L-threonine producing strain, construction method and application thereof

Abstract

The application provides an L-threonine production strain and a construction method and application thereof, and utilizes homologous recombination gene editing technology to knock out lysine transporter genes lysE and isoleucine transporter genes brnF, Firstly, threonine inhibition is overexpressed lysC (T311I) 、 hom (G378I) and thrB (A20G) ; overexpression thrC and aspB ; heterologous introduction and multiple copies of E. coli-derived thrA - thrB - thrC genes and efflux protein rhtC genes ， wherein thrA - thrB - thrC are synchronously and multiple-copied through an artificial operon P sod - thrA - thrB - thrC The constructed strain does not contain plasmids, is defect-free, does not need induction, has good genetic stability, and can efficiently synthesize L-threonine from scratch with glucose as a substrate, and has a high fermentation yield.

Description

Technical Field

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

[0002] L-Threonine, one of the three major amino acids, has significant applications in feed, food, pharmaceuticals, and cosmetics. Currently, the main production methods for L-Threonine include protein hydrolysis, chemical synthesis, and microbial fermentation. Protein hydrolysis suffers from high raw material costs, cumbersome separation and purification steps, and low yields. Chemical synthesis faces challenges such as complex reaction pathways, difficulty in chiral resolution, and severe environmental pollution, making it difficult to meet the demands of green manufacturing. In contrast, microbial fermentation offers advantages such as mild reaction conditions, a wide range of raw material sources, and good environmental compatibility, and has become the mainstream technology for the industrial production of L-Threonine.

[0003] Currently, the production of L-threonine via microbial fermentation has become a mature, efficient, economical, and environmentally friendly industrial process, ranking third in global amino acid production volume after glutamic acid and lysine. This method typically uses renewable carbon sources such as glucose as substrates and employs metabolically engineered microbial strains for directed fermentation synthesis. In recent years, advancements in synthetic biology and systems metabolic engineering have significantly improved the fermentation performance of L-threonine by rationally modifying model strains such as *Escherichia coli* and *Corynebacterium glutamicum*, thereby mitigating feedback inhibition in the L-threonine biosynthesis pathway and enhancing precursor supply and cofactor regeneration systems. However, existing production strains still have room for improvement in L-threonine yield, carbon source conversion rate, and process stability. Therefore, constructing a microbial strain with a clear genetic background, stable production performance, and high L-threonine production remains a key technical challenge that urgently needs to be addressed in this field. Summary of the Invention

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

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

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

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An L-threonine-producing strain was obtained by modifying a Corynebacterium strain as the starting strain. The modification included knocking out the isoleucine transporter gene in the starting strain. brnFOverexpression of the exogenous efflux protein gene derived from L-threonine in Escherichia coli rhtC .

[0008] Preferably, the L-threonine producing strain is *Corynebacterium glutamicum*.

[0009] Preferably, the L-threonine producing strain mentioned above, wherein the *Corynebacterium glutamicum* is... C. glutamicum ATCC13032.

[0010] Preferably, the above-mentioned L-threonine-producing strain is modified by... brnF Site integration of exogenous efflux protein genes containing L-threonine from E. coli rhtC The artificial manipulator is implemented.

[0011] Preferably, the L-threonine-producing strain described above is modified by overexpressing the exogenous efflux protein gene of L-threonine from *Escherichia coli*. rhtC It also integrates an exogenous efflux protein gene containing L-threonine from E. coli at a pseudogene site. rhtC The artificial manipulator is implemented.

[0012] Preferably, in the L-threonine producing strain described above, the pseudogene site is the cg1742 pseudogene site.

[0013] Preferably, the L-threonine-producing strain mentioned above contains the exogenous efflux protein gene for L-threonine from *Escherichia coli*. rhtC Artificial operons also include promoters, and the specific gene sequence includes promoters, rhtC Named artificial operon P- rhtC .

[0014] Preferably, in the above-mentioned L-threonine producing strain, the promoter included in the artificial operon is selected from: P sod promoter, P trc promoter, P tuf promoter, P H36 promoter, P lac promoter, P trp promoter, P tac promoter, P lPL promoter or P pgk100 Any of the promoters.

[0015] Preferably, in the above-mentioned L-threonine producing strain, the promoter in the artificial operon is P. sod The promoter is named artificial operator P. sod - rhtC P sod and rhtC Connect the first and last parts in order, where Psod The nucleotide sequence of the promoter is shown in SEQ ID NO.13 of the sequence listing. rhtC The nucleotide sequence is shown in SEQ ID NO.22, and the amino acid sequence is shown in SEQ ID NO.23, or the sequence is more than 95% identical to the sequence and originates from the same species.

[0016] Preferably, in the above-mentioned L-threonine producing strain, the promoter in the artificial operon is P. H36 The promoter is named artificial operator P. H36 - rhtC P H36 and rhtC Connect the first and last parts in order, where P H36 The nucleotide sequence of the promoter is shown in SEQ ID NO.24 of the sequence listing. rhtC The nucleotide sequence is shown in SEQ ID NO.22, and the amino acid sequence is shown in SEQ ID NO.23, or the sequence is more than 95% identical to the sequence and originates from the same species.

[0017] Preferably, in the above-mentioned L-threonine producing strain, the artificial operon is controlled by an artificial promoter to initiate transcription, and the natural terminator in the genome controls the termination of transcription.

[0018] Preferably, in the above-mentioned L-threonine producing strain, the modification further includes: knocking out the lysine transporter gene in the starting strain. lysE Overexpression of the threonine synthase gene from Escherichia coli thrC Homoserine kinase gene thrB A dual-function aspartate kinase / homoserine dehydrogenase gene thrA .

[0019] Preferably, the above-mentioned L-threonine-producing strain is modified by... lysE Integrating a threonine synthase gene from E. coli at the site thrC Homoserine kinase gene thrB A dual-function aspartate kinase / homoserine dehydrogenase gene thrA The artificial manipulator is implemented.

[0020] Preferably, the L-threonine-producing strain described above contains a threonine synthase gene from *Escherichia coli*. thrC Homoserine kinase gene thrB A dual-function aspartate kinase / homoserine dehydrogenase gene thrA Artificial operons also include promoters, and the specific gene sequence includes promoters, thrA , thrB and thrC Named artificial operon P- thrA - thrB - thrC .

[0021] Preferably, in the above-mentioned L-threonine producing strain, the promoter included in the artificial operon is selected from: P sod promoter, P trc promoter, P tuf promoter, P H36 promoter, P lac promoter, P trp promoter, P tac promoter, P lPL promoter or P pgk100 Any of the promoters.

[0022] Preferably, in the above-mentioned L-threonine producing strain, the promoter in the artificial operon is P. sod Promoter (promoter P) sod The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing, and it is named artificial operon P. sod - thrA - thrB - thrC Its nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing.

[0023] Preferably, in the L-threonine producing strains described above, the artificial operon is controlled by an artificial promoter to initiate transcription, and by a natural terminator in the genome to terminate transcription.

[0024] Preferably, the L-threonine-producing strain described above is obtained through further modification, wherein the further modification includes: overexpressing a threonine synthase gene derived from Corynebacterium glutamicum. thrC The homoserine kinase gene carrying a mutation site, derived from Corynebacterium glutamicum. thrB (A20G) Homoserine dehydrogenase gene carrying a mutation site man (G378E) Aspartate semialdehyde dehydrogenase gene asd Aspartate kinase gene carrying mutation sites lysC (T311I) and aspartate transaminase gene aspB .

[0025] Preferably, the L-threonine producing strain described above is modified entirely on the chromosomal genome of the starting strain using streptomycin selection vector homologous recombination editing technology.

[0026] Preferably, the above-mentioned L-threonine producing strain, wherein thrB (A20G) yes thrB Gene (the one mentioned) thrB The gene is derived from Corynebacterium glutamicum, whose nucleotide sequence is shown in SEQ ID NO.27 and amino acid sequence is shown in SEQ ID NO.28. Alternatively, a gene with more than 95% similarity to this sequence and 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. man (G378E) yes man 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; lysC (T311I) yes lysC The gene underwent a point mutation, specifically changing the base at position 932 from c to t, resulting in the amino acid residue at position 311 changing from threonine to isoleucine; this is the dual-function aspartate kinase / homoserine dehydrogenase gene derived from *E. coli* W3110. thrA .

[0027] Preferably, the above-mentioned L-threonine-producing strain is produced using the artificial promoter P. sod Replacement of threonine synthase gene thrC Homoserine dehydrogenase gene man (G378E) and aspartate kinase gene lysC (T311I) The natural promoter, using the artificial promoter P tuf Replacement of aspartate transaminase gene aspB The natural promoter.

[0028] Preferably, in the above-mentioned L-threonine-producing strain, the threonine synthase gene... thrC The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2, or is more than 95% identical to this sequence and originates from the same species; the homoserine dehydrogenase gene man (G378E) The nucleotide sequence is shown in SEQ ID NO. 3 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO. 4 of the sequence listing, or the sequence is 95% or more identical to the sequence and originates from the same species. manThe nucleotide sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6, or is more than 95% identical to this sequence and originates from the same species); the aspartate kinase gene lysC (T311I) The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.8 of the sequence listing, or the sequence is 95% or more identical to the sequence and originates from the same species. lysC The nucleotide sequence is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10, or is more than 95% identical to this sequence and originates from the same species); the aspartate transaminase gene aspB The nucleotide sequence is shown in SEQ ID NO. 11, and the amino acid sequence is shown in SEQ ID NO. 12, or is more than 95% identical to this sequence and originates from the same species; the promoter P sod The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing, and the promoter P... tuf The nucleotide sequence is shown in the sequence listing SEQ ID NO.14.

[0029] Preferably, the L-threonine producing strain mentioned above contains the threonine synthase gene derived from *Escherichia coli* W3110. thrC The nucleotide sequence is shown in SEQ ID NO.16, 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; homoserine kinase gene thrB The nucleotide sequence is shown in SEQ ID NO.18 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.19 of the sequence listing, or the sequence is more than 95% identical to the sequence and originates from the same species; thrB (A20G) The nucleotide sequence is shown in SEQ ID NO. 25, and the amino acid sequence is shown in SEQ ID NO. 26, or the sequence is more than 95% identical to the SEQ ID NO. 26 and originates from the same species; a dual-function aspartate kinase / homoserine dehydrogenase gene. thrA The nucleotide sequence is shown in SEQ ID NO. 20 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO. 21 of the sequence listing, or the sequence is more than 95% identical to the sequence and originates from the same species, and is promoted by the artificial promoter P. sod Regulation.

[0030] Preferably, the L-threonine producing strain described above contains the threonine transporter gene derived from *Escherichia coli* W3110. rhtCThe nucleotide sequence is shown in SEQ ID NO.22 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.23 of the sequence listing, or the sequence is more than 95% identical to the sequence and originates from the same species, and is promoted by the artificial promoter P. H36 Regulation, the artificial promoter P H36 The nucleotide sequence is shown in SEQ ID NO.24 of the sequence listing. brnF Site integration artificial operon P sod - rhtC The threonine transporter gene of Escherichia coli W3110 rhtC Using artificial promoter P sod Regulation.

[0031] A method for constructing an L-threonine-producing strain involves using a Corynebacterium spp. strain as the starting strain and knocking out the isoleucine transporter gene in the starting strain. brnF Overexpression of the exogenous efflux protein gene derived from L-threonine in Escherichia coli rhtC .

[0032] Preferably, the method for constructing the above-mentioned L-threonine-producing strain, in brnF Site integration of exogenous efflux protein genes containing L-threonine from E. coli rhtC Artificial operon; integration of an exogenous efflux protein gene containing L-threonine from E. coli at a pseudogene site. rhtC Artificial manipulator.

[0033] Preferably, the method for constructing the L-threonine-producing strain further includes knocking out the lysine transporter gene in the starting strain. lysE Overexpression of the threonine synthase gene from Escherichia coli thrC Homoserine kinase gene thrB A dual-function aspartate kinase / homoserine dehydrogenase gene thrA .

[0034] Preferably, the method for constructing the above-mentioned L-threonine-producing strain, in lysE Integrating a threonine synthase gene from E. coli at the site thrC Homoserine kinase gene thrB A dual-function aspartate kinase / homoserine dehydrogenase gene thrA The artificial operon was further modified by overexpressing the threonine synthase gene. thrC Homoserine kinase genes carrying mutation sites thrB (A20G) Homoserine dehydrogenase gene carrying a mutation site man (G378E)Aspartate semialdehyde dehydrogenase gene asd Aspartate kinase gene carrying mutation sites lysC (T311I) and aspartate transaminase gene aspB .

[0035] Preferably, the method for constructing the above-mentioned L-threonine-producing strain includes the following specific steps: (1) Knock out the isoleucine transporter gene in the genome of the above-mentioned starting strain. brnF, Integrating the artificial operator P at cg1742 site H36 - rhtC ,exist brnF Site integration artificial operon P sod - rhtC It enhances the efflux capacity of L-threonine; (2) By using a strong promoter P sod replace lysC (T311I) , man (G378E) and thrB (A20G) The natural promoter relieves feedback inhibition; (3) By using a strong promoter P sod replace thrC strong starter P tuf replace aspB The natural promoter; (4) Knockout of lysine transporter gene lysE ,exist lysE Integrating artificial operon P- at the site thrA - thrB - thrC Or artificially manipulated P sod - thrA - thrB - thrC This enhances the synthesis pathway of L-threonine.

[0036] Preferably, in the method for constructing the L-threonine producing strain described above, the starting strain is Corynebacterium glutamicum.

[0037] Preferably, in the method for constructing the above-mentioned L-threonine-producing strain, the starting strain is *Corynebacterium glutamicum*. C. glutamicum ATCC 13032.

[0038] Application of the above-mentioned L-threonine producing strains in the fermentation production of L-threonine.

[0039] Preferably, the above-mentioned L-threonine producing strain is used to produce L-threonine via shake-flask fermentation, and the specific steps are as follows: (1) Seed activation and seed culture: After the inoculated bacterial solution is evenly spread on the activation slant and cultured, it is transferred to the seed culture medium for seed culture; (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium, shake culture, add calcium carbonate, and intermittently add sodium hydroxide to control the pH at around 7.0.

[0040] Preferably, the above-mentioned L-threonine producing strain is used to produce L-threonine via shake-flask fermentation, and the specific steps are as follows: (1) Seed activation and seed culture: The bacterial solution was inoculated from the preservation tube and evenly spread on the activation slant. It was cultured at 32℃ for 12h, transferred to the activation slant and cultured for another 10h. Then it was transferred to a shaker containing 5mL of seed culture medium for seed culture. (2) Fermentation culture: 1.5 mL of seed liquid was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium at a 5% inoculation rate. The flask was sealed with gauze and placed on a circulating shaker (200 r / min). The flask was shaken and cultured at 31.5 °C for 70 h. 2% calcium carbonate was added and sodium hydroxide was added intermittently to control the pH at around 7.0.

[0041] Preferably, in the application of the above-mentioned L-threonine producing strain, the slant culture medium used for seed activation is as follows: glucose 1.0-3.0 g / L, peptone 8.0-12.0 g / L, yeast extract 4.0-6.0 g / L, urea 1.0-3.0 g / L, potassium dihydrogen phosphate 1.0-2.0 g / L, magnesium sulfate 0.3-0.5 g / L, vitamins H, VB1, VB3, and VB5 each 1.0-3.0 mg / L, agar powder 20-30 g / L, pH 7.0-7.2.

[0042] Preferably, in the application of the above-mentioned L-threonine producing strain, the slant culture medium used for seed activation is as follows: glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, urea 2.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.4 g / L, VH, VB1, VB3, and VB5 each 2.0 mg / L, agar powder 25 g / L, pH 7.0–7.2.

[0043] Preferably, in the application of the above-mentioned L-threonine producing strain, the seed culture medium used in the seed culture is as follows: glucose 25.0-35.0 g / L, yeast powder 0.5-1.5 g / L, corn steep liquor powder 4.0-6.0 g / L, ammonium sulfate 1.0-3.0 g / L, magnesium sulfate 0.6-1.0 g / L, potassium dihydrogen phosphate 2.0-4.0 g / L, L-isoleucine 0.4-0.6 g / L, ferrous sulfate 8.0-12.0 mg / L, manganese sulfate 8.0-12.0 mg / L, biotin 8.0-12.0 mg / L, VB1, VB3, and VB5 each 1.0-3.0 mg / L, L-methionine 0.4-0.6 g / L, antifoaming agent 0.4-0.6 mg / L, pH 7.0-7.2.

[0044] Preferably, in the application of the above-mentioned L-threonine producing strain, the seed culture medium used in the seed culture is as follows: glucose 30.0 g / L, yeast powder 1.0 g / L, corn steep liquor powder 5.0 g / L, ammonium sulfate 2.0 g / L, magnesium sulfate 0.8 g / L, potassium dihydrogen phosphate 3.0 g / L, L-isoleucine 0.5 g / L, ferrous sulfate 10.0 mg / L, manganese sulfate 10.0 mg / L, biotin 10.0 mg / L, VB1, VB3, and VB5 each 2.0 mg / L, L-methionine 0.5 g / L, antifoaming agent 0.5 mg / L, pH 7.0–7.2.

[0045] Preferably, in the application of the above-mentioned L-threonine producing strain, the fermentation medium used in the fermentation culture is as follows: glucose 35.0-45.0 g / L, molasses 8.0-12.0 g / L, corn steep liquor powder 8.0-12.0 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 4.0-6.0 g / L, magnesium sulfate 1.0-2.0 g / L, L-methionine 0.1-0.3 g / L, manganese sulfate 8.0-12.0 mg / L, zinc sulfate 1.0-3.0 mg / L, ferrous sulfate 8.0-12.0 mg / L, VB1, VB3, and VB5 each 1.0-3.0 mg / L, biotin 0.01-0.03 mg / L, L-isoleucine 0.5-0.7 g / L, antifoaming agent 0.4-0.6 mg / L, inoculum size 20-40%, pH 7.0-7.2.

[0046] Preferably, in the application of the above-mentioned L-threonine producing strain, the fermentation medium used in the fermentation culture is as follows: glucose 40.0 g / L, molasses 10.0 g / L, corn steep liquor powder 10.0 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 5.0 g / L, magnesium sulfate 1.5 g / L, L-methionine 0.2 g / L, manganese sulfate 10.0 mg / L, zinc sulfate 2.0 mg / L, ferrous sulfate 10.0 mg / L, VB1, VB3, and VB5 each 2.0 mg / L, biotin 0.02 mg / L, L-isoleucine 0.6 g / L, antifoaming agent 0.5 mg / L, inoculum size 30%, pH 7.0–7.2.

[0047] All of the above-mentioned culture media can be prepared using standard methods.

[0048] Beneficial effects: The L-threonine-producing strains described above used homologous recombination gene editing technology to knock out the lysine transporter gene. lysE and isoleucine transporter gene brnF, Firstly, overexpression of threonine inhibits... lysC (T311I) , man (G378I) and thrB (A20G) Overexpression thrC and aspB Heterogeneously introduced and multi-copy E. coli-derived thrA - thrB - thrC Genes and efflux proteins rhtC Gene ， in thrA - thrB - thrC Through artificial manipulation of P sod - thrA - thrB - thrC 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-threonine. The strain efficiently synthesizes L-threonine de novo using glucose as a substrate. After 36 hours of shake-flask fermentation, the L-threonine yield can reach up to 45 g / L. Attached Figure Description

[0049] Figure 1 This diagram illustrates the method for targeted modification of L-threonine-producing strains. Green text represents overexpression, blue text represents exogenous genes, and red text represents knockout. Detailed Implementation

[0050] 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.

[0051] 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.

[0052] The starting strain used in the examples was a uridine-producing strain. C. glutamicum ATCC 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.

[0053] 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.

[0054] Table 1 Primers used in strain construction

[0055]

[0056]

[0057] Example 1 like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows: Starting strain C. glutamicum ATCC 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.).

[0058] 1.1 lysC-asd Promoter replacement (P) lysC ::P sod - lysC ) by C. glutamicum Using the ATCC 13032 genome as a template, primer P was used. sod -lysC-UF and P sod -lysC-UR,P sod -lysC-DF and P sod -lysC-DR,P sod -F-1 and P sod -R-1 was subjected to PCR amplification to obtain the upstream homologous arm P sod -lysC-UP, promoter fragment P sod and downstream homologous arm P sod After recovering the above gene fragment using a DNA purification kit, the fragment was seamlessly ligated with plasmid pK18 to construct plasmid pK18-1. Finally, the pK18-1 plasmid was electroporated into... C.glutamicum Electrocompetent cells of ATCC 13032; then P sod -lysC-1 and P sod -lysC-2 was used as the identification primer to screen for positive transformants and obtain the strain. C. glutamicum Thr-1.

[0059] 1.2 Genes thrB mutation by C.glutamicum Using the ATCC 13032 genome as a template, PCR amplification was performed using primers thrB-up1 and thrB-up2, and thrB-dn1 and thrB-dn2, respectively, to obtain the upstream homologous arm thrB-(A20G)-UP and the downstream homologous arm thrB(A20G)-DW. The gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-2 plasmid. Finally, the pK18-2 plasmid was electroporated into… C.glutamicum Thr-1 electrocompetent cells; then, using hom-mut-1 and thrB-mut-2 as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-2.

[0060] 1.3 Genes man mutation Using the C. glutamicum ATCC 13032 genome as a template, PCR amplification was performed using primers hom-up1 and hom-up2, and hom-dn1 and hom-dn2, respectively, to obtain the upstream homologous arm hom-(G378E)-UP and the downstream homologous arm hom-(G378E)-DW. The gene fragments were recovered using a DNA purification kit and seamlessly ligated into plasmid pK18 to construct the pK18-3 plasmid. Finally, the pK18-3 plasmid was electroporated into… C.glutamicum Thr-2 electrocompetent cells; then, using hom-mut-1 and thrB-mut-2 as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-3.

[0061] 1.4 Artificial operon gene P H36 - rhtC Integration (Δ1742::P) H36 - rhtC (integration) Using the C. glutamicum ATCC 13032 genome as a template, primers pk18-1742-up and 1742-P were used. H36 PCR was performed using primers -dn, ph-36-rhtc-f, and 1742-pk18-bk-DN to obtain the upstream homologous arm 1742-UP and the downstream homologous arm 1742-DW; primers P H36 -2 and P H36 -3, perform PCR to obtain the artificial promoter P H36 Using a usable concentration of E. coli W3110 genome as a template, primer 1742-P was used. H36 -up and ph-36-rhtc-r were used for PCR to obtain gene fragments. rhtC The gene fragments described above were recovered using a DNA purification kit and then seamlessly ligated with plasmid pK18 to construct plasmid pK18-4. Finally, the pK18-4 plasmid was electroporated into… C.glutamicum The strain was obtained by electrocompetentizing Thr-3 cells; positive transformants were screened using Geno-1742-up-F and Geno-1742-dn-R as identification primers. C.glutamicum Thr-4.

[0062] 1.5 thrC Promoter replacement (P) thrC ::P sod - thrC ) Using the C. glutamicum ATCC 13032 genome as a template, primer P was used. sod-thrC-UF and P sod -thrC-UR,P sod -thrC-DF and P sod -thrC-DR,P sod -F-2 and P sod -R-2 were used for PCR amplification to obtain the upstream homologous arm P. sod -thrC-UP, promoter fragment P sod and downstream homologous arm P sod After recovering the above gene fragment using a DNA purification kit, the fragment was seamlessly ligated with plasmid pK18 to construct plasmid pK18-5. Finally, the pK18-5 plasmid was electroporated into... C.glutamicum In Thr-4 electrocompetent cells; then with P sod -thrC-1 and P sod -thrC-2 was used as the identification primer to screen for positive transformants and obtain the strain. C.glutamicum Thr-5.

[0063] 1.6 genes lysC mutation Using the C. glutamicum ATCC 13032 genome as a template, primer lysC was used. (T311I) -up1 and lysC (T311I) -up2, lysC (T311I) -dn1 and lysC (T311I) -dn2 was subjected to PCR amplification to obtain the upstream homologous arm lysC (T311I) -UP and downstream homologous arm lysC (T311I) -DW, the above gene fragment was recovered using a DNA purification kit and seamlessly ligated with plasmid pK18 to construct plasmid pK18-6. Finally, pK18-6 plasmid was electroporated to... C.glutamicum Thr-5 electrocompetent cells; then, using lysC-mut-1 and lysC-mut-2 as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-6.

[0064] 1.7 Artificial operon gene P H36 - rhtC Integration (Δ) brnF ::P H36 - rhtC (integration) Using the C. glutamicum ATCC 13032 genome as a template, primers pk18-bk-brnup-F and brnE-up-R, brnE-up-F and brnE-P were used. H36-R,brnE-P H36 PCR was performed using primers -F and rhtc-brndn-R to obtain the upstream homologous arm brnF-UP, gene fragment brnE, and downstream homologous arm brnF-DW. Using a usable concentration of pK18-4 plasmid as a template, PCR was performed using primers rhtc-brndn-F and brndn-pk18-bk-R to obtain gene fragment P. H36 - rhtC The gene fragments described above were recovered using a DNA purification kit and then seamlessly ligated with plasmid pK18 to construct the pK18-7 plasmid. Finally, the pK18-7 plasmid was electroporated into… C. glutamicum Thr-6 electrocompetent cells; then, using Geno-brnF-F and Geno-brnF-R as identification primers, positive transformants were screened to obtain the strain. C. glutamicum Thr-7.

[0065] 1.7.1 Artificial operon gene P H36 - rhtC Integration (Δ1890::P) H36 - rhtC (integration) Using the C. glutamicum ATCC 13032 genome as a template, PCR was performed using primers cg1890-UF and cg1890-UR, and cg1890-DF and cg1890-DR, respectively, to obtain the upstream homologous arm 1890-UP and the downstream homologous arm 1890-DW. Using a usable concentration of pK18-4 plasmid as a template, PCR was performed using primer P... H36 -F and rhtC-R were used for PCR to obtain gene fragment P. H36 - rhtC The gene fragments described above were recovered using a DNA purification kit and then seamlessly ligated with plasmid pK18 to construct the pK18-7-1 plasmid. Finally, the pK18-7-1 plasmid was electroporated into… C. glutamicum The strain was obtained by electrocompetentizing Thr-6 cells; positive transformants were screened using Geno-1890-F and Geno-1890-R primers. C. glutamicum Thr-7-1.

[0066] 1.8 hom-thrB Promoter replacement (P) hom ::P sod -hom) Using the C. glutamicum ATCC 13032 genome as a template, primers pk18-homup-F and UTR-P were used. sod -R,UTR-P sod -F and Psod -hom-R,P sod -hom-F and hom-thrB-R were respectively subjected to PCR amplification to obtain the upstream homologous arm P sod -hom-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 the pK18-8 plasmid. Finally, the pK18-8 plasmid was electroporated into... C. glutamicum Electrocompetent cells of Thr-7 were used; positive transformants were screened using Geno-hom-up-F and Geno-hom-dn-R as identification primers to obtain the strain. C. glutamicum Thr-8.

[0067] 1.9 aspB Promoter replacement (P) aspB ::P tuf - aspB ) Using the C. glutamicum 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, and Ptuf-F and Ptuf-R, 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 pK18-9 plasmid. The pK18-9 plasmid was then electroporated into... C. glutamicum Thr-8 electrocompetent cells; then, using Ptuf-aspB-1 and Ptuf-aspB-2 as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-9.

[0068] 1.10 Artificial Operator Genes thrABC Integration (△) lysE ::P sod - thrA - thrB - thrC (integration) Using the C. glutamicum ATCC 13032 genome as a template, PCR was performed using primers pk-bk-lysEup-F and lysEup-sod-R, rhtC-UTR-F and lysEdn-bk-pk-R, respectively, to obtain the upstream homologous arm. lysE -UP, downstream homologous arm lysE -DW; using a usable concentration of E. coli W3110 genome as a template, primer P sod -thrA-F and P sod -thrA-R, thrC(E)-UTR-F and thrC(E)-UTR-R, P sod -F-3 and P sod -R-1, PCR was performed separately to obtain the promoter fragment P. sod The gene fragment thrABC was recovered using a DNA purification kit and seamlessly ligated with plasmid pK18 to construct plasmid pK18-10. Finally, the pK18-10 plasmid was electroporated into... C.glutamicum Thr-9 electrocompetent cells; then, using Geno-lysE-up-F and Geno-lysE-dn-R as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-10.

[0069] 1.10.1 Artificial Operator Genes thrABC Integration (Δ2068::P) sod - thrA - thrB - thrC (integration) Using the C. glutamicum ATCC 13032 genome as a template, PCR was performed using primers cg2068-UF and cg2068-UR, and cg2068-DF and cg2068-DR, respectively, to obtain the upstream homologous arm 2068-UP and the downstream homologous arm 2068-DW; using a usable concentration of E. coli W3110 genome as a template, primer P... sod -thrA-F and P sod -thrA-R, thrC(E)-UTR-F and thrC(E)-UTR-R, P sod -F-3 and P sod -R-1, PCR was performed separately to obtain the promoter fragment P. sod The gene fragment thrABC was recovered using a DNA purification kit and seamlessly ligated with plasmid pK18 to construct the pK18-10-1 plasmid. Finally, the pK18-10-1 plasmid was electroporated into... C.glutamicum Thr-9 electrocompetent cells; then, using Geno-2068-F and Geno-2068-R as identification primers, positive transformants were screened to obtain the strain. C.glutamicum Thr-10-1.

[0070] The strains involved in the above construction process are shown in Table 2.

[0071] The strains listed in Table 2

[0072] Example 2 Using the L-threonine producing strain described in Example 1 C.glutamicum Thr -10 was used in shake-flask fermentation to produce L-threonine.

[0073] 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, urea 2.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.4 g / L, vitamins H, VB1, VB3, and VB5 each 2.0 mg / L, agar powder 25 g / L, pH 7.0–7.2, sterilized at 0.1 MPa for 20 min.

[0074] 2.1.2 Seed Culture Medium Glucose 30.0 g / L, yeast powder 1.0 g / L, corn steep liquor powder 5.0 g / L, ammonium sulfate 2.0 g / L, magnesium sulfate 0.8 g / L, potassium dihydrogen phosphate 3.0 g / L, L-isoleucine 0.5 g / L, ferrous sulfate 10.0 mg / L, manganese sulfate 10.0 mg / L, biotin 10.0 mg / L, vitamins VB1, VB3, and VB5 each 2.0 mg / L, L-methionine 0.5 g / L, defoamer 0.5 mg / L, pH 7.0–7.2, sterilized at 0.1 MPa for 15 min.

[0075] 2.1.3 Fermentation medium Glucose 40.0 g / L, molasses 10.0 g / L, corn steep liquor powder 10.0 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 5.0 g / L, magnesium sulfate 1.5 g / L, L-methionine 0.2 g / L, manganese sulfate 10.0 mg / L, zinc sulfate 2.0 mg / L, ferrous sulfate 10.0 mg / L, vitamins B1, B3, and B5 each 2.0 mg / L, biotin 0.02 mg / L, L-isoleucine 0.6 g / L, defoamer 0.5 mg / L, inoculum 30%, pH 7.0–7.2, sterilized at 0.1 MPa for 15 min, the remainder being water.

[0076] 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 hours, transferred to the activation slant and incubated for another 10 hours, before being transferred to a shaker containing 5 mL of seed culture medium for seed culture.

[0077] 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).

[0078] After 36 hours of shake-flask fermentation, the yield of L-threonine can reach up to 45 g / L.

[0079] Table 3 Results of shake-flask fermentation of threonine-producing strains

[0080] thrB (A20G) , man (G378E) , lysC (T311I) This indicates that the modification relieved the feedback inhibition of threonine and opened up the threonine production pathway. thrC , man The strong promoter was replaced, enhancing threonine synthesis. rhtC It acts as a threonine efflux pump to enhance threonine transport. lysC (T311I) , aspB The strong promoter was replaced, enhancing precursor synthesis. Knockout. brnF and lysE Reduce competition for branched-chain amino acid efflux and enhance targeted secretion of threonine. C.glutamicum Thr-7-1 and C.glutamicum Thr-10-1 was used as a control; knockout of the strain resulted in a certain increase in threonine production. Synchronous multiple copying was performed using an artificial operon. thrA - thrB - thrC Gene stacking significantly increased threonine production to as high as 45.1 g / L.

[0081] 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. A method for constructing an L-threonine-producing strain, characterized in that: Using Corynebacterium glutamicum as the starting strain, the specific steps are as follows: (1) By using a strong promoter P sod replace lysC The natural promoter of P sod The nucleotide sequence is shown in SEQ ID NO. 13 of the sequence listing; (2) Genes thrB Mutation thrB (A20G) The thrB (A20G) The nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing; (3) Genes hom Mutation hom (G378E) The hom (G378E) The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; (4) Integration of artificial operon gene P H36 - rhtC The artificial operon gene P H36 - rhtC China P H36 and rhtC Connect the first and last parts in order, where P H36 The nucleotide sequence of the promoter is shown in SEQ ID NO.24 of the sequence listing. rhtC The nucleotide sequence is shown in SEQ ID NO.22 of the sequence listing; (5) Using a strong promoter P sod replace thrC The natural promoter, the thrC The nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing; (6) Genes lysC Mutation lysC (T311I) The lysC (T311I) The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; (7) Knockout of the isoleucine transporter gene brnF ; and then reintegrate the artificial operon gene P. H36 - rhtC .

2. The method for constructing the L-threonine-producing strain according to claim 1, characterized in that: In step (4), the artificial operon gene P is integrated at the cg1742 pseudogene site. H36 - rhtC In step (7) brnF Site integration of artificial operon gene P H36 - rhtC .

3. The method for constructing the L-threonine-producing strain according to claim 1, characterized in that: It also includes the following steps: 1) Based on step (7), use the strong promoter P sod replace hom (G378E) The natural promoter, the hom (G378E) The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; 2) Strong promoter P tuf replace aspB The natural promoter, the aspB The nucleotide sequence is shown in SEQ ID NO. 11 of the sequence listing; 3) Knock out lysine transporter gene lysE ; 4) Integration of artificial operon gene P sod - thrA - thrB - thrC The artificial operon gene P sod - thrA - thrB - thrC The nucleotide sequence is shown in the sequence listing SEQ ID NO.

15.

4. The method for constructing the L-threonine-producing strain according to claim 3, characterized in that: exist lysE Site integration of artificial operon gene P sod - thrA - thrB - thrC .

5. An L-threonine-producing strain, characterized in that: It is obtained by modifying the construction method described in any one of claims 1-4.

6. The application of the L-threonine-producing strain according to claim 5 in the fermentation production of L-threonine.

Images