Application of NCgl1764 protein or mutant thereof in increasing yield of isoleucine
By modifying the NCgl1764 protein with specific amino acid mutants, the technical problems in the synthesis of L-isoleucine were solved. This addresses the technical challenges or needs that the patent application aims to solve.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-27
AI Technical Summary
In existing technologies, the synthesis of L-isoleucine in Corynebacterium glutamicum is limited by the precise regulatory mechanism of the bacteria itself, resulting in low yield and the generation of by-products. Traditional breeding methods are highly random and time-consuming, making it difficult to meet industrial needs.
By performing specific mutations in the amino acid sequence of the NCgl1764 protein, NCgl1764I21L and L66P mutants were obtained and introduced into Corynebacterium glutamicum to break endogenous regulation and increase L-isoleucine production.
It significantly increased the L-isoleucine yield of Corynebacterium glutamicum, reduced byproduct formation, and simplified the difficulty and cost of downstream separation and purification.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to application of NCgl1764 protein or mutant thereof in improving isoleucine yield. BACKGROUND
[0002] L-isoleucine, as one of the three branched-chain amino acids, has extremely important application value in the fields of medicine, nutritional health products and animal feed. At present, the industrial production of L-isoleucine mainly relies on microbial fermentation method, and corynebacterium glutamicum is widely used as a production strain because of its simple nutritional requirements, rapid growth and being recognized as a safe industrial microorganism.
[0003] However, in wild type or unmodified corynebacterium glutamicum, the synthetic metabolic pathway of L-isoleucine is subject to multiple constraints of the precise regulation mechanism of the cell itself. This endogenous regulation aims to maintain the normal growth and metabolic balance of the cell, but seriously limits the efficient accumulation of L-isoleucine. Specifically, the yield of the target product is low, and the generation of other structural similar amino acids and other by-products is often accompanied during the fermentation process, which not only reduces the yield of the final product, but also greatly increases the difficulty and cost of downstream separation and purification.
[0004] Traditional strain improvement methods, such as physical or chemical mutagenesis, can improve the yield to some extent, but they are random, long cycle, and non-directional mutations may introduce negative traits that are not conducive to industrial fermentation. Therefore, there is still an urgent need for a more precise and efficient breeding strategy to overcome the inherent metabolic bottleneck of corynebacterium glutamicum in L-isoleucine synthesis, so as to meet the growing market demand. SUMMARY
[0005] The technical problem to be solved by the present application is how to improve the L-isoleucine yield of the recipient strain. The technical problem to be solved by the present application is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description. To solve the above technical problem, the present application provides the following technical solution: The present application provides a mutant protein, which can include at least one of the following: a1), a protein obtained by mutating the 21st and / or 66th amino acid residue sites in the sequence shown in SEQ ID NO. 2 in the amino acid sequence; a2), a protein obtained by substituting, deleting and / or adding amino acid residues in the amino acid sequence shown in a1); a3), a protein having more than 98% identity with the amino acid sequence shown in a1) and having the same function; a4) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1), a2), or a3).
[0006] In this application, the mutant protein comprises a protein obtained by mutating the isoleucine residue at position 21 of the sequence shown in SEQ ID NO.2 to a leucine residue and / or mutating the leucine residue at position 66 to a proline residue.
[0007] In this application, the mutant protein may include the amino acid sequence shown in SEQ ID No. 4 and / or SEQ ID No. 6 and / or SEQ ID No. 8.
[0008] In this application, the amino acid sequence of the mutant protein may be as shown in SEQ ID No. 4, SEQ ID No. 6 and / or SEQ ID No. 8.
[0009] In this application, SEQ ID No. 4, SEQ ID No. 6, or SEQ ID No. 8 consists of 74 amino acid residues.
[0010] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.
[0011] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.
[0012] Further, the connection described in a4) can be a peptide bond formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the aforementioned protein. Alternatively, the connection described in a4) can be a peptide bond formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the aforementioned protein.
[0013] The tag may also include a linker peptide between itself and the protein. The linker peptide undergoes dehydration condensation with the N-terminus of the tag and the C-terminus of the protein to form a peptide bond. Alternatively, the linker peptide undergoes dehydration condensation with the C-terminus of the tag and the N-terminus of the protein to form a peptide bond.
[0014] This application also provides biological materials related to the above-mentioned mutant proteins, said biological materials may contain at least one of the following A1)-A5): A1) Nucleic acid molecules encoding the aforementioned mutant proteins, A2), an expression cassette containing the nucleic acid molecule described in A1), A3), a recombinant vector containing the nucleic acid molecule described in A1), and / or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), recombinant microorganisms containing the expression cassette described in A2), and / or recombinant microorganisms containing the recombinant vector described in A3); A5) A whole-cell catalyst containing the nucleic acid molecule described in A1), a whole-cell catalyst containing the expression cassette described in A2), and / or a whole-cell catalyst containing the recombinant vector described in A3), and / or a whole-cell catalyst containing the recombinant microorganism described in A4).
[0015] In the aforementioned biological materials, the nucleic acid molecule described in A1) may include at least one of the following: B1) DNA molecules whose coding sequences include SEQ ID No. 3 and / or SEQ ID No. 5 and / or SEQ ID No. 7; B2) is a DNA molecule that shares more than 85% identity with the DNA molecule described in B1) and encodes the same protein mutant.
[0016] Among the aforementioned biological materials, the microorganisms are at least bacteria.
[0017] Furthermore, the bacteria are at least Corynebacterium glutamicum.
[0018] This application also provides the use of the above-mentioned mutant protein or related biological materials and / or NCgl1764 protein or related biological materials in at least one of the following: C1) Application in the preparation of L-isoleucine; C2) Egg is used in the preparation of products containing L-isoleucine; C3) Application in increasing L-isoleucine production in recipient bacteria; C4) Application in constructing engineered bacteria that produce L-isoleucine; The NCgl1764 protein may be at least one of the following proteins: b1) A protein whose amino acid sequence includes the sequence shown in SEQ ID NO.2; b2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in b1). b3) Proteins with an amino acid sequence that is more than 98% identical to that shown in b1) and have the same function; b4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of b1), b2) or b3).
[0019] In the above applications, the relevant biomaterials for the NCgl1764 protein may be at least one of the following: D1) The nucleic acid molecule encoding the NCgl1764 protein. D2), an expression cassette containing the nucleic acid molecule described in D1), D3), a recombinant vector containing the nucleic acid molecule described in D1), and / or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), recombinant microorganisms containing the expression cassette described in D2), and / or recombinant microorganisms containing the recombinant vector described in D3); D5) A whole-cell catalyst containing the nucleic acid molecule described in D1), a whole-cell catalyst containing the expression cassette described in D2), and / or a whole-cell catalyst containing the recombinant vector described in D3) and / or a whole-cell catalyst containing the recombinant microorganism described in D4).
[0020] In the above applications, the nucleic acid molecule described in D1) comprises at least one of the following: E1), a DNA molecule whose coding sequence contains SEQ ID No. 1; E2) is a DNA molecule that has more than 85% identity with the DNA molecule described in E1 and encodes the NCgl1764 protein.
[0021] Furthermore, the nucleic acid molecule described in D1) may be a DNA molecule with a coding sequence as shown in SEQ ID No. 1.
[0022] This application provides a method for increasing the L-isoleucine production of microorganisms, the method comprising at least one of the following: F1) Express the gene encoding the mutant protein mentioned above into the target microorganism; F2) Introduce the above-mentioned biological materials into the target microorganism; F3) Mutate the coding gene of the NCgl1764 protein in the target microorganism to the coding gene of the mutant protein. F4) The gene encoding the NCgl1764 protein overexpressed in microorganisms.
[0023] The target microorganism may contain the gene encoding the NCgl1764 protein. In this application, the overexpression can be achieved by additionally introducing the gene encoding the NCgl1764 protein into the microorganism.
[0024] The target microorganism is any one of the following: E1) Microorganisms capable of producing L-isoleucine; E2), bacteria; E3), Gram-positive bacteria; E4), Corynebacterium spp.; E5), Corynebacterium glutamicum.
[0025] In this application, the term "microorganism capable of producing L-isoleucine" refers to a microorganism possessing the following capabilities: the ability to produce and accumulate L-isoleucine within its own body using external substances (such as culture medium), and further including the ability to secrete L-isoleucine into the culture system. Thus, L-isoleucine can be collected when the microorganism is cultured in the culture medium.
[0026] In some embodiments of this application, the Corynebacterium glutamicum may be Corynebacterium glutamicum ATCC13032.
[0027] In some embodiments of this application, the Corynebacterium glutamicum may also be Corynebacterium glutamicum CGMCC20437.
[0028] This application also provides recombinant Corynebacterium glutamicum, wherein the recombinant Corynebacterium glutamicum is at least one of the following: G1) Recombinant Corynebacterium glutamicum containing the gene encoding the above-mentioned mutant protein; G2) Recombinant Corynebacterium glutamicum obtained by enhancing the expression of the gene encoding the NCgl1764 protein containing the amino acid sequence of SEQ ID NO.2 in recipient bacteria.
[0029] The recipient bacteria may contain the gene encoding the NCgl1764 protein.
[0030] The recombinant Corynebacterium glutamicum described in G1) can be obtained by mutating the encoding gene of the NCgl1764 protein of the recipient bacterium to the encoding gene of the mutated protein.
[0031] The recombinant Corynebacterium glutamicum described in G1 can also be obtained by introducing the gene encoding the mutant protein into the recipient bacteria.
[0032] The recombinant Corynebacterium glutamicum described in G2 contains a gene encoding the NCgl1764 protein in more than one copy.
[0033] This application also provides compositions that may contain the above-described recombinant Corynebacterium glutamicum.
[0034] The active ingredient of the above composition may be the recombinant Corynebacterium glutamicum and / or the metabolites of the recombinant Corynebacterium glutamicum and / or the culture of the recombinant Corynebacterium glutamicum.
[0035] The culture can be a substance obtained by culturing the recombinant Corynebacterium glutamicum in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the recombinant Corynebacterium glutamicum and a substance secreted into a liquid culture medium, or a solid fermentation product containing the recombinant Corynebacterium glutamicum and a substance secreted into a solid culture medium).
[0036] In the above text, the metabolite may be a product obtained by removing the recombinant Corynebacterium glutamicum from the culture, such as culturing the recombinant Corynebacterium glutamicum in a liquid fermentation medium, collecting the fermentation broth (containing the recombinant Corynebacterium glutamicum and substances secreted into the liquid culture medium), removing the recombinant Corynebacterium glutamicum from the fermentation broth, collecting the remaining components of the fermentation broth, and obtaining the metabolite of the recombinant Corynebacterium glutamicum.
[0037] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.
[0038] The above composition may be the culture described above. The above composition may also be a microbial agent.
[0039] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.
[0040] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.
[0041] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.
[0042] This application also provides a method for preparing L-isoleucine, the method comprising the steps of preparing L-isoleucine using recombinant cells or recombinant microorganisms expressing the above-described mutant protein and / or using the above-described recombinant Corynebacterium glutamicum.
[0043] In this application, the method further includes the steps of culturing the recombinant microorganism or composition with a culture medium, collecting the culture product, and obtaining L-amino acids.
[0044] The term "culture product" refers to the collective term for liquid or solid products (all substances within the culture container) that have grown a microbial community after artificial inoculation and cultivation. In other words, it is the product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. It can also be a mixture containing a certain amount of culture medium, microbial cell metabolites, and with the microbial cells removed.
[0045] In this application, the improvement or enhancement is a comparison performed under comparable conditions. "Comparable conditions" refers to the same or similar environmental and fermentation conditions used to make meaningful comparisons between two or more microbial genotypes, such that neither the environmental nor fermentation conditions significantly promote or explain any differences observed between the two or more microbial genotypes.
[0046] The beneficial technical effects achieved by this application are as follows: This application obtains a protein mutant (NCgl1764) by point mutation of the NCgl1764 protein. I21L和 / 或L66P The NCgl1764 protein of the recipient bacteria was mutated to NCgl1764. I21L和 / 或L66P Or introduce the NCgl1764 into the recipient bacteria. I21L 和 / 或L66P Functional verification of the NCgl1764 protein showed that point mutations in the coding region of the NCgl1764 gene in Corynebacterium glutamicum resulted in the formation of NCgl1764. I21L和 / 或L66P Alternatively, introducing the coding sequence of the NCgl1764 gene can significantly increase L-isoleucine production; while knocking out or weakening the gene can significantly reduce L-isoleucine accumulation.
[0047] Preservation Instructions Classification and nomenclature: Corynebacterium glutamicum ( Corynebacterium glutamicum ) Strain number: YPILE001 Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Abbreviation of depositary institution: CGMCC Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101 Date of preservation: August 17, 2020 CGMCC Registration Number: 20437 Detailed Implementation I. Terminology in this application: Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.
[0048] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.
[0049] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide 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, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.
[0050] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency. More specifically, the above 80% consistency can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency.
[0051] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.
[0052] As is generally understood in the art, the term "promoter" typically refers to a DNA molecule containing an RNA polymerase binding site and / or a transcription start site that assists or promotes the transcription of transcribed DNA. In prokaryotes, the promoter sequence is located at the 5' end of the transcription start site (TSS), covering a region approximately 40 bp in length, and structurally generally includes the transcription start site (denoted as +1). District 35 District 10 and 35 districts and The spacer region between the 10 regions. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application can include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.
[0053] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. Promoters that drive expression in all or most tissues of the receptor are called “constitutive” promoters. Promoters that drive expression at certain times or stages of development are called “developmental” promoters. “Inducible” promoters are promoters that initiate transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, and synthetic.
[0054] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.
[0055] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.
[0056] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.
[0057] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).
[0058] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.
[0059] The term "microorganism capable of producing glutamate" refers to a microorganism that possesses the ability to produce and accumulate glutamate within itself using external substances (such as culture medium), and may further include the ability to secrete glutamate into the culture system. Thus, glutamate can be collected when the microorganism is cultured in a culture medium.
[0060] The term "culture product" refers to the collective term for liquid or solid products (all substances within the culture container) that have grown a microbial community after artificial inoculation and cultivation. In other words, it is the product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. It can also be a mixture containing a certain amount of culture medium, microbial cell metabolites, and with the microbial cells removed.
[0061] II. Implementation Examples The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.
[0062] 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.
[0063] Corynebacterium glutamicum ATCC13032, also known as ATCC13032, was purchased from the American Type Culture Collection (ATCC), with strain number 13032; Corynebacterium glutamicum ( Corynebacterium glutamicum CGMCC No. 20437, also known as Corynebacterium glutamicum CGMCC20437, is a strain that has been deposited through a patent procedure with the accession number CGMCC NO. 20437.
[0064] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.
[0065] Example 1: Construction of a recombinant vector containing a point-mutated NCgl1764 gene coding region fragment According to the NCBI's published data on Corynebacterium glutamicum ( Corynebacterium glutamicum The ATCC 13032 genome sequence was used to design and synthesize a pair of primers to amplify the coding region of the NCgl1764 gene (Gene ID: 1019796, 2020-3-6). Allelic substitution was employed in Corynebacterium glutamicum (C. glutamicum). Corynebacterium glutamicumA point mutation was introduced into the coding region (SEQ ID No. 1) of the NCgl1764 gene of CGMCC 20437 (which was confirmed by sequencing to retain the wild-type NCgl1764 gene on its chromosome). The point mutation was to mutate adenine (A) at position 61 of the nucleotide sequence (SEQ ID No. 1) of the NCgl1764 gene to cytosine (C), obtaining a DNA molecule with the coding sequence shown in SEQ ID No. 3; to mutate thymine (T) at position 197 to cytosine (C), obtaining a DNA molecule with the coding sequence shown in SEQ ID No. 5; and simultaneously to mutate adenine (A) at position 61 to cytosine (C) and thymine (T) at position 197 to cytosine (C), obtaining a DNA molecule with the coding sequence shown in SEQ ID No. 7.
[0066] The DNA molecule shown in SEQ ID No. 1 encodes a protein with the amino acid sequence of SEQ ID No. 2 (the protein is named protein NCgl1764).
[0067] The DNA molecule shown in SEQ ID No. 3 encodes a mutant protein with the amino acid sequence of SEQ ID No. 4, and this mutant protein is named NCgl1764. I21L The mutant protein NCgl1764 I21L The leucine (L) at position 21 in the amino acid sequence SEQ ID No.4 is derived from isoleucine (I) by mutation.
[0068] The DNA molecule shown in SEQ ID No. 5 encodes a mutant protein with the amino acid sequence of SEQ ID No. 6, and this mutant protein is named NCgl1764. L66P The mutant protein NCgl1764 L66P The proline (P) at position 66 in the amino acid sequence SEQ ID No.4 is a mutation of leucine (L).
[0069] The DNA molecule shown in SEQ ID No. 7 encodes a mutant protein with the amino acid sequence of SEQ ID No. 8, and this mutant protein is named NCgl1764. I21L、L66P The mutant protein NCgl1764 I21L、L66P The leucine (L) at position 21 in the amino acid sequence SEQ ID No.4 is derived from isoleucine (I) by mutation, and the proline (P) at position 66 is derived from leucine (L) by mutation.
[0070] Vectors were constructed using NEBuilder recombination technology, and the primers were designed as follows (synthesized by Invitrogen Shanghai). In the primers, bolded bases indicate mutation sites, and lowercase nucleotide sequences are those found on the pK18mobsacB plasmid. P1: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagCAGCCGTATTTCGGTCAGTCAGCA-3'; P2: 5'-CATACACTAGGCACATCGTGAGATGGACTATCGCAACAGG-3'; P3: 5'-ACGATGTGCCTAGTGTATGAGCTAATAAAGCTTAGACCTC-3'; P4: 5'-GCACCCATCGGCAAAAAAAGGTACAGCGTTGTTGGCTTT-3' P5: 5'-tttttttgcCgatgggtgcaacgaagacggagttatgatg-3' P6: 5'-cagctatgaccatgattacgaattcgagctcggtacccCCATCACCCTCAATGGGTCAGGCC-3'.
[0071] NCgl1764 I21L Construction method: Using Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P1 and P2, and P3 and P6, respectively, to obtain two NCgl1764 primers with mutant bases, with sizes of 685 bp and 662 bp, respectively. I21L DNA fragment in the gene coding region (NCgl1764) I21L Up and NCgl1764 I21L Down).
[0072] NCgl1764 L66P Construction method: Using Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P1 and P4, and P5 and P6, respectively, to obtain two NCgl1764 primers with mutant bases, with sizes of 821 bp and 526 bp, respectively. L66P DNA fragment in the gene coding region (NCgl1764) L66P Up and NCgl1764 L66P Down).
[0073] NCgl1764 I21L、L66PConstruction method: Using Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P1 and P2, P3 and P4, and P5 and P6, respectively, to obtain three NCgl1764 molecules with sizes of 685 bp, 155 bp, and 526 bp. L66P DNA fragment in the gene coding region (NCgl1764) I21L、L66P Up、NCgl1764 I21L、L66P and NCgl1764 I21L 、L66P Down).
[0074] The PCR amplification system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+ 4 μL of (25 mM) primers (10 pM) each, 2 μL of Ex Taq (5 U / μL), and 0.25 μL of ddH2O to a total volume of 50 μL; The PCR amplification reaction program was as follows: 94 ℃ pre-denaturation for 5 min, (94 ℃ denaturation for 30 s; 52 ℃ annealing for 30 s; 72 ℃ extension for 90 s; 30 cycles), and 72 ℃ final extension for 10 min.
[0075] After the seven DNA fragments were separated and purified by agarose gel electrophoresis, they were divided into three groups according to the mutation sites (NCgl1764). I21L Up and NCgl1764 I21L Down;NCgl1764 L66P Up and NCgl1764 L66P Down;NCgl1764 I21L、 L66P Up、NCgl1764 I21L、L66P and NCgl1764 I21L、L66P Down) and enzyme digestion ( Xbal I / BamH I) The purified pK18mobsacB plasmid (Addgene product, catalog number Plasmid #177839, abbreviated as pK18msB, containing a kanamycin resistance marker) was ligated with NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation product was transformed into DH5α, and the resulting single clones were identified by PCR using M13 primers (M13F and M13R) to obtain the positive recombinant vector pK18-NCgl1764. I21L pK18-NCgl1764 L66P and pK18-NCgl1764 I21L、L66PThe correctly digested recombinant plasmid was sent to a sequencing company for sequencing and identification, and the recombinant vector pK18-NCgl1764 containing the correct point mutation (AC) was sent to the sequencing company. I21L The recombinant vector pK18-NCgl1764 containing the correct point mutation (TC) L66P and the recombinant vector pK18-NCgl1764 containing the correct point mutations (AC; TC) I21L、L66P Save for future use.
[0076] The sequences of M13F and M13R are as follows: M13F: 5'-TGTAAAACGACGGCCAGT-3'; M13R: 5'-CAGGAAACAGCTATGACC-3'.
[0077] Recombinant vector pK18-NCgl1764 I21L NCgl1764 I21L Up-down DNA size 1328 bp, NCgl1764 I21L The Up-Down sequence (as shown in SEQ ID No. 9) contains a mutation site that causes the 61st adenine (A) in the coding region of the NCgl1764 gene in strain Corynebacterium glutamicum CGMCC 20437 to be mutated to cytosine (C), which ultimately leads to the 21st isoleucine (I) in the encoded protein being mutated to leucine (L).
[0078] Recombinant vector pK18-NCgl1764 L66P NCgl1764 L66P Up-down DNA size 1328 bp, NCgl1764 L66P The Up-Down sequence (as shown in SEQ ID No. 10) contains a mutation site that causes the thymine (T) at position 197 of the coding region of the NCgl1764 gene in strain Corynebacterium glutamicum CGMCC 20437 to be mutated to cytosine (C), which ultimately leads to the leucine (L) at position 66 of the encoded protein being mutated to proline (P).
[0079] Recombinant vector pK18-NCgl1764 I21L、L66P NCgl1764 I21L、L66P Up-down DNA size 1328 bp, NCgl1764 I21L、L66PThe up-down sequence (as shown in SEQ ID No. 11) contains a mutation site that causes the 61st adenine (A) in the coding region of the NCgl1764 gene in strain Corynebacterium glutamicum CGMCC 20437 to be mutated to cytosine (C), which ultimately leads to the 21st isoleucine (I) in the encoded protein being mutated to leucine (L); the 197th thymine (T) in the encoded protein is mutated to cytosine (C), which ultimately leads to the 66th leucine (L) in the encoded protein being mutated to proline (P).
[0080] The sequences in SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11 are as follows: positions 1 to 36 are the sequence on the pK18mobsacB plasmid; positions 37 to 579 are the upstream homologous arm sequence of the Ncgl1764 gene; and positions 616 to 840 are the Ncgl1764 gene sequences. I21L NCgl1764 L66P and NCgl1764 I21L、L66P The coding sequence, positions 841 to 1290 are the downstream homologous arm sequences of the Ncgl1764 gene, and positions 1291 to 1328 are the sequences on the pK18mobsacB plasmid.
[0081] The recombinant plasmid pK18-NCgl1764 I21L pK18-NCgl1764 L66P and pK18-NCgl1764 I21L、L66P The result is: the pK18mobsacB plasmid... Xbal I and BamH The recombinant vector was obtained by replacing the fragments (small fragments) between the I recognition sites with the DNA fragments shown at positions 37-1290 of SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11 in the sequence listing, while keeping the other sequences of the pK18mobsacB vector unchanged.
[0082] The recombinant vector pK18-NCgl1764 I21L pK18-NCgl1764 L66P and pK18-NCgl1764 I21L、L66P DNA molecules containing the mutant genes shown in SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 respectively, at positions 1-225.
[0083] Example 2: Constructing a structure containing the gene NCgl1764 I21L engineered strains Construction method: The allelic substitution plasmid (pK18-NCgl1764) from Example 1 was used. I21LpK18-NCgl1764 L66P and pK18-NCgl1764 I21L、L66P ) through electroporation to convert into Corynebacterium glutamicum ( Corynebacterium glutamicum After being cultured in CGMCC 20437 and ATCC 13032, the cultures were incubated in culture media. The composition and conditions of the culture media are shown in Table 1. Single colonies produced were picked and cultured on media containing 15% sucrose. These single colonies were then cultured on media containing and without kanamycin. Strains that grew on media without kanamycin but not on media containing kanamycin were selected for further PCR identification using the following primers (synthesized by Invitrogen Shanghai): P7: 5'-ACCTCACCGCGATAATGGCATC-3'; P8: 5'-ACATTAATGTCTCCCAAATA-3'.
[0084] The obtained PCR amplification products (346 bp) were sequenced. Sequence alignment revealed that recombinant strains of *Corynebacterium glutamicum* CGMCC 20437 and ATCC 13032 with adenine (A) mutated to cytosine (C) at position 61 were named YPI-1764-1 and YPI-1764-2, respectively, both containing the mutation sequence shown in SEQ ID No. 3; recombinant strains of *Corynebacterium glutamicum* CGMCC 20437 and ATCC 13032 with thymine (T) mutated to cytosine (C) at position 197 were named YPI-1764-3 and YPI-1764-4, respectively, both containing the mutation sequence shown in SEQ ID No. 5; CGMCC 20437 and ATCC... The recombinant strains of 13032 with adenine (A) at position 61 mutated to cytosine (C) and thymine (T) at position 197 mutated to cytosine (C) were named YPI-1764-5 and YPI-1764-6, respectively, both containing the mutated sequence shown in SEQ ID No. 7.
[0085] Table 1. Composition and culture conditions of the culture medium
[0086] Example 3: Constructing a genome overexpressing the NCgl1764 gene. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P engineered strains of genes Vectors were constructed using NEBuilder recombination technology. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, three pairs of amplified upstream and downstream homologous arm fragments and the NCgl1764 gene were designed and synthesized. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P Primers for the gene coding region and promoter region were used to introduce the NCgl1764 gene into Corynebacterium glutamicum CGMCC 20437 and ATCC 13032 via homologous recombination. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P Gene.
[0087] The primers were designed as follows (synthesized by Invitrogen Shanghai), and the nucleotide sequences in lowercase format are the sequences on the pK18mobsacB plasmid: P9:5'-cagtgccaagcttgcatgcctgcaggtcgactctagCATTGCCATCAATCCATGC-3', P10:5'-TTCCGGCTCAGAAAGCGGTGTGATTGATACACCTGCTGTT-3', P11:5'-AACAGCAGGTGTATCAATCACACCGCTTTCTGAGCCGGAA-3', P12:5'-CATAACGTTGAGGAGTTCAGTCATAACTCCGTCTTCGTTG-3', P13:5'-CAACGAAGACGGAGTTATGACTGAACTCCTCAACGTTATG-3', P14:5'-cagctatgaccatgattacgaattcgagctcggtacccGCACCAAGTGAAGCGATTC-3'.
[0088] Using genomic DNA from Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed with primers P9 and P10 to obtain an 860 bp upstream homologous arm fragment (sequence shown in SEQ ID No. 12); PCR amplification was performed with primers P11 and P12 to obtain the NCgl1764 gene and its promoter fragment of 315 bp (sequence shown in SEQ ID No. 13); PCR amplification was performed with primers P13 and P14 to obtain an 817 bp downstream homologous arm fragment (sequence shown in SEQ ID No. 17).
[0089] Using the genomic DNA of YPI-1764-1 as a template, PCR amplification was performed with primers P11 and P12 to obtain NCgl1764. I21L The gene and its promoter fragment 315 bp (sequence shown in SEQ ID No. 14).
[0090] Using the genomic DNA of YPI-1764-3 as a template, PCR amplification was performed with primers P11 and P12 to obtain NCgl1764. L66P The gene and its promoter fragment 315 bp (sequence shown in SEQ ID No. 15); Using the genomic DNA of YPI-1764-5 as a template, PCR amplification was performed with primers P11 and P12 to obtain NCgl1764. I21L、L66P The gene and its promoter fragment 315 bp (sequence shown in SEQ ID No. 16); After the PCR reaction, the amplified fragments were recovered by electrophoresis using a column-based DNA gel extraction kit. The NCgl1764 gene and its promoter fragment, NCgl1764... I21L Genes and their promoter fragments, NCgl1764 L66P Genes and their promoter fragments and NCgl1764 I21L、L66P The gene and its promoter fragment are combined with upstream and downstream homologous arm fragments, respectively, and then combined with... Xbal I / BamH The pK18mobsacB plasmid purified after I enzyme digestion was ligated with NEBuilder enzyme (purchased from NEB) at 50℃ for 30 min. The ligation product was transformed into DH5α, and the resulting single clones were identified by PCR using M13 primers (M13F and M13R) to obtain positive integrative plasmids (recombinant vectors), namely pK18-NCgl1764OE and pK18-NCgl1764. I21L OE, pK18-NCgl1764 L66P OE and pK18-NCgl1764 I21L、L66POE, with four positive integration plasmids containing kanamycin resistance markers, can be used to select recombinants whose plasmids have integrated into the genome through kanamycin screening.
[0091] The PCR reaction system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+ 4 μL of (25 mM) primers, 2 μL each of (10 pM) primers, 0.25 μL of Ex Taq (5 U / μL), and ddH2O added to a total volume of 50 μL.
[0092] The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s; 52 °C annealing for 30 s; 72 °C extension for 60 s (30 cycles), and 72 °C final extension for 10 min.
[0093] The correctly sequenced integration plasmids (pK18-NCgl1764OE, pK18-NCgl1764) were identified. I21L OE, pK18-NCgl1764 L66P OE and pK18-NCgl1764 I21L、L66P OE) was electroporated into Corynebacterium glutamicum CGMCC 20437 and ATCC 13032, respectively, and cultured in culture medium. The composition and culture conditions of the culture medium are shown in Table 1. Single colonies produced by culture were identified by PCR using primers P15 and P16. Strains that amplified by PCR containing a fragment of size 1113 bp (SEQ ID No. 18) were positive strains, while those that could not amplify the fragment were original strains.
[0094] The positive strain was cultured on a medium containing 15% sucrose. Single colonies produced were further identified by PCR using primers P17 and P18. The amplified colony (SEQ ID No. 19) was identified as containing the NCgl1764 gene. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P Positive strains integrated into the recipient bacterial genome. Positive strains (recombinant bacteria) obtained from *Corynebacterium glutamicum* CGMCC 20437 were named YPI-1764-07 (without point mutations), YPI-1764-08, YPI-1764-09, and YPI-1764-10, respectively; modified strains obtained from ATCC 13032 were named YPI-1764-11 (without point mutations), YPI-1764-12, YPI-1764-13, and YPI-1764-14, respectively.
[0095] The recombinant bacteria YPI-1764-07 and YPI-1764-11 contain two copies of the NCgl1764 gene shown in SEQ ID No. 1. Specifically, the recombinant bacteria YPI-1764-07 and YPI-1764-11 are obtained by replacing the coding region of the poxB gene (Gene ID: 1020557, 2025-12-12) in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC13032 with the NCgl1764 gene, while keeping other nucleotides in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC 13032 unchanged. The recombinant bacteria containing two copies of the NCgl1764 gene can significantly and stably increase the expression level of the NCgl1764 gene.
[0096] The recombinant strains YPI-1764-08 and YPI-1764-12 contain the mutated NCgl1764 shown in SEQ ID No. 3. I21L Specifically, the recombinant bacteria YPI-1764-8 and YPI-1764-12 are formed by replacing the poxB gene coding region in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC13032 with NCgl1764. I21L The recombinant bacteria were obtained by keeping other nucleotides in the genomes of Corynebacterium glutamicum CGMCC20437 and ATCC 13032 unchanged.
[0097] The recombinant strains YPI-1764-09 and YPI-1764-13 contain the mutated NCgl1764 shown in SEQ ID No. 5. L66P Specifically, the recombinant bacteria YPI-1764-09 and YPI-1764-13 are formed by replacing the poxB gene coding region in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC13032 with NCgl1764. L66P The recombinant bacteria were obtained by keeping other nucleotides in the genomes of Corynebacterium glutamicum CGMCC20437 and ATCC 13032 unchanged.
[0098] Recombinant strains YPI-1764-10 and YPI-1764-14 contain the mutated NCgl1764 shown in SEQ ID No. 7. I21L 、L66P Specifically, the recombinant bacteria YPI-1764-10 and YPI-1764-14 are formed by replacing the poxB gene coding region in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC 13032 with NCgl1764. I21L、L66PThe recombinant bacteria were obtained by keeping other nucleotides in the genomes of Corynebacterium glutamicum CGMCC 20437 and ATCC 13032 unchanged.
[0099] The PCR identification primers are shown below: P15:5'-GCCAGAAAATGGGTTGCGTGA-3', P16:5'-AAAGGTACAGCGTTGTTGG-3', P17:5'-CTGAATTCAGAGATCTACAA-3', P18:5'-TCAAACGTAATGCCGGATCAG-3'.
[0100] Example 4: Constructing a plasmid to overexpress the NCgl1764 gene. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P engineered strains Vectors were constructed using NEBuilder recombination technology. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, a pair of amplified NCgl1764 genes were designed and synthesized. L66P Gene, NCgl1764 I21L Genes and NCgl1764 I21L、L66P Primers for the coding and promoter regions were designed as follows (synthesized by Invitrogen Shanghai). The lowercase nucleotide sequences are the sequences from pXMJ19: P19:5'-gcttgcatgcctgcaggtcgactctagaggatccccCACCGCTTTCTGAGCCGG-3'; P20:5'-atcaggctgaaaatcttctctcatccgccaaaacTCATAACTCCGTCTTCGTTG-3'; M13R(-48)5'-AGCGGATAACAATTTCACACAGGA-3'.
[0101] Using genomic DNA from *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification was performed using primers P19 and P20 to obtain the NCgl1764 gene and its promoter fragment (sequence shown in SEQ ID No. 20). Using *Corynebacterium glutamicum* YPI-1764-1, YPI-1764-3, and YPI-1764-5 as templates, PCR amplification was performed using primers P19 and P20 to obtain NCgl1764, respectively.I21L Gene and its promoter fragment (sequence shown in SEQ ID No. 21), NCgl1764 L66P Gene and its promoter fragment (sequence shown in SEQ ID No. 22), NCgl1764 I21L、L66P Gene and its promoter fragment (sequence shown in SEQ ID No. 23).
[0102] The amplification products were subjected to electrophoresis and purified using a column-based DNA gel extraction kit. The recovered DNA fragments were compared with those obtained from the electrophoresis process. EcoR I / Kpn The shuttle plasmid pXMJ19 (Biovector pXMJ19, containing chloramphenicol resistance as a selection marker), recovered by enzyme digestion, was ligated with NEBuilder enzyme (NEB) at 50 °C for 30 min. The ligation product was transformed into DH5α competent cells, and the resulting single clones were identified by PCR using primers M13R(-48) and P20 (taking the sequence SEQ ID No. 24 without point mutations as an example). Positive overexpression plasmids pXMJ19-NCgl1764 (containing the NCgl1764 gene) and pXMJ19-NCgl1764 were obtained. I21L (Contains NCgl1764) I21L (gene), pXMJ19-NCgl1764 L66P (Contains NCgl1764) L66P (gene) and pXMJ19-NCgl1764 I21L、L66P (Contains NCgl1764) I21L、L66P The plasmid (containing a gene) was sent for sequencing. Because the plasmid contains a chloramphenicol resistance marker, chloramphenicol can be used to screen whether the plasmid has been transformed into a bacterial strain.
[0103] The PCR reaction system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+ 4 μL of (25 mM) primers, 2 μL each of (10 pM) primers, 0.25 μL of Ex Taq (5 U / μL), and ddH2O added to a total volume of 50 μL.
[0104] The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s; 52 °C annealing for 30 s; 72 °C extension for 60 s (30 cycles), and 72 °C final extension for 10 min.
[0105] pXMJ19-NCgl1764 and pXMJ19-NCgl1764 were correctly sequenced. I21LpXMJ19-NCgl1764 L66P and pXMJ19-NCgl1764 I21L、L66P The plasmids were electroporated into Corynebacterium glutamicum CGMCC 20437 and ATCC 13032, respectively, and cultured in culture media. The culture medium composition and conditions are shown in Table 1. Single colonies were identified by PCR using primers M13R(-48) and P20. Strains amplified by PCR containing a 384 bp fragment were considered positive (using the sequence SEQ ID No. 24 without point mutations as an example). The recombinant bacteria obtained from CGMCC 20437 were named YPI-1764-15 (without point mutations), YPI-1764-16, YPI-1764-17, and YPI-1764-18, respectively; those obtained from ATCC 13032 or as modified strains were named YPI-1764-19 (without point mutations), YPI-1764-20, YPI-1764-21, and YPI-1764-22, respectively.
[0106] The recombinant bacteria YPI-1764-15 and YPI-1764-19 contain the recombinant plasmid pXMJ19-NCgl1764. The recombinant plasmid pXMJ19-NCgl1764 contains the NCgl1764 gene shown in SEQ ID No. 1 and can express the NCgl1764 protein shown in SEQ ID No. 2.
[0107] Recombinant bacteria YPI-1764-16 and YPI-1764-20 contain the recombinant plasmid pXMJ19-NCgl1764 I21L Recombinant plasmid pXMJ19-NCgl1764 I21L NCgl1764 containing the mutation shown in SEQ ID No. 3 I21L The gene can express NCgl1764 as shown in SEQ ID No. 4. I21L Mutant protein.
[0108] Recombinant bacteria YPI-1764-17 and YPI-1764-21 contain the recombinant plasmid pXMJ19-NCgl1764 L66P Recombinant plasmid pXMJ19-NCgl1764 L66P NCgl1764 containing the mutation shown in SEQ ID No. 5 L66P The gene can express NCgl1764 as shown in SEQ ID No. 6. L66P Mutant protein.
[0109] Recombinant bacteria YPI-1764-18 and YPI-1764-22 contain the recombinant plasmid pXMJ19-NCgl1764 I21L、L66P Recombinant plasmid pXMJ19-NCgl1764 I21L、L66P NCgl1764 containing the mutation shown in SEQ ID No. 7 I21L、L66P The gene can express NCgl1764, as shown in SEQ ID No. 8. I21L、L66P Mutant protein.
[0110] Example 5: Constructing an engineered strain with the NCgl1764 gene deleted from its genome. Vectors were constructed using NEBuilder recombination technology. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the segments at both ends of the coding region of the NCgl1764 gene, serving as upstream and downstream homologous arms. The primers were designed as follows (synthesized by Invitrogen Shanghai): P21:5'-cagtgccaagcttgcatgcctgcaggtcgactctagTTTCAGGTGACTTATGAAG-3'; P22:5'-AAGTCTTGATTCGTCGTTCCTTCATTGTCTTTCGATGCCA-3'; P23:5'-TGGCATCGAAAGACAATGAAGGAACGACGAATCAAGACTT-3'; P24:5'-cagctatgaccatgattacgaattcgagctcggtacccTTCGATTCTTCCCGTGCTGTGAAAGT-3'.
[0111] Construction method: Using Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed with primers P21 and P22 to obtain the upstream homologous arm fragment of NCgl1764, which is 660 bp; PCR amplification was performed with primers P23 and P24 to obtain the downstream homologous arm fragment of NCgl1764, which is 654 bp.
[0112] The amplified products were subjected to electrophoresis and purified using a column-based DNA gel extraction kit. The recovered DNA fragments were compared with those obtained through electrophoresis. Xbal I / BamHThe pK18mobsacB plasmid purified after I enzyme digestion was ligated with NEBuilder enzyme (purchased from NEB) at 50 °C for 30 min. The single clones grown after transformation were identified by PCR using M13 primers to obtain the positive knockout vector pK18-ΔNCgl1764. This recombinant plasmid pK18-ΔNCgl1764 contains a 1274 bp up-down DNA fragment of ΔNCgl1764 (sequence shown in SEQ ID No. 25).
[0113] The plasmid was sent for sequencing. The correctly sequenced knockout plasmid pK18-ΔNCgl1764 was electroporated into Corynebacterium glutamicum CGMCC 20437 and cultured in culture medium. The composition and culture conditions of the culture medium are shown in Table 1. Single colonies generated by culture were identified by PCR using the following primers (synthesized by Invitrogen Shanghai): P25:5'-TTTCAGGTGACTTATGAAGG-3', P26:-5'-TTCGATCTTCCCGTGCTGTGAAAGT-3'.
[0114] The strains that simultaneously amplified bands of 1200 bp and 1425 bp using the above PCR were identified as positive strains, while the strains that amplified only the 1425 bp band were identified as the original strain. Positive strains were screened on 15% sucrose medium and then cultured on media containing and without kanamycin. Strains that grew on the kanamycin-free medium but not on the kanamycin-containing medium were further identified by PCR using primers P25 and P26. Strains amplifying a 1200 bp band were identified as positive strains with the NCgl1764 gene coding region knocked out. The NCgl1764 fragment of the positive strains was amplified again by PCR using primers P25 and P26 and sequenced. The correctly sequenced strain was named YPI-1764-23 (NCgl1764 gene knocked out in the genome of Corynebacterium glutamicum CGMCC 20437).
[0115] Example 6: L-Isoleucine Fermentation Experiment The strains constructed in the above examples and the original strain Corynebacterium glutamicum CGMCC 20437 were fermented in a BLBIO-5GC-4-H fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) using fermentation medium and the control process shown in Table 2. Each strain was replicated three times, and the results are shown in Table 3.
[0116] The results are shown in Table 3. Point mutations were performed on the coding region of the NCgl1764 gene in Corynebacterium glutamicum. I21LOverexpression or insertion of the coding sequence of the NCgl1764 gene can significantly increase L-isoleucine production, while knockout or attenuation of the gene can significantly reduce L-isoleucine accumulation.
[0117] The fermentation medium formula is as follows: glucose 5.0 g / L, phosphate 0.38 g / L, magnesium sulfate 1.85 g / L, potassium chloride 1.6 g / L, biotin 550 μg / L, vitamin biotin B1 300 μg / L, ferrous sulfate 10 mg / L, manganese sulfate 10 g / dL, KH2PO4 2.8 g / L, vitamin C 0.75 mg / L, vitamin B12 2.5 μg / L, para-aminobenzoic acid 0.75 mg / L, antifoaming agent 0.0015 ml / dL, betaine 1.5 g / L, sugarcane molasses 7 ml / L, corn steep liquor 77 ml / L, aspartic acid 1.7 g / L, hair powder 2 g / L, and water as the solvent.
[0118] Table 2. Fermentation Control Process
[0119] Table 3. Results of L-Isoleucine Fermentation Experiment
[0120] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A mutant protein, characterized in that: The mutant protein includes at least one of the following: a1) Proteins whose amino acid sequences contain mutations at amino acid residues at positions 21 and / or 66 as shown in SEQ ID NO.2; a2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in a1). a3) Proteins with more than 95% identity to the amino acid sequence shown in a1) and with the same function; a4) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1), a2), or a3).
2. The mutant protein according to claim 1, characterized in that, The mutant protein comprises a protein obtained by mutating the isoleucine residue at position 21 of the sequence shown in SEQ ID NO.2 to a leucine residue and / or mutating the leucine residue at position 66 to a proline residue.
3. The mutant protein according to any one of claims 1-2, characterized in that, The mutant protein comprises the amino acid sequence shown in SEQ ID No. 4 and / or SEQ ID No. 6 and / or SEQ ID No.
8.
4. A biomaterial, said biomaterial comprising at least one of the following A1)-A5): A1) A nucleic acid molecule encoding any of the mutant proteins described in claims 1-3. A2), an expression cassette containing the nucleic acid molecule described in A1), A3), a recombinant vector containing the nucleic acid molecule described in A1), and / or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), recombinant microorganisms containing the expression cassette described in A2), and / or recombinant microorganisms containing the recombinant vector described in A3); A5) A whole-cell catalyst containing the nucleic acid molecule described in A1), a whole-cell catalyst containing the expression cassette described in A2), a whole-cell catalyst containing the recombinant vector described in A3), and / or a whole-cell catalyst containing the recombinant microorganism described in A4).
5. The biomaterial according to claim 4, characterized in that: A1) The nucleic acid molecule described herein includes at least one of the following: B1) DNA molecules whose coding sequences include SEQ ID No. 3 and / or SEQ ID No. 5 and / or SEQ ID No. 7; B2) is a DNA molecule that shares more than 85% identity with the DNA molecule described in B1) and encodes the same protein mutant.
6. Application, characterized in that: The application is the use of the mutant protein of any one of claims 1-3 or the biological material of claim 4 or 5 and / or the NCgl1764 protein or related biological materials in at least one of the following: C1) Application in the preparation of L-isoleucine; C2) Used in the preparation of products containing L-isoleucine; C3) Application in increasing L-isoleucine production in recipient bacteria; C4) Application in constructing engineered bacteria that produce L-isoleucine.
7. A method for increasing the L-isoleucine production of microorganisms, characterized in that: The method includes at least one of the following: F1) Expressing the gene encoding the mutant protein of any one of claims 1-3 into the target microorganism; F2) Introducing the biomaterial described in claim 4 or 5 into the target microorganism; F3) Mutate the coding gene of NCgl1764 protein in the target microorganism to the coding gene of any of the mutant proteins described in claims 1-3; F4), the gene encoding the NCgl1764 protein in overexpressed microorganisms.
8. Recombinant Corynebacterium glutamicum, characterized in that, The recombinant Corynebacterium glutamicum is at least one of the following: G1) Recombinant Corynebacterium glutamicum containing the gene encoding the mutant protein of any one of claims 1-3; G2) Recombinant Corynebacterium glutamicum obtained by enhancing the expression of the gene encoding the NCgl1764 protein containing the amino acid sequence of SEQ ID NO.2 in the recipient bacteria.
9. A composition, characterized in that: The composition contains the recombinant Corynebacterium glutamicum as described in claim 8.
10. A method for preparing L-isoleucine, characterized in that: The method includes the step of preparing L-isoleucine using recombinant cells or recombinant microorganisms expressing the mutant protein of claim 1 or 2 and / or using recombinant Corynebacterium glutamicum of claim 9.