Recombinant microorganisms and methods for their construction and use

Abstract

The present invention relates to the field of microbial engineering technology, specifically to recombinant microorganisms and the construction and use thereof. The present invention constructs a strain with inactivated phosphate acetyltransferase and uses it to produce threonine, thereby significantly improving the threonine production ability of the strain, and the threonine production of the strain is significantly improved compared with that of the unmodified strain. By combining with weakening or inactivating the expression of acetate kinase, HTH transcription factor, etc., and enhancing the activity of pyruvate carboxylase and enzymes in the pathway related to threonine synthesis, the threonine production is further improved. The above modification can be used for fermentation production of threonine and has good use value.

Description

[Technical field]

[0001] The present invention relates to the field of microbial engineering, specifically to recombinant microorganisms and methods for their construction and use. [Background technology]

[0002] Threonine (β-hydroxy-α-aminobutyric acid) has the molecular formula C4H9NO3 and a relative molecular mass of 119.12. It is an essential amino acid and is mainly used in medicines, chemicals, food supplements, feed additives, etc.

[0003] Corynebacterium glutamicum is an important producer of threonine fermentation. In Corynebacterium glutamicum, oxaloacetate is converted to threonine through five catalytic steps catalyzed by aspartokinase (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). Currently, reports on threonine production using Corynebacterium glutamicum have focused mainly on its synthetic pathway, but there are few reports on metabolic engineering modifications to the precursor supply of threonine and the overflow metabolism of pyruvate in the threonine synthesis process. Summary of the Invention

[0004] The present invention aims to provide a recombinant microorganism capable of producing threonine, which has improved threonine production capacity by inactivating phosphate acetyltransferase. The present invention also provides a method for constructing and using the recombinant microorganism.

[0005] Currently, metabolic engineering modifications for threonine synthesis using Corynebacterium glutamicum are mainly focused on the synthetic pathway from oxaloacetate to threonine. As an important intermediate metabolic product in the microbial metabolic network, pyruvate mainly enters the tricarboxylic acid cycle to provide energy and precursor substances for the growth of the bacterial cell, but when the upstream and downstream metabolic pathways become unbalanced, it causes overflow metabolism of pyruvate, which results in waste of pyruvate. The precursor of threonine is oxaloacetate, which can be produced by catalyzing pyruvate with pyruvate carboxylase, or by pyruvate entering the tricarboxylic acid cycle and undergoing a series of enzyme-catalyzed reactions. In the course of research into threonine metabolic engineering, the present invention discovered that by reducing the overflow metabolism of pyruvate, the flow rate of pyruvate to oxaloacetate, the precursor of threonine synthesis, can be improved, and thus the synthesis of threonine can be promoted. Compared with other methods for reducing pyruvate overflow metabolism, reducing or eliminating the activity of phosphate acetyltransferase is obviously more effective, and reducing or eliminating the activity of phosphate acetyltransferase can effectively reduce pyruvate overflow metabolism, improve the supply of threonine synthesis precursors, and significantly improve the threonine synthesis ability of the strain.

[0006] In order to achieve the object of the present invention, in a first aspect, the present invention provides a modified Corynebacterium microorganism having reduced or lost phosphate acetyltransferase activity compared to an unmodified microorganism and having enhanced threonine production ability compared to an unmodified microorganism.

[0007] Preferably, the reference sequence number on NCBI for the phosphate acetyltransferase is NP_601948.1, or an amino acid sequence having 90% identity thereto and having an equivalent function.

[0008] Furthermore, the reduction or loss of phosphate acetyltransferase activity in the microorganism can be achieved by reducing the expression of a gene encoding phosphate acetyltransferase or by knocking out a gene encoding an endogenous phosphate acetyltransferase.

[0009] Through methods of mutagenesis, site-directed mutagenesis or homologous recombination, expression of the gene encoding the phosphate acetyltransferase can be reduced or the gene encoding the endogenous phosphate acetyltransferase can be knocked out.

[0010] Furthermore, the activity of pyruvate carboxylase in the microorganism is enhanced and / or feedback inhibition is relieved, as compared to an unmodified microorganism.

[0011] Preferably, the reference sequence number on NCBI for pyruvate carboxylase is WP_011013816.1, or an amino acid sequence having 90% identity thereto and having an equivalent function.

[0012] Furthermore, the microorganism has reduced or lost activity of any one or two of the following enzymes (1) to (2) as compared to an unmodified microorganism: (1) Acetate kinase (2) HTH transcription factor

[0013] Preferably, the reference sequence numbers on NCBI for acetate kinase and HTH transcription factor are WP_003862874.1 and WP_003859703.1, respectively, or amino acid sequences having 90% identity thereto and equivalent functions.

[0014] Furthermore, the microorganism has enhanced activity of an enzyme involved in a threonine synthesis pathway in the body and / or has feedback inhibition relieved, compared to an unmodified microorganism, wherein the enzyme involved in the threonine synthesis pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, and threonine synthase.

[0015] Preferably, the reference sequence numbers on NCBI for aspartokinase, homoserine dehydrogenase, and threonine synthase are WP_003855724.1, WP_003855724.1, and WP_011014964.1, respectively, or amino acid sequences having 90% identity thereto and equivalent functions.

[0016] Preferably, the microorganism is one of the following: 1 )~( 4 ) ( 1 ) A microorganism in which the activity of phosphate acetyltransferase is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase are enhanced and / or feedback inhibition is eliminated. ( 2 ) A microorganism in which the activity of phosphate acetyltransferase and / or acetate kinase is reduced or lost, and the activity of aspartokinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase is enhanced and / or feedback inhibition is eliminated. ( 3 ) A microorganism in which the activity of phosphate acetyltransferase and / or HTH transcription factor is reduced or lost, and the activity of aspartokinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase is enhanced and / or feedback inhibition is released. ( 4A microorganism in which at least one of the activities of phosphate acetyltransferase, acetate kinase, and HTH transcription factor is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, threonine synthase, and / or pyruvate carboxylase are enhanced and / or feedback inhibition is eliminated.

[0017] The enhancement of the enzyme activity can be achieved by the following 1) to 6) or any combination thereof. 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome. 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome. 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme. 5) Enhancing the enzyme by modifying its amino acid sequence 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

[0018] Preferably, the enhancement of the activity of an enzyme is achieved by replacing the original promoter of the gene encoding the enzyme with a stronger promoter with stronger activity and / or by mutating the start codon of the gene to ATG.

[0019] Here, the strong promoter comprises Psod or PcspB.

[0020] The nucleotide sequences of the promoters Psod and PcspB are shown in SEQ ID NOs. 1 and 2, respectively.

[0021] Preferably, the enhancement of the activities of pyruvate carboxylase, aspartokinase and threonine synthase is achieved by replacing their original promoters with the Psod promoter.

[0022] Enhancement of the activity of homoserine dehydrogenase is achieved by replacing its original promoter with the PcspB promoter.

[0023] The release of feedback inhibition as described above is preferably achieved by the following mutations:

[0024] Relief of feedback inhibition of pyruvate carboxylase is achieved by mutating the gene encoding pyruvate carboxylase so as to generate a P458S mutation in the pyruvate carboxylase encoded thereby.

[0025] Relief of feedback inhibition of aspartokinase is achieved by mutating the gene encoding aspartokinase to generate a T311I mutation in the encoded aspartokinase.

[0026] The release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the gene encoding homoserine dehydrogenase so as to generate a G378E mutation in homoserine dehydrogenase.

[0027] Preferably, the microorganism according to the present invention is Corynebacterium glutamicum, including ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), with Corynebacterium glutamicum ATCC 13032 being more preferred.

[0028] In a second aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: A. Attenuating a gene encoding phosphate acetyltransferase in a Corynebacterium having amino acid production ability (including knocking out or reducing the expression of the phosphate acetyltransferase-encoding gene) to obtain a gene-attenuated strain, and / or B. enhancing the activity of pyruvate carboxylase and / or relieving its feedback inhibition, and / or C. Reducing or eliminating the activity of any one or more of the following enzymes (1)-(2): (1) Acetate kinase (2) HTH transcription factor and / or D. enhancing the activity of an enzyme involved in the threonine synthesis pathway selected from at least one of aspartokinase, homoserine dehydrogenase, and threonine synthase and / or relieving the feedback inhibition thereof; The method for enhancing the activity is selected from the following 1) to 6), or any combination thereof: 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome. 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome. 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme. 5) Enhancing the enzyme by modifying its amino acid sequence 6) Enhancement by modifying the nucleotide sequence encoding the enzyme and / or the reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or by knocking out the gene encoding the enzyme; Methods for constructing threonine-producing strains are provided.

[0029] In a third aspect, the present invention provides a method for producing a composition comprising the steps of: a) culturing the microorganism to obtain a culture of the microorganism; b) recovering the produced threonine from the culture obtained in step a).

[0030] In a fourth aspect, the present invention provides the use of reduced or lost enzymatic activity of phosphate acetyltransferase for the fermentative production of threonine or for improving the fermentative production yield of threonine.

[0031] Preferably, the reduction or loss of phosphate acetyltransferase activity in the microorganism is achieved by reducing the expression of a gene encoding phosphate acetyltransferase or by knocking out a gene encoding an endogenous phosphate acetyltransferase.

[0032] Furthermore, the fermentation production of threonine is improved by inactivating phosphate acetyltransferase in Corynebacterium having amino acid production ability.

[0033] Preferably, the Corynebacterium according to the present invention is Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), with Corynebacterium glutamicum ATCC 13032 being more preferred.

[0034] In a fifth aspect, the present invention provides use of the modified Corynebacterium microorganism or a threonine-producing strain constructed by the method described above for the fermentative production of threonine or for improving the fermentative production yield of threonine.

[0035] The modification methods for the above strains, including gene strengthening and weakening, are all known to those skilled in the art, and can be seen in the following: Man Zaiwei, Systematic Pathway Engineering Modification of Corynebacterium crenatum with High L-Arginine Production [D], Jiangnan University, 2016; Cui Yi, Metabolic Engineering Modification of Corynebacterium glutamicum for L-Leucine Production [D], Tianjin University of Science and Technology; Xu Guodong, Construction of a Strain Producing L-Isoleucine and Optimization of Fermentation Conditions, Tianjin University of Science and Technology, 2015.

[0036] The present invention has the following beneficial effects. By inactivating phosphate acetyltransferase, the present invention reduces the overflow metabolism of pyruvate, reduces the production of overflow metabolites, and reduces the waste of pyruvate, allowing more pyruvate to flow in the direction of oxaloacetate, a precursor for threonine synthesis, thereby significantly improving the threonine production ability of the strain, and the threonine production of the strain is significantly improved compared to that of a non-modified strain. By combining with weakening or inactivating the expression of acetate kinase, HTH transcriptional regulator, etc., and enhancing the activity of pyruvate carboxylase and enzymes in the pathway related to threonine synthesis, the production of threonine is further improved. The above modification can be used for fermentation production of threonine and has good use value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. Information on the proteins and their encoding genes in the following examples is as follows: Phosphate acetyltransferase: coding gene name pta, NCBI number: cg3048, Cgl2753, NCgl2657. Aspartokinase: coding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247. Homoserine dehydrogenase: coding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136. Threonine synthase: coding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139. Pyruvate carboxylase: coding gene name pyc, NCBI number: cg0791, Cgl0689, NCgl0659. Acetate kinase: coding gene name ackA, NCBI number: cg3047, Cgl2752, NCgl2656. HTH transcription factor RamB: coding gene name ramB, NCBI number: cg0444, Cgl0369, NCgl0358. EXAMPLES

[0038] Example 1 Construction of a plasmid for modifying the genome of a strain

[0039] 1. Aspartokinase Expression Enhancement Plasmid pK18mobsacB-P sod -lysC g1a-T311I Construction Using the ATCC13032 genome as a template, PCR amplification was performed with the P21 / P22 primer set to obtain the upstream homologous arm up, PCR amplification was performed with the P23 / P24 primer set to obtain the promoter fragment Psod, and PCR amplification was performed with the P25 / P26 primer set to obtain lysC g1a-T311I The downstream homologous arm dn was obtained by PCR amplification using the P27 / P28 primer set. The up and Psod fragments were used as templates to perform fusion PCR using the P21 / P24 primer set to obtain the up-Psod fragment. g1a-T311I Fusion PCR was performed using dn as a template and the P21 / P28 primer set to obtain the full-length fragment up-Psod-lysC g1a-T311I -dn was obtained. pK18mobsacB was digested with BamHI / HindIII. The digested up-Psod-lysC g1a-T311I pK18mobsacB-dn and pK18mobsacB were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I was obtained.

[0040] 2. Homoserine dehydrogenase expression-enhancing plasmid pK18mobsacB-P cspB -hom G378E Construction The plasmid construction method was as described above in 1, and the primers used were P29, P30, P31, P32, P33, P34, P35, and P36.

[0041] 3. Threonine synthase expression enhancing plasmid pk18mobsacB-Psod-thrC g1a Construction The plasmid construction method was as described above in 1, and the primers used were P37, P38, P39, P40, P41, and P42.

[0042] 4. Pyruvate carboxylase expression enhancing plasmid pK18mobsacB-Psod-pyc P458S Construction The plasmid construction method was as described above in 1, and the primers used were P13, P14, P15, P16, P17, P18, P19, and P20.

[0043] 5. Construction of phosphate acetyltransferase inactivating plasmid pK18mobsacB-△pta Using the ATCC13032 genome as a template, PCR amplification was performed with the P67 / P68 primer set to obtain the upstream homologous arm up, and PCR amplification was performed with the P69 / P70 primer set to obtain the downstream homologous arm dn. Using up and dn as templates, fusion PCR was performed with the P67 / P70 primer set to obtain the fragment up-dn. pK18mobsacB was cleaved with BamHI / HindIII. The cleaved up-dn and pK18mobsacB were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pk18mobsacB-△pta.

[0044] 6. Construction of acetate kinase inactivation plasmid pk18mobsacB-△ackA The plasmid construction method was as described above in 5. The primers used were P165, P166, P167, and P168.

[0045] 7. Construction of the phosphate acetyltransferase and acetate kinase co-inactivating plasmid pk18mobsacB-△pta-△ackA The plasmid construction method was as described above in 5. The primers used were pta-ackAup1, pta-ackAup2q, pta-ackAdn1q, and P168.

[0046] 8. HTH transcription factor RamB weakened plasmid pk18mobsacB-ramB a1g Construction The plasmid construction method was as described above in 5. The primers used were P115, P116, P117, and P118.

[0047] 9. Construction of the HTH transcription factor inactivation plasmid pk18mobsacB-△ramB The plasmid construction method was as described above in 5. The primers used were P119, P120, P121, and P122.

[0048] The primers used in the above plasmid construction process are shown in Table 1.

[0049] [Table 1]

[0050] Example 2 Construction of genome modified strain

[0051] 1. Construction of an aspartokinase enhanced expression strain ATCC13032 competent cells were prepared according to the classical method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). The recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311Itransformed the competent cells by electroporation and selected transformants in which the target gene had been integrated into the chromosome by homology in a selective medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in a normal liquid brain-heart infusion medium, cultured at 30°C and shaken at 220 rpm in a rotary shaker. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome by gene exchange. The culture was serially diluted (10 -2 From 10 -4 The strain was serially diluted to 1000 μg / ml, and the dilutions were spread onto standard solid brain heart infusion medium containing 10% sucrose and incubated at 33°C for 48 h. The genome of the colonies grown on the sucrose medium did not carry the integrated vector sequence. The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis, and the target mutant strain obtained was named SMCT121. Compared to the ATCC13032 strain, this strain has a mutation in the start codon of the lysC gene from GTG to ATG, the 311th amino acid in the encoded amino acid, threonine, was mutated to isoleucine, and the promoter of the lysC gene was replaced with the Psod promoter.

[0052] 2. Construction of homoserine dehydrogenase expression-enhanced strain The strain construction method was as described above in 1. Starting from SMCT121, the plasmid pK18mobsacB-P cspB -hom G378E was introduced into this strain to enhance homoserine dehydrogenase expression, and the resulting modified strain was named SMCT122. Compared to the starting strain SMCT121, this strain has a G378E mutation in the protein encoded by the hom gene, and the hom gene promoter has been replaced with the PcspB promoter.

[0053] 3. Construction of Threonine Synthase Overexpression Strain The strain construction method was as described above in 1. Starting from SMCT122, the plasmid pk18mobsacB-Psod-thrC wasg1a was introduced into this strain to enhance threonine synthase expression, and the resulting modified strain was named SMCT123. Compared to the starting strain SMCT122, this strain has a mutated start codon of the thrC gene from GTG to ATG, and the promoter of the thrC gene has been replaced with the Psod promoter.

[0054] 4. Construction of Pyruvate Carboxylase Enhanced Expression Strain The strain construction method was as described above in 1. Starting from SMCT123, the plasmid pK18mobsacB-Psod-pyc P458S was introduced into this strain to enhance pyruvate carboxylase expression, and the resulting modified strain was named SMCT124. Compared to the starting strain SMCT123, this strain has a pyc gene mutation that results in a P458S mutation in the encoded protein, and the pyc gene promoter has been replaced with the Psod promoter.

[0055] 5. Construction of Acetate Kinase Inactivated Strain The strain was constructed as described above in 1. Starting from SMCT124, the plasmid pk18mobsacB-△ackA was introduced into the strain to inactivate acetate kinase, and the resulting modified strain was named SMCT125. Compared to the starting strain SMCT124, this strain has ackA knocked out.

[0056] 6. Construction of mutants with weakened HTH transcription factors The strain construction method was as described above in 1. Starting from SMCT124, the plasmid pk18mobsacB-ramB a1g was introduced into this strain to weaken the ramB gene, and the resulting mutant strain was named SMCT126, in which the start codon of the ramB gene was mutated to GTG compared to the starting strain SMCT124.

[0057] 7. Construction of HTH transcription factor inactivated mutant The strains were constructed as described above in 1. Starting from SMCT124 and SMCT125, pk18mobsacB-ΔramB was introduced into the starting strains to inactivate ramB, and the resulting modified strains were named SMCT127 and SMCT128. These two strains have ramB knocked out compared to the corresponding starting strains.

[0058] 8. Construction of Phosphate Acetyltransferase Inactivated Strain The strains were constructed as described above in 1. Starting strains were SMCT124, SMCT125, SMCT126, SMCT127, and SMCT128, which were modified to inactivate phosphate acetyltransferase with pk18mobsacB-△pta or pk18mobsacB-△pta-△ackA plasmids, and the resulting modified strains were named SMCT129, SMCT130, SMCT131, SMCT132, and SMCT133. These strains have the pta gene knocked out compared to the corresponding starting strains.

[0059] The genotype information of the obtained strains is shown in Table 2.

[0060] [Table 2]

[0061] Example 3 Shake flask fermentation validation of strains For each strain constructed in Example 2, a shake flask fermentation test was carried out, specifically as follows.

[0062] 1. Culture Medium Seed activation medium: BHI 3.7%, agar 2%, pH 7. Seed medium: peptone 5 / L, yeast extract 5g / L, sodium chloride 10g / L, ammonium sulfate 16g / L, urea 8g / L, potassium dihydrogen phosphate 10.4g / L, dipotassium hydrogen phosphate 21.4g / L, biotin 5mg / L, magnesium sulfate 3g / L, glucose 50g / L, pH 7.2. Fermentation medium: Corn steep liquor 50mL / L, glucose 30g / L, ammonium sulfate 4g / L, MOPS 30g / L, potassium dihydrogen phosphate 10g / L, urea 20g / L, biotin 10mg / L, magnesium sulfate 6g / L, ferrous sulfate 1g / L, VB1·HCl 40mg / L, calcium pantothenate 50mg / L, nicotinamide 40mg / L, manganese sulfate 1g / L, zinc sulfate 20mg / L, copper sulfate 20mg / L, pH 7.2.

[0063] 2. L-Threonine production by shake flask fermentation of engineered fungi (1) Seed culture: A loopful of slant culture seed of the strains SMCT121, SMCT122, SMCT123, SMCT124, SMCT125, SMCT126, SMCT127, SMCT128, SMCT129, SMCT130, SMCT131, SMCT132 and SMCT133 was taken and inoculated into a 500 mL Erlenmeyer flask containing 20 mL of seed medium, and cultured with shaking at 30°C and 220 r / min for 16 hours to obtain a seed solution. (2) Fermentation culture: 2 mL of the seed solution was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and cultured at 33°C and 220 r / min for 24 hours with shaking to obtain a fermentation liquid. (3) 1 mL of the fermentation liquid was taken and centrifuged (12,000 rpm, 2 min) to recover the supernatant, and L-threonine in the fermentation liquid of the engineered bacteria and the control bacteria was measured by HPLC.

[0064] The fermentation results of the strains with rudimentary threonine synthesis ability are shown in Table 3.

[0065] [Table 3]

[0066] As can be seen from Table 3, in the strain in which aspartokinase was modified from the wild-type strain ATCC13032, the amount of threonine produced was initially accumulated, and with the enhancement of the expression of enzymes in the threonine synthesis pathway (homoserine dehydrogenase, threonine synthase), the amount of threonine produced was further improved, and threonine could be accumulated at 3.0 g / L.

[0067] Furthermore, the threonine accumulation status in the strains in which the phosphate acetyltransferase-encoding gene was modified is shown in Table 4.

[0068] [Table 4]

[0069] As can be seen from Table 4, the strains that have optimized carbon metabolic flow and reduced carbon loss by inactivating the pta gene have different levels of threonine production, of which SMCT133 is 30% higher than the control SMCT128. This shows that after the threonine terminal synthesis pathway is penetrated, the overflow metabolic flux is reduced, and more carbon flows into threonine synthesis, which can significantly improve the threonine production ability of the strain. In addition, the inactivation of the pta gene and the modification targets such as pyruvate carboxylase coding gene pyc, acetate kinase coding gene ackA, HTH transcription factor coding gene ramB, aspartokinase coding gene lysC, homoserine dehydrogenase coding gene hom and threonine synthase coding gene thrC can all be modified to further improve threonine production.

[0070] Although the present invention has been described in detail above using general descriptions and specific embodiments, it is clear to those skilled in the art that various modifications or improvements based on the present invention are possible. Therefore, any of these modifications or improvements made without departing from the spirit of the present invention are included in the scope of the claims of the present invention.

Claims

1. A modified Corynebacterium microorganism, characterized in that the activity of phosphate acetyltransferase is reduced or eliminated compared to that of an unmodified microorganism, and that the ability to produce threonine is enhanced compared to that of an unmodified microorganism.

2. The microorganism described in claim 1, characterized in that the reduction or loss of phosphate acetyltransferase activity within the microorganism is achieved by reducing the expression of a gene encoding the phosphate acetyltransferase or by knocking out a gene encoding an endogenous phosphate acetyltransferase.

3. The microorganism described in claim 2, characterized in that the expression of a gene encoding a phosphate acetyltransferase is reduced or the gene encoding an endogenous phosphate acetyltransferase is knocked out by mutagenesis, site-specific mutation, or homologous recombination.

4. 2. The microorganism according to claim 1, wherein the activity of pyruvate carboxylase in the microorganism is enhanced and / or feedback inhibition is relieved compared to that in an unmodified microorganism.

5. The microorganism according to claim 1 or 4, characterized in that the activity of any one or two of the following enzymes (1) to (2) is reduced or lost compared to an unmodified microorganism: (1) Acetate kinase (2) HTH transcription factor

6. The microorganism has enhanced activity of an enzyme involved in the threonine synthesis pathway in its body and / or feedback inhibition is relieved compared to an unmodified microorganism, 2. The microorganism according to claim 1, wherein the enzyme involved in the threonine synthesis pathway is selected from at least one of aspartokinase, homoserine dehydrogenase, and threonine synthase.

7. The microorganism is any one of the following (1) to (4): (1) A microorganism in which the activity of phosphate acetyltransferase is reduced or lost, and the activities of aspartokinase, homoserine dehydrogenase, threonine synthase, and / or pyruvate carboxylase are enhanced and / or feedback inhibition is eliminated. (2) A microorganism in which the activity of phosphate acetyltransferase and / or acetate kinase is reduced or lost, and the activity of aspartokinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase is enhanced and / or feedback inhibition is eliminated. (3) A microorganism in which the activity of phosphate acetyltransferase and / or HTH transcription factor is reduced or lost, and the activity of aspartokinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase is enhanced and / or feedback inhibition is deactivated. (4) A microorganism in which the activity of at least one of phosphate acetyltransferase, acetate kinase, and HTH transcription factor is reduced or lost, and the activity of aspartokinase, homoserine dehydrogenase, threonine synthase, and / or pyruvate carboxylase is enhanced and / or feedback inhibition is desensitized. The microorganism according to claim 6, wherein the activity of the enzyme is preferably enhanced by the following 1) to 6), or any combination thereof: 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme 5) Enhancement by modifying the amino acid sequence of the enzyme 6) Enhancement by modifying the nucleotide sequence encoding the enzyme

8. 2. The microorganism according to claim 1, wherein the microorganism is Corynebacterium glutamicum.

9. A. Attenuating a gene encoding phosphate acetyltransferase in a Corynebacterium capable of producing amino acids (including knocking out or reducing the expression of the phosphate acetyltransferase-encoding gene) to obtain a gene-attenuated strain; and / or B. Increasing the activity of pyruvate carboxylase and / or relieving its feedback inhibition, and / or C. Reducing or eliminating the activity of one or more of the following enzymes (1)-(2): (1) Acetate kinase (2) HTH transcription factor and / or D. It comprises enhancing the activity of an enzyme involved in the threonine synthesis pathway selected from at least one of aspartokinase, homoserine dehydrogenase, and threonine synthase and / or relieving the feedback inhibition thereof; The method for enhancing the activity is selected from the following 1) to 6), or any combination thereof: 1) Enhancement by introducing a plasmid carrying a gene encoding the enzyme 2) Enhancement by increasing the copy number of the gene encoding the enzyme in the chromosome 3) Enhancement by modifying the promoter sequence of the gene encoding the enzyme in the chromosome 4) Enhancement by operably linking a strong promoter to the gene encoding the enzyme 5) Enhancement by modifying the amino acid sequence of the enzyme 6) A method for constructing a threonine-producing strain, characterized in that the enhancement and / or reduction or loss of the activity is achieved by modifying the nucleotide sequence encoding the enzyme, or by reducing the expression of the gene encoding the enzyme or by knocking out the gene encoding the enzyme.

10. 10. The microorganism according to claim 9, wherein the Corynebacterium is Corynebacterium glutamicum.

11. A method for producing threonine, comprising the steps of: a) culturing the microorganism according to claim 1 or the threonine-producing strain constructed by the method according to claim 9 or 10, and obtaining a culture of the microorganism or the threonine-producing strain; b) recovering the produced threonine from the culture obtained in step a).