A recombinant microorganism, its construction method and application

Abstract

This invention relates to the field of microbial engineering technology, specifically to a recombinant microorganism, its construction method, and its application. This invention significantly improves the threonine production capacity of a strain by constructing a phosphorylacetyltransferase-inactivated strain and applying it to threonine production. The threonine yield of this strain is significantly higher than that of the unmodified strain. Furthermore, by weakening or inactivating the expression of acetate kinase, HTH transcription factors, etc., and enhancing the activity of pyruvate carboxylase and enzymes related to threonine synthesis pathways, the threonine yield is further increased. The above modifications can be used in the fermentation production of threonine and have good application value.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, specifically to a recombinant microorganism, its construction method, and its application. 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 medicine, chemical reagents, food fortifiers, and feed additives.

[0003] *Corynebacterium glutamicum* is an important producer of threonine through fermentation. In *Corynebacterium glutamicum*, oxaloacetate is converted to threonine via a five-step catalytic reaction involving aspartate kinase (lysC-encoded), aspartate semialdehyde dehydrogenase (asd-encoded), homoserine dehydrogenase (hom-encoded), homoserine kinase (thrB-encoded), and threonine synthase (thrC-encoded). Current reports on threonine production using *Corynebacterium glutamicum* mainly focus on its synthetic pathway; however, there are very few reports on metabolic engineering modifications related to threonine precursor supply and the metabolic spillover of pyruvate during threonine synthesis. Summary of the Invention

[0004] The purpose of this invention is to enhance the ability of a strain to produce threonine by inactivating phosphoacetyltransferase, thereby providing a recombinant microorganism for threonine production. This invention also provides a method for constructing and applying this recombinant microorganism.

[0005] Current metabolic engineering studies on threonine synthesis using Corynebacterium glutamicum mainly focus on the oxaloacetate-to-threonine synthesis pathway. Pyruvate, an important intermediate metabolite in microbial metabolic networks, primarily enters the tricarboxylic acid cycle to provide energy and precursors for bacterial growth. However, imbalances in upstream and downstream metabolic pathways can lead to pyruvate metabolic overflow, resulting in pyruvate waste. Although oxaloacetate is a precursor to threonine, it can be converted from pyruvate via pyruvate carboxylase catalysis, or it can be converted from pyruvate via a series of enzymatic reactions in the tricarboxylic acid cycle. This invention, in its research on threonine metabolic engineering, discovered that reducing pyruvate metabolic overflow can increase the flow of pyruvate to oxaloacetate, the precursor for threonine synthesis, thereby promoting threonine synthesis. Compared to other methods of reducing pyruvate metabolic overflow, reducing or eliminating phosphoacetyltransferase activity is significantly more effective. By reducing or eliminating phosphoacetyltransferase activity, pyruvate metabolic overflow can be effectively reduced, increasing the supply of threonine precursors and thus significantly improving the strain's threonine synthesis capacity.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a modified Corynebacterium microorganism, wherein the microorganism has reduced or lost phosphorylacetyltransferase activity compared to the unmodified microorganism, and the microorganism has enhanced threonine production capacity compared to the unmodified microorganism.

[0007] Preferably, the reference sequence number of phosphoacetyltransferase on NCBI is NP_601948.1, or an amino acid sequence that is 90% similar to it and has the same function.

[0008] Furthermore, the reduction or loss of phosphorylacetyltransferase activity in the microorganism is achieved by reducing the expression of the gene encoding phosphorylacetyltransferase or knocking out the endogenous gene encoding phosphorylacetyltransferase.

[0009] Mutagenesis, site-directed mutagenesis, or homologous recombination can be used to reduce the expression of genes encoding phosphoacetyltransferase or to knock out endogenous genes encoding phosphoacetyltransferase.

[0010] Furthermore, compared to unmodified microorganisms, the microorganisms exhibit enhanced pyruvate carboxylase activity and / or relief of feedback inhibition.

[0011] Preferably, the reference sequence number of pyruvate carboxylase on NCBI is WP_011013816.1, or an amino acid sequence that is 90% similar to it and has the same function.

[0012] Furthermore, compared to unmodified microorganisms, the activity of any one or two of the following enzymes (1) to (2) is reduced or lost:

[0013] (1) Acetylkinase;

[0014] (2) HTH transcriptional regulatory factors.

[0015] Preferably, the reference sequence numbers of acetate kinase and HTH transcription regulatory factor on NCBI are WP_003862874.1 and WP_003859703.1, respectively, or amino acid sequences that are 90% similar to them and have the same function.

[0016] Furthermore, compared to unmodified microorganisms, the microorganism exhibits enhanced activity and / or relief of feedback inhibition of enzymes related to the threonine synthesis pathway; wherein the enzymes related to the threonine synthesis pathway are selected from at least one of aspartate kinase, homoserine dehydrogenase, and threonine synthase.

[0017] Preferably, the reference sequence numbers of aspartate kinase, homoserine dehydrogenase, and threonine synthase on NCBI are WP_003855724.1, WP_003855724.1, and WP_011014964.1, respectively, or amino acid sequences that are 90% similar to them and have equivalent functions.

[0018] Preferably, the microorganism is any one of the following ① to ④:

[0019] ① Microorganisms with reduced or lost phosphoacetyltransferase activity and enhanced and / or relieved feedback inhibition activities of aspartate kinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase.

[0020] ② Microorganisms with reduced or lost phosphoacetyltransferase and / or acetate kinase activity and enhanced aspartate kinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase activity and / or relief of feedback inhibition.

[0021] ③ Microorganisms with reduced or lost activity of phosphorylated acetyltransferase and / or HTH transcriptional regulatory factors, and enhanced and / or relieved feedback inhibition of aspartate kinase, homoserine dehydrogenase, threonine synthase and / or pyruvate carboxylase.

[0022] ④ Microorganisms with reduced or lost activity of at least one of phosphorylated acetyltransferase, acetate kinase, and HTH transcriptional regulatory factor, and enhanced and / or relieved activity of aspartate kinase, homoserine dehydrogenase, threonine synthase, and / or pyruvate carboxylase.

[0023] The enhanced activity of the above enzymes is achieved by selecting from 1) to 6) below, or an optional combination thereof:

[0024] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;

[0025] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;

[0026] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

[0027] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;

[0028] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme;

[0029] 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme.

[0030] Preferably, the enzyme activity is enhanced by replacing the original promoter of the gene encoding the enzyme with a stronger promoter, and / or by mutating the start codon of the gene to ATG.

[0031] The strong promoters include Psod or PcspB.

[0032] The nucleotide sequences of promoters Psod and PcspB are shown in SEQ ID NO.1 and 2, respectively.

[0033] Preferably, the enhanced activity of pyruvate carboxylase, aspartate kinase, and threonine synthase is achieved by replacing their original promoters with the Psod promoter;

[0034] The enhanced activity of homoserine dehydrogenase is achieved by replacing its original promoter with the PcspB promoter.

[0035] The above-mentioned relief of feedback inhibition is preferably achieved through the following mutation: the relief of feedback inhibition of pyruvate carboxylase is achieved by mutating the gene encoding pyruvate carboxylase, so that the encoded pyruvate carboxylase undergoes a P458S mutation.

[0036] The release of feedback inhibition of aspartate kinase is achieved by mutating the aspartate kinase encoding gene, resulting in a T311I mutation in the encoded aspartate kinase.

[0037] The release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the homoserine dehydrogenase encoding gene, resulting in a G378E mutation in homoserine dehydrogenase.

[0038] Preferably, the microorganism described in this invention is *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.

[0039] Secondly, the present invention provides a method for constructing a threonine-producing strain, the method comprising:

[0040] A. Attenuating the gene encoding phosphorylated acetyltransferase in Corynebacterium species capable of amino acid production to obtain gene-attenuated strains; the attenuation includes knocking out or reducing the expression of the phosphorylated acetyltransferase-encoding gene; and / or

[0041] B. Enhance the activity of pyruvate carboxylase and / or relieve its feedback inhibition; and / or

[0042] C. Reduce the activity of any one or more of the following enzymes (1)-(2) or cause them to lose their enzyme activity:

[0043] (1) Acetylkinase;

[0044] (2) HTH transcriptional regulators; and / or

[0045] D. Enhance the activity of enzymes related to the threonine synthesis pathway and / or relieve their feedback inhibition, wherein the enzymes related to the threonine synthesis pathway are selected from at least one of aspartate kinase, homoserine dehydrogenase, and threonine synthase.

[0046] The pathways for enhancing activity are selected from the following 1) to 6), or any combination thereof:

[0047] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;

[0048] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;

[0049] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

[0050] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;

[0051] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme;

[0052] 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme;

[0053] And / or, the reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out the gene encoding the enzyme.

[0054] Thirdly, the present invention provides a method for producing threonine, the method comprising the following steps:

[0055] a) Cultivate the microorganisms to obtain a culture of the microorganisms;

[0056] b) Collect the resulting threonine from the culture obtained in step a).

[0057] Fourthly, the present invention provides the application of reduced or lost enzyme activity of phosphoacetyltransferase in threonine fermentation production or to increase threonine fermentation yield.

[0058] Preferably, the reduction or loss of phosphorylacetyltransferase activity in the microorganism is achieved by reducing the expression of the gene encoding phosphorylacetyltransferase or knocking out the endogenous gene encoding phosphorylacetyltransferase.

[0059] Furthermore, the fermentation yield of threonine was increased by inactivating phosphoacetyltransferase in Corynebacterium, a bacterium capable of producing amino acids.

[0060] Preferably, the Corynebacterium described in this 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), and more preferably Corynebacterium glutamicum ATCC 13032.

[0061] Fifthly, the present invention provides the application of the modified Corynebacterium genus microorganism or the threonine-producing strain constructed according to the above method in the production of threonine by fermentation or in increasing the yield of threonine by fermentation.

[0062] The aforementioned methods for modifying the strains, including gene enhancement and weakening, are all modification techniques 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 of L-Isoleucine-Producing Strains and Optimization of Fermentation Conditions. Tianjin University of Science and Technology, 2015.

[0063] The beneficial effects of this invention are as follows: By inactivating phosphoacetyltransferase, this invention reduces the metabolic overflow of pyruvate, decreases the production of overflow metabolites, and reduces the waste of pyruvate, allowing more pyruvate to flow to oxaloacetate, the precursor for threonine synthesis. This significantly improves the strain's ability to produce threonine, resulting in a significantly higher threonine yield compared to the unmodified strain. Furthermore, the weakening or inactivation of the expression of acetate kinase, HTH transcription factors, and other enzymes, along with the enhanced activity of pyruvate carboxylase and enzymes related to threonine synthesis pathways, further increases threonine production. The above modifications can be used in the fermentation production of threonine and have good application value. Detailed Implementation

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0065] The information on the proteins and their encoding genes involved in the following examples is as follows:

[0066] Phosphoacetyltransferase, encoding gene name pta, NCBI number: cg3048, Cgl2753, NCgl2657.

[0067] Aspartate kinase, encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.

[0068] Homoserine dehydrogenase, encoded by gene name hom, NCBI ID: cg1337, Cgl1183, NCgl1136.

[0069] Threonine synthase, encoded by gene name thrC, NCBI ID: cg2437, Cgl2220, NCgl2139.

[0070] Pyruvate carboxylase, encoded by gene pyc, NCBI IDs: cg0791, Cgl0689, NCgl0659.

[0071] Acetyl kinase, encoded by gene ackA, NCBI ID: cg3047, Cgl2752, NCgl2656.

[0072] The HTH transcriptional regulator RamB, whose encoded gene name is ramB, has the NCBI numbers cg0444, Cgl0369, and NCgl0358.

[0073] Example 1: Construction of plasmids for strain genome modification

[0074] 1. Aspartate kinase expression enhancement plasmid pK18mobsacB-P sod -lysC g1a-T311I Construction

[0075] 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 dn as a template, fusion PCR was performed to obtain the full-length fragment up-Psod-lysC. g1a-T311I -dn. pK18mobsacB was digested with BamHI / HindIII. The digested up-Psod-lysC g1a-T311I-dn and pK18mobsacB were assembled using a seamless cloning kit, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-P was obtained. sod -lysC g1a-T311I .

[0076] 2. Homoserine dehydrogenase expression enhancement plasmid pK18mobsacB-P cspB -hom G378E Construction

[0077] The plasmid construction method is the same as described in section 1 above, and the primers used are P29, P30, P31, P32, P33, P34, P35, and P36.

[0078] 3. Threonine synthase expression enhancement plasmid pk18mobsacB-Psod-thrC g1a Construction

[0079] The plasmid construction method is the same as described in section 1 above, and the primers used are P37, P38, P39, P40, P41, and P42.

[0080] 4. Pyruvate carboxylase expression enhancement plasmid pK18mobsacB-Psod-pyc P458S Construction

[0081] The plasmid construction method is the same as described in section 1 above, and the primers used are P13, P14, P15, P16, P17, P18, P19, and P20.

[0082] 5. Construction of the phosphoacetyltransferase inactivation plasmid pK18mobsacB-Δpta

[0083] Using the ATCC13032 genome as a template, PCR amplification with primers P67 / P68 yielded the upstream homologous arm 'up', and PCR amplification with primers P69 / P70 yielded the downstream homologous arm 'dn'. Fusion PCR was then performed using primers P67 / P70 with 'up' and 'dn' as templates to obtain the 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-Δpta.

[0084] 6. Construction of the kinase-inactivating plasmid pk18mobsacB-△ackA

[0085] The plasmid construction method is the same as described in section 5 above, and the primers used are P165, P166, P167, and P168.

[0086] 7. Construction of the co-inactivation plasmid pk18mobsacB-△pta-△ackA for phosphoacetyltransferase and acetate kinase

[0087] The plasmid construction method is the same as described in section 5 above, and the primers used are pta-ackAup1, pta-ackAup2q, pta-ackAdn1q, and P168.

[0088] 8. The HTH transcriptional regulator RamB weakens the plasmid pk18mobsacB-ramB. a1g Construction

[0089] The plasmid construction method is the same as described in section 5 above, and the primers used are P115, P116, P117, and P118.

[0090] 9. Construction of the HTH transcription factor inactivation plasmid pk18mobsacB-ΔramB

[0091] The plasmid construction method is the same as described in section 5 above, and the primers used are P119, P120, P121, and P122.

[0092] The primers used in the construction of the above plasmids are shown in Table 1.

[0093] Table 1 Primer sequences

[0094]

[0095]

[0096] Example 2 Construction of Genome-Modified Strains

[0097] 1. Construction of strains with enhanced expression of aspartate kinase

[0098] ATCC13032 competent cells were prepared according to the classic method for *Corynebacterium glutamicum* (C. glutamicum Handbook, Charter 23). Recombinant plasmid pK18mobsacB-P was used. sod -lysC 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 sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT121. Compared to strain ATCC13032, this strain exhibits a mutation in the start codon of the lysC gene, changing from GTG to ATG, and replacing threonine at position 311 with isoleucine. Additionally, the promoter of the lysC gene was replaced with the Psod promoter.

[0099] 2. Construction of strains for enhancing homoserine dehydrogenase expression

[0100] The strain construction method is the same as described in section 1 above, using SMCT121 as the starting strain, and the plasmid pK18mobsacB-P... cspB -hom G378E The strain was introduced into the mutant strain and modified to enhance the expression of homoserine dehydrogenase. The resulting mutant strain was named SMCT122. Compared with the original strain SMCT121, 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.

[0101] 3. Construction of threonine synthase overexpression strains

[0102] The strain construction method is the same as described in section 1 above, using SMCT122 as the starting strain, and the plasmid pk18mobsacB-Psod-thrC is constructed. g1a The strain was introduced into the threonine synthase expression enhancement modification, and the resulting modified strain was named SMCT123. Compared with the original strain SMCT122, the start codon of the thrC gene in this strain was mutated from GTG to ATG, and the promoter of the thrC gene was replaced with the Psod promoter.

[0103] 4. Construction of strains with enhanced expression of pyruvate carboxylase

[0104] The strain construction method is the same as described in section 1 above, using SMCT123 as the starting strain, and the plasmid pK18mobsacB-Psod-pyc is constructed. P458S The strain was introduced into the strain and modified to enhance the expression of pyruvate carboxylase. The resulting modified strain was named SMCT124. Compared with the original strain SMCT123, the pyc gene of this 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.

[0105] 5. Construction of Acetylkinase Inactivating Strains

[0106] The strain construction method is the same as described in 1 above. SMCT124 was used as the starting strain. The plasmid pk18mobsacB-△ackA was introduced into the strain to inactivate acetylkinase. The resulting modified strain was named SMCT125. Compared with the starting strain SMCT124, the ackA of this strain was knocked out.

[0107] 6. Construction of strains with weakened HTH transcription factor regulation

[0108] The strain construction method is the same as described in section 1 above, using SMCT124 as the starting strain, and the plasmid pk18mobsacB-ramB is constructed. a1g The strain was introduced and modified by weakening ramB. The resulting modified strain was named SMCT126. Compared with the original strain SMCT124, the start codon of the ramB gene in this strain was mutated to GTG.

[0109] 7. Construction of strains inactivating HTH transcription factor regulation

[0110] The strain construction method is the same as described in 1 above. SMCT124 and SMCT125 were used as the starting strains, and pk18mobsacB-△ramB was introduced into the starting strains to inactivate ramB. The resulting modified strains were named SMCT127 and SMCT128. Compared with their corresponding starting strains, ramB was knocked out in these two strains.

[0111] 8. Construction of phosphoacetyltransferase-inactivated strains

[0112] The strain construction method is the same as described in 1 above. SMCT124, SMCT125, SMCT126, SMCT127 and SMCT128 were used as the starting strains, and the pk18mobsacB-△pta or pk18mobsacB-△pta-△ackA plasmids were used to modify the strains to inactivate phosphoacetyltransferase. The modified strains were named SMCT129, SMCT130, SMCT131, SMCT132 and SMCT133. Compared with their corresponding starting strains, the pta gene was knocked out in these strains.

[0113] The genotypic information of the strains obtained above is shown in Table 2.

[0114] Table 2. Strain Genotype Information

[0115]

[0116]

[0117] Example 3: Shake-flask fermentation verification of the strain

[0118] The strains constructed in Example 2 were verified by shake-flask fermentation, as follows:

[0119] 1. Culture medium

[0120] Seed activation medium: BHI 3.7%, agar 2%, pH 7.

[0121] 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.

[0122] 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 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.

[0123] 2. Production of L-threonine by shake-flask fermentation with engineered bacteria

[0124] (1) Seed culture: 1 slant seed of strains SMCT121, SMCT122, SMCT123, SMCT124, SMCT125, SMCT126, SMCT127, SMCT128, SMCT129, SMCT130, SMCT131, SMCT132 and SMCT133 was looped into a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium and cultured at 30 °C and 220 r / min for 16 h to obtain seed liquid.

[0125] (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.

[0126] (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.

[0127] The fermentation results of strains that initially demonstrated threonine synthesis capabilities are shown in Table 3.

[0128] Table 3 Fermentation detection results (1)

[0129] strain number <![CDATA[OD 562 ]]> L-Threonine (g / L) SMCT121 23 1.2 SMCT122 23 2.4 SMCT123 23 3.0

[0130] As shown in Table 3, the strain modified with aspartate kinase based on wild strain ATCC13032 initially accumulated threonine. With the enhanced expression of enzymes in the threonine synthesis pathway (homoserine dehydrogenase and threonine synthase), the threonine yield was further increased, and 3.0 g / L of threonine could be accumulated.

[0131] The threonine accumulation of strains that underwent further modification, such as the modification of the phosphorylacetyltransferase encoding gene, is shown in Table 4.

[0132] Table 4 Fermentation test results (2)

[0133] strain number <![CDATA[OD 562 ]]> L-Threonine (g / L) strain number <![CDATA[OD 562 ]]> L-Threonine (g / L) SMCT124 23 3.6 SMCT129 23 4.3 SMCT125 23 4.2 SMCT130 23 5.3 SMCT126 23 4.0 SMCT131 23 5.0 SMCT127 23 4.2 SMCT132 23 5.3 SMCT128 23 5.0 SMCT133 23 6.5

[0134] As shown in Table 4, the threonine production of strains optimized by inactivating the pta gene and reducing carbon loss was improved to varying degrees. Among them, SMCT133 showed a 30% increase compared to the control strain SMCT128. This indicates that after the threonine terminal synthesis pathway is opened, reducing the overflow metabolic flux allows more carbon to flow to threonine synthesis, which can significantly improve the strain's ability to produce threonine. Moreover, different combinations of inactivating the pta gene with modification targets such as the pyruvate carboxylase encoding gene pyc, the acetate kinase encoding gene ackA, the HTH transcription factor encoding gene ramB, the aspartate kinase encoding gene lysC, the homoserine dehydrogenase encoding gene hom, and the threonine synthase encoding gene thrC can further improve threonine production.

[0135] 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> A recombinant microorganism, its construction method and application <130> KHP211124467.8 <160> 51 <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> 46 <212> DNA <213> Artificial Sequence <400>​​​​​​​<212> DNA <213> Artificial Sequence <400> 4 cccggaataa ttggcagcta tagagtaatt attcctttca 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <400> 5 tgaaaggaat aattactcta tagctgccaa ttatccggg 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <400> 6 gaagatgtgt gagtcgacac gggtaaaaaa tcctttcgta 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <400> 7 tacgaaagga ttttttaccc gtgtcgactc acacatcttc 40 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <400> 8 ggtggagcct gaaggaggtg cgagtgatcg gcaatgaatc cgg 43 <210> 9 <211> 43 <212> DNA <213> Artificial Sequence <400> 9 ccggattcat tgccgatcac tcgcacctcc ttcaggctcc acc 43 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <400> 10 gtaaaacgac ggccagtgcc aagcttcgcg gcagacggag tctggg 46 <210> 11 <211> 46 <212> DNA <213> Artificial Sequence <400> 11 aattcgagct cggtacccgg ggatccagcg acaggacaag cactgg 46 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <400> 12 cccggaataa ttggcagcta tgtgcacctt tcgatctacg 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <400> 13 cgtagatcga aaggtgcaca tagctgccaa ttatccggg 40 <210> 14 <211> 41 <212> DNA <213> Artificial Sequence <400> 14 tttctgtacg accagggcca tgggtaaaaa atcctttcgt a 41 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <400> 15 tacgaaagga ttttttaccc atggccctgg tcgtacagaa a 41 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <400> 16 tcggaacgag ggcaggtgaa ggtgatgtcg gtggtgccgt ct 42 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <400> 17 agacggcacc accgacatca ccttcacctg ccctcgttcc ga 42 <210> 18 <211> 46 <212> DNA <213> Artificial Sequence <400> 18 gtaaaacgac ggccagtgcc aagcttagcc tggtaagagg aaacgt 46 <210> 19 <211> 46 <212> DNA <213> Artificial Sequence <400> 19 aattcgagct cggtacccgg ggatccctgc gggcagatcc ttttga 46 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <400> 20 atttctttat aaacgcaggt catatctacc aaaactacgc 40 <210> twenty one <211> 40 <212> DNA <213> Artificial Sequence <400> twenty one gcgtagtttt ggtagatatg acctgcgttt ataaagaaat 40 <210> twenty two <211> 40 <212> DNA <213> Artificial Sequence <400> twenty two gtatatctcc ttctgcagga ataggtatcg aaagacgaaa 40 <210> twenty three <211> 40 <212> DNA <213> Artificial Sequence <400> twenty three tttcgtcttt cgatacctat tcctgcagaa ggagatatac 40 <210> twenty four <211> 41 <212> DNA <213> Artificial Sequence <400> twenty four tagccaattc agccaaaacc cccacgcgat cttccacatc c 41 <210> 25 <211> 41 <212> DNA <213> Artificial Sequence <400> 25 ggatgtggaa gatcgcgtgg gggttttggc tgaattggct a 41 <210> 26 <211> 46 <212> DNA <213> Artificial Sequence <400> 26 gtaaaacgac ggccagtgcc aagcttgctg gctcttgccg tcgata 46 <210> 27 <211> 46 <212> DNA <213> Artificial Sequence <400> 27 attcgagctc ggtacccggg gatccgccgt tgatcattgt tcttca 46 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <400> 28 cccggaataa ttggcagcta ggatataaccctatcccaag 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <400> 29 cttgggatag ggttatatcc tagctgccaa ttatccggg 40 <210> 30 <211> 41 <212> DNA <213> Artificial Sequence <400> 30 acgcgtcgaa atgtagtcca tgggtaaaaa atcctttcgt a 41 <210> 31 <211> 41 <212> DNA <213> Artificial Sequence <400> 31 tacgaaagga ttttttaccc atggactaca tttcgacgcg t 41 <210> 32 <211> 46 <212> DNA <213> Artificial Sequence <400> 32 gtaaaacgac ggccagtgcc aagcttgaat acgcggattc cctcgc 46 <210> 33 <211> 38 <212> DNA <213> Artificial Sequence <400> 33 agctcggtac ccggggatcc tgtccaactg cggtgatt 38 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <400> 34 ggtagacaag caaggcaaca ggcaaatgtg tttatcttcc 40 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <400> 35 ggaagataaa cacatttgcc tgttgccttg cttgtctacc 40 <210> 36 <211> 38 <212> DNA <213> Artificial Sequence <400> 36 caatacggag cggttacaaa gcttggcact ggccgtcg 38 <210> 37 <211> 55 <212> DNA <213> Artificial Sequence <400> 37 catgattacg aattcgagct cggtacccgg ggatcccgtg cgtggattgt cagca 55 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <400> 38 gatcacgcta tagttgcgcc gtgggaaaga catatgtggg 40 <210> 39 <211> 40 <212> DNA <213> Artificial Sequence <400> 39 cccacatatg tctttcccac ggcgcaacta tagcgtgatc 40 <210> 40 <211> 57 <212> DNA <213> Artificial Sequence <400> 40 tcacgacgtt gtaaaacgac ggccagtgcc aagcttcacg gtcgggattt ctaacag 57 <210> 41 <211> 57 <212> DNA <213> Artificial Sequence <400> 41 catgattacg aattcgagct cggtacccgg ggatccagta gacacctcga acgctac 57 <210> 42 <211> 42 <212> DNA <213> Artificial Sequence <400> 42 gatcacgcta tagttgcgcc gaaaaggagc ttgctttacg ac 42 <210> 43 <211> 42 <212> DNA <213> Artificial Sequence <400> 43 gtcgtaaagc aagctccttt tcggcgcaac tatagcgtga tc 42 <210> 44 <211> 57 <212> DNA <213> Artificial Sequence <400> 44 tcacgacgtt gtaaaacgac ggccagtgcc aagcttcacg gtcgggattt ctaacag 57 <210> 45 <211> 52 <212> DNA <213> Artificial Sequence <400> 45 cgagctcggt acccggggat ccacccgggt gtggcgcgca agaagatgcc ag 52 <210> 46 <211> 46 <212> DNA <213> Artificial Sequence <400> 46 taaatgttgt acgcggacca gaacaagatt ccgccgtgga ccacgc 46 <210> 47 <211> 43 <212> DNA <213> Artificial Sequence <400> 47 ggcggaatct tgttctggtc cgcgtacaac atttacatac acc 43 <210> 48 <211> 51 <212> DNA <213> Artificial Sequence <400> 48 gtaaaacgac ggccagtgcc aagcttagca aggtgttaga gcaaattttc g 51 <210> 49 <211> 47 <212> DNA <213> Artificial Sequence <400> 49 gaattcgagc tcggtacccg gggatccaac ttgtaaccgc tccgtat 47 <210> 50 <211> 48 <212> DNA <213> Artificial Sequence <400> 50 ggtgtatgta aatgttgtac gcggaccagt gcttgccttg cttgtcta 48 <210> 51 <211> 48 <212> DNA <213> Artificial Sequence <400> 51 tagacaagca aggcaagcac tggtccgcgt acaacattta catacacc 48

Claims

1. A modified Corynebacterium glutamicum, characterized in that, Compared to unmodified Corynebacterium glutamicum, its phosphorylacetyltransferase activity is reduced or lost, and it has enhanced threonine production capacity; In the modified Corynebacterium glutamicum, the activities of the following enzymes are enhanced and / or feedback inhibition is relieved: pyruvate carboxylase, aspartate kinase, homoserine dehydrogenase, and threonine synthase.

2. The modified Corynebacterium glutamicum according to claim 1, characterized in that, The reduction or loss of phosphoacetyltransferase activity is achieved by reducing the expression of the gene encoding phosphoacetyltransferase or knocking out the endogenous gene encoding phosphoacetyltransferase.

3. The modified Corynebacterium glutamicum according to claim 2, characterized in that, Methods such as site-directed mutagenesis or homologous recombination can be used to reduce the expression of genes encoding phosphoacetyltransferase or to knock out endogenous genes encoding phosphoacetyltransferase.

4. The modified Corynebacterium glutamicum according to any one of claims 1 to 3, characterized in that, In the modified Corynebacterium glutamicum, the activity of any one or two of the following enzymes (1) to (2) is reduced or lost: (1) Acetylkinase; (2) HTH transcriptional regulators.

5. The modified Corynebacterium glutamicum according to any one of claims 1 to 3, characterized in that, The enhancement of enzyme activity 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. A method for constructing threonine-producing Corynebacterium glutamicum, characterized in that, The method includes: A. Weaken the gene encoding phosphorylated acetyltransferase in Corynebacterium glutamicum, which has the ability to produce amino acids, to obtain gene-weakened strains; the weakening includes knocking out or reducing the expression of the phosphorylated acetyltransferase encoding gene. B. Enhance the activity of pyruvate carboxylase and / or relieve its feedback inhibition; C. Enhance the activity of enzymes related to the threonine synthesis pathway and / or relieve their feedback inhibition, wherein the enzymes related to the threonine synthesis pathway are selected from aspartate kinase, homoserine dehydrogenase and threonine synthase. The pathways for enhancing enzyme activity are selected from the following 1) to 6), or any 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.

7. The method for constructing threonine-producing Corynebacterium glutamicum according to claim 6, characterized in that, The method further includes: reducing or eliminating the activity of any one or two of the following enzymes (1)-(2): (1) Acetylkinase; (2) HTH transcriptional regulators.

8. The method for constructing Corynebacterium threonine-producing bacteria according to claim 7, characterized in that, Reducing enzyme activity or causing it to lose its activity can be achieved by reducing the expression of the gene encoding the enzyme or knocking out the gene encoding the enzyme.

9. A method for producing threonine, characterized in that, The method includes the following steps: a) Culturing the modified Corynebacterium glutamicum according to any one of claims 1-5 to obtain a culture of the modified Corynebacterium glutamicum; b) Collect the resulting threonine from the culture obtained in step a).

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