A method for constructing a recombinant microorganism and the use of reducing or inactivating the activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase in a microorganism for the fermentative production of threonine

Abstract

The present application relates to the technical field of genetic engineering, and specifically discloses a method for constructing a recombinant microorganism and application of reducing or inactivating activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase in the fermentation production of threonine. The present application finds that reducing or inactivating activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase in a microorganism can improve the fermentation effect of threonine, and thus proposes a method for constructing a recombinant microorganism, which makes the activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase in the recombinant microorganism lower or inactivated compared with a starting strain. The present application proposes a new method for constructing a threonine production strain and a method for improving the yield of threonine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a method for constructing recombinant microorganisms and the application of reducing or inactivating the activity of 3-deoxy-D-arabinohepulose-7-phosphate synthase in microorganisms in the fermentation production of threonine. Background Technology

[0002] L-Threonine is a white orthorhombic or crystalline powder. It is odorless and has a slightly sweet taste. It possesses various physiological functions, including promoting muscle growth, accelerating fat metabolism, and improving anxiety symptoms. It has been widely used in food, animal feed, and pharmaceutical industries.

[0003] Fermentation is currently the mainstream method for producing threonine, typically using recombinant microorganisms. Various methods exist for modifying these microorganisms, such as removing feedback inhibition of the product, enhancing key enzymes in the terminal synthesis pathway, strengthening the supply of cofactors for related enzymes, optimizing carbon flux allocation in the central metabolism, and blocking competing pathways. However, due to the highly complex metabolic networks of microorganisms, besides these established modification strategies, other unknown mutations can interfere with both anabolism and catabolism, causing either positive or negative effects. Therefore, further research is necessary. Summary of the Invention

[0004] One of the objectives of this invention is to provide a novel method for constructing threonine-producing strains.

[0005] This invention provides a method for constructing recombinant microorganisms, which reduces or inactivates the activity of 3-deoxy-D-arabinohepenosose-7-phosphate synthase in the recombinant microorganisms compared to the starting strain.

[0006] Based on years of research, this invention discovered that the deletion or weakening of the 3-deoxy-D-arabinohepenoyl-7-phosphate (DAHP) synthase gene can have a positive effect on the accumulation of L-threonine metabolites in Corynebacterium glutamicum, and this was further verified, thus completing this invention.

[0007] In the method of the present invention, the activity of the 3-deoxy-D-arabinohepenosose-7-phosphate synthase is reduced or inactivated by one or more base mutations or deletions in the coding region, or by replacing the weak promoter.

[0008] In the method of the present invention, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the recombinant microorganism is relieved compared with the starting strain, and the gene thrABC for the threonine terminal synthesis pathway is expressed; preferably, compared with the starting strain, the 311th position of the lysC protein sequence is mutated from threonine to isoleucine, and the 378th position of the hom protein is mutated from glycine to glutamic acid. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.

[0009] Specifically, this invention uses three Corynebacterium glutamicum model strains, ATCC 13032, ATCC 13869, and ATCC 14067, as test subjects. Through genetic engineering, the L-threonine terminal synthesis pathway was introduced and the feedback inhibition of key genes was relieved, resulting in three L-threonine-producing strains, named SMCT301, SMCT302, and SMCT303, respectively. These genetically engineered mutant strains were then used for shake-flask fermentation to produce L-threonine.

[0010] Further, using model strains ATCC 13032, ATCC 13869, and ATCC 14067, and three threonine-producing strains SMCT301, SMCT302, and SMCT303 as subjects, genetic engineering methods were used to inactivate or weaken the DAHP synthase gene in the producing strains. After shake-flask fermentation, the modified strains showed an increased L-threonine yield.

[0011] That is, Corynebacterium glutamicum with inactivated DAHP synthase or carrying weakened DAHP synthase can be used to produce L-amino acids, especially L-threonine.

[0012] Corynebacterium glutamicum ATCC 13032, ATCC 13869, and ATCC 14067 are model strains of Corynebacterium glutamicum, well-known in the field. These strains can be purchased publicly or obtained from relevant research institutes. Their genome sequences are publicly available and can be found on the NCBI website. Escherichia coli MG1655 is a model strain of Escherichia coli, well-known in the field. This strain can be purchased publicly or obtained from relevant research institutes. Its genome sequence is publicly available and can be found on the NCBI website.

[0013] The present invention also provides a recombinant microorganism, which is constructed by the above method.

[0014] The present invention also provides a DNA molecule having a nucleotide sequence as shown in any one of SEQ ID No. 25-27.

[0015] The DNA molecules encode DAHP synthase mutants with amino acid sequences as shown in SEQ ID No. 31-33.

[0016] The present invention also provides biological materials containing the above-mentioned DNA molecules, wherein the biological materials are expression cassettes, vectors or host cells.

[0017] The present invention also provides a 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase mutant, the amino acid sequence of which is shown in any one of SEQ ID No. 31-33.

[0018] The mutant of this invention can weaken the activity of DAHP synthase.

[0019] The present invention also provides the use of the above-mentioned recombinant microorganisms, or DNA molecules, or biological materials, or 3-deoxy-D-arabinohepulose-7-phosphate synthase mutants in any of the following aspects: (1) L-threonine fermentation production; (2) Genetic breeding of microorganisms for L-threonine fermentation production; (3) Increase the fermentation yield of L-threonine.

[0020] The present invention also provides the application of reducing or inactivating the activity of 3-deoxy-D-arabinohepulose-7-phosphate synthase in microorganisms in order to increase the yield of L-threonine produced by microbial fermentation. Preferably, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the microorganism is further relieved, and the gene thrABC for the threonine terminal synthesis pathway is expressed; And / or, the microorganism can ferment to produce threonine, preferably Corynebacterium glutamicum.

[0021] The present invention also provides a fermentation production method for L-threonine, which uses the above-mentioned recombinant microorganisms for fermentation production.

[0022] The beneficial effects of this invention are at least as follows: This invention provides a novel method for constructing recombinant microorganisms for threonine fermentation production. The activity of DAHP synthase in these recombinant microorganisms is weakened or inactivated, which can improve production efficiency when used for threonine production. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0025] The key gene for threonine synthesis described in this invention, lysC (NCBI ID NCgl0247 in ATCC13032, BBD29_01500 in ATCC13869, and CEY17_01500 in ATCC14067), encodes aspartate kinase, and hom (NCBI ID NCgl1136 in ATCC13032, BBD29_06325 in ATCC13869, and C in ATCC14067) encodes aspartate kinase. EY17_0645 encodes homoserine dehydrogenase, which is relieved by point mutation of threonine feedback inhibition. The threonine terminal synthesis pathway is achieved by referencing the thrABC operon from E. coli MG1655 (thrA, thrB, and thrC are numbered IEU92_RS00010, IEU92_RS00015, and IEU92_RS00020 in NCBI). Among them, thrA encodes a bifunctional enzyme, namely aspartate kinase and homoserine dehydrogenase, thrB encodes homoserine kinase, and thrC encodes threonine synthase.

[0026] The gene encoding the DAHP synthase described in this invention is numbered NCgl0950 in ATCC13032, and its corresponding amino acid sequence is shown in SEQ ID No. 28; it is numbered BBD29_05380 in ATCC13869, and its corresponding amino acid sequence is shown in SEQ ID No. 29; it is numbered CEY17_05570 in ATCC14067, and its corresponding amino acid sequence is shown in SEQ ID No. 30. Primer sequence information used in the examples is shown in Table 1. The plasmid pk18mobsacB-speC used in the specific embodiments of this invention was prepared by metabolically engineering the kanamycin resistance gene of the pK18mobsacB plasmid (GenBank: FJ1287239.1; available for purchase from public sources) to replace it with the spectinomycin resistance gene.

[0027] The embodiments of this invention are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0028] Table 1 Primer sequence information (SEQ ID No. 1-24) Example 1: Construction of an engineered plasmid carrying the thrABC metabolic pathway (threonine terminal pathway). Using the whole genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using the thrABC-f / thrABC-r primer pair. The PCR products were purified by gel electrophoresis and gel extraction. The PCR product contained the thrABC sequence carrying the threonine terminal metabolic pathway.

[0029] The plasmid vector was pVWEx1 (GenBank: MF034723.1; purchased from public sources). This vector was linearized by PCR amplification using the pVWEx1-f / pVWEx1-r primer pair. The PCR product was purified by gel electrophoresis and gel extraction for later use.

[0030] The vectors carrying the thrABC sequence and linearized were then circularized and assembled. The assembly method used was a single-fragment assembly kit from Novizan. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on kanamycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. The correctly sequenced plasmid was named pVWEx1-thrABC.

[0031] Example 2: Carrying the key gene lysC for threonine synthesis T311I Construction of 3 engineered plasmids Using the fully synthesized lysC gene sequence of ATCC13032 as a template, PCR amplification was performed using the lysC-f-1 / lysC-r-1 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a lysC gene carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. T311I (It encodes a mutant of the lysC protein). Using the fully synthesized lysC gene sequence of ATCC13869 as a template, PCR amplification was performed using the lysC-f-2 / lysC-r-2 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was lysC carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. T311I (It encodes a mutant of the lysC protein). Using the fully synthesized lysC gene sequence of ATCC14067 as a template, PCR amplification was performed using the lysC-f-3 / lysC-r-3 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was lysC carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC14067.T311I (It encodes a mutant lysC protein). In all of the above lysC protein mutants, the wild-type lysC protein has a threonine residue at position 311 that is mutated to isoleucine.

[0032] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.

[0033] The lysC carrying the above-mentioned homologous recombination sequences of ATCC13032, ATCC13869 and ATCC14067 T311I The linearized vectors were then circularized and assembled. Assembly was performed using a single-fragment assembly kit from Novizan. Specific instructions were provided in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. LysC vectors correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were identified. T311I The plasmids were named pK18mobsacB-speC-lysC, respectively. T311I -1、pK18mobsacB-speC-lysC T311I -2 and pK18mob sacB-speC-lysC T311I -3.

[0034] Example 3: Carrying the key gene hom for threonine synthesis G378E Construction of 3 engineered plasmids Using the fully synthesized hom gene sequence of ATCC13032 as a template, PCR amplification was performed using the hom-f-1 / hom-r-1 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom gene carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. G378E (It encodes a mutant hom protein). Using the fully synthesized hom gene sequence of ATCC13869 as a template, PCR amplification was performed using the hom-f-2 / hom-r-2 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom protein carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. G378E(It encodes a mutant hom protein). Using the fully synthesized hom gene sequence of ATCC13869 as a template, PCR amplification was performed using the hom-f-3 / hom-r-3 primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product was a hom protein carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. G378E (It encodes a mutant hom protein). In all of the above hom protein mutants, the wild-type hom protein has a glycine-to-glutamic acid mutation at position 378.

[0035] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.

[0036] The above-mentioned homogeneous recombination sequences carrying ATCC13032, ATCC13869 and ATCC14067 were used. G378E The linearized vectors were then circularized and assembled. Assembly was performed using Novizan's single-fragment assembly kit. Specific instructions were found in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB broth. Plasmids were extracted the following day and sequenced. Transformants correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were identified. G378E The plasmids were named pK18mobsacB-speC-hom respectively. G378E -1、pK18mobsacB-speC-hom G378E -2、pK18mobsacB-speC-hom G378E -3.

[0037] Example 4: Construction of three engineered plasmids inactivated by DAHP synthase Using the genomic sequences of Corynebacterium glutamicum ATCC13032, ATCC13869, and ATCC14067 as templates, PCR amplification was performed using primers P1 / P2 to obtain upper homologous arm fragments UP-1, UP-2, and UP-3. PCR amplification was then performed using primers P3 / P4 to obtain lower homologous arm fragments DN-1, DN-2, and DN-3. The PCR products were purified by gel electrophoresis and gel extraction. The PCR products were DAHP-UP-1, DAHP-UP-2, DAHP-UP-3 and DAHP-DN-1, DAHP-DN-2, DAHP-DN-3, respectively, carrying homologous recombination sequences.

[0038] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.

[0039] The DAHP-UP-1 and DAHP-DN-1 / DAHP-UP-2 and DAHP-DN-2 / DAHP-UP-3 and DAHP-DN-3 carrying homologous recombination sequences were circularized and assembled with linearized vectors, respectively. Assembly was performed using the Novizan multi-fragment assembly kit. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on spectinomycin plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. Plasmids correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were named pK18mobsacB-speC-ΔDAHP-1, pK18mobsacB-speC-ΔDAHP-2, and pK18mobsacB-speC-ΔDAHP-3, respectively.

[0040] Example 5 Construction of engineered plasmids with weakened DAHP synthase Using the genomic sequences of Corynebacterium glutamicum ATCC13032, ATCC13869, and ATCC14067 as templates, PCR amplification was performed using primers P5 / P6 to obtain upper homologous arm fragments UP-1', UP-2', and UP-3'. PCR amplification was then performed using primers P7 / P8 to obtain lower homologous arm fragments DN-1', DN-2', and DN-3'. The PCR products were purified by gel electrophoresis and gel extraction. The PCR products were DAHPr-UP-1, DAHPr-UP-2, DAHPr-UP-3 and DAHPr-DN-1, DAHPr-DN-2, DAHPr-DN-3, respectively, carrying homologous recombination sequences.

[0041] The plasmid vector was pK18mobsacB-speC, which had been pre-linearized by double enzyme digestion. The restriction endonucleases were EcoRI and HindIII from NEB. The vector was purified by enzyme digestion and then used for later use.

[0042] The DAHPr-UP-1 and DAHPr-DN-1 / DAHPr-UP-2 and DAHPr-DN-2 / DAHPr-UP-3 and DAHPr-DN-3 carrying homologous recombination sequences were circularized and assembled with linearized vectors, respectively. Assembly was performed using the Novizan multi-fragment assembly kit. Specific instructions for the kit can be found in the kit's manual. Transformants were screened on spectinomyces plates, and the resulting transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. Plasmids correctly sequenced and carrying homologous recombination sequences of ATCC13032, ATCC13869, or ATCC14067 were named pK18mobsacB-speC-DAHPr-1, pK18mobsacB-speC-DAHPr-2, and pK18mobsacB-speC-DAHPr-3, respectively.

[0043] The nucleotide sequence of the DAHP synthase encoding gene of the attenuated ATCC13032 is shown in SEQ ID No. 25, and the corresponding amino acid sequence is shown in SEQ ID No. 31. The nucleotide sequence of the DAHP synthase encoding gene of the attenuated ATCC13869 is shown in SEQ ID No. 26, and the corresponding amino acid sequence is shown in SEQ ID No. 32. The nucleotide sequence of the DAHP synthase encoding gene of the attenuated ATCC14067 is shown in SEQ ID No. 27, and the corresponding amino acid sequence is shown in SEQ ID No. 33.

[0044] Example 6: The threonine terminal pathway thrABC was introduced into the model bacteria ATCC 13032, ATCC 13869, and ATCC 14067, respectively. Competent cells of C. glutamicum model bacteria ATCC13032, ATCC13869, and ATCC14067 were prepared according to the method described in the C. glutamicum Handbook (Charpter 23) and exogenous genes were expressed.

[0045] The expression plasmid pVWEx1-thrABC was transformed into ATCC13032, ATCC13869, and ATCC14067 competent cells using electroporation, and transformants were screened on BHI selective medium containing 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed as the final results. The resulting modified strains were named 13032-thrABC, 13869-thrABC, and 14067-thrABC, respectively.

[0046] Example 7: lysC was introduced into 13032-thrABC, 13869-thrABC, and 14067-thrABC, respectively. T311I and hom G378E Competent cells of 13032-thrABC, 13869-thrABC, and 14067-thrABC were prepared and their genes were recombined according to the method in the C. glutamicum Handbook (Charpter 23).

[0047] The recombinant plasmid pK18mobsacB-speC-lysC was electroporated. T311I -1、pK18mobsacB-speC-lysC T311I -2 and pK18mobsacB-speC-lysC T311I Transformations were performed on 13032-thrABC, 13869-thrABC, and 14067-thrABC competent cells, respectively, and transformants were screened on BHI selective media containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The selected transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The cultures were serially diluted (to 10⁻⁶ ppm). -2The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated statically at 30°C for 48 h. The resulting transformants should carry the target mutation and not the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed as the final result. The resulting modified strains were named 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C, respectively.

[0048] Following the methods described in the C. glutamicum Handbook (Charpter 23), competent cells of 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C were prepared and gene recombination was performed.

[0049] The recombinant plasmid pK18mobsacB-speC-hom was electroporated. G378E -1、pK18mobsacB-speC-hom G378E -2、pK18mobsacB-speC-hom G378E Transformations were performed on 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C competent cells, and transformants were screened on BHI selective media containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The selected transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The cultures were serially diluted (to 10⁻⁶ ppm). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated statically at 30°C for 48 h. The resulting transformants should carry the target mutation and not the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the final results. The resulting modified strains were named SMCT301, SMCT302, and SMCT303, respectively.

[0050] Example 8: Shake-flask test of fermentation performance of three model strains after introduction of the threonine terminal pathway and removal of threonine feedback inhibition. The culture medium used in the shake flask test is as follows: Plate activation medium: BHI 37 g / L, 20 g / L agar powder.

[0051] 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.

[0052] 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.

[0053] Fermentation method: 1. Seed activation: Take the strain to be verified from the cryopreservation tube, streak it on seed activation medium, and incubate at 30℃ for 24h; 2. Seed culture: Pick 1 plate of activated seeds and transfer it to a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Incubate at 30 °C and 230 r / min for 6 h with shaking. 3. Fermentation culture: Inoculate 6 mL of seed culture into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 30 °C and 150 r / min for 24 h with shaking. Perform 3 replicates for each strain.

[0054] 4. OD 562 Measurement: The fermentation broth was diluted 100 times, and the absorbance was measured at a wavelength of 562 nm using a spectrophotometer. Each strain was tested in triplicate, and the average value was calculated. The results are shown in Table 2, where the data represents the average of the three parallel tests.

[0055] 5. Amino acid concentration determination: Centrifuge 2 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and detect it using Agilent high-performance liquid chromatography (HPLC). Three replicates were performed for both the recombinant strain and the control strain, and the average value was calculated. The results are shown in Table 2.

[0056] Table 2 Comparison of amino acid yield detection results of recombinant strains The amino acid content in the fermentation broth was analyzed, and it was found that the threonine concentration of Corynebacterium glutamicum, which carried the threonine terminal pathway and removed the threonine terminal restriction, was significantly increased. Table 2 shows that compared to the wild-type strain ATCC13032, SMCT301 increased threonine production from 2.5 g / L to 7.6 g / L, an increase of 240.8%; compared to the wild-type strain ATCC13869, SMCT302 increased threonine production from 2.3 g / L to 7.1 g / L, an increase of 246.2%; and compared to the wild-type strain ATCC14067, SMCT303 increased threonine production from 2.2 g / L to 7.0 g / L, an increase of 262.3%. This indicates that opening the threonine terminal synthesis pathway significantly improves the strain's threonine production capacity. Therefore, it can be proven that after metabolic engineering modification, SMCT301, SMCT302, and SMCT303 are better threonine-producing strains.

[0057] Example 9: Inactivation or attenuation modification of DAHP synthase in the model strain ATCC13032 of Corynebacterium glutamicum and the threonine-producing strain SMCT301. Competent cells of the C. glutamicum model strain ATCC13032 and the threonine-producing strain SMCT301 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).

[0058] The recombination method was the same as in Example 7, i.e., inactivated and weakened recombinant plasmids (pK18mobsacB-speC-ΔDAHP-1 and pK18mobsacB-speC-DAHPr-1) were transformed into ATCC13032 and SMCT301 competent cells, respectively, using electroporation. Transformants were screened on BHI selective media containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ oz). -2The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The resulting transformants were amplified by PCR, and nucleotide sequencing analysis was performed as the final results. The modified strains with inactivated DAHP synthase were named SMCT331 (originating strain ATCC13032) and SMCT337 (originating strain SMCT301), respectively, while the modified strains with weakened DAHP synthase were named SMCT332 (originating strain ATCC13032) and SMCT338 (originating strain SMCT301), respectively.

[0059] Example 10: Inactivation or attenuation modification of DAHP synthase in the model strain ATCC13869 of Corynebacterium glutamicum and the threonine-producing strain SMCT302. Competent cells of the C. glutamicum model strain ATCC13869 and the threonine-producing strain SMCT302 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).

[0060] The recombination method was the same as in Example 7, i.e., inactivated or weakened recombinant plasmids (pK18mobsacB-speC-ΔDAHP-2 and pK18mobsacB-speC-DAHPr-2) were transformed into ATCC13869 and SMCT302 competent cells, respectively, using electroporation. Transformants were screened on BHI selective medium containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ ppm). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The resulting transformants were amplified by PCR, and nucleotide sequencing analysis was performed as the final results. The modified strains with inactivated DAHP synthase were named SMCT333 (originating strain ATCC13869) and SMCT339 (originating strain SMCT302), respectively, while the modified strains with weakened DAHP synthase were named SMCT334 (originating strain ATCC13869) and SMCT340 (originating strain SMCT302), respectively.

[0061] Example 11: Inactivation or attenuation modification of DAHP synthase in the model strain ATCC14067 of Corynebacterium glutamicum and the threonine-producing strain SMCT303. Competent cells of the C. glutamicum model strain ATCC14067 and the threonine-producing strain SMCT303 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).

[0062] The recombination method was the same as in Example 7, i.e., inactivated or weakened recombinant plasmids (pK18mobsacB-speC-ΔDAHP-3 and pK18mobsacB-speC-DAHPr-3) were transformed into ATCC14067 and SMCT303 competent cells, respectively, using electroporation. Transformants were screened on BHI selective medium containing 100 mg / L spectinomycin and 25 mg / L kanamycin. The screened transformants were cultured overnight in BHI liquid medium containing 25 mg / L kanamycin at 30°C with shaking at 200 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (the original solution was serially diluted to 10⁻⁶ ppm). -2 The diluted solution was spread onto BHI solid medium containing 10% sucrose and 25 mg / L kanamycin, and incubated at 30°C for 48 h. The transformed strains were amplified by PCR, and nucleotide sequencing analysis was performed as the final results. The modified strains that obtained DAHP synthase inactivation were named SMCT335 (originating strain ATCC14067) and SMCT341 (originating strain SMCT303), respectively, and the modified strains that obtained DAHP synthase attenuation were named SMCT336 (originating strain ATCC14067) and SMCT342 (originating strain SMCT303), respectively.

[0063] Example 12: Shake-flask test of the fermentation performance of the above-mentioned *Corynebacterium glutamicum* model strain and threonine-producing strain after DAHP synthase inactivation or attenuation modification. The culture medium and fermentation method used in the shake flask test were the same as in Example 8, and the test results are shown in Table 3.

[0064] Table 3 Comparison of amino acid yield detection results of recombinant strains The amino acid content in the fermentation broth was analyzed, and it was found that the threonine concentration in the culture medium of strains modified with inactivated or weakened DAHP synthase was increased to varying degrees. This result indicates that the DAHP synthase-inactivated or weakened strains provided in this invention promote both the threonine yield and productivity of Corynebacterium.

[0065] 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 are within the scope of protection claimed by the present invention.

Claims

1. A method for constructing recombinant microorganisms, characterized in that, The recombinant microorganism exhibits reduced or inactivated activity of 3-deoxy-D-arabinohepenosyl-7-phosphate synthase compared to the original strain.

2. The method according to claim 2, characterized in that, The activity of the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase can be reduced or inactivated by mutations or deletions of one or more bases in the coding region, or by replacing the weak promoter.

3. The method according to claim 1 or 2, characterized in that, Compared with the starting strain, the recombinant microorganism has the feedback inhibition of the key genes lysC and hom for threonine synthesis relieved and expresses the thrABC gene for the threonine terminal synthesis pathway; preferably, compared with the starting strain, the lysC protein sequence has a mutation at position 311 from threonine to isoleucine and the hom protein sequence has a mutation at position 378 from glycine to glutamic acid. And / or, the starting strain is a Corynebacterium that can ferment to produce threonine, preferably Corynebacterium glutamicum.

4. A recombinant microorganism, characterized in that, It is constructed by the method described in any one of claims 1-3.

5. A DNA molecule, characterized in that, The nucleotide sequence is shown in any one of SEQ ID No. 25-27.

6. A biological material containing the DNA molecule of claim 5, wherein the biological material is an expression cassette, a vector, or a host cell.

7. A mutant of 3-deoxy-D-arabinohepenoyl-7-phosphate synthase, characterized in that, The amino acid sequence is shown in any one of SEQ ID No. 31-33.

8. The use of the recombinant microorganism of claim 4, or the DNA molecule of claim 5, or the biomaterial of claim 6, or the 3-deoxy-D-arabinohepulose-7-phosphate synthase mutant of claim 7 in any of the following aspects: (1) L-threonine fermentation production; (2) Genetic breeding of microorganisms for L-threonine fermentation production; (3) Increase the fermentation yield of L-threonine.

9. The application of reducing or inactivating the activity of 3-deoxy-D-arabinohepulose-7-phosphate synthase in microorganisms in increasing the yield of L-threonine produced by microbial fermentation; Preferably, the feedback inhibition of the key genes lysC and hom for threonine synthesis in the microorganism is further relieved, and the gene thrABC for the threonine terminal synthesis pathway is expressed; And / or, the microorganism can ferment to produce threonine, preferably Corynebacterium glutamicum.

10. A fermentation method for producing L-threonine, characterized in that, Fermentation production using the recombinant microorganisms described in claim 4.

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