Bifunctional (p) ppGpp synthetase or hydrolase mutant, biological material and application thereof in preparation of L-threonine

Abstract

The invention discloses a double-function (p) ppGpp synthetase or hydrolase mutant, a biological material and application of the double-function (p) ppGpp synthetase or hydrolase mutant to preparation of L-threonine, and belongs to the technical field of gene engineering. The technical problem to be solved by the invention is how to improve the yield of L-threonine. Relative to a wild type, the bifunctional (p) ppGpp synthetase or hydrolase mutant comprises one or more of the following mutations: K1) mutation of 174th amino acid of SEQ ID NO: 2 into cysteine, K2) mutation of 529th amino acid of SEQ ID NO: 2 into phenylalanine, and K3) insertion of histidine and aspartic acid between 84th amino acid and 85th amino acid of SEQ ID NO: 2. The bifunctional (p) ppGpp synthetase or hydrolase mutant provided by the invention can be used for improving the yield of L-threonine of Escherichia coli.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to bifunctional (p)ppGpp synthase or hydrolase mutants, biomaterials and their application in the preparation of L-threonine. Background Technology

[0002] L-Threonine is a limiting amino acid widely used in the pharmaceutical, food, and feed industries. The application of *E. coli* in threonine production dates back to the late 20th and early 21st centuries. With advancements in biotechnology, scientists discovered that metabolic engineering of *E. coli* can efficiently produce L-threonine. The application of metabolic engineering has made *E. coli* the primary strain for industrial L-threonine production due to its short growth cycle, high cell strength, and low equipment requirements.

[0003] In Escherichia coli, the synthesis of L-threonine begins with aspartic acid and is gradually converted to L-threonine through a series of enzymatic reactions. These reactions are controlled by a variety of enzymes, and changes in each step of the metabolic pathway affect the system's parameters and metabolic flux.

[0004] Threonine is formed when aspartic semialdehyde is converted to homoserine by NADPH under the catalysis of homoserine dehydrogenase; then it is phosphorylated by ATP under the catalysis of homoserine kinase to generate O-phosphohomoserine, and finally converted to threonine under the catalysis of threonine synthase.

[0005] Methionine is produced by the reaction of homoserine with succinyl-CoA catalyzed by homoserine succinyltransferase to generate O-succinylhomoserine. Then, O-succinylhomoserine reacts with cysteine ​​under the catalysis of cystathionine-γ-synthase to generate cystathionine. Cystathionine is then cleaved under the catalysis of cystathionine-β-lyase to produce homocysteine ​​and ketobutyrate as byproducts. This step also requires PLP. Finally, homocysteine ​​is converted from N-methionine to methionine under the catalysis of methionine synthase. 5 -Methyltetrahydrofolate acquires a methyl group, ultimately forming methionine.

[0006] Although threonine and methionine both originate from aspartic acid, they differ significantly in key regulatory nodes, key enzymes, energy consumption, feedback mechanisms, and their association with other pathways. Their synthesis processes also differ in key enzymes and cofactors. The threonine pathway lacks sulfur atoms and methylation steps; methionine requires the introduction of sulfur (from cysteine) and methylation (requiring vitamin B12). 12 (folic acid); threonine is inhibited by threonine / isoleucine synergistic feedback; methionine is strongly inhibited by its own or S-adenosylmethionine feedback; threonine is mainly used in protein synthesis and isoleucine precursor; methionine is a protein component and a general methyl donor precursor.

[0007] Bifunctional (p)ppGpp synthases or hydrolases possess both synthetic and hydrolytic activities, catalyzing the production or degradation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp. These molecules are key signaling molecules for bacteria in response to environmental stresses such as nutrient deficiency and antibiotic stress. However, they are not key enzymes or proteins in the methionine or threonine metabolic pathways. Therefore, while these enzymes and their mutants may affect methionine production, their impact on threonine production remains scientifically inconclusive. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide mutants that can improve L-threonine preparation or increase L-threonine yield. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a bifunctional (p)ppGpp synthase or hydrolase mutant, wherein the bifunctional (p)ppGpp synthase or hydrolase mutant comprises any one of the following: A1) Its amino acid sequence is the sequence obtained by inserting a histidine residue and an aspartic acid residue between the 84th and 85th amino acid residues of SEQ ID NO:2; A2) A protein having more than 95% identity and the same function as the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1). A3) A fusion protein with the same function is obtained by attaching a tag protein to the N-terminus and / or C-terminus of the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1) or A2).

[0010] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:4.

[0011] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:4.

[0012] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:3.

[0013] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:3.

[0014] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0015] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0016] In this document, the 98% or higher degree of identity may be at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the degree of identity.

[0017] In the above method, the bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 174th amino acid residue of the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutant to a cysteine ​​residue.

[0018] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:11.

[0019] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:11.

[0020] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:10.

[0021] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:10.

[0022] In the above method, the bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 529th amino acid residue of the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutant to a phenylalanine residue.

[0023] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:13.

[0024] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:13.

[0025] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:12.

[0026] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:12.

[0027] In the above method, the bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 174th amino acid residue of the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutant to a cysteine ​​residue and mutating the 529th amino acid residue to a phenylalanine residue.

[0028] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:15.

[0029] In one specific embodiment of the present invention, the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:15.

[0030] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant includes SEQ ID NO:14.

[0031] In one specific embodiment of the present invention, the coding sequence (CDS) encoding a bifunctional (p)ppGpp synthase or hydrolase mutant is SEQ ID NO:14.

[0032] The present invention also provides biomaterials, said biomaterials comprising any of the following: B1) Nucleic acid molecules encoding the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutants; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3) or the recombinant microorganism described in B4).

[0033] In the aforementioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to DNA capable of expressing the proteins described above in host cells. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for expressing any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in suitable host cells. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein into the secretion pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those systems that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification.

[0034] In some embodiments of the present invention, the whole-cell catalyst is a recombinant cell.

[0035] In the aforementioned biological materials, the nucleic acid molecule described in B1) includes any one of b11-b13). b11) A nucleic acid molecule containing a coding sequence, said coding sequence including any one of SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; b12) Nucleic acid molecules having a similarity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or higher to the coding sequences described in b11); b13) Hybridizes with the nucleic acid molecule defined by b11) under strict conditions and encodes the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutant nucleic acid molecule.

[0036] The present invention also provides the application of the aforementioned bifunctional (p)ppGpp synthase or hydrolase mutant and / or the aforementioned biological material and / or the aforementioned whole-cell catalyst, wherein the application is selected from at least one of the following: K1) The application of the aforementioned mutants or biomaterials in the preparation of L-threonine; K2) The application of the aforementioned mutants or biomaterials in the preparation of products containing L-threonine; K3) The application of the aforementioned mutants or biological materials in enhancing the enzyme activity of the aforementioned proteins; K4) The application of the aforementioned mutants or biological materials in the construction of recombinant microorganisms for the production of L-threonine; K5) The application of the aforementioned mutants or biological materials in regulating the production of L-threonine by microorganisms.

[0037] The present invention also provides a method for preparing recombinant microorganisms, wherein the recombinant microorganisms are prepared by means of the following steps: introducing the aforementioned nucleic acid molecules, expression cassettes or recombinant vectors into a recipient microorganism, wherein the recipient microorganism includes Escherichia coli.

[0038] The present invention also provides the application of the aforementioned proteins and / or the aforementioned biomaterials and / or the aforementioned whole-cell catalysts in lysine, threonine, tryptophan, arginine, valine, glycine, alanine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutarate, citric acid, ornithine, and citrulline.

[0039] This invention also provides a method for preparing L-threonine, comprising the following steps: Step 1: Construct recombinant cells or recombinant microorganisms that can express the coding genes of the aforementioned mutants; Step 2: Cultivate the recombinant cells or recombinant microorganisms to obtain L-threonine.

[0040] The present invention also provides a method for increasing L-threonine production, comprising at least one of the following D1) or D2): The D1) includes the following steps: inserting the gene encoding the aforementioned protein into the genome of the target bacteria to increase the production of L-threonine in the target bacteria, wherein the target bacteria include Escherichia coli; The D2) includes the following steps: increasing the content and / or activity of the aforementioned proteins in the target bacteria, or increasing the expression level of the coding gene of the bifunctional (p)ppGpp synthase or hydrolase mutant in the target bacteria, thereby increasing the L-threonine production in the target bacteria, wherein the target bacteria include Escherichia coli.

[0041] The improvement is a comparison conducted under comparable conditions. The term "comparable conditions" refers to identical or similar environmental conditions. Environmental conditions include, for example, in vitro culture conditions such as culture temperature, culture medium, and gas environment.

[0042] The present invention provides recombinant Escherichia coli containing the aforementioned mutant or containing the aforementioned nucleic acid molecules.

[0043] In the above method, the recipient microorganism, target bacteria, recombinant microorganism, or recombinant Escherichia coli includes prokaryotic microorganisms.

[0044] In the above method, the prokaryotic microorganisms specifically include Gram-negative bacteria.

[0045] In the above method, the Gram-negative bacteria or target bacteria may specifically include Escherichia coli.

[0046] In the above method, the Escherichia spp. bacteria may specifically include Escherichia coli.

[0047] In the above method, the Escherichia coli bacteria specifically refers to Escherichia coli. Escherichia coli .

[0048] The present invention provides a composition containing the aforementioned recombinant microorganisms or containing recombinant microorganisms prepared by the aforementioned method.

[0049] This invention spoT mutant spoT D84_M85 ins HD 、spoT D84_M85 ins HD、G174C 、spoT D84_M85 ins HD、L529F 、 spoT D84_M85 ins HD、G174C、L529F All genes significantly increased the concentration of L-threonine in strain CGMCC No. 25404.

[0050] Preservation Instructions Chinese name of the bacterial strain: Escherichia coli; Latin scientific name: Escherichia coli ; Classification and nomenclature: Escherichia coli Escherichia coli ; Number: YP0158; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Abbreviation of depositary institution: CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Date of deposit: July 25, 2022; Registered with the China National Collection Center (CGMCC) No. 25404. Attached Figure Description

[0051] Figure 1 This is a map of the pREDCas9 plasmid.

[0052] Figure 2 This is a pGRB plasmid map. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] The term "water" as used in this invention includes any feasible type of water that can be used in the art, such as deionized water, distilled water, ion-exchange water, double-distilled water, high-purity water, and purified water.

[0056] The term "and / or" as used in this invention refers to and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in an alternative manner ("or").

[0057] In this invention, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any phosphate ester analogue thereof, such as single-stranded or double-stranded helical thiophosphates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The "product" of this disclosure comprises one or more nucleic acids as described herein.

[0058] As is known in the art, the term "identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, "identity" also refers to the degree of sequence correlation between polypeptide or polynucleotide sequences, as determined by matching strings of such sequences. "Identity" can be readily calculated using known methods, including but not limited to those described in: *Computational Molecular Biology* (edited by Lesk, AM), Oxford University Press, New York (1988); *Biocomputing: Informatics and Genome Projects* (edited by Smith, DW), Academic Press, New York (1993); *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); *Sequence Analysis in Molecular Biology* (edited by von Heinje, G.), Academic Press (1987); and *Sequence Analysis Primer* (edited by Gribskov, M. and Devereux, J.), Stockton Press, New York (1991). Preferred methods for determining identity are designed to yield the best match between the tested sequences. Methods for determining identity have been incorporated into publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE Bioinformatics Computing Suite (DNASTAR, Madison, WI), the GCG program suite (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, 1228 S. Park St. Madison, WI 53715 USA). In the context of this application, it will be understood that, when using sequence analysis software for analysis, unless otherwise stated, the results of the analysis will be based on the “default values” of the cited program. As used herein, “default values” refers to any set of values ​​or parameters initially loaded with the software upon initial initialization.

[0059] The aforementionedspoT The gene encodes a bifunctional (p)ppGpp synthase or hydrolase (amino acid sequence SEQ ID NO:2), wherein... spoT The gene contains the nucleotide sequence described in SEQ ID NO:1.

[0060] The following examples use R language to process the data, with three batch biological replicates for each strain, and significance tests are used. t-test method, p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference.

[0061] Example 1: Genetically engineered bacteria YPThr- spoT D84_M85 ins HD and W3110- spoT D84_M85 ins HD Construction This embodiment uses the CRISPR-Cas9 gene editing method to produce high-yield L-threonine bacteria in Escherichia coli CGMCC No. 25404. spoT A mutant was constructed by inserting a nucleotide 5'-CATGAT-3' between the nucleotides encoding amino acids 84 and 85. spoT D84_M85 ins HD , the mutant spoT D84_M85 ins HD Replace wild type spoT Constructing an engineered Escherichia coli strain YPThr that produces high levels of L-threonine -spoT D84_M85 ins HD .

[0062] The two plasmid maps used in this method are attached. Figure 1 and Figure 2 The pREDCas9 expression plasmid carries gRNA, contains the elimination system of pGRB, the Red recombination system of λ phage, and the Cas9 protein expression system. It is also incubated with azithromycin resistance (working concentration: 100 mg / L) at 32°C. pGRB uses pUC18 as its backbone and includes the promoter J23119, the gRNA-Cas9 binding region sequence, and the terminator sequence. It is also incubated with ampicillin resistance (working concentration: 100 mg / L) at 37°C.

[0063] 1.1 spoT- pGRB plasmid construction Constructing plasmids spoT-The purpose of pGRB is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and to achieve a double-strand break in the target DNA through base pairing and PAM recognition of the target gene site. pGRB was purchased from Addgene (catalog number #71539) and was constructed using a recombination method involving a DNA fragment containing the target sequence and a linearized vector fragment. spoT- pGRB plasmid.

[0064] (1) Target sequence and primer design The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools, and primers for amplifying the sgRNA fragment were designed for the target sequence (forward primer F structure: 5'-linearized vector terminal sequence (34bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (34bp)-3', reverse primer R structure: inversely complementary to forward primer F), as detailed below: gRNA-F:5'-tgacagctagctcagtcctaggtataatactagtctctttacggttgccgcgcggttttagagctagaaatagcaagttaaaataagg-3'; gRNA-R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcgcgcggcaaccgtaaagagACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3'.

[0065] In the primers mentioned above, the lowercase letters are: spoT Target sequence, corresponding to spoT Nucleotides 614 to 633 of gene SEQ ID NO:1.

[0066] (2) Preparation of DNA fragments containing target sequences DNA fragments containing the target sequence were prepared by annealing single-stranded DNA. Reaction conditions: pre-denaturation 95℃, 5 min; annealing 30-50℃, 1 min. Annealing reaction mixture: 10 μL gRNA-F (10 μmol / L), 10 μL gRNA-R (10 μmol / L), yielding DNA fragments containing the target sequence.

[0067] (3) Preparation of linear carriers The vector was linearized using inverse PCR amplification. The amplification primers are as follows: pGRB-F:5'-actagtattatacctaggactgagc-3'; pGRB-R:5'-gttttagagctagaaatagcaagtt-3'.

[0068] The PCR reaction system is shown in Table 1.

[0069] PCR reaction procedure (Takara Bio PrimeSTAR HS enzyme): pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintain (4℃) to obtain the linearized cloning vector.

[0070] (4) Recombination reaction The recombination system is shown in the table below. All recombinant enzymes used were from the ClonExpress® II One Step Cloning Kit series. Recombination conditions: 37℃, 30 min, to obtain the reaction solution.

[0071] Table 1. Reorganization System

[0072] (5) Plasmid transformation Take 10 μL of the reaction solution from “(4) Recombination Reaction” and add it to 100 μL of DH5α-transformed competent cells. After gently mixing, incubate on ice for 20 min, heat shock at 42℃ for 45-90 s, immediately incubate on ice for 2-3 min, add 900 μL of SOC, and revive at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, and resuspend the bacterial cells in about 200 μL. Spread the resuspended cells onto a plate containing 100 mg / L ampicillin, invert the plate, and incubate overnight at 37℃.

[0073] (6) Cloning identification The above ampicillin-resistant colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight. After preservation, plasmids were extracted and identified by enzyme digestion.

[0074] The plasmid identified as correctly inserted was named spoT- pGRB. Plasmid spoT- pGRB is a recombinant vector obtained by inserting the DNA fragment represented by nucleotides 35 to 54 of gRNA-F into the HS restriction enzyme recognition site of the pGRB plasmid while keeping other nucleotide sequences unchanged. spoT-pGRB can transcribe gRNA with the nucleotide sequence SEQ ID NO:5, thereby forming a complex with the Cas9 protein. It then recognizes the target gene site through base pairing and PAM, achieving a double-strand break in the target DNA. The gRNA targeting strain CGMCC No. 25404 is located on the genome of this strain. spoT Nucleotides 614 to 633 of gene SEQ ID NO:1.

[0075] 1.2 spoT D84_M85 ins HD Fabrication of the integrated frame For spoT D84_M85 ins HD The integration frame consists of an upstream homologous arm and a downstream homologous arm. Primer design software Primer5 was used to design primers... spoT D84_M85 ins HD Primers were designed using the upstream and downstream homologous arm sequences of the mutant as templates, as follows: spoT -F:5'-ggcacgcgtaactgttcagg-3'; spoT -R3:5'-ttccatATCATGatcctggtaggtggcggg-3'; spoT -F3:5'-accaggatCATGATatggaacagctttttg-3'; spoT -R:5'-gcgcatgatattcgccagatg-3'.

[0076] Using the amplification system shown in Table 2, and with the genomic DNA of engineered strain CGMCC No. 25404 as a template, ... spoT -F / spoT -R3 and spoT -F3 / spoT -R amplification spoT D84_M85 ins HD Upstream and downstream homologous arms of a gene.

[0077] Table 2. PCR reaction system

[0078] Table 3. Overlap PCR Reaction System

[0079] Using the overlapping PCR reaction system in Table 3, the upstream primers of the aforementioned upstream homologous arms... spoT- F and downstream primers of downstream homologous arms spoT-R represents the amplification primers. Two mutant segments, one upstream and one downstream homologous, serve as templates for overlapping PCR to prepare the amplification material for homologous recombination. spoT D84_M85 ins HD The integration frame, with the nucleotide sequence SEQ ID NO:6, wherein nucleotides 1 to 556 are... spoT D84_M85 ins HD The upstream homologous arm of the gene (556 bp), nucleotides 537 to 2356 are spoT D84 _M85 ins HD Downstream homologous arm of the gene (1820 bp). spoT D84_M85 ins HD The amino acid sequence is similar to that of wild type spoT The difference in amino acid sequences lies in the fact that, in the wild type... spoT Histidine H and aspartic acid D are inserted between amino acids 84 and 85 of (SEQ ID NO:2), i.e., in the wild type spoT A 5'-CATGAT-3' insertion was made between nucleotides 252 and 253 of the nucleotide sequence (SEQ ID NO:1). spoT D84_M85 ins HD The amino acid sequence contains 704 amino acid residues, as shown in SEQ ID NO:4, and the nucleotide sequence is shown in SEQ ID NO:3 (2115 bp).

[0080] 1.3 Transformation of pREDCas9 plasmid pREDCas9 was purchased from Addgene, catalog number #71541. It carries the elimination system of gRNA expression plasmid pGRB, the Red recombination system of λ phage, and the Cas9 protein expression system. It is resistant to zirconia (working concentration: 100 mg / L) and cultured at 32°C.

[0081] (1) Conversion of pREDCas9 The pREDCas9 plasmid was electroporated into the electrocompetent cells of L-threonine-producing strain CGMCC No. 25404. After cell resuscitation and culture, the cells were plated on LB agar plates containing zirconia and incubated overnight at 32°C. Single colonies growing on the resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0082] (2) Preparation of electrotransformation competent cells of target strain containing pREDCas9 The positive recombinants in “(1) pREDCas9 transformation” were cultured at 32℃ until OD 600nm When the concentration reaches 0.1–0.2, add 0.1 M IPTG (to bring the final concentration to 0.1 mM) and continue culturing until OD reaches 0.2. 600nmCompetent cells were prepared when the pH was 0.6–0.7. The purpose of adding IPTG was to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process for competent cells were performed according to standard operating procedures, resulting in CGMCC No. 25404 electroporated competent cells containing pREDCas9.

[0083] 1.4 spoT- pGRB plasmid and recombinant spoT D84_M85 ins HD Conversion of integration box plasmid spoT- pGRB and recombinant spoT D84_M85 ins HD The integrase frame was simultaneously electroporated into CGMCC No. 25404 electroporated competent cells containing pREDCas9 obtained in step 1.3. The electroporated and revived cells were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Screening was performed by sequencing. spoT D84_M85 ins HD Positive mutants and preserved bacteria.

[0084] 1.5 Plasmid Elimination (1) spoT Elimination of -pGRB plasmid The above-mentioned positive recombinants were cultured overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread on LB plates containing zirconia-resistant bacteria and incubated overnight at 32°C. Single colonies were picked and streaked one-to-one onto LB plates containing ampicillin and zirconia-resistant bacteria, respectively. Single colonies that did not grow on ampicillin-resistant plates but grew on zirconia-resistant plates were selected and preserved to obtain positive recombinants.

[0085] (2) Elimination of pREDCas9 plasmid Will" spoT The positive recombinants obtained by "eliminating -pGRB" were transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, they were plated on antibiotic-free LB plates and incubated overnight at 37°C. Single colonies were picked and streaked one-to-one onto LB plates containing dysmycin resistance and those without resistance. Single colonies that did not grow on dysmycin-resistant plates but grew on antibiotic-free plates were selected for preservation to obtain positive recombinants.

[0086] The above-mentioned positive mutants were eliminated using the plasmid elimination method described in Example 1. spoT -pGRB and pREDCas9 plasmids were obtained and sequenced again to identify them. spoT Point mutant engineered bacteria YPThr- spoT D84_M85 ins HD Recombinant bacteria YPThr- spoT D84 _M85 ins HDRecombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain CGMCC No. 25404 (as shown in SEQ ID NO:6) with nucleotides 293-2356 of SEQ ID NO:1, while retaining the other nucleotide sequences of the CGMCC No. 25404 genome unchanged. The recombinant strain YPThr- spoT D84_M85 ins HD The genome contains only spoT mutant spoT D84_M85 ins HD (SEQ ID NO:3), does not contain wild-type spoT (SEQ ID NO:1).

[0087] Following the aforementioned method, using wild-type strain W3110 as the starting strain, recombinant strain W3110- was obtained. spoT D84 _M85 ins HD Recombinant strain W3110 -spoT D84_M85 ins HD Recombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain W3110 (as shown in SEQ ID NO:6) with nucleotides 293-2356 of SEQ ID NO:1, while retaining the other nucleotide sequences of the W3110 genome unchanged. spoT D84_M85 ins HD The genome contains only spoT mutant spoT D84_M85 ins HD (SEQ ID NO:3), does not contain wild-type spoT (SEQ ID NO:1).

[0088] Example 2, Genetically Engineered Bacteria YPThr- spoT D84_M85 ins HD、G174C and W3110- spoT D84_M85 ins HD、G174C Construction This embodiment uses the CRISPR-Cas9 gene editing method to modify the L-threonine-producing strain of *Escherichia coli*, CGMCC No. 25404. spoTA mutant was constructed by inserting a 5'-CATGAT-3' nucleotide between the 84th and 85th amino acids and mutating the C at position 1585 to T. spoT D84_M85 ins HD、G174C , the mutant spoT D84_M85 ins HD、G174C Replace wild type spoT Constructing a high-yield engineered strain YPThr -spoT D84_M85 ins HD、G174C .

[0089] 2.1 spoT D84_M85 ins HD、G174C Fabrication of the integrated frame For spoT D84_M85 ins HD、G174C The integration box of point mutations consists of upstream homologous arms, spoT It consists of a mutation region and a downstream homologous arm. Primer design software Primer5 was used to... spoT D84_M85 ins HD、G174C Primers were designed using the upstream and downstream homologous arm sequences of the mutant as templates, as follows: spoT -F:5'-aggggcaaacacgttcaagc-3'; spoT -R3:5'-ttccatATCATGatcctggtaggtggcggg-3'; spoT -F3:5'-accaggatCATGATatggaacagctttttg-3'; spoT -R1:5'-ttaatgtggtggatacAtaaacggtgcg-3'; spoT -F1:5'-gctggcgcaccgtttaTgtatccaccac-3'; spoT -R:5'-gcgcatgatattcgccagatg-3'.

[0090] Using the amplification system shown in Table 2, and with the genomic DNA of engineered strain CGMCC No. 25404 as a template, ... spoT -F / spoT -R3、 spoT -F3 / spoT -R1 and spoT -F1 / spoT -R amplification spoT D84_M85 ins HD、G174C upstream homologous arms of genes spoT Mutation region and downstream homologous arm.

[0091] Using the overlapping PCR reaction system in Table 3, the upstream primers of the aforementioned upstream homologous arms... spoT -F and downstream homologous arm downstream primers spoT -R represents the amplification primer, the upstream homologous arm, spoT The mutant region and downstream homologous arm were used as amplification templates for overlap PCR to prepare materials for homologous recombination. spoT D84_M85 ins HD、G174C The integration frame, with the nucleotide sequence SEQ ID NO:7, wherein nucleotides 1 to 556 are... spoT D84_M85 ins HD、G174C The upstream homologous arm of the gene (556 bp), nucleotides 537 to 834 are spoT The mutation region (298 bp), nucleotides 802 to 2356 are spoT D84_M85 ins HD、G174C Downstream homologous arm of the gene (1555 bp). spoT D84_M85 ins HD、G174C The amino acid sequence is similar to that of wild type spoT The difference in amino acid sequences lies in the fact that, in the wild type... spoT Histidine H and aspartic acid D are inserted between amino acids 84 and 85 of (SEQ ID NO:2), i.e., in the wild type spoT The nucleotide sequence (SEQ ID NO:1) contains an insertion of 5'-CATGAT-3' between nucleotides 252 and 253, and the amino acid 174 of SEQ ID NO:2 is mutated from glycine G (codon GGT) to cysteine ​​C (codon TGT), thus creating the wild-type... spoT The nucleotide sequence (SEQ ID NO:1) at position 520 is mutated from G to T. spoT D84_M85 ins HD、G174C The amino acid sequence is SEQ ID NO:11, and its amino acid sequence differs from SEQ ID NO:4 in that... spoT D84_M85 ins HD、G174C The 176th amino acid is cysteine ​​C, and the remaining amino acids are the same as those in SEQ ID N:4. spoT D84_M85 ins HD、G174C The nucleotide sequence contains 2115 nucleotides and is SEQ ID NO:10. The difference between it and SEQ ID NO:3 is that the 526th nucleotide is T, and the remaining nucleotides are the same as those in SEQ ID NO:3.

[0092] 2.2 spoT- pGRB plasmid and recombinant spoT D84_M85 ins HD、G174C Conversion of integration box plasmid spoT- pGRB and recombinant spoTD84_M85 ins HD、G174C The integrase frame was simultaneously electroporated into CGMCC No. 25404 electroporated competent cells containing pREDCas9 obtained in step 1.3. The electroporated and revived cells were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Screening was performed by sequencing. spoT G174C、D84_M85 ins HD Positive mutants and preserved bacteria.

[0093] 2.3 Plasmid Elimination The above-mentioned positive mutants were eliminated using the plasmid elimination method described in Example 1. spoT -pGRB and pREDCas9 plasmids were obtained and sequenced again to identify them. spoT Point mutant engineered bacteria YPThr- spoT D84_M85 ins HD、G174C Recombinant bacteria YPThr- spoT D84_M85 ins HD、G174C Recombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:7, and replacing nucleotides 1-2058 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:7 with nucleotides 293-2356 of SEQ ID NO:7, while keeping the other nucleotide sequences of the CGMCC No. 25404 genome unchanged. The recombinant strain YPThr- spoT D84_M85 ins HD、G174C The genome contains only spoT mutant spoT D84 _M85 ins HD、G174C (The nucleotide sequence is SEQ ID NO:10, and the encoded amino acid sequence is SEQ ID NO:11), and it does not contain wild-type. spoT (SEQ ID NO:1).

[0094] Following the aforementioned method, using wild-type strain W3110 as the starting strain, recombinant strain W3110- was obtained. spoT D84 _M85 ins HD、G174C Recombinant strain W3110 -spoT D84_M85 ins HD、G174CRecombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain W3110 (as shown in SEQ ID NO:7) with nucleotides 293-2356 of SEQ ID NO:7, and replacing nucleotides 1-2058 of the genome of strain W3110 (as shown in SEQ ID NO:1) with nucleotides 293-2356 of SEQ ID NO:7, while keeping the other nucleotide sequences of the W3110 genome unchanged. Recombinant strain W3110- spoT DD84_M85 ins HD、G174C The genome contains only spoT mutant spoT D84_M85 ins HD、G174C (The nucleotide sequence is SEQ ID NO:10, and the encoded amino acid sequence is SEQ ID NO:11), and it does not contain wild-type. spoT (SEQ IDNO:1).

[0095] Example 3: Genetically engineered bacteria YPThr- spoT D84_M85 ins HD、L529F and W3110- spoT D84_M85 ins HD、L529F Construction This embodiment uses the CRISPR-Cas9 gene editing method to modify the L-threonine-producing strain of *Escherichia coli*, CGMCC No. 25404. spoT A mutant was constructed by inserting a 5'-CATGAT-3' nucleotide between the 84th and 85th amino acids and mutating the C at position 1585 to T. spoT D84_M85 ins HD、L529F , the mutant spoT D84_M85 ins HD、L529F Replace wild type spoT Constructing an engineered Escherichia coli strain YPThr that produces high levels of L-threonine -spoT D84_M85 ins HD、L529F .

[0096] 3.1 spoT D84_M85 ins HD、L529F Fabrication of the integrated frame For spoT D84_M85 ins HD、L529F The integration box of point mutations consists of upstream homologous arms, spoT It consists of a mutation region and a downstream homologous arm. Primer design software Primer5 was used to... spoT D84_M85 ins HD、L529F Primers were designed using the upstream and downstream homologous arm sequences of the mutant as templates, as follows: spoT -F:5'-aggggcaaacacgttcaagc-3'; spoT-R3:5'-ttccatATCATGatcctggtaggtggcggg-3'; spoT -F3:5'-accaggatCATGATatggaacagctttttg-3'; spoT- R2:5'-cattgcgttaccaaAtccgatttctgcc-3'; spoT -F2:5'-cagaaatcggaTttggtaacgcaatgag-3'; spoT -R:5'-gcgcatgatattcgccagatg-3'.

[0097] Using the amplification system shown in Table 2, and with the genomic DNA of engineered strain CGMCC No. 25404 as a template, ... spoT -F / spoT -R3、 spoT -F3 / spoT -R2 and spoT -F2 / spoT -R amplification spoT D84_M85 ins HD、L529F upstream homologous arms of genes spoT Mutation region and downstream homologous arm.

[0098] Using the overlapping PCR reaction system in Table 3, the upstream primers of the aforementioned upstream homologous arms... spoT -F and downstream homologous arm downstream primers spoT -R represents the amplification primer, the upstream homologous arm, spoT The mutant region and downstream homologous arm were used as amplification templates for overlap PCR to prepare materials for homologous recombination. spoT D84_M85 ins HD、L529F The integration frame, with the nucleotide sequence SEQ ID NO:8, wherein nucleotides 1 to 556 are... spoT D84_M85 ins HD、L529F The upstream homologous arm of the gene (556 bp), nucleotides 537 to 1897 are spoT The mutation region (1361 bp), nucleotides 1872 to 2356 are... spoT D84_M85 ins HD、L529F Downstream homologous arm of the gene (485 bp). spoT D84_M85 ins HD、L529F amino acids and wild type spoT The difference lies in the wild type. spoT Histidine H and aspartic acid D are inserted between amino acids 84 and 85 of (SEQ ID NO:2), i.e., in the wild type spoTA 5'-CATGAT-3' was inserted between nucleotides 252 and 253 of the nucleotide sequence (SEQ ID NO:1), and amino acid 529 of SEQ ID NO:2 was mutated from leucine L (codon CTT) to phenylalanine F (codon TTT). spoT D84_M85 ins HD、L529F The amino acid sequence is SEQ ID NO:13, and its amino acid sequence differs from SEQ ID NO:4 in that... spoT D84_M85 ins HD、L529F The 529th amino acid is phenylalanine F, and the remaining amino acids are the same as those in SEQ ID N:4. spoT D84_M85 ins HD、L529F The nucleotide sequence contains 2115 nucleotides. It differs from SEQ ID NO:3 in that the 1591st nucleotide is T. The remaining nucleotides are the same as SEQ ID NO:3. Its specific sequence is SEQ ID NO:12.

[0099] 3.2 spoT- pGRB plasmid and recombinant spoT D84_M85 ins HD、L529F Conversion of integration box plasmid spoT- pGRB and recombinant spoT D84_M85 ins HD、L529F The integrase frame was simultaneously electroporated into CGMCC No. 25404 electroporated competent cells containing pREDCas9 obtained in step 1.3. The electroporated and revived cells were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Screening was performed by sequencing. spoT D84_M85 ins HD、L529F Positive mutants and preserved bacteria.

[0100] 3.3 Plasmid elimination The above-mentioned positive mutants were eliminated using the plasmid elimination method described in Example 1. spoT -pGRB and pREDCas9 plasmids were obtained and sequenced again to identify them. spoT Point mutant engineered bacteria YPThr- spoT D84_M85 ins HD、L529F Recombinant bacteria YPThr- spoT D84_M85 ins HD、L529FRecombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:8, and replacing nucleotides 1-2058 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:8, with nucleotides 293-2356 of SEQ ID NO:8, while keeping the other nucleotide sequences of the CGMCC No. 25404 genome unchanged. The recombinant strain YPThr- spoT D84_M85 ins HD、L529F The genome contains only spoT mutant spoT D84 _M85 ins HD、L529F (The nucleotide sequence is SEQ ID NO:12, and the encoded amino acid sequence is SEQ ID NO:13), and it does not contain wild-type. spoT (SEQ ID NO:1).

[0101] Following the aforementioned method, using wild-type strain W3110 as the starting strain, recombinant strain W3110- was obtained. spoT D84 _M85 ins HD、L529F Recombinant strain W3110 -spoT D84_M85 ins HD、L529F Recombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain W3110 (as shown in SEQ ID NO:8) with nucleotides 293-2356 of SEQ ID NO:1, while keeping the other nucleotide sequences of the W3110 genome unchanged. spoT D84_M85 ins HD、L529F The genome contains only spoT mutant spoT D84_M85 ins HD、L529F (The nucleotide sequence is SEQ ID NO:12, and the encoded amino acid sequence is SEQ ID NO:13), and it does not contain wild-type. spoT (SEQ IDNO:1).

[0102] Example 4: Genetically engineered bacteria YPThr- spoT D84_M85 ins HD、G174C、L529F and W3110- spoT D84 _M85 ins HD、G174C、L529F Construction This embodiment uses the CRISPR-Cas9 gene editing method to modify the L-threonine-producing strain of *Escherichia coli*, CGMCC No. 25404. spoT The mutant was constructed by inserting a 5'-CATGAT-3' between the nucleotides encoding amino acids 84 and 85, mutating the G at position 520 to T, and mutating the C at position 1585 to T. spoT D84_M85 ins HD、G174C、L529F , the mutant spoT D84_M85 ins HD、G174C、L529F Replace wild type spoT Constructing an engineered Escherichia coli strain YPThr that produces high levels of L-threonine - spoT D84_M85 ins HD、G174C、L529F .

[0103] 4.1 spoT D84_M85 ins HD、G174C、L529F Fabrication of the integrated frame For spoT D84_M85 ins HD、G174C、L529F The integration box of point mutations consists of upstream homologous arms, spoT It consists of a mutation region and a downstream homologous arm. Primer design software Primer5 was used to... spoT D84_M85 ins HD、G174C、L529F Primers were designed using the upstream and downstream homologous arm sequences of the mutant as templates, as follows: spoT -F:5'-aggggcaaacacgttcaagc-3'; spoT -R3:5'-ttccatATCATGatcctggtaggtggcggg-3'; spoT -F3:5'-accaggatCATGATatggaacagctttttg-3'; spoT -R1:5'-ttaatgtggtggatacAtaaacggtgcg-3'; spoT -F1:5'-gctggcgcaccgtttaTgtatccaccac-3'; spoT- R2:5'-cattgcgttaccaaAtccgatttctgcc-3'; spoT -F2:5'-cagaaatcggaTttggtaacgcaatgag-3'; spoT -R:5'-gcgcatgatattcgccagatg-3'.

[0104] Using the amplification system shown in Table 2, and with the genomic DNA of engineered strain CGMCC No. 25404 as a template, ... spoT -F / spoT -R3、 spoT -F3 / spoT -R1、 spoT -F1 / spoT -R2, and spoT -F2 / spoT -R amplification spoT G174C 、D84_M85 ins HD、L529F upstream homologous arms of genes spoT Mutation region and downstream homologous arm.

[0105] Using the overlapping PCR reaction system in Table 3, the upstream primers of the aforementioned upstream homologous arms... spoT -F and downstream homologous arm downstream primers spoT -R represents the amplification primer, the upstream homologous arm, spoT The mutant region and downstream homologous arm were used as amplification templates for overlap PCR to prepare materials for homologous recombination. spoT D84_M85 ins HD、G174C、L529F The integration frame, with the nucleotide sequence SEQ ID NO:9, wherein nucleotides 1 to 556 are... spoT G174C、D84_M85 ins HD、L529F The upstream homologous arm of the gene (556 bp), nucleotides 537 to 1897 are spoT The mutation region (1361 bp), nucleotides 1872 to 2356 are... spoT G174C、D84_M85 ins HD、L529F Downstream homologous arm of the gene (485 bp). spoT D84_M85 ins HD、G174C、L529F amino acids and wild type spoT The difference lies in the wild type. spoT Histidine H and aspartic acid D are inserted between amino acids 84 and 85 of (SEQ ID NO:2), i.e., in the wild type spoT The nucleotide sequence (SEQ ID NO:1) contains an insertion of 5'-CATGAT-3' between nucleotides 252 and 253, and the amino acid 174 of SEQ ID NO:2 is mutated from glycine G (codon GGT) to cysteine ​​C (codon TGT), thus creating the wild-type... spoT The nucleotide sequence (SEQ ID NO:1) at position 520 is mutated from G to T, and the wild-type... spoT Amino acid at position 529 of (SEQ ID NO:2) is mutated from leucine L (codon CTT) to phenylalanine F (codon TTT), which is close to the wild type. spoTThe nucleotide sequence (SEQ ID NO:1) has a C-to-T mutation at position 1585. spoT D84_M85 ins HD、G174C、L529F The specific amino acid sequence is SEQ ID NO:15, and its amino acid sequence differs from SEQ ID NO:4 in that... spoT D84_M85 ins HD、G174C、L529F The 176th amino acid is cysteine ​​(C), the 531st amino acid is phenylalanine (F), and the remaining amino acids are the same as those in SEQ ID N:4. spot D84_M85 ins HD、G174C、L529F The nucleotide sequence contains 2115 nucleotides. It differs from SEQ ID NO:3 in that the 526th nucleotide is T and the 1591st nucleotide is T. The remaining nucleotides are the same as SEQ ID NO:3. Its specific sequence is SEQ ID NO:14.

[0106] 4.2 spot- pGRB plasmid and recombinant spot D84_M85 ins HD、G174C、L529F Conversion of integration box plasmid spot- pGRB and recombinant spot D84_M85 ins HD、G174C、L529F The integrase frame was simultaneously electroporated into CGMCC No. 25404 electroporated competent cells containing pREDCas9 obtained in step 1.3. The electroporated and revived cells were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Screening was performed by sequencing. spot D84_M85 ins HD、G174C、L529F Positive mutants and preserved bacteria.

[0107] 4.3 Plasmid elimination The above-mentioned positive mutants were eliminated using the plasmid elimination method described in Example 1. spot -pGRB and pREDCas9 plasmids were obtained and sequenced again to identify them. spot Point mutant engineered bacteria YPThr- spot D84_M85 ins HD、G174C、L529F Recombinant bacteria YPThr- spot D84_M85 ins HD、G174C、L529F Recombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:9, and replacing nucleotides 1-2058 of the genome of strain CGMCC No. 25404 as shown in SEQ ID NO:9, with nucleotides 293-2356 of SEQ ID NO:9, while keeping the other nucleotide sequences of the CGMCC No. 25404 genome unchanged. The recombinant strain YPThr- spot D84_M85 ins HD、G174C、L529F The genome contains only spot mutant spot D84 _M85 ins HD、G174C、L529F (The nucleotide sequence is SEQ ID NO:14, and the encoded amino acid sequence is SEQ ID NO:15), and it does not contain wild-type. spot (SEQ ID NO:1).

[0108] Following the aforementioned method, using wild-type strain W3110 as the starting strain, recombinant strain W3110- was obtained. spot D84 _M85 ins HD、G174C、L529F Recombinant strain W3110 -spot D84_M85 ins HD、G174C、L529F Recombinant *E. coli* was obtained by replacing nucleotides 1-292 of the genome of strain W3110 (as shown in SEQ ID NO:9) with nucleotides 293-2356 of SEQ ID NO:9, and replacing nucleotides 1-2058 of the genome of strain W3110 with nucleotides 293-2356 of SEQ ID NO:9, while keeping the other nucleotide sequences of the W3110 genome unchanged. Recombinant strain W3110- spot D84_M85 ins HD、G174C、L529F The genome contains only spot mutant spot D84 _M85 ins HD、G174C、L529F (The nucleotide sequence is SEQ ID NO:14, and the encoded amino acid sequence is SEQ ID NO:15), and it does not contain wild-type. spot (SEQ ID NO:1).

[0109] Example 5: L-Threonine Fermentation Experiment The experiment was repeated 3 times, with each repetition as follows: The W3110 strain, CGMCC No. 25404 strain, and its engineered strain W3110- were compared. spot D84_M85 ins HD W3110- spot D84_M85 ins HD、G174C W3110- spot D84_M85 ins HD、L529F W3110- spot D84_M85 ins HD、G174C、L529F YPThr- spot D84_M85 ins HD YPThr- spot D84_M85 ins HD、G174C YPThr- spot D84_M85 ins HD、L529F YPThr- spot D84 _M85 ins HD、G174C、L529F The culture was streaked onto slant agar and cultured at 37℃ for 12 h. A loopful of seed culture was then inoculated into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. The flask was sealed with nine layers of gauze and cultured at 37℃ and 200 rpm for 7–10 h. Then, 1 mL of each strain's culture was inoculated into a 50 mL shake flask fermentation medium and fermented at 37℃ and 200 rpm for 36 h. The L-threonine content was determined by HPLC. The chromatographic conditions were as follows: Method: 2,4-dinitrofluorobenzene (DNFB) pre-column derivatization HPLC; Mobile phase: 55% acetonitrile-water solution (phase B), 5.44 g / L KH₂PO₄ solution (pH = 7.20) (phase D); Flow rate: 1.0 mL / min; Detection wavelength: 360 nm. Three replicates were performed for each strain, and the average value was calculated. The results are shown in Table 4.

[0110] Slant culture medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L, agar 15-20 g / L, the remainder is water, pH 7.0-7.2; The seed culture medium consists of: glucose 1-5 g / L, peptone 5-10 g / L, beef extract 5-10 g / L, yeast extract 1-5 g / L, NaCl 1-2.5 g / L, and the remainder is water, with a pH of 7.0-7.2.

[0111] Shake-flask fermentation medium: glucose 40 g / L, (NH4)2SO4 12 g / L, KH2PO4 0.8 g / L, MgSO4•7H2O 0.8 g / L, FeSO4•7H2O 0.01 g / L, MnSO4•H2O 0.01 g / L, FM902 yeast extract 1.5 g / L, calcium carbonate 0.5 g / L, the remainder being water, and sodium hydroxide to adjust the pH to 7.0.

[0112] Table 4. Fermentation results of engineered strains

[0113] The results are shown in Table 4. For both the high-L-threonine-producing strain CGMCC No. 25404 and the model strain W3110, spot All mutants increased L-threonine production, with highly significant differences.

[0114] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A bifunctional (p)ppGpp synthase or hydrolase mutant, characterized in that, The bifunctional (p)ppGpp synthase or hydrolase mutant includes any one of the following: A1) Its amino acid sequence is the sequence obtained by inserting a histidine residue and an aspartic acid residue between the 84th and 85th amino acid residues of SEQ ID NO:2; A2) A protein having more than 95% identity and the same function as the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1). A3) A fusion protein with the same function is obtained by attaching a tag protein to the N-terminus and / or C-terminus of the bifunctional (p)ppGpp synthase or hydrolase mutant described in A1) or A2).

2. The bifunctional (p)ppGpp synthase or hydrolase mutant according to claim 1, characterized in that, The bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 174th amino acid residue of the bifunctional (p)ppGpp synthase or hydrolase mutant of claim 1 to a cysteine ​​residue.

3. The bifunctional (p)ppGpp synthase or hydrolase mutant according to claim 1, characterized in that, The bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 529th amino acid residue of the bifunctional (p)ppGpp synthase or hydrolase mutant of claim 1 to a phenylalanine residue.

4. The bifunctional (p)ppGpp synthase or hydrolase mutant according to claim 1, characterized in that, The bifunctional (p)ppGpp synthase or hydrolase mutant further includes mutating the 174th amino acid residue of the bifunctional (p)ppGpp synthase or hydrolase mutant of claim 1 to a cysteine ​​residue and mutating the 529th amino acid residue to a phenylalanine residue.

5. The bifunctional (p)ppGpp synthase or hydrolase mutant according to any one of claims 1-4, characterized in that, The bifunctional (p)ppGpp synthase or hydrolase mutant contains any one of the amino acid sequences SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:

15.

6. A biomaterial, characterized in that, The biomaterial includes any of the following: B1) A nucleic acid molecule encoding a bifunctional (p)ppGpp synthase or hydrolase mutant as described in any one of claims 1-5; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3) or the recombinant microorganism described in B4).

7. The biomaterial according to claim 6, characterized in that, B1) The nucleic acid molecule described herein contains any one of the nucleotide sequences SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:

14.

8. Application, characterized in that, The application is selected from at least one of the following: K1) The use of the bifunctional (p)ppGpp synthase or hydrolase mutant of any one of claims 1-5 or / and the biomaterial of claim 6 or 7 in the preparation of L-threonine; K2) The use of the bifunctional (p)ppGpp synthase or hydrolase mutant of any one of claims 1-5 or / and the biomaterial of claim 6 or 7 in the preparation of products containing L-threonine; K3) The use of any of the bifunctional (p)ppGpp synthase or hydrolase mutants according to claims 1-5 or / and the biomaterials according to claims 6 or 7 in improving the activity of the bifunctional (p)ppGpp synthase or hydrolase mutants; K4) The use of any of the bifunctional (p)ppGpp synthase or hydrolase mutants of claims 1-5 or / and the biomaterials of claims 6 or 7 in the construction of recombinant microorganisms that produce L-threonine; K5) The use of the bifunctional (p)ppGpp synthase or hydrolase mutant of claims 1-5 and / or the biomaterial of claim 6 or 7 in regulating the production of L-threonine by microorganisms.

9. A method for preparing L-threonine, characterized in that, Includes the following steps: Step 1: Construct recombinant cells or recombinant microorganisms that express the encoding gene of any of the bifunctional (p)ppGpp synthase or hydrolase mutants as described in claims 1-5; Step 2: Cultivate the recombinant cells or recombinant microorganisms to obtain L-threonine.

10. Recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli contains a bifunctional (p)ppGpp synthase or hydrolase mutant as described in any one of claims 1-5, or contains a nucleic acid molecule as described in claim 6 or 7.

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