Transcriptional regulator mutant and application thereof in constructing amino acid production strain
By mutating transcription factors at specific sites in Corynebacterium glutamicum, mutant transcription factors were constructed, solving the problem of underutilization of transcription factors, improving threonine production efficiency and conversion rate, and reducing production costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MEIHUA BIOTECH LANGFANG CO LTD
- Filing Date
- 2024-12-09
- Publication Date
- 2026-06-09
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Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to mutant transcription regulatory factors and their application in the construction of amino acid-producing bacteria. Background Technology
[0002] Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium glutamicum is a Gram-positive bacterium characterized by rapid growth, non-pathogenicity, and weak ability to degrade its own metabolites. As a traditional industrial microorganism, Corynebacterium glutamicum is widely used in the production of various amino acids, nucleotides, and organic acids.
[0003] The production performance of fermentation strains is a key factor affecting the production efficiency and cost of biochemicals such as amino acids. Improving the production performance of fermentation strains, such as the yield of target biochemicals, is of great significance for improving production efficiency and reducing production costs.
[0004] Complex transcriptional regulatory mechanisms exist in Corynebacterium glutamicum, and some transcriptional regulatory factors have been identified and used to regulate the synthesis and accumulation of biochemicals such as amino acids. However, a large number of unknown transcriptional regulatory factors remain to be studied, and their utilization needs to be expanded. Summary of the Invention
[0005] This invention provides transcriptional regulatory factor mutants and their application in constructing amino acid-producing bacteria.
[0006] Specifically, the present invention provides the following technical solutions.
[0007] In a first aspect, the present invention provides a transcription factor mutant, wherein the transcription factor mutant, compared with the wild-type transcription factor of Corynebacterium glutamicum, contains a mutation in which the 120th amino acid is mutated to an amino acid other than proline; or, the transcription factor mutant, compared with the wild-type transcription factor of Corynebacterium glutamicum, contains a mutation in which the 120th amino acid is mutated to an amino acid other than proline, and the 75th amino acid is mutated to valine. The Corynebacterium glutamicum wild-type transcriptional regulatory factor has an amino acid sequence as shown in SEQ ID NO.5, 7 or 9.
[0008] This invention reveals that mutating the 120th amino acid of the transcriptional regulatory factor in the wild-type Corynebacterium glutamicum can effectively promote the synthesis and extracellular accumulation of threonine, significantly increasing the threonine yield and conversion rate of the strain. Furthermore, combining other mutations (e.g., the 75th amino acid mutation) with the 120th amino acid mutation can further enhance the threonine production capacity of the strain.
[0009] The amino acids mentioned above, excluding proline, include, but are not limited to, serine.
[0010] Preferably, the transcription factor mutant contains a mutation in which amino acid 120 is changed to serine, compared to the wild-type transcription factor of Corynebacterium glutamicum; or, the transcription factor mutant contains a mutation in which amino acid 120 is changed to serine and amino acid 75 is changed to valine, compared to the wild-type transcription factor of Corynebacterium glutamicum.
[0011] Preferably, the transcription factor mutant contains a mutation (P120S) in which proline at position 120 is changed to serine, compared to the wild-type transcription factor of Corynebacterium glutamicum; or, the transcription factor mutant contains a mutation (G75V, P120S) in which proline at position 120 is changed to serine and glycine at position 75 is changed to valine, compared to the wild-type transcription factor of Corynebacterium glutamicum.
[0012] As a preferred embodiment of the present invention, the amino acid sequence of the transcription factor mutant is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.5 to serine; (2) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.7 to serine; (3) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.9 to serine.
[0013] As another preferred embodiment of the present invention, the amino acid sequence of the transcription factor mutant is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.5 to serine and the 75th amino acid to valine; (2) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.7 to serine and the 75th amino acid to valine; (3) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.9 to serine and the 75th amino acid to valine.
[0014] This invention experimentally verifies that the above-mentioned transcription factor mutant can significantly improve the threonine production capacity of the strain. Those skilled in the art will understand that adding one or more amino acid residues that do not affect protein function to the N-terminus and / or C-terminus of the above-mentioned transcription factor mutant will not significantly affect the function of the mutant. For example, adding protein tags, signal peptides, linker peptides, or other amino acid residues to the N-terminus and / or C-terminus of the above-mentioned transcription factor mutant can still achieve the aforementioned effect of improving threonine production capacity, and therefore is also within the scope of protection of this invention.
[0015] Secondly, the present invention provides biomaterials, said biomaterials comprising any one of the following: (1) Nucleic acid molecules encoding mutants of the transcriptional regulatory factors described above; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A vector comprising the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1) or the expression cassette described in (2) or the vector described in (3).
[0016] For the nucleic acid molecules described in (1) above, based on the amino acid sequence of the transcription factor mutant provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned transcription factor mutant. Due to the degeneracy of codons, the nucleotide sequence encoding a transcription factor mutant is not unique, and all nucleic acid molecules capable of encoding the above-mentioned transcription factor mutant are within the scope of protection of this invention.
[0017] In some specific embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.3 or 4.
[0018] The expression cassette in (2) above can be a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.
[0019] The vectors mentioned in (3) above include, but are not limited to, plasmid vectors, viral vectors, and transposons.
[0020] The host cell in (4) above is any cell capable of carrying the above nucleic acid molecules or expressing the mutant of the transcription regulatory factor, including microbial cells, preferably Escherichia coli, Corynebacterium, or Bacillus.
[0021] Thirdly, the present invention provides any of the following applications of the above-described transcriptional regulatory factor mutants or the biological materials: (1) Application in regulating the production capacity of bacterial amino acids or their derivatives; (2) Application in the construction of bacteria for the production of amino acids or their derivatives; (3) Applications in the production of amino acids or their derivatives; (4) Application in increasing the yield and / or conversion rate of bacterial amino acids or their derivatives; (5) Application as a marker in screening bacteria for the production of amino acids.
[0022] In the above applications, the amino acid is preferably threonine.
[0023] In the above applications, the bacteria are preferably Corynebacterium or Escherichia. Specifically, the Corynebacterium species is preferably Corynebacterium glutamicum; and the Escherichia species is preferably Escherichia coli.
[0024] In this invention, the production capacity refers to the ability of bacteria to synthesize and / or accumulate amino acids, including but not limited to the yield, conversion rate, and production intensity of amino acids.
[0025] In this invention, the transcription regulatory factor is a transcription regulatory factor of Corynebacterium glutamicum, which has the NCBI number NCgl1859 in the wild-type strain ATCC13032 of Corynebacterium glutamicum, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 6, and the protein sequence is shown in SEQ ID NO. 5; the NCBI number BBD29_09175 in the wild-type strain ATCC13869 of Corynebacterium glutamicum, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 8, and the protein sequence is shown in SEQ ID NO. 7; the NCBI number CEY17_09170 in the wild-type strain ATCC14067 of Corynebacterium glutamicum, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 10, and the protein sequence is shown in SEQ ID NO. 9.
[0026] Fourthly, the present invention provides a recombinant bacterium that expresses the above-described transcriptional regulatory factor mutant or contains a nucleic acid molecule encoding the transcriptional regulatory factor mutant.
[0027] Preferably, the recombinant bacteria are Corynebacterium or Escherichia. The Corynebacterium species is preferably Corynebacterium glutamicum; the Escherichia species is preferably Escherichia coli.
[0028] The recombinant bacteria described above are able to accumulate amino acids, and their threonine production and / or conversion rate are increased by expressing the mutants of the transcriptional regulatory factors described above or containing nucleic acid molecules encoding the mutants of the transcriptional regulatory factors described above.
[0029] In this invention, the conversion rate refers to the ratio of substrate to threonine. For example, when glucose is used as the substrate, the conversion rate is the glucose-acid conversion rate.
[0030] Preferably, the wild-type transcriptional regulatory factor of the recombinant bacteria is not expressed or is inactivated.
[0031] Preferably, the wild-type transcription factor of the recombinant bacteria is mutated to the transcription factor mutant described above.
[0032] The originating strain of the recombinant bacteria is not particularly limited in principle in this invention and can be any bacteria, preferably Corynebacterium or Escherichia. Preferably, the originating strain can be any strain capable of synthesizing and accumulating amino acids (especially threonine), including but not limited to wild-type strains, amino acid-producing strains obtained through genetic engineering or mutagenesis breeding.
[0033] The recombinant bacteria described above can be obtained by mutating the gene of the wild-type transcription factor in the starting strain so that it encodes the mutant of the transcription factor.
[0034] Preferably, the recombinant bacteria is Corynebacterium glutamicum, wherein the gene of the wild-type transcription regulatory factor is mutated so that it encodes the above-mentioned transcription regulatory factor mutant; The wild-type transcriptional regulator has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.
[0035] As a preferred embodiment of the present invention, a recombinant Corynebacterium glutamicum is provided, which expresses the above-described transcriptional regulatory factor mutant or contains a nucleic acid molecule encoding the transcriptional regulatory factor mutant. The amino acid yield (especially threonine) of the recombinant Corynebacterium glutamicum is significantly improved and / or the conversion rate is increased.
[0036] In some specific embodiments of the present invention, a recombinant Corynebacterium glutamicum is provided, wherein the gene sequence of the wild-type transcriptional regulatory factor, as shown in SEQ ID NO. 5, 7 or 9, is mutated, resulting in the protein it encodes being mutated into the transcriptional regulatory factor mutant described above.
[0037] The originating strain of the recombinant Corynebacterium glutamicum is not particularly limited in principle in this invention and can be any Corynebacterium glutamicum. Preferably, it is a Corynebacterium glutamicum capable of synthesizing and accumulating amino acids (especially threonine), including but not limited to wild-type Corynebacterium glutamicum, and amino acid-producing Corynebacterium glutamicum obtained through genetic engineering or mutagenesis breeding.
[0038] In some specific embodiments of the present invention, the starting strain is wild-type Corynebacterium glutamicum, and the wild-type Corynebacterium glutamicum is ATCC 13032, ATCC 13869 or ATCC 14067.
[0039] In some specific embodiments of the present invention, the starting strain is a threonine-producing Corynebacterium glutamicum genetically engineered bacterium, which, compared with wild-type Corynebacterium glutamicum, has an enhanced threonine-terminal synthesis pathway and relieves feedback inhibition on key genes at the threonine terminus.
[0040] Optionally, the genetically engineered bacteria comprises one or more of the following modifications: (1) Expression or overexpression of the thrABC gene; (2) Mutate the lysC gene so that the 311th amino acid of its encoded protein is changed from T to I (lysC). T311I ); (3) Mutate the hom gene so that the 378th amino acid of its encoded protein changes from G to E (hom G378E ).
[0041] In (1) above, the expression refers to the expression of a heterologous (preferably from Escherichia coli) thrABC gene; the overexpression refers to the enhancement of the expression of the endogenous thrABC gene.
[0042] It should be noted that the above modifications are merely examples, intended to endow the starting strain with a certain threonine production capacity, and do not constitute a restriction on the genetic background of the starting strain. Those skilled in the art can choose any strain with threonine synthesis and accumulation capacity as the starting strain.
[0043] This invention constructed recombinant strains expressing the aforementioned transcriptional regulatory factor mutants using Corynebacterium glutamicum with different genetic backgrounds as starting strains. Verification showed that these recombinant strains exhibited significantly increased threonine production and conversion efficiency. Therefore, the effect of the transcriptional regulatory factor mutant of this invention on enhancing the threonine production capacity of strains is independent of the genetic background of the aforementioned strains; the genetic modifications to the strains are solely intended to enable them to produce a certain amount of threonine. Thus, the effect of the transcriptional regulatory factor mutant of this invention on enhancing the threonine production capacity of strains is universally applicable to starting strains capable of synthesizing and accumulating threonine, and this mutant can be introduced into other Corynebacterium glutamicum.
[0044] Fifthly, the present invention provides a method for constructing recombinant bacteria, the method comprising: modifying the bacteria to express the above-described transcriptional regulatory factor mutants.
[0045] Preferably, the recombinant bacteria are Corynebacterium or Escherichia. The Corynebacterium species is preferably Corynebacterium glutamicum; the Escherichia species is preferably Escherichia coli.
[0046] As a preferred embodiment of the present invention, the bacteria is Corynebacterium glutamicum, and the method includes: mutating the gene of a wild-type transcriptional regulatory factor of Corynebacterium glutamicum to encode the above-described transcriptional regulatory factor mutant; the wild-type transcriptional regulatory factor has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.
[0047] Sixthly, the present invention provides the application of the above-described recombinant bacteria in the production of amino acids or their derivatives.
[0048] Preferably, the amino acid is threonine.
[0049] In this invention, the derivatives of the amino acid can be compounds synthesized using the amino acid as a precursor or intermediate. For threonine, its derivatives include isoleucine.
[0050] In a seventh aspect, the present invention provides a method for producing amino acids or their derivatives, the method comprising: culturing the recombinant bacteria described above, and collecting the amino acids or their derivatives from the culture.
[0051] Preferably, the method includes: seed culture of the recombinant bacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for fermentation culture to obtain fermentation broth, and separating and extracting the amino acid or its derivative from the fermentation broth.
[0052] Preferably, the amino acid is threonine.
[0053] Eighthly, the present invention provides a method for increasing the threonine yield and / or conversion rate of a strain, the method comprising: modifying the strain to express the above-described transcriptional regulatory factor mutant.
[0054] Preferably, the method includes: using genetic engineering methods to mutate the gene of the wild-type transcriptional regulatory factor of the strain, so that the encoded protein is mutated to the above-described transcriptional regulatory factor mutant; the wild-type transcriptional regulatory factor has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.
[0055] The beneficial effects of this invention include at least the following: the transcriptional regulatory factor mutant provided by this invention can significantly improve the production capacity of amino acids such as threonine in bacteria, and has good application prospects in amino acid strain modification. Based on this mutant, the threonine yield and conversion rate of the recombinant bacteria constructed by this invention are significantly improved, providing a new method and strategy for strain modification of amino acids such as threonine. Detailed Implementation
[0056] This invention provides a transcriptional regulatory factor mutant, which is a mutation of the 120th amino acid of the amino acid sequence shown in SEQ ID NO. 5, 7 or 9 to a serine (named cg2118 respectively). P120S -1、cg2118 P120S -2、cg2118 P120S -3), or mutate the 120th amino acid of the amino acid sequence shown in SEQ ID NO. 5, 7 or 9 to serine and the 75th amino acid to valine (named cg2118 respectively). G75V，P120S -1、cg2118 G75V，P120S -2、cg2118 G75V，P120S -3) obtained.
[0057] The present invention also provides a recombinant Corynebacterium glutamicum that expresses the above-described transcriptional regulatory factor mutant.
[0058] Corynebacterium glutamicum carrying the above-mentioned transcriptional regulatory factor mutants can be used to produce amino acids, especially threonine, with higher production efficiency.
[0059] Preferably, the recombinant Corynebacterium glutamicum is obtained by mutating the wild-type transcription factor gene of the starting strain to encode the transcription factor mutant.
[0060] Preferably, the starting strain is Corynebacterium glutamicum.
[0061] Preferably, the starting strain is obtained by introducing the thrABC gene of the threonine terminal synthesis pathway from Escherichia coli into Corynebacterium glutamicum and relieving the feedback inhibition of the key genes lysC and hom for threonine synthesis.
[0062] As an example, this invention uses three Corynebacterium glutamicum model strains as test subjects. The thrABC gene from the threonine terminal synthesis pathway of Escherichia coli was introduced using genetic engineering methods, and the feedback inhibition of the key threonine synthesis genes lysC and hom was relieved, resulting in three threonine-producing strains, named SMCT301, SMCT302, and SMCT303, which, along with the three Corynebacterium glutamicum model strains, served as starting strains. Based on the above six starting strains, the coding sequence of the wild-type transcription factor gene of the starting strains was mutated using genetic engineering methods, causing a mutation at amino acid position 120 of the protein sequence, changing proline to serine, or changing proline at position 120 of the protein sequence to serine while simultaneously changing glycine at position 75 to valine, thereby obtaining recombinant Corynebacterium glutamicum. Fermentation experiments verified that, compared with the starting strains, the threonine yield and conversion rate of the recombinant Corynebacterium glutamicum were significantly improved.
[0063] The *Corynebacterium glutamicum* strains ATCC 13032, ATCC 13869, and ATCC 14067 used in this invention are all model strains of *Corynebacterium glutamicum*, which are publicly available. Their genome sequences are publicly available and can be found on the NCBI website. *Escherichia coli* MG1655 is a model strain of *Escherichia coli*, which is also publicly available. Its genome sequence is publicly available and can be found on the NCBI website.
[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0065] In the following examples, the key gene lysC for threonine synthesis encodes aspartate kinase, and hom encodes homoserine dehydrogenase. Point mutations are used to remove the feedback inhibition of threonine on this enzyme. The threonine terminal synthesis pathway is achieved by referencing the thrABC operon from E. coli MG1655, where thrA encodes a bifunctional enzyme, namely aspartate kinase and homoserine dehydrogenase, thrB encodes homoserine kinase, and thrC encodes threonine synthase. Among them, lysC has the NCBI number NCgl0247 in Corynebacterium glutamicum ATCC13032, BBD29_01500 in ATCC13869, and CEY17_01500 in ATCC14067; hom has the NCBI number NCgl1136 in ATCC13032, BBD29_06325 in ATCC13869, and CEY17_06455 in ATCC14067; thrA, thrB, and thrC have the NCBI numbers IEU92_RS00010, IEU92_RS00015, and IEU92_RS00020, respectively.
[0066] In the following examples, the NCBI number of the gene encoding the wild-type transcriptional regulator in Corynebacterium glutamicum wild-type strain ATCC13032 is NCgl1859, the nucleotide sequence of which is shown in SEQ ID NO.6, and the protein sequence is shown in SEQ ID NO.5. The amino acid sequence of the mutant obtained based on this wild-type transcriptional regulator is shown in SEQ ID NO.1 or 2, and the gene sequence of which is shown in SEQ ID NO.3 or 4. The NCBI number of the gene encoding the wild-type transcriptional regulator in Corynebacterium glutamicum wild-type strain ATCC13869 is BBD29_09175, the nucleotide sequence of which is shown in SEQ ID NO.8, and the protein sequence is shown in SEQ ID NO.7. The NCBI number of the gene encoding the wild-type transcriptional regulator in Corynebacterium glutamicum wild-type strain ATCC14067 is CEY17_09170, the nucleotide sequence of which is shown in SEQ ID NO.10, and the protein sequence is shown in SEQ ID NO.9. The coding genes of transcription factor mutants can be obtained by fusion PCR or by whole-genome synthesis by a gene synthesis company. In this invention, the coding genes of the transcription factor mutants were obtained by whole-genome synthesis by a third-party gene synthesis company.
[0067] In the following embodiments, unless specific techniques or conditions are specified, they were performed in accordance with 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 through legitimate channels.
[0068] The plasmid pk18mobsacB-speC used in the following examples was prepared by genetically engineering the kanamycin resistance gene of the pK18mobsacB plasmid (GenBank: FJ1287239.1; available for purchase from public channels) to replace it with the spectinomycin resistance gene.
[0069] The primer sequence information used in the following examples is shown in Table 1.
[0070] Table 1 Primer sequence information
[0071] Example 1: Construction of an engineered plasmid carrying the thrABC gene, a threonine-terminal metabolic 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 product was purified by gel electrophoresis and gel extraction. The PCR product was a sequence fragment carrying the thrABC gene, a metabolic pathway gene with a threonine terminus.
[0072] The plasmid vector used 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 products were purified by gel electrophoresis and gel extraction for later use.
[0073] The sequence fragment carrying thrABC and the linearized vector were circularized and assembled. Assembly was performed using a single-fragment assembly kit from Novizan. Specific instructions for the kit can be found in the manufacturer's manual. Transformants were screened using kanamycin-resistant plates. The transformed individuals were cultured overnight in LB broth, and plasmids were extracted the following day for sequencing analysis. The correctly sequenced plasmid was named pVWEx1-thrABC.
[0074] Example 2: Carrying the key gene lysC for threonine synthesis T311I Construction of 3 engineered plasmids lysC of ATCC13032 synthesized from the whole genome T311I Using the sequence as a template, PCR amplification was performed with 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 sequence carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. T311I (It encodes a mutant of the lysC protein). The lysC protein of ATCC13869 was synthesized using the whole genome. T311IUsing the gene sequence as a template, PCR amplification was performed with the lysC-f-2 / lysC-r-2 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 ATCC13869. T311I (It encodes a mutant of the lysC protein). The lysC protein of ATCC14067 was synthesized using the whole genome. T311I Using the gene sequence as a template, PCR amplification was performed with 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 of the lysC protein); all mutants differ from the wild-type lysC protein in that the mutation at position 311 is a change from threonine to isoleucine.
[0075] The plasmid vector used 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.
[0076] The lysCs carrying the homologous recombination sequences ATCC13032, ATCC13869, and ATCC14067 were respectively... T311I The linearized vector was 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-resistant plates. The obtained transformants were cultured overnight in LB liquid medium, and plasmids were extracted the following day for sequencing analysis. LysC vectors correctly sequenced, carrying homologous recombination sequences of ATCC13032, ATCC13869, and ATCC14067, respectively. T311I The plasmid was named pK18mobsacB-speC-lysC T311I -1、pK18mobsacB-speC-lysC T311I -2 and pK18mob sacB-speC-lysC T311I -3.
[0077] Example 3: Carrying the key gene hom for threonine synthesis G378E Construction of 3 engineered plasmids homogeneous synthesis of ATCC13032 G378EUsing the sequence as a template, PCR amplification was performed with 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-f-1 primer carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13032. G378E (It encodes a mutant of the hom protein). The hom protein of ATCC13869 was synthesized using the whole genome. G378E Using the sequence as a template, PCR amplification was performed with 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-f-2 primer carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC13869. G378E (It encodes a mutant of the hom protein). The hom protein of ATCC14067 was synthesized using the whole genome. G378E Using the sequence as a template, PCR amplification was performed with 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-f-3 primer carrying the homologous recombination sequence of the Corynebacterium glutamicum model strain ATCC14067. G378E (It encodes a mutant hom protein). All mutants differ from the wild-type hom protein in that the mutation at position 378 is a change from G to E.
[0078] The plasmid vector used 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.
[0079] The above-mentioned homogeneous recombination sequences carrying ATCC13032, ATCC13869 and ATCC14067 were respectively... G378E The linearized vector was then circularized and assembled. Assembly was performed using the Novizan single-fragment assembly kit. Specific instructions were provided in the kit's manual. Transformants were screened on spectinomycin-resistant plates. The obtained transformants were cultured overnight in LB liquid medium, and plasmids were extracted the following day for sequencing analysis. Homologous recombination sequences carrying ATCC13032, ATCC13869, and ATCC14067 were correctly sequenced. G378E The plasmid was named pK18mobsacB-speC-hom G378E -1、pK18mobsacB-speC-hom G378E -2 and pK18mob sacB-speC-hom G378E -3.
[0080] Example 4 Construction of engineered plasmids carrying transcription factor mutants Transcription regulator mutants synthesized from the whole genome (the amino acid sequence of the mutant is obtained by mutating the 120th amino acid of the amino acid sequence shown in SEQ ID NO. 5, 7 or 9 to serine) were named cg2118 respectively. P120S -1、cg2118 P120S -2、cg2118 P120S -3), or mutate the 120th amino acid of the amino acid sequence shown in SEQ ID NO. 5, 7 or 9 to serine and the 75th amino acid to valine (named cg2118 respectively). G75V，P120S -1、cg2118 G75V ，P120S -2、cg2118 G75V，P120S -3) obtained; among which, the mutant cg2118 was obtained by mutating the 120th amino acid of SEQ ID NO.5 to serine. P120S The gene sequence of -1 is shown in SEQ ID NO.3. The mutant cg2118 was obtained by mutating amino acid position 120 of SEQ ID NO.5 to serine and amino acid position 75 to valine. G75V，P120S Using the gene sequence of cg2118 (as shown in SEQ ID NO.4) as a template, PCR amplification was performed with the cg2118-f / cg2118-r primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product is cg2118 carrying homologous recombination sequences. P120S and cg2118 G75V，P120S .
[0081] The plasmid vector used 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.
[0082] The above-mentioned cg2118 carrying homologous recombination sequences P120S -1、cg2118 P120S -2、cg2118 P120S -3、cg2118 G75V，P120S -1、cg2118 G75V，P120S -2、cg2118 G75V，P120S -3 were then circularized and assembled with the linearized vector. Assembly was performed using the Novizan single-fragment assembly kit. Specific instructions were provided in the kit's manual. Transformants were screened on spectinomycin resistance plates, and the obtained transformants were cultured overnight in LB liquid medium. Plasmids were extracted the following day and sequenced. The correctly sequenced plasmids were named pK18mobsacB-speC-cg2118. P120S-1、pK18mobsacB-speC-cg2118 P120S -2、pK18mobsacB-speC-cg2118 P120S -3 and pK18mobsacB-speC-cg2118 G75V，P120S -1、pK18mobsacB-speC-cg2118 G75V，P120S -2、pK18mobsacB-speC-cg2118 G75V ，P120S -3.
[0083] Example 5: Introduction of the thrABC gene for the threonine terminal pathway into the model strains of Corynebacterium glutamicum ATCC 13032, ATCC 13869, and ATCC 14067. Competent cells of the C. glutamicum model strains ATCC 13032, ATCC 13869, and ATCC 14067 were prepared and exogenous genes were expressed according to the method described in the C. glutamicum Handbook (Charpter 23).
[0084] The expression plasmid pVWEx1-thrABC was transformed into ATCC 13032, ATCC 13869, and ATCC 14067 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 result. The resulting modified strains were named 13032-thrABC, 13869-thrABC, and 14067-thrABC, representing the strains of ATCC13032, ATCC 13869, and ATCC 14067 carrying the pVWEx1-thrABC expression plasmid, respectively.
[0085] 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 genetic recombination was performed according to the method in the C. glutamicum Handbook (Charpter 23).
[0086] 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. 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 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, representing strains obtained by mutating the lysC gene in 13032-thrABC, 13869-thrABC, and 14067-thrABC, resulting in the T311I mutation in their encoded protein.
[0087] Following the methods outlined in the C. glutamicum Handbook (Charpter 23), competent cells of 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C were prepared and subjected to gene recombination.
[0088] The recombinant plasmid pK18mobsacB-speC-hom was electroporated. G378E -1、pK18mobsacB-speC-hom G378E -2 and pK18mobsacB-speC-hom G378ETransformations 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 as the final result. The resulting modified strains were named SMCT301, SMCT302, and SMCT303, respectively. These strains were created by mutating the hom gene in strains 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C, resulting in the G378E mutation in the encoded protein.
[0089] Example 7: Shake-flask fermentation test of the fermentation performance of three model strains after the introduction of the threonine terminal pathway gene and the removal of threonine feedback inhibition. The culture medium used in the shake-flask fermentation test is as follows: Plate activation medium: BHI 37 g / L, 20 g / L agar powder.
[0090] 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.
[0091] 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.
[0092] The fermentation method is as follows: 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 6-8 h with shaking. 3. Fermentation culture: Inoculate 6-8 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.
[0093] 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.
[0094] 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 bacteria (test bacteria) and the control bacteria, and the average value was calculated. The results are shown in Table 2.
[0095] Table 2. Detection of amino acid yield in recombinant bacteria
[0096] The amino acid content in the fermentation broth was analyzed, and it was found that *Corynebacterium glutamicum* carrying the threonine terminal pathway gene and having its threonine terminal restriction removed significantly increased the threonine concentration. Specifically, SMCT301 showed a 240.8% increase in threonine production compared to the wild-type strain ATCC 13032 (from 2.5 g / L to 7.6 g / L); SMCT302 showed a 246.1% increase in threonine production compared to the wild-type strain ATCC 13869 (from 2.3 g / L to 7.1 g / L); and SMCT303 showed a 262.2% increase in threonine production compared to the wild-type strain ATCC 14067 (from 2.2 g / L to 7.0 g / L). These results indicate that the ability of the strains to produce threonine was significantly enhanced after the threonine terminal synthesis pathway was opened. Therefore, it can be demonstrated that SMCT301, SMCT302, and SMCT303, after metabolic engineering modification, became threonine-producing strains.
[0097] Example 8: The transcriptional regulator mutant cg2118 was introduced into the Corynebacterium glutamicum model strain ATCC 13032 and the threonine-producing strain SMCT301, respectively. P120S -1 and cg2118 G75V，P120S -1 Competent cells of the C. glutamicum model strain ATCC 13032 and the threonine-producing strain SMCT301 were prepared and recombined according to the method in the C. glutamicum Handbook (Charpter 23).
[0098] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-cg2118 is recombined by electroporation. P120S -1 and pK18mobsacB-speC-cg2118 G75V，P120S Transformations were performed on ATCC 13032 and SMCT301 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, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The cultures were serially diluted (to 10⁻⁶ oz) to different concentrations. -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 as the final result. The final mutant cg2118, expressing the transcriptional regulatory factor, was obtained from the ATCC 13032 strain. P120S The target strain, named SMCT496, was modified to express the transcriptional regulatory factor mutant cg2118. G75V，P120S The target strain was named SMCT497; a transcription factor mutant cg2118 was obtained from the SMCT301 strain. P120S The target modified strain, named SMCT503, expressed the transcriptional regulatory factor mutant cg2118. G75V，P120S The modified strain with the target strain was named SMCT504.
[0099] Example 9: The transcriptional regulator mutant cg2118 was introduced into the Corynebacterium glutamicum model strain ATCC 13869 and the threonine-producing strain SMCT302, respectively. P120S -2 and cg2118 G75V，P120S -2 Competent cells of the C. glutamicum model strain ATCC 13869 and the threonine-producing strain SMCT302 were prepared and recombined according to the method in the C. glutamicum Handbook (Charpter 23).
[0100] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-cg2118 is recombined by electroporation. P120S -2 and pK18mobsacB-speC-cg2118 G75V，P120S Transformations were performed on ATCC 13869 and SMCT302 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 final result was a transcription factor mutant cg2118 expressing the transcription regulator, derived from strain ATCC 13869. P120S The target modified strain, named SMCT498, expressed the transcriptional regulatory factor mutant cg2118. G75V ，P120S The target strain was named SMCT499; a transcription factor mutant cg2118 was obtained from the starting strain SMCT302. P120S The target modified strain, named SMCT505, expressed the transcriptional regulatory factor mutant cg2118. G75V，P120S The modified strain was named SMCT506.
[0101] Example 10: The transcriptional regulator mutant cg2118 was introduced into the Corynebacterium glutamicum model strain ATCC 14067 and the threonine-producing strain SMCT303, respectively. P120S -3 and cg2118 G75V，P120S -3 Competent cells of the C. glutamicum model strain ATCC 14067 and the threonine-producing strain SMCT303 were prepared and recombined according to the method in the C. glutamicum Handbook (Charpter 23).
[0102] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-cg2118 is recombined by electroporation. P120S -3 and pK18mobsacB-speC-cg2118 G75V，P120S Transformations were performed on ATCC 14067 and SMCT303 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, the transformants underwent a second recombination, 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 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 final result was cg2118, a transcription factor mutant expressing a transcription regulator, derived from strain ATCC 14067. P120S The target strain, named SMCT500, was modified to express the transcriptional regulatory factor mutant cg2118. G75V ，P120S The target strain was named SMCT502; a transcription factor mutant cg2118 was obtained from the SMCT303 strain. P120S The target modified strain, named SMCT507, expressed the transcriptional regulatory factor mutant cg2118. G75V，P120S The modified strain with -3 was named SMCT508.
[0103] Example 11: Shake-flask fermentation test of the fermentation performance of three threonine-producing strains after introducing transcription factor mutants. The culture medium and fermentation method used in the shake-flask fermentation test were the same as in Example 7, as detailed below: Plate activation medium: BHI 37 g / L, 20 g / L agar powder.
[0104] 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.
[0105] 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.
[0106] The fermentation method is as follows: 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 6-8 h with shaking. 3. Fermentation culture: Inoculate 6-8 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.
[0107] 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. Three replicates were performed for each strain, and the average value was calculated. The results are shown in Table 3, where the data represents the average of the three replicates.
[0108] 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 bacteria (test bacteria) and the control bacteria, and the average value was calculated. The results are shown in Table 3.
[0109] Table 3. Detection of amino acid yield in recombinant bacteria
[0110] Note: The P-value after threonine production (g / L) is the P-value for analyzing the significant difference in threonine production between each test strain and its corresponding control strain; the P-value after conversion rate (%) is the P-value for analyzing the significant difference in conversion rate between each test strain and its corresponding control strain.
[0111] Analysis of the amino acid content in the fermentation broth revealed the presence of a transcriptional regulatory factor mutant, cg2118. P120S The threonine concentration in the culture medium of Corynebacterium glutamicum was increased to varying degrees, suggesting an increased efficiency in threonine synthesis or secretion; the transcriptional regulatory factor mutant cg2118 G75V，P120S Simultaneously carrying two mutations, it was found that the threonine concentration in the culture medium of *Corynebacterium glutamicum* carrying this mutant was significantly higher than that of the mutant cg2118. P120S The improvement was even more significant. The aforementioned increase in threonine production and conversion rate was due to the mutation of transcriptional regulatory factors.
[0112] In summary, the transcription factor mutants provided by this invention promote the production and yield of threonine in Corynebacterium. The mutated transcription factor is more conducive to driving the synthesis of threonine or promoting its secretion into the extracellular space. Furthermore, the transcription factor mutants of this invention are universally applicable and can improve the synthesis efficiency of different strains and various amino acids.
[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A mutant of a transcriptional regulatory factor, characterized in that, The mutant transcription factor, compared to the wild-type transcription factor of Corynebacterium glutamicum, contains a mutation at amino acid position 120 that replaces all amino acids except proline. Alternatively, the mutant transcription factor contains a mutation at amino acid position 120 (other than proline) and amino acid position 75 (valine) compared to the wild-type transcription factor of Corynebacterium glutamicum. The Corynebacterium glutamicum wild-type transcriptional regulatory factor has an amino acid sequence as shown in SEQ ID NO.5, 7 or 9.
2. The transcriptional regulatory factor mutant according to claim 1, characterized in that, The mutant transcription factor contains a mutation at amino acid position 120, which is a serine residue compared to the wild-type transcription factor of Corynebacterium glutamicum. Alternatively, the mutant transcription factor contains a mutation in which amino acid position 120 is changed to serine and amino acid position 75 is changed to valine, compared to the wild-type transcription factor of Corynebacterium glutamicum.
3. The transcriptional regulatory factor mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the transcription factor mutant is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.5 to serine; (2) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.7 to serine; (3) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.9 to serine.
4. The transcriptional regulatory factor mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the transcription factor mutant is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.5 to serine and the 75th amino acid to valine; (2) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.7 to serine and the 75th amino acid to valine; (3) The amino acid sequence obtained by mutating the 120th amino acid of the sequence shown in SEQ ID NO.9 to serine and the 75th amino acid to valine.
5. A biomaterial, characterized in that, The biomaterial includes any one of the following: (1) A nucleic acid molecule encoding a mutant of the transcriptional regulatory factor according to any one of claims 1 to 4; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A vector comprising the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1) or the expression cassette described in (2) or the vector described in (3).
6. Any of the following applications of the transcriptional regulatory factor mutant according to any one of claims 1 to 4 or the biological material according to claim 5: (1) Application in regulating the production capacity of bacterial amino acids or their derivatives; (2) Application in the construction of bacteria for the production of amino acids or their derivatives; (3) Applications in the production of amino acids or their derivatives; (4) Application in increasing the yield and / or conversion rate of bacterial amino acids or their derivatives; (5) Its application as a marker in screening bacteria for amino acid production; Preferably, the amino acid is threonine, and / or the bacteria are Corynebacterium or Escherichia.
7. A recombinant bacterium, characterized in that, The recombinant bacteria express the transcriptional regulatory factor mutant of any one of claims 1 to 4 or a nucleic acid molecule containing the transcriptional regulatory factor mutant of any one of claims 1 to 4.
8. The recombinant bacteria according to claim 7, characterized in that, The recombinant bacteria are either Corynebacterium or Escherichia; Preferably, the recombinant bacteria is Corynebacterium glutamicum, wherein the gene of the wild-type transcription regulatory factor is mutated so that it encodes a mutant of the transcription regulatory factor according to any one of claims 1 to 4; The wild-type transcriptional regulator has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.
9. A method for constructing recombinant bacteria, characterized in that, The method includes: modifying bacteria to express the transcriptional regulatory factor mutant according to any one of claims 1 to 4; Preferably, the bacteria is Corynebacterium glutamicum, and the method includes: mutating the gene of a wild-type transcriptional regulatory factor of Corynebacterium glutamicum to encode a mutant of the transcriptional regulatory factor according to any one of claims 1 to 4; The wild-type transcriptional regulator has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.
10. A method for producing amino acids or their derivatives, characterized in that, The method includes: culturing the recombinant bacteria according to claim 7 or 8, and collecting the amino acid or its derivative from the culture; Preferably, the amino acid is threonine.