Plasmid, alpha-ketoglutaric acid engineering strain and construction method and application thereof

Abstract

The invention provides a plasmid, an alpha-ketoglutaric acid engineering strain and a construction method and application of the alpha-ketoglutaric acid engineering strain, the strain is obtained by adopting a directional transformation method, transcriptional levels of katE, katG, sodB and grxA genes are up-regulated, a catR gene and a katA gene are heterologously expressed, a high-copy plasmid pETKG is carried, and the plasmid simultaneously expresses L-glutamate oxidase LGOX genes from Streptomyces sp and Chain A; the strain self-synthesized catalase eliminates H2O2 generated in the process of producing alpha-ketoglutaric acid through enzyme catalysis, engineering bacteria of L-glutamate oxidase LGOX expressed by pETKG plasmids are used for performing whole-cell catalysis on L-sodium glutamate to synthesize alpha-ketoglutaric acid, and the engineering strain is a high-level engineering strain capable of industrially and stably producing alpha-ketoglutaric acid.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and fermentation engineering, in particular to plasmids, α-ketoglutaric acid engineering strains, and their construction methods and applications. Background Art

[0002] α-Ketoglutaric acid (α-KG), also known as α-oxosuccinate or α-oxoglutarate, is a white powdery substance at room temperature, soluble in water and alcohol, but insoluble in ether, and turns light gray-yellow after long-term storage. α-Ketoglutaric acid is an intermediate metabolite in the tricarboxylic acid energy metabolism and plays a crucial role in the synthesis of amino acids and peptides. It has the effects of preventing osteoporosis, stabilizing the immune system, improving weight gain, extending lifespan, regulating aging, etc., and has broad application prospects in the fields of medicine, medical aesthetics, cosmetics, food, health products, etc.

[0003] Currently, there are three methods for producing α-ketoglutaric acid: chemical synthesis, microbial production, and enzymatic conversion. The method of chemical synthesis from diethyl succinate and diethyl oxalate has been successfully used for the large-scale industrial production of α-ketoglutaric acid, but the chemical synthesis has low yields and produces toxic chemicals and solvents harmful to the environment. The microbial production of α-ketoglutaric acid has been studied for decades and has made great progress, but it still produces different by-products and requires a long fermentation period, which is not conducive to industrial production. Enzymatic conversion is environmentally friendly, requires less energy, has high selectivity, produces fewer by-products and is non-toxic, and has received increasing attention.

[0004] Although the enzymatic conversion method has significant advantages in terms of environmental friendliness and reaction specificity, there are still several key bottleneck problems that have not been solved in its industrial application. The most important of these problems is that the large generation of by-product hydrogen peroxide (H2O2) during the reaction process will seriously inhibit the activity of L-glutamate oxidase (LGOX), thereby leading to enzyme inactivation and reduced conversion efficiency. At the same time, engineering bacteria are prone to oxidative stress accumulation when converting high-concentration substrates, causing cell damage and enzyme system instability, which limits their application under high-load and long-term reaction conditions. To achieve the efficient and stable production of α-ketoglutaric acid, it is urgent to construct a new type of engineering strain with strong antioxidant ability, high enzyme expression level, and good system stability to overcome the technical problems of the existing enzyme catalytic system in antioxidant defense and enzyme continuous activity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a plasmid.

[0006] Another technical problem to be solved by the present invention is to provide an α-ketoglutaric acid engineering strain obtained by applying the above plasmid.

[0007] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned α-ketoglutaric acid engineering strain.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned α-ketoglutaric acid engineering strain.

[0009] To solve the above technical problems, the technical solution of the present invention is: A plasmid, namely plasmid pETKG, whose nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing.

[0010] The above plasmid carries plasmid elements such as a replication origin, a kanamycin resistance gene, a T7 promoter, a terminator, etc., and at the same time carries the L-glutamate oxidase LGOX gene derived from Streptomyces sp and Chain A. The T7 promoter is selected to enhance transcription and strengthen the accumulation of L-glutamate oxidase. The T7 promoter is used on the plasmid to express the L-glutamate oxidase LGOX gene derived from Streptomyces sp and Chain A, providing a large amount of L-glutamate oxidase for the whole-cell catalysis of synthesizing α-ketoglutaric acid from sodium L-glutamate.

[0011] Among them, the nucleotide sequence of the LGOX Str gene is shown in SEQ ID NO.8 of the sequence listing, and the nucleotide sequence of the LGOX Cha gene is shown in SEQ ID NO.9 of the sequence listing, and the sequence of the T7 promoter is shown in SEQ ID NO.10 of the sequence listing.

[0012] An α-ketoglutaric acid engineering strain, namely strain KG7, uses E.coli W3110 as the chassis strain, and up-regulates the transcriptional levels of the katE, katG, sodB, and grxA genes ， heterologously expresses the catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis, and at the same time carries the above plasmid pETKG.

[0013] Preferably, the above-mentioned α-ketoglutaric acid engineering strain uses E.coli W3110 as the chassis strain, uses the trc promoter to strengthen the katE gene, and integrates it at the genomic ylbE gene locus; uses the trc promoter to strengthen katGGenes were integrated at the genomic gapC gene locus; the sodB gene was enhanced using the trc promoter and integrated at the genomic rph gene locus; the grxA gene was enhanced using the trc promoter and integrated at the genomic yeeL gene locus; the catR gene derived from Aspergillus niger was enhanced using the trc promoter and integrated at the genomic ycgH gene locus; the katA gene derived from Bacillus subtilis was enhanced using the trc promoter and integrated at the genomic yjgX gene locus.

[0014] Preferably, for the above α-ketoglutaric acid engineering strain, the E.coli W3110 is E.coli W3110 ATCC27325.

[0015] Preferably, for the above α-ketoglutaric acid engineering strain, the nucleotide sequence of the katE gene is as shown in SEQ ID NO.1 in the sequence listing, the nucleotide sequence of the katG gene is as shown in SEQ ID NO.2 in the sequence listing, the nucleotide sequence of the sodB gene is as shown in SEQ ID NO.3 in the sequence listing, the nucleotide sequence of the grxA gene is as shown in SEQ ID NO.4 in the sequence listing, the nucleotide sequence of the catR gene is as shown in SEQ ID NO.5 in the sequence listing, the nucleotide sequence of the katA gene is as shown in SEQ ID NO.6 in the sequence listing, and the sequence of the trc promoter is as shown in SEQ ID NO.7 in the sequence listing.

[0016] The construction method of the above α-ketoglutaric acid engineering strain involves directional modification based on the starting strain E.coli W3110, and the specific steps are as follows: (1) Enhancement of catalase accumulation: Increase katE the expression level of the gene, increase the expression level of the katG gene, heterologously express the catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis, and enhance catalase accumulation; (2) Reduction of catalase consumption: Increase sodB the expression level of the gene, increase the expression level of the grxA gene, promptly scavenge the superoxide anion radicals generated by the engineering bacteria, inhibit the production of hydrogen peroxide, and reduce catalase consumption; (3) Production of L-glutamate oxidase using the pETKG plasmid system: Transform the complete pETKG plasmid into the engineering bacteria.

[0017] Use of the above-mentioned α-ketoglutaric acid engineering strain in the conversion production of α-ketoglutaric acid.

[0018] Preferably, in the use of the above-mentioned α-ketoglutaric acid engineering strain, it is fermented and cultured in a medium for the conversion production of α-ketoglutaric acid.

[0019] Preferably, in the use of the above-mentioned α-ketoglutaric acid engineering strain, the medium includes but is not limited to carbon source, nitrogen source, inorganic salts, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

[0020] Preferably, the specific steps of the use of the above-mentioned α-ketoglutaric acid engineering strain are as follows: ①Slant culture: Inoculate the α-ketoglutaric acid engineering strain on a slant medium and culture it at 32 - 35 °C for 12 - 16 h. The slant medium is preferably a general LB solid medium; ②Seed culture: Inoculate the solid slant strain into a shake flask medium for fermentation. The culture time is 7 - 10 h, the temperature (T) is 35 °C, the pH is 6.5 - 6.8, the rotation speed (R) is 200 rpm, the ventilation (W) is 6 L / min, and the pressure (P) is 0.05 Mpa; ③Fermentation culture: The fermentation inoculation amount is 15 - 20%, the temperature (T) is 35 °C, the pH is 6.9, the rotation speed (R) is 200 rpm, the ventilation (W) is 6 L / min, and the pressure (P) is 0.05 Mpa; ④After the fermentation is completed, centrifuge to collect the thalli. The centrifugation conditions are 5000–6000 r / min. After collecting the thalli and diluting them to 10 - 15 g / L with phosphate buffer solution (PBS), store them at -20 °C for 12 h for standby; ⑤Conversion process: Use a 5 L bioreactor with a liquid loading of 2 L. Add sodium glutamate with a concentration of 100 g / L (as the conversion reaction substrate), adjust the pH to 6.5 and the temperature to 32 °C to the predetermined values. Add the frozen thalli in step ④, control the air volume at 15 - 20 L / min, the rotation speed (R) at 200 - 400 rpm, and the tank pressure (P) at 0.05 Mpa. Use the biocatalytic membrane reactor SBA-40E to detect the content of sodium glutamate until the reaction stops when there is no substrate residue.

[0021] Preferably, the seed medium used in step ② is: glucose 25 g / L, yeast 3.5 g / L, peptone 1.3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 2 g / L, citric acid 1.5 g / L, MnSO4·H2O 5 mg / L, and the rest is water.

[0022] Preferably, for the application of the above α-ketoglutaric acid engineering strain, the fermentation medium used in step ③ is: glucose 15 g / L, yeast powder 3 g / L, peptone 1 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 5 g / L, MgSO4·7H2O 2 g / L, MnSO4·H2O 10 mg / L, FeSO4·7H2O 30 mg / L, and the rest is water.

[0023] The above media can all be prepared by standard methods.

[0024] Beneficial effects: The above α-ketoglutaric acid engineering strain is obtained by a directed modification method, up-regulating the kat E, kat G, sod B, grx transcription levels of genes A, heterologously expressing the catR gene derived from Aspergillus niger and the catA gene derived from Bacillus subtilis and the kat A gene, and carrying a high-copy plasmid pETKG, the plasmid uses the T7 promoter to simultaneously express the Streptomyces sp and Chain L-glutamate oxidase LGOX gene of A, the glutamate oxidase gene carried by the plasmid efficiently accumulates L-glutamate oxidase, provides sufficient L-glutamate oxidase for the whole-cell catalysis of L-glutamate sodium to synthesize α-ketoglutaric acid during the conversion process, improves the catalytic efficiency, can effectively improve the synthesis efficiency of α-ketoglutaric acid, improves the production level of α-ketoglutaric acid, and realizes the high-efficient production of α-ketoglutaric acid; the strain itself synthesizes catalase to eliminate the H2O2 generated during the enzymatic production of α-ketoglutaric acid, and uses the engineered bacterium expressing L-glutamate oxidase LGOX with the pETKG plasmid for the whole-cell catalysis of L-glutamate sodium to synthesize α-ketoglutaric acid. After 14 h of conversion, the yield of α-ketoglutaric acid is 64.85 g / L, and the conversion rate is 83.15%. It is an engineering strain that can stably produce α-ketoglutaric acid at a high level and can be industrialized, and has excellent industrial application prospects.

[0025] The above α-ketoglutaric acid engineering strain has good catalase synthesis ability, stable performance, and high enzyme production efficiency. Specifically: (1) By increasing the katE expression level of the gene, increasing the expression level of the katG gene, heterologously expressing the catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis, and strengthening the accumulation of catalase.

[0026] (2)By increasingsodB The expression level of the gene, improve the expression level of the grxA gene, timely scavenge the superoxide anion radicals produced by the engineered bacteria, inhibit the production of hydrogen peroxide, and reduce the consumption of catalase.

[0027] (3) The pETKG plasmid carries plasmid elements such as the replication origin, kanamycin resistance gene, T7 promoter, and terminator, and at the same time carries the L-glutamate oxidase (LGOX) gene from Streptomyces sp and Chain A; the T7 promoter is selected to enhance transcription, strengthening the accumulation of L-glutamate oxidase. Description of the Drawings

[0028] Figure 1 It is a schematic diagram of the structure of plasmid pETKG.

[0029] Figure 2 It is a schematic diagram of the structure of plasmid pET-28a.

[0030] Figure 3 It is a schematic diagram of the transformation of the engineered strain KG7 and enzyme conversion. Among them, at the stage of cell enzyme production, when the OD reaches 30, IPTG is added to start inducing the expression of the L-glutamate oxidase gene from Streptomyces sp and Chain A by the T7 promoter, realizing the large accumulation of L-glutamate oxidase. After 8 hours of induction, the fermentation ends. After the fermentation ends, the cells are collected by centrifugation under the conditions of 5000 - 6000 r / min. The collected cells are diluted to 10 - 15 g / L with phosphate buffer (PBS) and then stored at -20 °C for 12 h for standby; in the glutamate conversion stage, the conversion process is to use a 5 L bioreactor with a liquid loading of 2 L, add sodium glutamate with a concentration of 100 g / L as the conversion reaction substrate, adjust the Ph = 6.5 and the temperature = 32 °C to the predetermined values, add the frozen cells, control the air volume at 15 - 20 L / min, the rotation speed R = 300 rpm, the tank pressure P = 0.05 Mpa, and use the biocatalytic membrane reactor SBA-40E to detect the content of sodium glutamate until the reaction stops when there is no substrate residue. Detailed Embodiments

[0031] In order 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 in conjunction with the specific embodiments.

[0032] The percentage "%" involved in the embodiments, unless otherwise specified, refers to the mass percentage. The percentage of the solution refers to the number of grams of the solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25 °C.

[0033] The starting strain (chassis strain) used in the embodiments is E.coliW3110 ATCC 27325, the corresponding promoter and gene etc. can be found in the sequence listing. The primers used in the strain construction process are shown in Table 1.

[0034] Table 1 Primers Involved in the Strain Construction Process Primer Name Sequence Number Primer Sequence (5'-3') pGRB-ylbE-S SEQ ID NO.12 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-A SEQ ID NO.13 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ylbE-US SEQ ID NO.14 ACCCAACCTTACGCAACCAGTTAA ylbE-UA SEQ ID NO.15 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCATTG ylbE-DS SEQ ID NO.16 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAACA ylbE-DA SEQ ID NO.17 GGGCGTAACTCAGCAGGCAG katE-S SEQ ID NO.18 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCGCAACATAACGAAAAGAACCCA katE-A SEQ ID NO.19 ACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAGGCAGGAATTTTGTCAATCTTAGGAA pGRB-gapC-S SEQ ID NO.20 AGTCCTAGGTATAATACTAGTAGGTCATGCGCAACGCGTGCGTTTTAGAGCTAGAA pGRB-gapC-A SEQ ID NO.21 TTCTAGCTCTAAAACGCACGCGTTGCGCATGACCTACTAGTATTATACCTAGGACT gapC-US SEQ ID NO.22 TGGGAAGAAACCACGAAACTCCA gapC-UA SEQ ID NO.23 TATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGAA gapC-DS SEQ ID NO.24 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG gapC-DA SEQ ID NO.25 TTATCCGCCGACATTGCTGC katG-S SEQ ID NO.26 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCACGTCAGACGATATCCAT katG-A SEQ ID NO.27 CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCAGGTCGAAACGGTCG pGRB-rph-S SEQ ID NO.28 AGTCCTAGGTATAATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA pGRB-rph-A SEQ ID NO.29 TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT rph-US SEQ ID NO.30 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.31 TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACAC rph-DS SEQ ID NO.32 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACATGAA rph-DA SEQ ID NO.33 ACAGCGGTTGTGGTGGCAAT sodB-S SEQ ID NO.34 ATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCATTCGAATTACCTGCACTACCA sodB-A SEQ ID NO.35 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATGCAGCGAGATTTTTCGCTACGA pGRB-yeeL-S SEQ ID NO.36 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-yeeL-A SEQ ID NO.37 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT yeeL-US SEQ ID NO.38 TTCATCGGGACGAGTGGAGA yeeL-UA SEQ ID NO.39 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGA yeeL-DS SEQ ID NO.40 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGCC yeeL-DA SEQ ID NO.41 CATTCCCTCTACAGAACTAGCCCTT grxA-S SEQ ID NO.42 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCAAACCGTTATTTTTGGTCGTTC grxA-A SEQ ID NO.43 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAGGCGTCCAGATTTTCTTTCAC pGRB-ycgH-S SEQ ID NO.44 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA pGRB-ycgH-A SEQ ID NO.45 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT ycgH-US SEQ ID NO.46 TAAACTCGTCAGCGGCACAAC ycgH-UA SEQ ID NO.47 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTGAA ycgH-DS SEQ ID NO.48 TTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCGA ycgH-DA SEQ ID NO.49 GATTCAGGTTGCCATTTACGCCA catR-S SEQ ID NO.50 CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGTCATTTCTGGCTTTTGCC catR-A SEQ ID NO.51 TTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTCATCCAGCGCAAACCGG pGRB-yjgX-S SEQ ID NO.52 AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-A SEQ ID NO.53 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGAC yjgX-US SEQ ID NO.54 GGAAGTCAACGGGTTATGCGG yjgX-UA SEQ ID NO.55 TTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATCGC yjgX-DS SEQ ID NO.56 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.57 GGCGAAGGATACCATCAAGCTG katA-S SEQ ID NO.58 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGTTCAAATAAACTGACAACTAGCTGGG katA-A SEQ ID NO.59 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAGAATCTTTTTTAATCGGCAATCCAAGGCC pet-XZ-A SEQ ID NO.60 AGCCTTCAACCCAGTCAGCT pet-XZ-S SEQ ID NO.61 ACTGAGATCCGGCTGCTAACA <![CDATA[LGOX cha -pet-S]]> SEQ ID NO.62 CTAAAATTCAGTTAGGAGGTATTTGATGGGTTGGCCAACGAAATGAC <![CDATA[LGOX cha -pet-A]]> SEQ ID NO.63 GTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGTTACCCGCCGCCGCGGTCACACCGGTGTCGC <![CDATA[LGOX str -pet-S]]> SEQ ID NO.64 AACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAATGACCGACACCGCGC <![CDATA[LGOX str -pet-A]]> SEQ ID NO.65 CTAACTGAATTTTAGCGAGGTCAGGGCCTCCTC In the examples, the α-ketoglutaric acid engineering strain was constructed through the following 3 modules: (1) By increasing katE the expression level of the gene, increasing the expression level of the katG gene, heterologously expressing the catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis, and strengthening the accumulation of catalase.

[0035] (2) By increasing sodB the expression level of the gene, increasing the expression level of the grxA gene, timely scavenging the superoxide anion radicals produced by the engineering bacteria, inhibiting the production of hydrogen peroxide, and reducing the consumption of catalase.

[0036] (3) Transforming the complete pETKG plasmid into the engineering bacteria. The pETKG plasmid carries plasmid elements such as the replication origin, kanamycin resistance gene, T7 promoter, terminator, etc., and at the same time carries the L-glutamate oxidase (LGOX) gene derived from Streptomyces sp and Chain A. The T7 promoter was selected to enhance transcription and strengthen the accumulation of L-glutamate oxidase.

[0037] The gene editing method used in the above gene operations refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). For the professional terms involved in the present invention, unless otherwise noted, they can all be explained in this article. "Introduction" means inserting the foreign gene into the engineering bacteria genome after connecting it with the promoter and terminator.

[0038] Example 1 This example aims to illustrate the specific construction steps of strain KG6. In particular, if there are gene operation methods of the same type in the examples, only one time will be provided and annotated, and no more details will be given.

[0039] ① Overexpress the katE gene under the control of the trc promoter at the ylbE pseudogene locus: Using E.coliUsing the W3110 genome as a template, with ylbE-US, ylbE-UA and ylbE-DS, ylbE-DA, katE-S , katE-A as primers, the upstream homologous arm, downstream homologous arm and katE gene fragment were obtained by PCR amplification. Then, using the upstream and downstream homologous arms and the katE gene fragment as templates, with ylbE-US and ylbE-DA as primers, an overlapping fragment was obtained by overlapping PCR amplification; using pGRB-ylbE-S and pGRB-ylbE-A as primers, a gRNA fragment was obtained by annealing and ligated it to the pGRB vector to obtain ylbE-pGRB; prepare E.coli W3110 electrocompetent cells, electrotransform the overlapping fragment and ylbE-pGRB into the competent cells together, and screen for positive transformants to obtain strain KG1.

[0040] ② Overexpress the katG gene using the trc promoter at the gapC pseudogene locus: Using E.coli the W3110 genome as a template, with gapC-US, gapC-UA and gapC-DS, gapC-DA, katG-S, katG-A as primers, the upstream homologous arm, downstream homologous arm and katG gene fragment were obtained by PCR amplification. Then, using the upstream and downstream homologous arms and the katG gene fragment as templates, with gapC-US and gapC-DA as primers, an overlapping fragment was obtained by overlapping PCR amplification; using pGRB-gapC-S and pGRB-gapC-A as primers, a gRNA fragment was obtained by annealing and ligated it to the pGRB vector to obtain gapC-pGRB; prepare E.coli W3110 electrocompetent cells, electrotransform the overlapping fragment and gapC-pGRB into the competent cells together, and screen for positive transformants to obtain strain KG2.

[0041] ③ Overexpress the sodB gene using the trc promoter at the rph pseudogene locus: Using E.coli the W3110 genome as a template, with rph-US, rph-UA and rph-DS, rph-DA, sodB-S, sodB-A as primers, the upstream homologous arm, downstream homologous arm and sodB gene fragment were obtained by PCR amplification. Then, using the upstream and downstream homologous arms and the sodB gene fragment as templates, with rph-US and rph-DA as primers, an overlapping fragment was obtained by overlapping PCR amplification; using pGRB-rph-S and pGRB-rph-A as primers, a gRNA fragment was obtained by annealing and ligated it to the pGRB vector to obtain rph-pGRB; prepare E.coliThe electrocompetent cells of W3110 were used. The overlapping fragment and rph-pGRB were co-electroporated into the electrocompetent cells, and positive transformants were screened to obtain the strain KG3.

[0042] ④ Overexpression of the grxA gene was controlled by the trc promoter at the yeeL pseudogene locus: Using E.coli the W3110 genome as a template, the upstream homologous arm, downstream homologous arm, and grxA gene fragment were amplified by PCR using yeeL-US, yeeL-UA, yeeL-DS, yeeL-DA, grxA-S, and grxA-A as primers. Then, using the upstream and downstream homologous arms and the grxA gene fragment as templates, and yeeL-US and yeeL-DA as primers, an overlapping fragment was amplified by overlapping PCR; gRNA fragments were obtained by annealing using pGRB-yeeL-S and pGRB-yeeL-A as primers, and they were ligated to the pGRB vector to obtain yeeL-pGRB; Preparation of E.coli the electrocompetent cells of W3110. The overlapping fragment and yeeL-pGRB were co-electroporated into the electrocompetent cells, and positive transformants were screened to obtain the strain KG4.

[0043] ⑤ Overexpression of the catR gene was controlled by the trc promoter at the ycgH pseudogene locus: Using E.coli the W3110 genome as a template, the upstream homologous arm, downstream homologous arm, and catR gene fragment were amplified by PCR using ycgH-US, ycgH-UA, ycgH-DS, ycgH-DA, catR-S, and catR-A as primers. Then, using the upstream and downstream homologous arms and the catR gene fragment as templates, and ycgH-US and ycgH-DA as primers, an overlapping fragment was amplified by overlapping PCR; gRNA fragments were obtained by annealing using pGRB-ycgH-S and pGRB-ycgH-A as primers, and they were ligated to the pGRB vector to obtain ycgH-pGRB; Preparation of E.coli the electrocompetent cells of W3110. The overlapping fragment and ycgH-pGRB were co-electroporated into the electrocompetent cells, and positive transformants were screened to obtain the strain KG5.

[0044] ⑥ Overexpression of the katA gene was controlled by the trc promoter at the yjgX pseudogene locus: Using E.coliUsing the W3110 genome as a template, the upstream homologous arm, downstream homologous arm, and katA gene fragment were amplified by PCR using yjgX-US, yjgX-UA and yjgX-DS, yjgX-DA, katA-S, and katA-A respectively. Then, using the upstream and downstream homologous arms and the katA gene fragment as templates and yjgX-US and yjgX-DA as primers, an overlapping fragment was amplified by overlapping PCR; using pGRB-yjgX-S and pGRB-yjgX-A as primers, a gRNA fragment was annealed and ligated to the pGRB vector to obtain yjgX-pGRB; prepare E.coli electrocompetent cells of W3110, co-electrotransform the overlapping fragment and yjgX-pGRB into the electrocompetent cells, and screen for positive transformants to obtain strain KG6.

[0045] ⑦ Transform plasmid pETKG to obtain engineering strain KG7: Transform the complete plasmid pETKG into KG6 electrocompetent cells by electroporation (or other methods such as chemical transformation) to obtain engineering strain KG7.

[0046] Example 2 This example aims to illustrate the construction method of plasmid pETKG in Example 1 (as Figure 1 shown), and the specific steps are as follows: Using Streptomyces sp genome as a template and LGOX str -pet-S and LGOX str -pet-A as primers, the LGOX str gene fragment was amplified; using Chain A genome as a template and LGOX cha -pet-S and LGOX cha -pet-A as primers, the LGOX cha gene fragment was amplified; then, using the LGOX str gene fragment, the LGOX cha gene fragment, and LGOX str -pet-S and LGOX cha -pet-A as primers, an overlapping fragment was amplified by overlapping PCR; using the pET-28a plasmid (as Figure 2 shown, purchased from EasyGo Biotechnology (Tianjin) Co., Ltd.) as a template and pet-XZ-S and pet-XZ-A as primers, a linear vector fragment was amplified; the above-mentioned genes were overexpressed in the plasmid through the T7 promoter.

[0047] The gene fragment is ligated to the linearized plasmid vector by a recombinase to obtain a complete plasmid, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO.11. Any recombinase can be selected for the ligation method. In this example, the ClonExpress® rapid cloning technology of Nanjing Novozymes Biotech Co., Ltd. is used to construct the overexpression plasmid.

[0048] Example 3 This example aims to illustrate the fermentation application of engineering bacterium KG7. As Figure 3 shown, the specific steps are as follows: ① Slant culture: Inoculate the α-ketoglutaric acid engineering strain on the slant medium and culture it at 32 - 35 °C for 14 h. The slant medium is a general LB solid medium. ② Seed culture: Inoculate the solid slant strain into the shake flask medium for fermentation. The culture time is 8 h, T = 35 °C, pH = 6.5 - 6.8, R = 200 rpm, W = 6 L / min, P = 0.05 Mpa. The seed medium used is: glucose 25 g / L, yeast 3.5 g / L, peptone 1.3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 2 g / L, citric acid 1.5 g / L, MnSO4·H2O 5 mg / L, and the rest is water.

[0049] ③ Fermentation culture: The fermentation inoculation amount is 15 - 20%, T = 35 °C, PH = 6.9, R = 200 rpm, W = 6 L / min, P = 0.05 Mpa. The fermentation medium used is: glucose 15 g / L, yeast powder 3 g / L, peptone 1 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 5 g / L, MgSO4·7H2O 2 g / L, MnSO4·H2O 10 mg / L, FeSO4·7H2O 30 mg / L, and the rest is water. Before induction, control the good growth environment of the bacteria, express the catalase gene, and achieve the accumulation of catalase. When OD reaches 30, add IPTG to start inducing the expression of the L-glutamate oxidase gene derived from Streptomyces sp and Chain A by the T7 promoter to achieve the large accumulation of L-glutamate oxidase. After 8 hours of induction, end the fermentation.

[0050] ④ After ending the fermentation, centrifuge to collect the bacteria. The centrifugation conditions are 5000 - 6000 r / min. Dilute the collected bacteria with phosphate buffer (PBS) to 10 - 15 g / L and then store them at -20 °C for 12 h for standby.

[0051] ⑤Conversion process: Use a 5L bioreactor with a liquid filling volume of 2L. Add sodium glutamate with a concentration of 100g / L as the conversion reaction substrate. Adjust the pH to 6.5 and the temperature to 32°C to the predetermined values. Add the frozen bacteria in step ④. Control the air volume at 15 - 20L / min, the rotation speed R = 300rpm, and the tank pressure P = 0.05Mpa. Use the biocatalytic membrane reactor SBA-40E to detect the content of sodium glutamate. Stop the reaction until there is no substrate residue. The yield of catalytic synthesis of α-ketoglutaric acid reaches 64.85g / L, the conversion rate of glutamic acid reaches 83.15%, and the enzyme catalytic cycle is shortened to 14h.

[0052] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made. The improvements and refinements such as strain transformation by those skilled in this technical field using the method of the present invention or based on this method are regarded as the protection scope of the present invention.

Claims

1. A plasmid, characterized in that: Its nucleotide sequence is as shown in Sequence Listing SEQ ID NO.

11.

2. An α-ketoglutaric acid engineering strain, characterized in that: Using E.coli W3110 as the chassis strain, the transcriptional levels of the katE, katG, sodB, and grxA genes were upregulated ， The catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis were heterologously expressed, and the plasmid pETKG described in claim 1 was carried at the same time.

3. The α-ketoglutaric acid engineering strain according to claim 2, characterized in that: Using E.coli W3110 as the chassis strain, the katE gene was strengthened using the trc promoter and integrated at the ylbE gene locus of the genome; the katG gene was strengthened using the trc promoter and integrated at the gapC gene locus of the genome; the sodB gene was strengthened using the trc promoter and integrated at the rph gene locus of the genome; the grxA gene was strengthened using the trc promoter and integrated at the yeeL gene locus of the genome; the catR gene derived from Aspergillus niger was strengthened using the trc promoter and integrated at the ycgH gene locus; the katA gene derived from Bacillus subtilis was strengthened using the trc promoter and integrated at the yjgX gene locus.

4. The α-ketoglutaric acid engineering strain according to claim 2 or 3, characterized in that: The said E.coli W3110 is E.coli W3110 ATCC 27325.

5. The α-ketoglutaric acid engineering strain according to claim 2 or 3, characterized in that: The nucleotide sequence of the said katE gene is as shown in Sequence Listing SEQ ID NO. 1, the nucleotide sequence of the katG gene is as shown in Sequence Listing SEQ ID NO. 2, the nucleotide sequence of the sodB gene is as shown in Sequence Listing SEQ ID NO. 3, the nucleotide sequence of the grxA gene is as shown in Sequence Listing SEQ ID NO. 4, the nucleotide sequence of the catR gene is as shown in Sequence Listing SEQ ID NO. 5, the nucleotide sequence of the katA gene is as shown in Sequence Listing SEQ ID NO. 6, and the sequence of the trc promoter is as shown in Sequence Listing SEQ ID NO.

7.

6. The method for constructing the α-ketoglutaric acid engineering strain according to any one of claims 2-5, characterized in that: Based on the starting strain E.coli W3110, the directed modification was carried out as follows: (1) Enhance the accumulation of catalase: Improve katE The expression level of the gene was improved, the expression level of the katG gene was improved, and the catR gene derived from Aspergillus niger and the katA gene derived from Bacillus subtilis were heterologously expressed; (2) Reduce the consumption of catalase: Improve sodB the expression level of genes and improve the expression level of the grxA gene; (3) Produce L-glutamate oxidase with the pETKG plasmid system: Transform the complete pETKG plasmid into the engineering bacteria.

7. Use of the α-ketoglutaric acid engineering strain as described in any one of claims 2 - 5 in the conversion production of α-ketoglutaric acid.

8. The application according to claim 7, wherein: The specific steps are as follows: ① Slant culture: Inoculate the α-ketoglutaric acid engineering strain on the slant medium and culture at 32 - 35 °C for 12 - 16 h. The slant medium is selected as the general LB solid medium; ② Seed culture: Inoculate the solid slant strain into the shake flask medium for fermentation. The culture time is 7 - 10 h, the temperature is 35 °C, the pH is 6.5 - 6.8, the rotation speed is 200 rpm, the ventilation is 6 L / min, and the pressure is 0.05 Mpa; ③ Fermentation culture: The fermentation inoculation amount is 15 - 20%, the temperature is 35 °C, the PH is 6.9, the rotation speed is 200 rpm, the ventilation is 6 L / min, and the pressure is 0.05 Mpa; ④ After the fermentation ends, centrifuge to collect the thallus. The centrifugation conditions are 5000–6000 r / min. After diluting the collected thallus with phosphate buffer to 10 - 15 g / L, store it at -20 °C for 12 h for standby; ⑤ Conversion process: Use a 5 L bioreactor with a liquid loading of 2 L. Add sodium glutamate with a concentration of 100 g / L, adjust the pH to 6.5 and the temperature to 32 °C to the preset values. Add the frozen thallus in step ④, control the air volume at 15 - 20 L / min, the rotation speed at 200 - 400 rpm, and the tank pressure at 0.05 Mpa. Use the biocatalytic membrane reactor SBA - 40E to detect the content of sodium glutamate until the reaction stops when there is no substrate residue.

9. The application according to claim 8, characterized in that: The seed medium used in step ② is: glucose 25 g / L, yeast 3.5 g / L, peptone 1.3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 2 g / L, citric acid 1.5 g / L, MnSO4·H2O 5 mg / L, and the rest is water.

10. The application according to claim 8, characterized in that: The fermentation medium used in step ③ is as follows: glucose 15 g / L, yeast powder 3 g / L, peptone 1 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 5 g / L, MgSO4·7H2O 2 g / L, MnSO4·H2O 10 mg / L, FeSO4·7H2O 30 mg / L, and the rest is water.

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