Mutated transcription factors and their use in amino acid production

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a mutant transcription factor and its application in amino acid production. The present application finds that the transcription factor rpoC can improve the production capacity of strains for threonine and other amino acids. The mutant transcription factor provided by the present application can effectively promote the synthesis and extracellular accumulation of threonine, improve the production capacity of strains for threonine, and significantly improve the threonine yield and conversion rate of recombinant bacteria expressing the mutant transcription factor. The new use of the transcription factor rpoC and its mutant provided by the present application provide a new strategy and method for improving the performance of amino acid production strains, and have a good application prospect in the modification of amino acid strains.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to mutated transcription factors and their application in amino acid production. Background Technology

[0002] The rpoC gene encodes the β' subunit protein of RNA polymerase, which can act as a transcription factor to regulate gene transcription. Although there have been some reports on the regulation of rpoC through mutations, there are few reports on its use in regulating amino acid production, and even fewer reports on rpoC mutants that can increase amino acid production.

[0003] Amino acids are widely used in food, feed, and pharmaceuticals, and market demand is increasing year by year. Among them, L-threonine is currently the third most produced amino acid industrially via microbial fermentation, after L-glutamic acid and L-lysine. Corynebacterium glutamicum (… Corynebacterium glutamicum Microorganisms are commonly used strains for amino acid production. Due to their rapid growth, non-pathogenicity, and weak ability to degrade their own metabolites, they have broad application prospects in the field of amino acid fermentation. To improve the amino acid production capacity of these strains, it is still necessary to develop new modification targets and strategies to increase amino acid synthesis efficiency and reduce production costs. Summary of the Invention

[0004] This invention provides mutated transcription factors and their application in amino acid production.

[0005] In the process of research on amino acid fermentation production, this invention discovered that regulating the transcription factor rpoC has a significant impact on the synthesis and extracellular accumulation of amino acids such as threonine, and can be used to construct amino acid-producing bacteria such as threonine. Furthermore, this invention developed a mutant transcription factor rpoC that can promote threonine synthesis and accumulation, and constructed a recombinant bacterium expressing this mutant transcription factor. Verification showed that the threonine yield and conversion rate of the recombinant bacterium were significantly improved.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides the application of transcription factors in enhancing the amino acid production capacity of bacteria or constructing amino acid-producing bacteria, said transcription factors having an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.

[0008] Specifically, the above applications include: regulating the transcription factors to improve the amino acid production capacity of bacteria or constructing amino acid-producing bacteria.

[0009] The regulation described above can refer to the regulation of expression levels or the regulation of enzyme activity levels.

[0010] Preferably, the amino acid is threonine.

[0011] Preferably, the bacteria or producing bacteria are Corynebacterium or Escherichia. Specifically, the Corynebacterium species is preferably Corynebacterium glutamicum; the Escherichia species is preferably Escherichia coli.

[0012] Secondly, the present invention provides a mutated transcription factor, which, compared with the wild-type transcription factor rpoC of Corynebacterium glutamicum, contains a mutation in which the 41st amino acid is mutated to serine or threonine.

[0013] This invention discovered that a specific amino acid mutation in the transcription factor rpoC has a positive promoting effect on the synthesis and extracellular accumulation of threonine (unless otherwise specified in this invention, all amino acids refer to L-amino acids). Based on this, this invention developed a transcription factor with the aforementioned mutation. Verification showed that mutating the 41st amino acid of the transcription factor rpoC to serine or threonine can effectively promote threonine synthesis, significantly increasing threonine yield and conversion rate.

[0014] Preferably, the mutated transcription factor contains a mutation at position 41 proline to serine or threonine, compared to the wild-type transcription factor rpoC from Corynebacterium glutamicum. The wild-type transcription factor rpoC of Corynebacterium glutamicum has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.

[0015] In this invention, the NCBI sequence of the wild-type transcription factor rpoC of Corynebacterium glutamicum is NCgl0472 in the wild-type strain ATCC13032, and its nucleotide sequence is shown in SEQ ID NO.6, while its protein sequence is shown in SEQ ID NO.5; in the wild-type strain ATCC13869, the NCBI sequence is BBD29_02890, and its nucleotide sequence is shown in SEQ ID NO.8, while its protein sequence is shown in SEQ ID NO.7; in the wild-type strain ATCC14067, the NCBI sequence is CEY17_02800, and its nucleotide sequence is shown in SEQ ID NO.10, while its protein sequence is shown in SEQ ID NO.9.

[0016] As a preferred embodiment of the present invention, the amino acid sequence of the mutated transcription factor is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.5 to serine or threonine; (2) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.7 to serine or threonine; (3) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.9 to serine or threonine.

[0017] For example, the mutant in (1) above has an amino acid sequence as shown in SEQ ID NO.1 or 2.

[0018] This invention experimentally verified that the mutated transcription factors shown in (1)-(3) above 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 amino acid sequences shown in (1)-(3) above will not have a significant impact on the function of the mutated transcription factor. For example, adding amino acid residues such as protein tags, signal peptides, and linker peptides to the N-terminus and / or C-terminus of the amino acid sequences shown in (1)-(3) above can still achieve the above-mentioned effect of improving threonine production capacity, and therefore is also within the scope of protection of this invention.

[0019] Thirdly, the present invention provides a nucleic acid molecule that encodes the above-described mutated transcription factor.

[0020] Based on the amino acid sequence of the mutated transcription factor provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the mutated transcription factor. Due to the degeneracy of codons, the nucleotide sequence encoding a mutated transcription factor is not unique, and all nucleic acid molecules capable of encoding the mutated transcription factor are within the scope of protection of this invention.

[0021] 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.

[0022] Fourthly, the present invention provides biological materials comprising the above-mentioned nucleic acid molecules, wherein the biological material is an expression cassette, a vector, or a host cell.

[0023] The expression cassette is a recombinant nucleic acid molecule obtained by operatively linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.

[0024] The vectors include, but are not limited to, plasmid vectors, viral vectors, and transposons.

[0025] The host cell is any cell capable of carrying the aforementioned nucleic acid molecules or expressing the aforementioned mutated transcription factors, including microbial cells, preferably Escherichia coli, Corynebacterium, or Bacillus.

[0026] Fifthly, the present invention provides any of the following applications of the mutated transcription factor, the nucleic acid molecule, or the biological material described above: (1) Application in the production of amino acids or their derivatives by microorganisms; (2) Applications in the production of amino acids or their derivatives; (3) Application in increasing the yield and / or conversion rate of amino acids or their derivatives in the producing bacteria; (4) Its application as a marker in screening high-yield amino acid-producing bacteria; In the above applications, the amino acid is preferably threonine.

[0027] 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.

[0028] In a sixth aspect, the present invention provides a recombinant bacterium that expresses the above-described mutated transcription factor or contains the nucleic acid molecule.

[0029] Preferably, the original transcription factor rpoC of the recombinant bacteria is not expressed or is inactivated.

[0030] Preferably, the transcription factor rpoC of the recombinant bacteria is mutated to the mutated transcription factor described above.

[0031] The transcription factor rpoC of the aforementioned recombinant bacteria refers to the original transcription factor rpoC of that bacteria.

[0032] Preferably, the recombinant bacteria are Corynebacterium or Escherichia. The Corynebacterium species is preferably Corynebacterium glutamicum; the Escherichia species is preferably Escherichia coli.

[0033] The recombinant strains described above exhibited increased threonine production and / or conversion rates compared to the original strains.

[0034] 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.

[0035] The present invention does not impose any special restrictions on the starting strain of the recombinant bacteria. It can be any strain capable of synthesizing and accumulating threonine, including but not limited to wild-type strains, threonine-producing strains obtained by genetic engineering or mutagenesis breeding.

[0036] The recombinant bacteria described above can be obtained by mutating the original transcription factor rpoC encoding gene in the starting strain so that it encodes the mutated transcription factor.

[0037] As a preferred embodiment of the present invention, a recombinant Corynebacterium glutamicum is provided, which expresses the above-described mutated transcription factor or contains the above-described nucleic acid molecule. The threonine yield and / or conversion rate of the recombinant Corynebacterium glutamicum are improved.

[0038] In some specific embodiments of the present invention, a recombinant Corynebacterium glutamicum is provided, wherein the gene sequence encoding the transcription factor rpoC is mutated, resulting in the protein it encodes being mutated into the aforementioned mutated transcription factor.

[0039] The present invention does not impose any particular restrictions on the starting strain of the recombinant Corynebacterium glutamicum. It can be any Corynebacterium glutamicum capable of synthesizing and accumulating threonine, including but not limited to wild-type Corynebacterium glutamicum, threonine-producing Corynebacterium glutamicum obtained through genetic engineering or mutagenesis breeding.

[0040] 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.

[0041] 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.

[0042] 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 ).

[0043] 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.

[0044] This invention constructed recombinant strains expressing the mutated transcription factor using Corynebacterium glutamicum with different genetic backgrounds as starting strains. Verification showed that the threonine production and conversion rate of these recombinant strains were significantly improved. Therefore, the mutated transcription factor of this invention enhances the threonine production capacity of the strains independently of the genetic background of the strains; the genetic modification of the strains is solely to enable them to produce a certain amount of threonine. Thus, the mutated transcription factor of this invention has universal applicability to starting strains capable of synthesizing and accumulating threonine, and its introduction into other threonine-producing strains can effectively promote the increase of threonine production and / or conversion rate.

[0045] In a seventh aspect, the present invention provides the application of the above-described recombinant bacteria in the production of amino acids or their derivatives.

[0046] Preferably, the amino acid is threonine.

[0047] In this invention, the derivatives of the amino acid can be compounds synthesized using the amino acid as a precursor. For threonine, its derivatives include isoleucine.

[0048] Eighthly, 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.

[0049] 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.

[0050] Preferably, the amino acid is threonine.

[0051] In a ninth aspect, the present invention provides a method for increasing the threonine yield and / or conversion rate of a bacterial strain, the method comprising: modifying the bacterial strain to express the mutated transcription factor described above.

[0052] Preferably, the method includes: using genetic engineering methods to mutate the transcription factor rpoC gene of the strain, so that the protein it encodes is mutated into the mutated transcription factor described above.

[0053] The beneficial effects of this invention include at least the following: This invention discovers that regulating the transcription factor rpoC can improve the production capacity of amino acids such as threonine in strains. The mutant transcription factor provided by this invention can effectively promote the synthesis and extracellular accumulation of threonine, thereby enhancing the threonine production capacity of strains. The threonine yield and conversion rate of recombinant bacteria expressing this mutant transcription factor are significantly improved. The novel uses of the transcription factor rpoC and its mutants provided by this invention offer new strategies and methods for improving the performance of amino acid-producing strains, and have good application prospects in the modification of amino acid strains. Detailed Implementation

[0054] This invention provides a mutated transcription factor whose amino acid is obtained by mutating proline at position 41 of the amino acid sequence shown in SEQ ID NO. 5, 7, or 9 to serine (named rpoC, respectively). P41S -1, rpoC P41S -2, rpoC P41S -3) or threonine (named rpoC respectively) P41T -1, rpoC P41T -2, rpoC P41T -3) obtained.

[0055] The present invention also provides a threonine-producing Corynebacterium glutamicum that expresses the above-described mutated transcription factor.

[0056] Corynebacterium glutamicum carrying the above-mentioned mutations can be used to produce amino acids, especially threonine, with higher production efficiency.

[0057] Preferably, the threonine-producing Corynebacterium glutamicum is obtained by mutating the rpoC gene of the starting strain to encode the mutated transcription factor.

[0058] 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.

[0059] 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 original transcription factor rpoC gene in the starting strains was mutated using genetic engineering methods, causing a mutation at position 41 of its protein sequence, changing proline to serine or threonine, thereby obtaining threonine-producing Corynebacterium glutamicum. Fermentation experiments verified that, compared with the starting strains, the threonine-producing Corynebacterium glutamicum showed significantly improved threonine yield and conversion rate.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In the following examples, the NCBI number of the wild-type transcription factor (rpoC) encoding gene in the wild-type Corynebacterium glutamicum strain ATCC13032 is NCgl0472, and its nucleotide sequence is shown in SEQ ID NO. 6, while its protein sequence is shown in SEQ ID NO. 5. The amino acid sequence of the mutant transcription factor (rpoC) obtained based on this wild-type is shown in SEQ ID NO. 1 or 2, and its encoding gene sequence is shown in SEQ ID NO. 3 or 4. The NCBI number of the wild-type transcription factor (rpoC) encoding gene in the wild-type Corynebacterium glutamicum strain ATCC13869 is BBD29_02890, and its nucleotide sequence is shown in SEQ ID NO. 8, while its protein sequence is shown in SEQ ID NO. 7. The NCBI number of the wild-type transcription factor (rpoC) encoding gene in the wild-type Corynebacterium glutamicum strain ATCC14067 is CEY17_02800, and its nucleotide sequence is shown in SEQ ID NO. 10, while its protein sequence is shown in SEQ ID NO. 5. As shown in NO.9. The encoding gene of the mutated transcription factor can be obtained by fusion PCR or by whole-genome synthesis by a gene synthesis company. The encoding gene of the mutated transcription factor of this invention was obtained by whole-genome synthesis by a third-party gene synthesis company.

[0064] 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.

[0065] 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.

[0066] The primer sequence information used in the following examples is shown in Table 1.

[0067] Table 1 Primer sequence information

[0068] 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.

[0069] The plasmid vector used was pVWEx1 (GenBank: MF034723.1; purchased from public sources). The 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.

[0070] 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 kit's manual. Transformants were screened using kanamycin-resistant plates. The transformed individuals were cultured overnight in LB liquid medium, and plasmids were extracted the following day for sequencing analysis. The correctly sequenced plasmid was named pVWEx1-thrABC.

[0071] Example 2: Carrying the key gene lysC for threonine synthesis T311I Construction of 3 engineered plasmids lysC of ATCC13032 synthesized from the whole genome T311IUsing 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. T311I Using 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). The mutation in all mutants compared to the wild-type lysC protein is a change from threonine to isoleucine at position 311.

[0072] The plasmid vector used was pK18mobsacB-speC, which was 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.

[0073] The lysCs carrying the homologous recombination sequences ATCC13032, ATCC13869, and ATCC14067 respectively were used to... T311I The linearized vector was then circularized and assembled. Assembly was performed using a single-fragment assembly kit from Novizan. Specific instructions for the kit were provided. Transformants were screened using 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.

[0074] Example 3: Carrying the key gene hom for threonine synthesis G378E Construction of 3 engineered plasmids homogeneous synthesis of ATCC13032 G378E Using 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.

[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 above-mentioned homologous recombination sequences carrying ATCC13032, ATCC13869 and ATCC14067 respectively were used to... 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 using 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.

[0077] Example 4 Construction of engineered plasmids carrying transcription factor mutants Transcription factor mutants synthesized from the whole genome (the amino acid sequences of the mutants are respectively obtained by mutating proline at position 41 of the amino acid sequences shown in SEQ ID NO. 5, 7, and 9 to serine) (named rpoC respectively) P41S -1, rpoC P41S -2, rpoC P41S -3) or threonine (named rpoC respectively) P41T -1, rpoC P41T -2, rpoC P41T -3) We obtained two mutants, rpoC, by mutating the proline at position 41 of the sequence shown in SEQ ID NO.5 to serine and threonine. P41S -1, rpoC P41T Using the gene sequence encoding -1 (as shown in SEQ ID NO. 3 and 4) as a template, PCR amplification was performed with the rpoC-f / rpoC-r primer pair. The PCR product was purified by gel electrophoresis and gel extraction. The PCR product is an rpoC gene carrying a homologous recombination sequence. P41S or rpoC P41T .

[0078] The plasmid vector used was pK18mobsacB-speC, which was 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 rpoC carrying homologous recombination sequences P41S -1, rpoC P41S -2, rpoC P41S -3, rpoC P41T -1, rpoC P41T -2, rpoC P41T -3 fragments were 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 using spectinomycin-resistant plates. The obtained transformants were cultured overnight in LB liquid medium, and plasmids were extracted the following day for sequencing analysis. The correctly sequenced plasmid was named pK18mobsacB-speC-rpoC. P41S -1、pK18mobsacB-speC-rpoC P41S -2、pK18mobsacB-speC-rpoCP41S -3 and pK18mobsacB-speC-rpoC P41T -1、pK18mobsacB-speC-rpoC P41T -2、pK18mobsacB-speC-rpoC P41T -3.

[0080] Example 5: Introduction of the thrABC gene into the Corynebacterium glutamicum model strains ATCC 13032, ATCC 13869, and ATCC 14067. Competent cells of Corynebacterium glutamicum model strains ATCC13032, ATCC13869, and ATCC14067 were prepared and exogenous genes were expressed according to the method in the C. glutamicum Handbook (Charpter 23).

[0081] 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.

[0082] 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).

[0083] 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 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 finally obtained modified strains were named 13032-thrABC-C, 13869-thrABC-C, and 14067-thrABC-C, respectively, which were obtained by mutating the lysC gene in 13032-thrABC, 13869-thrABC, and 14067-thrABC, resulting in the T311I mutation in the encoded protein.

[0084] 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.

[0085] 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 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.

[0086] 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.

[0087] 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.

[0088] 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 C 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Table 2. Detection of amino acid yield in recombinant bacteria

[0093] The amino acid content in the fermentation broth was analyzed, and it was found that the threonine concentration of Corynebacterium glutamicum carrying the threonine terminal pathway gene and removing the threonine terminal restriction was significantly increased. Specifically, SMCT301 increased threonine production from 2.5 g / L to 7.6 g / L compared to the wild-type strain ATCC 13032, with a conversion rate increase of 240.8%; SMCT302 increased threonine production from 2.3 g / L to 7.1 g / L compared to the wild-type strain ATCC13869, with a conversion rate increase of 246.1%; and SMCT303 increased threonine production from 2.2 g / L to 7.0 g / L compared to the wild-type strain ATCC 14067, with a conversion rate increase of 262.2%. These results indicate that opening the threonine terminal synthesis pathway significantly enhances the threonine production capacity of the strains. This demonstrates that through metabolic engineering, SMCT301, SMCT302, and SMCT303 have become threonine-producing strains.

[0094] Example 8: The transcription factor mutant rpoC was introduced into the Corynebacterium glutamicum model strain ATCC 13032 and the threonine-producing strain SMCT301, respectively. P41S -1, rpoC P41T -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).

[0095] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-rpoC is recombined by electroporation. P41S -1 and pK18mobsacB-speC-rpoC P41T 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 result was a transcription factor mutant rpoC expressed by the ATCC 13032 strain. P41S The target strain, named SMCT421, was modified to express the transcription factor mutant rpoC. P41T The target strain was named SMCT422; a transcription factor mutant rpoC was obtained from the starting strain SMCT301. P41S The target modified strain, named SMCT427, expressed the transcription factor mutant rpoC. P41T The modified strain with the target strain was named SMCT428.

[0096] Example 9: The transcription factor mutant rpoC was introduced into the Corynebacterium glutamicum model strain ATCC 13869 and the threonine-producing strain SMCT302, respectively. P41S -2, rpoC P41T -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).

[0097] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-rpoC is recombined by electroporation. P41S and pK18mobsacB-speC-rpoC P41T 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, 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 result was the transcription factor mutant rpoC expressed by the ATCC 13869 strain. P41S The target modified strain, named SMCT423, expressed the transcription factor mutant rpoC. P41T The target strain was named SMCT424; a transcription factor mutant rpoC was obtained from the starting strain SMCT302. P41S The target modified strain, named SMCT429, expressed the transcription factor mutant rpoC. P41T The modified strain was named SMCT430.

[0098] Example 10: The transcription factor mutant rpoC was introduced into the Corynebacterium glutamicum model strain ATCC 14067 and the threonine-producing strain SMCT303, respectively. P41S -3, rpoC P41T -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).

[0099] The recombination method is the same as in Example 6, that is, the recombinant plasmid pK18mobsacB-speC-rpoC is recombined by electroporation. P41S -3 and pK18mobsacB-speC-rpoC P41TTransformations 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 a transcription factor mutant rpoC expressed by the ATCC 14067 strain. P41S The target strain was named SMCT425 and expressed the transcription factor mutant rpoC. P41T The target strain was named SMCT426; a transcription factor mutant rpoC was obtained from the starting strain SMCT303. P41S The target modified strain, named SMCT431, expressed the transcription factor mutant rpoC. P41T The modified strain with -3 was named SMCT432.

[0100] Example 11: Shake-flask fermentation test of the fermentation performance of three wild-type strains and three threonine-producing strains after the introduction of 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Table 3. Detection of amino acid yield in recombinant bacteria

[0107] 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.

[0108] Analysis of the amino acid content in the fermentation broth revealed that the threonine concentration in the culture medium of *Corynebacterium glutamicum* carrying a transcription factor mutant was increased to varying degrees, suggesting an enhanced efficiency in threonine synthesis or secretion, which is attributed to the mutated transcription factor. Among different threonine-producing bacteria, those carrying the mutant rpoC... P41SThe recombinant strain showed a more significant increase in threonine production and yield.

[0109] In summary, the mutant transcription factor provided by this invention promotes the production and yield of threonine in Corynebacterium. The mutant transcription factor is more conducive to the synthesis of threonine or promotes its secretion into the extracellular space. In addition, the mutant transcription factor has universality and can improve the synthesis efficiency of different strains and various amino acids.

[0110] 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. The application of transcription factors in enhancing the amino acid production capacity of bacteria or constructing amino acid-producing bacteria, said transcription factors having an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9.

2. The application according to claim 1, characterized in that, The applications include: regulating the transcription factors to enhance the amino acid production capacity of bacteria or constructing amino acid-producing bacteria; Preferably, the amino acid is threonine, and / or the bacteria or producing bacteria are Corynebacterium or Escherichia.

3. A mutated transcription factor, characterized in that, The mutated transcription factor, compared to the wild-type transcription factor rpoC from Corynebacterium glutamicum, contains a mutation at amino acid position 41 that changes to serine or threonine.

4. The mutated transcription factor according to claim 3, characterized in that, The mutated transcription factor, compared to the wild-type transcription factor rpoC from Corynebacterium glutamicum, contains a mutation at position 41 where proline is changed to serine or threonine. The wild-type transcription factor rpoC of Corynebacterium glutamicum has an amino acid sequence as shown in SEQ ID NO. 5, 7 or 9; Preferably, the amino acid sequence of the mutated transcription factor is any one of the following (1)-(3): (1) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.5 to serine or threonine; (2) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.7 to serine or threonine; (3) The amino acid sequence obtained by mutating the 41st proline of the sequence shown in SEQ ID NO.9 to serine or threonine.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the mutated transcription factor as described in claim 3 or 4.

6. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule as described in claim 5; the biomaterial is an expression cassette, vector, or host cell.

7. Any of the following applications of the mutated transcription factor of claim 3 or 4, the nucleic acid molecule of claim 5, or the biological material of claim 6: (1) Application in the production of amino acids or their derivatives by microorganisms; (2) Applications in the production of amino acids or their derivatives; (3) Application in increasing the yield and / or conversion rate of amino acids or their derivatives in the producing bacteria; (4) Its application as a marker in screening high-yield amino acid-producing bacteria; Preferably, the amino acid is threonine, and / or the bacteria are Corynebacterium or Escherichia.

8. A recombinant bacterium, characterized in that, The recombinant bacteria express the mutated transcription factor as described in claim 3 or 4, or contain the nucleic acid molecule as described in claim 5.

9. The recombinant bacteria according to claim 8, characterized in that, The recombinant bacteria has a transcription factor rpoC mutation that is the mutated transcription factor as described in claim 3 or 4; Preferably, the recombinant bacteria are Corynebacterium or Escherichia.

10. A method for producing amino acids or their derivatives, characterized in that, The method includes: culturing the recombinant bacteria according to claim 8 or 9, and collecting the amino acid or its derivative from the culture; Preferably, the amino acid is threonine.

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