition to its direct application as a dietary supplement and therapeutic agent in pharmaceuticals and cosmetics, AKG is also used as a structural unit in polymers and in the synthesis of the platform chemical ethylene.

[0004] Currently, the main methods for producing α-ketoglutarate (AKG) include microbial fermentation, chemical synthesis, and biomass conversion. However, these methods suffer from drawbacks such as low conversion rates, high costs, and complex operations in industrial production. Therefore, we used *Escherichia coli* as the substrate strain and genetically modified it using genetic engineering techniques to provide a new method for producing engineered bacteria for α-ketoglutarate production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing and applying engineered bacteria for increasing the yield of α-ketoglutarate.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered bacteria that produce α-ketoglutarate.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned genetically engineered bacteria that produce α-ketoglutarate.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A genetically engineered bacterium that produces α-ketoglutaric acid has been named E. coli - Og12 This refers to artificially modified *E. coli* strains with a specific genotype. The modifications include: [the modifications are made to] the wild-type strain... E.coli w3110 Deletion in the genome ldhA, poxB, pflB Genes, downregulatedsucA The transcriptional level of the gene upregulated the strain's ppc and icd At the transcriptional level, PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae were heterologously introduced, as were YALI2_E01708g, YALI2_E01754g, and YALI2_D00183g from Yersinia lipophila.

[0009] Preferably, the starting strain of the α-ketoglutarate-producing genetically engineered bacterium is wild-type E. coli W3110 (ATCC 27325).

[0010] Preferably, the above-mentioned α-ketoglutaric acid producing strain, wherein ldhA, poxB, pflB The nucleotide sequences of the gene are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; sucA The natural promoter of the gene is replaced with P BBa_J23109 The sucA The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing, wherein P BBa_J23109 The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; in yeel Site integration ppc The gene, initiated by the Ptrc promoter, is described ppc The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing, wherein P trc The promoter nucleotide sequence is shown in SEQ ID NO.1; the icd gene is integrated at the ycdN site, and the nucleotide sequence of the icd gene is shown in SEQ ID NO.8.

[0011] Preferably, the above-mentioned α-ketoglutarate producing strain integrates the PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae into pseudogene sites yjiV, yghX, and ygaY, respectively. The nucleotide sequences of the PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae are shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, respectively. Preferably, the above-mentioned α-ketoglutarate producing strain is derived from Yersinia lipolytica. YALI2_E01708g, YALI2_E01754g and YALI2_D00183g The genes are integrated into the rph, yjgX, and ycgH sites, respectively, and are derived from *Yarrowia lipolytica*. YALI2_E01708g, YALI2_E01754g and YALI2_D00183g The nucleotide sequences of the gene are shown in the sequence listings SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively.

[0012] Preferably, the above-mentioned genetically engineered bacteria that produce α-ketoglutarate are wild-type. E.coli w3110 As the starting strain (chassis strain), gene editing was performed using metabolic engineering techniques. The specific steps are as follows: (1) Lactate dehydrogenase was knocked out in the genome. ldhA Gene (NCBI-Gene ID: 946315) ， Pyruvate dehydrogenase poxB Gene (NCBI-Gene ID: 946132), formate C-acetyltransferase pflB Gene (NCBI-Gene ID:945514); (2) Replace the natural promoter of the α-ketoglutarate dehydrogenase sucA gene (NCBI-Gene ID: 945303) encoded in the genome with P BBa_J23109 ; (3) The genome integrates the phosphoenolpyruvate carboxylase ppc gene (NCBI-Gene ID: 948457) and isocitrate dehydrogenase icd gene (NCBI-Gene ID: 945702) derived from Escherichia coli. (4) The genomes of PDB1 (NCBI-Gene ID: 852522), PDA1 (NCBI-Gene ID: 856925), and LAT1 (NCBI-Gene ID: 855653), which are derived from Saccharomyces cerevisiae and encode pyruvate dehydrogenase, were integrated. (5) Integrating the enzyme encoding isocitrate synthase from *Yersinia lipophila* into the genome. YALI2_ E01708g (NCBI-Gene ID: 2912112) , YALI2_E01754g (NCBI-Gene ID: 2912887) and aconitine hydratase YALI2_D00183g Gene (NCBI-Gene ID: 2910842).

[0013] The above-mentioned genetically engineered bacteria are used in the fermentation production of α-ketoglutarate.

[0014] Preferably, the above-mentioned α-ketoglutaric acid producing strain is used to produce α-ketoglutaric acid through fermentation in a fermenter, and the specific steps are as follows: (1) Seed activation: The strain was evenly spread on the activation slant and incubated at 36℃ for 15 h, then transferred to a new slant and incubated at 37℃ for 13 h; (2) Seed culture: The bacterial suspension was inoculated into the seed culture medium, pH 6.8, temperature constant at 37℃, dissolved oxygen at 35-50%, and cultured for 14h; (3) Fermentation culture: wait for the OD of the seed cell mass600 At around 20°C, inoculate the fermentation medium at a rate of 20% to begin fermentation. During fermentation, maintain the pH at around 6.8, the temperature at 37°C, and the dissolved oxygen at 35-50%. Once the glucose in the medium is depleted, add an 80% glucose solution to maintain the glucose concentration in the fermentation medium below 1 g / L.

[0015] Preferably, in the application of the above-mentioned α-ketoglutarate producing strain, the slant culture medium used in the seed activation is: glucose 4 g / L, peptone 4 g / L, yeast extract powder 8 g / L, sodium chloride 3 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 2 g / L, agar powder 20% (volume percentage), the remainder being water, pH 6.8.

[0016] Preferably, in the application of the above-mentioned α-ketoglutarate producing strain, the seed culture medium used in the seed culture is as follows: glucose 20 g / L, yeast extract powder 6 g / L, peptone 3 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate monohydrate 20 mg / L, vitamin H 0.5 mg / L, vitamin B1 1 mg / L, and the remainder is water. The medium is sterilized in a high-pressure steam autoclave at 115℃ for 20 min.

[0017] Preferably, in the application of the above-mentioned α-ketoglutaric acid producing strain, the fermentation medium used in the fermentation culture is: glucose 30 g / L, yeast extract powder 8 g / L, peptone 4 g / L, ammonium sulfate 6 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 2 g / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate monohydrate 10 mg / L, vitamin H 1 mg / L, vitamin B1 2 mg / L, and the remainder is water. Sterilization is performed in a high-pressure steam autoclave at 115℃ for 20 min.

[0018] All of the above-mentioned culture media can be prepared using standard methods.

[0019] The beneficial effects of this invention are: The aforementioned genetically engineered bacteria that produce α-ketoglutarate have a clear genetic background. By knocking out lactate dehydrogenase ldhA, pyruvate dehydrogenase poxB, and formic acid C-acetyltransferase pflB, the flow of pyruvate to lactate, acetic acid, and formic acid is blocked; and by using promoter P... BBa_J23109By replacing the natural promoter of the α-ketoglutarate dehydrogenase (sucA) gene, the α-ketoglutarate dehydrogenase was weakened, thus reducing the degradation pathway of α-ketoglutarate. Overexpression of phosphoenolpyruvate carboxylase (ppc) enhanced the flow of phosphoenolpyruvate to oxaloacetate. Overexpression of isocitrate dehydrogenase (icd) from *E. coli* promoted product synthesis. The introduction of pyruvate dehydrogenase from *Saccharomyces cerevisiae* promoted the flow of the important precursor pyruvate to acetyl-CoA. The introduction of citrate synthase and aconitate hydratase from *Yarrowia lipolytica* further enhanced the product synthesis pathway. The method for constructing α-ketoglutarate-producing strains provided by this invention is a rational method for strain construction, ultimately yielding… E. coli - Og12 It has promising application prospects. Attached Figure Description

[0020] Figure 1 α-Ketoglutarate engineered strain E. coli - Og12 A diagram illustrating the construction method.

[0021] Figure 2 Genetically engineered strain E. coli - Og12 Fermentation process curve. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art.

[0023] 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.

[0024] The corresponding promoters and genes in the examples are shown in the sequence listing. The primers used in the construction of the strains involved are shown in Table 1.

[0025] Table 1 Primers used in strain construction Example 1 like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows: 1.1 Genes ldhA Knockout Using the Escherichia coli W3110 genome as a template, primers were used... ldhA -1 and ldhA -2, ldhA -3 and ldhA -4 were subjected to PCR amplification to obtain the upstream homologous arm. [[ID=3(4]]ldhA -UP and downstream homologous armsldhA -DW, using the recovered upstream and downstream homologous arms as templates, with primers ldhA -1 and ldhA -4. Overlap PCR was performed to obtain the knockout gene. ldhA Required replacement fragment Δ ldhA Next, the primer pGRB- ldhA -s and pGRB- ldhA The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ldhA Finally, pGRB- ldhA Plasmids and overlapping fragments Δ ldhA Electroporation was performed into wild-type E. coli W3110 / pRed-Cas9 electrocompetent cells; then... ldhA -1 and ldhA -4 is the identification primer; positive transformants are screened to obtain the strain. E. coli -Og1.

[0026] 1.2 Genes poxB Knockout Using the Escherichia coli W3110 genome as a template, primers were used... poxB -1 and poxB -2, poxB -3 and poxB -4 were subjected to PCR amplification to obtain the upstream homologous arm. poxB -UP and downstream homologous arms poxB -DW, using the recovered upstream and downstream homologous arms as templates, with primers poxB -1 and poxB -4. Overlap PCR was performed to obtain the knockout gene. poxB Required replacement fragment Δ poxB Next, the primer pGRB- poxB -s and pGRB- poxB The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- poxB Finally, pGRB- poxB Plasmids and overlapping fragments Δ poxB Electric transfer to E. coli -Og1 / pRed-Cas9 electrocompetent cells; then with poxB -1 and poxB -4 is the identification primer; positive transformants are screened to obtain the strain. E. coli -Og2.

[0027] 1.3 Genes pflB Knockout Using the Escherichia coli W3110 genome as a template, primers were used... pflB -1 and pflB -2,pflB -3 and pflB -4 were subjected to PCR amplification to obtain the upstream homologous arm. pflB -UP and downstream homologous arms pflB -DW, using the recovered upstream and downstream homologous arms as templates, with primers pflB -1 and pflB -4. Overlap PCR was performed to obtain the knockout gene. pflB Required replacement fragment Δ pflB Next, the primer pGRB- pflB -s and pGRB- pflB The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- pflB Finally, pGRB- pflB Plasmids and overlapping fragments Δ pflB Electric transfer to E. coli -Og2 / pRed-Cas9 electrocompetent cells; then with pflB -1 and pflB -4 is the identification primer; positive transformants are screened to obtain the strain. E.coli -Og3.

[0028] 1.4 Genes sucA The weakening (P) BBa_J23109 - sucA ) pGRB- sucA -s、pGRB- sucA -a is a primer, used to construct pGRB- sucA Plasmid. Then with sucA -ups, sucA -up-a、 sucA -dn-s、 sucA Using -dn-a as a primer and the genome of E. coli W3110 as a template, upstream homologous arms were obtained. sucA -up, downstream homologous arm sucA -dn; then obtain the fusion fragment P BBa_J23109 - sucA Finally, pGRB- sucA Plasmids and overlapping fragments P BBa_J23109 - sucA Electric transfer to E. coli -Og3-electrocompetent cells; then with sucA -ups, sucA -dn-a is the identification primer; positive transformants are screened to obtain the strain. E. coli -Og4.

[0029] 1.5 genes ppc Integration ( yeeL ::Ptrc - ppc (integration) Using Escherichia coli W3110 as a template, primers were used... yeeL -1 and yeeL -trc-2, yeeL -3 and yeeL -4; ppc -s and ppc -a was used for PCR amplification to obtain the upper homologous arm. yeel -trc-UP, lower homologous arm yeeL -DW and intermediate destination fragments ppc Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... yeeL -1 and yeeL -4 The target fragment P required for integration was obtained through overlap PCR. trc - ppc Then, the primer pGRB- yeeL -s and pGRB- yeeL The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- yeeL Plasmid. Finally, the purified P... trc - ppc Integration fragment and plasmid pGRB- yeeL Simultaneously transferred via electroconversion E. coli In competent cells of -Og4 / pRed-Cas9, then with primers yeeL -1 and yeeL -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E. coli -Og5.

[0030] 1.6 genes icd Integration ( ycdN ::P trc - icd (integration) Using Escherichia coli W3110 as a template, primers were used... ycdN -1 and ycdN -trc-2, ycdN -3 and ycdN -4; icd -s and icd -a was used for PCR amplification to obtain the upper homologous arm. ycdN -trc-UP, lower homologous arm ycdN -DW and intermediate destination fragments icd Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ycdN -1 and ycdN -4 The target fragment P required for integration was obtained through overlap PCR. trc -icd Then, the primer pGRB- ycdN -s and pGRB- ycdN The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ycdN Plasmid. Finally, the purified P... trc - icd Integration fragment and plasmid pGRB- ​ Simultaneously transferred via electroconversion ​ In competent cells of -Og5 / pRed-Cas9, primers were then used. ​ -1 and ​ -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. ​ -Og6.

[0031] 1.7 genes ​ Integration ( ​ ::P trc - ​ (integration) Using the Saccharomyces cerevisiae genome as a template, primers were used... ​ -1 and ​ -trc-2, ​ -3 and ​ -4; ​ -s and ​ -a was used for PCR amplification to obtain the upper homologous arm. ​ -trc-UP, lower homologous arm ​ -DW and intermediate destination fragments ​ Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ​ -1 and ​ -4 The target fragment P required for integration was obtained through overlap PCR. trc - ​ Then, the primer pGRB- ​ -s and pGRB- ​ The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ​ Plasmid. Finally, the purified P... trc - ​ Integration fragment and plasmid pGRB- ​ Simultaneously transferred via electroconversion ​ In competent cells of -Og6 / pRed-Cas9, primers were then used. ​ -1 and ​ -4 indicates the primers used for identification to screen positive transformants and obtain the strain. ​ -Og7.

[0032] 1.8 genes ​ Integration (​ ::P trc - ​ (integration) Using the Saccharomyces cerevisiae genome as a template, primers were used... ​ -1 and ​ -trc-2, ​ -3 and ​ -4; ​ -s and ​ -a was used for PCR amplification to obtain the upper homologous arm. ​ -trc-UP, lower homologous arm ​ -DW and intermediate destination fragments ​ Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ​ -1 and ​ -4 The target fragment P required for integration was obtained through overlap PCR. trc - ​ Then, the primer pGRB- ​ -s and pGRB- ​ The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ​ Plasmid. Finally, the purified P... trc - ​ Integration fragment and plasmid pGRB- ​ Simultaneously transferred via electroconversion ​ In competent cells of -Og7 / pRed-Cas9, then with primers ​ -1 and ​ -4 indicates the primers used for identification to screen positive transformants and obtain the strain. ​ -Og8.

[0033] 1.9 genes ​ Integration ( ​ ::P trc - ​ (integration) Using the Saccharomyces cerevisiae genome as a template, primers were used... ygaY -1 and ygaY -trc-2, ygaY -3 and ygaY -4; LAT1 -s and LAT1 -a was used for PCR amplification to obtain the upper homologous arm. ygaY -trc-UP, lower homologous arm ygaY -DW and intermediate destination fragments LAT1 Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ygaY -1 and ygaY -4 The target fragment P required for integration was obtained through overlap PCR. trc- LAT1 Then, the primer pGRB- ygaY -s and pGRB- ygaY The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ygaY Plasmid. Finally, the purified P... trc - LAT1 Integration fragment and plasmid pGRB- ygaY Simultaneously transferred via electroconversion E. coli In competent cells of -Og8 / pRed-Cas9, then with primers ygaY -1 and ygaY -4 indicates the primers used for identification to screen positive transformants and obtain the strain. E. coli -Og9.

[0034] 1.10 genes YALI2_E01708g Integration ( rph ::P trc - YALI2_E01708g (integration) Using the genome of Yersinia lipophila as a template, primers were used... rph -1 and rph -trc-2, rph -3 and rph -4; YALI2_ E01708g -s and YALI2_E01708g -a was used for PCR amplification to obtain the upper homologous arm. rph -trc-UP, lower homologous arm rph -DW and intermediate destination fragments YALI2_E01708g Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... rph -1 and rph -4 The target fragment P required for integration was obtained through overlap PCR. trc - YALI2_E01708g Then, the primer pGRB- rph -s and pGRB- rph The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- rph Plasmid. Finally, the purified P... trc - YALI2_E01708g Integration fragment and plasmid pGRB- rph Simultaneously transferred via electroconversion E. coli In competent cells of -Og9 / pRed-Cas9, then with primers rph -1 and rph -4 indicates the primers used for identification to screen positive transformants and obtain the strain. E. coli -Og10.

[0035] 1.11 genesYALI2_E01754g Integration ( yjgX ::P trc - YALI2_E01754g (integration) Using the genome of Yersinia lipophila as a template, primers were used... yjgX -1 and yjgX -trc-2, yjgX -3 and yjgX -4; YALI2_E01754g -s and YALI2_E01754g -a was used for PCR amplification to obtain the upper homologous arm. yjgX -trc-UP, lower homologous arm yjgX -DW and intermediate destination fragments YALI2_E01754g Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... yjgX -1 and yjgX -4 The target fragment P required for integration was obtained through overlap PCR. trc - YALI2_E01754g Then, the primer pGRB- yjgX -s and pGRB- yjgX The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- yjgX Plasmid. Finally, the purified P... trc - YALI2_E01754g Integration fragment and plasmid pGRB- yjgX Simultaneously transferred via electroconversion E. coli In competent cells of -Og10 / pRed-Cas9, primers were then used. yjgX -1 and yjgX -4 indicates the primers used for identification to screen positive transformants and obtain the strain. E. coli -Og11.

[0036] 1.12 genes YALI2_D00183g Integration ( ycgH ::P trc - YALI2_E01754g (integration) Using the genome of Yersinia lipophila as a template, primers were used... ycgH -1 and ycgH -trc-2, ycgH -3 and ycgH -4; YALI2_E01754g -s and YALI2_E01754g -a was used for PCR amplification to obtain the upper homologous arm. ycgH -trc-UP, lower homologous arm ycgH -DW and intermediate destination fragments YALI2_E01754g Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ycgH -1 and ycgH-4 The target fragment P required for integration was obtained through overlap PCR. trc - YALI2_E01754g Then, the primer pGRB- ycgH -s and pGRB- ycgH The DNA fragment obtained by -a annealing was ligated onto plasmid pGRB to construct pGRB- ycgH Plasmid. Finally, the purified P... trc - YALI2_E01754g Integration fragment and plasmid pGRB- ycgH Simultaneously transferred via electroconversion E. coli In competent cells of -Og11 / pRed-Cas9, primers were then used. ycgH -1 and ​ -4 indicates the primers used for identification to screen positive transformants and obtain the strain. ​ -Og12.

[0037] 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.

[0038] Example 2 Using the α-ketoglutaric acid producing strain described in Example 1 ​ -Og12 was fermented in a 5 L fermenter to produce α-ketoglutaric acid.

[0039] 2.1 Culture medium 2.1.1 Slant Culture Medium Glucose 4 g / L, peptone 4 g / L, yeast extract 8 g / L, sodium chloride 3 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 2 g / L, agar powder 20% (volume percentage), the remainder being water, pH 7.0. Dispense into test tubes (10 ml / tube) and sterilize in a high-pressure steam oven at 115℃ for 25 min.

[0040] 2.1.2 Seed Culture Medium Glucose 20 g / L, yeast extract 6 g / L, peptone 3 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate monohydrate 20 mg / L, vitamin H 0.5 mg / L, vitamin B1 1 mg / L, and the remainder is water. Sterilize in a high-pressure steam autoclave at 115℃ for 20 min.

[0041] 2.1.3 Fermentation medium Glucose 30 g / L, yeast extract 8 g / L, peptone 4 g / L, ammonium sulfate 6 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 2 g / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate monohydrate 10 mg / L, vitamin H 1 mg / L, vitamin B1 2 mg / L, and the remainder is water. Sterilize in a high-pressure steam autoclave at 115℃ for 20 min.

[0042] 2.2 Cultivation Methods 2.2.1 Slant activation culture Take the bacterial culture from the preservation tube, spread it evenly on the activation slant, incubate at 36℃ for 15 h, then transfer to a new slant and continue incubation at 37℃ for 13 h; 2.2.2 Seed Culture Take an appropriate amount of sterile water into a slant test tube, inoculate the bacterial suspension into the seed culture medium, stabilize the pH at around 6.8, keep the temperature constant at 37℃, and keep the dissolved oxygen between 35% and 50%, and incubate for 14 hours. 2.2.3 Fermentation Culture Wait until the seed cell count reaches approximately 600 to 20, then inoculate with fresh fermentation medium at a rate of 20% to begin fermentation. During fermentation, maintain a stable pH of approximately 6.8, a temperature of 37°C, and dissolved oxygen levels between 35% and 50%. Once the glucose in the medium is depleted, add an 80% glucose solution to maintain the glucose concentration in the fermentation medium below 1 g / L. The fermentation cycle is 40 hours.

[0043] like ​ As shown, the yield of α-ketoglutaric acid reached 74.8 g / L after 40 h of fermentation in a 5 L fermenter.

[0044] 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 genetically engineered bacterium that produces α-ketoglutarate, named E. coli -Og12 blocks the flow of pyruvate to lactate, acetic acid, and formic acid, enhancing the precursor supply module for α-ketoglutarate; it replaces the natural promoter of the sucA gene with the weaker promoter P. BBa_J23109 This weakens the breakdown of α-ketoglutarate; enhances the supply of oxaloacetate by overexpressing phosphoenolpyruvate carboxylase ppc at the yeeL site of the E. coli W3110 genome; and strengthens the supply of oxaloacetate by overexpressing the isocitrate dehydrogenase gene icd at the ycdN site of the genome. trc Promoter initiation; via the E. coli W3110 genome yjiV, yghX, ygaY The pyruvate dehydrogenase genes PDB1, PDA1, and LAT1 from Saccharomyces cerevisiae were introduced at the site, by P trc Promoter initiation; the citrate synthase genes YALI2_E01708g, YALI2_E01754g and aconitate hydratase YALI2_D00183g from *Yergium lipophilum* were introduced into the *E. coli* W3110 genome at the rph, yjgX, and ycgH sites. All genes were initiated by P. trc Startup sub-boot. 2.The genetically engineered bacterium for producing alpha-ketoglutarate according to claim 1, characterized in that: The originating bacterium, Escherichia coli W3110, has the accession number ATCC 27325.

3. The genetically engineered bacterium for producing α-ketoglutaric acid according to claim 1, characterized in that: The strain was deleting the ldhA, poxB, and pflB genes, downregulating the transcription level of the sucA gene, upregulating the transcription levels of the ppc and icd genes, heterologously introducing the PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae, and introducing YALI2_E01708g, YALI2_E01754g, and YALI2_D00183g from Yacinthia spp.

4. The genetically engineered bacterium for producing α-ketoglutaric acid according to claim 1, characterized in that: The ldhA, poxB, and pflB genes were knocked out of the strain's genome; P was used BBa_J23109 Promoter replacement sucA The natural promoter of genes, weakened sucA Gene expression; PPC Genes are overexpressed onto the genome and their expression is enhanced using the TRC promoter; icd Genes were overexpressed onto the genome and their expression was enhanced using a trc promoter; PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae were integrated into the genome and their expression was enhanced using a trc promoter; in addition, YALI2_E01708g, YALI2_E01754g, and YALI2_D00183g genes from Yersinia lipolyticis were integrated into the genome and their expression was enhanced using a trc promoter.

5. The genetically engineered bacterium for producing α-ketoglutaric acid according to claim 1, characterized in that: The trc promoter nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing, and the P BBa_J23109 The promoter nucleotide sequence is shown in SEQ ID NO.2, the ldhA gene nucleotide sequence is shown in SEQ ID NO.3, the poxB gene nucleotide sequence is shown in SEQ ID NO.4, and the pflB gene nucleotide sequence is shown in SEQ ID NO.

5. sucA The gene nucleotide sequence is shown in SEQ ID NO. 6 of the sequence listing. PPC The gene nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing. icd The gene nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing.

6. The genetically engineered bacterium for producing α-ketoglutaric acid according to claim 1, characterized in that: The source of the brewing yeast PDB1, PDA1, LAT1 The gene nucleotide sequences are shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11 of the sequence listing. The nucleotide sequences of the YALI2_E01708g, YALI2_E01754g, and YALI2_D00183g genes from YALI2 are shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14 of the sequence listing.

7. A method for constructing a genetically engineered bacterium for producing α-ketoglutaric acid according to any one of claims 1-6, characterized in that: The specific steps are as follows: (1) Blocking precursor branching: Using wild-type E.coli.W3110 as the starting strain, the genes ldhA, poxB, and pflB encoding the production of lactic acid, acetic acid, and formic acid from pyruvate were knocked out; (2) Weaken the branch pathway of a-ketoglutarate: replace the native promoter of the gene with P sucA BBa_J23109 ;​ (3) Enhance the synthesis of α-ketoglutarate: Overexpress ppc and icd genes at the yeeL and ycdN sites in the genome, enhance expression using the trc promoter, integrate PDB1, PDA1, and LAT1 genes from Saccharomyces cerevisiae into the yjiV, yghX, and ygaY pseudogene sites in the genome, respectively, and start with the Ptrc promoter, and integrate YALI2_E01708g, YALI2_E01754g, and YALI2_D00183g genes from Yersinia lipolytica into the rph, yjgX, and ycgH sites in the genome, respectively, and start with the Ptrc promoter.

8. The application of a genetically engineered bacterium for producing α-ketoglutaric acid according to any one of claims 1-6 in a fermentation process, characterized in that: Fermentation is carried out using a mechanically stirred fermenter. The specific steps are as follows: (1) Slant culture: The α-ketoglutarate producing strain was inoculated on slant medium as the first generation slant culture and cultured at 36℃ for 15h; the colonies of the first generation slant culture were inoculated on a new slant medium as the second generation slant culture and cultured at 37℃ for 13h. (2) Seed culture: The bacterial growth in the second generation slant was washed with sterile water into the seed culture medium. The culture temperature was 37℃. The pH of the seed culture was maintained at 6.8±0.2 and the dissolved oxygen value was 35-50% by automatically adding ammonia solution through the bioreactor. When the OD600nm of the seed culture reached 20, the next fermentation culture was carried out. (3) Fermentation culture: The inoculum amount is 20%, the culture temperature is 37℃, and the pH of the seed culture is maintained at 6.8±0.2 and the dissolved oxygen value is 35-50% by automatically adding ammonia solution through a bioreactor. 9.The application of the genetically engineered bacteria producing α-ketoglutarate in fermentation process according to claim 8, characterized in that: The seed culture medium in step (2) is: glucose 20 g / L, yeast extract powder 6 g / L, peptone 3 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate monohydrate 20 mg / L, V H 0.5 mg / L, V B1 1 mg / L, the remainder being water. 10.The application of the genetically engineered bacteria producing α-ketoglutarate in fermentation process according to claim 8, characterized in that: The fermentation medium in step (3) is: glucose 30 g / L, yeast extract 8 g / L, peptone 4 g / L, ammonium sulfate 6 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 2 g / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate monohydrate 10 mg / L, V H 1mg / L, V B1 2 mg / L, the remainder being water.

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