Valine-producing strain and construction method and application thereof

Abstract

The application provides a valine production strain and a construction method and application thereof, a designed acetyl-hydroxy acid synthase mutant is ilvB The 88th base of the gene is changed from a to c, the 382nd base of the gene is changed from a to g, and the 413th base of the gene is changed from c to t, the gene order of the designed artificial operator comprises a promoter, a coding mutated acetyl-hydroxy acid synthase ilvB (A138V) gene or ilvB (Q30K，S128G，A138V) gene, ilvN (G20D，I21D，I22F) gene, a coding pyruvate kinase pyk gene and a terminator; by designing a specific acetyl-hydroxy acid synthase mutant and related biological materials and an artificial operator, using a pK18mob sacB The gene editing technology based on allele exchange has the advantages of good genetic stability, high fermentation yield and the like for the constructed strain through directional modification of the strain, and can stably produce valine.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering technology, and in particular to a valine-producing strain, its construction method, and its application. Background Technology

[0002] Valine, chemically known as 2-amino-3-methylbutyric acid, belongs to the branched-chain amino acids (BCAAs) and has wide applications in feed nutrition, pharmaceutical manufacturing, and the food industry. In recent years, with the promotion of soybean meal reduction technology and low-protein diet formulations in the feed industry, the demand for L-valine supplementation has increased significantly, making it the fourth largest amino acid product in the animal nutrition field, with a high compound annual growth rate.

[0003] In the industrial production of valine, microbial fermentation has become the mainstream technology due to its environmental friendliness and sustainability. Currently, globally, *Escherichia coli* is the primary microbial host used for L-valine production, and its technological development mainly focuses on the systematic modification of metabolic pathways to improve valine yield and sugar-acid conversion rate. However, *E. coli* produces endotoxins, which, in contrast... C. glutamicum This strain is not only a recognized safe strain, but also the most traditional amino acid production strain, and has the potential to become a highly efficient valine production strain in the future. Therefore, developing valine production strains with higher acid production capacity, higher conversion efficiency, and stronger environmental adaptability has significant practical value in production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a valine-producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned valine-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned valine-producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An acetylhydroxyl synthase mutant, wherein the amino acid sequence of the large subunit of the acetylhydroxyl synthase mutant is as shown in SEQ ID NO.16 (the nucleotide sequence is as shown in SEQ ID NO.15) or is more than 95% identical to the sequence and originates from the same species; and the amino acid sequence of the small subunit of the acetylhydroxyl synthase mutant is as shown in SEQ ID NO.20 (its nucleotide sequence is as shown in SEQ ID NO.19) or is more than 95% identical to the sequence and originates from the same species.

[0008] The biological material associated with the above-mentioned acetylhydroxyl synthase mutant is any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the above-mentioned acetylhydroxy acid synthase mutant; (a2) An expression cassette containing the nucleic acid molecule described in (a1): (a3) A recombinant vector comprising the nucleic acid molecule described in (a1) or the expression cassette described in (a2); (a4) A recombinant microorganism comprising the nucleic acid molecule of (a1), the expression cassette of (a2), or the recombinant vector of (a3).

[0009] Preferably, in the above-mentioned biological material, the nucleotide sequence of the nucleic acid molecule encoding the large subunit of the acetylhydroxyl synthase mutant in (a1) is as shown in SEQ ID NO.15 of the sequence listing or is more than 95% identical to the sequence and originates from the same species; the nucleotide sequence of the nucleic acid molecule encoding the small subunit of the acetylhydroxyl synthase mutant is as shown in SEQ ID NO.19 of the sequence listing or is more than 95% identical to the sequence and originates from the same species.

[0010] Preferably, in the above-mentioned biological materials, the vector skeleton of the recombinant vector in (a3) ​​includes pK18, pK19, pK18mobsacB, pNV18, or pNV19, etc.

[0011] Preferably, in the above-mentioned biological material, the recombinant microorganisms in (a4) include Escherichia coli, Corynebacterium glutamicum, or Bacillus, etc.

[0012] The above-mentioned acetylhydroxy acid synthase mutants or biological materials are used in the construction of artificial operons or valine-producing strains or in the fermentation production of valine.

[0013] An artificial operon is constructed by reordering and linking genes related to valine synthesis, wherein the genes related to valine synthesis are selected from... ilvB (A138V) Gene or ilvB (Q30K，S128G，A138V) Gene, ilvN (G20D，I21D，I22F) Genes and pyk Gene.

[0014] Preferably, the aforementioned artificial operon further includes a promoter and a terminator, and the specific gene sequence includes a promoter, a terminator encoding a mutated acetylhydroxyl synthase, and a terminator encoding a mutated acetylhydroxyl synthase. ilvB (A138V) Gene or ilvB (Q30K，S128G，A138V) Gene, ilvN (G20D，I21D，I22F) Genes encoding pyruvate kinase pykGenes and terminators: transcription is initiated by an artificial operon controlled by a promoter, and terminated by an artificial operon controlled by a terminator. The artificial operon is named... ilvB (Q30K，S128G，A138V) ilvN (G20D，I21D，I22F) - pyk .

[0015] Preferably, in the above-mentioned artificial manipulator, the promoter is connected to the... ilvB (A138V) Gene or ilvB (Q30K，S128G，A138V) The genes are directly linked end to end; ilvB (A138V) Gene or ilvB (Q30K，S128G，A138V) Genes and ilvN (G20D，I21D，I22F) The genes are linked using RBS-1; ilvN (G20D，I21D，I22F) Genes and pyk The genes are linked using RBS-2; pyk It is directly connected to the beginning and end of the terminator.

[0016] Preferably, in the above-mentioned artificial operon, the nucleotide sequence of RBS-1 is shown in SEQ ID NO.30, and the nucleotide sequence of RBS-2 is shown in SEQ ID NO.24.

[0017] Preferably, in the above-mentioned artificial manipulator, the promoter is a strong promoter, and the promoter is selected from: P tuf promoter, P lac promoter, P trp promoter, P tac promoter, P lPL promoter or P pgk100 Any of the promoters.

[0018] Preferably, in the above-mentioned artificial manipulator, the promoter is P. tuf The promoter, the P tuf The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.23.

[0019] Preferably, in the above-mentioned artificial manipulator, the terminator is a strong terminator, and the terminator is selected from any one of the following: trc terminator, T7 terminator, rrnB T1 / 2 terminator, BBa_B1006 terminator or L3S2P21 / P55 terminator.

[0020] Preferably, the terminator of the above-mentioned artificial operon is the rrnBT1 terminator, and the nucleotide sequence of the rrnBT1 terminator is shown in SEQ ID NO.25.

[0021] Preferably, the above-mentioned artificial operator is named P. tuf - ilvB (A138V) ilvN (G20D,I21D,I22F) - pyk Its nucleotide sequence is shown in SEQ ID NO.26 of the sequence listing.

[0022] Preferably, the above-mentioned artificial operator is named P. tuf- ilvB (Q30K，S128G，A138V) ilvN (G20D，I21D，I22F) - pyk Its nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing.

[0023] The above-mentioned artificial operon is used in the construction of valine-producing strains or in the fermentation production of valine.

[0024] A valine-producing strain, characterized by the integration of the aforementioned artificial operon.

[0025] Preferably, the above-mentioned valine-producing strain is named strain [strain name missing]. CVal-8 It utilizes targeted modification methods on the starting strain Corynebacterium glutamicum This was obtained through further modifications based on ATCC 13032, the modifications of which also include: Corynebacterium glutamicum The lactate dehydrogenase gene was knocked out on the ATCC 13032 genome. ldhA Overexpression of branched-chain amino acid efflux protein genes brnFE Acetylhydroxyl synthase gene obtained by point mutation in the genome. ilvB (A138V) and ilvN (G20D，I21D，I22F) and overexpression ilvB (A138V) and ilvN (G20D，I21D，I22F) It relieved valine feedback inhibition and enhanced affinity for pyruvate substrates.

[0026] The above acetylhydroxyl synthase gene ilvB (Q30K，S128G，A138V) and ilvN (G20D，I21D，I22F) yes ilvB and ilvN It is obtained after point mutation of the gene, that is ilvB The change from a to c at position 88 of the gene causes the amino acid residue at position 30 to change from lysine to glutamine; the change from a to g at position 382 causes the amino acid residue at position 128 to change from glycine to serine; and the change from c to t at position 413 causes the amino acid residue at position 138 to change from alanine to valine.ilvN Changing the base at position 59 from g to a and at position 60 from a to t causes the amino acid residue at position 20 to change from glycine to aspartic acid; changing the base at position 61 from a to g and at position 62 from t to a causes the amino acid residue at position 21 to change from isoleucine to aspartic acid; changing the base at position 64 from a to t causes the amino acid residue at position 22 to change from isoleucine to phenylalanine.

[0027] Preferably, in the above-mentioned valine-producing strain, the directional modification method utilizes pK18mob based on allele exchange. sacB The system starts with the strain Corynebacterium glutamicum The genome of chromosome 13032 of ATCC was modified.

[0028] Preferably, the above-mentioned valine-producing strain, in ldhA Integrating artificial operons at the site ilvB (Q30K，S128G，A138V) ilvN (G20D，I21D，I22F) - pyk and with promoter P tuf Regulation; the aforementioned pyk The amino acid sequence is shown in SEQ ID NO. 22 (its nucleotide sequence is shown in SEQ ID NO. 21) or is more than 95% identical to this sequence and originates from the same species; promoter P tuf The nucleotide sequence is shown in the sequence listing SEQ ID NO.23.

[0029] Preferably, the above-mentioned valine-producing strain, wherein ldhA The amino acid sequence is shown in SEQ ID NO.2 (its nucleotide sequence is shown in SEQ ID NO.1) or is more than 95% identical to the sequence and originates from the same species.

[0030] Preferably, the above-mentioned valine-producing strain uses promoter P sod replace ilvB (A138V) , ilvN (G20D，I21D，I22F) and brnFE The natural promoter, the ilvB The amino acid sequence is shown in SEQ ID NO.4 (its nucleotide sequence is shown in SEQ ID NO.3) or is more than 95% identical to this sequence and originates from the same species. ilvB (A138V) The amino acid sequence is shown in SEQ ID NO. 14 (its nucleotide sequence is shown in SEQ ID NO. 13) or is more than 95% identical to this sequence and originates from the same species.ilvB (Q30K，S128G，A138V) The amino acid sequence is shown in SEQ ID NO. 16 (the nucleotide sequence is shown in SEQ ID NO. 15) or is more than 95% identical to this sequence and originates from the same species. ilvN The amino acid sequence is shown in SEQ ID NO. 18 (its nucleotide sequence is shown in SEQ ID NO. 17) or is more than 95% identical to this sequence and originates from the same species. ilvN (G20D，I21D，I22F) The amino acid sequence is shown in SEQ ID NO. 20 (its nucleotide sequence is shown in SEQ ID NO. 19) or is more than 95% identical to this sequence and originates from the same species. brnFE The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing (wherein, brnF The amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.6. brnE The amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.8, or it is 95% identical to this sequence and originates from the same species. The promoter P sod The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing. ilvB natural promoter (P) ilvB The nucleotide sequence of the compound is shown in SEQ ID NO. 11 of the sequence listing. brnFE natural promoter (P) brnFE The nucleotide sequence of is shown in the sequence listing SEQ ID NO.12.

[0031] The specific steps for constructing the above-mentioned valine-producing strain are as follows: (1) In Corynebacterium glutamicum A point mutation in the ATCC 13032 genome yields the acetylhydroxyl synthase gene. ilvB (A138V) and ilvN (G20D，I21D，I22F) Relieve the effects of valine ilvBN Feedback inhibition, and enhanced affinity for pyruvate; (2) Using promoter P sod Replacement of acetylhydroxyl synthase gene ilvB (A138V) and ilvN (G20D，I21D，I22F) The natural promoter; (3) Using promoter P sod Replacement of branched-chain amino acid efflux genes brnFEThe natural promoter enhances the ability to efflux valine; (4) Knockout of lactate dehydrogenase gene ldhA and in ldhA The artificial operon is integrated at the site and promoted by the promoter P. tuf Regulation.

[0032] Application of the above-mentioned valine-producing strains in the fermentation production of valine.

[0033] Preferably, the above application follows these steps: (1) Seed activation and culture: After the bacterial solution is inoculated from the preservation tube and evenly spread on the activation slant for culture, it is then transferred to a shaker containing seed culture medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the Erlenmeyer flask containing the fermentation medium at an inoculation rate of 10-15%, seal the flask with gauze and shake to culture. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water. Add glucose solution to maintain the fermentation. The fermentation cycle is 30-32 h.

[0034] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 1.0-3.0 g / L, peptone 8.0-12.0 g / L, yeast extract 4.0-6.0 g / L, sodium chloride 2-3 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.1-0.3 g / L, agar powder 2.3-2.7%, with the remainder being water, pH 7.0-7.2.

[0035] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 2.5%, with the remainder being water, pH 7.0-7.2.

[0036] Preferably, in the above applications, the seed culture medium used in seed culture is: yeast extract 6.0-10.0 g / L, peptone 2.0-4.0 g / L, (NH4)2SO4 2.0-4.0 g / L, KH2PO4 2.0-4.0 g / L, V B1 V B2 V B3 V B5 V B12 1-3 mg / L each, V H 0.5-1.5 mg / L, MgSO4·7H2O 0.4-0.6 g / L, the remainder is water.

[0037] Preferably, in the above application, the seed culture medium used is: yeast extract 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0038] Preferably, in the above application, the fermentation medium used in the fermentation culture is: yeast extract 8.0-12.0 g / L, citric acid 2.0-4.0 g / L, glutamic acid 1.0-3.0 g / L, (NH4)2SO4 3.0-5.0 g / L, KH2PO4 6.0-10.0 g / L, MgSO4·7H2O 1.0-3.0 g / L, FeSO4·7H2O 35-45 mg / L, V B1 V B2 V B3 V B5 V B12 Each 0.5-1.5 mg / L, V H 0.08-0.12 mg / L, phenol red 1-3%, the remainder is water.

[0039] Preferably, in the above application, the fermentation medium used in the fermentation culture is: yeast extract 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 8.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0040] All of the above-mentioned culture media can be prepared using standard methods.

[0041] Beneficial effects: The above-mentioned valine-producing strain utilizes pK18mob based on allele exchange. sacB Systematic gene editing technology first removes the valine pair... ilvBN The feedback inhibition and enhanced affinity for pyruvate; at the same time ilvBN The natural promoter is replaced with the strong promoter P.sod This causes more pyruvate to flow to valine; knockout ldhA And construct an artificial manipulator at this location using the promoter P. tuf control ilvB (Q30K，S128G，A138V) ilvN (G20D，I21D，I22F) - pyk Simultaneous multicopy expression was performed; brnFE The natural promoter is replaced with the strong promoter P. sod This enhances the efflux capacity of valine. The constructed strain is plasmid-free, defect-free, requires no induction, and possesses advantages such as good genetic stability and high fermentation yield. It is an excellent strain for stable valine production. The strain efficiently synthesizes valine de novo using glucose as a substrate. After 32 hours of shake-flask fermentation, the valine yield can reach up to 16.5 g / L. Attached Figure Description

[0042] Figure 1 A diagram illustrating the method for targeted modification of valine-producing strains. Detailed Implementation

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0044] Unless otherwise specified, the percentage sign "%" in the examples refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 mL of solution.

[0045] The starting strain used in the examples was wild-type Corynebacterium glutamicum. Corynebacterium glutamicum ATCC 13032; see the sequence listing for the corresponding promoter and gene.

[0046] The gene editing method used was based on the literature (SUZUKIN, INUIM. Genome engineering of Corynebacterium glutamicum[M]. Berlin: Springer, 2013:89-105). The engineered plasmid pK18mob involved in this method... sacB It uses pK18 as its framework, including the promoter P tuf , sacB Genes and kanamycin resistance (working concentration: 10 mg / L). Technical terms such as gene integration and plasmid construction used in the following examples are explained in this article. Primers used in the strain construction process are shown in Table 1.

[0047] Table 1 Primers used in strain construction

[0048]

[0049] Example 1 like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows: Starting strain Corynebacterium glutamicum Electrocompetent cells were prepared according to the procedures described in Sambrook, J., FE, Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ATCC 13032.

[0050] 1.1 Genes ilvB Point mutation by Corynebacterium glutamicum Using the ATCC 13032 genome as a template, PCR amplification of the upstream homologous arm was performed using primers Up-ilvBA138V-F / Up-ilvB A138V-R, and PCR amplification of the downstream homologous arm was performed using primers Down-ilvB A138V-F / Down-ilvB A138V-R. The A138V mutation was introduced using these primers. Using the recovered upstream and downstream homologous arms containing the mutation as templates, overlap PCR was performed using primers Up-ilvB A138V-F and Down-ilvB A138V-R to obtain the point mutation. ilvB The required fragment is up-ilvB-down. Then, the up-ilvB-down fragment is combined with the linearized plasmid pK18mob. sacB Connect and build pK18mob sacB- up-ilvB-down plasmid.

[0051] pK18mob sacB- up-ilvN-down plasmid electroporation to Corynebacterium glutamicumElectroporation of ATCC13032 competent cells was performed. After electroporation, the cells were plated onto plates containing kanamycin-resistant (10 μg / mL) agar. Transformants growing on the kanamycin-resistant plates were amplified by colony PCR using primers pK18-CX1 and pK18-CX2. Single-crossover colonies were inoculated into BHI shakers containing kanamycin-resistant (10 μg / mL) agar and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto sucrose-containing plates. Single colonies growing on the plates were then used to spot sucrose and kanamycin-resistant (10 μg / mL) agar plates. The plates were inverted and incubated at 32°C. The identification primers G-ilvBA138V-F / G-ilvB were used. A138V-R was used to verify colonies that did not grow on kanamycin-resistant plates (i.e., the resistance gene was removed) but grew well on sucrose plates. Agarose gel electrophoresis showed that the fragment size was consistent with the theoretical value, and positive transformants were screened to obtain the strain. CVal -1.

[0052] 1.2 Genes ilvN Point mutation by Corynebacterium glutamicum Using the ATCC 13032 genome as a template, PCR amplification of the upstream homologous arm was performed using primers Up-ilvN-mut202122-F / Up-ilvN-mut202122-R, and PCR amplification of the downstream homologous arm was performed using primers Down-ilvN-mut202122-F / Down-ilvN-mut202122-R. G20D, I21D, and I22F mutations were introduced using primers. Using the recovered upstream and downstream homologous arms containing the mutations as templates, overlap PCR was performed using primers Up-ilvN-mut202122-F and Down-ilvN-mut202122-R to obtain the fragment up-ilvN-down required for the point mutation ilvN. Subsequently, the up-ilvN-down fragment was combined with the linearized plasmid pK18mob. sacB Connect and build pK18mob sacB- up-ilvN-down plasmid.

[0053] pK18mob sacB- up-ilvN-down plasmid electroporation to CValElectroporation was performed on competent cells at -1°C. After electroporation, the cells were plated on plates containing kanamycin-resistant (10 μg / mL) culture medium. Transformants grown on these plates were then amplified by colony PCR using primers pK18-CX1 and pK18-CX2. Single-exchange colonies were then inoculated into BHI culture medium containing kanamycin-resistant (10 μg / mL) culture medium. The culture was carried out in shake tubes at 32°C for 12 hours. The culture was then diluted 300-fold with BHIS resuscitation solution and spread onto sucrose-containing plates. Single colonies grown on the plates were then used to spot sucrose and kanamycin-resistant plates (10 μg / mL). The plates were inverted and incubated at 32°C. Colonies that did not grow on kanamycin-resistant plates (i.e., the resistance gene was removed) but grew well on sucrose plates were verified by colony PCR using the identification primers genome-seq-ilvNmut-F / genome-seq-ilvNmut-R. The fragment size obtained was consistent with the theoretical value, and positive transformants were screened to obtain the bacterial strain. CVal -2.

[0054] 1.3 Genes ilvB (A138V) N (G20D，I21D，I22F) Replace with a stronger starter by Corynebacterium glutamicum Using the ATCC13032 genome as a template, primer P was used. sod -F and P sod -R was used for PCR amplification to obtain the promoter sequence fragment sod; primers UP-P were used. sod -F and UP-P sod -ilvB-R amplifies the upstream homologous arm up-sod; using primers Down-P sod -ilvB-F and Down-P sod -ilvBN-R amplifies the downstream homologous arm sod-down. Using the recovered promoter sequence and upstream and downstream homologous arms as templates, primers UP-P are used. sod -F and Down-P sod Overlap PCR was performed using -ilvBN-R to obtain the overexpressed gene. ilvB The required fragment is up-sod-down. Then, the up-sod-down fragment is combined with the linearized plasmid pK18mob. sacB Connect and build pK18mob sacB- up-sod-down plasmid.

[0055] pK18mob sacB- up-sod-down plasmid electroporation to CValElectroporation was performed on competent cells at -2°C. After electroporation, the cells were plated onto plates containing kanamycin-resistant (10 μg / mL) agar. Transformants growing on the kanamycin-resistant plates were verified by colony PCR using primers pK18-CX1 and pK18-CX2. Single colonies with single-exchange were inoculated into BHI shakers containing kanamycin-resistant (10 μg / mL) agar and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto sucrose-containing plates. Single colonies growing on the plates were then used to spot sucrose and kanamycin-resistant (10 μg / mL) agar plates. The plates were inverted and incubated at 32°C. The results were analyzed using the identification primer genome-seq-P. sod -ilvBN-F / genome-seq-P sod -ilvBN-R colonies that did not grow on kanamycin-resistant plates (i.e., the resistance gene was removed) but grew well on sucrose plates were validated by colony PCR. Agarose gel electrophoresis showed that the fragment size was consistent with the theoretical value. Positive transformants were screened to obtain the bacterial strain. CVal -3.

[0056] 1.4 Genes brnFE Replace with a stronger starter by Corynebacterium glutamicum Using the ATCC 13032 genome as a template, and primer UP-P sod -BrnFE-F / UP-P sod -BrnFE-R was used for PCR to obtain the upstream homologous arm, and primer DN-P was used. sod -brnFE-F / DN-P sod PCR was performed using -brnFE-R to obtain the downstream homologous arm; primer P was used to obtain the downstream homologous arm. sod -F / P sod -R is used for PCR amplification to obtain P sod Fragment; with primer UP-P sod -BrnFE-F and DN-P sod -brnFE-R was used as a primer to obtain UP-P via overlap PCR. sod -brnFE-Down gene fragment, then, UP-P fragment sod -brnFE-Down and linearized plasmid pK18mob sacB Connect and build pK18mob sacB- UP-P sod -brnFE-Down plasmid.

[0057] pK18mob sacB- UP-P sod -brnFE-Down plasmid electroporation to CValElectroporation was performed on competent cells at -3°C. After electroporation, the cells were plated onto plates containing kanamycin-resistant (10 μg / mL) agar. Transformants grown on the kanamycin-resistant plates were verified by colony PCR using primers pK18-CX1 and pK18-CX2. Single colonies with single-exchange were inoculated into BHI shakers containing kanamycin-resistant (10 μg / mL) agar and incubated at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto sucrose-containing plates. Single colonies grown on the plates were then used to spot sucrose and kanamycin-resistant (10 μg / mL) agar plates. The plates were inverted and incubated at 32°C. The results were verified using identification primers UP-P. sod -BrnFE-F / UP-P sod -BrnFE-R performed colony PCR verification on colonies that did not grow on kanamycin-resistant plates (i.e., those with the resistance gene removed) but grew well on sucrose plates. Agarose gel electrophoresis showed that the fragment size was consistent with the theoretical value. Positive transformants were then screened to obtain the bacterial strain. CVal -4.

[0058] 1.5 genes ilvB (A138V) ilvN (G20D，I21D，I22F) and pyk Add copies respectively by CVal Using the LDH-UP-BNP-F / LDH-UP-BNP-R primers as a template, PCR was performed to obtain the upstream homologous arm, and PCR was performed using the LDH-DOWN-BNP-F / LDH-DOWN-BNP-R primers to obtain the downstream homologous arm; PCR was performed using primer P... tuf -F / P tuf -R amplification to obtain P tuf Fragment; obtained by amplification using primers ilvBN-F / ilvBN-R ilvB (A138V) N (G20D，I21D，I22F) Gene fragments were amplified by PCR using primers pyk-F / pyk-R to obtain the pyk gene fragment. The terminator rrnBT1 gene fragment was amplified by PCR using primers rrnBT-F / rrnBT-R. The fragment LDHup-P was amplified by overlap PCR using primers LDH-UP-BNP-F and LDH-DOWN-BNP-R. tuf -ilvB (A138V) N (G20D，I21D，I22F) -rrnBT1-P tuf -pyk-LDHdown, then the fragment is combined with the linearized plasmid pK18mob sacB Ligation, constructing plasmid pK18mobsacB- LDHup-P tuf -ilvB (A138V) N (G20D，I21D，I22F) -rrnBT1-P tuf -pyk-LDHdown.

[0059] The above plasmid was electroporated to CVal Electroporation was performed on competent cells at -4°C. After electroporation, the cells were plated on plates containing kanamycin-resistant cells (10 μg / mL). Transformants growing on the kanamycin-resistant plates were verified by colony PCR using primers pK18-CX1 and pK18-CX2. Single-colony amplification was performed to complete the single-exchange process. These colonies were then inoculated into BHI shakers containing kanamycin-resistant cells (10 μg / mL) and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto plates containing sucrose. The single colonies growing on the plates were then... Colonies were spotted onto sucrose and kanamycin-resistant plates (10 μg / mL), and the plates were inverted and incubated at 32°C. Colonies that did not grow on kanamycin-resistant plates (i.e., the resistance gene was removed) but grew well on sucrose plates were verified by colony PCR using the identification primers genome-seq-ldh-F / genome-seq-ldh-R. The fragment size obtained was consistent with the theoretical value, and positive transformants were screened to obtain the bacterial strain. CVal -5.

[0060] 1.6 Artificial Operator Genes ilvB (A138V) ilvN (G20D，I21D，I22F) - pyk Integration ldhA :: ilvB (A138V) ilvN (G20D，I21D，I22F) - pyk ) by CVal Using the LDH-UP-BNP-F2 / LDH-UP-BNP-R2 primers as a template, the upstream homologous arm was obtained by PCR, and the downstream homologous arm was obtained by PCR using the LDH-DOWN-BNP-F2 / LDH-DOWN-BNP-R2 primers; primer P... tuf -F / P tuf -R amplification to obtain P tuf Fragment; ilvB was obtained by amplification using primers ilvBN-F2 / ilvBN-R2. (A138V) N (G20D，I21D，I22F)The pyk gene fragment was obtained by PCR amplification using primers pyk-F2 / pyk-R2, and then further amplified by overlap PCR using primers LDH-UP-BNP-F2 and LDH-DOWN-BNP-R2 to obtain the LDHup-P fragment. tuf -ilvB (A138V) N (G20D，I21D，I22F) -pyk-LDHdown, using primers rrnBT-F2 / rrnBT-R2, PCR amplification was performed to obtain the terminator rrnBT1 gene fragment. Then, the fragment was used for LDHup-P tuf -ilvB (A138V) N (G20D，I21D，I22F) -pyk-LDHdown and linearized plasmid pK18mob sacB Connect and build pK18mob sacB- LDHup-P tuf -ilvB (A138V) N (G20D，I21D，I22F) -pyk-LDHdown plasmid.

[0061] pK18mob sacB- LDHup-P tuf -ilvB (A138V) N (G20D，I21D，I22F) -pyk-LDHdown plasmid electroporation to CVal Electroporation was performed on competent cells at -3°C. After electroporation, the cells were plated on plates containing kanamycin-resistant cells (10 μg / mL). Transformants growing on the kanamycin-resistant plates were verified by colony PCR using primers pK18-CX1 and pK18-CX2. Single-colony amplification was performed, and the resulting single-colony amplification was inoculated into BHI shakers containing kanamycin-resistant cells (10 μg / mL) and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto plates containing sucrose. The single colonies growing on the plates were then... Colonies were spotted onto sucrose and kanamycin-resistant plates (10 μg / mL), and the plates were inverted and incubated at 32°C. Colonies that did not grow on kanamycin-resistant plates (i.e., the resistance gene was removed) but grew well on sucrose plates were verified by colony PCR using the identification primers genome-seq-ldh-F / genome-seq-ldh-R. The fragment size obtained was consistent with the theoretical value, and positive transformants were screened to obtain the bacterial strain. CVal -6.

[0062] 1.7 Genetic analysis based on CVal-5 ilvB (A138V) Point mutations (Q30K, S128G) At Genewiz, a gene sequence fragment up-ilvBM-down containing mutations in Q30K, S128G, and A138V was synthesized. This fragment was then ligated to the linearized plasmid pK18mobsacB to construct the pK18mobsacB-up-ilvBM-down plasmid. The plasmid was then electroporated into… CVal -5, Following the same method described above, construct and screen to obtain positive transformants and obtain the bacterial strain. CVal -7.

[0063] 1.8 Genetic analysis based on CVal-6 ilvB (A138V) Point mutations (Q30K, S128G) At Genewiz, a gene sequence fragment up-ilvBM-down containing mutations in Q30K, S128G, and A138V was synthesized. This fragment was then ligated to the linearized plasmid pK18mobsacB to construct the pK18mobsacB-up-ilvBM-down plasmid. The plasmid was then electroporated into… CVal -6, Following the same method described above, construct and screen to obtain positive transformants and obtain the bacterial strain. CVal -8.

[0064] The strains involved in the above construction process are shown in Table 2.

[0065] The strains listed in Table 2

[0066] The aforementioned artificial manipulator P tuf - ilvB (Q30K, S128G，A138V) ilvN (G20D,I21D,I22F) The nucleotide sequence is shown in SEQ ID NO. 28 of the sequence listing; P tuf - pyk The nucleotide sequence is shown in the sequence listing SEQ ID NO.29.

[0067] Example 2 Valine was produced by shake-flask fermentation using the valine-producing strain described in Example 1.

[0068] 2.1 Culture medium 2.1.1 Slant Culture Medium Glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 2.5%, dissolved in water, then the pH was adjusted to 7.0-7.2 with sodium hydroxide, and the volume was brought to 500 mL. The solution was dispensed into test tubes (9 mL / tube) and flasks (45 mL / flask), and sterilized in an autoclave at 121℃ for 20 min.

[0069] 2.1.2 Seed Culture Medium Yeast extract 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0070] 2.1.3 Fermentation medium Yeast powder 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 8.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0071] 2.2.1 Seed activation and culture: The bacterial culture was inoculated from the preservation tube and evenly spread onto the activation slant. It was then incubated at 37°C for 12 hours, transferred to the activation slant and incubated for another 10 hours. Finally, it was transferred to a shaker containing 5 mL of seed culture medium for seed culture.

[0072] 2.2.2 Fermentation culture: Inoculate the seed culture at a rate of 15% into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL). Seal the flask with nine layers of gauze and incubate at 36°C with shaking at 220 rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia. Add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator; when the fermentation broth color no longer changes, it indicates a sugar deficiency; add 1-2 mL of 60% (m / v) glucose solution if a sugar deficiency is detected). The fermentation cycle is 32 h. No antibiotics or inducers are added during fermentation.

[0073] After 32 hours of shake-flask fermentation, the valine yield is shown in Table 3. The valine yield of strain CVal-8 can reach as high as 16.5 g / L.

[0074] Table 3

[0075] According to the experimental results in Table 3, strains CVal-1 to CVal-4 showed that the modification relieved the feedback inhibition of valine and opened up the valine production pathway. Although strain CVal-5 added the key gene ilvBN for valine synthesis and the key gene pyk for precursor pyruvate supply, the yield was not significantly improved. In contrast, strain CVal-6, which used one operon for simultaneous multiple copies, significantly improved the yield. On the other hand, strain CVal-7, which further rationally modified the key gene ilvB, also achieved a considerable increase in yield. Finally, strain CVal-8, which used one operon for simultaneous multiple copies and further rationally modified the key gene ilvB, had the highest valine yield, reaching 16.5 g / L.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An acetylhydroxy acid synthase mutant, characterized in that: The acetylhydroxyl synthase mutant is composed of a large subunit and a small subunit. The amino acid sequence of the large subunit of the acetylhydroxyl synthase mutant is shown in SEQ ID NO.16 of the sequence listing; the amino acid sequence of the small subunit of the acetylhydroxyl synthase mutant is shown in SEQ ID NO.20 of the sequence listing.

2. A biomaterial related to the acetylhydroxyl synthase mutant of claim 1, characterized in that: It can be any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the acetylhydroxyl synthase mutant; (a2) An expression cassette containing the nucleic acid molecule described in (a1): (a3) A recombinant vector comprising the nucleic acid molecule described in (a1) or the expression cassette described in (a2); (a4) A recombinant microorganism comprising the nucleic acid molecule of (a1), the expression cassette of (a2), or the recombinant vector of (a3).

3. The use of the acetylhydroxy acid synthase mutant of claim 1 or the biomaterial of claim 2 in the construction of artificial operons or valine-producing strains or in the fermentation production of valine.

4. An artificial manipulator, characterized in that: The gene sequence of the artificial operon includes a promoter and a sequence encoding a mutated acetylhydroxyl synthase. ilvB Gene, ilvN (G20D，I21D，I22F) Genes encoding pyruvate kinase pyk Gene and terminator, the promoter of which is associated with mutated acetylhydroxyl synthase ilvB Genes are directly linked end-to-end; mutated acetylhydroxyl synthase ilvB Genes and ilvN (G20D，I21D，I22F) Genes are linked using RBS-1; ilvN (G20D，I21D，I22F) Genes and pyk Genes are linked using RBS-2; pyk The gene and the terminator are directly linked head-to-tail; the mutated acetylhydroxyl synthase... ilvB The nucleotide sequence of the gene is shown in SEQ ID NO.15 of the sequence listing. ilvN (G20D，I21D，I22F) The nucleotide sequence of the gene is shown in SEQ ID NO.19 of the sequence listing, encoding pyruvate kinase. pyk The nucleotide sequences of the genes are shown in SEQ ID NO.21, the nucleotide sequences of RBS-1 are shown in SEQ ID NO.30, the nucleotide sequences of RBS-2 are shown in SEQ ID NO.24, the nucleotide sequences of the promoters are shown in SEQ ID NO.23, and the nucleotide sequences of the terminators are shown in SEQ ID NO.

25.

5. The artificial manipulator according to claim 4, characterized in that: Its nucleotide sequence is shown in the sequence listing SEQ ID NO.

27.

6. The use of the artificial operon of claim 4 or 5 in the construction of valine-producing strains or in the fermentation production of valine.

7. A valine-producing strain, characterized in that: It utilizes targeted modification methods on the starting strain Corynebacterium glutamicum Obtained through further modifications based on ATCC 13032: Corynebacterium glutamicum The lactate dehydrogenase gene was knocked out on the ATCC 13032 genome. ldhA Overexpression of branched-chain amino acid efflux protein genes brnFE Acetylhydroxyl synthase gene obtained by point mutation in the genome. ilvB (A138V) and ilvN (G20D，I21D，I22F) and overexpression ilvB (A138V) and ilvN (G20D，I21D，I22F) The artificial manipulator described in claim 4 or 5 is integrated.

8. The method for constructing the valine-producing strain according to claim 7, characterized in that: The specific steps are as follows: (1) In Corynebacterium glutamicum A point mutation in the ATCC 13032 genome yields the acetylhydroxyl synthase gene. ilvB (A138V) and ilvN (G20D，I21D，I22F) Relieve the effects of valine ilvBN Feedback inhibition, and enhanced affinity for pyruvate; (2) Using promoter P sod Replacement of acetylhydroxyl synthase gene ilvB (A138V) and ilvN (G20D，I21D，I22F) The natural promoter; (3) Using promoter P sod Replacement of branched-chain amino acid efflux genes brnFE The natural promoter enhances the ability to efflux valine; (4) Knockout of lactate dehydrogenase gene ldhA and in ldhA The artificial operon of claim 4 or 5 is integrated at the site, the artificial operon being promoted by promoter P. tuf Regulation.

9. The application of the valine-producing strain of claim 7 in the fermentation production of valine.

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