N-acetylglutamate kinase mutants and uses thereof

Abstract

The present disclosure relates to N-acetylglutamate kinase mutants and applications thereof, and belongs to the technical fields of biotechnology and genetic engineering. The present disclosure specifically relates to a high-throughput screening method for N-acetylglutamate kinase mutants, N-acetylglutamate kinase mutants obtained by the screening method, polynucleotides encoding the N-acetylglutamate kinase mutants, nucleic acid constructs, recombinant expression vectors, recombinant host cells, cultures, and methods for producing L-arginine. The screening method provided by the present disclosure can obtain a large number of diverse N-acetylglutamate kinase mutants, and has higher screening efficiency. Moreover, the N-acetylglutamate kinase mutants obtained by the screening method have significantly improved L-arginine production performance, and are suitable for industrial production of L-arginine or compounds related to the metabolic pathway thereof.

Description

Technical Field

[0001] This disclosure relates to N-acetylglutamate kinase mutants and their applications, specifically to polynucleotides, nucleic acid constructs, recombinant expression vectors, recombinant host cells, cultures, and methods for producing L-arginine, belonging to the fields of biotechnology and genetic engineering technology. Background Technology

[0002] L-Arginine is one of the semi-essential amino acids required by the human body. Due to its various important physiological functions, it has significant and wide-ranging applications in medicine, food, and health products. In the microbial synthesis of arginine, N-acetylglutamate kinase (ArgB, encoded by the argB gene) is the second enzyme in this process and also the key rate-limiting enzyme, catalyzing the conversion of N-acetylglutamate to N-acetyl-γ-glutamyl phosphate. In Corynebacterium glutamicum, the ArgB-catalyzed reaction is subject to feedback inhibition by the final product arginine. Therefore, overcoming the feedback inhibition of ArgB is the primary task in constructing arginine-producing strains.

[0003] Several studies have reported that mutants of Corynebacterium glutamicum ArgB have the effect of relieving feedback inhibition. For example, the N-terminal helix or nearby amino acid residues (E19, H26, R209, H268, and G287) are essential for the formation of arginine inhibition (Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum. Current Microbiology, 2012, 64(2):164-72. Monomeric Corynebacterium glutamicum N-acetyl glutamate kinase maintains sensitivity to L-arginine but has a lower intrinsic catalytic activity. Applied Microbiology and Biotechnology, 2016, 100(4):1789-1798.). Introducing ArgB into the chromosome of Corynebacterium glutamicum... A26V,M31VMutations can lead to the accumulation of arginine and its intermediate metabolite citrulline (Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer. Applied and Environmental Microbiology, 2009, 75(6):1635-41.). Other studies have identified potential binding sites for arginine and the residues essential for its inhibition through homology modeling, inhibitor docking, and site-directed mutagenesis, and have increased L-arginine production in Corynebacterium glutamicum ATCC 14067 by overexpressing the ArgB mutant (Mutational analysis to identify theresidues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum. Applied Microbiology and Biotechnology, 2015, 99(18):7527-37.).

[0004] Although several existing ArgB mutants can relieve the feedback inhibition of arginine, there is still a need in the field to develop other more efficient ArgB mutants in order to further increase the production of arginine and other metabolites by Corynebacterium glutamicum. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] Although several ArgB mutants capable of relieving arginine feedback inhibition have been reported in the prior art, the number of mutants remains relatively small, and the performance of these mutants in increasing arginine production in strains still needs further improvement. This disclosure provides a high-throughput screening method for efficiently screening ArgB mutants that relieve feedback inhibition. This method can obtain a large number of beneficial mutants, and the screened mutants can effectively relieve arginine feedback inhibition and significantly increase L-arginine production in strains.

[0007] Solution for solving the problem

[0008] This disclosure provides a high-throughput screening method for N-acetylglutamate kinase mutants. The method combines high-throughput random mutagenesis with single-point saturation mutagenesis. First, a random mutant library of N-acetylglutamate kinase mutants is constructed, and then the arginine-specific biosensor pLysG is used. F222IThe arginine production of the mutant was coupled with the fluorescence intensity. The fluorescence intensity of the mutant library was analyzed by flow cytometry. The region with the highest fluorescence intensity in the library strain was selected for initial and secondary screening of arginine production to obtain N-acetylglutamate kinase mutants. Subsequently, the high fluorescence intensity unit sites obtained by random mutation were subjected to saturation mutation to further obtain unit site mutants of N-acetylglutamate kinase.

[0009] Furthermore, this disclosure provides N-acetylglutamate kinase mutants obtained by the high-throughput screening method for N-acetylglutamate kinase mutants described above, wherein the N-acetylglutamate kinase mutants are selected from any one of the following groups (I)-(V):

[0010] (I) The N-acetylglutamate kinase mutant described herein, compared with the sequence shown in SEQ ID NO:1, has the following characteristics at positions 15, 17, 18, 20, 24, 25, 27, 43, 46, 47, 48, 49, 54, 55, 56, 66, 77, 85, 113, 122, 126, 136, 142, 145, 153, 160, 161, 162, 163, 164, 167, 168, 169, 170, and 172 corresponding to the sequence shown in SEQ ID NO:1. The mutation is present at one or more of the following positions: 173, 174, 188, 197, 200, 202, 204, 207, 208, 213, 215, 225, 231, 233, 235, 236, 242, 259, 261, 265, 266, 273, 275, 283, 286, 288, 289, 297; and it exhibits increased arginine production compared to the N-acetylglutamate kinase shown in SEQ ID NO:1.

[0011] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:1;

[0012] (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions:

[0013] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0014] (b) A full-length complementary polynucleotide to (a);

[0015] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still has N-acetylglutamate kinase activity;

[0016] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0017] In some embodiments, the N-acetylglutamate kinase mutant corresponds to an amino acid with a mutation at at least one of the following positions in the sequence shown in SEQ ID NO:1:

[0018] G153S, I169N, R174H, G122S, N160S, S265G, L24M, M54I, M77I, L213H, A259V, A 266T, A235P, F136L, A48T, D168E, E208G, A207T, I170M, Q188R, L24F, V66M, G28 9A, K47E, M142V, I288F, A197V, A200V, I233T, F297S, A20V, F27L, F56L, G85D, M167V, D225N, A18V, L242P, L283S, G286D, L17P, V55A, A202V, N15S, L46P, D162 N, R273H, A275V, D113N, V126A, A172T, S231P, D43G, G173R, N215D, V161I, V66 M, R261C, A200T, T236I, Q25R, I145V, A163T, S164P, A204V, L17K, L17S, L17R, L 17G, L17T, L17Q, V66Y, V66L, A163E, A163Q, A163I, A163W, A163R, A200I, A204 F, A204L, A204Y, L17A, V66F, A163C, A163V, A200D, L17V, A163P, A204G, A204I.

[0019] In some embodiments, the N-acetylglutamate kinase mutant corresponds to the sequence shown in SEQ ID NO:1 and has the following (m1) to (m2) sequences. 61 Any mutation shown in any of the following:

[0020] (m1)G153S, I169N;

[0021] (m2)R174H;

[0022] (m3)G122S, N160S, S265G;

[0023] (m4)L24M, L283S;

[0024] (m5)M54I、M77I、V161I、L213H;

[0025] (m6)A259V、A266T;

[0026] (m7)S231P、A235P;

[0027] (m8)F136L;

[0028] (m9)A48T、D168E、A207T、E208G;

[0029] (m 10 )A207T;

[0030] (m 11 )I170M、Q188R;

[0031] (m 12 )L24F、V55A、V66M、G289A;

[0032] (m 13 )K47E、M142V;

[0033] (m 14 )I288F;

[0034] (m 15 )A197V;

[0035] (m 16 )A49V、I233T、F297S;

[0036] (m 17 )A20V、F27L、F56L、G85D、M167V;

[0037] (m 18 )F136L、D225N;

[0038] (m 19 )A18V、L242P、L283S、G286D;

[0039] (m 20 )L17P、V55A;

[0040] (m 21 )A202V;

[0041] (m 22 )N15S、L46P;

[0042] (m 23 )V55A、D162N、R273H、A275V;

[0043] (m 24 )D113N、V126A;

[0044] (m 25 )A172T、S231P;

[0045] (m 26 )D43G、G173R、N215D;

[0046] (m 27 )V161I;

[0047] (m 28 )V66M、R261C;

[0048] (m 29 )A200T;

[0049] (m 30 )V66M、T236I;

[0050] (m 31 )L17P、Q25R、I145V;

[0051] (m 32 )V66M;

[0052] (m 33 )A163T、S164P、A204V;

[0053] (m 34 )L17P;

[0054] (m 35 )L17K;

[0055] (m 36 )L17S;

[0056] (m 37 )L17R;

[0057] (m 38 )L17G;

[0058] (m 39 )L17T;

[0059] (m 40 )L17Q;

[0060] (m 41 )V66Y;

[0061] (m 42 )V66L;

[0062] (m 43 )A163E;

[0063] (m 44 A163Q;

[0064] (m 45 A163I;

[0065] (m 46 A163W;

[0066] (m 47 A163R;

[0067] (m 48 A200I;

[0068] (m 49 A200V;

[0069] (m 50 A204F;

[0070] (m 51 A204L;

[0071] (m 52 A204Y;

[0072] (m 53 L17A;

[0073] (m 54 V66F;

[0074] (m 55 A163C;

[0075] (m 56 A163V;

[0076] (m 57 A200D;

[0077] (m 58 L17V;

[0078] (m 59 A163P;

[0079] (m 60 A204G;

[0080] (m 61 )A204I.

[0081] This disclosure provides an isolated polynucleotide encoding an N-acetylglutamate kinase mutant as described above.

[0082] This disclosure provides a nucleic acid construct comprising a polynucleotide as described above, the polynucleotide being operatively linked to one or more regulatory sequences that direct the production of a polypeptide in an expression host.

[0083] This disclosure provides a recombinant expression vector comprising isolated polynucleotides as described above, or nucleic acid constructs as described above.

[0084] This disclosure provides a recombinant host cell, wherein the recombinant host cell comprises the N-acetylglutamate kinase mutant as described above, the polynucleotide as described above, the nucleic acid construct as described above, or the recombinant expression vector as described above.

[0085] In some embodiments, the host cell is derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium.

[0086] Preferably, the host cell is derived from Corynebacterium; more preferably, the host cell is derived from Corynebacterium glutamicum.

[0087] This disclosure provides a cell culture comprising the recombinant host cells as described above.

[0088] This disclosure provides the use of the N-acetylglutamate kinase mutant, the isolated polynucleotide, the nucleic acid construct, the recombinant expression vector, the recombinant host cell, or the cell culture as described above in the production of arginine or its metabolites in the synthetic pathway.

[0089] Preferably, the arginine includes L-arginine;

[0090] Optionally, the metabolites in the arginine biosynthesis pathway include N-acetyl-γ-glutamyl phosphate, N-acetylornithine, ornithine, citrulline, and / or arginine succinate.

[0091] This disclosure provides a method for producing arginine or a metabolite in its synthetic pathway, wherein the method includes the steps of producing arginine or a metabolite in its synthetic pathway using the N-acetylglutamate kinase mutant as described above, the isolated polynucleotide as described above, the nucleic acid construct as described above, the recombinant expression vector as described above, the recombinant host cell as described above, or the cell culture as described above.

[0092] Optionally, it also includes a step of isolating arginine or its metabolites in its synthetic pathway;

[0093] Preferably, the arginine includes L-arginine;

[0094] Optionally, the metabolites in the arginine biosynthesis pathway include N-acetyl-γ-glutamyl phosphate, N-acetylornithine, ornithine, citrulline, and / or arginine succinate.

[0095] The effects of the invention

[0096] This disclosure presents a high-throughput screening method for N-acetylglutamate kinase mutants, which can obtain a large number and variety of N-acetylglutamate kinase mutants with higher screening efficiency.

[0097] This disclosure utilizes the above method to discover a large number of N-acetylglutamate kinase mutants, which can significantly increase the L-arginine production of the strain and are suitable for the industrial production of L-arginine or compounds related to its metabolic pathway. Attached Figure Description

[0098] Figure 1 The flow cytometry analysis of the ArgB mutant library is shown.

[0099] Figure 2 The initial screening of ArgB mutant fluorescence intensity is shown.

[0100] Figure 3 The flow cytometry analysis of the single-point saturated mutant library is shown. Detailed Implementation

[0101] Various exemplary embodiments, features, and aspects of this disclosure will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0102] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0103] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this disclosure should be understood to include systematic errors that are unavoidable in industrial production.

[0104] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0105] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0106] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0107] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0108] In this specification, the term "N-acetylglutamate kinase (NAGK, also known as ArgB)" belongs to the amino acid kinase family and is mainly involved in the biosynthesis of L-arginine, serving as a key enzyme in the synthetic pathway. It catalyzes the phosphorylation of N-acetylglutamate (NAG) to produce N-acetyl-γ-glutamyl phosphate (NAGP). In many organisms, the activity of ArgB is subject to feedback inhibition by L-arginine; this mechanism regulates arginine synthesis and prevents excessive accumulation.

[0109] As used in this disclosure, the terms "peptide" and "protein" are used interchangeably herein and refer to an amino acid polymer of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds). The polymer may be linear, branched, or cyclic, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, ubiquitination, glycosylation, amidation of C-terminal amino acids, or any other manipulation, such as conjugation with labeled components).

[0110] As used in this disclosure, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and their abbreviations and English abbreviations in this disclosure are as follows:

[0111] Histidine (His, H); Serine (S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0112] As used in this disclosure, the term "fragment" means a polypeptide or a catalytic or carbohydrate-binding module that has one or more (e.g., several) amino acids deleted from the amino and / or carboxyl ends of a mature polypeptide or domain. In the technical solutions of this disclosure, the fragment has N-acetylglutamate kinase activity.

[0113] As used in this disclosure, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms. The term "control" refers to the starting strain of this study, or the control group of the experiment.

[0114] As used in this disclosure, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Exemplarily, a "mutant" in this disclosure is a polypeptide having enhanced N-acetylglutamate kinase activity.

[0115] As used in this disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this disclosure, the term "mutation" refers to an alteration of a nucleotide sequence or amino acid sequence. In one specific embodiment, the term "mutation" refers to "substitution."

[0116] In this disclosure, "mutation" may also include the addition, deletion, or substitution of amino acids at one or more positions corresponding to the sequence shown in SEQ ID NO:1 that do not affect the activity of N-acetylglutamate kinase. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, does not substantially alter its biological activity; for example, appropriately replacing, adding, or deleting certain amino acids results in sequences that do not affect their activity.

[0117] As used herein, the terms “corresponding” and “corresponding” have the meanings commonly understood by those skilled in the art. Specifically, “corresponding” and “corresponding” refer to the positions in one sequence that correspond to a specified position in another sequence after homology or sequence identity alignment.

[0118] In some embodiments, the term "mutation" in this disclosure may be selected from "conservative mutation." In this disclosure, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0119] As used in this disclosure, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0120] As used in this disclosure, a "conservative substitution" typically involves exchanging one amino acid at one or more sites on a protein. This substitution can be conserved. Examples of substitutions considered conserved include, but are not limited to, substitutions of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0121] As used in this disclosure, the terms "sequence identity" or "percentage of identity" in the comparison of two nucleic acids or peptides refer to the percentage of identical sequences or having the same sequence when compared and aligned with the highest possible correspondence using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of the number of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion when two or more nucleotide or amino acid sequences are aligned in a manner that maximizes the number of identical nucleotides or amino acids, with gaps added as needed.

[0122] The methods disclosed herein for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, ed. Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, ed. Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, ed. Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, ed. Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0123] As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or as a component of a larger nucleotide sequence structure derived from a nucleotide sequence isolated at least once in number or concentration, capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, T, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (single fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0124] As used in this disclosure, the term "isolated" means a substance in a form or environment not naturally occurring. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance including, but not limited to, any enzyme, mutant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more naturally occurring components associated with it; (3) any substance artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples. For example, host cells may be genetically modified to express the polypeptides of this disclosure. Fermentation broth from host cells will contain isolated polypeptides. "Recombinant polynucleotide" is a type of "polynucleotide".

[0125] As used in this disclosure, the term "recombinant polynucleotide" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides may be included in a suitable vector, and the vector may be used for transformation into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0126] As used in this disclosure, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned relative to the coding sequence of a polynucleotide such that the regulatory sequence directs the expression of the coding sequence. Exemplarily, the regulatory sequence may be selected from sequences encoded by promoters and / or enhancers.

[0127] As used in this disclosure, the term "nucleic acid construct" comprises a polynucleotide encoding a polypeptide or domain or module efficiently linked to a suitable regulatory sequence necessary for polynucleotide expression in selected cells or strains. In this disclosure, transcriptional regulatory elements comprise promoters, and may further comprise enhancers, silencers, insulators, and other elements.

[0128] As used in this disclosure, the term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0129] As used in this disclosure, the term "expression vector" refers to a linear or circular DNA molecule containing a polynucleotide encoding a polypeptide and the polynucleotide is effectively linked to a control sequence for its expression.

[0130] As used in this disclosure, the term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies DNA.

[0131] In this disclosure, the term "host cell" refers to any cell type that is easily transformed, transfected, or transduced using a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector containing the mutant polypeptide of this disclosure. The term "recombinant host cell" encompasses a host cell that differs from its parent cell after the introduction of a polynucleotide encoding a mutant polypeptide or a recombinant expression vector; recombinant host cells are specifically achieved through transformation. The host cell of this disclosure can be a prokaryotic cell, as long as it is a cell capable of receiving the polynucleotide encoding the polypeptide or recombinant polypeptide of this disclosure that has N-acetylglutamate kinase activity. In one embodiment, the host cell refers to a prokaryotic cell. Specifically, the host cell originates from microorganisms suitable for fermenting and producing arginine or its metabolites in the synthetic pathway, such as microorganisms from the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, *Bacillus*, or *Corynebacterium*. In some preferred embodiments, the host cell originates from *Corynebacterium*, more preferably from *Corynebacterium glutamicum*, such as *Corynebacterium glutamicum* ATCC 13032, *Corynebacterium glutamicum* ATCC 14067, and *Corynebacterium glutamicum* ATCC 13869.

[0132] The terms “transformation,” “transfection,” and “transduction” in this disclosure have the meanings commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0133] The host cell culture disclosed herein can be performed according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0134] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0135] The technical solution of this disclosure is described in detail below:

[0136] Existing research has identified several ArgB mutants capable of relieving feedback inhibition, but their performance still has room for improvement. Therefore, the inventors employed a high-throughput random mutagenesis combined with saturation mutagenesis to discover an ArgB mutant with significantly improved arginine production performance compared to existing technologies. This provides more diverse and efficient synthetic pathways for microbial production of arginine and other metabolites in the arginine synthesis pathway.

[0137] <First Aspect>

[0138] Based on the above research, in the first aspect of this disclosure, a high-throughput screening method for N-acetylglutamate kinase mutants is provided. This method combines high-throughput random mutagenesis with single-point saturation mutagenesis. First, a random mutant library of N-acetylglutamate kinase mutants is constructed, and then the arginine-specific biosensor pLysG is used. F222I The arginine production of the mutant was coupled with the fluorescence intensity. The fluorescence intensity of the mutant library was analyzed by flow cytometry. The region with the highest fluorescence intensity in the library strain was selected for initial and secondary screening of arginine production to obtain N-acetylglutamate kinase mutants. Subsequently, the high fluorescence intensity unit sites obtained by random mutation were subjected to saturation mutation to further obtain unit site mutants of N-acetylglutamate kinase.

[0139] Furthermore, novel ArgB mutants with significantly improved performance obtained by the above screening method are provided, wherein the ArgB mutants are selected from any one of the following groups (I)-(V):

[0140] (I) Compared with the sequence shown in SEQ ID NO:1, the ArgB mutant has the following differences at positions 15, 17, 18, 20, 24, 25, 27, 43, 46, 47, 48, 49, 54, 55, 56, 66, 77, 85, 113, 122, 126, 136, 142, 145, 153, 160, 161, 162, 163, 164, 167, 168, 169, 170, 172, ... The mutation is present at one or more of the following positions: 173, 174, 188, 197, 200, 202, 204, 207, 208, 213, 215, 225, 231, 233, 235, 236, 242, 259, 261, 265, 266, 273, 275, 283, 286, 288, 289, 297; and it exhibits increased arginine production compared to the N-acetylglutamate kinase shown in SEQ ID NO:1.

[0141] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:1;

[0142] (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions:

[0143] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0144] (b) A full-length complementary polynucleotide to (a);

[0145] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still has N-acetylglutamate kinase activity;

[0146] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0147] In some embodiments, the ArgB mutant corresponds to an amino acid mutation at at least one of the following positions in the sequence shown in SEQ ID NO:1:

[0148] G153S, I169N, R174H, G122S, N160S, S265G, L24M, M54I, M77I, L213H, A259V, A 266T, A235P, F136L, A48T, D168E, E208G, A207T, I170M, Q188R, L24F, V66M, G28 9A, K47E, M142V, I288F, A197V, A200V, I233T, F297S, A20V, F27L, F56L, G85D, M167V, D225N, A18V, L242P, L283S, G286D, L17P, V55A, A202V, N15S, L46P, D162 N, R273H, A275V, D113N, V126A, A172T, S231P, D43G, G173R, N215D, V161I, V66 M, R261C, A200T, T236I, Q25R, I145V, A163T, S164P, A204V, L17K, L17S, L17R, L 17G, L17T, L17Q, V66Y, V66L, A163E, A163Q, A163I, A163W, A163R, A200I, A204 F, A204L, A204Y, L17A, V66F, A163C, A163V, A200D, L17V, A163P, A204G, A204I.

[0149] Furthermore, the ArgB mutant corresponds to the sequence shown in SEQ ID NO:1 and has the following characteristics (m1) to (m2). 61 Any mutation shown in any of the following:

[0150] (m1)G153S, I169N; (m2) R174H; (m3) G122S, N160S, S265G; (m4) L24M, L283S; (m5) M54I, M77I, V16 1I, L213H; (m6) A259V, A266T; (m7) S231P, A235P; (m8) F136L; (m9) A48T, D168E, A207T, E208G; (m 10 A207T; (m 11 I170M, Q188R; (m 12 )L24F, V55A, V66M, G289A; (m 13 K47E, M142V; (m 14 )I288F;(m 15 A197V; (m 16 A49V, I233T, F297S; (m 17)A20V、F27L、F56L、G85D、M167V;(m 18 )F136L、D225N;(m 19 )A18V、L242P、L283S、G286D;(m 20 )L17P、V55A;(m 21 )A202V;(m 22 )N15S、L46P;(m 23 )V55A、D162N、R273H、A275V;(m 24 )D113N、V126A;(m 25 )A172T、S231P;(m 26 )D43G、G173R、N215D;(m 27 )V161I;(m 28 )V66M、R261C;(m 29 )A200T;(m 30 )V66M、T236I;(m 31 )L17P、Q25R、I145V;(m 32 )V66M;(m 33 )A163T、S164P、A204V;(m 34 )L17P;(m 35 )L17K;(m 36 )L17S;(m 37 )L17R;(m 38 )L17G;(m 39 )L17T;(m 40 )L17Q;(m 41 )V66Y;(m 42 )V66L;(m 43 )A163E;(m 44 )A163Q;(m 45 )A163I;(m 46 )A163W;(m 47 )A163R;(m 48 )A200I;(m 49 )A200V;(m 50 )A204F;(m 51 )A204L;(m 52 )A204Y;(m 53 )L17A;(m 54 )V66F;(m 55 )A163C;(m 56 )A163V;(m 57 )A200D;(m58 L17V; (m 59 A163P; (m 60 A204G; (m 61 )A204I.

[0151] The ArgB mutants described above exhibit superior catalytic performance compared to WT and existing ArgB mutants. The arginine production of strains containing the ArgB mutants is at least 1.59 times higher than that of strains expressing wild-type ArgB and at least 14% higher than that of existing ArgB mutant strains.

[0152] <Second aspect>

[0153] In a second aspect of this disclosure, an isolated polynucleotide is provided, wherein the polynucleotide encodes an ArgB mutant as described in the first aspect of this disclosure.

[0154] The polynucleotides disclosed herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0155] The polynucleotides encoding the mutants disclosed herein include: a coding sequence that encodes only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0156] <Third aspect>

[0157] In a third aspect of this disclosure, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the polynucleotide described in the second aspect of this disclosure.

[0158] In some embodiments, the polynucleotide described in the second aspect is operatively linked to one or more heterologous regulatory sequences that control gene expression, thereby forming a recombinant polynucleotide capable of expressing a polypeptide.

[0159] <Fourth Aspect>

[0160] In a fourth aspect of this disclosure, a recombinant host cell is provided, wherein the recombinant host cell comprises the ArgB mutant described in the first aspect of this disclosure, the isolated polynucleotide described in the second aspect of this disclosure, or the recombinant expression vector described in the third aspect of this disclosure.

[0161] In some implementations, an expression vector containing a heteropolynucleotide encoding an ArgB mutant peptide is introduced into a suitable host cell to express the corresponding ArgB mutant peptide.

[0162] In some embodiments, the host cell is derived from the genus Corynebacterium.

[0163] In some preferred embodiments, the host cell is derived from Corynebacterium glutamicum.

[0164] In some exemplary embodiments, the host cell has the transcription factor ArgR and the transcription regulator FarR, which repress the arginine synthesis pathway.

[0165] <Fifth Aspect>

[0166] In a fifth aspect of this disclosure, a cell culture comprising the recombinant host cells described in the fourth aspect of this disclosure is provided.

[0167] <Sixth Aspect>

[0168] In a sixth aspect of this disclosure, the use of the ArgB mutant described in the first aspect of this disclosure, the isolated polynucleotide described in the second aspect of this disclosure, the recombinant expression vector described in the third aspect of this disclosure, or the recombinant host cell described in the fourth aspect of this disclosure in the production of arginine or metabolites in its synthetic pathway is provided. In some optional embodiments, the arginine comprises L-arginine; and the metabolites in the arginine synthetic pathway comprise N-acetyl-γ-glutamyl phosphate (NAGP), N-acetylornithine (NAO), ornithine (Orn), citrulline, and / or arginine succinate (ASA).

[0169] <Seventh Aspect>

[0170] In a seventh aspect of this disclosure, a method for producing arginine or a metabolite thereof in its synthetic pathway is provided, the method comprising the steps of producing arginine or a metabolite thereof using an ArgB mutant as described in a first aspect of this disclosure, an isolated polynucleotide as described in a second aspect of this disclosure, a recombinant expression vector as described in a third aspect of this disclosure, a recombinant host cell as described in a fourth aspect of this disclosure, or a cell culture as described in a fifth aspect of this disclosure.

[0171] In some alternative embodiments, the method further includes isolating arginine or its metabolites from its synthetic pathway.

[0172] In some optional embodiments, the arginine comprises L-arginine; metabolites in the arginine biosynthetic pathway include N-acetyl-γ-glutamyl phosphate (NAGP), N-acetyl-ornithine (NAO), ornithine (Orn), citrulline, and / or arginine succinate (ASA).

[0173] Example

[0174] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0175] The culture media and buffer solutions used in the examples are as follows:

[0176] The composition of LB liquid medium (g / L) is: peptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L.

[0177] The LB plate medium consists of the following components (g / L): peptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L; and agar powder, 15 g / L.

[0178] The composition of TSB liquid culture medium (g / L) is as follows: glucose, 5 g / L; yeast extract, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L.

[0179] The TSB plate medium consists of the following components (g / L): glucose, 5 g / L; yeast extract, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K₂HPO₄·3H₂O, 1 g / L; MgSO₄·7H₂O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L; and agar powder, 15 g / L.

[0180] The arginine seed culture medium consisted of: glucose, 20 g / L; peptone, 20 g / L; KH2PO4, 0.5 g / L; K2HPO4·3H2O, 1.5 g / L; MgSO4·7H2O, 0.5 g / L; yeast extract, 10 g / L; biotin, 5 mg / L; MOPS, 20 g / L; and an initial pH of 7.2.

[0181] The arginine fermentation medium consisted of: glucose, 80 g / L; soybean peptone, 20 g / L; urea, 10 g / L; K2HPO4·3H2O, 0.7 g / L; MgSO4·7H2O, 0.5 g / L; MOPS, 40 g / L; and an initial pH of 7.5.

[0182] CGXII+Y medium: yeast extract, 2 g / L; (NH4)2·SO4, 20 g / L; urea, 5 g / L; KH2PO4, 1 g / L; K2HPO4·3H2O, 1.3 g / L; MOPS, 42 g / L; CaCl2, 0.01 g / L; FeSO4·7H2O, 0.01 g / L; MnSO4·H2O, 0.01 g / L; ZnSO4·7H2O, 0.001 g / L; CuSO4, 0.0002 g / L; NiCl·6H2O, 0.00002 g / L; MgSO4·7H2O, 0.25 g / L; protocatechuic acid, 0.03 g / L; vitamin B1, 0.0001 g / L; biotin, 0.0002 g / L; initial pH 7.0.

[0183] Example 1: Construction of ArgB overexpression vector and high-throughput screening system

[0184] (1) Construction of L-arginine-producing strains

[0185] In Corynebacterium glutamicum, L-arginine biosynthesis is subject to multiple feedback regulation mechanisms. According to literature reports, the transcription factor ArgR binds to the arginine operon, thereby inhibiting L-arginine synthesis. Simultaneously, the transcription factor FarR also represses the expression of the arginine operon and Gdh (Metabolic engineering of Corynebacterium glutamicum for L-arginine production, Nature Communications, 2014, 5:4618.). Therefore, the proteins ArgR and FarR, which repress the arginine synthesis pathway, were knocked out in strain C. glutamicum ATCC 13869.

[0186] Based on the publicly available genome sequence of Corynebacterium glutamicum ATCC 13869 (GenBank: CP016335.1), primers ArgR-UH-F / R and ArgR-DH-F / R were designed. Using the ATCC 13869 genome as a template, the DNA fragment with ArgR knockout was obtained by PCR amplification using the above primers. Based on the sequence information of plasmid pK18mobsacB, primer pK-F / R was designed. Using plasmid pK18mobsacB as a template, the linearized vector fragment was obtained by reverse PCR amplification. The three fragments were recovered and recombined. The clones obtained after transformation were collected and plasmids were extracted to obtain the recombinant vector pK18-ΔArgR with ArgR knockout. C. glutamicum ATCC 13869 competent cells were prepared. 1 μg of pK18-ΔArgR plasmid was electroporated into the prepared competent cells. 1 mL of preheated (46℃) TSB medium was added, and the cells were incubated at 46℃ for 6 min, then at 30℃ for 3 h. The transformed cells were then spread onto TSB solid medium containing 25 μg / mL kanamycin and cultured at 30℃ for 1 day to obtain the first recombinant transformant. The correct transformant was transferred to TSB medium containing 5 g / L glucose and cultured overnight, then transferred to TSB medium containing 100 g / L sucrose and cultured at 30℃ for 6 h. Afterward, it was spread onto TSB medium supplemented with 100 g / L sucrose for screening to obtain the L-arginine-producing strain ATCC13869ΔArgR, i.e., CgArg5.

[0187] Based on the publicly available genome sequence of Corynebacterium glutamicum ATCC 13869 (GenBank: CP016335.1), primers FarR-UH-F / R and FarR-DH-F / R were designed. Using the ATCC 13869 genome as a template, the DNA fragments with FarR knockout were obtained by PCR amplification using the above primers. Primers pK-F / R were designed based on the sequence information of plasmid pK18mobsacB. Using plasmid pK18mobsacB as a template, linearized vector fragments were obtained by reverse PCR amplification. The three fragments were recovered, recombinated, and ligated. The clones obtained after transformation were collected, and plasmids were extracted to obtain the FarR knockout editing vector pK18-ΔFarR. 1 μg of pK18-ΔFarR plasmid was electroporated into CgArg5 competent cells. 1 mL of preheated (46°C) TSB medium was added, and the cells were incubated at 46°C for 6 min, then at 30°C for 3 h. The transformed cells were then spread onto TSB solid medium containing 25 μg / mL kanamycin and cultured at 30°C for 1 day to obtain the first recombinant transformant. The correct transformant was transferred to TSB medium containing 5 g / L glucose and cultured overnight, then transferred to TSB medium containing 100 g / L sucrose and cultured at 30°C for 6 h. Afterward, it was spread onto TSB medium supplemented with 100 g / L sucrose for screening to obtain the L-arginine-producing strain ATCC 13869ΔArgRΔFarR, i.e., CgArg6.

[0188] (2) Construction of wild-type and double-mutant ArgB overexpression vectors

[0189] Based on the publicly available genome sequence of Corynebacterium glutamicum ATCC 13869 (GenBank: CP016335.1), primers ArgB-1 / 2 were designed to amplify the wild-type argB gene fragment using the ATCC 13869 genome as a template. Using the pXMJ19 plasmid (Construction and application of new Corynebacterium glutamicum vectors. Biotechnology Techniques, 1999, 13(6), 437-441, cited in this article) as a template, the pXMJ19 plasmid backbone was amplified segmentally using primers p19-1 / 2 and p19-3 / 4, respectively. After the fragments were recovered and purified, they were cloned and ligated using a one-step recombination kit from Novizan to obtain the constitutive overexpression vector pXM-ArgB of wild-type ArgB. WT .

[0190] Amino acid sequences of ArgB derived from Corynebacterium glutamicum ATCC 13869 and Corynebacterium glutamicum ATCC 13032 were compared. The double mutation (the difference between ArgB derived from Corynebacterium glutamicum ATCC 13869 and ArgB derived from Corynebacterium glutamicum ATCC 13032 in the paper is only the start site of the annotation sequence) was introduced into the wild-type ArgB overexpression vector. Mutations were made at positions 49 (Alanine) and 54 (Methionine) in the wild-type ArgB (i.e., the ArgB in the paper). A26V ,M31V To obtain mutant overexpression vectors capable of relieving feedback inhibition and increasing L-arginine production, pXM-ArgB was used. WT Using the overexpression vector as a template, primers 2B-F / R were used to amplify ArgB-containing vectors. A49V,M54V The mutated fragment was terminally phosphorylated using T4 PNK and then ligated using T4 ligase to obtain the double-mutant ArgB overexpression vector, which was then used to construct pXM-ArgB. A49V ,M54V .

[0191] The primers used in this embodiment are shown in Table 1.

[0192] Table 1. Primers used in this embodiment

[0193]

[0194]

[0195] (3) Construction of ArgB high-throughput screening system

[0196] To test the fluorescence intensity of different ArgB cells in the arginine high-throughput screening system, CgArg6 competent cells were prepared using a method reported in the literature (Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnology Letters, 2015, 37(12):2445-2452., which is incorporated herein by reference). The overexpression plasmid pXM-ArgB was then applied to each cell. WT and pXM-ArgB A49V,M54V With arginine-specific biosensor plasmid pLysG F222I (pLysG plasmid) F222I The strain CgArg6 / pLysG was co-transformed into the competent cells described above and obtained from CN118063573A (which is incorporated herein by reference). F222I +pXM-ArgB WT and CgArg6 / pLysG F222I +pXM-ArgB A49V,M54V .

[0197] The constructed strain was inoculated into TSB liquid medium containing 25 μg / mL kanamycin and 5 μg / mL chloramphenicol, and cultured overnight in a shaker at 30°C and 220 rpm. The seed culture was then cultured according to the initial OD... 600 0.5 μL of the culture medium was transferred to 24-well plates containing 40 g / L glucose, 25 μg / mL kanamycin, and 5 μg / mL chloramphenicol in 800 μL CGXⅡ+Y medium. After incubation at 30°C and 800 rpm for 24 h on a shaker, the fluorescence intensity of the strain was detected using a microplate reader, and the L-arginine production was simultaneously measured. The excitation wavelength for fluorescence measurement was 488 nm, and the emission wavelength was 520 nm; the OD of the bacterial culture was also measured simultaneously. 600 The fluorescence intensity of the strains was calculated, and the results are shown in Table 2.

[0198] Table 2. Fluorescence intensity and yield of strains overexpressing different ArgB mutants

[0199]

[0200] As can be seen from Table 2, ArgB A49V,M54VThe fluorescence intensity and L-arginine production of the mutant overexpressing strain were 4.08 times and 7.40 times that of the wild-type ArgB overexpressing strain, respectively, indicating that the ArgB mutant, after relieving feedback inhibition, significantly increased fluorescence intensity and L-arginine production under this system. This high-throughput screening system can distinguish different ArgB overexpressing strains and can be used for high-throughput screening of ArgB mutants.

[0201] Example 2: Construction and sorting of ArgB random mutant libraries

[0202] (1) Construction of ArgB random mutation library based on manganese ions

[0203] Using the overexpression vector pXM-ArgB containing the wild-type argB gene WT Using ArgB-M-1 / 2 as a template, the mutated argB gene fragment was amplified by error-prone PCR under conditions of 0.1 mM MnCl2 addition. Simultaneously, pXM-ArgB... WT Using pXM-F / R primers as templates, a linearized vector backbone was obtained by reverse PCR amplification. The two fragments were recovered, recombinated, and chemically transformed into E. coli Trans1-T1 competent cells, and cultured overnight at 37°C. After single colonies grew, colonies were scraped from the plate, and the mixed plasmid was extracted to obtain an ArgB random mutant plasmid library.

[0204] The arginine-specific biosensor plasmid pLysG F222I (pLysG plasmid) F222I Disclosed in CN118063573A, which is incorporated herein by reference, the strain CgArg6 / pLysG was obtained by electroporation into CgArg6 competent cells. F222I Then, strain CgArg6 / pLysG was prepared. F222I The above plasmid library was electroporated into competent cells, and 1 mL of TSB medium preheated to 46℃ was added. After incubation at 46℃ for 6 min, the cells were thawed at 30℃ for 2 h. The thawed solution was then transferred to TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol and cultured at 30℃ for 16 h to obtain the strain library for screening.

[0205] The primers used in this embodiment are shown in Table 3.

[0206] (2) Construction of ArgB random mutant library based on mutation kit

[0207] To construct a randomized mutation library with greater mutation diversity, Beyotime's QuickMutation was used. TMA random mutation library of ArgB was constructed using a gene random mutation kit. The overexpression vector pXM-ArgB containing the wild-type argB gene was used. WT Using pXM-ArgB as a template, primers argB-TB-F / R were used, and an appropriate amount of template plasmid was added. The mutated argB gene fragment was then amplified by PCR. WT Using argB-ZT-F / R as a template, a linearized vector backbone was obtained by reverse PCR amplification. The two fragments were recovered, recombinated, and chemically transformed into *E. coli* Trans1-T1 competent cells, and cultured overnight at 37°C. After single colonies emerged, colonies were scraped from the plate, and the mixed plasmid was extracted to obtain an ArgB random mutant plasmid library. This plasmid library was then electroporated into CgArg6 / pLysG. F222I Competent cells were added to 1 mL of TSB medium preheated to 46℃, incubated at 46℃ for 6 min, and then recovered at 30℃ for 2 h. The recovery solution was then transferred to TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol, and cultured at 30℃ for 16 h to obtain a strain library for screening.

[0208] The primers used in this embodiment are shown in Table 3.

[0209] Table 3. Primers used in this embodiment

[0210] Primers nucleotide sequence pXM-F agcttggctgttttggcg pXM-R ctgtttcctgtgtgaaattgttatccg ArgB-M-1 cggataacaatttcacacaggaaacagatg ArgB-M-2 ccgccaaaacagccaagcttta argB-TB-F atgaatgacttgatcaaaga argB-TB-R ttacagttccccatccttgt argB-ZT-F acaaggatggggaactgtaaaagcttggctgttttggcg argB-ZT-R tctttgatcaagtcattcatctgtttcctgtgtgaaattgttatccgc

[0211] (3) ArgB random mutant library sorting by flow cytometry

[0212] The control strain CgArg6 / pLysG F222I +pXM-ArgB WT The two mutant strain libraries were inoculated into TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol, and cultured overnight at 30°C. The seed culture was then measured at the initial OD value. 600 0.5 μg / mL cells were transferred to CGXII+Y liquid medium containing 15 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 40 g / L glucose. After incubation at 30°C for 24 h, the cell concentration was diluted to OD using PBS buffer. 600 The value was 0.05, and the fluorescence intensity of the above strains was analyzed by flow cytometry. Figure 1 The region with the highest fluorescence intensity in the library strains was selected for sorting and obtaining single clones.

[0213] Example 3: Screening for ArgB mutants

[0214] (1) Initial screening of ArgB mutants

[0215] The single-cell colonies obtained by flow cytometry were randomly selected, and a portion of the colonies were inoculated into four 96-well plates containing 200 μL of TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol. At the same time, the wild-type control strain CgArg6 / pLysG was inoculated. F222I +pXM-ArgB WT The culture was incubated overnight at 30°C and 800 rpm in a shaker. The seed culture was then transferred at 5% (v / v) to 200 μL of CGXII+Y liquid medium containing 15 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 40 g / L glucose into 96-well plates. After incubation for 24 h at 30°C and 800 rpm in a shaker, the bacterial culture was diluted 20-fold with PBS buffer. The fluorescence intensity and OD of eYFP were then detected using a microplate reader (SpectraMax M5, Molecular Devices, λexcitation = 488 nm, λemission = 520 nm). 600nm ,like Figure 2 As shown, mutants with stronger fluorescence values ​​than wild-type control strains were sent for sequencing to analyze their mutation sites.

[0216] (2) Rescreening of ArgB mutants

[0217] Based on the fluorescence intensity and sequencing results from the initial screening, mutants with high fluorescence intensity and high enrichment were selected for retesting (Table 4), and CgArg6 / pLysG was used for further analysis. F222I +pXM-ArgB WT and CgArg6 / pLysG F222I +pXM-ArgB A49V,M54V The selected strain served as a control. Selected monoclonal strains were inoculated into 96-well plates containing 200 μL of arginine seed medium (15 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 20 g / L glucose). After 8 h of culture, the culture was transferred at 5% (V / V) to 96-well plates containing 200 μL of arginine fermentation medium (15 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 80 g / L glucose). Three replicates were set for each clone. After culturing in a shaker at 30℃ and 800 rpm for 24 h, the L-arginine yield was measured, and the results are shown in Table 5. The results show that the fluorescence intensity of the screened ArgB mutant strains was 2.06–6.39 times that of the wild-type ArgB control strain, and the corresponding L-arginine yield was 4.70–27.59 times that of the wild-type ArgB control strain. Both were also significantly higher than the reported levels of the ArgB mutant strains in the literature. A49V,M54V (for ArgB) A49V,M54VThe results showed that the selected ArgB mutants could achieve different levels of L-arginine accumulation and had better catalytic performance (1.14-6.72 times higher than the original).

[0218] Table 4. Summary of ArgB mutant information

[0219]

[0220]

[0221] Table 5. Increase in L-arginine production by ArgB-selected mutant strains compared to wild type

[0222]

[0223] Example 4: Construction and sorting of ArgB single point mutation libraries

[0224] (1) Construction of ArgB single-site saturation mutant library

[0225] Based on the above results, the mutant ArgB was selected. WT1-10 ArgB WT2-47 ArgB WT1-48 ArgB WT1-67 Single-point saturation mutations were performed on the sites in the gene, specifically saturation mutation libraries were constructed at sites L17, Q25, V66, I145, A163, S164, A200, A204, and T236. pXM-ArgB was used as the base. WT Using plasmids as templates, a "NNK (N=A / T / C / G, K=G / T)" mutation sequence was designed at the target site for saturation mutagenesis. Primers L17-F / R, Q25-F / R, V66-F / R, I145-F / R, A163-F / R, S164-F / R, A200-F / R, A204-F / R, and T236-F / R were designed. Fragments containing the target site saturation mutation were obtained by reverse PCR. After purification and recovery, the fragments were terminally phosphorylated with T4PNK and ligated with T4 ligase to obtain a single-site overexpression vector library pXM-ArgB. L17 pXM-ArgB Q25 pXM-ArgB V66 pXM-ArgB I145 pXM-ArgB A163 pXM-ArgB S164 pXM-ArgB A200 pXM-ArgB A204 pXM-ArgB T236 .

[0226] Preparation of CgArg6 / pLysGF222I Competent cells were electroporated with 2 μg of the single-point saturation mutant library plasmid pXM-ArgB. L17 pXM-ArgB Q25 pXM-ArgB V66 pXM-ArgB I145 pXM-ArgB A163 pXM-ArgB S164 pXM-ArgB A200 pXM-ArgB A204 pXM-ArgB T236 Add 1 mL of TSB medium preheated to 46℃, incubate at 46℃ for 6 min, then revive at 30℃ for 2 h. Transfer the revival solution to TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol, and incubate at 30℃ for 16 h to obtain a single-site saturated mutant strain library for screening.

[0227] The primers used in this embodiment are shown in Table 6.

[0228] Table 6. Primers used in this embodiment

[0229] Primers nucleotide sequence L17-F cgcaaatgtcNNKgctgaggcgttgccatggtt L17-R cgcacctcagagcctaaatc Q25-F gccatggttgNNKcatttccgcgacaagattgt Q25-R aacgcctcagcgaggacatt V66-F aaaaccagtgNNKgtgcacggtggtggacctca V66-R gcgcccacggtgcgcaagaa I145-F catggtcaacNNKgatggcgtaccccactgatat I145-R cgcttctgcgcggtaaacag A163-F taatgtcgatNNKtcttccttgatggatatcat A163-R atgatgtctccgaccaaacc S164-F tgtcgatgccNNKtccttgatggatatcatcga S164-R ttaatgatgtctccgaccaa A200-F cgcagcgggtNNKttggctgcagcgattggtgc A200-R gtatcggcgttgatgttgtaaa A204-F tttggctgcaNNKattggtgcagaacgcctgct A204-R gcacccgctgcggtatcg T236-F gatcaaggccNNKgagctggaggccattcttcc T236-R ttggacaccagtgagctctt

[0230] (2) ArgB single-site saturation mutant library sorting by flow cytometry

[0231] The control strain CgArg6 / pLysG F222I +pXM-ArgB WT Nine site-saturated mutant strain libraries were inoculated into TSB liquid medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol, and cultured overnight at 30°C. The seed culture was then measured at the initial OD value. 600 0.5 μg / mL cells were transferred to CGXII+Y liquid medium containing 15 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 40 g / L glucose. After incubation at 30°C for 24 h, the cell concentration was diluted to OD using PBS buffer. 600 The value was 0.05, and the fluorescence intensity of the above strains was analyzed by flow cytometry. Figure 3 Using the control strain as a reference, clones with fluorescence intensity greater than that of the wild-type strain were selected and their proportion of the total number of clones was calculated. Mutant libraries (L17, V66, A163, A200, A204) with a proportion greater than 1% were selected for sorting and obtaining single clones.

[0232] Example 5: Screening for ArgB unit point mutants

[0233] Forty single clones were randomly selected from the sorting plates of each point mutation library for sequencing, and their mutation sites were counted. The results are shown in Table 7. It can be seen that the diversity of mutants is high at most sites, indicating that there may be multiple mutants for each amino acid residue that can relieve the feedback inhibition of arginine.

[0234] Table 7. Mutants obtained from screening unit point saturation mutation libraries

[0235] site mutant L17 T, R, S, Q, G, A, P, K, V V66 L, Y, M, F A163 V, W, E, R, C, P, I, Q, T A200 I, T, V, S, H, D A204 L, F, I, Y, G, V

[0236] Example 6: Effect of the single-site ArgB mutant on arginine synthesis

[0237] With CgArg6 / pLysG F222I +pXM-ArgB WT and CgArg6 / pLysG F222I +ArgB A49V,M54V As control strains, strains expressing different unit point mutants were tested using plate fermentation. The constructed strains were inoculated into arginine seed medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol and cultured for 8 h. The culture was then used as seed culture and inoculated at a 5% inoculum into 96-well plates containing 200 μL of arginine fermentation medium containing 15 μg / mL kanamycin and 5 μg / mL chloramphenicol per well. The plates were shaken at 800 rpm, with three replicates for each strain. Fermentation was carried out at 30℃ for 24 h, and the L-arginine yield was measured. The results showed that, except for ArgB... A163T ArgB A200S ArgB A200H ArgB A204V Except for the mutants, whose L-arginine production was less than double that of the wild type, the other mutants all showed an increase of more than double the L-arginine production compared to the wild type, ranging from 1.59 to 38.65. This indicates that these mutants can improve L-arginine production to varying degrees and have good application prospects in the production of L-arginine and its derivatives.

[0238] Table 8. Multiples of L-arginine production in single-point mutant strains compared to wild type

[0239]

[0240] Example 7: Effect of ArgB mutant on arginine synthesis

[0241] (1) Construction of strains for L-arginine production using ArgB mutant

[0242] To verify the effect of ArgB mutants on arginine synthesis, two ArgB mutants with higher fluorescence intensity were randomly selected from the screened ArgB mutants.WT1-48 ArgB WT1-67 The arginine-producing strain CgArg6 was integrated into the gene. Primers ArgBM-UF / R and ArgBM-DF / R were designed based on the published genome sequence of Corynebacterium glutamicum ATCC 13869 (GenBank: CP016335.1). Using the ATCC13869 genome as a template, the upstream and downstream homologous arm fragments of ArgB were obtained by PCR amplification using the above primers, respectively. The fragments were then used to carry pXM-ArgB. WT1-48 pXM-ArgB WT1-67 Using the cloned mutant plasmid as a template, ArgB was amplified using primers ArgBM-F / R. WT1-48 ArgB WT1-67 The mutant gene fragment was obtained; primers pK-F / R were designed based on the sequence information of plasmid pK18mobsacB, and linearized vector fragments were obtained by reverse PCR using plasmid pK18mobsacB as a template; the above four fragments were recovered and recombined; the clones obtained after transformation were collected and plasmids were extracted to obtain ArgB. WT1-48 ArgB WT1-67 Mutated recombinant vector pK18-ArgB WT1-48 pK18-ArgB WT1-67 .

[0243] CgArg6 competent cells were prepared, and 1 μg of pK18-ArgB was electroporated into each of the competent cells prepared above. WT1-48 pK18-ArgB WT1-67 The plasmid was added to 1 mL of preheated (46°C) TSB medium and incubated at 46°C for 6 min, then at 30°C for 3 h. The resulting transformants were then spread onto TSB solid medium containing 25 μg / mL kanamycin and cultured at 30°C for 1 day to obtain the first recombinant transformants. The correct transformants were then transferred to TSB medium containing 5 g / L glucose and cultured overnight, followed by transfer to TSB medium containing 100 g / L sucrose and cultured at 30°C for 6 h. After culturing, the transformants were spread onto TSB medium supplemented with 100 g / L sucrose for selection to obtain the strain CgArg6-ArgB. WT1-48 CgArg6-ArgB WT1-67 .

[0244] The primers used in this embodiment are shown in Table 9.

[0245] Table 9. Primers used in this embodiment

[0246] Primers nucleotide sequence ArgBM-UF tgacatgattacgaattcGGTAATGCTAATGCGTGTAATGGTC ArgBM-UR gcctgtacccttttccctgctc ArgBM-DF atgagcacgctggaaacttg ArgBM-DR cgacggccagtgccaagcttAGAAAGCGTCATCGACAACAGACAG ArgBM-F agggaaaagggtacaggcatgaatgacttgatcaaagatttaggct ArgBM-R agtttccagcgtgctcatttacagttccccatccttgtcg

[0247] (2) Effects of ArgB mutant on arginine synthesis

[0248] The strain CgArg6-ArgB constructed above WT1-48 CgArg6-ArgB WT1-67 The control strain CgArg6 was inoculated into arginine seed medium and cultured for 8 h. The culture was used as seed culture and inoculated into 24-well plates containing 800 μL of arginine fermentation medium per well at a 5% inoculation rate. The plate was shaken at 800 rpm, with 3 replicates for each strain. Fermentation was carried out at 30 °C for 24 h. The yield of L-arginine and the amount of glucose consumed were measured, and the sugar-acid conversion rate from glucose to L-arginine was calculated. The results are shown in Table 10.

[0249] Table 10. L-arginine production and sugar-acid conversion rate of ArgB mutant strains

[0250] strain L-arginine (g / L) Sugar-acid conversion rate (g / g, %) CgArg6 0.00±0.00 0.00±0.00 <![CDATA[CgArg6-ArgB WT1-48 ]]> 5.00±0.15 6.25±0.19 <![CDATA[CgArg6-ArgB WT1-67 ]]> 6.05±0.29 7.56±0.36

[0251] As shown in Table 10, the ArgB mutant can significantly increase the yield of L-arginine and the sugar-acid conversion rate, indicating that these ArgB mutants can relieve the feedback inhibition to varying degrees and have good application prospects in the production of L-arginine and its derivatives.

[0252] SEQ ID NO:1, ArgB wild-type amino acid sequence:

[0253] MNDLIKDLGSEVRANVLAEALPWLQHFRDKIVVVKYGGNAMVDDDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDII NVDASSLMDIIEAGRIPVVSTIAPGEDGQIYNINADTAAGALAAAIGAERLLVLTNVEGLYTDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL*

[0254] SEQ ID NO:2, ArgB wild-type nucleotide sequence:

[0255] atgaatgacttgatcaaagatttaggctctgaggtgcgcgcaaatgtcctcgctgaggcgttgccatggttgcagcatttccgcgacaagattgttgtcgtgaaatatggcggaaacgccatggtggatgatgatctcaaggctgcttttgctgccgacatggtcttcttgcgcaccgtgggcgcaaaaccagtggtggtgcacggtggtggacctcagatttctgagatgctaaaccgtgtgggtctccagggcgagttcaagggtggtttccgtgtgaccactcctgaggtcatggacattgtgcgcatggtgctctttggtcaggtcggtcgcgatttagttggtttgatcaactctcatggcccttacgctgtgggaacctccggtgaggatgccggcctgtttaccgcgcagaagcgcatggtcaacatcgatggcgtacccactgatattggtttggtcggagacatcattaatgtcgatgcctcttccttgatggatatcatcgaggccggtcgcattcctgtggtctctacgattgctccaggcgaagacggccagatttacaacatcaacgccgataccgcagcgggtgctttggctgcagcgattggtgcagaacgcctgctggttctcaccaatgtggaaggtctgtacaccgattggcctgataagagctcactggtgtccaagatcaaggccaccgagctggaggccattcttccgggacttgattccggcatgattccaaagatggagtcttgcttgaatgcggtgcgtgggggagtaagcgctgctcatgtcattgacggccgcatcgcgcactcggtgttgctggagcttttgaccatgggtggaattggcacgatggtgctgccggatgtttttgatcgggagaattatccggaaggcaccgtttttagaaaagacgacaaggatggggaactgtaa。

Claims

1. N-acetylglutamate kinase mutant, among which, The N-acetylglutamate kinase mutant is selected from any one of the following groups (I)-(V): (I) Compared with the sequence shown in SEQ ID NO:1, the N-acetylglutamate kinase mutant has the following characteristics at positions 15, 17, 18, 20, 24, 25, 27, 43, 46, 47, 48, 49, 54, 55, 56, 66, 77, 85, 113, 122, 126, 136, 142, 145, 153, 160, 161, 162, 163, 164, 167, 168, 169, 170, 172, ... The mutation is present at one or more of the following positions: 173, 174, 188, 197, 200, 202, 204, 207, 208, 213, 215, 225, 231, 233, 235, 236, 242, 259, 261, 265, 266, 273, 275, 283, 286, 288, 289, 297; and it exhibits increased arginine production compared to the N-acetylglutamate kinase shown in SEQ ID NO:

1. (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:1; (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions: (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I); (b) A full-length complementary polynucleotide to (a); (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses N-acetylglutamate kinase activity; (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

2. The N-acetylglutamate kinase mutant according to claim 1, wherein, The N-acetylglutamate kinase mutant corresponds to an amino acid with a mutation at at least one of the following positions in the sequence shown in SEQ ID NO:1: G153S, I169N, R174H, G122S, N160S, S265G, L24M, M54I, M77I, L213H, A259V, A266T, A235P, F136L, A48T, D168E, E208G, A207T, I170M, Q188R, L24F, V66M, G 289A, K47E, M142V, I288F, A197V, A200V, I233T, F297S, A20V, F27L, F56L, G85 D. M167V, D225N, A18V, L242P, L283S, G286D, L17P, V55A, A202V, N15S, L46P, D 162N, R273H, A275V, D113N, V126A, A172T, S231P, D43G, G173R, N215D, V161I, R261C, A200T, T236I, Q25R, I145V, A163T, S164P, A204V, L17K, L17S, L17R, L1 7G, L17T, L17Q, V66Y, V66L, A163E, A163Q, A163I, A163W, A163R, A200I, A204F , A204L, A204Y, L17A, V66F, A163C, A163V, A200D, L17V, A163P, A204G, A204I.

3. The N-acetylglutamate kinase mutant according to claim 1 or 2, wherein, The N-acetylglutamate kinase mutant corresponds to the sequence shown in SEQ ID NO:1 and has the following (m1)~(m) sequence. 61 Any mutation shown in any of the following: (m1)G153S, I169N; (m2)R174H; (m3)G122S, N160S, S265G; (m4)L24M, L283S; (m5)M54I, M77I, V161I, L213H; (m6)A259V, A266T; (m7)S231P, A235P; (m8)F136L; (m9)A48T, D168E, A207T, E208G; (m 10 )A207T; (m 11 )I170M、Q188R; (m 12 )L24F、V55A、V66M、G289A; (m 13 )K47E、M142V; (m 14 )I288F; (m 15 )A197V; (m 16 )A49V、I233T、F297S; (m 17 )A20V、F27L、F56L、G85D、M167V; (m 18 )F136L、D225N; (m 19 )A18V、L242P、L283S、G286D; (m 20 )L17P、V55A; (m 21 )A202V; (m 22 )N15S、L46P; (m 23 )V55A、D162N、R273H、A275V; (m 24 )D113N、V126A; (m 25 )A172T、S231P; (m 26 )D43G、G173R、N215D; (m 27 )V161I; (m 28 )V66M、R261C; (m 29 )A200T; (m 30 )V66M、T236I; (m 31 )L17P、Q25R、I145V; (m 32 )V66M; (m 33 )A163T、S164P、A204V;(m 34 )L17P; (m 35 )L17K; (m 36 )L17S; (m 37 )L17R; (m 38 )L17G; (m 39 )L17T; (m 40 )L17Q; (m 41 )V66Y; (m 42 )V66L; (m 43 )A163E; (m 44 )A163Q; (m 45 )A163I; (m 46 )A163W; (m 47 )A163R; (m 48 )A200I; (m 49 )A200V; (m 50 )A204F; (m 51 )A204L; (m 52 )A204Y; (m 53 )L17A; (m 54 )V66F; (m 55 )A163C; (m 56 )A163V; (m 57 )A200D; (m 58 )L17V; (m 59 )A163P; (m 60 )A204G; (m 61 )A204I.

4. An isolated polynucleotide encoding an N-acetylglutamate kinase mutant as described in any one of claims 1 to 3.

5. A nucleic acid construct, wherein, The nucleic acid construct comprises the polynucleotide as described in claim 4, the polynucleotide being operatively linked to one or more regulatory sequences that direct the production of the polypeptide in the expression host.

6. A recombinant expression vector comprising the isolated polynucleotide as described in claim 4, or the nucleic acid construct as described in claim 5.

7. A recombinant host cell, wherein, The recombinant host cell comprises the N-acetylglutamate kinase mutant as described in any one of claims 1 to 3, the polynucleotide as described in claim 4, the nucleic acid construct as described in claim 5, or the recombinant expression vector as described in claim 6.

8. The recombinant host cell according to claim 7, wherein, The host cells are derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium. Preferably, the host cell is derived from Corynebacterium; more preferably, the host cell is derived from Corynebacterium glutamicum.

9. A cell culture comprising the recombinant host cells as described in claim 7 or 8.

10. The use of the N-acetylglutamate kinase mutant as described in any one of claims 1 to 3, the isolated polynucleotide as described in claim 4, the nucleic acid construct as described in claim 5, the recombinant expression vector as described in claim 6, the recombinant host cell as described in claim 7 or 8, or the cell culture as described in claim 9 in the production of arginine or its metabolites in the synthetic pathway; Preferably, the arginine includes L-arginine; Optionally, the metabolites in the arginine biosynthesis pathway include N-acetyl-γ-glutamyl phosphate, N-acetylornithine, ornithine, citrulline, and / or arginine succinate.

11. A method for producing arginine or a metabolite thereof in its synthetic pathway, wherein, The method includes the steps of producing arginine or its metabolites in the synthetic pathway using the N-acetylglutamate kinase mutant as described in any one of claims 1 to 3, the isolated polynucleotide as described in claim 4, the nucleic acid construct as described in claim 5, the recombinant expression vector as described in claim 6, the recombinant host cell as described in claim 7 or 8, or the cell culture as described in claim 9. Optionally, it also includes a step of isolating arginine or its metabolites in its synthetic pathway; Preferably, the arginine includes L-arginine; Optionally, the metabolites in the arginine biosynthesis pathway include N-acetyl-γ-glutamyl phosphate, N-acetylornithine, ornithine, citrulline, and / or arginine succinate.

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