Construction, screening and application of aspartate kinase lysc full gene deep mutation

Abstract

This disclosure provides the construction of a LysC scanning library for aspartate kinase using Retrotron editing technology, the screening of mutants, and their applications, belonging to the fields of biotechnology and genetic engineering technology. Specifically, this disclosure constructs a mutant library of an in situ whole-genome deep mutant library of aspartate kinase using novel Retrotron editing technology. The mutants in the library can significantly increase the production of amino acids such as lysine, threonine, and isoleucine.

Description

Technical Field

[0001] This disclosure belongs to the fields of biotechnology and genetic engineering technology, specifically relating to the construction, screening, and application of a novel gene editing technology—Retron editing technology—for the in situ full-genome deep mutation library of aspartate kinase LysC. Background Technology

[0002] In Corynebacterium species, aspartate kinase (AKIII), encoded by the lysC gene, catalyzes the production of aspartate phosphate from L-aspartic acid. This is the first step in the biosynthetic pathway of L-aspartic acid family amino acids, the rate-limiting step in lysine production, and a key enzyme in the production of threonine, isoleucine, methionine, and other amino acids. The activity of aspartate kinase is subject to feedback inhibition by lysine. Obtaining an aspartate kinase that effectively overcomes feedback inhibition is of great significance for breeding high-yielding strains of L-aspartic acid family amino acids.

[0003] In the prior art, several aspartate kinase mutants that have relieved feedback inhibition have been reported. For example, EP1590463A2 discloses aspartate kinase mutants derived from Corynebacterium glutamicum, including 279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I, and S381F. Although the aforementioned AK III mutants can relieve the feedback inhibition of lysine on aspartate kinase, their efficiency in lysine production varies considerably. Among them, T311I is the mutant with the best effect in lysine production, significantly increasing the lysine yield of the strain. The applicant previously disclosed several mutants derived from Corynebacterium glutamicum that had been freed from feedback inhibition in CN113201514A. These mutants included those with amino acid 293 changed from isoleucine (I) to serine (S), glycine (G), glutamic acid (E), proline (P), tryptophan (W), tyrosine (Y), histidine (H), methionine (M), glutamine (Q), cysteine ​​(C), or arginine (R); amino acid 294 changed from aspartic acid (D) to tyrosine (Y), tryptophan (W), or phenylalanine (F); and amino acid 307 changed from threonine (T) to tyrosine (Y), glycine (G), or phenylalanine (F). Several of these mutants showed better lysine production than T311I. However, these studies often used traditional random mutation or site-saturated mutation methods, resulting in a very limited number of mutants and failing to obtain mutants with low enrichment but significant effects.

[0004] Therefore, utilizing the precise genome retrotron editing technology of Corynebacterium glutamicum to construct a genome-wide scanning library of aspartate kinase LysC, thereby screening for dominant mutants of aspartate kinase in metabolic pathways, increasing the yield of target compounds, and enhancing the competitiveness of the bio-fermentation industry, is an important problem that urgently needs to be solved in the field of microbial fermentation. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] In view of the technical problems existing in the prior art, the Retron editing technology is used to construct a genome-wide in situ deep mutant library to screen for advantageous mutants, thereby improving the activity of key enzymes in the target compound synthesis pathway.

[0007] Solution for solving the problem

[0008] The first aspect of this disclosure provides a method for constructing an in situ full-genome deep mutation library of aspartate kinase, the method comprising the following steps:

[0009] (a) Mutation steps: The aspartate kinase encoding gene shown in SEQ ID NO:4 was mutated using the Retron editing system to obtain mutants with mutations at any site relative to the sequence shown in SEQ ID NO:5;

[0010] (b) Screening steps: Screening mutants that increase lysine production compared to the aspartate kinase shown in SEQ ID NO:5 to obtain aspartate kinase mutants;

[0011] The Retron editing system includes:

[0012] (i) Plasmids expressing Retron; and

[0013] (ii) Plasmids expressing the recombinase RecT;

[0014] Preferably, the plasmid expressing Retron contains a sequence as shown in SEQ ID NO:3 or SEQ ID NO:6;

[0015] Preferably, the plasmid expressing the recombinase RecT contains the sequence shown in SEQ ID NO:2.

[0016] In some embodiments, the plasmid expressing Retron further contains a sequence of a target nucleic acid, which includes a mutated sequence at the site to be edited, and upstream and downstream homologous arms of the site to be edited. In some specific embodiments, the target nucleic acid sequence contains a codon sequence of the target mutation, which may be one or more consecutive sequences corresponding to the site to be edited in the target gene. In some specific embodiments, the total length of the upstream and downstream homologous arm sequences of the site to be edited is 67 bp to 127 bp, for example, it may be 67 bp, 70 bp, 73 bp, 76 bp, 79 bp, 82 bp, 85 bp, 88 bp, 91 bp, 94 bp, 97 bp, 100 bp, 103 bp, 106 bp, 109 bp, 112 bp, 115 bp, 118 bp, 121 bp, 124 bp, or 127 bp, preferably 67 bp.

[0017] In some more specific embodiments, the sequence of the target nucleic acid includes the sequence shown in SEQ ID NO:9 or SEQ ID NO:10.

[0018] The second aspect of this disclosure provides an in situ full-genome deep mutant library of aspartate kinase, wherein the mutant library is obtained using the construction method described above.

[0019] A third aspect of this disclosure provides a mutant derived from the mutant library described above, wherein the mutant is selected from any one of the group consisting of (i) to (iii):

[0020] (i) The aspartate kinase corresponding to SEQ ID NO:5 contains a mutation at any of the following positions: 201, 203, 221, 275, 277, 283, 295, 296, 298, 299, 305, 308, 311, 341, 345, 352, 374, 377, 381, 384, 390, 84, 292, 301, 310, 312, 313, 322, 359, 365, and 383;

[0021] (ii) A polypeptide having 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);

[0022] (iii) A polypeptide with an amino acid sequence as shown in (i) or (ii) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0023] In some embodiments, the aspartate kinase mutant corresponds to the sequence shown in SEQ ID NO:5 and has any one of the following (m1) to (m2) sequences. 54 Any mutation shown in any of the following:

[0024] (m1)M201V; (m2)R221T; (m3)K275A; (m4)G277Y; (m5)G277C; (m6)G277L; (m7)G277M; (m8)G277P; (m9)F283P; (m 10 M295R; (m 11 V296W; (m 12 Q298A; (m 13 Q298T; (m 14 N299S; (m 15 N299I; (m 16 N299A; (m 17 D305P; (m 18 T308F; (m 19 T308P; (m 20 T311A; (m 21 )D341Y;(m 22 G345Y; (m 23 G345W; (m 24 A352S; (m 25 N374T; (m 26 L377Y; (m 27 L377F; (m 28 L377G; (m 29 S381G; (m 30 S381L; (m 31 S384Y; (m 32 R390W; (m 33 A84S; (m 34 E203I; (m 35 K275G; (m 36 G277T; (m 37 N292G; (m 38 N292Q;(m 39 Q298P; (m 40 Q298S; (m 41 S301V; (m 42 )I310F;(m 43 F312W; (m 44 T313R; (m 45 T313V; (m 46A322L; (m 47 D341F; (m 48 A352H; (m 49 G359F; (m 50 M365Y; (m 51 L377N; (m 52 )I383M;(m 53 )I383L;(m 54 )R384I.

[0025] The fourth aspect of this disclosure provides an isolated polynucleotide encoding any mutant from the mutant library described above or a mutant as described above.

[0026] A fifth aspect of 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.

[0027] The sixth aspect of this disclosure provides a recombinant expression vector comprising isolated polynucleotides as described above, or nucleic acid constructs as described above.

[0028] The seventh aspect of this disclosure provides a recombinant host cell, wherein the recombinant host cell comprises a mutant from the mutant library described above, a mutant described above, an isolated polynucleotide described above, or a nucleic acid construct described above, or a recombinant expression vector described above.

[0029] In some embodiments, the host cell is derived from the genus Corynebacterium;

[0030] Preferably, the host cell is derived from Corynebacterium glutamicum.

[0031] The eighth aspect of this disclosure provides a cell culture comprising the recombinant host cells as described above.

[0032] The ninth aspect of this disclosure provides the use of the mutants, isolated polynucleotides, nucleic acid constructs, recombinant expression vectors, recombinant host cells, or cell cultures described above in the production of amino acids or their derivatives; preferably, the amino acids are selected from at least one of lysine, threonine, isoleucine, homoserine, and amino acid derivatives; optionally, the amino acid derivatives include at least one of pentanediamine, 5-aminovaleric acid, glutaric acid, and hydroxyisoleucine.

[0033] The tenth aspect of this disclosure provides a method for producing amino acids and their derivatives, wherein the method includes the steps of producing amino acids and their derivatives using the mutants described above, the isolated polynucleotides described above, the nucleic acid constructs described above, the recombinant expression vectors described above, and the recombinant host cells described above;

[0034] Optionally, it also includes the step of separating and obtaining amino acids and their derivatives;

[0035] Preferably, the amino acid is selected from at least one of lysine, threonine, isoleucine, homoserine, and amino acid derivatives; optionally, the amino acid derivative includes at least one of pentanediamine, 5-aminovaleric acid, glutaric acid, and hydroxyisoleucine.

[0036] The effects of the invention

[0037] In some embodiments, this disclosure utilizes precise retrotron editing technology of Corynebacterium glutamicum to obtain an in situ full-genome deep mutant library of aspartate kinase. This mutant library exhibits rich mutation diversity, and these mutants possess high aspartate kinase activity. Applying this library to lysine production can significantly increase lysine yield, achieving stable and efficient lysine production. Furthermore, the aspartate kinase mutants of this disclosure can also efficiently produce aspartate family amino acids and other amino acids, such as threonine, isoleucine, and methionine, as well as amino acid derivatives such as pentanediamine, 5-aminovaleric acid, glutaric acid, and hydroxyisoleucine. Attached Figure Description

[0038] Figure 1 A diagram showing the metabolic pathway catalyzed by aspartate kinase in Corynebacterium glutamicum is presented.

[0039] Figure 2 This demonstrates the diversity of genomic mutation libraries of the lysC gene;

[0040] Figure 3 The flow cytometry analysis and partitioning diagram of the lysC mutant library are shown. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0048] 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.

[0049] While the disclosed content supports the definition of the term "or" as a substitute, in this specification, the term "or" when used to connect two or more options should be understood to mean any one of the options or any two or more of the options.

[0050] When used in claims or description, the optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered in the middle of the numerical endpoints relative to the aforementioned numerical endpoints.

[0051] The terms “preferred” and “ideal” as used herein are not intended to limit the scope of the claimed disclosure or to imply that certain features are alternatives or additional features that can or cannot be used in embodiments of the claimed disclosure’s structure or function, whether they are critical, necessary, or even important. Rather, these terms are merely used to emphasize alternatives or additional features that can or cannot be used in particular embodiments of the disclosure.

[0052] 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.

[0053] As used in this disclosure, the term "aspartate kinase" and its abbreviation "AKIII" refer to a polypeptide (enzyme) that catalyzes the phosphorylation of aspartate, converting aspartate into aspartic acid phosphate. As the first key enzyme in the lysine biosynthesis pathway, aspartate kinase controls the biosynthesis pathways of all amino acids in the aspartate family.

[0054] 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).

[0055] 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:

[0056] 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).

[0057] 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 aspartate kinase activity.

[0058] 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.

[0059] 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 aspartate kinase activity.

[0060] 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."

[0061] 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:5 that do not affect the activity of aspartate 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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] In this disclosure, the mutation type of an amino acid site includes all codon types corresponding to the mutated amino acid. For example, G33A represents a mutation of glycine to alanine at position 33 of the aspartate kinase. In the specific base sequence, the base sequence corresponding to the mutated alanine site can be GCU, GCC, GCA, or GCG. All four codons can represent different base sequence types of the mutation at this site.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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".

[0071] 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."

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 the 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 or eukaryotic cell, as long as it is a cell capable of introducing the polypeptide or recombinant polypeptide polynucleotide of this disclosure that encodes aspartate kinase activity. In one implementation, the host cell refers to a prokaryotic cell. Specifically, the host cell is derived from microorganisms suitable for fermenting and producing amino acids of the aspartic acid family, such as those derived from Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium. In some preferred embodiments, the host cell is derived from the genus Corynebacterium, more preferably Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032 (also known as Corynebacterium glutamicum 13032), Corynebacterium glutamicum ATCC 14067, and Corynebacterium glutamicum ATCC 13869.

[0078] 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.

[0079] Example

[0080] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0081] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0082] The culture media and buffer solutions used in this disclosure are as follows:

[0083] The TSB medium consists of: 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; and MOPS, 20 g / L.

[0084] The LBG medium consisted of: glucose, 5 g / L; yeast extract, 5 g / L; tryptone, 10 g / L; and NaCl, 10 g / L.

[0085] Fermentation medium A consisted of: glucose, 80 g / L; yeast extract, 1 g / L; soybean peptone, 1 g / L; NaCl, 1 g / L; ammonium sulfate, 1 g / L; urea, 10 g / L; K₂HPO₄·3H₂O, 1 g / L; MgSO₄·7H₂O, 0.45 g / L; FeSO₄·7H₂O, 0.05 g / L; biotin, 0.4 mg / L; vitamin B1, 0.1 mg / L; MOPS, 40 g / L; and an initial pH of 7.2. Kanamycin and zizomycin were added to the medium to a final concentration of 25 μg / mL and 100 μg / mL, respectively.

[0086] The fermentation medium B consisted of: glucose, 80 g / L; yeast extract, 1 g / L; soybean peptone, 1 g / L; NaCl, 1 g / L; ammonium sulfate, 1 g / L; urea, 6 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.45 g / L; FeSO4·7H2O, 0.05 g / L; biotin, 0.4 mg / L; vitamin B1, 0.1 mg / L; MOPS, 40 g / L; and an initial pH of 7.2.

[0087] The PBS buffer composition (g / L) is: potassium dihydrogen phosphate, 2.69 g / L; dipotassium hydrogen phosphate trihydrate, 18.24 g / L; initial pH 7.4.

[0088] Example 1. Construction of chassis strains

[0089] (1) Construction of recombinant enzyme plasmid pRecT-sen containing biosensor

[0090] This disclosure describes a method for coupling a lysine biosensor to a gene mutation library via fluorescence screening. First, a recombinase plasmid containing the lysine biosensor is constructed, using the lysine biosensor N146D mutant plasmid (pLysWT) reported in a prior patent. N146D Using the previously constructed pRecT-1 plasmid (pRecT-1, whose sequence is shown in SEQ ID NO:1) as a template, the RecT expression cassette was amplified using rec-1 / rec-2 primers. The amplified plasmid backbone and RecT expression cassette fragment were then cloned and ligated using a one-step recombinant kit from Novizan to obtain the recombinase plasmid pRecT-sen containing the biosensor. The primers are shown in Table 1.

[0091] pRecT-1: SEQ ID NO:1 (The bold part is the recT gene sequence, and the italic part is the per gene sequence)

[0092]

[0093]

[0094] pRecT-sen plasmid sequence (SEQ ID NO:2): (lowercase bold text indicates the RecT expression box, lowercase sequence indicates the lysG-Eyfp expression box)

[0095]

[0096]

[0097]

[0098] Table 1

[0099] Primers nucleotide sequence Serial Number sen-1 AATTAATTCCGCTAGATGACGTGCG SEQ ID NO:13 sen-2 CATGAGCGGATACATATTTG SEQ ID NO:14 rec-1 CAAATATGTATCCGCTCATGGTGCGTGGCGAGTTTTACAAAG SEQ ID NO:15 rec-2 GTCATCTAGCGGAATTAATTAGAGTTTGTAGAAACGCAAAAAGGCC SEQ ID NO:16

[0100] (2) Construction of chassis strains

[0101] The plasmid pRecT-sen was electroporated into Corynebacterium glutamicum 13032, plated onto TSB solid plates supplemented with 25 μg / mL kanamycin, and cultured to obtain the final chassis strain.

[0102] Example 2. Construction of control plasmid and control strain

[0103] This disclosure uses plasmid pRetronEc73-2 as a template, amplifies the fragment with the rpsL-70bp removed using WT-1 and NK-1 primers, and amplifies the plasmid backbone fragment using WT-2 and NK-2 primers. Both fragments are cloned and ligated using a one-step recombination kit from Novizan to obtain the control plasmid pRetron-no70bp-spe. The primers used are shown in Table 2.

[0104] pRetron-no70bp-spe plasmid sequence (SEQ ID NO:3):

[0105] (Bold text indicates RT sequences, underlined text indicates ncRNA (70bp removed), italic text indicates zirconia gene sequences):

[0106]

[0107]

[0108]

[0109] Table 2

[0110] Primers nucleotide sequence Serial Number WT-1 GTACACGCCGTTTCAGATGTTGG SEQ ID NO:17 WT-2 AACGACGGGCATAGCTAAAATAC SEQ ID NO:18 NK-1 GTATTTTAGCTAGCCCGTCGTTATCGACGTGCTCAAGTAGGT SEQ ID NO:19 NK-2 CCAACATCTGAAACGGCGTGTAC SEQ ID NO:20

[0111] This disclosure further describes the electroporation of the constructed control plasmid pRetron-no70bp-spe into the chassis strain from Example 1, followed by plating onto TSB agar plates supplemented with 25 μg / mL kanamycin and 100 μg / mL zidimecrolimus, and incubation at 30°C to obtain the control strain Retron-WT-spe. Single clones were inoculated into 24-well plates containing 800 μL of TSB medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zidimecrolimus, and incubated at 30°C for 24 h with the plate shaker speed at 800 rpm and humidity at 90%. 600 When the temperature reaches approximately 5, add an equal volume of 50% glycerol and store at -80°C.

[0112] Example 3. Construction of a lysC genome in situ whole-genome mutation library using the Retron editing system

[0113] (1) Construction of lysC gene mutation editing plasmid library

[0114] This disclosure utilizes the Retron editing tool to construct an in situ full-gene deep mutant library of the lysC genome. To achieve high-throughput construction of the edit plasmid, the tool plasmid pRetron-2-ccdB was first constructed, and a type II restriction endonuclease BspQI (5'…GCTCTTC(N)1…3') restriction site was introduced. Using plasmid pRetronEc73-2 as a template, the plasmid backbone was amplified using pRetron-3 / 4 primers; using pCas9gRNA-proB2 plasmid (disclosed in patent CN112111469B, which is incorporated herein by reference) as a template, the ccdB fragment was amplified using ccdB-1 / ccdB-2 primers. The two fragments were cloned and ligated using the Novizan one-step recombination kit to obtain the correct plasmid pRetron-2-ccdB. This plasmid can be efficiently cloned using goldengate ligation for the construction of mutant library plasmids. After construction, the plasmid is replaced only at the recombination template (i.e., the 70nt sequence described in this paper) with the sequence used for the corresponding target site.

[0115] pRetron-2-ccdB sequence (SEQ ID NO:6):

[0116] (Bold text indicates RT sequences, underlined text indicates ncRNA, underlined and bold text indicates ccdB, and italic text indicates zithromycin gene sequences):

[0117]

[0118]

[0119] The aspartate kinase (AK) encoded by the lysC gene consists of 422 amino acids, the nucleotide sequence of which is shown in SEQ ID NO:4, and the amino acid sequence of which is shown in SEQ ID NO:5. A 70nt fragment (25200 fragments in total) was designed by mutating each of the 420 amino acids of AK (with the start and stop codons removed) using 60 different codons (with 3 stop codons and the self-codon removed). To facilitate the amplification of the primer pool fragment, YW-3 and BspQI restriction sites (GACATGCGCTTGGCGCATCGCTCTTCTGTT, SEQ ID NO:7) were added to the 5' end of the 70nt sequence, and YW-4 and BspQI restriction sites (ATCTGAAGAGCGTGGACAGACCAGCCAGT, SEQ ID NO:8) were added to the 3' end of the 70nt sequence. Taking the second codon GCC of lysC as an example: Select the 34th base to the left of GCC and the 33rd base to the right of GCC in the lagging sequence, and replace GCC with one of 60 codons excluding the three stop codons (TGA, TAA, TAG). The position corresponding to GCC is replaced with NNN, resulting in:

[0120] TGTCAGCACGTAGATCGAAAGGTGCACAAAGGTG NNN CTGGTCGTACAGAAATATGGCGGTTCCTCGC TT (SEQ ID NO:9, N = A, C, G or T, NNN refers to the codon replacing GCC), based on this, the above-mentioned YW-3 and BspQI restriction site sequences and YW-4 and BspQI restriction site sequences are added to the 5' and 3' ends of the 70nt sequence, respectively, that is, the sequence is:

[0121] GACATGCGCTTGGCGCATCGCTCTTCTGTT TGTCAGCACGTAGATCGAAAGGTGCACAAAGGTG NNN C TGGTCGTACAGAAATATGGCGGTTCCTCGCTT ATCTGAAGAGCGTGGACAGACCAGCCAGT (SEQ ID NO:10, 129 nt). Primer design for each codon was completed in this manner, and the company further synthesized the whole genome primer pool for this gene.

[0122] lysC gene sequence: SEQ ID NO:4

[0123] GTG GCC

[0124] Aspartate kinase amino acid sequence: SEQ ID NO:5

[0125] MALVVQKYGGSSLESAERIRNVAERIVATKKAGNDVVVVCSAMGDTTDELLELAAAVNPVPPAREMDMLLTAGERISNALVAMAIESLGAEAQSFTGSQAGVLTTERHGNARIVDVTPGRVREALDEGKICIVAGFQGVNKETRDVTTLGRGGSDTTAVALAAALNADVCEIYSDVDGVYTADPRIVPNAQKLEKLSFEEMLELAAVGSKI LVLRSVEYARAFNVPLRVRSSYSNDPGTLIAGSMEDIPVEEAVLTGVATDKSEAKVTVLGISDKPGEAAKVFRALADAEINIDMVLQNVSSVEDGTTDITFTCPR SDGRRAMEILKKLQVQGNWTNVLYDDQVGKVSLVGAGMKSHPGVTAEFMEALRDVNVNIELISTSEIRISVLIREDDLDAAARALHEQFQLGGEDEAVVYAGTGR*

[0126] This disclosure further describes the construction of a plasmid library using the Golden Gate method: Using a synthesized primer pool as a template, the mutant library fragment is amplified using YW-3 / YW-4 primers. The obtained mutant fragment is then combined with the constructed tool plasmid pRetron-2-ccdB to construct a mutant plasmid library using the Golden Gate method. The 15 μL reaction system consists of: pRetron-2-ccdB, 100 ng; mutant library fragment, 30 ng; T7 ligase, 1 μL; T7 buffer, 4.25 μL; BspQ I restriction endonuclease, 1 μL; NE buffer. TM 1.5 μL of r3.1 (BspQ I enzyme buffer) and 3.75 μL of BSA (bovine serum albumin) were added, with the remaining volume made up with deionized water. The reaction program was: (43℃, 10 min; 25℃, 10 min) * 12 cycles; 80℃, 5 min. A library of approximately 500,000 *E. coli* transformants was obtained, covering approximately 20 times the theoretical mutation count. A mutant plasmid library was obtained through plasmid extraction. Primers are listed in Table 3.

[0127] Table 3

[0128]

[0129]

[0130] To assess the quality of the plasmid library, a mutant plasmid library was obtained as a template, and the plasmid was amplified using GK-1 / 2 primers.

[0131] The ncRNA fragments from the library were recovered, purified, and subjected to PE150 next-generation sequencing. The sequencing results of 10 G sequencing data were compared and analyzed, and it was found that it could cover 98.0% of all 60 designed codon mutation types. Each site corresponds to 19 amino acid mutation types, which can cover 99.4% of the theoretical types, indicating that a high-quality plasmid library was successfully constructed (Table 4).

[0132] Table 4

[0133] Statistical parameters codon mutation Amino acid mutation Types of theories 25200 8400 Actual species 24705 8354 Coverage 98.0％ 99.4％

[0134] (2) Construction of lysC genome editing library

[0135] This disclosure further constructs a Corynebacterium glutamicum genome editing library, and electroporates the obtained editing library plasmid into the chassis strain constructed in Example 1, yielding approximately 3 × 10⁻⁶ pclogs. 6 One transformant was used to cover approximately 120-fold of the theoretical library. All incubated bacteria were then transferred to 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zizomycin. The plates were shaken at 800 rpm and 90% humidity for 24 h at 30°C. Subsequently, the transformants were transferred to 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin, 100 μg / mL zizomycin, and 0.5 mM IPTG (inoculum size 12 μL). The plates were then shaken at 800 rpm and 30°C for 24 h, followed by another 24 h at 90% humidity. This process was repeated twice more to induce library editing, yielding the corresponding lysC genome editing library. A portion of the edited bacterial culture was used to extract the genome of the edited library bacteria. Using the genome as a template, the lysC fragment was amplified using primers lys-Y3 / lys-Y4 (lys-Y3:GAGTTGAGCGGGTAACTGTCAG, SEQ ID NO:11; lys-Y4:CACCAACAACTGCGATGGTGG, SEQ ID NO:12) and sent to the company for PE150 next-generation sequencing. At the same time, the remaining edited bacterial culture was added with an equal volume of 50% glycerol and stored at -80℃.

[0136] The sequencing results of the lysC gene-editing library are shown in Table 5. Analysis of approximately 40 G of sequencing data revealed that it covers 78.6% of all 60 designed codon mutation types, and the 19 amino acid mutation types corresponding to each site cover 92.4% of the theoretical range (Table 5). Figure 2 This indicates that a high-quality lysC genome editing library was successfully constructed.

[0137] Table 5

[0138] Statistical parameters codon mutation Amino acid mutation Types of theories 25200 8400 Actual species 19799 7761 Coverage 78.6％ 92.4％

[0139] Example 4. Screening of lysC genome editing libraries and characterization of mutant yield.

[0140] To screen for dominant mutants from the library, the mutant library constructed in Example 3 was cultured under fermentation conditions. Fermentation medium A was used, with 25 μg / mL kanamycin and 100 μg / mL zizomycin supplemented throughout the culture. The preserved whole-genome mutant library and the control strain were inoculated into TSB liquid medium and cultured for 8 h. The culture was then used as seed culture to inoculate 12 μL into each well of a 24-well plate containing 800 μL of fermentation medium A, and cultured for 6 h to allow the mutants to synthesize lysine and achieve a biosensor response. The shaker conditions for the well plates were 30°C, 800 rpm, and 90% humidity.

[0141] After centrifuging the bacterial culture for 6 h, remove the supernatant culture medium, resuspend the culture in an appropriate amount of PBS buffer, and dilute to the OD value of the bacterial culture. 600 The concentration was approximately 0.05 (measured by spectrophotometer). The cells were sonicated in a washing sonicator for 6 minutes to disperse them into single cells. Flow cytometry was then used for analysis, with excitation light at 488 nm and fluorescence detection at 515 ± 10 nm. Analysis with control cells showed that the fluorescence value was higher in approximately 5% of the total cells in the library compared to the control. Figure 3 A). Simultaneously, the first 0.01% of the high-fluorescence region is outlined ( Figure 3 B).

[0142] To screen for more mutants of aspartate kinase that increase amino acid production, this disclosure employs two sorting methods to obtain as many beneficial mutations as possible. Method 1: Direct single-cell sorting of high-fluorescence regions; Method 2: Regional collection of high-fluorescence regions, mixed-cell next-generation sequencing to obtain high-frequency mutations in high-fluorescence regions.

[0143] (1) Characterization of lysine production and sequence analysis in single-cell sorting mutants

[0144] High-fluorescence regions of the library were selected, and the first 0.01% of the strains were sorted onto TSB solid plates (…). Figure 3B) The culture medium was supplemented with 25 μg / mL kanamycin and 100 μg / mL zeaxanthin, and cultured at 30°C until clones grew, eventually obtaining about 200 clones.

[0145] To test the lysine production level of the sorted mutants, clones from the plates were evaluated using 96-well plate fermentation. All clones grown from the plates, along with the control strain, were inoculated using toothpicks into 96-well deep-well plates containing 200 μL of TSB liquid medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zizomycin per well, and cultured for 8 h. The inoculum was then transferred to fermentation medium A using an automated high-throughput pipetting station, with 25 μg / mL kanamycin and 100 μg / mL zizomycin added to medium A at an inoculum volume of 12 μL, and cultured for 24 h. The plate shaker conditions were 30°C, 800 rpm, and 90% humidity. The L-lysine yield at the fermentation endpoint was detected using Echo-MS, and the OD was measured using a microplate reader. 600 Using primers lys-Y3 / Y4, the lysC gene of the mutants with increased lysine production was amplified and sequenced, resulting in 45 mutant types with increased lysine production (Table 6).

[0146] Table 6

[0147]

[0148]

[0149] (2) Next-generation sequencing analysis of lysC library partitioning and verification of mutant reconstruction

[0150] Due to the limited number of direct single-strain sorts, next-generation sequencing was used to further screen for more mutation types from the edited library. The first 0.01% of the high-fluorescence region was collected, resulting in 1500 clones. The cells were collected in sterile flow cytometry tubes, thoroughly dissolved in 100 μL ddH2O, and incubated in boiling water for 15 min to fully lyse the cells. These lysed cells were then used as templates to amplify a 70 bp region on the plasmid using primers GKK-1 / GKK-2. The final fragment was then subjected to PE150 next-generation sequencing. Primers are listed in Table 7.

[0151] Further data processing yielded the number of reads for each mutation. Analysis of this high-fluorescence region identified 935 mutation types with read counts ranging from 1 to 36,055. To demonstrate data accuracy, this disclosure selected 279 mutation types with sequencing read counts >100, including 38 of the 50 mutation types screened by sorting single clones. This disclosure considers mutants highly enriched in the mixed-bacteria sequencing results of this region to be highly likely to be beneficial mutations. Therefore, 24 mutants with different read counts were randomly selected from the mixed-bacteria sequencing library for reconstruction (Table 9).

[0152] First, 24 mutants were constructed using the Retron editing method. Based on the pRetron-2-ccdB plasmid, editing template plasmids for the 24 mutants were constructed. Complementary primers were designed for each of the 24 mutants, and an annealing reaction system (10 μL) was prepared: 4 μL of 10 mM stock solution was added to each of the two complementary primers, and the remaining volume was made up with deionized water. The mixture was then denatured at 98 °C for 10 min, and then naturally cooled to room temperature for annealing to double strands. The mutant plasmids were further constructed using the Golden Gate method. The obtained editing plasmids were electroporated into the chassis strain constructed in Example 1 to obtain single clones, which were then inoculated into 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zizomycin and cultured for 24 h. The culture was then transferred to 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin, 100 μg / mL zizomycin, and 0.5 mM IPTG, with an inoculum size of 12 μl. The plates were then subcultured twice using the same method to induce mutant editing. The well conditions were 800 rpm, 90% humidity, and 30°C. The edited bacterial culture was diluted and plated. Different transformants were picked, and the gene fragment was amplified by PCR using primers lys-Y3 / Y4 and sequenced to obtain the correct mutant. All primers are listed in Table 8.

[0153] Table 7

[0154] Primers nucleotide sequence Serial Number GKK-1 CCATGAGTCATGGTTTCGCC SEQ ID NO:26 GKK-2 CAGAGCCAAACCTACTTGAGC SEQ ID NO:28

[0155] The obtained mutant and control strains were inoculated into 96-well deep-well plates containing 200 μL of TSB liquid medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zizomycin, respectively, and cultured for 8 h. The inoculum was then transferred to fermentation medium A using an automated high-throughput pipetting station, with 25 μg / mL kanamycin and 100 μg / mL zizomycin added to fermentation medium A at an inoculum volume of 12 μL, and cultured for 24 h. The shaker conditions for the well plates were 30℃, 800 rpm, and 90% humidity. The L-lysine yield at the fermentation endpoint was detected using Echo-MS, and the OD was measured using a microplate reader. 600 .

[0156] Table 8

[0157]

[0158]

[0159]

[0160] By measuring lysine production, all 24 mutant types in Table 9 were found to produce more than 2 g / L of lysine, including 17 sites. The results indicate that combining high-throughput sequencing can further screen for mutants that increase production.

[0161] Table 9

[0162]

[0163] Example 5. Application of the superior mutant in the production of other amino acids

[0164] To further verify the application of the above-mentioned lysC gene mutant in the production of aspartic acid family amino acids, this disclosure verifies the application effects of the G345Y and E203I mutants in threonine, homoserine, glycine and isoleucine.

[0165] The first step was to construct the chassis strain. The ZcglT4 strain was created by sequentially introducing the feedback-relief aspartate kinase lysC gene T311I mutation (base mutation ACC→ATC), the homoserine dehydrogenase hom gene G378E mutation (GGG→GAG), the lysC gene start codon GTG to ATG, and the artificial strong promoter P before the start codon into the *Corynebacterium glutamicum* ATCC13032 genome. pyc -20(P pyc -20 is published in CN113755492A and incorporated herein by reference, hom gene start codon insertion of artificial strong promoter P gpmA -16(P gpmA-16 is published in CN115506035A and is incorporated herein by reference. The resulting strain was named ZcglT4. To verify the amino acid production potential of the mutant in this study, the T311I mutation (base mutation ACC→ATC) in the aspartate kinase lysC gene of the ZcglT4 strain was first restored to the wild type, that is, the codon ATC was changed to ACC, to obtain the ZcglT4-lysC-WT strain as a verification chassis. The editing-assisted plasmid pRecT-1 (shown in SEQ ID NO:1) was electroporated into the ZcglT4-lysC-WT strain to obtain the editing chassis ZcglT4-lysC-WT(pRecT-1) strain.

[0166] To introduce the lysC mutation of this disclosure into the genome of the ZcglT4-lysC-WT strain, two editing template plasmids for mutants were first constructed based on the pRetron-2-ccdB plasmid. Primers G345Y-F / R and E203I-F / R were designed, respectively. In a 10 μL reaction system, 4 μL of each of the two complementary primers (10 mM) was added, and the remaining volume was made up with deionized water. The mixture was then denatured at 98 °C for 10 min, followed by natural cooling to room temperature for annealing to double strands. The mutant plasmids were then constructed using the Golden Gate method. The obtained editing plasmids were electroporated into the ZcglT4-lysC-WT(pRecT-1) strain to obtain single clones. These clones were then inoculated into 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin and 100 μg / mL zizomycin and cultured for 24 h. The culture was then transferred to 24-well plates containing 0.8 mL of LBG medium supplemented with 25 μg / mL kanamycin, 100 μg / mL zizomycin, and 0.5 mM IPTG, with an inoculum size of 12 μL. The plates were then subcultured twice using the same method to induce mutant editing. The plate culture conditions were 800 rpm, 90% humidity, and 30°C. The edited bacterial mixture was diluted and plated. Different transformants were picked, and the gene fragment was amplified using primers lys-Y3 / Y4 and sequenced to obtain the correct mutants. By repeatedly subculturing in antibiotic-free medium and losing two editing helper plasmids, the correctly introduced mutants ZcglT4-lysC-WT-G345Y and ZcglT4-lysC-WT-E203I were obtained. All primers are listed in Table 10.

[0167] Table 10

[0168]

[0169] The above strains were fermented in 24-well plates. The strains listed in the table were activated on TSB solid plates and cultured at 30°C for 12 hours. Then, they were inoculated into 24-well plates with 800 μL of TSB liquid medium per well and cultured at 30°C and 800 rpm for 12 hours, with the same initial OD. 600 The cultures were transferred to 24-well plates, with 800 μL of fermentation medium B in each well. Each strain was cultured in triplicate at 30°C and 800 rpm for 30 h. The yields of various amino acids were then determined by liquid chromatography. The results are shown in Table 11. Compared with the control strain ZcglT4-lysC-WT, the yields of all four amino acids in the two mutant strains were significantly increased. These results indicate that the aspartate kinase mutants obtained in this study have the potential for producing aspartic acid family amino acids.

[0170] Table 11

[0171]

Claims

1. A method for constructing an in situ full-genome deep mutant library of aspartate kinase, the method comprising the following steps: (a) Mutation steps: The aspartate kinase encoding gene shown in SEQ ID NO:4 was mutated using the Retron editing system to obtain mutants with mutations at any site relative to the sequence shown in SEQ ID NO:5; (b) Screening steps: Screening mutants that increase lysine production compared to the aspartate kinase shown in SEQ ID NO:5 to obtain aspartate kinase mutants; in, The Retron editing system includes: (i) Plasmids expressing Retron; and (ii) Plasmids expressing the recombinase RecT; Preferably, the plasmid expressing Retron contains a sequence as shown in SEQ ID NO:3 or SEQ ID NO:6; Preferably, the plasmid expressing the recombinase RecT contains the sequence shown in SEQ ID NO:

2.

2. A library of in situ deep mutants of the entire aspartate kinase genome, wherein, The mutant library was obtained using the construction method described in claim 1.

3. A mutant derived from the mutant library as described in claim 2, wherein, The mutant is selected from any one of the following groups (i) to (iii): (i) The aspartate kinase corresponding to SEQ ID NO:5 contains a mutation at any of the following positions: 201, 203, 221, 275, 277, 283, 295, 296, 298, 299, 305, 308, 311, 341, 345, 352, 374, 377, 381, 384, 390, 84, 292, 301, 310, 312, 313, 322, 359, 365, and 383; (ii) A polypeptide having 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); (iii) A polypeptide with an amino acid sequence as shown in (i) or (ii) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

4. The mutant according to claim 3, wherein, The aspartate kinase mutant corresponds to the sequence shown in SEQ ID NO:5 and has any of the following (m1) to (m 54 Any mutation shown in any of the following: (m1)M201V; (m2)R221T; (m3)K275A; (m4)G277Y; (m5)G277C; (m6)G277L; (m7)G277M;(m8)G277P;(m9)F283P;(m 10 )M295R;(m 11 )V296W; (m 12 )Q298A;(m 13 )Q298T;(m 14 )N299S;(m 15 )N299I;(m 16 )N299A;(m 17 )D305P;(m 18 )T308F;(m 19 )T308P;(m 20 )T311A;(m 21 )D341Y;(m 22 )G345Y;(m 23 )G345W;(m 24 )A352S;(m 25 )N374T;(m 26 )L377Y;(m 27 )L377F;(m 28 )L377G;(m 29 )S381G;(m 30 )S381L;(m 31 )S384Y;(m 32 )R390W;(m 33 )A84S;(m 34 )E203I;(m 35 )K275G;(m 36 )G277T; (m 37 )N292G; (m 38 )N292Q; (m 39 )Q298P; (m 40 )Q298S; (m 41 )S301V; (m 42 )I310F; (m 43 )F312W; (m 44 )T313R; (m 45 )T313V; (m 46 )A322L; (m 47 )D341F; (m 48 A352H; (m 49 )G359F; (m 50 )M365Y; (m 51 L377N; (m 52 )I383M; (m 53 )I383L; (m 54 )R384I。 5. An isolated polynucleotide encoding any mutant in the mutant library as described in claim 2 or a mutant as described in claim 3 or 4.

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

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

8. A recombinant host cell, wherein, The recombinant host cell comprises a mutant from the mutant library as described in claim 2, a mutant as described in claim 3 or 4, an isolated polynucleotide as described in claim 5, a nucleic acid construct as described in claim 6, or a recombinant expression vector as described in claim 7.

9. The recombinant host cell according to claim 8, wherein, The host cells are derived from the genus Corynebacterium; Preferably, the host cell is derived from Corynebacterium glutamicum.

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

11. The use of mutants from the mutant library of claim 2, mutants of claim 3 or 4, isolated polynucleotides of claim 5, nucleic acid constructs of claim 6, recombinant expression vectors of claim 7, recombinant host cells of claim 8 or 9, or cell cultures of claim 10 in the production of amino acids or their derivatives; preferably, the amino acid is selected from at least one of lysine, threonine, isoleucine, homoserine, and amino acid derivatives; optionally, the amino acid derivative includes at least one of pentanediamine, 5-aminovaleric acid, glutaric acid, and hydroxyisoleucine.

12. A method for producing amino acids or their derivatives, wherein, The method includes the steps of producing amino acids or their derivatives using mutants from the mutant library as described in claim 2, mutants as described in claim 3 or 4, isolated polynucleotides as described in claim 5, nucleic acid constructs as described in claim 6, recombinant expression vectors as described in claim 7, or recombinant host cells as described in claim 8 or 9. Optionally, it also includes the step of separating and obtaining an amino acid or a derivative thereof; Preferably, the amino acid is selected from at least one of lysine, threonine, isoleucine, homoserine, and amino acid derivatives; optionally, the amino acid derivative includes at least one of pentanediamine, 5-aminovaleric acid, glutaric acid, and hydroxyisoleucine.

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