Construction method of recombinant microorganism, recombinant microorganism and fermentation process of recombinant microorganism

Abstract

The invention discloses a construction method of a recombinant microorganism, the recombinant microorganism and a fermentation process thereof, and belongs to the technical field of genetic engineering. In order to solve the technical problems of low conversion rate and poor fermentation performance of L-tryptophan, L-threonine and L-valine in the prior art, the invention provides a construction method of a recombinant microorganism, and the recombinant microorganism is obtained by respectively inserting glf, glk and galp genes subjected to point mutation treatment into escherichia coli of original strains CGMCC NO.11073, CGMCC NO.16144 and CGMCC NO.29960 and knocking out ptsI and crr genes. The recombinant microorganisms WB-B, TH-B and VB-B provided by the invention are respectively combined with fermentation processes thereof, so that the yields and conversion rates of L-tryptophan, L-threonine and L-valine are respectively improved, the production efficiency is improved, and the requirements of large-scale industrial production are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and relates to a method for constructing a recombinant microorganism, the recombinant microorganism and its fermentation process. Background Art

[0002] L-tryptophan, L-threonine and L-valine belong to 8 kinds of essential amino acids for the human body, and are widely used in fields such as food, medicine and feed; in food, they can be used as nutritional supplements, food fortifiers or preservatives, etc.; in the pharmaceutical manufacturing industry, they are used in health products, biological medicines and pharmaceutical raw materials, etc.; in feed manufacturing, they can be used as additives.

[0003] Compared with the traditional industrial production methods of L-tryptophan, L-threonine and L-valine, the microbial fermentation method for producing L-tryptophan, L-threonine and L-valine has the advantages of low raw material price, simple process control and high product quality. Among them, Escherichia coli has the advantages of clear genetic background and simple operation, etc., and is the main microorganism for industrial production of amino acids. Therefore, Escherichia coli is often used as the starting strain in microbial fermentation.

[0004] Those skilled in the art select Escherichia coli as the starting strain and develop a fermentation strain with a high conversion rate of L-tryptophan, L-threonine and L-valine and good fermentation performance for producing L-tryptophan, L-threonine and L-valine to meet the needs of large-scale industrial production. Summary of the Invention

[0005] In order to solve the technical problems of low conversion rates of L-tryptophan, L-threonine and L-valine and poor fermentation performance in the prior art, the present invention provides a method for constructing a recombinant microorganism, the recombinant microorganism and its fermentation process.

[0006] One of the purposes of the present invention is to provide a method for constructing a recombinant microorganism, and the construction method includes the following steps:

[0007] S1: Transfer the vector into the competent cells of the starting strain and make electrocompetent cells to obtain the strain to be transformed;

[0008] S2: Use the CRISPR gene editing tool, take the P26D plasmid as the template, insert the point-mutated glf,

[0009] glk and galp genes, and knock out the ptsI and crr genes to obtain a recombinant plasmid;

[0010] S3: Transform the recombinant plasmid obtained in S2 into the strain to be transformed obtained in S1 by electrotransformation to obtain a recombinant microorganism.

[0011] In a preferred embodiment of the present invention, when the starting strain in S1 is Escherichia coli CGMCC NO.11073, the constructed recombinant microorganism is used for producing L-tryptophan;

[0012] When the starting strain in S1 is Escherichia coli CGMCC NO.16144, the constructed recombinant microorganism is used for producing L-threonine;

[0013] When the starting strain in S1 is Escherichia coli CGMCC No.29960, the constructed recombinant microorganism is used for producing L-valine.

[0014] In a preferred embodiment of the present invention, the nucleotide sequence of the ptsI gene in S2 is as shown in SEQ ID No.1; the nucleotide sequence of the crr gene is as shown in SEQ ID No.2; the glf gene is derived from Zymomonas mobilis, and the nucleotide sequence of the glf gene is as shown in SEQ ID No.3; the glk and galp genes are derived from Escherichia coli MG1655, the nucleotide sequence of the glk gene is as shown in SEQ ID No.4, and the nucleotide sequence of the galp gene is as shown in SEQ ID No.5.

[0015] In a preferred embodiment of the present invention, the point mutation treatment in S2 is to replace the 95th amino acid of the glk gene with proline and replace the 24th amino acid of the galp gene with proline.

[0016] The second object of the present invention is to provide a recombinant microorganism WB-B, which is obtained by the above construction method. The starting strain of the WB-B is Escherichia coli CGMCC NO.11073, and the WB-B is used for producing L-tryptophan.

[0017] The third object of the present invention is to provide a recombinant microorganism TH-B, which is obtained by the above construction method. The starting strain of the TH-B is Escherichia coli CGMCC NO.16144, and the TH-B is used for producing L-threonine.

[0018] The fourth object of the present invention is to provide a recombinant microorganism VB-B, which is obtained by the above construction method. The starting strain of the VB-B is Escherichia coli CGMCC No.29960, and the VB-B is used for producing L-valine.

[0019] A fifth object of the present invention is to provide a fermentation process for producing L-tryptophan, wherein the fermentation process comprises inoculating the above-mentioned recombinant microorganism WB-B into a flat solid slant culture medium and culturing at 36°C for 16 hours; inoculating the above-mentioned cultured WB-B into a seed liquid shake flask and culturing at 36°C to the logarithmic growth phase; inoculating the above-mentioned WB-B cultured to the logarithmic growth phase into a fermentation medium, and fermenting and culturing for 32 hours to 40 hours at 36°C, pH = 7.0, dissolved oxygen 25%-30%, and tank pressure 0.02MPa. During the fermentation process, the feed concentration is 50%-60% glucose, and the residual sugar content is controlled at 0.04% to obtain an L-tryptophan fermentation liquid.

[0020] The sixth object of the present invention is to provide a fermentation process for producing L-threonine, wherein the fermentation is carried out at 36.5°C for 19 hours; the cultured TH-B is inoculated into a sterile saline solution containing glass beads, the glass beads are used to break up the bacteria, and the OD value is obtained. 660 The TH-B strain suspension was diluted to 10 -5 -10 -6 , 0.3 ml of the diluted recombinant microbial TH-B strain suspension was applied to the plate, cultured at 36.5 ° C for 3 hours, and then inverted and cultured for 24 hours to obtain activated TH-B; the activated TH-B was transferred to a seed tank containing seed culture medium and cultured for 7-8 hours until the OD 660 The inoculum concentration reached 0.7, and then 30% of the inoculum amount was inoculated into the fermentation medium. The fermentation culture was carried out at 37° C., the pH was automatically maintained at 7.2 using 25% ammonia water, the pressure was adjusted to 0.05 MPa, the rotation speed was adjusted to 400 rpm, the air volume was adjusted to 0.2-1 m3 / h, and the dissolved oxygen was controlled at ≥30%. Liquid glucose was added 5 hours after the start of fermentation to maintain the residual sugar concentration in the fermentation tank ≤1.0 g / L. The fermentation time was 29 hours to obtain L-threonine fermentation liquid.

[0021] A seventh object of the present invention is to provide a fermentation process for producing L-valine, wherein the fermentation process comprises inoculating the above-mentioned recombinant microorganism VB-B into a slant culture medium and culturing at 32°C for 30 hours; eluting and breaking up the above-mentioned solid slant culture medium with physiological saline to obtain a VB-B bacterial suspension; inoculating the above-mentioned VB-B bacterial suspension into a stainless steel fermenter containing 3L of seed culture medium, and culturing the recombinant microorganism VB-B to maturity at 37°C by adjusting the pressure, speed, and air volume to maintain dissolved oxygen ≥30%; the pressure adjustment range is 0.04-0.1MPa, the speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1m 3 / h; The VB-B seed liquid cultured to the mature stage is inoculated into the fermentation medium at an inoculation amount of 15%-20%, and aerobic fermentation is carried out at 35°C, pH = 6.7, and dissolved oxygen of 25%-30%; during the fermentation process, when the glucose in the medium is consumed, an 80% (m / v) glucose solution needs to be added continuously to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L; after 6 h of the above aerobic fermentation, anaerobic fermentation is carried out, and the dissolved oxygen is controlled at 5%-10%, and the total fermentation cycle is 28 h to obtain the L-valine fermentation broth.

[0022] The beneficial effects of the present invention: During the processes of Escherichia coli synthesizing L-tryptophan, L-threonine, and L-valine respectively, PEP (phosphoenolpyruvate) is an important precursor for synthesizing L-tryptophan and L-threonine, and PEP also plays an important role in the process of synthesizing L-valine and is an essential precursor for the tricarboxylic acid cycle, especially during the microaerobic fermentation process. However, 1 mol of phosphoenolpyruvate is consumed for transporting 1 mol of glucose in the PTS system, resulting in a large consumption of PEP; therefore, reducing the transport of the PTS system plays an important role in increasing the yields of L-tryptophan, L-threonine, and L-valine with phosphoenolpyruvate.

[0023] The present invention provides a method for constructing a recombinant microorganism, the recombinant microorganism and its fermentation process. By knocking out the ptsI (nucleotide sequence shown in SEQ ID No. 1) and crr (nucleotide sequence shown in SEQ ID No. 2) genes of the starting strains Escherichia coli CGMCC NO.11073, CGMCC NO.16144, and CGMCC No.29960 respectively, and inserting plasmids of glf (nucleotide sequence shown in SEQ ID No. 3), glk* and galp* genes, the glk* and galp* genes are subjected to point mutation treatment. The 95th amino acid of the glk* gene is replaced by proline from leucine, and the nucleotide sequence is shown in SEQ ID No. 4; the 24th amino acid of the galp* gene is replaced by proline from leucine, and the nucleotide sequence of the galp gene is shown in SEQ ID No.5.

[0024] The present invention introduces point mutations to the glk* and galp* genes, thereby improving the glucose consumption ability and growth ability of the recombinant microorganism during the fermentation process, and solving the problem that the recombinant microorganism cannot grow normally during the fermentation process due to insufficient glucose consumption ability during the fermentation process.

[0025] The recombinant microorganism WB-B provided by the present invention is combined with a fermentation process to maximize the ability of WB-B to produce L-tryptophan. The yield of L-tryptophan produced by it is 46.07 g / L, and the productivity is 21.21%; the recombinant microorganism TH-B provided by the present invention is combined with a fermentation process to maximize the ability of TH-B to produce L-threonine. The yield of L-threonine produced by it is 118.07 g / L, and the productivity is 59.21%; the recombinant microorganism VB-B provided by the present invention is combined with a fermentation process to maximize the ability of VB-B to produce L-valine. The yield of L-valine produced by it is 95.07 g / L, and the productivity is 54.21%.

[0026] It can be seen that the recombinant microorganisms WB-B, TH-B and VB-B provided by the present invention, and their corresponding fermentation processes, respectively have the ability to increase the yields and conversion rates of L-tryptophan, L-threonine and L-valine, improve the production efficiency, so as to meet the requirements of large-scale industrial production. Detailed implementation manners

[0027] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0028] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation manners. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0029] The starting strains used in the present invention:

[0030] The Escherichia coli CGMCC NO.11073 is disclosed in the invention patent with the authorization announcement number CN116376852B;

[0031] The Escherichia coli CGMCC NO.16144 is disclosed in the invention patent with the authorization announcement number CN118406630B;

[0032] The Escherichia coli CGMCC NO.29960 is disclosed in the invention patent with the authorization announcement number CN 118389395B.

[0033] In the following examples, the glk gene subjected to point mutation is denoted as glk*, and the galp gene subjected to point mutation is denoted as galp*.

[0034] Example 1: Preparation and transformation of competent cells

[0035] (1) Transformation of plasmid p118

[0036] Take out the bacterial suspensions of Escherichia coli CGMCC NO.11073, Escherichia coli CGMCC NO.16144, and Escherichia coli CGMCC NO.29960 from the cryotubes, streak them onto LB solid plates respectively, and incubate statically overnight (12h - 16h) at 37°C; pick single colonies from the above LB solid plates, inoculate them into 5 mL of LB liquid medium, and incubate with shaking overnight (12h - 16h) at 30°C and 200 rpm; take 500 μL of the above overnight-cultured bacterial suspension and inoculate it into 500 mL of LB liquid medium containing kanamycin, and incubate with shaking at 37°C and 200 rpm until OD 660 = 0.5 - 0.6 (the culture time is 3 - 4 h); place the above OD 600 qualified bacterial suspension on ice for 20 - 30 min for pre-cooling, then transfer it to a pre-cooled 50 mL centrifuge tube, centrifuge at 4°C and 4500 rpm for 10 min, carefully discard the supernatant; first add 5 mL of ice-cold 10% glycerol to the obtained bacterial cell pellet, gently pipette and mix evenly, then make up to 30 mL with 10% glycerol, centrifuge at 4°C and 4500 rpm for 10 min, carefully discard the supernatant, and repeat the above steps twice; add 1 mL of ice-cold 10% glycerol to the obtained precipitated bacterial cells to resuspend and mix evenly, aliquot into pre-cooled 1.5 mL EP tubes at a volume of 100 μL per tube, and use immediately or store at -80°C;

[0037] Transform the plasmid p118 containing the kanamycin resistance gene into the above-obtained Escherichia coli CGMCC NO.11073 to obtain Escherichia coli CGMCC NO.11073 competent cells; spread the above competent cells on an LB agar plate containing kanamycin and culture at 37°C to obtain single colonies of Escherichia coli CGMCC NO.11073, Escherichia coli CGMCC NO.16144, and Escherichia coli CGMCC NO.29960, and name the strains of the above colonies as Escherichia coli CGMCC NO.11073-p118 strain, Escherichia coli CGMCC NO.16144-p118 strain, and Escherichia coli CGMCC NO.29960-p118 strain respectively.

[0038] (2) Preparation of electrocompetent cells of the strain

[0039] Separate the bacterial suspensions of CGMCC NO.11073-p118 strain, CGMCC NO.16144-p118 strain and CGMCC NO.29960-p118 strain obtained in (1) from the cryotubes, streak them on LB solid plates respectively, and statically culture overnight (12h - 16h) at 37°C; pick single colonies from the above LB solid plates, inoculate them into 5 mL of LB liquid medium, and shake culture overnight (12h - 16h) at 30°C and 200 rpm; take 500 μL of the above overnight-cultured bacterial suspension and inoculate it into 500 mL of LB liquid medium containing kanamycin, shake culture at 37°C and 200 rpm until OD 660 When = 0.1, add 0.1 M IPTG, and culture the strain until OD 600 = 0.7 - 0.9 (the culture time is 4 - 6 h);

[0040] Place the above OD 600 qualified bacterial suspension on ice for 20 - 30 min for pre-cooling, then transfer it to a pre-cooled 50 mL centrifuge tube, centrifuge at 4°C and 4500 rpm for 10 min, carefully discard the supernatant; first add 5 mL of ice-cold 10% glycerol to the obtained bacterial cell precipitate, gently pipette and mix evenly, then make up to 30 mL with 10% glycerol, centrifuge at 4°C and 4500 rpm for 10 min, carefully discard the supernatant, and repeat the above steps twice; add 500 μL of ice-cold 10% glycerol to resuspend the obtained precipitated bacteria, aliquot them into pre-cooled 1.5 mL EP tubes at a volume of 90 μL per tube respectively to obtain electrocompetent cells of CGMCC NO.11073-p118 strain, CGMCC NO.16144-p118 strain and CGMCC NO.29960-p118 strain, and use immediately or store at -80°C.

[0041] Example 2: Construction of p26D-△ptsI△crr-glf-glk-galp plasmid

[0042] Obtain the genomic sequence of Escherichia coli MG1655 through NCBI. Using the P26D plasmid as a template, insert the original glf, glk and gal p genes without point mutation treatment, and knockout the ptsI and crr genes to obtain a recombinant plasmid; the nucleotide sequence of the ptsI gene is shown as SEQ ID No.1; the nucleotide sequence of the crr gene is shown as SEQ ID No.2.

[0043] The specific steps are as follows:

[0044] Using the genome of Escherichia coli MG1655 as a template, and using the high-fidelity enzyme 2×Phanta Flash MasterMix, with P1 (shown as SEQ ID No.6) and P2 (shown as SEQ ID No.7) as primers, the upstream homologous arm was amplified to obtain a target fragment with a length of 496 bp, denoted as F1;

[0045] The puc57-glf-glk-galp plasmid expressing the glf, glk, and galp genes was synthesized by GenScript Biotech Corporation; the glf gene uses the Pamyl promoter, and the glk and galp genes use the Ptac promoter; the glf gene is derived from Zymomonas mobilis; the glf gene sequence is shown as SEQ ID No.3, the glk gene sequence is shown as SEQ ID No.4, and the galp gene sequence is shown as SEQ ID No.5;

[0046] Using the puc57-glf-glk-galp plasmid as a template, and using the high-fidelity enzyme 2×Phanta Flash MasterMix, with P3 (shown as SEQ ID No.8) and P4 (shown as SEQ ID No.9) as primers, the glf-glk-galp gene was amplified to obtain a target fragment with a length of 3985 bp, denoted as F2;

[0047] Using the genome of Escherichia coli MG1655 as a template, and using the high-fidelity enzyme 2×Phanta Flash MasterMix, with P5 (shown as SEQ ID No.10) and P6 (shown as SEQ ID No.11) as primers, a target fragment with a length of 518 bp was amplified, denoted as F3;

[0048] Using the P26D plasmid as a template, and using the high-fidelity enzyme 2×Phanta Flash Master Mix, with P7 (shown as SEQ IDNo.12) and P8 (shown as SEQ ID No.13) as primers, a target fragment with a length of 3185 bp was amplified; after digesting the above amplification product with DpnI enzyme, the target fragment was recovered using a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) and denoted as F4;

[0049] Using the Seamless Cloning Kit (purchased from Beyotime) for plasmid ligation, the four target fragments F1, F2, F3, and F4 obtained above were ligated. The ligation reaction conditions were 50°C for 60 min in a 20 μL system. The above ligation fragments were transformed into Escherichia coli DH5α competent cells to obtain the p26D-ΔptsIΔcrr-glf-glk-galp plasmid.

[0050] The PCR reaction system was: 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of plasmid DNA / template, 25 μL of PFU enzyme, and 19 μL of water; the reaction program was: 98°C for 30 s, 98°C for 10 s, 55°C for 10 s, 72°C for 5 s / kb, 72°C for 1 min, and 4°C ∞, for 35 cycles.

[0051] The DpnI digestion system was: 50 μL of plasmid PCR product, 5 μL of 10x Buffer, and 1 μL of DpnI, and it was digested at 37°C for 1 h to remove circular plasmid DNA.

[0052] Example 3: Construction of p26D-ΔptsIΔcrr-glf-glk*-galp* plasmid

[0053] The genomic sequence of Escherichia coli MG1655 was obtained through NCBI. Using the CRISPR / Cas9 gene editing technology, with the P26D plasmid as the template, the glf, glk, and galp genes treated with point mutations were inserted, and the ptsI and crr genes were knocked out to obtain a recombinant plasmid; the nucleotide sequence of the ptsI gene is shown as SEQ ID No.1; the nucleotide sequence of the crr gene is shown as SEQ ID No.2; the point mutation treatment was to replace the 95th amino acid of the glk gene from leucine to proline (denoted as: glk*), and the 24th amino acid of the galp gene from leucine to proline (galp*); the glf gene uses the Pamyl promoter, and the glk and galp genes use the Ptac promoter; the glf gene is derived from Zymomonas mobilis; the glf gene sequence is shown as SEQ ID No.3, the glk gene sequence is shown as SEQ ID No.4, and the galp gene sequence is shown as SEQ ID No.5;

[0054] Using the p26D-△ptsI△crr-glf-glk-galp obtained in Example 2 as a template, and using the high-fidelity enzyme 2×Phanta Flash Master Mix, with P11 (shown as SEQ ID No.14) and P12 (shown as SEQ ID No.15) as primers, amplify a target fragment with a length of 788bp; after digesting the above amplification product with DpnI enzyme, use a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to recover the target fragment and record it as F5;

[0055] Through the CRISPR / Cas9 gene editing technology, using the p26D-△ptsI△crr-glf-glk-gal p obtained in Example 2 as a template, and using the high-fidelity enzyme 2×Phanta Flash Master Mix, with P13 (shown as SEQ ID No.16) and P14 (shown as SEQ ID No.17) as primers, amplify a target fragment with a length of 788bp; after digesting the above amplification product with DpnI enzyme, use a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to recover the target fragment and record it as F6;

[0056] Connect the above-obtained two target fragments F5 and F6 through the plasmid ligation Seamless Cloning Kit (purchased from Beyotime), and the ligation reaction conditions are 50℃, 60min, 20μL system; transform the above ligation fragment into Escherichia coli DH5α competent cells to obtain the p26D-△ptsI△crr-glf-glk*-galp* plasmid.

[0057] Example 4: Construction of recombinant microorganism WB-B

[0058] S1: Transform the p26D-△ptsI△crr-glf-glk*-galp* plasmid obtained in Example 3 into the electrocompetent cells of the CGMCC NO.11073-p118 strain obtained in Example 1 by electroporation method. After centrifuging and enriching the cells recovered and cultured after the above electroporation, coat them on an LB plate containing kanamycin and ampicillin, and culture overnight at 30℃. Pick the above single colonies and inoculate them into 5mL LB liquid medium containing kanamycin and ampicillin, culture at 30℃ and 220rpm for 3 hours, then add 50μL IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ IDNo.19) as primers to identify the positive transformants of the above single colonies;

[0059] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Perform three-zone streaking on a kanamycin LB plate and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an LB plate containing both kanamycin and ampicillin. Preserve the strains that grow only on the kanamycin LB plate.

[0060] S3: Transfer the strains that grow only on the kanamycin LB plate in S2 to an antibiotic-free LB medium (added with 200 μL of 50% sucrose) and culture overnight at 37°C. Perform three-zone streaking on an antibiotic-free LB plate and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an antibiotic-free LB plate. Select and preserve the strains that do not grow on the kanamycin plate but grow on the antibiotic-free plate to obtain the recombinant microorganism WB-B.

[0061] Example 5: Construction of the recombinant microorganism TH-B

[0062] S1: Transform the p26D-△ptsI△crr-glf-glk*-galp* plasmid obtained in Example 3 into the electrocompetent cells of the CGMCC NO.16144-p118 strain obtained in Example 1 by electrotransformation. After centrifugally enriching the cells recovered and cultured after the above electrotransformation, coat them on an LB plate containing both kanamycin and ampicillin and culture overnight at 30°C. Pick the above single colonies and inoculate them into 5 mL of an LB liquid medium containing kanamycin and ampicillin, and culture at 30°C and 220 rpm for 3 hours, then add 50 μL of IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ ID No.19) as primers to identify the positive transformants from the above single colonies.

[0063] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Coat them on a kanamycin LB plate and perform three-zone streaking, then culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an LB plate containing both kanamycin and ampicillin. Preserve the strains that grow only on the kanamycin LB plate.

[0064] S3: Transfer the strains that grow only on the kanamycin LB plate in S2 to an antibiotic-free LB medium (added with 200 μL of 50% sucrose) and culture overnight at 37°C. Perform three-zone streaking on an antibiotic-free LB plate and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an antibiotic-free LB plate. Select and preserve the strains that do not grow on the kanamycin plate but grow on the antibiotic-free plate to obtain the recombinant microorganism TH-B.

[0065] Example 6: Construction of the recombinant microorganism VB-B

[0066] S1: Transform the p26D-ΔptsIΔcrr-glf-glk*-galp* plasmid obtained in Example 3 into the electrocompetent cells of CGMCC NO.29960-p118 strain obtained in Example 1 by electroporation. After centrifuging and enriching the cells recovered and cultured after the above electroporation, spread them on an LB plate containing kanamycin and ampicillin, and culture overnight at 30°C. Pick the above single colonies and inoculate them into 5 mL of LB liquid medium containing kanamycin and ampicillin, culture for 3 hours at 30°C and 220 rpm, then add 50 μL of IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ ID No.19) as primers to identify the positive transformants from the above single colonies;

[0067] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Streak in three zones on a kanamycin LB plate and culture overnight at 37°C. Spot culture on a kanamycin LB plate and an LB plate containing kanamycin and ampicillin, and preserve the strains that only grow on the kanamycin LB plate;

[0068] S3: Transfer the strains that only grow on the kanamycin LB plate in S2 to a non-resistant LB medium (added with 200 μL of 50% sucrose) and culture overnight at 37°C. Streak in three zones on a non-resistant LB plate and culture overnight at 37°C. Spot culture on a kanamycin LB plate and a non-resistant LB plate, and pick and preserve the strains that do not grow on the kanamycin plate but grow on the non-resistant plate to obtain the recombinant microorganism VB-B.

[0069] Comparative Example 1: Construction of the recombinant microorganism WB-A

[0070] S1: Transform the p26D-ΔptsIΔcrr-glf-glk-galp plasmid obtained in Example 2 into the electrocompetent cells of CGMCC NO.11073-p118 strain obtained in Example 1 by electroporation. After centrifuging and enriching the cells recovered and cultured after the above electroporation, spread them on an LB plate containing kanamycin and ampicillin, and culture overnight at 30°C. Pick the above single colonies and inoculate them into 5 mL of LB liquid medium containing kanamycin and ampicillin, culture for 3 hours at 30°C and 220 rpm, then add 50 μL of IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ ID No.19) as primers to identify the positive transformants from the above single colonies;

[0071] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Streak the culture on a kanamycin LB plate in three zones and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an LB plate containing both kanamycin and ampicillin, and preserve the strains that grow only on the kanamycin LB plate.

[0072] S3: Transfer the strains that grow only on the kanamycin LB plate in S2 to an antibiotic-free LB medium (supplemented with 200 μL of 50% sucrose) and culture overnight at 37°C. Streak the culture on an antibiotic-free LB plate in three zones and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an antibiotic-free LB plate, and select and preserve the strains that do not grow on the kanamycin plate but grow on the antibiotic-free plate to obtain the recombinant microorganism WB-A.

[0073] Comparative Example 2: Construction of recombinant microorganism TH-A

[0074] S1: Transform the p26D-ΔptsIΔcrr-glf-glk-galp plasmid obtained in Example 2 into the electrocompetent cells of the CGMCC NO.16144-p118 strain obtained in Example l by electrotransformation. After centrifugally enriching the cells recovered and cultured after the above electrotransformation, spread them on an LB plate containing both kanamycin and ampicillin and culture overnight at 30°C. Pick the above single colonies and inoculate them into 5 mL of an LB liquid medium containing both kanamycin and ampicillin, culture at 30°C and 220 rpm for 3 hours, then add 50 μL of IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ ID No.19) as primers to identify the positive transformants of the above single colonies.

[0075] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Streak the culture on a kanamycin LB plate in three zones and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an LB plate containing both kanamycin and ampicillin, and preserve the strains that grow only on the kanamycin LB plate.

[0076] S3: Transfer the strains that grow only on the kanamycin LB plate in S2 to an antibiotic-free LB medium (supplemented with 200 μL of 50% sucrose) and culture overnight at 37°C. Streak the culture on an antibiotic-free LB plate in three zones and culture overnight at 37°C. Spot-culture on a kanamycin LB plate and an antibiotic-free LB plate, and select and preserve the strains that do not grow on the kanamycin plate but grow on the antibiotic-free plate to obtain the recombinant microorganism TH-A.

[0077] Comparative Example 3: Construction of recombinant microorganism VB-A

[0078] S1: Transform the p26D-ΔptsIΔcrr-glf-glk-galp plasmid obtained in Example 2 into the electrocompetent cells of the CGMCC NO.29960-p118 strain obtained in Example 1 by electroporation. After centrifugally enriching the cells recovered and cultured after the above electroporation, coat them on an LB plate containing kanamycin and ampicillin, and culture overnight at 30°C. Pick the above single colonies and inoculate them into 5 mL of LB liquid medium containing kanamycin and ampicillin, culture for 3 hours at 30°C and 220 rpm, then add 50 μL of IPTG and culture for 1 hour. Use P9 (shown as SEQ ID No.18) and P10 (shown as SEQ ID No.19) as primers to identify the positive transformants among the above single colonies;

[0079] S2: Transfer the positive transformants identified in S1 to an LB medium containing kanamycin and culture overnight. Streak in three zones on a kanamycin LB plate and culture overnight at 37°C. Spot culture on a kanamycin LB plate and an LB plate containing kanamycin and ampicillin, and preserve the strains that only grow on the kanamycin LB plate;

[0080] S3: Transfer the strains that only grow on the kanamycin LB plate in S2 to a non-resistant LB medium (added with 200 μL of 50% sucrose) and culture overnight at 37°C. Streak in three zones on a non-resistant LB plate and culture overnight at 37°C. Spot culture on a kanamycin LB plate and a non-resistant LB plate, and pick and preserve the strains that do not grow on the kanamycin plate but grow on the non-resistant plate to obtain the recombinant microorganism VB-A.

[0081] Effect experiment:

[0082] 1. A fermentation process for producing L-tryptophan, L-threonine and L-valine

[0083] (1) Fermentation process for producing L-tryptophan

[0084] Inoculate the recombinant microorganism WB-B in Example 4, the recombinant microorganism WB-A in Comparative Example 1, and Escherichia coli CGMCC NO.11073 onto a solid slant medium of a plate according to an inoculum size of 2 loops, and culture at 36°C for 16 h; respectively pick 2 loops of the cultured WB-B, WB-A and Escherichia coli CGMCC NO.11073 and inoculate them into a seed liquid shake flask, and culture at 36°C until the logarithmic growth phase;

[0085] The WB-B, WB-A, and Escherichia coli CGMCC NO.11073 cultured to the growth stage above were respectively inoculated into a fermentation medium and fermented and cultured at 36°C, pH = 7.0, dissolved oxygen of 25%-30%, and tank pressure of 0.02 MPa for 32 h - 40 h. During the fermentation process, glucose with a concentration of 50%-60% was added dropwise, and the residual sugar content was controlled at 0.04% to obtain the corresponding L-tryptophan fermentation broth, with each strain repeated 3 times.

[0086] The slant medium (g / L): tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, anhydrous glucose 1 - 4 g / L, KH2PO4 1 - 5 g / L, the rest is water, pH = 6.8 - 7.2;

[0087] The seed liquid medium (g / L): dipotassium hydrogen phosphate 20 g / L, potassium dihydrogen phosphate 12 g / L, yeast powder 18 g / L, ammonium sulfate 4 g / L. Use a 45% sodium hydroxide solution to adjust the pH to 7.15, add an antifoaming agent, and sterilize at 121°C for 20 min to obtain the medium; dissolve 1.5 g / L of magnesium sulfate and 35 g / L of glucose in water and sterilize at 115°C for 15 min to obtain a mixed solution of glucose and magnesium sulfate; dispense 45 mL of the medium and 5 mL of the mixed solution of glucose and magnesium sulfate into each baffle flask, wrap it, and set it aside;

[0088] The fermentation medium (g / L): glucose 10 - 20 g / L, yeast powder 4 - 6 g / L, citric acid 1 - 4 g / L, (NH4)2SO4 2 - 8 g / L, KH2PO4 4 - 6 g / L, MgSO4·7H2O 1 - 2 g / L, FeSO4·7H2O 50 - 80 mg / L, VB1 5 - 7 mg / L, VH 0.2 - 0.6 mg / L, trace element mixed solution 1 - 2 mL / L;

[0089] The components of the trace element mixed solution are: CoSO4·7H2O 0.4 - 0.8 g / L, ZnSO4·7H2O 6 - 8 g / L, CuSO4·5H2O 4 - 6 g / L, Al2(SO4)3·18H2O 2 - 4 g / L, MnSO4·H2O 4 - 6 g / L, Na2MoO4·2H2O 2 - 4 g / L, NiSO4·6H2O 2 - 4 g / L, H3BO3 1 - 2 g / L.

[0090] (2) Fermentation process for producing L-threonine

[0091] The recombinant microorganism TH-B in Example 5, the recombinant microorganism TH-A in Comparative Example 2, and Escherichia coli CGMCC NO. 16144 were respectively inoculated onto a solid slant medium at an inoculum size of 3 loops for streaking, and cultured at 36.5 °C for 19 h;

[0092] The cultured TH-B, TH-A, and Escherichia coli CGMCC NO. 16144 were respectively inoculated into 50 mL of sterilized physiological saline containing glass beads, and the cell bodies were dispersed using the glass beads to obtain suspensions of TH-B and TH-A strains. The OD 660 of the strain suspension was 0.3 - 0.4, and its concentration was diluted to 10 -5 -10 -6 . 0.3 mL of the diluted suspensions of the recombinant microorganisms TH-B, TH-A, and Escherichia coli CGMCC NO. 16144 were respectively taken and spread on plates, cultured at 36.5 °C for 3 h, and then inverted and cultured continuously until 24 h to obtain the activated TH-B, TH-A, and Escherichia coli CGMCC NO. 16144;

[0093] The activated TH-B, TH-A, and Escherichia coli CGMCC NO. 16144 were respectively transferred to a seed tank containing a seed medium and cultured for 7 - 8 h until the OD 660 reached 0.7, and then inoculated into a fermentation medium at an inoculum size of 30%. The culture was carried out at 37 °C, the pH was automatically controlled at 7.2 using 25% ammonia water, the pressure was adjusted to 0.05 MPa, the rotation speed was adjusted to 400 rpm, the air volume was adjusted to 0.2 - 1 m 3 / h, and the dissolved oxygen was controlled at ≥ 30% for fermentation culture. Liquid glucose was supplemented 5 h after the start of fermentation to maintain the residual sugar concentration in the fermentation tank ≤ 1.0 g / L. The fermentation time was 29 h, and the corresponding L-threonine fermentation broths were respectively obtained, with each strain repeated 3 times.

[0094] The slant medium (g / L): glucose 2.0 g, ammonium chloride 1.0 g, KH2PO4 1.5 g, NaHPO4 3.5 g, MgSO4·7H2O 0.1 g, and agar 20 g, pH value = 7.0 - 7.2;

[0095] The seed medium (g / L): glucose 40.0 g, (NH4)2SO4 10.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.5 g, yeast extract 2.0 g, and MOPS 15.0 g;

[0096] The fermentation medium (g / L): glucose 80.0 g, (NH4)2SO4 25.0 g, KH2PO4 2.0 g, MgSO4·7H2O 1.0 g, yeast extract 4.0 g, FeSO4·5H2O 0.5 g, MnSO4·5H2O 0.5 g, and MOPS 30.0 g.

[0097] (3) Fermentation process for producing L-valine

[0098] The recombinant microorganism VB-B in Example 6, the recombinant microorganism VB-A in Comparative Example 3, and Escherichia coli of CGMCC NO.29960 were respectively inoculated into the slant medium in an eggplant bottle according to the inoculum size of 3 loops, and cultured at 32 °C for 30 h;

[0099] 200 mL of normal saline was used to wash and disperse the solid slant medium in the eggplant bottle to obtain the suspensions of VB-B, VB-A, and Escherichia coli of CGMCC NO.29960; the suspensions of VB-B, VB-A, and Escherichia coli of CGMCC NO.29960 were respectively inoculated into a stainless-steel fermenter containing 3 L of seed medium, and under the condition of 37 °C, the dissolved oxygen was maintained at ≥30% by adjusting the pressure, rotation speed, and air volume; the pressure adjustment range was 0.04 - 0.1 MPa, the rotation speed adjustment range was 200 - 600 rpm, and the air volume adjustment range was 0.2 - 1 m 3 / h;

[0100] The seed solutions of VB-B, VB-A, and Escherichia coli of CGMCC NO.29960 cultured to the mature stage were respectively inoculated into the fermentation medium according to the inoculum size of 15% - 20%, and aerobic fermentation was carried out at 35 °C, pH = 6.7, and dissolved oxygen of 25% - 30%; during the fermentation process, when the glucose in the medium was consumed completely, an 80% (m / v) glucose solution needed to be fed to maintain the glucose concentration in the fermentation medium at 0.1 - 1 g / L; after 6 h of the above aerobic fermentation, anaerobic fermentation was carried out, and the dissolved oxygen was controlled at 5% - 10%, and the total fermentation period was 28 h to obtain the corresponding L-valine fermentation broth respectively, and each strain was repeated 3 times.

[0101] Slant medium (g / L): peptone 10 g / L, beef extract 10 g / L, glucose 5 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, agar powder 20 g / L, the balance being water, pH = 6.5;

[0102] Seed culture medium (g / L): Glucose 75 g / L, yeast powder 5 g / L, K2HPO4 4 g / L, (NH4)2SO4 2.5 g / L, citric acid 2 g / L, MgSO4·7H2O 1.5 g / L, VB1, VB3, VB5, VB12, VH each 2 mg / L, FeSO4·7H2O 2.8 mg / L, MnSO4 1.2 mg / L, the balance is water, pH = 7.0 - 7.5;

[0103] Fermentation medium (g / L): Glucose 30 g / L, K2HPO4 7.5 g / L, (NH4)2SO4 3 g / L, yeast powder 2 g / L, citric acid 2 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 30 mg / L, MnSO4 10 mg / L, VB1, VB3, VB5, VB12, VH each 1 mg / L, the balance is water, pH = 6.5 - 7.0.

[0104] 2. Fermentation verification of recombinant microorganisms

[0105] (1) Analysis of the results of L-tryptophan fermentation verification in a 5 L tank

[0106] In this example, liquid chromatography and a Kjeldahl nitrogen analyzer were used to detect the relevant indexes of L-tryptophan in the fermentation broth obtained by fermenting recombinant microorganisms WB-B, WB-A, and Escherichia coli CGMCC NO.11073. The results are shown in Table 1.

[0107] The OD value of the recombinant microorganism WB-A without introduced point mutation was only 7.6, indicating that it could not grow normally during fermentation and had a serious shortage of sugar consumption ability, thus terminating fermentation in advance; moreover, the L-tryptophan yield of the recombinant microorganism WB-B was 46.07 g / L, which was 44.29 g / L higher than the L-tryptophan yield of the recombinant microorganism WB-A. At the same time, the conversion rate increased from 17.05% to 21.21%; on the premise of the same fermentation cycle, the L-tryptophan yield of the recombinant microorganism WB-B was 8.25 g / L higher than the L-tryptophan yield of Escherichia coli CGMCC NO.11073, and the conversion rate increased from 17.73% to 21.21%; it can be seen that the recombinant microorganism WB-B provided by the present invention has a high L-tryptophan yield and a high L-tryptophan conversion rate, and the yield and conversion rate of L-tryptophan are improved compared with the original starting strain.

[0108] Table 1

[0109]

[0110]

[0111] (2) Analysis of Fermentation Verification Results of 10L Tank for L-Threonine

[0112] In this example, high performance liquid chromatography and Kjeldahl nitrogen analyzer were used to detect the relevant indexes of L-threonine in the fermentation broth obtained by fermenting recombinant microorganisms TH-B, TH-A and Escherichia coli CGMCC NO.16144. The results are shown in Table 2.

[0113] On the premise of the same fermentation cycle, the L-threonine yield of recombinant microorganism TH-B was 118.07 g / L, which was 36.95 g / L higher than that of recombinant microorganism TH-A. At the same time, the conversion rate increased from 41.34% to 59.21%. The L-threonine yield of recombinant microorganism TH-B was 3.98 g / L higher than that of Escherichia coli CGMCC NO.16144, and the conversion rate increased by 2.6%. It can be seen that

[0114] The sugar consumption ability of recombinant microorganism TH-A without introduced point mutation was weak during the fermentation process. The sugar consumption ability and growth of the recombinant microorganism TH-B provided by the present invention returned to the normal level during the fermentation process and were slightly higher than those of the starting strain Escherichia coli CGMCC NO.16144, indicating that the recombinant microorganism TH-B provided by the present invention had a high L-threonine yield and conversion rate, and the yield and conversion rate of L-threonine were improved compared with the original starting strain.

[0115] Table 2

[0116] - TH-B TH-A Escherichia coli CGMCC NO.16144 L-Threonine (g / L) 118.07 81.12 109.33 Conversion rate (%) 59.21 41.34 55.23 OD 66.32 45.61 63.72 Cycle (h) 29 29 29

[0117] (3) Analysis of Fermentation Verification Results of 5L Tank for L-Valine

[0118] In this example, high performance liquid chromatography and Kjeldahl nitrogen analyzer were used to detect the relevant indexes of L-valine in the fermentation broth obtained by fermenting recombinant microorganisms VB-B, VB-A and Escherichia coli CGMCC NO.29960. The results are shown in Table 3.

[0119] The OD value of the recombinant microorganism VB-A without introduced point mutations was only 7.6, indicating that it could not grow normally during fermentation, and its sugar consumption ability during fermentation was severely insufficient, thus terminating fermentation in advance; moreover, on the premise of the same fermentation cycle, the L-valine yield of the recombinant microorganism VB-B was 95.07 g / L, which was 56.97 g / L higher than the L-valine yield of the recombinant microorganism VB-A, and the conversion rate increased by 11.21%; the L-valine yield of the recombinant microorganism VB-B was 1.46 g / L higher than the L-valine yield of Escherichia coli CGMCC NO. 29960, and the conversion rate increased by 5.98%; it can be seen that the recombinant microorganism VB-B provided by the present invention has a high L-valine yield and conversion rate, and the L-valine yield and conversion rate are improved compared with the original starting strain.

[0120] Table 3

[0121] - VB-B VB-A Escherichia coli CGMCC NO.29960 L-Valine (g / L) 95.07 38.1 93.61 Conversion rate (%) 54.21 42.97 51.23 OD 31.3 15.6 29.5 Cycle (h) 28 28 28

[0122] The content not described in detail in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for constructing a recombinant microorganism, characterized in that, The construction method includes the following steps: S1: Transfer the vector into the competent cells of the starting strain to prepare electrocompetent cells, and obtain the strain to be modified; S2: Using the CRISPR gene editing tool, with the P26D plasmid as a template, insert the point-mutated glf, glk, and galp genes, and knockout the ptsI and crr genes to obtain a recombinant plasmid; S3: Transform the recombinant plasmid obtained in S2 into the strain to be modified obtained in S1 by electrotransformation to obtain a recombinant microorganism.

2. The construction method according to claim 1, wherein when the starting strain in S1 is Escherichia coli CGMCC NO.11073, the constructed recombinant microorganism is used for producing L-tryptophan; when the starting strain in S1 is Escherichia coli CGMCC NO.16144, the constructed recombinant microorganism is used for producing L-threonine; when the starting strain in S1 is Escherichia coli CGMCC No.29960, the constructed recombinant microorganism is used for producing L-valine.

3. The construction method according to claim 1, characterized in that, The nucleotide sequence of the ptsI gene in S2 is shown as SEQ ID No.1; the nucleotide sequence of the crr gene is shown as SEQ ID No.2; the glf gene is derived from Zymomonas mobilis, and the nucleotide sequence of the glf gene is shown as SEQ ID No.3; the glk and galp genes are derived from Escherichia coli MG1655, the nucleotide sequence of the glk gene is shown as SEQ ID No.4, and the nucleotide sequence of the galp gene is shown as SEQ ID No.

5.

4. The construction method according to claim 1, characterized in that The point mutation treatment in S2 is to replace the 95th amino acid of the glk gene from leucine to proline, and replace the 24th amino acid of the galp gene from leucine to proline.

5. A recombinant microorganism WB-B, characterized in that, The recombinant microorganism WB-B is obtained by using the construction method according to any one of claims 1 to 4. The starting strain of the WB-B is Escherichia coli CGMCC NO.11073, and the WB-B is used for producing L-tryptophan.

6. A recombinant microorganism TH-B, characterized in that, The recombinant microorganism TH-B is obtained by using the construction method according to any one of claims 1 to 4. The starting strain of the TH-B is Escherichia coli CGMCC NO.16144, and the TH-B is used for producing L-threonine.

7. A recombinant microorganism VB-B, characterized in that, The recombinant microorganism VB-B is obtained by using the construction method according to any one of claims 1 to 4. The starting strain of the VB-B is Escherichia coli CGMCC No.29960, and the VB-B is used for producing L-valine.

8. A fermentation process for producing L-tryptophan, characterized in that, The fermentation process is as follows: inoculate the recombinant microorganism WB-B described in claim 5 into a flat solid slant medium and culture it at 36°C for 16 h; inoculate the WB-B after the above culture into a seed liquid shake flask and culture it at 36°C until the logarithmic growth phase; inoculate the WB-B cultured to the logarithmic growth phase into a fermentation medium and ferment and culture it at 36°C, pH = 7.0, dissolved oxygen 25%-30%, and tank pressure 0.02 MPa for 32 h-40 h. During the fermentation process, glucose with a concentration of 50%-60% is added dropwise, and the residual sugar content is controlled at 0.04% to obtain an L-tryptophan fermentation broth.

9. A fermentation process for producing L-threonine, characterized in that, The fermentation process is to streak the recombinant microorganism TH-B described in claim 6 onto a solid slant medium and culture it at 36.5 °C for 19 h; inoculate the cultured TH-B into sterilized physiological saline containing glass beads, and break up the bacterial cells using the glass beads to obtain a suspension of TH-B strain with an OD 660 of 0.3 - 0.

4. Dilute its concentration to 10 -5 -10 -6 . Pipette 0.3 ml of the diluted suspension of the recombinant microorganism TH-B strain onto a plate, culture it at 36.5 °C for 3 h, and continue to culture it in an inverted position until 24 h to obtain the activated TH-B; transfer the activated TH-B to a seed tank containing a seed medium and culture it for 7 - 8 h until the OD 660 reaches 0.7, then inoculate it into the fermentation medium at an inoculation amount of 30%, and perform fermentation culture at 37 °C, automatically control the pH to 7.2 using 25% ammonia water, adjust the pressure to 0.05 MPa, adjust the rotation speed to 400 rpm, adjust the air volume to 0.2 - 1 m3 / h, and control the dissolved oxygen at ≥ 30%. Start adding liquid glucose 5 h after the start of fermentation, maintain the residual sugar concentration in the fermentation tank ≤ 1.0 g / L, and the fermentation time is 29 h to obtain the L-threonine fermentation broth.

10. A fermentation process for producing L-valine, characterized in that, The fermentation process is to inoculate the recombinant microorganism VB-B described in claim 7 into a slant medium and culture it at 32 °C for 30 h; use physiological saline to elute and disperse the above solid slant medium to obtain a VB-B bacterial suspension; inoculate the above VB-B bacterial suspension into a stainless steel fermenter containing 3 L of seed medium, and under the condition of 37 °C, maintain the dissolved oxygen ≥ 30% by adjusting the pressure, rotation speed, and air volume, and culture the recombinant microorganism VB-B until it reaches the mature stage; the pressure adjustment range is 0.04 - 0.1 MPa, the rotation speed adjustment range is 200 - 600 rpm, and the air volume adjustment range is 0.2 - 1 m 3 / h; inoculate the above VB-B seed liquid cultured to the mature stage into the fermentation medium at an inoculation amount of 15% - 20%, and perform aerobic fermentation at 35 °C, pH = 6.7, and dissolved oxygen of 25% - 30%; during the fermentation process, when the glucose in the medium is consumed, an 80% (m / v) glucose solution needs to be added continuously to maintain the glucose concentration in the fermentation medium at 0.1 - 1 g / L; after 6 h of the above aerobic fermentation, anaerobic fermentation is carried out, and the dissolved oxygen is controlled at 5% - 10%, and the total fermentation cycle is 28 h to obtain the L-valine fermentation broth.

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