Alpha-ketoisocaproic acid producing strain as well as construction method and application thereof

Abstract

The invention provides a strain for producing alpha-ketoisocaproic acid as well as a construction method and application thereof. The strain is obtained by taking C.glutamicum Val19 as an initial strain through a gene modification method and comprises the following steps: knocking out an ltbR gene, and introducing a leuB gene at an NcgCgl0021a site; the method comprises the following steps: introducing a leuCD gene at a site NcgCgl0998a to obtain a strain KlC3; the method comprises the following steps: linearizing a pXtuf plasmid by using Hind III and BamH I, replacing a Ptuf promoter with a Ptac promoter, then connecting a euA gene segment with the Ptac promoter, and electrically transferring the obtained pXtuf-euA plasmid into a KIC3 competent state; the constructed strain can produce alpha-ketoisocaproic acid at high yield, exceeds the previous fermentation result and most of the whole cell catalysis process, and highlights the industrial application potential of alpha-ketoisocaproic acid.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to an alpha-ketoisocaproic acid producing strain and a construction method and application thereof. Background Art

[0002] α-Ketoisocaproic acid (KIC) is an organic compound that plays a key role in many fields. In the medical field, they can be used to treat chronic kidney disease; in the food industry, they can be used as a nitrogen-free substitute for the corresponding branched-chain amino acids (BCAAs); in terms of biosynthesis, α-ketoisocaproic acid has the effects of reducing protein catabolism, stimulating protein synthesis, inhibiting glucagon secretion, and enhancing insulin secretion. In addition, they can also be catalyzed into branched higher alcohols such as isobutanol and isopentanol through specific pathways. These alcohols have excellent physical and chemical properties and show broad application prospects in the field of new biofuels.

[0003] At present, the production of α-ketoisocaproic acid is mainly through chemical synthesis, but this method has many disadvantages. On the one hand, it relies on non-renewable resources, needs to be carried out under harsh conditions during the production process, uses toxic reactants, and produces dangerous by-products, causing serious damage to the environment. On the other hand, chemical synthesis requires expensive catalysts, special starting materials, and numerous subsequent purification steps, resulting in high production costs.

[0004] As the research on the production of KIC by biotechnology continues to develop, the bioconversion of L-leucine to KIC by expressing L-amino acid oxidase or L-amino acid deaminase with whole-cell catalysts has been widely studied. However, due to the extremely high cost of pure L-leucine (about $5,000 / kg), while the price of the final product KIC is only $22 / kg, this conversion method lacks economic feasibility. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an alpha-ketoisocaproic acid producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned α-ketoisocaproic acid producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned α-ketoisocaproic acid producing strain.

[0008] In order to solve the above technical problems, the technical solution of the present invention is:

[0009] An α-ketoisocaproic acid producing strain is obtained by genetic modification using C. glutamicum Val19 as a starting strain: the ltbR gene (KEGG: cgl486) is knocked out, the leuB gene encoding 3-isopropylmalate dehydrogenase (KEGG [EC: 1.1.1.85]) is introduced at the NcgCglOO21a site, and the P tuf Promoter start; introduce part of the leucine biosynthesis operon gene leuCD (KEGG [EC: 4.2.1.334.2.1.35]) at the NcgCgl0998a site, by P tuf The promoter was activated to obtain strain KIC3;

[0010] The pXtuf plasmid (derived from pXMJ19 (GenBank: AJ133195.1), constructed by referring to Gui Yongli, Ma Yuechao, Xu Qingyang, et al. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese L-leucine producing strain [J]. Journal of Biotechnology, 2016, (Null): 1-2.) was linearized with Hind III and BamH I, and P tac The promoter was replaced by P tuf Promoter, and then use ClonExpress II One Step Cloning Kit (Nanjing Vazyme Biotech Company) to connect the leuA gene fragment amplified from the genome of C. glutamicum CP with it through homologous recombination, and the obtained pXtuf-leuA plasmid is electroporated into KIC3 competent cells to obtain the final strain.

[0011] Preferably, in the above-mentioned α-ketoisocaproic acid producing strain, the nucleotide sequence of the leuA gene is shown in the sequence listing SEQ ID NO.25.

[0012] The method for constructing the above-mentioned α-ketoisocaproic acid producing strain uses CRISPR / Cas9-mediated gene editing technology for gene modification, and the specific steps are as follows:

[0013] (1) Take C.glutamicum ATCC 13032 rpsL K43R The genome was used as a template to amplify the upstream and downstream homology arms of ΔItbR by PCR. The overlapping fragments ΔItbR were obtained by overlapping PCR and then cloned into the linearized vector pK18mobrpsl (Ptuf-rpsL cassette, kanR Resistance) homologous recombination and chemical transformation were used to construct the plasmid pK18mobrpsL-ΔltbR, which was electroporated into the competent cell of C. glutamicum Val19 to obtain the ItbR gene knockout strain KIC1;

[0014] (2) The leuB gene was integrated into the NcgCg / 0021a site of strain KIC1, and the P tuf Promoter was used to construct plasmid pK18mobrpsL-NCgI0021a::Ptuf-leuB and electrotransformed to obtain strain KIC2 with LeuB gene integrated;

[0015] (3) The leuCD gene was integrated into the NcgCgl0998a locus of strain KIC2 by P tuf Promoter was used to construct plasmid pK18mobrpsL-Ncgl0998a::Ptuf-leuCD and electrotransformed to obtain strain KIC3;

[0016] (4) The pXtuf plasmid was linearized with Hind III and BamH I, the Ptac promoter was replaced with the Ptuf promoter, and the leuA gene fragment amplified from the C. glutamicum CP genome was ligated to it using homologous recombination. The pXtuf-leuA plasmid was electroporated into the KIC3 competent cell to obtain the final strain.

[0017] Application of the α-ketoisocaproic acid producing strain in producing α-ketoisocaproic acid.

[0018] Preferably, the α-ketoisocaproic acid producing strain is used to produce α-ketoisocaproic acid by a fermentation culture method.

[0019] Preferably, in the application of the above-mentioned α-ketoisocaproic acid producing strain, the fermentation culture is fermentation in a fermenter.

[0020] Preferably, the specific steps of the fermentation in the fermenter are as follows:

[0021] (1) Activation of bacterial strains: Take bacteria from the -80°C bacterial preservation tube and place them on an LB chloramphenicol resistance plate (LB plate containing 12 mg / L chloramphenicol), divide them into three zones, culture them at 32°C for 32-36 h, spot them on the LB chloramphenicol resistance plate for 14-18 h, inoculate chloramphenicol in a BHI shake tube, take bacteria from the BHI shake tube and inoculate them on a slant, culture them at 32°C for 16-18 h, and you can get a first-generation activated slant; transfer the first-generation slant to an eggplant-shaped flask and culture them at 32°C for 8-12 h to get a second-generation activated slant;

[0022] (2) Seed culture: The cells cultured on the second generation activated slant were transferred to a fermenter (Baoxing, Shanghai, China) containing 3 L of seed culture medium to start seed culture; the pH value was automatically maintained at 7.0 by adding ammonia water; the dissolved oxygen content was maintained at 25-35% and the temperature was maintained at 32 ± 0.2 °C by adjusting the stirring speed and ventilation; seed culture was carried out;

[0023] (3) Fermentation culture: Seed culture until OD 600 When the pH reaches about 20-25, 600 mL of culture medium is retained for fed-batch fermentation; during the fed-batch fermentation, the pH value is adjusted to 7.0 by ammonia water 24 hours before fermentation, and sodium acetate is added after 24 hours to adjust the pH value to 7.0, and the temperature is controlled at 32°C; when the glucose is exhausted, 80% (w / v) glucose solution is added to maintain the glucose concentration above 3 g / L to ensure a continuous supply of glucose.

[0024] Preferably, in the application of the above-mentioned α-ketoisocaproic acid producing strain, the slant culture medium and eggplant flask culture medium used in the step (1) are: peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, NaCl 2.5 g / L, corn steep liquor 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, agar 2%, pH 7.0-7.2, and the rest is water.

[0025] The sterilization conditions of the above slant culture medium and eggplant flask culture medium are: 121°C, 20min; the working concentration of chloramphenicol is 12mg / L, which is added after sterilization to maintain the working concentration of plasmid PXtuf-IeuA.

[0026] Preferably, in the application of the above-mentioned α-ketoisocaproic acid producing strain in a fermentation tank, the seed culture medium used in the step (2) is: 40g / L glucose, 5g / L corn syrup powder, 5g / L yeast extract, 5g / L peptone, 3g / L (NH4)2SO4, 1.5g / L KH2PO4, 0.4g / L MgSO4·7H2O, 0.5g / L valine, 0.5g / L leucine, 0.5g / L isoleucine, 0.5g / L methionine, 1g / L glutamic acid, 1g / L citric acid, 10mg / L FeSO4·7H2O, 10mg / L MnSO4·7H2O, VB1, VB3, VB5, VB 12 0.4 mg / L, 0.1 mg / L VH (pH 7.0-7.2), and the rest is water.

[0027] Preferably, in the application of the above-mentioned α-ketoisocaproic acid producing strain in a fermentation tank, the fermentation medium (pH 7.0-7.2) used in step (3) is: 80 g / L glucose, 20 g / L corn flour, 3 g / L yeast extract, 3 g / L peptone, 5 g / L (NH4)2SO4, 2.5 g / L KH2PO4, 1.6 g / L MgSO4·7H2O, 1 g / L valine, 1 g / L leucine, 1 g / L isoleucine, 1 g / L methionine, 5 g / L glutamic acid, 2 g / L citric acid, 10 mL / L soybean meal extract, 20 mg / L FeSO4·7H2O, 20 mg / L MnSO4·7H2O, VB1, VB3, VB5, VB 12 0.3 mg / L each, the rest is water.

[0028] The above culture media can be prepared using standard methods.

[0029] Beneficial effects:

[0030] The above-mentioned α-ketoisocaproic acid producing strain is a strain that can produce a high amount of α-ketoisocaproic acid, which is obtained by deleting the transcriptional repressor LtbR and increasing the gene copy number to overexpress the genes in the L-leucine biosynthesis operon (IeuA, IeuCD and IeuB) under a strong promoter; during the fermentation and cultivation of α-ketoisocaproic acid, the supply of acetyl-coenzyme A is enhanced by the supplementation of acetate, thereby increasing the production of α-ketoisocaproic acid and reducing the accumulation of KIV. The α-ketoisocaproic acid accumulated by the strain in a 5L fermenter exceeds previous fermentation results and most of the entire cell catalytic processes, highlighting its potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the fermentation yield diagram of the α-ketoisocaproate producing strain. DETAILED DESCRIPTION

[0032] 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 is further described in detail below in conjunction with specific implementation methods.

[0033] For information on the strain C. glutamicum Val19, corresponding promoters, terminators and genes used in the following examples, please refer to the references Wang F, Cai N, Leng Y, Wu C, Wang Y, Tian S, Zhang C, Xu Q, Peng H, Chen N, Li Y. Metabolic Engineering of Corynebacterium glutamicum for the High-Level Production of I-Valine under Aerobic Conditions. ACS Synth Biol. 2024 Sep 20; 13(9): 2861-2872. doi: 10.1021 / acssynbio.4c00278. Epub 2024 Jun 30. PMID: 38946081. The source of C. glutamicum CP is Gui Yongli, Ma Yuechao, Xu Qingyang, et al. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese L-Ieucine productionstrain[J]. Journal of Biotechnology, 2016, (Null): 1-2. The pXtuf plasmid is a chloramphenicol-resistant plasmid derived from pXMJ19 (GenBank: AJ133195.1, for construction methods, see Gui Yongli, Ma Yuechao, Xu Qingyang, et al. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese L-leucine producing strain [J]. Journal of Biotechnology, 2016, (Nul1): 1-2. pK18mobrpsL is a plasmid that replaces the sacB gene of pK18mobsacB with Ptuf-rpsL. pK18mobsacB (a suicide vector containing the sacB gene of Bacillus subtilis; kanR resistance) is referenced by Tauch, A., Kirchner, 0., B., Gotker, S., A., and Kalinowski, J. (2002) Efficient electrotransformation of Corynebacterium diphtheriae with a mini-repliconderived from the Corynebacterium glutamicum plasmid pGA1. CurrMicrobiol 45: 362-367.

[0034] The primers used in the construction of the strains involved are shown in Table 1.

[0035] Table 1 Primers involved in strain construction

[0036]

[0037]

[0038] In the strain construction process in the following examples, CRISPR / Cas9-mediated gene editing technology was used to gradually transform C. glutamicum Val19. The specific steps were based on an updated suicide plasmid-mediated genome editing system (Wang T, Li Y, Li J, Zhang D, Cai N, Zhao G, Ma H, Shang C, Ma Q, Xu Q, Chen N. An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum. Microb Biotechnol. 2019 Sep; 12 (5): 907-919. doi: 10.1111 / 1751-7915.13444. Epub 2019 Jun 10. PMID: 31180185; PMCID: PMC6680612.)

[0039] Example 1

[0040] This example is intended to illustrate the specific steps for constructing an α-ketoisocaproic acid producing strain.

[0041] 1. Construction of strain KIC3

[0042] C.glutamicum ATCC 13032 rpsL K43R (rpsL of Corynebacterium glutamicum ATCC 13032 K43RThe mutant was endowed with streptomycin resistance) genome as a template for PCR amplification, and the primers ΔItbP-up-1 (SEQ ID NO.3) and ΔItbR-up-2 (SEQ ID NO.4) were designed using Primer Premier 5.0 software to amplify the upstream homology arm, and the primers ΔItbR-down-1 (SEQ ID NO.5) and ΔItbR-down-2 (SEQ ID NO.6) were used to amplify the downstream homology arm. Then, overlapping PCR was performed, and the overlapping fragment ΔItbR was amplified using primers ΔItbR-up-1 (SEQ ID NO.3) and ΔItbR-down-2 (SEQ ID NO.6), and then purified and recovered after verification by agarose gel electrophoresis. The vector pK18mobrps / selected two restriction sites, Xba I and Kpn I, for double restriction digestion and recovery. The overlapping fragment was homologously recombined with the linearized vector pK18mobrpsl for transformation. The correct transformant was verified by colony PCR, which was the constructed plasmid pK18mobrpsL-ΔItbR. The constructed plasmid pK18mobrpsL-ΔItbR was extracted and electrotransformed into C. glutamicum Val19 competent cells. After one round of screening and two rounds of screening and colony PCR verification, strain KIC1 was obtained. Primers ΔItbR-identifying-1 (SEQID NO.7) and ΔItbR-identifying-2 (SEQ ID NO.8) were used for identification during screening. If the band size was identified as 1063bp, it was the correct strain. If the band size was identified as 1520bp or there was no band, it was restored to the original strain.

[0043] The strain KIC1 is a ItbR knockout strain, into which leuB was integrated. The first step was to use C. glutamicum ATCC 13032 rpsL K43RThe genome was used as a template for PCR amplification. Primer 5.0 software was used to design primers NCgl0021a-up-1 (SEQ ID NO.17) and NCgl0021a-up-2 (SEQ ID NO.18) for amplifying the upstream homology arm, and primers NCgl0021a-down-1 (SEQ ID NO.23) and NCgl0021a-down-2 (SEQ ID NO.24) for amplifying the downstream homology arm. Primers NCgl0021a-Ptuf-1 (SEQ ID NO.19) and NCgl0021a-Ptuf-2 (SEQ ID NO.20) were used to amplify the Ptuf promoter, and primers leuB-1 (SEQ ID NO.21) and leuB-2 (SEQ ID NO.22) were used to amplify the leuB target gene fragment, and then overlapping PCR was performed using primers NCgl0021a-up-1 (SEQ ID NO.17) and NCgl0021a-down-2 (SEQ ID NO.24) to amplify the overlapping fragment NCgl0021a::Ptuf-leuB, which was purified and recovered after verification by agarose gel electrophoresis. The vector pK18mobrps1 selected two restriction sites, Xba I and Kpn I, for double restriction digestion and recovery. The overlapping fragments were homologously recombined with the linearized vector pK18mobrps1 for transformation. The correct transformant was verified by colony PCR, that is, the constructed plasmid pK18mobrpsL-NCgl0021a::Ptuf-leuB was extracted and electroporated into the KIC1 competent cell. After one and two rounds of screening and colony PCR verification, strain KIC2 was obtained.

[0044] The strain KIC2 is a strain with LeuB integrated, and leuCD was integrated into this strain. K43RThe genome was used as a template for PCR amplification. Primer 5.0 software was used to design primers Ncgl0998a-up-1 (SEQ ID NO.9) and Ncgl0998a-up-2 (SEQ ID NO.10) for amplifying the upstream homology arm, and primers Ncgl0998a-down-1 (SEQ ID NO.15) and NcglO998a-down-2 (SEQ ID NO.16) for amplifying the downstream homology arm. Primers Ncgl0998a-Ptuf-1 (SEQ ID NO.11) and Ncgl0998a-Ptuf-2 (SEQ ID NO.12) were used to amplify the Ptuf promoter, primers leuCD-1 (SEQ ID NO.13) and leuCD-2 (SEQ ID NO.14) were used to amplify the leuCD target gene, and then overlapping PCR was performed using primers Ncgl0998a-up-1 (SEQ ID NO.9) and Ncgl0998a-down-2 (SEQ ID NO.16) to amplify the overlapping fragment Ncgl0998a::Ptuf-leuCD, which was purified and recovered after verification by agarose gel electrophoresis. Two restriction sites, Xba I and Kpn I, were selected for double restriction digestion recovery in the vector pK18mobrps / . The overlapping fragments were homologously recombined with the linearized vector pK18mobrpsl for transformation. The correct transformant was verified by colony PCR, that is, the constructed plasmid pK18mobrpsL-Ncgl0998a::Ptuf-leuCD was extracted and electrotransformed into the KIC2 competent cell. After two rounds of screening by single and double exchange and colony PCR verification, strain KIC3 was obtained.

[0045] The pXtuf plasmid was linearized using Hind III and BamH I, and the Ptac promoter was replaced by the Ptuf promoter). Primers leuA-F (SEQ ID NO.1) and leuA-R (SEQ ID NO.2) were designed using Primer 5.0 software to amplify the leuA gene fragment of the genome of C. glutamicum CP. Then, the amplified leuA gene fragment was connected to it by homologous recombination using the ClonExpress II One Step Cloning Kit (Nanjing Vazyme Biotech Company), and the pXtuf-leuA plasmid was electroporated into the KIC3 competent cell to obtain the final strain α-ketoisocaproic acid producing strain.

[0046] Example 2

[0047] The α-ketoisocaproic acid producing strain constructed in Example 1 was used for fermentation, and the specific steps were as follows:

[0048] 1.1 Activation of bacteria: Take bacteria from the -80C bacteria preservation tube and place them on LB chloramphenicol resistance plates (LB plates containing 12 mg / L chloramphenicol), divide them into three zones, culture at 32C for 32-36 hours, spot on LB chloramphenicol resistance plates for 14-18 hours, inoculate 12 mg / L chloramphenicol in BHI shake tubes, take bacteria from the BHI shake tubes and inoculate on slants, culture at 32C for 16-18 hours to obtain the first-generation activated slants; transfer the first-generation slants to eggplant-shaped bottles and culture at 32C for 8-12 hours to obtain the second-generation activated slants.

[0049] 1.2 Seed culture: An appropriate amount of cells from the second generation activated slant culture were transferred to a fermenter (Baoxing, Shanghai, China) containing 3 L of seed culture medium to start seed culture; the pH value was automatically maintained at 7.0 by adding ammonia water; the temperature was maintained at 32 ± 0.2 °C, and the dissolved oxygen was maintained at 25%-35% by adjusting the stirring speed and ventilation for seed culture;

[0050] 1.3 Fermentation culture: Seed culture until OD 600 When the pH reaches about 20-25, 600 mL of the culture solution is retained for fed-batch fermentation; during the fed-batch fermentation, the pH value is adjusted to 7.0 by ammonia water 24 hours before fermentation, and sodium acetate is added after 24 hours to adjust the pH value to 7.0, and the temperature is controlled at 32°C; when the glucose is exhausted, 80% (w / v) glucose solution is added to maintain the glucose concentration above 3 g / L to ensure a continuous supply of glucose, thereby preparing KIC.

[0051] In the above fermentation tank culture process, the slant medium and eggplant flask medium used are: peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, NaCI 2.5g / L, corn steep liquor 10g / L, KH2PO4 1g / L, MgSO4·7H2O 0.5g / L, agar 2%, pH 7.0-7.2, and the rest is water. Sterilization conditions: 121°C, 20min. Chloramphenicol working concentration 12mg / L is added after sterilization.

[0052] The seed culture medium used was: 40 g / L glucose, 5 g / L corn syrup powder, 5 g / L yeast extract, 5 g / L peptone, 3 g / L (NH4)2SO4, 1.5 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.5 g / L valine, 0.5 g / L leucine, 0.5 g / L isoleucine, 0.5 g / L methionine, 1 g / L glutamic acid, 1 g / L citric acid, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, VB1, VB3, VB5, VB 12 0.4 mg / L, 0.1 mg / L VH (pH 7.0-7.2), and the rest is water.

[0053] The fermentation medium (pH 7.0-7.2) used was: 80 g / L glucose, 20 g / L corn flour, 3 g / L yeast extract, 3 g / L peptone, 5 g / L (NH4)2SO4, 2.5 g / L KH2PO4, 1.6 g / L MgSO4·7H2O, 1 g / L valine, 1 g / L leucine, 1 g / L isoleucine, 1 g / L methionine, 5 g / L glutamic acid, 2 g / L citric acid, 10 mL / L soybean meal extract, 20 mg / L FeSO4·7H2O, 20 mg / L MnSO4·7H20, VB1, VB3, VB5, VB 12 0.3 mg / L each, the rest is water.

[0054] Cell growth (biomass) was monitored by measuring the optical density at 600 nanometers (OD600) using a spectrophotometer. The concentrations of glucose, acetate, KIV (α-ketoisovalerate) and KIC (α-ketoisocaproate) were quantitatively measured using an HPLC system (1200 series; Agilent Technologies, Inc., USA) equipped with an Aminex HPX-87H column. For specific detection methods, see reference Siyu Tian, ​​Guihong Zhao, Gengcheng Lv, Chen Wu, Rui Su, Feiao Wang, Zeting Wang, Yuexiang Liu, Ning Chen, and Yanjun Li Journal of Agricultural and Food Chemistry 202472 (14), 8039-8051 DOI: 10.1021 / acs.jafc.4c00914.

[0055] like Figure 1 As shown, KIC was produced by fermentation in a 5L fermenter. After 76 h, the OD of the strain was 600 At 182, the KIC production reached 79.79 g / L, laying the foundation for the subsequent fermentation production of KIC.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. An α-ketoisocaproic acid producing strain, characterized in that: The strain KIC3 was obtained by genetic modification using C. glutamicum Val19 as the starting strain: the ItbR gene was knocked out, the leuB gene was introduced at the NcgCg / 0021a site, and the leuCD gene was introduced at the NcgCg / 0998a site. The pXtuf plasmid was linearized with Hind III and BamH I, and the P tac The promoter was replaced by P tuf The promoter was then ligated with the leuA gene fragment, and the resulting pXtuf- / euA plasmid was electroporated into the KIC3 competent cells.

2. The α-ketoisocaproic acid producing strain according to claim 1, characterized in that: The nucleotide sequence of the leuA gene is shown in the sequence listing SEQ ID NO.

25.

3. The method for constructing the α-ketoisocaproic acid producing strain according to claim 1 or 2, characterized in that: Gene modification was performed using CR1SPR / Cas9-mediated gene editing technology. The specific steps are as follows: (1) Take C.glutamicum ATCC 13032 rpsL K43R The genome was used as a template to PCR amplify the upstream and downstream homologous arms of ΔItbR. The overlapping fragments ΔItbR were obtained by overlapping PCR, and homologous recombination and transformation with the linearized vector pK18mobrpsl recovered by double restriction digestion were used to construct the plasmid pK18mobrpsL-ΔItbR, which was electroporated into the competent cell of C. glutamicum Vai19 to obtain the strain KIC1 with the ItbR gene knocked out. (2) The leuB gene was integrated into the NcgCg / 0021a locus of strain KIC1, and the P tuf Promoter was used to construct plasmid pK18mobrpsL-NCgl0021a::Ptuf-leuB and electrotransformed to obtain strain KIC2 with LeuB gene integrated; (3) The leuCD gene was integrated into the NcgCg / 0998a locus of strain KIC2 by P tuf Promoter was used to construct plasmid pK18mobrpsL-Ncgl0998a::Ptuf-leuCD and electrotransformed to obtain strain KIC3; (4) The pXtuf plasmid was linearized with Hind III and BamH I, the Ptac promoter was replaced with the Ptuf promoter, and the leuA gene fragment amplified from the C. glutamicum CP genome was ligated to it using homologous recombination. The pXtuf-leuA plasmid was electroporated into the KIC3 competent cell to obtain the final strain.

4. Use of the α-ketoisocaproic acid producing strain according to claim 1 or 2 in producing α-ketoisocaproic acid.

5. The use according to claim 4, characterized in that: The α-ketoisocaproic acid is produced by a fermentation culture method.

6. The use according to claim 5, characterized in that: The fermentation culture is fermentation in a fermenter.

7. The use according to claim 6, characterized in that: The specific steps of the fermentation tank fermentation are as follows: (1) Activation of strains: Take the strain and divide it into three zones on an LB chloramphenicol resistance plate, culture it at 32°C for 32-36 hours, spot it on an LB chloramphenicol resistance plate for 14-18 hours, inoculate chloramphenicol in a BHI shake tube, take the strain from the BHI shake tube and inoculate it on a slant, culture it at 32°C for 16-18 hours, and you can get a first-generation activated slant; transfer the first-generation slant to an eggplant-shaped bottle, culture it at 32°C for 8-12 hours, and you can get a second-generation activated slant; (2) Seed culture: The cells cultured on the second generation activated slant were transferred to a fermenter containing a seed culture medium to start seed culture; the pH value was automatically maintained at 7.0 by adding ammonia water; the dissolved oxygen content was maintained at 25-35% and the temperature was maintained at 32±0.2°C by adjusting the stirring speed and ventilation; and seed culture was performed; (3) Fermentation culture: Seed culture until OD 600 When the pH reaches 20-25, the culture medium is retained for fed-batch fermentation; during the fed-batch fermentation, the pH value is adjusted to 7.0 by ammonia water 24 hours before fermentation, and sodium acetate is added after 24 hours to adjust the pH value to 7.0, and the temperature is controlled at 32°C; when the glucose is exhausted, glucose solution is added to maintain the glucose concentration above 3g / L.

8. The use according to claim 7, characterized in that: The slant culture medium and eggplant flask culture medium used in the step (1) are: peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, NaCl 2.5g / L, corn steep liquor 10g / L, KH2PO4 1g / L, MgSO4·7H2O 0.5g / L, agar 2%, pH 7.0-7.2, and the rest is water.

9. The use according to claim 7, characterized in that: The working concentration of chloramphenicol is 12 mg / L.

10. The use according to claim 7, characterized in that: The seed culture medium used was: 40 g / L glucose, 5 g / L corn syrup powder, 5 g / L yeast extract, 5 g / L peptone, 3 g / L (NH4)2SO4, 1.5 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.5 g / L valine, 0.5 g / L leucine, 0.5 g / L isoleucine, 0.5 g / L methionine, 1 g / L glutamic acid, 1 g / L citric acid, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, VB1, VB3, VB5, VB 12 0.4 mg / L each, 0.1 mg / L VH (pH 7.0-7.2), and the rest is water; the fermentation medium used is: 80 g / L glucose, 20 g / L corn flour, 3 g / L yeast extract, 3 g / L peptone, 5 g / L (NH4)2SO4, 2.5 g / L KH2PO4, 1.6 g / L MgSO4·7H2O, 1 g / L valine, 1 g / L leucine, 1 g / L isoleucine, 1 g / L methionine, 5 g / L glutamic acid, 2 g / L citric acid, 10 mL / L soybean meal extract, 20 mg / LFeSO4·7H2O, 20 mg / L MnSO4·7H2O, VB1, VB3, VB5, VB 12 0.3 mg / L each, the rest is water.

Images