Construction and application of a glutamic acid corynebacterium mutant strain with high yield of L-homoserine

Abstract

The application discloses a high-yield L-homoserine Corynebacterium glutamicum mutant strain and application thereof, and belongs to the technical field of fermentation engineering. The application takes Corynebacterium glutamicum ATCC13032 as a starting strain, knocks out the regulatory protein McbR, amino acid intracellular transport protein MetD, homoserine kinase, phosphoenolpyruvate carboxykinase, cysteine beta cleavage enzyme, O-acetylhomoserine sulfhydrylase, weakens isocitric acid dehydrogenase and dihydrodipicolinic acid synthase, and overexpresses pyruvate carboxylase gene mutant, aspartate kinase mutant, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, branched-chain amino acid transport protein BrnF, branched-chain amino acid transport protein BrnE, aspartate aminotransferase, bifunctional aspartate kinase / homoserine dehydrogenase 1 mutant, NADP + dependent glycerol-3-phosphate dehydrogenase. The recombinant bacteria provided by the application can reach 63.5g / L of L-homoserine yield after 96h of culture in a fermenter.

Description

TECHNICAL FIELD

[0001] The application relates to construction and application of a high-yield L-homoserine Corynebacterium glutamicum mutant, and belongs to the technical field of fermentation engineering. BACKGROUND

[0002] L-homoserine is an important platform compound, which can be used not only for synthesizing L-methionine, but also as a precursor for synthesizing spermine. Recently, L-homoserine has also been used for synthesizing the herbicide L-glufosinate. Most commercial glufosinate is a mixture of D- and L-glufosinate, but only L-glufosinate has herbicidal activity. If all D-glufosinate is converted into L-glufosinate, the amount and cost of glufosinate can be reduced, thereby reducing the harm to the soil and crops. Therefore, the market demand for L-homoserine is increasing.

[0003] In Corynebacterium glutamicum, glucose is converted into pyruvate through glycolysis, in which NADH is produced by the catalysis of glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.12; gapA encoding), and ATP is produced by the catalysis of phosphoglycerate kinase (EC: 2.7.2.3; pgk encoding); pyruvate is converted into oxaloacetate under the catalysis of pyruvate carboxylase (EC: 6.4.1.1; pyc encoding), which consumes ATP and absorbs carbon dioxide; oxaloacetate is converted into aspartate under the catalysis of aspartate dehydrogenase (EC: 2.6.1.1; aspB encoding), which consumes one glutamate, and the synthesis of glutamate consumes NADPH; aspartate is converted into aspartate-4-phosphate under the catalysis of aspartate kinase (EC: 2.7.2.4; lysC encoding), which consumes ATP; aspartate-4-phosphate is converted into aspartate-4-semialdehyde under the catalysis of aspartate-semialdehyde dehydrogenase (EC: 1.2.1.11; asd encoding), which consumes NADPH; and aspartate-4-semialdehyde is converted into the target product L-homoserine under the catalysis of homoserine dehydrogenase (EC: 1.1.1.3; hom encoding), which consumes NADPH. It is found that the synthesis of L-homoserine from glucose as a carbon source consumes a large amount of NADPH, and a large amount of NADH is generated at the same time.

[0004] Corynebacterium glutamicum is a safe and robust industrial microorganism, which can be used to produce high-value chemicals from cheap substrates. Currently, there are reports on the synthesis of L-homoserine by Corynebacterium glutamicum, but the yield is not high (Construction and application of a mutant strain of Corynebacterium glutamicum producing L-homoserine. Patent No. ZL202010439183.2; Li et al., Optimization of CRISPR-Cas9 through promoter replacement and efficient production of L-homoserine in Corynebacterium glutamicum. Biotechnology Journal, published in 2021). It is speculated from the literature that the yield of L-homoserine synthesized by Corynebacterium glutamicum is limited due to the imbalance of co-factor supply. SUMMARY

[0005] To solve the above problems, the present application constructs a recombinant Corynebacterium glutamicum strain, so that Corynebacterium glutamicum can use glucose as a raw material for efficient production of L-homoserine.

[0006] The present application provides a recombinant Corynebacterium glutamicum strain, wherein the coding gene of regulatory protein McbR (mcbR), the coding gene of amino acid intracellular transport protein MetD (metD), the coding gene of homoserine kinase (thrB), the coding gene of phosphoenolpyruvate carboxykinase (pck), the NCgl2360 site gene, the NCgl2688 site gene and the coding gene of O-acetylhomoserine thiolase (metY) are knocked out; the coding gene of isocitrate dehydrogenase (icd) and the coding gene of dihydrodipicolinate synthase (dapA) are weakened; the coding gene of pyruvate carboxylase gene mutant (pyc P458S ), the coding gene of aspartate kinase mutant (lysC T311I ), the coding gene of aspartate semialdehyde dehydrogenase (asd), the coding gene of homoserine dehydrogenase (hom), the coding gene of branched-chain amino acid transport protein BrnF (brnF), the coding gene of branched-chain amino acid transport protein BrnE (brnE), the coding gene of aspartate aminotransferase (aspC), and the coding gene of bifunctional aspartate kinase / homoserine dehydrogenase 1 mutant (thrA S345 ) are integrated on the genome.

[0007] In one embodiment, the recombinant strain is further modified according to (a) or (b) as follows:

[0008] (a) knocking out the NCgl2360 site and integrating a gene expressing NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase at the NCgl2360 site;

[0009] (b) knocking out the metY site and integrating a gene expressing NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase at the metY site.

[0010] In one embodiment, the genes at the NCgl2360 site and the NCgl2688 site both encode the cysteine beta-lyase encoding gene metB.

[0011] In one embodiment, the weakened expression refers to replacing the ATG start codon of icd with GTG.

[0012] In one embodiment, after knocking out pck, a gene aspC is integrated at the pck site; after knocking out the NCgl2360 site, a gene expressing thrA S345 is integrated at the NCgl2360 site.

[0013] In one embodiment, the NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase encoding gene is expressed from the promoter P tuf , P sod , or P NCgl1676 .

[0014] In one embodiment, the genes lysC sod , pyc T311I , asd, hom, thrA S345 are expressed from the promoter P P458S .

[0015] In one embodiment, the recombinant bacteria also express thrA S345 and / or the NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase encoding gene in free form.

[0016] In one embodiment, the NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase encoding gene comprises gapN or gapC.

[0017] In one embodiment, the NADP trc dependent glycerolaldehyde-3-phosphate dehydrogenase encoding gene is expressed from the promoter P + .

[0018] In an embodiment, the nucleotide sequence of the mcbR is shown as SEQ ID NO. 1; the nucleotide sequence of the metD is shown as SEQ ID NO. 2; the nucleotide sequence of the thrB is shown as SEQ ID NO. 3; the nucleotide sequence of the pck is shown as SEQ ID NO. 4; the nucleotide sequence of the metY is shown as SEQ ID NO. 7;

[0019] In an embodiment, the nucleotide sequence of the icd is shown as SEQ ID NO. 8; the nucleotide sequence of the dapA is shown as SEQ ID NO. 9.

[0020] In an embodiment, the nucleotide sequence of the pyc P458S is shown as SEQ ID NO. 10; the nucleotide sequence of the lysC T311I is shown as SEQ ID NO. 11; the nucleotide sequence of the asd is shown as SEQ ID NO. 12; the nucleotide sequence of the hom is shown as SEQ ID NO. 13; the nucleotide sequence of the brnF is shown as SEQ ID NO. 14, the nucleotide sequence of the brnE is shown as SEQ ID NO. 15; the nucleotide sequence of the aspC is shown as SEQ ID NO. 16; the nucleotide sequence of the thrA S345 is shown as SEQ ID NO. 17.

[0021] In an embodiment, the nucleotide sequence of the Cgl1066 site is shown as SEQ ID NO. 18, the nucleotide sequence of the gapN is shown as SEQ ID NO. 19; the nucleotide sequence of the gapC is shown as SEQ ID NO. 20.

[0022] In an embodiment, the nucleotide sequence of the NCgl2360 site gene is shown as SEQ ID NO. 5, the nucleotide sequence of the NCgl2688 site gene is shown as SEQ ID NO. 6.

[0023] In an embodiment, the nucleotide sequence of the promoter P tuf is shown as SEQ ID NO. 21, the nucleotide sequence of the promoter P sod is shown as SEQ ID NO. 22, the nucleotide sequence of the promoter P NCgl1676 is shown as SEQ ID NO. 23.

[0024] In an embodiment, the Corynebacterium glutamicum ATCC 13032 is used as the starting strain.

[0025] The application provides a method for efficiently producing L-homoserine, and the recombinant bacteria are used as fermentation strains to produce L-homoserine.

[0026] In an embodiment, the recombinant bacteria are used to produce L-homoserine by fermentation with glucose as the main carbon source and corn slurry, yeast powder and peptone as supplements.

[0027] In an embodiment, the recombinant bacteria are inoculated into the fermentation medium when the OD 600 is 15-30, and the glucose concentration is maintained at 10-20 g / L.

[0028] In an embodiment, the fermentation tank fermentation is carried out at 30-35℃, 400-600 rpm, 0.8-4.0vvm ventilation, and 15%-40% dissolved oxygen.

[0029] The application also protects the use of the recombinant C. glutamicum bacteria or the method for producing L-homoserine in the preparation of L-homoserine or products containing L-homoserine in the biological and chemical fields.

[0030] The application has the following beneficial effects:

[0031] The application reengineers the genome of C. glutamicum, knocks out or overexpresses the related genes in the L-homoserine metabolic pathway, and balances the supply of coenzymes, thereby obtaining a recombinant C. glutamicum bacteria that can efficiently produce L-homoserine. The application uses C. glutamicum ATCC 13032 as the starting strain, knocks out the regulatory protein McbR, the amino acid intracellular transport protein MetD, the homoserine kinase, the phosphoenolpyruvate carboxykinase, the cysteine beta cleavage enzyme, the O-acetylhomoserine thiol-lyase, weakens the isocitrate dehydrogenase and the dihydrodipicolinate synthase, integrates and expresses the pyruvate carboxylase gene mutant, the aspartate kinase mutant, the aspartate semialdehyde dehydrogenase, the homoserine dehydrogenase, the branched-chain amino acid transport protein BrnF, the branched-chain amino acid transport protein BrnE, the aspartate aminotransferase, the bifunctional aspartate kinase / homoserine dehydrogenase 1 mutant, the NADP + dependent glycerol-3-phosphate dehydrogenase, and further, separately expresses the bifunctional aspartate kinase / homoserine dehydrogenase 1 mutant and / or the NADP + dependent glycerol-3-phosphate dehydrogenase. The recombinant bacteria in the application are currently the highest level of C. glutamicum for synthesizing L-homoserine. The L-homoserine yield can reach 60.3-63.5 g / L when the recombinant bacteria are cultured in a system with cheap glucose as the substrate for 96 h. DETAILED DESCRIPTION

[0032] The specific embodiments of the present application will be described in detail below with specific examples.

[0033] (I) Plasmid

[0034] Plasmid pK18mobsacB (described in the literature Schafer A, A Tauch, W Jager, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene, 1994, 145(1): 69-73.).

[0035] pEC-XK99E (described in the literature Kirchner, O. et al. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. Journal of Biotechnology, 2003, 104(1-3): 287-99.).

[0036] (II) Culture medium

[0037] LBHIS culture medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, 91 g / L of D-sorbitol. Add 20 g / L agar strip to prepare LBHIS solid culture medium.

[0038] Seed culture medium: 25 g / L of D-glucose, 1.25 g / L of urea, 20 g / L of corn syrup, 1 g / L of KH2PO4, 0.5 g / L of MgSO4, and use ammonia water to adjust the pH to 7.0.

[0039] Fermentation culture medium: 100 g / L of D-glucose, 20 g / L of corn syrup, 20 g / L of (NH4)2SO4, 1 g / L of KH2PO4, 0.5 g / L of MgSO4, 0.01 g / L of MnSO4·H2O, 0.01 g / L of FeSO4·7H2O, 1 mg / L of vitamin B1, 6 mg / L of vitamin B6, 4 mg / L of vitamin B 12 , 0.025 mg / L of biotin, 2 g / L of yeast powder, 2 g / L of proteose peptone, and use ammonia water to adjust the pH to 7.0.

[0040] (III) Assay method

[0041] Determination of cell concentration: a certain amount of bacterial suspension was diluted with deionized water, and the OD value was determined at 660 nm using a UV 7500 type visible spectrophotometer.

[0042] Determination of glucose: high performance liquid chromatography (HPLC). Instrument: LC-20AT high performance liquid chromatograph (equipped with a differential refractive index detector), chromatographic conditions: chromatographic column: Aminex HPX-87H ion exchange column, mobile phase: 5mM H2SO4, flow rate: 0.6mL / min, column temperature: 40℃, sample size: 10μL, differential refractive index detector: glucose detection, sample preparation: 1mL of fermentation broth was centrifuged at 12,000rpm for 5min, the supernatant was appropriately diluted and filtered through a 0.22μL filter membrane, and then subjected to high performance liquid chromatography analysis.

[0043] Determination of amino acids: high performance liquid chromatography (HPLC). Instrument: LC-20AT high performance liquid chromatograph (equipped with an ultraviolet detector and an automatic sampler). Chromatographic conditions: chromatographic column: C18 column; mobile phase: A phase (disodium hydrogen phosphate 9.0g, sodium tetraborate decahydrate 9.5g, add water 2000mL, adjust pH to 8.2 with 36% hydrochloric acid, filter with 0.45μm filter membrane); B phase (methanol:acetonitrile:water = 45:45:10, filter with 0.45μm filter membrane); before injection, use borate buffer and OPA reagent for derivatization.

[0044] (IV) Methods involved in strain construction

[0045] Gibson assembly method: see Gibson et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods, 2009, 6(5):343-5 for specific steps.

[0046] Table 1. Strains involved in the following examples

[0047]

[0048] Table 2. Primers used in Examples 1-8

[0049]

[0050]

[0051]

[0052] Table 3. Plasmids required for Examples 1-8

[0053]

[0054] Example 1: Construction of strain Cg13

[0055] Corynebacterium glutamicum CglO (described in Li N, Xu S, Du G, et al. Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux [J]. Biochemical Engineering Journal, 2020, 161: 107665.) was used as the starting strain. The NCgl2360 site was knocked out, and thrA was integrated at the NCgl2360 site. S345F The NCgl2688 site was knocked out, and metY was knocked out to obtain strain Cg13.

[0056] The restriction enzymes HindIII and BamHI were used to digest the plasmid pK18mobsacB to obtain a linearized pK18mobsacB fragment. Corynebacterium glutamicum CglO was used as the template to amplify the upstream homologous arm fragment, the promoter fragment P sod , and the downstream homologous arm fragment using primers NCgl2360-UP-F / R, primers Psod-F / R, and NCgl2360-DOWN-F / R, respectively. S345F The restriction enzymes HindIII and BamHI were used to digest the plasmid pK18mobsacB to obtain a linearized pK18mobsacB fragment. Corynebacterium glutamicum CglO was used as the template to amplify the upstream homologous arm fragment, the promoter fragment P S345F , and the downstream homologous arm fragment using primers NCgl2360-UP-F / R, primers Psod-F / R, and NCgl2360-DOWN-F / R, respectively. sod The restriction enzymes HindIII and BamHI were used to digest the plasmid pK18mobsacB to obtain a linearized pK18mobsacB fragment. Corynebacterium glutamicum CglO was used as the template to amplify the upstream homologous arm fragment, the promoter fragment P S345F , and the downstream homologous arm fragment using primers NCgl2360-UP-F / R, primers Psod-F / R, and NCgl2360-DOWN-F / R, respectively. sod The restriction enzymes HindIII and BamHI were used to digest the plasmid pK18mobsacB to obtain a linearized pK18mobsacB fragment. Corynebacterium glutamicum CglO was used as the template to amplify the upstream homologous arm fragment, the promoter fragment P S345F (P).

[0057] Using the same method, the knockout plasmid pK-NCgl2688 was constructed using primers NCgl2688-U-F / NCgl2688-U-R and NCgl2688-D-F / NCgl2688-D-R, and the knockout plasmid pK-metY was constructed using primers LN191-metY-UP-F / LN192-metY-UP-R and primers LN193-metY-DOWN-F / LN194-metY-DOWN-R.

[0058] The above obtained integrated plasmid pK-P sod -thrA S345F (P), knockout plasmid pK-NCgl2688 and knockout plasmid pK-metY were introduced into strain CglO in turn to obtain strain Cgl3.

[0059] Example 2: Construction of strains Cgl3-22, Cgl3-23

[0060] Plasmid pEC-XK99E was used as a template, and restriction enzymes EcoR I and BamHI were used to linearize plasmid pEC-XK99E;

[0061] Using E. coli K12-MG1655 as a template, primers thrAl-F / R and thrA2-F / R were used to amplify fragments 1 and 2 of the thrA site-directed mutation; Gibson assembly was used to assemble fragments 1 and 2 of the thrA site-directed mutation and linearized pEC-XK99E to obtain heterologous expression plasmid pEC-thrA S345F _Ec.

[0062] Plasmid pEC-thrA S345F _Ec was used as a template, and primers P trc -F / R were used to amplify P trc promoter fragment; gapN fragment was amplified using primers LN51-gapN-F / LN52-gapN-R with the synthesized gapN gene as a template; plasmid pEC-thrA S345F _Ec was linearized using restriction enzymes Xba I and BamHI; Gibson assembly was used to assemble P trc promoter fragment, gapN fragment and linearized pEC-thrA S345F _Ec to obtain heterologous expression plasmid pEC-thrA S345F _Ec-P trc -gapN.

[0063] Plasmid pEC-thrA S345F _Ec was used as a template, and primers P trc -F / R were used to amplify P trc promoter fragment; gapC fragment was amplified using primers LN59-gapC-F / LN60-gapC-R with the synthesized gapC gene as a template; plasmid pEC-thrA S345F _Ec was linearized using restriction enzymes Xba I and BamHI; Gibson assembly was used to assemble P trcPromoter fragment, gapC fragment and linearized pEC-thrA S345F The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. S345F The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. trc The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA.

[0064] The constructed plasmid pEC-thrA was transformed into the strain Cg13 constructed in Example 1 by electroporation, and the transformed bacterial liquid was respectively coated on LBHIS medium containing 15 μL / mL of kanamycin and cultured at 30°C until single colonies were formed; single colonies were picked and cultured in LBHIS medium containing 15 μL / mL of kanamycin at 30°C and 220 rpm for 12 h to obtain bacterial liquid, 2 μL of which was coated on a plate of LBHIS medium containing 100 g / L sucrose and cultured at 30°C for 48 h, and single colonies were picked for verification to obtain strains Cg13-22 and Cg13-23. S345F The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. trc The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. S345F The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. trc The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA.

[0065] Example 3: Construction of strains Cg18, Cg19, Cg20 and Cg21

[0066] The plasmid pK18mobsacB was digested with restriction endonucleases HindIII and BamHI to obtain a linearized pK18mobsacB fragment;

[0067] Cg10 as a template and LN78-Cgl1066-UP-F / LN79-Cgl1066-UP-R, LN80-Ptuf-F / LN81-Ptuf-R and LN589-Cgl1066-DOWN-F / LN590-Cgl1066-DOWN-R as primers, respectively, to obtain the upstream homologous arm, the promoter fragment P tuf tuf and the downstream homologous arm fragment, the promoter fragment Ptuf and the gapN fragment were assembled by Gibson assembly method to obtain the integration plasmid pK18trc-Cgl1066(Ptuf-gapN) with the linearized pK18mobsacB. S345F The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA. trc The heterologous expression plasmid pEC-thrA was assembled by assembling the promoter fragment, the gapC fragment and the linearized pEC-thrA.

[0068] Using Cg10 as a template, and with primers LN86-metY-UP-F / LN592-metY-UP-R, LN80-Ptuf-F / LN81-Ptuf-R, and LN84-metY-DOWN-F / LN85-metY-DOWN-R, the upstream homologous arm and promoter fragment P were amplified. tuf Downstream homologous arm; with pEC-thrA S345F _Ec-P trc Using -gapN as a template, and LN82-gapN-F / LN83-gapN-R as templates, the gapN fragment is amplified. The upstream homologous arm fragment, downstream homologous arm fragment, and promoter fragment P are then amplified. tuf The gapN fragment was assembled with linearized pK18mobsacB using the Gibson assembly method to obtain the integration plasmid pK18trc-metY(Ptuf-gapN).

[0069] Using Cg10 as a template, and with primers LN86-metY-UP-F / LN87-metY-UP-R, LN88-Psod-F / LN89-Psod-R, and LN84-metY-DOWN-F / LN85-metY-DOWN-R, the upstream homologous arm and promoter fragment P were amplified. sod Downstream homologous arm; using pEC-thrA from Example 2 S345F _Ec-P trc Using -gapN as a template, and LN90-gapN-F / LN83-gapN-R as templates, the gapN fragment was amplified. The upstream homologous arm fragment, downstream homologous arm fragment, and promoter fragment P were then amplified. sod The gapN fragment was assembled with linearized pK18mobsacB using the Gibson assembly method to obtain the integration plasmid pK18trc-metY (Psod-gapN).

[0070] Using Cg10 as a template, and with primers LN86-metY-UP-F / LN91-metY-UP-R, LN92-PNCgl1676-F / LN32-PNCgl1676-R, and LN84-metY-DOWN-F / LN85-metY-DOWN-R, the upstream homologous arm and promoter fragment P were amplified, respectively. NCgl1676 Downstream homologous arm; using pEC-thrA from Example 2 S345F _Ec-P trc Using -gapN as a template, and LN93-gapN-F / LN83-gapN-R as templates, the gapN fragment is amplified. The upstream homologous arm fragment, downstream homologous arm fragment, and promoter fragment P are then amplified. NCgl1676The gapN fragment was assembled with linearized pK18mobsacB by Gibson assembly to obtain the integration plasmid pK18trc-metY(PNCgl1676-gapN).

[0071] The gapN integration expression plasmids pK18trc-Cgl1066(Ptuf-gapN), pK18trc-metY(Ptuf-gapN), pK18trc-metY(Psod-gapN), pK18trc-metY(PNCgl1676-gapN) were transformed into the strain Cg13 constructed in Example 1, respectively, and sucrose counter- selection was performed to obtain strains Cgl8, Cgl9, Cg20, Cg21, respectively.

[0072] Example 4: Construction of strains Cgl8-1, Cgl9-1, Cg20-1, Cg21-1

[0073] The pEC-thrA S345F The Cg18, Cgl9, Cg20, Cg21 were respectively electroporated with the pEC-thrA of Example 2, and the obtained bacterial liquid was spread on LBHIS medium containing 15 μL / mL kanamycin and cultured at 30°C until single colonies were formed; single colonies were picked and cultured in LBHIS medium containing 15 μL / mL kanamycin at 30°C, 220 rpm for 12 h to obtain bacterial liquid, 2 μL of which was spread on LBHIS medium containing 100 g / L sucrose and cultured at 30°C for 48 h, and single colonies were picked for verification to obtain strains Cgl8-1, Cgl9-1, Cg20-1, Cg21-1.

[0074] Example 5: Fermentation experiment of mutant strains

[0075] The single colonies of the recombinant strains prepared in Examples 1-4 and Corynebacterium glutamicum ATCC 13032 containing empty plasmid pEC-XK99E were inoculated on seed culture medium for activation, and cultured for 20 h to obtain seed liquid with OD 600 The seed liquid was inoculated into a 5 L fermenter containing 2500 mL fermentation medium at a 10% (v / v) inoculation amount. The initial rotation speed was set at 400 rpm, the initial aeration amount was set at 0.8 vvm, the culture temperature was constant at 30°C, and ammonia was used to maintain the pH at 6.5. Then the rotation speed and aeration amount were changed to maintain the dissolved oxygen at 25%. The glucose was maintained at about 15 g / L by glucose addition. The fermentation was carried out for 96 h, and the L-homoserine yield of the recombinant strains was shown in Table 1, which could reach up to 63.5 g / L.

[0076] Table 4 Fermentation results of mutant strains

[0077] Strain L-homoserine production (g / L) Glucose consumption (g / L) OD 600 ]]> Cg13-22 62.8 280.2 60.5 Cg13-23 40.1 270.1 63.4 Cg18-1 63.5 250.1 62.2 Cg19-1 62.1 290.5 63.4 Cg20-1 60.3 281.4 61.2 Cg21-1 61.4 271.2 60.5

[0078] While the application has been disclosed in its preferred embodiments with reference to specific details, it is to be understood that various modifications and substitutions can be made by one skilled in the art without departing from the spirit and scope of the application, as defined by the following claims.

Claims

1. A recombinant strain of Corynebacterium glutamicum, characterized in that, The recombinant bacteria knock out the coding gene of regulatory protein McbR of Corynebacterium glutamicum ATCC13032 mcbR , the coding gene of amino acid intracellular transport protein MetD metD , the coding gene of homoserine kinase thrB , the coding gene of phosphoenolpyruvate carboxykinase pck , NCgl2360 site gene, NCgl2688 site gene, the coding gene of O-acetylhomoserine thiolase metY ; the coding gene of weak isocitrate dehydrogenase icd , the coding gene of dihydrodipicolinate synthase dapA ; A gene encoding a mutant of acetohydroxy acid synthase is integrated into the genome pyc P458S A gene encoding a mutant of aspartate kinase lysC T311I A gene encoding a mutant of aspartate semialdehyde dehydrogenase asd A gene encoding a mutant of homoserine dehydrogenase hom A gene encoding a branched-chain amino acid transporter BrnF brnF A gene encoding a branched-chain amino acid transporter BrnE brnE Knocking out Cgl1066 a site, and integrating a gene encoding NADP + dependent glycerolaldehyde-3-phosphate dehydrogenase at the site gapN Knocking out pck After that, a gene pck is integrated into the site of the ilvA gene aspC ; knockout NCgl2360 after the site, in NCgl2360 site integration expression thrA S345F , The mcbR The nucleotide sequence is shown in SEQ ID NO.1; metD The nucleotide sequence is shown in SEQ ID NO.2; thrB The nucleotide sequence is shown in SEQ ID NO.3; pck The nucleotide sequence is shown in SEQ ID NO.4; NCgl2360 The nucleotide sequence of the site gene is shown in SEQ ID NO.

5. NCgl2688 The nucleotide sequence of the site gene is shown in SEQ ID NO.

6. metY The nucleotide sequence is shown in SEQ ID NO.7; icd The nucleotide sequence is shown in SEQ ID NO. 8; dapA The nucleotide sequence is shown in SEQ ID NO. 9; pyc P458S The nucleotide sequence is shown in SEQ ID NO.10; lysC T311I The nucleotide sequence is shown in SEQ ID NO.11; asd The nucleotide sequence is shown in SEQ ID NO.12; hom The nucleotide sequence is shown in SEQ ID NO.13; brnF The nucleotide sequence is shown in SEQ ID NO.

14. brnE The nucleotide sequence is shown in SEQ ID NO.15; aspC The nucleotide sequence is shown in SEQ ID NO.16; thrA S345F The nucleotide sequence is shown in SEQ ID NO.17; Cgl1066 The nucleotide sequence of the site is shown in SEQ ID NO.18; gapN The nucleotide sequence is shown in SEQ ID NO.

19.

2. The recombinant bacteria of claim 1, wherein, The NADP + The coding gene for the glycerinaldehyde-3-phosphate dehydrogenase dependent on the promoter P tuf , P sod or P NCgl1676 initiates expression.

3. The recombinant bacteria of claim 2, wherein The nucleotide sequence of said promoter P tuf The nucleotide sequence of said promoter P sod The nucleotide sequence of said promoter P NCgl1676 The nucleotide sequence of said promoter P 4. The recombinant bacteria of claim 1, wherein The recombinant bacteria also express, in isolation thrA S345F .

5. A method for efficiently producing L-homoserine, characterized by, The recombinant bacteria of any one of claims 1-4 are used as fermentation strains to produce L-homoserine by fermentation.

6. The method of claim 5, wherein, The recombinant bacteria of any one of claims 1-4 are used as fermentation strains to produce L-homoserine by fermentation, with glucose as the main carbon source, supplemented with corn steep liquor, yeast powder and peptone.

7. The recombinant C. glutamicum bacteria of any one of claims 1-4, or the method of claim 5 or 6, for use in the biological and chemical fields to prepare L-homoserine or products containing L-homoserine.

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