A recombinant microorganism and application of lactic acid dehydrogenase mutant in fermentative production of threonine
By genetically engineering Corynebacterium glutamicum to mutate its lactate dehydrogenase and remove the feedback inhibition of lysC and hom, the thrABC gene was expressed, solving the problem of insufficient threonine production in microbial fermentation and achieving a significant increase in threonine yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2026-06-09
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to the application of a recombinant microorganism and a lactate dehydrogenase mutant in the fermentation production of threonine. Background Technology
[0002] L-Threonine, with the molecular formula C4H9NO3, is a white orthorhombic or crystalline powder. It is an essential amino acid with various physiological functions and is widely used in food, feed, and pharmaceuticals, with market demand increasing year by year.
[0003] Microbial fermentation is a low-cost and low-pollution method suitable for the industrial production of amino acids. Microorganisms used in fermentation production can have their amino acid production increased through various genetic modifications, such as removing feedback inhibition of the product, strengthening key enzymes in the terminal synthesis pathway, enhancing the supply of cofactors for related enzymes, optimizing the carbon flow distribution in central metabolism, and blocking competing pathways.
[0004] However, due to the highly complex metabolic networks of microorganisms, in addition to the established modification strategies mentioned above, there are still other unknown mutations, including point mutations and gene deletions, that can interfere with microbial anabolic and catabolistic metabolism, causing either positive or negative effects. Therefore, further research is still necessary. Summary of the Invention
[0005] One of the objectives of this invention is to provide a new microorganism capable of efficiently producing threonine.
[0006] The present invention provides a recombinant microorganism in which the alanine at position 265 of the wild-type lactate dehydrogenase (D-lactate dehydrogenase, FAD / FMN dehydrogenase) of the recombinant microorganism is mutated to threonine or serine; the amino acid sequence of the wild-type lactate dehydrogenase is shown in any one of SEQ ID No. 25-27, and the starting strain of the recombinant microorganism is a Corynebacterium that can ferment to produce threonine.
[0007] Based on years of research, this invention discovered that mutations in certain lactate dehydrogenases can have a positive effect on the accumulation of L-threonine metabolites in Corynebacterium glutamicum, and this was further verified, thus completing this invention.
[0008] The recombinant microorganism of the present invention, compared with the starting strain, has the feedback inhibition of the key genes lysC and hom for threonine synthesis relieved, and expresses the gene thrABC for the threonine terminal synthesis pathway; Preferably, compared with the starting strain, the recombinant microorganism has a threonine mutation at position 311 of the lysC protein sequence to isoleucine, and a glycine mutation at position 378 of the hom protein to glutamic acid. And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.
[0009] Specifically, this invention used three Corynebacterium glutamicum model strains, ATCC 13032, ATCC 13869, and ATCC 14067, as test subjects. Through genetic engineering methods, the coding sequence of the wild-type lactate dehydrogenase was mutated as described above. After fermentation experiments, it was found that the threonine production was increased.
[0010] This invention also introduces the L-threonine terminal synthesis pathway into the above-mentioned model strains using genetic engineering methods and removes feedback inhibition of key genes, obtaining 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.
[0011] This invention further uses three threonine-producing strains, SMCT301, SMCT302, and SMCT303, as the implementation objects. Through genetic engineering, the coding sequence of the wild-type lactate dehydrogenase in these strains is mutated, causing a mutation at position 265 of the protein sequence, changing alanine to threonine or serine. Shake-flask fermentation using these mutant strains showed an increased L-threonine yield.
[0012] Corynebacterium glutamicum carrying the above-mentioned lactate dehydrogenase mutant can be used to produce L-amino acids, especially L-threonine.
[0013] 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.
[0014] The present invention also provides a lactate dehydrogenase mutant having an amino acid sequence as shown in any one of SEQ ID NO.28-33.
[0015] The present invention also provides a nucleic acid encoding the above-mentioned lactate dehydrogenase mutant.
[0016] The nucleic acid of the present invention has a nucleotide sequence as shown in any one of SEQ ID NO.19-24.
[0017] The present invention also provides biological materials containing the above-mentioned nucleic acids, wherein the biological materials are expression cassettes, vectors or host cells.
[0018] The present invention also provides any of the following applications of the above-mentioned recombinant microorganisms, or lactate dehydrogenase mutants, or nucleic acids, or biological materials: (1) Application in the fermentation production of L-threonine; (2) Application in microbial genetic breeding for the production of L-threonine; (3) Application in increasing the yield of L-threonine produced by fermentation.
[0019] The present invention also provides a method for producing L-threonine, which includes a step of fermentation culture with recombinant microorganisms as described above.
[0020] The present invention also provides a method for constructing recombinant microorganisms, comprising the step of expressing a lactate dehydrogenase mutant in the recombinant microorganisms, the lactate dehydrogenase mutant being as described above; the starting strain of the recombinant microorganisms is a Corynebacterium capable of fermenting to produce threonine.
[0021] The method of the present invention further includes the steps of relieving the feedback inhibition of the key genes lysC and hom for threonine synthesis in the recombinant microorganism and expressing the threonine terminal synthesis pathway gene thrABC. Preferably, the method includes the step of mutating position 311 of the lysC protein sequence of the recombinant microorganism from threonine to isoleucine, and position 378 of the hom protein from glycine to glutamic acid. And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.
[0022] The beneficial effects of this invention are at least as follows: This invention provides a novel recombinant microorganism that can efficiently ferment and produce threonine, offering a new method for the genetic breeding of threonine-producing strains and the fermentation production of threonine. 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 wild-type lactate dehydrogenase described in this invention is designated NCgl0865 in ATCC13032, with the corresponding amino acid sequence shown in SEQ ID No. 25; designated BBD29_04965 in ATCC13869, with the corresponding amino acid sequence shown in SEQ ID No. 26; and designated CEY17_04880 in ATCC14067, with the corresponding amino acid sequence shown in SEQ ID No. 27. The nucleotide sequence of the mutant can be obtained by fusion PCR or by whole-genome synthesis by a gene synthesis company.
[0027] The primer sequence information used in the examples is shown in Table 1. The plasmid pk18mobsacB-speC used in the specific embodiments of the present invention was prepared by replacing the kanamycin resistance gene of the pK18mobsacB plasmid (GenBank: FJ1287239.1; available for purchase from public channels) with the spectinomycin resistance gene through metabolic engineering.
[0028] 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.
[0029] Table 1 Primer sequence information (SEQ ID No. 1-18) 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.
[0030] 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.
[0031] 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.
[0032] 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-mentioned mutant lysC proteins, the wild-type lysC protein has a threonine residue at position 311 that is mutated to isoleucine.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] Example 4: Construction of three engineered plasmids carrying lactate dehydrogenase mutants Using the wild-type dld gene sequences of ATCC13032, ATCC13869, and ATCC14067 synthesized from whole genomes as templates, PCR amplification was performed using primer pairs P1 / P2. The PCR products were purified by gel electrophoresis and gel extraction. The PCR products were dld sequences carrying homologous recombination sequences of ATCC13032, ATCC13869, and ATCC14067, respectively. A265T and dld A265S .
[0039] dld carrying the ATCC13032 homologous recombination sequence A265T and dld A265SAs shown in SEQ ID No. 19 and SEQ ID No. 20 respectively, the encoded amino acid sequences are shown in SEQ ID No. 28 and SEQ ID No. 29 respectively. The dld carrying the ATCC13869 homologous recombination sequence... A265T and dld A265S As shown in SEQ ID No. 21 and SEQ ID No. 22 respectively, the encoded amino acid sequences are shown in SEQ ID No. 30 and SEQ ID No. 31 respectively. The dld carrying the homologous recombination sequence of ATCC14067... A265T and dld A265S As shown in SEQ ID No. 23 and SEQ ID No. 24 respectively, the encoded amino acid sequences are shown in SEQ ID No. 32 and SEQ ID No. 33 respectively.
[0040] 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.
[0041] The above-mentioned dld carrying homologous recombination sequences of ATCC13032, ATCC13869 and ATCC14067 A265T dld A265S 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 broth. Plasmids were extracted the following day and sequenced. DLDs carrying the homologous recombination sequences ATCC13032, ATCC13869, and ATCC14067 were correctly sequenced. A265T dld A265S The plasmids were named pK18mobsacB-speC-dld, respectively. A265T -1、pK18mobsacB-speC-dld A265S -1;pK18mobsacB-speC-dld A265T -2、pK18mobsacB-speC-dld A265S -2;pK18mobsacB-speC-dld A265T -3、pK18mobsacB-speC-dld A265S -3.
[0042] Example 5: 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 and exogenous genes were expressed according to the method in the C. glutamicum Handbook (Charpter 23).
[0043] 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.
[0044] Example 6: 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).
[0045] The recombinant plasmid pK18mobsacB-speC-lysC was electroporated. T311I -1、pK18mobsacB-speC-lysC T311I -2 and pK18mobsacB-speC-lysC T311ITransformations 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). -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 as the final result. The resulting modified strains were named 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C, respectively.
[0046] Following the methods outlined in the C. glutamicum Handbook (Charpter 23), we continued to prepare competent cells of 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C and performed gene recombination.
[0047] 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). -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 to obtain the final results. The resulting modified strains were named SMCT301, SMCT302, and SMCT303, respectively.
[0048] Example 7: 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 for the shake flask test is as follows: Plate activation medium: BHI 37 g / L, 20 g / L agar powder.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] Example 8: Lactate dehydrogenase mutant 1 and lactate dehydrogenase mutant 2 were introduced into the model strain of Corynebacterium glutamicum ATCC13032 and the threonine-producing strain SMCT301, respectively. Competent cells of Corynebacterium glutamicum ATCC13032 and threonine-producing strain SMCT301 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0056] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-dld is recombined by electroporation. A265T -1 and pK18mobsacB-speC-dld A265STransformants carrying the ATCC13032 homologous recombination sequence were transformed into ATCC13032 and SMCT301 competent cells, respectively. Transformants were screened on BHI selective medium 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 (the original solution was serially diluted to 10⁻⁶ oz.) -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 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 result. The final lactate dehydrogenase mutant 1 (dld) was obtained. A265T The modified strains were named SMCT376 (originating strain ATCC13032) and SMCT382 (originating strain SMCT301) to obtain lactate dehydrogenase mutant 2 (dld A265S The modified strains were named SMCT377 (originating strain ATCC13032) and SMCT383 (originating strain SMCT301), respectively.
[0057] Example 9: Lactate dehydrogenase mutant 1 and lactate dehydrogenase mutant 2 were introduced into the model strain of Corynebacterium glutamicum ATCC13869 and the threonine-producing strain SMCT302, respectively. Competent cells of Corynebacterium glutamicum ATCC13869 and threonine-producing strain SMCT302 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0058] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-dld is recombined by electroporation. A265T -2 and pK18mobsacB-speC-dld A265STransformations of ATCC13869 and SMCT302 competent cells were performed using a gene transfer medium (carrying the homologous recombination sequence of ATCC13869), and the transformed cells were screened on BHI selective medium 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 a rotary shaker 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 (the original solution was serially diluted to 10⁻⁶ oz.) -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 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 result. The final lactate dehydrogenase mutant 1 (dld) was obtained. A265T The modified strains were named SMCT378 (originating strain ATCC13869) and SMCT384 (originating strain SMCT302) to obtain lactate dehydrogenase mutant 2 (dld A265S The modified strains were named SMCT379 (originating strain ATCC13869) and SMCT385 (originating strain SMCT302), respectively.
[0059] Example 10: Lactate dehydrogenase mutant 1 and lactate dehydrogenase mutant 2 were introduced into the model strain of Corynebacterium glutamicum ATCC14067 and the threonine-producing strain SMCT303, respectively. Competent cells of Corynebacterium glutamicum ATCC14067 and threonine-producing strain SMCT303 were prepared and their genes were recombined according to the methods in the C. glutamicum Handbook (Charpter 23).
[0060] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-dld is recombined by electroporation. A265T -3 and pK18mobsacB-speC-dld A265STransformants carrying the ATCC14067 homologous recombination sequence were transformed into ATCC14067 and SMCT303 competent cells, respectively. Transformants were screened on BHI selective medium 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 culture was serially diluted (the original solution was serially diluted to 10...) -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 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 result. The final lactate dehydrogenase mutant 1 (dld) was obtained. A265T The modified strains were named SMCT380 (originating strain ATCC14067) and SMCT386 (originating strain SMCT303) to obtain lactate dehydrogenase mutant 2 (dld A265S The modified strains were named SMCT381 (originating strain ATCC14067) and SMCT387 (originating strain SMCT303), respectively.
[0061] Example 11: Shake-flask test of the fermentation performance of the above-mentioned *Corynebacterium glutamicum* model strain and threonine-producing strain after introducing lactate dehydrogenase mutants. The culture medium and fermentation method used in the shake flask test were the same as in Example 7, and the test results are shown in Table 3.
[0062] Table 3 Comparison of amino acid yield detection results of recombinant strains Analysis of the amino acid content in the fermentation broth revealed that the threonine concentration in the culture medium of *Corynebacterium glutamicum* carrying both lactate dehydrogenase mutant 1 and lactate dehydrogenase mutant 2 was increased to varying degrees, suggesting an improved efficiency in the synthesis or secretion of threonine by these bacteria. Among the different threonine-producing bacteria, the modified bacteria carrying mutant 1 showed a more significant increase in threonine yield and productivity.
[0063] The results indicate that the lactate dehydrogenase mutant provided in this invention promotes the production and yield of threonine in Corynebacterium. The mutated lactate dehydrogenase is more conducive to the synthesis of threonine or promotes its secretion into the extracellular space. Furthermore, the related mutants are universal and can improve the synthesis efficiency of various amino acids.
[0064] 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.
Claims
1. A recombinant microorganism, characterized in that, The wild-type lactate dehydrogenase of the recombinant microorganism has a mutation at position 265, where alanine is replaced with threonine or serine; the amino acid sequence of the wild-type lactate dehydrogenase is shown in any one of SEQ ID No. 25-27; and the starting strain of the recombinant microorganism is a Corynebacterium that can ferment to produce threonine.
2. The recombinant microorganism according to claim 1, characterized in that, Compared with the original strain, the recombinant microorganism showed that the feedback inhibition of the key genes lysC and hom for threonine synthesis was relieved, and the gene thrABC for the threonine terminal synthesis pathway was expressed. Preferably, compared with the starting strain, the recombinant microorganism has a threonine mutation at position 311 of the lysC protein sequence to isoleucine, and a glycine mutation at position 378 of the hom protein to glutamic acid. And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.
3. A lactate dehydrogenase mutant, characterized in that, It has an amino acid sequence as shown in any one of SEQ ID NO.28-33.
4. The nucleic acid encoding the lactate dehydrogenase mutant of claim 3.
5. The nucleic acid according to claim 4, characterized in that, It has a nucleotide sequence as shown in any one of SEQ ID NO.19-24.
6. A biological material containing the nucleic acid of claim 4 or 5, wherein the biological material is an expression cassette, a vector, or a host cell.
7. Any of the following applications of the recombinant microorganism of claim 1 or 2, or the lactate dehydrogenase mutant of claim 3, or the nucleic acid of claim 4 or 5, or the biomaterial of claim 6: (1) Application in the fermentation production of L-threonine; (2) Application in microbial genetic breeding for the production of L-threonine; (3) Application in increasing the yield of L-threonine produced by fermentation.
8. A method for producing L-threonine, characterized in that, It includes the step of fermenting and culturing with recombinant microorganisms as described in claim 1 or 2.
9. A method for constructing recombinant microorganisms, characterized in that, The method includes the step of expressing a lactate dehydrogenase mutant in the recombinant microorganism, the lactate dehydrogenase mutant as described in claim 3; the starting strain of the recombinant microorganism is a Corynebacterium that can ferment to produce threonine.
10. The method according to claim 9, characterized in that, The steps include relieving the feedback inhibition of the key genes lysC and hom in the threonine synthesis of the recombinant microorganism and expressing the thrABC gene of the threonine terminal synthesis pathway. Preferably, the method includes the step of mutating position 311 of the lysC protein sequence of the recombinant microorganism from threonine to isoleucine, and position 378 of the hom protein from glycine to glutamic acid. And / or, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.