Modified corynebacterium microorganism, methods of making and using same
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- MEIHUA BIOTECH LANGFANG CO LTD
- Filing Date
- 2022-01-30
- Publication Date
- 2026-06-16
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial engineering technology, specifically to modified Corynebacterium microorganisms, their construction methods, and applications. Background Technology
[0002] L-Threonine, chemically known as β-hydroxy-α-aminobutyric acid, has the molecular formula C4H9NO3 and a relative molecular mass of 119.12. It is an essential amino acid primarily used in pharmaceuticals, chemical reagents, food fortifiers, and feed additives.
[0003] Corynebacterium glutamicum is an important industrial microorganism. In Corynebacterium glutamicum, the production of threonine from oxaloacetate requires five catalytic steps, and the catalytic enzymes for these five steps are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), homoserine dehydrogenase (encoded by hom), homoserine kinase (encoded by thrB), and threonine synthase (encoded by thrC). In the development of threonine-producing Corynebacterium glutamicum, Hermann Sahm et al. obtained the feedback-resistant hom gene (Reinscheid DJ, Eikmanns BJ, Sahm H. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J]. Journal of Bacteriology, 1991, 173(10):3228-3230.) and the lysC gene (Eikmanns BJ, Eggeling L, Sahm H. Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum.[J]. Antonie Van Leeuwenhoek, 1993, 64(2):145-163.). Lothar Eggling et al. increased threonine production by weakening the gene encoding glyA in the threonine utilization pathway and overexpressing the threonine exporter ThrE (Simic P,Willuhn J,Sahm H, et al. Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2002, 68(7):3321-3327.).
[0004] Current reports on the production of threonine using Corynebacterium glutamicum mainly focus on the modification of its synthetic pathway. Studies on the knockout of non-essential genes mainly focus on changes in strain growth after knockout, whether nutritional deficiencies occur, and whether they are beneficial for gene modification. There are few reports on the impact on threonine synthesis. cg1507-cg1524 are genes encoding a prophage membrane protein and are non-essential genes of Corynebacterium glutamicum (Baumgart, M.; Unthan, S.; Ruckert, C.; Sivalingam, J.; Grunberger, A.; Kalinowski, J.; Bott, M.; Noack, S.; Frunzke, J. Construction of a prophage-free variant of Corynebacterium glutamicum ATCC13032 for use as a platform strain for basic research and industrial biotechnology. Appl. Environ. Microbiol. 2013, 79, 6006-6015.). Summary of the Invention
[0005] The purpose of this invention is to enhance the ability of a strain to produce threonine by inactivating non-essential genes, thereby providing a modified Corynebacterium microorganism, its construction method, and its application.
[0006] The present invention aims to develop Corynebacterium microorganisms that produce threonine. During the research and development process, it was discovered that knocking out non-essential genes cg1507-cg1524 (Cgl1336-Cgl1352 or NCgl1281-NCgl1298) in Corynebacterium glutamicum can significantly increase the threonine production of the strain and improve the growth performance of the strain.
[0007] Based on the above findings, the present invention provides the following technical solution:
[0008] This invention provides the application of weakening or inactivating non-essential genes in increasing threonine production in microorganisms or constructing microorganisms that produce threonine; the non-essential genes are cg1507-cg1524, Cgl1336-Cgl1352, or NCgl1281-NCgl1298.
[0009] Taking cg1507-cg1524 as an example, the cg1507-cg1524 mentioned above are chromosome segments composed of multiple genes corresponding to NCBI numbers cg1507 to cg1524.
[0010] The weakening described above can be achieved through any one or both of the following methods (1) to (2):
[0011] (1) Use elements with lower transcription or translation initiation and regulation activity to regulate the transcription or translation of non-essential genes, thereby reducing their expression levels;
[0012] (2) Mutate non-essential genes to reduce their expression levels.
[0013] In (1) above, having lower activity means having lower activity compared to the original transcription or translation elements of non-essential genes.
[0014] Transcriptional elements include promoters and enhancers; translational elements include ribosome binding sites and 5'-UTR.
[0015] In (2) above, the mutation may be to mutate the start codon of a non-essential gene to a start codon other than ATG (e.g., GTG or TTG).
[0016] The inactivation described above can be achieved by deleting, inserting, or replacing one or more nucleotides in a non-essential gene, causing the non-essential gene to cease expression.
[0017] Preferably, the above-described application is achieved by knocking out the non-essential gene.
[0018] In the applications described above, the microorganism is a Corynebacterium bacterium, preferably Corynebacterium glutamicum.
[0019] Furthermore, the present invention provides a modified Corynebacterium microorganism, wherein the non-essential genes of the microorganism are weakened or inactivated compared with the unmodified microorganism; the non-essential genes are cg1507-cg1524, Cgl1336-Cgl1352 or NCgl1281-NCgl1298.
[0020] The weakening described above can be achieved through any one or both of the following methods (1) to (2):
[0021] (1) Use elements with lower transcription or translation initiation and regulation activity to regulate the transcription or translation of non-essential genes, thereby reducing their expression levels;
[0022] (2) Mutate non-essential genes to reduce their expression levels.
[0023] In (1) above, having lower activity means having lower activity compared to the original transcription or translation elements of non-essential genes.
[0024] Transcriptional elements include promoters and enhancers; translational elements include ribosome binding sites and 5'-UTR.
[0025] In (2) above, the mutation may be to mutate the start codon of a non-essential gene to a start codon other than ATG (e.g., GTG or TTG).
[0026] The inactivation described above can be achieved by deleting, inserting, or replacing one or more nucleotides in a non-essential gene, causing the non-essential gene to cease expression.
[0027] Preferably, the non-essential genes in the microorganism are knocked out or inactivated.
[0028] Preferably, the microorganism has enhanced threonine production capacity compared to the unmodified microorganism.
[0029] This invention reveals that inactivation of the aforementioned non-essential gene enhances threonine synthesis in both wild-type strains and strains capable of threonine accumulation. Comparatively, the increase in threonine synthesis is greater in strains capable of threonine accumulation.
[0030] Preferably, compared with the unmodified microorganism, the microorganism exhibits enhanced activity and / or relief of feedback inhibition of any one or more of the following enzymes (1) to (7):
[0031] (1) Aspartate kinase;
[0032] (2) Aspartate semialdehyde dehydrogenase;
[0033] (3) Homoserine dehydrogenase;
[0034] (4) Homoserine kinase;
[0035] (5) Threonine efflux protein;
[0036] (6) Pyruvate carboxylase;
[0037] (7) Glucose-6-phosphate dehydrogenase.
[0038] The above-mentioned activity enhancement is achieved by selecting from 1) to 6) below, or an optional combination thereof:
[0039] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;
[0040] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;
[0041] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
[0042] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;
[0043] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme;
[0044] 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme.
[0045] The reference sequence numbers of the above-mentioned aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, homoserine kinase, pyruvate carboxylase, and glucose-6-phosphate dehydrogenase on NCBI are WP_003855724.1, WP_003854900.1, WP_011013506.1, WP_011014183.1, WP_011013816.1, and NP_600790.1, respectively, or amino acid sequences that have 90% similarity to the above reference sequences and have equivalent functions.
[0046] The threonine export protein is preferably a threonine export protein derived from Escherichia coli, whose reference sequence number on NCBI is YP_026264.1, or an amino acid sequence that has 90% similarity to the above reference sequence and has the same function.
[0047] Preferably, compared with unmodified microorganisms, the activity of any one or more of the following enzymes (1) to (3) is reduced or lost:
[0048] (1) Diaminopimelic acid dehydrogenase;
[0049] (2) 4-Hydroxytetrahydropyridinedicarboxylic acid synthase;
[0050] (3) Citric acid synthase.
[0051] Preferably, the reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out an endogenous gene encoding the enzyme.
[0052] The reference sequence numbers for the above-mentioned diaminopimelic acid dehydrogenase, 4-hydroxytetrahydropyridine dicarboxylic acid synthase, and citrate synthase on NCBI are WP_011015254.1, WP_011014792.1, and WP_011013914.1, respectively, or amino acid sequences that have 90% similarity to the above reference sequences and have equivalent functions.
[0053] The modifications that enhance enzyme activity and relieve feedback inhibition can act alone or in combination with the gene targets that reduce enzyme activity or inactivate it. The resulting strains all accumulate threonine to varying degrees. In these strains, inactivation of non-essential genes can increase threonine production to varying degrees.
[0054] As a preferred embodiment of the present invention, the microorganism is any one of the following:
[0055] (1) Microorganisms with inactivated non-essential genes and enhanced enzyme activity of aspartate kinase and / or relief of feedback inhibition;
[0056] (2) Microorganisms with inactivated non-essential genes and enhanced enzyme activity of aspartate kinase and / or aspartate semialdehyde dehydrogenase and / or relief of feedback inhibition.
[0057] (3) Microorganisms with inactivated non-essential genes and enhanced enzyme activity and / or relief of feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase.
[0058] (4) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase and homoserine kinase.
[0059] (5) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine efflux protein.
[0060] (6) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein and pyruvate carboxylase.
[0061] (7) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein, pyruvate carboxylase and glucose-6-phosphate dehydrogenase.
[0062] (8) Microorganisms with inactivation of non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein, pyruvate carboxylase and glucose-6-phosphate dehydrogenase, while the enzyme activity of diaminopimelic acid dehydrogenase is reduced or lost.
[0063] (9) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein, pyruvate carboxylase and glucose-6-phosphate dehydrogenase, while the enzyme activity of diaminopimelic acid dehydrogenase and / or 4-hydroxytetrahydropyridine dicarboxylic acid synthase synthase is reduced or lost.
[0064] (10) Microorganisms with inactivated non-essential genes and enhanced and / or relieved feedback inhibition of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein, pyruvate carboxylase and glucose-6-phosphate dehydrogenase, while having reduced or lost enzyme activity of diaminopimelic acid dehydrogenase, 4-hydroxytetrahydropyridine dicarboxylic acid synthase and / or citrate synthase.
[0065] Preferably, the above-mentioned enhancement of enzyme activity is achieved through any one or more of the following methods:
[0066] (1) Replace the original promoter of the target gene with a strong promoter;
[0067] (2) Mutate the start codon of the target gene to ATG;
[0068] (3) Insert one or more copies of the target gene into the chromosome.
[0069] The strong promoters include Psod or PcspB.
[0070] The nucleotide sequences of promoters Psod and PcspB are shown in SEQ ID NO.1 and 2, respectively.
[0071] Preferably, the enhanced enzyme activities of aspartate kinase, aspartate semialdehyde dehydrogenase, pyruvate carboxylase, and glucose-6-phosphate dehydrogenase are achieved by replacing their original promoters with Psod promoters.
[0072] The enhanced enzyme activity of homoserine dehydrogenase and homoserine kinase is achieved by replacing their original promoters with PcspB promoters;
[0073] The enhanced enzymatic activity of E. coli-derived threonine export protein was achieved by inserting a copy of the E. coli-derived threonine export protein encoding gene rhtC into the genome.
[0074] Preferably, the release of feedback inhibition of aspartate kinase is achieved by mutating the aspartate kinase encoding gene, causing the encoded aspartate kinase to undergo a T311I mutation;
[0075] The release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the gene encoding homoserine dehydrogenase, resulting in a G378E mutation in homoserine dehydrogenase.
[0076] The release of feedback inhibition of pyruvate carboxylase is achieved by mutating the gene encoding pyruvate carboxylase, resulting in a P458S mutation in the encoded pyruvate carboxylase.
[0077] The release of feedback inhibition of glucose-6-phosphate dehydrogenase is achieved by mutating the gene encoding glucose-6-phosphate dehydrogenase, resulting in the A243T mutation of the encoded glucose-6-phosphate dehydrogenase.
[0078] The inactivation described above is achieved by deleting, inserting, or replacing one or more nucleotides of the target gene, so that the target gene is no longer expressed.
[0079] Preferably, the diaminopimelic acid dehydrogenase encoding gene is inactivated, and the start codons of the 4-hydroxytetrahydropyridine dicarboxylic acid synthase and citrate synthase encoding genes are mutated to GTG.
[0080] The microorganisms described in this invention are preferably Corynebacterium glutamicum. Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), and more preferably Corynebacterium glutamicum ATCC 13032.
[0081] The present invention also provides a method for constructing a threonine-producing strain, the method comprising: weakening or inactivating non-essential genes in Corynebacterium bacteria with amino acid production capacity to obtain a gene-weakened strain; wherein the non-essential genes are cg1507-cg1524, Cgl1336-Cgl1352 or NCgl1281-NCgl1298.
[0082] Preferably, the method further includes: enhancing the activity of any one or more enzymes from (1) to (7) below and / or relieving their feedback inhibition:
[0083] (1) Aspartate kinase;
[0084] (2) Aspartate semialdehyde dehydrogenase;
[0085] (3) Homoserine dehydrogenase;
[0086] (4) Homoserine kinase;
[0087] (5) Threonine efflux protein;
[0088] (6) Pyruvate carboxylase;
[0089] (7) Glucose-6-phosphate dehydrogenase;
[0090] The enhancement of activity is achieved by selecting from 1) to 6) below, or an optional combination thereof:
[0091] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;
[0092] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;
[0093] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
[0094] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;
[0095] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme;
[0096] 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme;
[0097] And / or, the method further includes: reducing or eliminating the activity of any one or more of the following enzymes (1) to (3):
[0098] (1) Diaminopimelic acid dehydrogenase;
[0099] (2) 4-Hydroxytetrahydropyridinedicarboxylic acid synthase synthase;
[0100] (3) Citrate synthase;
[0101] Preferably, the reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out an endogenous gene encoding the enzyme.
[0102] The aforementioned methods for modifying the strains, including gene enhancement, are all modifications known to those skilled in the art. See Man Zaiwei, Systematic Pathway Engineering of High-Yielding L-Arginine Corynebacterium [D]. Jiangnan University, 2016; Cui Yi, Metabolic Engineering of Corynebacterium Glutamate for L-Leucine Production [D]. Tianjin University of Science and Technology; Xu Guodong, Construction and Fermentation Condition Optimization of L-Isoleucine-Producing Strains. Tianjin University of Science and Technology, 2015.
[0103] This invention provides any of the following applications of the microorganisms described above:
[0104] (1) Application in the fermentation production of threonine or its derivatives;
[0105] (2) Application as a starting strain in the production strain for constructing threonine or its derivatives;
[0106] (3) Application in increasing the yield and / or conversion of threonine or its derivatives.
[0107] The threonine derivatives described in this invention can be compounds synthesized using threonine as a precursor, including isoleucine, glycine, etc.
[0108] The present invention also provides a method for producing threonine or its derivatives by fermentation, comprising the steps of culturing the microorganisms described above and isolating threonine or its derivatives from the culture.
[0109] Specifically, the above method includes: inoculating the microorganism into a seed culture medium for seed culture to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation broth; and separating and extracting the fermentation broth to obtain threonine or its derivatives.
[0110] Preferably, the fermentation medium comprises the following components: corn steep liquor 45-55 mL / L, glucose 25-35 g / L, ammonium sulfate 3-5 g / L, MOPS 25-35 g / L, potassium dihydrogen phosphate 8-12 g / L, urea 15-25 g / L, biotin 8-12 mg / L, magnesium sulfate 5-7 g / L, ferrous sulfate 0.5-1.5 g / L, vitamin B1·HCl 35-45 mg / L, calcium pantothenate 45-55 mg / L, nicotinamide 35-45 mg / L, manganese sulfate 0.5-1.5 g / L, zinc sulfate 15-25 mg / L, copper sulfate 15-25 mg / L, pH 7.0-7.2.
[0111] The beneficial effects of this invention are as follows: By inactivating the non-essential genes cg1507-cg1524 and by strengthening or weakening enzymes such as aspartate kinase and homoserine dehydrogenase, this invention significantly improves the strain's ability to produce threonine. The threonine yield of the strain is significantly higher than before the modification, while also enhancing the strain's growth performance. The modification of the non-essential genes cg1507-cg1524 can be used in the fermentation production of threonine and has good application value. Detailed Implementation
[0112] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0113] The information regarding the proteins and their encoding genes involved in this invention is as follows:
[0114] Non-essential genes, NCBI IDs: cg1507-cg1524, Cgl1336-Cgl1352, NCgl1281-NCgl1298;
[0115] Aspartate kinase, encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247;
[0116] Aspartate semialdehyde dehydrogenase, encoding gene name asd, NCBI number: cg0307, Cgl0252, NCgl0248;
[0117] Homoserine dehydrogenase, encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136;
[0118] Diaminopimelic acid dehydrogenase, encoding gene name ddh, NCBI number: cg2900, Cgl2617, NCgl2528;
[0119] Pyruvate carboxylase, encoding gene name pyc, NCBI number: cg0791, Cgl0689, NCgl0659;
[0120] Glucose-6-phosphate dehydrogenase, encoding gene name zwf, NCBI number: cg1778, Cgl1576, NCgl1514;
[0121] Homoserine kinase, encoding gene name thrB, NCBI number: cg1338, Cgl1184, NCgl1137;
[0122] The threonine efflux protein from Escherichia coli is encoded by the gene rhtC, with NCBI number b3823.
[0123] 4-Hydroxytetrahydropyridinedicarboxylic acid synthase, encoding gene name dapA, NCBI number: cg2161, Cgl1971, NCgl1895;
[0124] Citrate synthase, encoding gene name gltA, NCBI number: cg0949, Cgl0829, NCgl0795.
[0125] Example 1: Construction of plasmids for strain genome modification
[0126] 1. Construction of the cg1507-cg1524 knockout plasmid pK18mobsacB-Δcg1507-cg1524
[0127] Using the ATCC13032 genome as a template, PCR amplification with PCT72 / PCT73 primers yielded the upstream homologous arm up, and PCR amplification with PCT74 / PCT75 primers yielded the downstream homologous arm dn. Fusion PCR using the up and dn fragments as templates with PCT72 / PCT75 primers obtained the full-length fragment up-dn. pK18mobsacB was digested with BamHI / HindIII. The digested up-dn and pK18mobsacB were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-Δcg1507-cg1524.
[0128] 2. Aspartate kinase expression enhancement plasmid pK18mobsacB-Psod-lysC g1a-T311I Construction
[0129] Using the ATCC13032 genome as a template, PCR amplification with primers P21 / P22 yielded the upstream homologous arm up; PCR amplification with primers P23 / P24 yielded the promoter fragment Psod; and PCR amplification with primers P25 / P26 yielded lysC. g1a-T311I PCR amplification was performed using primers P27 / P28 to obtain the downstream homologous arm dn. Fusion PCR was then performed using primers P21 / P24 with up and Psod as templates to obtain the up-Psod fragment. Finally, PCR amplification was performed using primers P21 / P28 with up-Psod and lysC... g1a-T311I Using pK18mobsacB as a template, fusion PCR was performed to obtain the full-length fragment up-Psod-lysCV1M-T311I-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-Psod-lysCV1M-T311I-dn. g1a-T311I .
[0130] 3. Construction of aspartate aminotransferase expression enhancement plasmid pK18mobsacB-Psod-asd
[0131] The plasmid construction method is the same as described in section 2 above, and the primers used are P1, P2, P3, P4, P5, and P6.
[0132] 4. Homoserine dehydrogenase expression enhancement plasmid pK18mobsacB-PcspB-hom G378E Construction
[0133] The plasmid construction method is the same as described in section 2 above, and the primers used are P29, P30, P31, P32, P33, P34, P35, and P36.
[0134] 5. Construction of the homoserine kinase expression enhancement plasmid pK18mobsacB-PcspB-thrB
[0135] The plasmid construction method is the same as described in section 2 above, and the primers used are P7, P8, P9, P10, P11, and P12.
[0136] 6. Construction of the enhancement plasmid pK18mobsacB-cg2899::rhtC for the expression of threonine export protein derived from E. coli
[0137] Using the ATCC13032 genome as a template, PCR amplification with primers P157 / P158 yielded the upstream homologous arm up. PCR amplification with primers P159 / P160 yielded the promoter fragment Psod. Using the E. coli MG1655 genome as a template, PCR amplification with primers P161 / P162 yielded rhtC. Using the ATCC13032 genome as a template, PCR amplification with primers P163 / P164 yielded the downstream homologous arm dn. Fusion PCR was performed using primers P157 / P160 with up and Psod as templates to obtain the up-Psod fragment. Fusion PCR was performed using primers P157 / P164 with up-Psod, rhtC, and dn as templates to obtain the full-length fragment up-Psod-rhtC-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-Psod-rhtC was obtained.
[0138] 7. Pyruvate carboxylase expression enhancement plasmid pK18mobsacB-Psod-pyc P458S Construction
[0139] The plasmid construction method is the same as described in section 2 above, and the primers used are P13, P14, P15, P16, P17, P18, P19, and P20.
[0140] 8. Glucose-6-phosphate dehydrogenase expression enhancement plasmid pK18mobsacB-Psod-zwf A243T Construction
[0141] The plasmid construction method is the same as described in section 2 above, and the primers used are P129, P130, P131, P132, P133, P134, P135, and P136.
[0142] 9. Construction of the diamino dehydrogenase expression attenuated plasmid pK18mobsacB-Δddh
[0143] The plasmid construction method is the same as described in section 1 above, and the primers used are P99, P100, P101, and P102.
[0144] 10. The expression attenuated plasmid pK18mobsacB-dapA for 4-hydroxytetrahydropyridine dicarboxylic acid synthase a1g Construction
[0145] The plasmid construction method is the same as described in section 1 above, and the primers used are P75, P76, P77, and P78.
[0146] 11. Citrate synthase expression attenuated plasmid pK18mobsacB-gltA a1g Construction
[0147] The plasmid construction method is the same as described in section 1 above, and the primers used are P153, P154, P155, and P156.
[0148] The primers used in the construction of the above plasmids are shown in Table 1.
[0149] Table 1 Primer sequences
[0150]
[0151]
[0152]
[0153] Example 2 Construction of Genome-Modified Strains
[0154] 1. Construction of strains with enhanced expression of aspartate kinase
[0155] ATCC13032 competent cells were prepared according to the classic method for *Corynebacterium glutamicum* (C. glutamicum Handbook, Charter 23). The recombinant plasmid pK18mobsacB-Psod-lysC was then used. g1a-T311I Competent cells were transformed by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the target gene was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The genomes of the colonies growing on the sucrose medium did not carry the inserted vector sequence. The target fragment was amplified by PCR and analyzed by nucleotide sequencing, resulting in the target mutant strain named SMCT196. Compared to strain ATCC13032, this strain showed a mutation in the lysC gene start codon from GTG to ATG, a change from threonine to isoleucine at position 311, and the lysC gene promoter was replaced by the Psod promoter.
[0156] 2. Construction of strains with enhanced expression of aspartate semialdehyde dehydrogenase
[0157] The strain construction method is the same as described in 1 above. Using SMCT196 as the starting strain, the plasmid pK18mobsacB-Psod-asd was introduced into the strain SMCT196 to enhance the expression of aspartate semialdehyde dehydrogenase. The resulting modified strain was named SMCT197. Compared with strain SMCT196, the promoter of the asd gene in this strain was replaced with the Psod promoter.
[0158] 3. Construction of strains enhanced with homoserine dehydrogenase expression
[0159] The strain construction method is the same as described in section 1 above, using SMCT197 as the starting strain, and the plasmid pK18mobsacB-PcspB-hom is constructed. G378E The strain SMCT197 was introduced into the strain to enhance the expression of homoserine dehydrogenase. The resulting modified strain was named SMCT198. Compared with strain SMCT197, the hom gene of this strain was mutated, resulting in the G378E mutation in the protein it encodes. At the same time, the promoter of the hom gene was replaced with the PcspB promoter from strain ATCC14067.
[0160] 4. Construction of strains with enhanced expression of homoserine kinase
[0161] The strain construction method is the same as described in 1 above. Using SMCT198 as the starting strain, the plasmid pK18mobsacB-PcspB-thrB was introduced into the strain SMCT198 to enhance the expression of high serine kinase. The resulting modified strain was named SMCT199. Compared with strain SMCT198, the promoter of the thrB gene in this strain was replaced with the PcspB promoter.
[0162] 5. Construction of strains with enhanced expression of threonine efflux protein from *E. coli*
[0163] The strain construction method is the same as described in section 1 above. Using SMCT199 as the starting strain, the plasmid pK18mobsacB-cg2899::rhtC was introduced into the strain SMCT199 to attenuate the expression of threonine efflux protein from E. coli. The resulting modified strain was named SMCT200. Compared with strain SMCT199, this strain has an insertion of one copy of the rhtC gene after the last base of the cg2899 gene.
[0164] 6. Construction of strains for enhanced expression of pyruvate carboxylase
[0165] The strain construction method is the same as described in section 1 above, using SMCT200 as the starting strain, and the plasmid pK18mobsacB-Psod-pyc is constructed. P458S The strain SMCT200 was introduced and modified to enhance pyruvate carboxylase. The resulting modified strain was named SMCT201. Compared with strain SMCT200, the pyc gene of the strain was mutated, resulting in the P458S mutation in the encoded protein. At the same time, the promoter of the pyc gene was replaced with the Psod promoter.
[0166] 7. Construction of strains for enhanced expression of glucose-6-phosphate dehydrogenase
[0167] The strain construction method is the same as described in section 1 above, using SMCT201 as the starting strain, and the plasmid pK18mobsacB-Psod-zwf is constructed. A243T The strain SMCT201 was introduced into the strain to enhance the expression of glucose-6-phosphate dehydrogenase. The resulting modified strain was named SMCT202. Compared with strain SMCT201, the amino acid sequence encoded by the zwf gene of this strain has undergone the A243T mutation, and the promoter of the zwf gene has been replaced by the Psod promoter.
[0168] 8. Construction of a strain expressing attenuated diaminopimelic acid dehydrogenase
[0169] The strain construction method is the same as described in 1 above. Using SMCT202 as the starting strain, the plasmid pK18mobsacB-Δddh was introduced into the strain SMCT202 to modify the expression of diaminopimelic acid dehydrogenase. The resulting modified strain was named SMCT203. Compared with strain SMCT202, the ddh gene of this strain was knocked out.
[0170] 9. Construction of a strain with weakened expression of 4-hydroxytetrahydropyridine dicarboxylic acid synthase
[0171] The strain construction method is the same as described in section 1 above, using SMCT203 as the starting strain, and the plasmid pK18mobsacB-dapA is constructed. a1gThe SMCT203 strain was introduced to weaken the expression of 4-hydroxytetrahydropyridine dicarboxylic acid synthase. The resulting modified strain was named SMCT204. Compared with SMCT203, the start codon of the dapA gene in this strain was mutated to GTG.
[0172] 10. Construction of a strain with weakened citrate synthase expression
[0173] The strain construction method is the same as described in section 1 above, using SMCT204 as the starting strain, and the plasmid pK18mobsacB-gltA is constructed. a1g The citrate synthase expression was weakened in strain SMCT204, and the resulting modified strain was named SMCT205. Compared with strain SMCT204, the start codon of the gltA gene in this strain was mutated to GTG.
[0174] 11. Construction of inactivated strains cg1507-cg1527
[0175] The strain construction method is the same as described in section 1 above. ATCC13032, SMCT196, SMCT197, SMCT198, SMCT199, SMCT200, SMCT201, SMCT202, SMCT203, SMCT204, and SMCT205 were used as starter strains. The plasmid pK18mobsacB-Δcg1507-cg1524 was introduced into these starter strains, and the cg1507-cg1524 gene was inactivated to obtain the modified strains SMCT206, SMCT207, SMCT208, SMCT209, SMCT210, SMCT211, SMCT212, SMCT213, SMCT214, SMCT215, and SMCT216. Compared with their corresponding starter strains, the cg1507-cg1524 gene was knocked out in the genome of these modified strains.
[0176] The genotypic information of the strains obtained above is shown in Table 2.
[0177] Table 2. Strain Genotype Information
[0178]
[0179]
[0180] Example 3: Shake-flask fermentation verification of the constructed strain
[0181] The strains constructed in Example 2 were verified by shake-flask fermentation, as follows:
[0182] 1. Culture medium
[0183] Seed activation medium: BHI 3.7%, agar 2%, pH 7.
[0184] Seed culture medium: peptone 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ammonium sulfate 16 g / L, urea 8 g / L, potassium dihydrogen phosphate 10.4 g / L, dipotassium hydrogen phosphate 21.4 g / L, biotin 5 mg / L, magnesium sulfate 3 g / L, glucose 50 g / L, pH 7.2.
[0185] Fermentation medium: corn steep liquor 50 mL / L, glucose 30 g / L, ammonium sulfate 4 g / L, MOPS 30 g / L, potassium dihydrogen phosphate 10 g / L, urea 20 g / L, biotin 10 mg / L, magnesium sulfate 6 g / L, ferrous sulfate 1 g / L, vitamin C B1 • HCl 40 mg / L, calcium pantothenate 50 mg / L, nicotinamide 40 mg / L, manganese sulfate 1 g / L, zinc sulfate 20 mg / L, copper sulfate 20 mg / L, pH 7.2.
[0186] 2. Production of L-threonine by shake-flask fermentation with engineered bacteria
[0187] (1) Seed culture: Pick ATCC13032, SMCT196, SMCT197, SMCT198, SMCT199, SMCT200, SMCT201, SMCT202, SMCT203, SMCT204, SMCT205, SMCT206, SMCT207, SMCT208, SMCT209, SMCT210, SMCT211, SMCT212, SMCT213, SMCT214, SMCT215, and SMCT216 slant seeds and loop them into a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium. Culture at 30 °C and 220 r / min for 16 h with shaking to obtain seed liquid.
[0188] (2) Fermentation culture: 2 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation culture medium and cultured at 33 °C and 220 r / min for 24 h to obtain fermentation broth.
[0189] (3) Take 1 mL of fermentation broth and centrifuge (12000 rpm, 2 min), collect the supernatant, and use HPLC to detect L-threonine in the fermentation broth of engineered bacteria and control bacteria.
[0190] The results of the detection of the threonine production capacity of each strain are shown in Table 3.
[0191] Table 3 Fermentation detection results
[0192] strain number <![CDATA[OD 562 ]]> Threonine (g / L) strain number <![CDATA[OD 562 ]]> Threonine (g / L) ATCC13032 25 - SMCT206 26 0.2 SMCT196 24 1.2 SMCT207 25 1.3 SMCT197 24 1.5 SMCT208 25 1.7 SMCT198 24 2.4 SMCT209 25 2.7 SMCT199 23 2.8 SMCT210 25 3.2 SMCT200 23 3.8 SMCT211 25 4.5 SMCT201 22 4.4 SMCT212 24 5.3 SMCT202 23 5.3 SMCT213 24 6.5 SMCT203 24 6.4 SMCT214 23 7.9 SMCT204 22 8.3 SMCT215 25 10.5 SMCT205 21 9.9 SMCT216 23 12.7
[0193] Note: In Table 3, "-" indicates that threonine was not detected.
[0194] As shown in Table 3, the modified strains with cg1507-cg1524 knockout exhibited varying degrees of threonine production increase compared to strains without cg1507-cg1524 inactivation, with increases ranging from 10% to 28%. Simultaneously, strain growth also showed some improvement. Furthermore, when the cg1507-cg1524 knockout was combined with modifications that enhanced the expression of at least one of the following enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein from *E. coli*, pyruvate decarboxylase, and glucose-6-phosphate dehydrogenase, removed feedback inhibition, and weakened the expression of at least one of the following enzymes: diaminopimelic acid dehydrogenase, 4-hydroxytetrahydropyridine dicarboxylic acid synthase, and citrate synthase, threonine production was further increased. The combination of inactivation of 7-cg1524 and modification of the above-mentioned sites also favors threonine production; in addition, enhanced expression of at least one of the following enzymes, namely aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine efflux protein from E. coli, pyruvate decarboxylase, glucose-6-phosphate dehydrogenase, removal of feedback inhibition, and weakened expression of at least one of the following enzymes, namely diaminopimelic acid dehydrogenase, 4-hydroxytetrahydropyridine dicarboxylic acid synthase, and citrate synthase, all favor threonine production by the strain.
[0195] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> Modified Corynebacterium microorganisms, their construction methods and applications <130> KHP211124937.0 <160> 70 <170> SIPOSequenceListing 1.0 <210> 1 <211> 192 <212> DNA <213> Artificial Sequence <400> 1 tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60 ttcgttgcaa tatcaacaaa aaggcctatc attgggaggt gtcgcaccaa gtacttttgc 120 gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180 gattttttac cc 192 <210> 2 <211> 260 <212> DNA <213> Artificial Sequence <400> 2 acctgcgttt ataaagaaat gtaaacgtga tcggatcgat ataaaagaaa cagtttgtac 60 tcaggtttga agcattttct ccaattcgcc tggcaaaaat ctcaattgtc gcttacagtt 120 tttctcaacg acaggctgct aagctgctag ttcggtggcc tagtgagtgg cgtttacttg 180 gataaaagta atcccatgtc gtgatcagcc attttgggtt gtttccatag catccaaagg 240 tttcgtcttt cgatacctat 260 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 agctcggtac ccggggatcc cgcatttgtg gccaatttga 40 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <400> 4 catgttaacc ctatggctga aacgcaatag ttgcattttt ag 42 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <400> 5 ctaaaaatgc aactattgcg tttcagccat aggggttaaca tg 42 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <400> 6 cgacggccag tgccaagctt gtagatgcac agttgatggg gttc 44 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 aattcgagct cggtacccgg ggatccgaca agtccgaagc caaagt 46 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <400> 8 cccggaataa ttggcagcta tgtaaaacta ctcctttaaa 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 tttaaaggag tagttttaca tagctgccaa ttatccggg 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <400> 10 acaactgcga tggtggtcat gggtaaaaaa tcctttcgta 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <400> 11 tacgaaagga ttttttaccc atgaccacca tcgcagttgt 40 <210> 12 <211> 46 <212> DNA <213> Artificial Sequence <400> 12 gtaaaacgac ggccagtgcc aagcttcacc tgcaagacca gaaccg 46 <210> 13 <211> 46 <212> DNA <213> Artificial Sequence <400> 13 aattcgagct cggtacccgg ggatccgctc gcgtgcaccc gactct 46 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <400> 14 atttctttat aaacgcaggt gtcagtaaaa ttagtccctt 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <400> 15 aagggactaa ttttactgac acctgcgttt ataaagaaat 40 <210> 16 <211> 41 <212> DNA <213> Artificial Sequence <400> 16 gacgttcagt tcaattgcca tataggtatc gaaagacgaa a 41 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <400> 17 tttcgtcttt cgatacctat atggcaattg aactgaacgt c 41 <210> 18 <211> 46 <212> DNA <213> Artificial Sequence <400> 18 gtaaaacgac ggccagtgcc aagcttgcat actgtggctg gctctt 46 <210> 19 <211> 46 <212> DNA <213> Artificial Sequence <400> 19 aattcgagct cggtacccgg ggatcctgac agttgctgat ctggct 46 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <400> 20 cccggaataa ttggcagcta tagagtaatt attcctttca 40 <210> twenty one <211> 40 <212> DNA <213> Artificial Sequence <400> twenty one tgaaaggaat aattactcta tagctgccaa ttatccggg 40 <210> twenty two <211> 40 <212> DNA <213> Artificial Sequence <400> twenty two gaagatgtgt gagtcgacac gggtaaaaaa tcctttcgta 40 <210> twenty three <211> 40 <212> DNA <213> Artificial Sequence <400> twenty three tacgaaagga ttttttaccc gtgtcgactc acacatcttc 40 <210> twenty four <211> 43 <212> DNA <213> Artificial Sequence <400> twenty four ggtggagcct gaaggaggtg cgagtgatcg gcaatgaatc cgg 43 <210> 25 <211> 43 <212> DNA <213> Artificial Sequence <400> 25 ccggattcat tgccgatcac tcgcacctcc ttcaggctcc acc 43 <210> 26 <211> 46 <212> DNA <213> Artificial Sequence <400> 26 gtaaaacgac ggccagtgcc aagcttcgcg gcagacggag tctggg 46 <210> 27 <211> 46 <212> DNA <213> Artificial Sequence <400> 27 aattcgagct cggtacccgg ggatccagcg acaggacaag cactgg 46 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <400> 28 cccggaataa ttggcagcta tgtgcacctt tcgatctacg 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <400> 29 cgtagatcga aaggtgcaca tagctgccaa ttatccggg 40 <210> 30 <211> 41 <212> DNA <213> Artificial Sequence <400> 30 tttctgtacg accagggcca tgggtaaaaa atcctttcgt a 41 <210> 31 <211> 41 <212> DNA <213> Artificial Sequence <400> 31 tacgaaagga ttttttaccc atggccctgg tcgtacagaa a 41 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <400> 32 tcggaacgag ggcaggtgaa ggtgatgtcg gtggtgccgt ct 42 <210> 33 <211> 42 <212> DNA <213> Artificial Sequence <400> 33 agacggcacc accgacatca ccttcacctg ccctcgttcc ga 42 <210> 34 <211> 46 <212> DNA <213> Artificial Sequence <400> 34 gtaaaacgac ggccagtgcc aagcttagcc tggtaagagg aaacgt 46 <210> 35 <211> 46 <212> DNA <213> Artificial Sequence <400> 35 aattcgagct cggtacccgg ggatccctgc gggcagatcc ttttga 46 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <400> 36 atttctttat aaacgcaggt catatctacc aaaactacgc 40 <210> 37 <211> 40 <212> DNA <213> Artificial Sequence <400> 37 gcgtagtttt ggtagatatg acctgcgttt ataaagaaat 40 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <400> 38 gtatatctcc ttctgcagga ataggtatcg aaagacgaaa 40 <210> 39 <211> 40 <212> DNA <213> Artificial Sequence <400> 39 tttcgtcttt cgatacctat tcctgcagaa ggagatatac 40 <210> 40 <211> 41 <212> DNA <213> Artificial Sequence <400> 40 tagccaattc agccaaaacc cccacgcgat cttccacatc c 41 <210> 41 <211> 41 <212> DNA <213> Artificial Sequence <400> 41 ggatgtggaa gatcgcgtgg gggttttggc tgaattggct a 41 <210> 42 <211> 46 <212> DNA <213> Artificial Sequence <400> 42 gtaaaacgac ggccagtgcc aagcttgctg gctcttgccg tcgata 46 <210> 43 <211> 57 <212> DNA <213> Artificial Sequence <400> 43 catgattacg aattcgagct cggtacccgg ggatcccaag ccaaacaagg tttagtg 57 <210> 44 <211> 42 <212> DNA <213> Artificial Sequence <400> 44 gaagaaggta accttgaact ctgtgagcac aggtttaaca gc 42 <210> 45 <211> 42 <212> DNA <213> Artificial Sequence <400> 45 gctgttaaac ctgtgctcac agagttcaag gttaccttct tc 42 <210> 46 <211> 56 <212> DNA <213> Artificial Sequence <400> 46 tcacgacgtt gtaaaacgac ggccagtgcc aagctttga gtctcggttc gctttc 56 <210> 47 <211> 41 <212> DNA <213> Artificial Sequence <400> 47 gagctcggta cccggggatc ctctgcaact ggcatgttgg a 41 <210> 48 <211> 47 <212> DNA <213> Artificial Sequence <400> 48 tcgagctaaa ccttgttggg ctagttgtcc tccttttttc cgtagcc 47 <210> 49 <211> 47 <212> DNA <213> Artificial Sequence <400> 49 ggctacggaa aaaaggagga caactagccc aacaaggttt agctcga 47 <210> 50 <211> 39 <212> DNA <213> Artificial Sequence <400> 50 acgacggcca gtgccaagct tactcaacgg cgattgcgg 39 <210> 51 <211> 56 <212> DNA <213> Artificial Sequence <400> 51 catgattacg aattcgagct cggtacccgg ggatccgatg aggctttggc tctgcg 56 <210> 52 <211> 42 <212> DNA <213> Artificial Sequence <400> 52 agcccggaat aattggcagc tagatggtag tgtcacgatc ct 42 <210> 53 <211> 42 <212> DNA <213> Artificial Sequence <400> 53 aggatcgtga cactaccatc tagctgccaa ttatccggg ct 42 <210> 54 <211> 39 <212> DNA <213> Artificial Sequence <400> 54 gggtcgtgtt tgtgctcatg ggtaaaaaat cctttcgta 39 <210> 55 <211> 42 <212> DNA <213> Artificial Sequence <400> 55 tacgaaagga ttttttaccc atgagcacaa acacgacccc ct 42 <210> 56 <211> 44 <212> DNA <213> Artificial Sequence <400> 56 cacccaagcc aatatcttca gtcatggtga tctggacgtg gtca 44 <210> 57 <211> 44 <212> DNA <213> Artificial Sequence <400> 57 tgaccacgtc cagatcacca tgactgaaga tattggcttg ggtg 44 <210> 58 <211> 54 <212> DNA <213> Artificial Sequence <400> 58 tcacgacgtt gtaaaacgac ggccagtgcc aagcttcgaa tcacgatggc gttt 54 <210> 59 <211> 46 <212> DNA <213> Artificial Sequence <400> 59 aattcgagct cggtacccgg ggatccttca atttctaggt tgttaa 46 <210> 60 <211> 41 <212> DNA <213> Artificial Sequence <400> 60 cacgatatcc ctttcaaaca catttgttcg gaaaaaaact c 41 <210> 61 <211> 41 <212> DNA <213> Artificial Sequence <400> 61 gagttttttt ccgaacaaat gtgtttgaaa gggatatcgt g 41 <210> 62 <211> 46 <212> DNA <213> Artificial Sequence <400> 62 gtaaaacgac ggccagtgcc aagcttgttg ccttatcaag ctgtgc 46 <210> 63 <211> 56 <212> DNA <213> Artificial Sequence <400> 63 tacgaattcg agctcggtac ccggggatcc agttaactcc accgaccggg tactgc 56 <210> 64 <211> 43 <212> DNA <213> Artificial Sequence <400> 64 aagcccggaa taattggcag ctatgtcttc gctggaccaa gag 43 <210> 65 <211> 43 <212> DNA <213> Artificial Sequence <400> 65 ctcttggtcc agcgaagaca tagctgccaa ttatccggg ctt 43 <210> 66 <211> 44 <212> DNA <213> Artificial Sequence <400> 66 gacggtgaga aataacatca acatgggtaa aaaatccttt cgta 44 <210> 67 <211> 44 <212> DNA <213> Artificial Sequence <400> 67 tacgaaagga ttttttaccc atgttgatgt tatttctcac cgtc 44 <210> 68 <211> 48 <212> DNA <213> Artificial Sequence <400> 68 tgcctctttt agccttttca gagggtcacc gcgaaataat caaatgaa 48 <210> 69 <211> 48 <212> DNA <213> Artificial Sequence <400> 69 ttcatttgat tatttcgcgg tgaccctctg aaaaggctaa aagaggca 48 <210> 70 <211> 51 <212> DNA <213> Artificial Sequence <400> 70 gttgtaaaac gacggccagt gccaagctta aaaggcagtc cagtacaccc t 51
Claims
1. Application of inactivation of cg1507-cg1524 in increasing threonine production in Corynebacterium glutamicum; The Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032; Alternatively, the *Corynebacterium glutamicum* is obtained by enhancing the activity of any one or more enzymes in *Corynebacterium glutamicum* ATCC13032 and / or removing feedback inhibition, and / or reducing or eliminating the activity of any one or more enzymes in *Corynebacterium glutamicum* ATCC13032: (1) Aspartate kinase; (2) Aspartate semialdehyde dehydrogenase; (3) Homoserine dehydrogenase; (4) Homoserine kinase; (5) Threonine efflux protein; (6) Pyruvate carboxylase; (7) Glucose-6-phosphate dehydrogenase; (8) Diaminopimelic acid dehydrogenase; (9) 4-Hydroxytetrahydropyridinedicarboxylic acid synthase; (10) Citrate synthase.
2. The application according to claim 1, characterized in that, The activity enhancement is achieved by selecting from the following 1) to 6), or an optional combination thereof: 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme; 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome; 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome; 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme; 5) Enhancement is achieved by altering the amino acid sequence of the enzyme; 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme.
3. The application according to claim 1, characterized in that, The reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out an endogenous gene encoding the enzyme.
4. Any of the following applications of modified Corynebacterium glutamicum: (1) Application in the fermentation production of threonine; (2) Application in increasing threonine production; in, Compared to the unmodified *Corynebacterium glutamicum*, the modified *Corynebacterium glutamicum* showed inactivation of cg1507-cg1524. The modified Corynebacterium glutamicum has an enhanced threonine production capacity compared to the unmodified Corynebacterium glutamicum. The unmodified Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032; Alternatively, the unmodified Corynebacterium glutamicum is obtained by enhancing the activity of any one or more of the following enzymes (1) to (7) and / or removing feedback inhibition in Corynebacterium glutamicum ATCC13032, and / or reducing or losing the activity of any one or more of the following enzymes (8) to (10): (1) Aspartate kinase; (2) Aspartate semialdehyde dehydrogenase; (3) Homoserine dehydrogenase; (4) Homoserine kinase; (5) Threonine efflux protein; (6) Pyruvate carboxylase; (7) Glucose-6-phosphate dehydrogenase; (8) Diaminopimelic acid dehydrogenase; (9) 4-Hydroxytetrahydropyridinedicarboxylic acid synthase; (10) Citrate synthase.
5. The application according to claim 4, characterized in that, The activity enhancement is achieved by selecting from the following 1) to 6), or an optional combination thereof: 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme; 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome; 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome; 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme; 5) Enhancement is achieved by altering the amino acid sequence of the enzyme; 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme.
6. The application according to claim 4, characterized in that, The reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out an endogenous gene encoding the enzyme.
7. A method for producing threonine by fermentation, characterized in that, The process includes culturing modified Corynebacterium glutamicum and isolating threonine from the culture; The modified Corynebacterium glutamicum is as described in any one of claims 4 to 6.