L-isoleucine production strain as well as construction method and application thereof

Abstract

The invention provides a strain for producing L-isoleucine and a construction method and application of the strain. Pck genes of the strain are knocked out, ilvBN (G20D, I21D and I22F), ilvA (V140M, F383A), lysC (T311I), hom (G378I), thrB (A20G), ppc, aspB, thrC, asd and brnFE are over-expressed, and the strain is prepared through operons Psod-ilvBN (G20D, I21D and I22F)-ilvA (V140M, F383A)-Ptuf-thrA-thrB-thrC and Psod-ppc-aspB-Ptuf

Description

Technical Field

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

[0002] L-Isoleucine is an essential branched-chain amino acid that cannot be synthesized by organisms and must be obtained through exogenous intake or artificial supplementation. As a basic building block of proteins, it not only participates in the construction and repair of body tissues but also plays a crucial role in physiological processes such as energy metabolism, immune function regulation, and hemoglobin synthesis. Furthermore, L-isoleucine has wide applications in medicine, nutritional supplements, and animal feed. With the continued growth in market demand, developing efficient L-isoleucine production processes has become a research hotspot.

[0003] Currently, the main methods for producing L-isoleucine include protein hydrolysis extraction, chemical synthesis, and microbial fermentation. Protein hydrolysis suffers from high raw material costs, cumbersome separation and purification steps, and low yields; chemical synthesis is hampered by complex reaction steps, difficulties in chiral resolution, and significant environmental pollution, making it unsuitable for green manufacturing. In contrast, microbial fermentation, with its advantages of mild conditions, wide availability of raw materials, and environmental friendliness, has gradually become the mainstream method for industrial L-isoleucine production. This method typically uses renewable carbon sources such as glucose as substrates and utilizes metabolically engineered microbial strains for directed fermentation synthesis. In recent years, with the development of synthetic biology and systems metabolic engineering, researchers have effectively eliminated feedback inhibition in the L-isoleucine biosynthesis pathway by rationally modifying model strains such as *Escherichia coli* and *Corynebacterium glutamicum*, enhancing precursor supply and cofactor regeneration, and further improving fermentation performance. However, existing production strains still have room for improvement in terms of yield, carbon source conversion rate, and process stability. Therefore, constructing a high-yield L-isoleucine production strain with a clear genetic background and stable performance is a pressing technical problem to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an L-isoleucine producing strain.

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

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

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An artificial operon is constructed by rearranging and linking genes related to L-isoleucine synthesis, wherein the L-isoleucine synthesis-related genes are selected from... ilvA (V140M、F383A) Gene, ilvBN (G20D，I21D，I22F) Genes and thrA Gene, thrB Gene, thrC Gene, ppc Genes and aspB Two or more genes.

[0008] Preferably, in the above-mentioned artificial operon, the L-isoleucine synthesis-related gene is... ilvA (V140M、F383A) Gene, ilvBN (G20D，I21D，I22F) Gene, thrA Gene, thrB Genes and thrC Genes, including promoter 1 and promoter 2, and the specific gene sequence of the operon includes promoter 1, promoter 2, promoter 3, promoter 4, promoter 5, promoter 6, promoter 7, promoter 8, promoter 9, promoter 10, promoter 11, promoter 12, promoter 13, promoter 14, promoter 15, promoter 16, promoter 17, promoter 18, promoter 19, promoter 12, promoter 13, promoter 14, promoter 1 ilvBN (G20D，I21D，I22F) Gene, ilvA (V140M、F383A) Gene, promoter 2, thrA Gene, thrB Genes and thrC The gene was named the artificial operon P1- ilvBN (G20D，I21D，I22F) -ilvA (V140M、F383A) - P2- thrA - thrB - thrC .

[0009] Preferably, the aforementioned artificial operon further includes RBS and a terminator, and the specific gene sequence of the operon includes promoter 1, ... ilvBN (G20D，I21D，I22F) Genes, RBS1, ilvA (V140M、F383A) Gene, terminator, promoter 2 thrA Gene, thrB Genes and thrC The gene, the nucleotide sequence of which is RBS1 is shown in the sequence listing SEQ ID NO.32.

[0010] Preferably, in the above-mentioned artificial manipulator, the promoter is a strong promoter, and promoter 1 and promoter 2 are respectively selected from: P tuf promoter, P sod promoter, P lac promoter, P trp promoter, P tac promoter, P lPLpromoter or P pgk100 Any of the promoters.

[0011] Preferably, in the above-mentioned artificial manipulator, the promoter 1 is P. sod The promoter, the P sod The nucleotide sequence of the promoter is shown in SEQ ID NO.8, and promoter 2 is P. tuf The promoter, the P tuf The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.9.

[0012] Preferably, in the above-mentioned artificial manipulator, the terminator is a strong terminator, and the terminator is selected from any one of the following: T7 terminator, rrnB T1 / 2 terminator, BBa_B1006 terminator, or L3S2P21 / P55 terminator.

[0013] 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.31.

[0014] Preferably, the above-mentioned artificial operator is named artificial operator P. sod - ilvBN (G20D，I21D，I22F) - ilvA (V140M、F383A) - P tuf - thrA - thrB - thrC Its nucleotide sequence is shown in SEQ ID NO.25 of the sequence listing.

[0015] Preferably, the above-mentioned artificial operon is constructed by the following method: from P tuf and P sod Artificial promoters control the initiation of transcription by operons, while natural terminators in the genome control the termination of transcription by operons. This is achieved by optimizing gene linkage and the location of genes within the operon.

[0016] Preferably, the above-mentioned artificial manipulator, wherein thrA Gene, thrB Gene, thrC Genes originating from E. coli thrA Gene, thrB Gene, thrC Gene.

[0017] Preferably, in the above-mentioned artificial operon, the L-isoleucine synthesis-related gene is... ilvA (V140M、F383A) Gene, ppcGene, aspB Genes and ilvBN (G20D，I21D，I22F) Genes, including promoter 1 and promoter 2, and the specific gene sequence of the operon includes promoter 1, promoter 2, promoter 3, promoter 4, promoter 5, promoter 6, promoter 7, promoter 8, promoter 9, promoter 10, promoter 11, promoter 12, promoter 13, promoter 14, promoter 15, promoter 16, promoter 17, promoter 18, promoter 19, promoter 12, promoter 13, promoter 14, promoter 1 ppc Gene, aspB Gene, promoter 2, ilvA (V140M、F383A) Genes and ilvBN (G20D，I21D，I22F) The gene was named the artificial operon P1- ppc-aspB- P2- ilvA (V140M、F383A) -ilvBN (G20D，I21D，I22F) .

[0018] Preferably, the aforementioned artificial operon further includes RBS and a terminator, and the specific gene sequence of the operon includes promoter 1, ... ppc Genes, RBS1, aspB Gene, terminator, promoter 2 ilvA (V140M、F383A) Genes, RBS2, ilvBN (G20D，I21D，I22F) The genes, the nucleotide sequence of RBS1 is shown in SEQ ID NO.32, and the nucleotide sequence of RBS2 is shown in SEQ ID NO.33.

[0019] Preferably, in the above-mentioned artificial manipulator, the promoter is a strong promoter, and promoter 1 and promoter 2 are respectively selected from: P tuf promoter, P sod promoter, P lac promoter, P trp promoter, P tac promoter, P lPL promoter or P pgk100 Any of the promoters.

[0020] Preferably, in the above-mentioned artificial manipulator, the promoter 1 is P. sod The promoter, the P sod The nucleotide sequence of the promoter is shown in SEQ ID NO.8, and promoter 2 is P. tuf The promoter, the P tuf The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.9.

[0021] Preferably, in the above-mentioned artificial manipulator, the terminator is a strong terminator, and the terminator is selected from any one of the following: T7 terminator, rrnB T1 / 2 terminator, BBa_B1006 terminator, or L3S2P21 / P55 terminator.

[0022] 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.31.

[0023] Preferably, the above-mentioned artificial operator is named artificial operator P. sod - ppc-aspB- P tuf - ilvA (V140M、F383A) - ilvBN (G20D，I21D，I22F) Its nucleotide sequence is shown in SEQ ID NO.28 of the sequence listing.

[0024] Preferably, the above-mentioned artificial operon is constructed by the following method: from P tuf and P sod Artificial promoters control the initiation of transcription by operons, while natural terminators in the genome control the termination of transcription by operons. This is achieved by optimizing gene linkage and the location of genes within the operon.

[0025] The application of the above-mentioned artificial operons in constructing L-isoleucine-producing strains or in the fermentation production of L-isoleucine.

[0026] An L-isoleucine-producing strain, integrated with the aforementioned artificial operon P1- ilvBN (G20D，I21D，I22F) - ilvA (V140M、F383A) - P2- thrA - thrB - thrC Or artificially manipulated P sod - ilvBN (G20D，I21D，I22F) -ilvA (V140M、F383A) - P tuf - thrA - thrB - thrC and / or artificial manipulator P1- ppc-aspB- P2- ilvA (V140M、F383A) -ilvBN (G20D，I21D，I22F) Or artificially manipulated P sod - ppc-aspB- P tuf - ilvA (V140M、F383A) -ilvBN (G20D，I21D，I22F) .

[0027] Preferably, the L-isoleucine-producing strain described above is produced by targeted modification of the starting strain. C.glutamicum This strain was obtained through further modification based on ATCC 13032 (wild-type strain), the modification of which further includes: C.glutamicum ATCC 13032 genome knockout pck Gene, overexpression of branched-chain amino acid efflux protein gene brnFE Threonine dehydratase gene carrying mutation points ilvA (V140M、F383A) acetolactate synthase gene carrying mutation points ilvBN (G20D、I21D、I22F) Threonine synthase gene thrC Homoserine kinase genes carrying mutation sites thrB (A20G) Homoserine dehydrogenase gene carrying a mutation site hom (G378E) Aspartate semialdehyde dehydrogenase gene asd Aspartate kinase gene carrying mutation sites lysC (T311I) and aspartate transaminase gene [[ID=8 .

[0028] Preferably, the above-mentioned L-isoleucine-producing strain carries the threonine dehydratase gene with a mutation point. ​ (V140M、F383A) acetyllactate synthase gene ​ (G20D、I21D、I22F) And the threonine synthase gene derived from Escherichia coli W3110 ​ Homoserine kinase gene ​ and dual-function aspartate kinase / homoserine dehydrogenase gene ​ Through artificial manipulation of P sod - ​ (G20D，I21D，I22F) ​ (V140M、F383A) - P tuf - ​ - ​ - ​ To perform synchronous multiple copies; threonine dehydratase gene carrying mutation points. ​ (V140M、F383A) acetyllactate synthase gene ​ (G20D、I21D、I22F) and phosphoenolpyruvate carboxylase gene ​ Aspartate transaminase gene ​ Through artificial manipulation of P sod - ​ P tuf - ​(V140M、F383A) ​ (G20D，I21D，I22F) To perform synchronous multiple copies.

[0029] The above ​ (V140M、F383A) yes ​ The gene was obtained by point mutation, that is, the 418th base changed from g to a, the 1147th base changed from t to g, the 1148th base changed from t to c, and the 1149th base changed from t to a, resulting in the 140th amino acid residue changing from valine to methionine and the 383rd amino acid residue changing from phenylalanine to alanine. ​ (G20D、I21D、I22F) yes ​ The gene underwent point mutations, specifically changing the 59th base from g to a, the 60th base from a to t, the 61st base from a to g, the 62nd base from t to a, and the 64th base from a to t. This resulted in the 20th amino acid residue changing from glycine to aspartic acid, the 21st amino acid residue changing from isoleucine to aspartic acid, and the 22nd amino acid residue changing from isoleucine to phenylalanine. ​ (A20G) It is derived from Corynebacterium glutamicum. ​ The gene (nucleotide sequence as shown in SEQ ID NO.36, amino acid sequence as shown in SEQ ID NO.37, or with more than 95% identity to the sequence and originating from the same species) was obtained by point mutation, that is, the 59th base was changed from c to g and the 60th base was changed from a to c, resulting in the 20th amino acid residue being changed from alanine to glycine. ​ (G378E) yes ​ The gene was obtained by point mutation, that is, the base at position 1133 changed from g to a and the base at position 1134 changed from g to a, resulting in the amino acid residue at position 378 changing from glycine to glutamic acid; ​ (T311I) yes ​ The gene underwent a point mutation, specifically changing the 932nd base from c to t, which resulted in the 311th amino acid residue changing from threonine to isoleucine.

[0030] Preferably, in the above-mentioned L-isoleucine-producing strain, the directional modification method is homologous recombination gene editing technology, which utilizes streptomycin selection vector homologous recombination editing technology to completely modify the starting strain. ​ The genome of chromosome 13032 of ATCC was modified.

[0031] Preferably, the above-mentioned L-isoleucine-producing strain is produced using the artificial promoter P. sod Replacement of branched-chain amino acid efflux protein genes ​ acetyllactate synthase gene​ (G20D、I21D、I22F) Threonine synthase gene ​ and aspartate kinase gene ​ (T311I) The natural promoter, using the artificial promoter P tuf Replacement of threonine dehydratase gene ​ (V140M、F383A) Homoserine dehydrogenase gene ​ (G378E) and aspartate transaminase gene ​ The natural promoter; the artificial operator P1- is integrated at the cg1895 site. ​ (G20D，I21D，I22F) ​ (V140M、F383A) - P2- ​ - ​ - ​ Or artificially manipulated P sod - ​ (G20D、I21D、I22F) ​ (V140M、F383A) - P tuf - ​ - ​ - ​ ,exist ​ Site integration artificial operon P1- ​ P2- ​ (V140M、F383A) ​ (G20D，I21D，I22F) Or artificially manipulated P sod - ​ P tuf - ​ (V140M、F383A) ​ (G20D、I21D、I22F) .

[0032] Preferably, in the above-mentioned L-isoleucine-producing strain, the branched-chain amino acid efflux protein gene... ​ The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.15, or the sequence is more than 95% identical to the SEQ ID NO.1 and originates from the same species; the acetolactate synthase gene ​ (G20D、I21D、I22F) The nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.16, or the sequence is 95% identical to the sequence and originates from the same species (wherein, ​The nucleotide sequence is shown in SEQ ID NO.42 and the amino acid sequence is shown in SEQ ID NO.43, or the sequence is more than 95% identical to the sequence and originates from the same species; ​ The nucleotide sequence is shown in SEQ ID NO.44 and the amino acid sequence is shown in SEQ ID NO.45, or the sequence is more than 95% identical to the sequence and originates from the same species; ​ (G20D、I21D、I22F) The nucleotide sequence is shown in SEQ ID NO. 46, and the amino acid sequence is shown in SEQ ID NO. 47, or the sequence is 95% identical to the sequence and originates from the same species); the threonine synthase gene from Corynebacterium glutamicum. ​ The nucleotide sequence is shown in SEQ ID NO.3 and the amino acid sequence is shown in SEQ ID NO.17, or the sequence is more than 95% identical to the SEQ ID NO.17 and originates from the same species; the aspartate kinase gene ​ (T311I) The nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.18, or the sequence is 95% identical to the sequence and originates from the same species; the threonine dehydratase gene ​ (V140M、F383A) The nucleotide sequence is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 19, or the sequence is more than 95% identical to the sequence and originates from the same species; the homoserine dehydrogenase gene ​ (G378E) The nucleotide sequence is shown in SEQ ID NO. 6, and the amino acid sequence is shown in SEQ ID NO. 20, or the sequence is 95% identical to the sequence and originates from the same species; the aspartate transaminase gene ​ The nucleotide sequence is shown in SEQ ID NO.7 and the amino acid sequence is shown in SEQ ID NO.21, or the sequence is more than 95% identical to the sequence and originates from the same species; threonine synthase gene from *E. coli*. ​ The nucleotide sequence is shown in SEQ ID NO. 12, and the amino acid sequence is shown in SEQ ID NO. 22, or the sequence is more than 95% identical to the above sequence and originates from the same species; the homoserine kinase gene from *E. coli*. ​The nucleotide sequence of the aspartate kinase / homoserine dehydrogenase gene thrA from *E. coli* is shown in SEQ ID NO. 13, and the amino acid sequence is shown in SEQ ID NO. 23, or the sequence is more than 95% identical to the thrA gene and originates from the same species; the nucleotide sequence of the aspartate kinase / homoserine dehydrogenase gene thrA from *E. coli* is shown in SEQ ID NO. 14, and the amino acid sequence is shown in SEQ ID NO. 24, or the sequence is more than 95% identical to the thrA gene and originates from the same species; the phosphoenolpyruvate carboxylase gene ​ The nucleotide sequence is shown in SEQ ID NO.34, and the amino acid sequence is shown in SEQ ID NO.35, or the sequence is more than 95% identical to the sequence and originates from the same species; the homoserine kinase gene carrying the mutation site from Corynebacterium glutamicum. ​ (A20G) The nucleotide sequence is shown in SEQ ID NO.38, and the amino acid sequence is shown in SEQ ID NO.39, or the sequence is more than 95% identical to the sequence and originates from the same species; aspartate semialdehyde dehydrogenase gene ​ The nucleotide sequence is shown in SEQ ID NO.40 and the amino acid sequence is shown in SEQ ID NO.41, or the sequence is more than 95% identical to the sequence and originates from the same species.

[0033] Preferably, the above-mentioned L-isoleucine-producing strain, wherein ​ The nucleotide sequence of the gene is shown in SEQ ID NO. 29, and the amino acid sequence is shown in SEQ ID NO. 30, or the sequence is more than 95% identical to the sequence and originates from the same species.

[0034] Preferably, in the above-mentioned L-isoleucine-producing strain, the artificial promoter P sod The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing, and the artificial promoter P tuf The nucleotide sequence is shown in the sequence listing SEQ ID NO.9.

[0035] The specific steps for constructing the above-mentioned L-isoleucine-producing strain are as follows: (1) In ​ On the ATCC 13032 genome, via promoter P sod replace ​ The natural promoter enhances the efflux capacity of L-isoleucine; (2) By using a strong promoter P sod replace ​ (G20D、I21D、I22F) and ​ (T311I)The natural promoter, promoter P tuf replace ​ (V140M、F383A) , ​ (G378E) and ​ (A20G) The natural promoter relieves feedback inhibition (the ​ (G378E) and ​ (A20G) (Sharing a single promoter) (3) By using promoter P sod replace ​ The natural promoter, promoter P tuf replace ​ The natural promoter; (4) Integration of the artificial operon P1- at cg1895 site ​ (G20D，I21D，I22F) ​ (V140M、F383A) - P2- ​ - ​ - ​ Or artificially manipulated P sod - ​ (G20D，I21D，I22F) ​ (V140M、F383A) - P tuf - ​ - ​ - ​ This enhances the synthesis pathway of L-isoleucine; (5) Knockout ​ And integrate the artificial operon P1- at this site ​ P2- ​ (V140M、F383A) - ​ (G20D，I21D，I22F) Or artificially manipulated P sod - ​ P tuf - ​ (V140M、F383A) ​ (G20D，I21D，I22F) It blocks the reflux of isoleucine precursor oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) and enhances the carbon flow of phosphoenolpyruvate (PEP) directly to the isoleucine synthesis pathway.

[0036] Preferably, in the method for constructing the above-mentioned L-isoleucine-producing strain, each of the artificial operons is controlled by an artificial promoter to initiate transcription and by a terminator to terminate transcription, and is obtained by optimizing the gene connection method and the location of the gene in the operon.

[0037] Application of the above-mentioned L-isoleucine-producing strains in the fermentation production of L-isoleucine.

[0038] Preferably, the above application follows these steps: (1) Seed activation and seed culture: After the bacterial solution is evenly spread on the activation slant for culture, it is transferred to the activation slant for further culture, and then transferred to the shake tube containing seed culture medium for seed culture. (2) Fermentation culture: The seed liquid was inoculated into the fermentation medium and shaken for culture. Calcium carbonate was added and sodium hydroxide was added intermittently to control the pH at around 7.0.

[0039] Preferably, the above application follows these steps: (1) Seed activation and seed culture: The bacterial solution was inoculated from the preservation tube and evenly spread onto the activation slant. It was cultured at 32℃ for 12h, then transferred to the activation slant and cultured for another 10h. Finally, it was transferred to a shaker containing 5 mL of seed culture medium for seed culture. (2) Fermentation culture: 1.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium at a 5% inoculation rate. The flask was sealed with 9 layers of gauze and placed on a circulating shaker (200 r / min) for shaking culture at 31.5℃ for 70 h. 2% calcium carbonate was added and sodium hydroxide was added intermittently to control the pH at around 7.0.

[0040] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 0.5-1.5 g / L, peptone 8-12 g / L, beef extract 8-12 g / L, yeast powder 4-6 g / L, NaCl 2-3 g / L, agar 23-27 g / L, pH 7.0-7.2.

[0041] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 1 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, NaCl 2.5 g / L, agar 25 g / L, pH 7.0~7.2.

[0042] Preferably, in the above application, the seed culture medium used in seed culture is as follows: sucrose 18.0-22.0 g / L, corn steep liquor 18.0-22.0 mL / L, MnSO4·H2O 2.0-4.0 g / L, MgSO4·7H2O 0.3-0.5 g / L, KH2PO4 0.8-1.2 g / L, L-alanine 0.3-0.4 g / L, monosodium glutamate 0.08-0.12 g / L, MnSO4·H2O 0.008-0.012 g / L, MnSO4·H2O 0.008-0.012 g / L, biotin 45-55 μg / L, nicotinamide 4-6 mg / L, thiamine 0.1-0.3 mg / L, pH 7.0-7.2.

[0043] Preferably, in the above application, the seed culture medium used in seed culture is: sucrose 20.0 g / L, corn steep liquor 20.0 mL / L, MnSO4·H2O 3.0 g / L, MgSO4·7H2O 0.4 g / L, KH2PO4 1.0 g / L, L-alanine 0.35 g / L, monosodium glutamate 0.1 g / L, MnSO4·H2O 0.01 g / L, biotin 50 μg / L, nicotinamide 5 mg / L, thiamine 0.2 mg / L, pH 7.0~7.2.

[0044] Preferably, in the above application, the fermentation medium used in the fermentation culture is: glucose 100.0-150.0 g / L, corn steep liquor 35.0-45.0 mL / L, MnSO4·H2O 40.0-50.0 g / L, KH2PO4·3H2O 2.0-4.0 g / L, MgSO4·7H2O 1.0-2.0 g / L, MnSO4·H2O 0.01-0.02 g / L, MnSO4·H2O 0.01-0.02 g / L, biotin 0.7-0.9 mg / L, nicotinamide 0.7-0.9 mg / L, thiamine 120-180 μg / L, CaCO3 25-35 g / L (sterilized separately), pH 7.0-7.2, phenol red 1-3%, and the remainder is water.

[0045] Preferably, in the above application, the fermentation medium used in the fermentation culture is: glucose 120.0 g / L, corn steep liquor 40.0 mL / L, MnSO4·H2O 45.0 g / L, KH2PO4·3H2O 3.0 g / L, MgSO4·7H2O 1.5 g / L, MnSO4·H2O 0.015 g / L, MnSO4·H2O 0.015 g / L, biotin 0.8 mg / L, nicotinamide 0.8 mg / L, thiamine 150 μg / L, CaCO3 30 g / L (sterilized separately), pH 7.0~7.2, phenol red 2%, and the remainder is water.

[0046] All of the above-mentioned culture media can be prepared using standard methods.

[0047] Beneficial effects: The aforementioned L-isoleucine-producing strain, using homologous recombination gene editing technology, first overexpressed a non-L-isoleucine-repressed gene. ​ (G20D、I21D、I22F) and ​ (V140M、F383A) Overexpression of non-threonine-inhibited ​ (T311I) , ​ (G378I) and ​ (A20G) Overexpression ​ , ​ , ​ , ​ Escherichia coli W3110 ​ and efflux proteins ​ Knockout ​ Genes, among which ​ (G20D、I21D、I22F) and ​ (V140M、F383A) Escherichia coli W3110 ​ Genes are generated through artificial operon P sod - ​ (G20D、I21D、I22F) ​ (V140M、F383A) - P tuf - ​ - ​ - ​ To perform synchronous multiple copies, ​ , ​ , ​ (V140M、F383A) , ​ (G20D、I21D、I22F) Through artificial manipulation of P sod - ​ ​ P tuf -​ (V140M、F383A) ​ (G20D、I21D、I22F) This strain was used for synchronous multi-copying. The constructed strain is plasmid-free, defect-free, requires no induction, and has advantages such as good genetic stability and high fermentation yield. It is an excellent strain for stable production of L-isoleucine. The strain efficiently synthesizes L-isoleucine de novo using glucose as a substrate. After 48 h of shake-flask fermentation, the L-isoleucine yield can reach up to 10.37 g / L. Attached Figure Description

[0048] ​ This diagram illustrates the method for targeted modification of L-isoleucine-producing strains, where the green portion represents the overexpressed gene. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] The starting strain used in the examples was a uridine-producing strain. ​ 13032, the corresponding gene mutation sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd.; the corresponding promoter and gene are shown in the sequence listing.

[0052] The gene editing method used was homologous recombination. The engineered plasmid pK18 used in this method had pk18mobsacb as its backbone, with the sucrose selection marker replaced by a streptomycin resistance selection marker. The technical terms related to gene integration and plasmid construction used in the following examples can be explained in the article {Ying Yu. Metabolic engineering of Corynebacterium glutamicum for L-leucine fermentation [D]. Wuxi: Jiangnan University, 2020.}. The primers used in the strain construction process are shown in Table 1.

[0053] Table 1 Primers used in strain construction

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Example 1 like ​ As shown, the specific process for constructing genetically engineered strains is as follows: Starting strain ​ 13032 Electrocompetent cells were prepared according to the standard competent cell preparation method (Ying Yu. Metabolic engineering of Corynebacterium glutamicum for L-leucine fermentation [D]. Wuxi: Jiangnan University, 2020.).

[0060] 1.1 brnFE Promoter replacement (P) brnFE ::P sod - brnFE ) by C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers Psod-brnFE-UF and Psod-brnFE-UR, and Psod-brnFE-DF and Psod-brnFE-DR, respectively, to obtain the upstream homologous arm Psod-brnFE-UP and the downstream homologous arm Psod-brnFE-DW. The recovered upstream and downstream homologous arms were then used as templates. C. glutamicum Using the ATCC 13032 genome as a template, PCR was performed using primers Psod-F(1) and Psod-R(1) to amplify P. sod Promoter; the above gene fragment was recovered using a DNA purification kit and seamlessly ligated with plasmid pK18 to construct plasmid pK18-01. Finally, pK18-01 plasmid was electroporated to... C. glutamicum The strain was obtained by electrotransferring ATCC 13032 into electrocompetent cells; positive transformants were then screened using Psod-brnFE-1 and Psod-brnFE-2 as identification primers. C. glutamicum Ile - 01.

[0061] 1.2 Genes ilvN mutation At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers ilvN-(mut20-22)-UF and ilvN-(mut20-22)-UR, ilvN-(mut20-22)-DF and ilvN-(mut20-22)-DR, respectively, to obtain the upstream homologous arm ilvN-(mut20-22)-UP and the downstream homologous arm ilvN-(mut20-22)-DW. The gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct plasmid pK18-02. Finally, plasmid pK18-02 was electroporated into... C. glutamicum Ile - 01 electrocompetent cells; then, using ilvNmut-1 and ilvNmut-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 02.

[0062] 1.3 Genes ilva mutation At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers ilvA-dn2 and ilvA(V140M)-dn1, ilvA(V140M)-up2 and ilvA(F383A)-dn1, and ilvA(F383A)-up2 and ilvA-up1, respectively, yielding the upstream homologous arm ilvA-(V140M / F383A)-UP, the mutant fragment ilvA-(V140M / F383A), and the downstream homologous arm ilvN-(V140M / F383A)-DW. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-03 plasmid. Finally, the pK18-03 plasmid was electroporated into… C. glutamicum Ile - 02 electrocompetent cells; then, using ilvAmut-1 and ilvAmut-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 03.

[0063] 1.4 Genes man and thrB mutation At available concentration C. glutamicumUsing the ATCC 13032 genome as a template, PCR amplification was performed using primers hom(G378E)-up1 and hom(G378E)-up2, hom(G378E)-dn1 and thrB(A20G)-up2, and thrB(A20G)-dn1 and thrB(A20G)-dn2, respectively, yielding the upstream homologous arm hom-(G378E)-UP, the mutant fragment hom / thrB-(G378E / A20G), and the downstream homologous arm thrB(A20G)-DW. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-04 plasmid. Finally, the pK18-04 plasmid was electroporated into… C. glutamicum Ile - 03 electrocompetent cells; then, using hom-mut-1 and thrB-mut-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 04.

[0064] 1.5 genes lysC mutation At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers lysC(T311I)-up1 and lysC(T311I)-up2, lysC(T311I)-dn1 and lysC(T311I)-dn2, respectively, to obtain the upstream homologous arm lysC(T311I)-UP and the downstream homologous arm lysC(T311I)-DW. The gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-05 plasmid. Finally, the pK18-05 plasmid was electroporated into… C. glutamicum Ile - 04 electrocompetent cells; then, using lysCmut-1 and lysCmut-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 05.

[0065] 1.6 ilvBN Promoter replacement (P) ilvBN ::P sod - ilvBN ) At available concentration C. glutamicum Using the ATCC 13032 genome as a template, primer P was used. sod -ilvBN-UF and P sod -ilvBN-UR,P sod -ilvBN-DF and P sod-ilvBN-DR,P sod -F(1) and P sod -R(1) were subjected to PCR amplification to obtain the upstream homologous arm P sod -ilvBN-UP, promoter fragment P sod and downstream homologous arm P sod After recovering the gene fragment using a DNA purification kit, the above gene fragment was seamlessly ligated with plasmid pK18 to construct plasmid pK18-06. Finally, plasmid pK18-06 was electroporated into... C. glutamicum Ile - 0.05 electrocompetent cells; then with P sod -ilvBN-1 and P sod -ilvBN-2 was used as the identification primer to screen for positive transformants and obtain the strain. C. glutamicum Ile - 06.

[0066] 1.7 ilva Promoter replacement (P) ilvA ::P tuf - ilva ) At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers Ptuf-ilvA-UF and Ptuf-ilvA-UR, Ptuf-ilvA-DF and Ptuf-ilvA-DR, Ptuf-F(1) and Ptuf-R(1), respectively, to obtain the upstream homologous arm Ptuf-ilvA-UP and the promoter fragment P. tuf The gene fragments were recovered using a DNA purification kit and seamlessly ligated with the downstream homologous arm Ptuf-ilvA-DW to construct the pK18-07 plasmid. The pK18-07 plasmid was then electroporated into... C. glutamicum Ile - 06 electrocompetent cells; then, using Ptuf-ilvA-1 and Ptuf-ilvA-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 07.

[0067] 1.8 thrC Promoter replacement (P) thrC ::P sod - thrC ) At available concentration C. glutamicumUsing the ATCC 13032 genome as a template, PCR amplification was performed using primers Psod-thrC-UF and Psod-thrC-UR, Psod-F(2) and Psod-R(2), Psod-thrC-DF and Psod-thrC-DR, respectively, to obtain the upstream homologous arm Psod-thrC-UP and the promoter fragment P. sod The gene fragments were recovered using a DNA purification kit and seamlessly ligated with the downstream homologous arm Psod-thrC-DW to construct the pK18-08 plasmid. The pK18-08 plasmid was then electroporated into… C. glutamicum Ile - 07 electrocompetent cells; then, using Psod-thrC-1 and Psod-thrC-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 08.

[0068] 1.9 hom-thrB Promoter replacement (P) hom ::P tuf -hom) At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers Ptuf-hom-UF and Ptuf-hom-UR, Ptuf-hom-DF and Ptuf-hom-DR, Ptuf-F(1) and Ptuf-R(1), respectively, to obtain the upstream homologous arm Ptuf-hom-UP and the promoter fragment P. tuf The gene fragment was recovered using a DNA purification kit and seamlessly ligated with plasmid pK18 via a translocator, along with the downstream homologous arm Ptuf-hom-DW, to construct plasmid pK18-09. Finally, plasmid pK18-09 was electroporated into… C. glutamicum Ile - 08 electrocompetent cells; then, using Ptuf-hom-1 and Ptuf-hom-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 09.

[0069] 1.10 lysC-asd Promoter replacement (P) lysC ::P sod - lysC (T311I) ) At available concentration C. glutamicumUsing the ATCC 13032 genome as a template, PCR amplification was performed using primers Psod-lysC-UF and Psod-lysC-UR, Psod-lysC-DF and Psod-lysC-DR, Psod-F(1) and Psod-R(1), respectively, to obtain the upstream homologous arm Psod-lysC-UP and the promoter fragment P. sod The gene fragments were recovered using a DNA purification kit and seamlessly ligated with the downstream homologous arm Psod-lysC-DW to construct the pK18-10 plasmid. The pK18-10 plasmid was then electroporated into… C. glutamicum Ile - 09 electrocompetent cells; then, using Psod-lysC-1 and Psod-lysC-2 as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Ile - 10.

[0070] 1.11 aspB Promoter replacement (P) aspB ::P tuf - aspB ) At available concentration C. glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers Ptuf-aspB-UF and Ptuf-aspB-UR, Ptuf-aspB-DF and Ptuf-aspB-DR, Ptuf-F(2) and Ptuf-R(2), respectively, to obtain the upstream homologous arm Ptuf-aspB-UP and the promoter fragment P. tuf The gene fragment was recovered using a DNA purification kit and seamlessly ligated with the downstream homologous arm Ptuf-aspB-DW to construct the pK18-11 plasmid. The pK18-11 plasmid was then electroporated into... C. glutamicum Ile - 10 electrocompetent cells were used; positive transformants were screened using Ptuf-aspB-1 and Ptuf-aspB-2 as identification primers to obtain the strain. C. glutamicum Ile-11.

[0071] 1.12-1 Artificial Operator Gene ilvBN (G20D、I21D、I22F) - ilva (V140M、F383A) and thrABC Integration (cg1895::P) sod - ilvBN (G20D、I21D、I22F) - ilva (V140M、F383A) Integration, cg1890::Ptuf - thrA - thrB - thrC (integration) Corynebacterium glutamicum at usable concentration C. glutamicum Using the Ile-03 genome as a template, PCR amplification was performed using primers Psod-ilvBNA-UF and Psod-ilvBNA-UR, Psod-F(1) and Psod-R(1), ilvBN-F(1) and ilvBN-R(1), ilvA-F(1) and ilvA-R(1), Psod-ilvBNA-DF and Psod-ilvBNA-DR, respectively, to obtain the upstream homologous arm Psod-ilvBNA-UP and the promoter fragment P. sod The ilvBN fragment, ilvA fragment, and downstream homologous arm Psod-ilvBNA-DW were extracted. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-12-1-1 plasmid. Finally, the pK18-12-1-1 plasmid was electroporated into… C. glutamicum Ile-11 was electrocompetent in cells; positive transformants were screened using Psod-ilvBNA-1 and Psod-ilvBNA-2 as identification primers to obtain the strain. C. glutamicum Ile - 12-1-1.

[0072] Using a usable concentration of *E. coli* W3110 genome as a template, PCR was performed using primers Ptuf-thrABC-UF and Ptuf-thrABC-UR, Ptuf-thrABC-DF and Ptuf-thrABC-DR, Ptuf-F(1) and Ptuf-R(1), and thrABC-F and thrABC-R, respectively, to obtain the upstream homologous arm Ptuf-thrABC-UP, the downstream homologous arm Ptuf-thrABC-DW, the promoter fragment Ptuf, and the gene fragment thrABC. The gene fragments were then recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-12-2 plasmid. Finally, the pK18-12-2 plasmid was electroporated into… C. glutamicum Ile-11 was electrocompetent in cells; positive transformants were screened using Ptuf-thrABC-1 and Ptuf-thrABC-2 as identification primers to obtain the strain. C. glutamicum Ile - 12-1.

[0073] 1.12-2 Artificial Operator Genes ilvBN (G20D、I21D、I22F) - ilva (V140M、F383A) -thrA - thrB - thrC Integration (cg1895::P) sod - ilvBN (G20D、I21D、I22F) - ilva (V140M、F383A) - P tuf - thrA - thrB - thrC (integration) Corynebacterium glutamicum at usable concentration C. glutamicum Using the Ile-12-1 genome as a template, PCR amplification was performed using primers cg1895-UF and cg1895-UR, Psod-F(1) and Psod-R(1), ilvA-F(2) and ilvA-R(2), ilvBN-F(2) and ilvBN-R(2), T1-F(1) and T1-R(1), Ptuf-F(1) and Ptuf-R(1), thrABC(ec)-F and thrABC(ec)-R, cg1895-DF and cg1895-DR, respectively, to obtain the upstream homologous arm cg1895::ilvABN-thrABC-UP and the promoter fragment P. sod ilvA fragment, ilvBN fragment, T1 terminator fragment, P tuf The promoter fragment, thrABC fragment, and downstream homologous arm cg1895::ilvABN-thrABC-DW were extracted. After recovery using a DNA purification kit, these gene fragments were seamlessly ligated into plasmid pK18 to construct the pK18-12-3 plasmid. Finally, the pK18-12-3 plasmid was electroporated into… C. glutamicum Ile-11 was electrocompetent in cells; positive transformants were screened using cg1895::ilvABN-thrABC-1 and cg1895::ilvABN-thrABC-2 as identification primers to obtain the strain. C.glutamicum Ile - 12-2.

[0074] 1.13-1 Artificial Operon Genes ppc-aspB and ilva (V140M、F383A) -ilvBN (G20D、I21D、I22F) integration (△) pck ::P sod - ppc-aspB integration 、 cg1960::P tuf - ilva (V140M、F383A) -ilvBN(G20D、I21D、I22F) (integration) Corynebacterium glutamicum at usable concentration C. glutamicum Using the Ile-12-2 genome as a template, PCR amplification was performed using primers pck::Psod-ppc-aspB-UF and pck::Psod-ppc-aspB-UR, Psod-F(1) and Psod-R(1), ppc-F(1) and ppc-R(1), aspB-F and aspB-R, T1-F(2) and T1-R(2), pck::Psod-ppc-aspB-DF and pck::Psod-ppc-aspB-DR, respectively, to obtain the upstream homologous arm pck::Psod-ppc-aspB-UP and the promoter fragment P. sod The ppc fragment, aspB fragment, terminator fragment, and downstream homologous arm pck::Psod-ppc-aspB-DW were extracted. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-13-1-1 plasmid. Finally, the pK18-13-1-1 plasmid was electroporated into… C. glutamicum Ile-12-2 was electrocompetent in cells; positive transformants were screened using pck::Psod-ppc-aspB-1 and pck::Psod-ppc-aspB-2 as identification primers to obtain the strain. C. glutamicum Ile - 13-1-1.

[0075] Corynebacterium glutamicum at usable concentration C. glutamicum Using the Ile-12-2 genome as a template, PCR amplification was performed using primers, 1960::Ptuf-ilvABN-UF and 1960::Ptuf-ilvABN-UR, Ptuf-F(1) and Ptuf-R(1), ilvA-F(3) and ilvA-R(3), ilvBN-F(3) and ilvBN-R(3), 1960::Ptuf-ilvABN-DF and 1960::Ptuf-ilvABN-DR, respectively, to obtain the upstream homologous arm 1960::Ptuf-ilvABN-UP and the promoter fragment P. tuf The ilvA fragment, ilvBN fragment, and downstream homologous arm 1960::Ptuf-ilvABN-DW were extracted. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-13-1 plasmid. Finally, the pK18-13-1 plasmid was electroporated into… C.glutamicumIle-12-2 was electrocompetent in cells; positive transformants were screened using 1960::Ptuf-ilvABN-1 and 1960::Ptuf-ilvABN-2 as identification primers to obtain the strain. C. glutamicum Ile - 13-1.

[0076] 1.13-2 Artificial Operator Genes ppc-aspB-ilvA (V140M、F383A) -ilvBN (G20D、I21D、I22F) integration (△) pck ::P sod - ppc-aspB- P tuf - ilva (V140M、F383A) -ilvBN (G20D、I21D、I22F) (integration) Corynebacterium glutamicum at usable concentration C. glutamicum Using the Ile-12-2 genome as a template, PCR amplification was performed using primers pck::Psod-ppc-aspB-UF and pck::Psod-ppc-aspB-UR, Psod-F(1) and Psod-R(1), ppc-F(2) and ppc-R(2), aspB-F(2) and aspB-R, T1-F(3) and T1-R(3), Ptuf-F(1) and Ptuf-R(1), ilvA-F(4) and ilvA-R(4), ilvBN-F(4) and ilvBN-R(4), pck::Psod-ppc-aspB-DF and pck::Psod-ppc-aspB-DR, respectively, to obtain the upstream homologous arm pck::Psod-ppc-aspB-UP and the promoter fragment P. sod , ppc fragment, aspB fragment, terminating sub-fragment, P tuf The promoter fragment, ilvA fragment, ilvBN fragment, and downstream homologous arm pck::Psod-ppc-aspB-DW were extracted. These gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-13-2 plasmid. Finally, the pK18-13-2 plasmid was electroporated into… C. glutamicum Ile-12-2 was electrocompetent in cells; positive transformants were screened using pck::Psod-ppc-aspB-1 and pck::Psod-ppc-aspB-2 as identification primers to obtain the strain. C. glutamicum Ile - 13-2.

[0077] The strains involved in the above construction process are shown in Table 2.

[0078] The strains listed in Table 2 name Genome Ile01 <![CDATA[P brnFE ::P sod - brnFE ]]> Ile-02 <![CDATA[ C. glutamicum With - 01, ilvN :: ilvN (G20D、I21D、I22F) ]]> Ile-03 <![CDATA[ C.glutamicum With - 02, ilva :: ilva (V140M，F383A) ]]> Ile-04 <![CDATA[ C.glutamicum Ile - 03, man :: man (G378E) ,thrB ::thrB (A20G) ]]> Ile-05 <![CDATA[ C.glutamicum With - 04, lysC :: lysC (T311I) ]]> Ile-06 <![CDATA[ C.glutamicum authorities - 05,P ilvBN ::P sod - ilvBN (G20D，I21D，I22F) ]]> Ile-07 <![CDATA[ C.glutamicum authorities - 06,P ilvA ::P tuf - ilvA (V140M，F383A) ]]> Ile-08 <![CDATA[ C.glutamicum authorities - 07,P thrC ::P sod - thrC ]]> Ile-09 <![CDATA[ C.glutamicum How many - 08,P hom ::P tuf -hom (G378E) ]]> Ile-10 <![CDATA[ C.glutamicum authorities - 09,P lysC ::P sod - lysC (T311I) ]]> Ile-11 <![CDATA[C.glutamicum Ile-10,P aspB ::P tuf - aspB ]]> Ile-12-1-1 <![CDATA[C.glutamicum Ile-11,cg1895::P sod - ilvBN (G20D、I21D、I22F) - ilvA (V140M、F383A) ]]> Ile-12-1 <![CDATA[C.glutamicum Ile-11,cg1895::P sod - ilvBN (G20D、I21D、I22F) - ilvA (V140M、F383A) ,cg1890::P tuf - thrA - thrB - thrC ]]> Ile-12-2 <![CDATA[C.glutamicum Ile-11,cg1895::P sod -ilvBN (G20D、I21D、I22F) - ilvA (V140M、F383A) - P tuf - thrA - thrB - thrC ]]> Ile-13-1 <![CDATA[C.glutamicum Ile-11,cg1895::P sod - ilvBN (G20D、I21D、I22F) - ilvA (V140M、F383A) - P tuf - thrA - thrB - thrC ,Δ pck ::P sod - ppc-aspB,cg1960 ::P tuf - ilvA (V140M、F383A) - ilvBN (G20D、I21D、I22F) ]]> Ile-13-2 <![CDATA[C.glutamicum Ile-11,cg1895::P sod - ilvBN (G20D、I21D、I22F) -ilvA (V140M、F383A) - P tuf - thrA - thrB - thrC ,Δ pck ::P sod - ppc-aspB- P tuf - ilvA (V140M、F383A) - ilvBN (G20D、I21D、I22F) ]]>

[0079] The aforementioned artificial manipulator P sod - ilvBN (G20D、I21D、I22F) - ilvA (V140M、F383A) The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing, and the artificial operon P... tuf - thrA - thrB - thrC The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing, and the artificial operon P... sod - ppc-aspB The nucleotide sequence is shown in SEQ ID NO.26 of the sequence listing, and the artificial operon P tuf - ilvA (V140M、F383A) -ilvBN (G20D、I21D、I22F) The nucleotide sequence is shown in the sequence listing SEQ ID NO.27.

[0080] Example 2 L-Isoleucine producing strain as described in Example 1 C.glutamicum Ile-13 was used in shake-flask fermentation to produce L-isoleucine.

[0081] 2.1 Culture medium 2.1.1 Slant Culture Medium Glucose 1 g / L, peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, NaCl 2.5 g / L, agar 25 g / L, pH 7.0~7.2, sterilized at 0.1 MPa for 20 min.

[0082] 2.1.2 Seed Culture Medium Sucrose 20.0 g / L, corn steep liquor 20.0 mL / L, MnSO4·H2O 3.0 g / L, MgSO4·7H2O 0.4 g / L, KH2PO4 1.0 g / L, L-alanine 0.35 g / L, monosodium glutamate 0.1 g / L, MnSO4·H2O 0.01 g / L, MnSO4·H2O 0.01 g / L, biotin 50 μg / L, nicotinamide 5 mg / L, thiamine 0.2 mg / L, pH 7.0~7.2, sterilized at 0.1 MPa for 15 min.

[0083] 2.1.3 Fermentation medium Glucose 120.0 g / L, corn steep liquor 40.0 mL / L, MnSO4·H2O 45.0 g / L, KH2PO4·3H2O 3.0 g / L, MgSO4·7H2O 1.5 g / L, MnSO4·H2O 0.015 g / L, MnSO4·H2O 0.015 g / L, biotin 0.8 mg / L, nicotinamide 0.8 mg / L, thiamine 150 μg / L, CaCO3 30 g / L (sterilized separately), pH 7.0~7.2, sterilized at 0.1 MPa for 15 min, phenol red 2%, the remainder being water.

[0084] 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 32°C for 12 h, transferred to the activation slant and incubated for another 10 h, before being transferred to a shaker containing 5 mL of seed culture medium for seed culture.

[0085] 2.2.2 Fermentation culture: Inoculate 1.5 mL of seed culture (5% inoculum) into a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium. Seal the flask with nine layers of gauze and place it on a circulating shaker (200 rpm) at 31.5 °C for 70 h. Add 2% calcium carbonate and intermittently add sodium hydroxide to maintain the pH at approximately 7.0 (every 4 h).

[0086] After 30 hours of shake-flask fermentation, the yield of L-isoleucine can reach up to 10.37 g / L.

[0087] Example 3 L-Isoleucine producing strain as described in Example 1 C.glutamicum Ile-13 was used to produce L-isoleucine by shake-flask fermentation, using the same fermentation method as in Example 2. The L-isoleucine yield of each strain is shown in Table 3.

[0088] Table 3. L-Isoleucine yield from shake-flask fermentation of each strain.

[0089] Strains Ile-01 to Ile-13 demonstrated that the modification relieved feedback inhibition of isoleucine and threonine, thus opening up the isoleucine production pathway. Strain Ile-12-1 added key genes for isoleucine synthesis. ilvIH、ilvA Copy number and key genes for threonine synthesis thrABC (E. coli source), yield increased to some extent; in contrast, strain Ile-12-2 used artificial operons for synchronous multiple copying. ilvIH、ilvA and thrABC (E. coli source) The gene significantly increased yield. Subsequently, based on this strain, the gene was... pck Delete and use manual manipulators for multiple copies ppc , aspB , ilvA and ilvBN This further increases the yield, allowing strain Ile-13-2 to produce up to 10.37 g / L of isoleucine.

[0090] 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 artificial manipulator, characterized in that: It is constructed by rearranging and linking L-isoleucine synthesis-related genes, which are selected from... ilvA (V140M、F383A) Gene, ilvBN (G20D，I21D，I22F) Genes and thrA Gene, thrB Gene, thrC Gene, PPC Genes and aspB Two or more genes.

2. The artificial manipulator according to claim 1, characterized in that: The L-isoleucine synthesis-related gene is ilvA (V140M、F383A) Gene, ilvBN (G20D，I21D，I22F) Gene, thrA Gene, thrB Genes and thrC Genes, including promoter 1 and promoter 2, and the specific gene sequence of the operon includes promoter 1, promoter 2, promoter 3, promoter 4, promoter 5, promoter 6, promoter 7, promoter 8, promoter 9, promoter 10, promoter 11, promoter 12, promoter 13, promoter 14, promoter 15, promoter 16, promoter 17, promoter 18, promoter 19, promoter 12, promoter 13, promoter 14, promoter 1 ilvBN (G20D，I21D，I22F) Gene, ilvA (V140M、F383A) Gene, promoter 2, thrA Gene, thrB Genes and thrC Gene.

3. The artificial manipulator according to claim 1 or 2, characterized in that: Its nucleotide sequence is shown in the sequence listing SEQ ID NO.

25.

4. The artificial manipulator according to claim 1, characterized in that: The L-isoleucine synthesis-related gene is ilvA (V140M、F383A) Gene, PPC Gene, aspB Genes and ilvBN (G20D，I21D，I22F) Genes, including promoter 1 and promoter 2, and the specific gene sequence of the operon includes promoter 1, promoter 2, promoter 3, promoter 4, promoter 5, promoter 6, promoter 7, promoter 8, promoter 9, promoter 10, promoter 11, promoter 12, promoter 13, promoter 14, promoter 15, promoter 16, promoter 17, promoter 18, promoter 19, promoter 12, promoter 13, promoter 14, promoter 1 PPC Gene, aspB Gene, promoter 2, ilvA (V140M、F383A) Genes and ilvBN (G20D，I21D，I22F) Gene.

5. The artificial manipulator according to claim 1 or 4, characterized in that: Its nucleotide sequence is shown in the sequence listing SEQ ID NO.

28.

6. The use of the artificial operon according to any one of claims 1-5 in constructing L-isoleucine-producing strains or in the fermentation production of L-isoleucine.

7. An L-isoleucine-producing strain, integrated with the artificial operon of claim 2 or 3 and / or the artificial operon of claim 4 or 5.

8. The L-isoleucine-producing strain according to claim 7, characterized in that: It utilizes targeted modification methods on the starting strain C. glutamicum This was obtained through further modifications based on ATCC 13032, the modifications of which also include: C. glutamicum ATCC 13032 genome knockout pck Gene, overexpression of branched-chain amino acid efflux protein gene brnFE Threonine dehydratase gene carrying mutation points ilvA (V140M、F383A) acetolactate synthase gene carrying mutation points ilvBN (G20D、I21D、I22F) Threonine synthase gene thrC Homoserine kinase genes carrying mutation sites thrB (A20G) Homoserine dehydrogenase gene carrying a mutation site hom (G378E) Aspartate semialdehyde dehydrogenase gene asd Aspartate kinase gene carrying mutation sites lysC (T311I) and aspartate transaminase gene aspB .

9. The method for constructing the L-isoleucine-producing strain according to claim 7 or 8, comprising the following specific steps: (1) In Corynebacterium glutamicum On the ATCC 13032 genome, via promoter P sod replace brnFE The natural promoter; (2) By using a strong promoter P sod replace ilvBN (G20D、I21D、I22F) and lysC (T311I) The natural promoter, promoter P tuf replace ilvA (V140M、F383A) , hom (G378E) and thrB (A20G) The natural promoter; (3) By using promoter P sod replace thrC The natural promoter, promoter P tuf replace aspB The natural promoter; (4) Integrating the artificial cytokine of claim 2 or 3 at the cg1895 site; (5) Knockout pck And integrate the artificial manipulator as described in claim 4 or 5 at that site.

10. The use of the L-isoleucine-producing strain according to claim 7 or 8 in the fermentation production of L-isoleucine.

Images