A mutant of transcriptional regulatory factor LysG and its application

Abstract

The present disclosure belongs to the field of genetic engineering and molecular biology, and relates to a mutant of a transcriptional regulatory factor LysG, a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell, a biosensor and uses, a method for regulating the expression of a target gene, a method for producing alkaline amino acids, a strain with high production of alkaline amino acids, and a method for screening proteins or protein encoding genes related to alkaline amino acid synthesis. The mutant of the transcriptional regulatory factor LysG disclosed in the present disclosure is a mutant of the transcriptional regulatory factor LysG after mutation of the Linker Helix region. The mutant has an increased intensity of response to alkaline amino acid induction, and can achieve high-sensitivity detection of alkaline amino acid high-production strains and proteins related to alkaline amino acid synthesis or their encoding genes, which is of great significance for high-throughput screening of alkaline amino acid high-production strains and proteins related to synthesis pathways, and the expression regulation of key enzymes in the construction of alkaline amino acid high-production strains.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and molecular biology, and specifically relates to a mutant of a transcriptional regulatory factor LysG, a polynucleotide encoding the mutant, a nucleic acid construct, a recombinant expression vector, a recombinant host cell, a biosensor and uses thereof, a method for regulating the expression of a target gene, a method for producing basic amino acids, a strain with high basic amino acid production, and a method for screening proteins related to basic amino acid synthesis. Background Art

[0002] LysR-type transcriptional regulators (LLTRs) are widely distributed in various genera of microorganisms. The genes they regulate have diverse functions, involving anabolism, quorum sensing, virulence, etc. [1] With the development of bioinformatics and protein structure, more and more LysR-type transcriptional regulatory factors have been discovered. So far, LysR family transcriptional regulatory factors have become the largest family of transcription factors in prokaryotes.

[0003] LysG identified in Corynebacterium glutamicum belongs to the typical LysR family transcriptional regulatory factor, with a DNA-binding Helix-turn-helix motif (HTH) at the N-terminus and an effector binding region (Effector binding domain, EBD) at the C-terminus, and a flexible Linker Helix connecting the two. [2] The transcriptional regulatory factor LysG can respond to three basic amino acids (lysine, arginine and histidine) in the cell, and regulate the lysE promoter to activate the expression of the downstream transporter LysE, thereby maintaining the balance of basic amino acids in the cell. [3] .

[0004] Due to the sensing properties of transcriptional regulators, biosensors based on transcriptional regulators have been widely used in the detection of intracellular small molecule metabolite concentrations and high-throughput screening of small molecule metabolite high-yield strains, such as high-throughput screening of amino acid high-yield strains and key enzymes in their synthesis pathways. [3-5] However, biosensors based on natural transcriptional regulators often have low response intensity to small molecules and are difficult to apply to high-throughput screening of high-yield strains. Therefore, how to transform natural transcriptional regulators and optimize the response intensity of biosensors is an important issue that needs to be solved urgently.

[0005] The transcriptional regulator LysG can regulate the expression of the downstream target gene, amino acid transporter, through the lysE promoter [6]Its transcriptional regulation in response to basic amino acids is of great significance for improving the regulatory performance of response elements. However, the wild-type LysG has a low response intensity to the three basic amino acids, and its transcriptional activation effect on downstream amino acid transporters and other proteins is limited, so it cannot be used for a wider range of gene expression regulation.

[0006] References:

[0007] [1] Maddocks, SE and Oyston, PCF (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology, 154, 3609-3623.

[0008] [2]Della Corte, D., van Beek, HL, Syberg, F., Schallmey, M., Tobola, F., Cormann, KU, Schlicker, C., Baumann, PT, Krumbach, K., Sokolowsky, S. et al. (2020) Engineering and application of a biosensor with focused ligandspecificity. Nat. Commun., 11, 4851.

[0009] [3]Binder,S.,Schendzielorz,G., N.,Krumbach,K.,Hoffmann,K.,Bott,M.and Eggeling,L.(2012)A high-throughput approach to identify genomicvariants of bacterial metabolite producers at the single-cell level.Genomebiology,13,R40.

[0010] [4] Kortmann, M., Mack, C., Baumgart, M. and Bott, M. (2019) Pyruvatecarboxylase variants enabling improved lysine production from glucose identified by biosensor-based high-throughput fluorescence-activated cell sorting screening. ACS Synth. Biol., 8, 274-281.

[0011] [5]Schendzielorz,G.,et al.(2014)Taking control over control:use ​​ofproduct sensing in single cells to remove flux control at key enzymes inbiosynthesis pathways.ACS Synth.Biol.,3,21-29.

[0012] [6] Zhou LB, Zeng AP. (2015) Engineering a lysine-ON riboswitch formetabolic control of lysine production in Corynebacterium glutamicum.ACSSynth.Biol.,4,1335-1340. Summary of the invention

[0013] Problem that the invention aims to solve

[0014] In view of the problems existing in the prior art, for example, the wild-type LysG has a low response intensity to basic amino acids, cannot efficiently regulate the expression of target genes, and cannot construct a biosensor with high sensitivity. To this end, the present disclosure provides a polypeptide having transcriptional regulatory factor activity, which is a mutant obtained by mutating the Linker Helix region of the transcriptional regulatory factor LysG. Compared with the wild-type transcriptional regulatory factor LysG, the mutant has an improved induced response intensity to basic amino acids, and can achieve high-sensitivity detection of biosensors for high-yield strains of basic amino acids and protein encoding genes related to basic amino acid synthesis, which is of great significance for the construction of high-yield strains of basic amino acids and high-throughput screening of strains or proteins related to basic amino acid synthesis.

[0015] Solutions for solving problems

[0016] (1) A mutant of a transcriptional regulatory factor LysG, wherein the mutant has one or more mutated amino acids located in a linker helix region; and, compared with a wild-type transcriptional regulatory factor LysG, the mutant has an increased basic amino acid response intensity;

[0017] (2) The mutant of the transcriptional regulatory factor LysG according to (1), wherein the mutant is selected from any one of the following groups consisting of (i) to (iii):

[0018] (i) a mutant comprising a polypeptide having a sequence as shown in SEQ ID NO: 1, wherein the mutant has a mutated amino acid at one or more positions corresponding to positions 57 to 89 of the sequence shown in SEQ ID NO: 1;

[0019] (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);

[0020] (iii) A polypeptide having an amino acid sequence as shown in (i) or (ii) wherein one or more amino acids are added or deleted at at least one of the N-terminus and the C-terminus.

[0021] (3) The mutant of the transcriptional regulatory factor LysG according to (1) or (2), wherein the mutant corresponds to the sequence shown in SEQ ID NO: 1 and has a mutated amino acid at one or more of the following positions:

[0022] 58th, 59th, 61st, 64th, 65th, 66th, 67th, 70th, 73rd, 75th, 76th, 77th, 78th, 79th, 80th, 83rd, 84th, 85th, 86th, 87th, 88th, 89th;

[0023] Preferably, the mutant has a mutated amino acid at at least one, at least two or at least three positions corresponding to the sequence shown in SEQ ID NO:1.

[0024] (4) A mutant of the transcriptional regulatory factor LysG according to any one of (1) to (3), wherein the mutant corresponds to the sequence shown in SEQ ID NO: 1 and has the following characteristics: (a 1 )-(a 22 ) at any one position in the group consisting of, or at any two or more positions:

[0025] (a 1 )E58V;

[0026] (a 2 )A59S;

[0027] (a 3 )E61K or E61V;

[0028] (a 4 )V64A;

[0029] (a 5 )Q65R;

[0030] (a 6 )A66T;

[0031] (a 7 )A67T or A67S;

[0032] (a 8 )A70I;

[0033] (a 9 )L73I;

[0034] (a 10 )A75G;

[0035] (a 11 )E76D, E76C, E76K or E76W;

[0036] (a 12 )T77A;

[0037] (a 13 )K78E or K78R;

[0038] (a 14 )A79V, A79T or A79R;

[0039] (a 15 )Q80R or Q80K;

[0040] (a 16 )G83R;

[0041] (a 17 )R84H;

[0042] (a 18 ) L85P, L85A, L85D, L85G, L85A, L85S, L85T or L85Y;

[0043] (a 19 )A86R or A86D;

[0044] (a20 )E87G, E87D, E87R, E87N, E87H, E87K or E87S;

[0045] (a 21 )I88V, I88T, I88A, I88N, I88G, I88H, I88K, I88P or I88S;

[0046] (a 22 )P89Q.

[0047] (5) A mutant of the transcriptional regulatory factor LysG according to any one of (1) to (4), wherein the mutant corresponds to the sequence shown in SEQ ID NO: 1 and has the following characteristics (m 1 )-(m 51 ) in any one of the mutated amino acids:

[0048] (m 1 )E58V;(m 2 )A59S and Q80R; (m 3 )E61V;(m 4 )E61K and K78E;

[0049] (m 5 )V64A and E87D; (m 6 )Q65R;(m 7 )A66T;(m 8 )A67T and A79V

[0050] (m 9 )A67T;(m 10 )A67S and T77A; (m 11 )M70I;(m 12 )L73I;(m 13 )L73V;

[0051] (m 14 )A75G;(m 15 )E76D;(m 16 )E76C;(m 17 )E76K;(m 18 )E76W

[0052] (m 19 )T77A, K78R and G83R; (m 20 )T77A;(m 21 )A79V;(m 22 )A79R;

[0053] (m 23 )A79T and G83R; (m24 )Q80R;(m 25 )Q80K and R84H; (m 26 )L85P;

[0054] (m 27 )L85A;(m 28 )L85D;(m 29 )L85G;(m 30 )L85M;(m 31 )L85S;

[0055] (m 32 )L85T;(m 33 )L85Y;(m 34 )A86R;(m 35 )A86D;(m 36 )E87G;

[0056] (m 37 )E87R;(m 38 )E87N;(m 39 )E87H;(m 40 )E87K;(m 41 )E87S;

[0057] (m 42 )I88V;(m 43 )I88T;(m 44 )I88A;(m 45 )I88N;(m 46 )I88G;

[0058] (m 47 )I88H;(m 48 )I88K;(m 49 )I88P;(m 50 )I88S;(m 51 )P89Q.

[0059] (6) A mutant of the transcriptional regulatory factor LysG according to any one of (1) to (5), wherein the mutant has a basic amino acid response intensity that is 1.4-5 times higher than that of the polypeptide of the sequence shown in SEQ ID NO: 1.

[0060] (7) An isolated polynucleotide, wherein the polynucleotide encodes a mutant of the transcriptional regulatory factor LysG as described in any one of (1) to (6).

[0061] (8) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide described in (7), wherein the polynucleotide is operably linked to a LysG-regulated promoter, and the LysG-regulated promoter directs the expression of the polypeptide.

[0062] (9) A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide described in (7) or the nucleic acid construct described in (8).

[0063] (10) A recombinant host cell, wherein the recombinant host cell comprises a mutant of the transcriptional regulatory factor LysG as described in any one of (1) to (6), a polynucleotide as described in (7), a nucleic acid construct as described in (8), or a recombinant expression vector as described in (9).

[0064] (11) The recombinant host cell according to (10), wherein the host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium or Corynebacterium;

[0065] Preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067 strain or a derivative strain of any of the above.

[0066] (12) A biosensor, wherein the biosensor comprises a mutant of the transcriptional regulatory factor LysG as described in any one of (1) to (6), or a polynucleotide as described in (7), a nucleic acid construct as described in (8), a recombinant expression vector as described in (9), or a polypeptide expressed by a recombinant host cell as described in any one of (10) to (11).

[0067] (13) Use of the mutant of the transcriptional regulatory factor LysG according to any one of (1) to (6), the polynucleotide according to (7), the nucleic acid construct according to (8), the recombinant expression vector according to (9), or the recombinant host cell according to any one of (10) to (11) in at least one of the following (a) to (d):

[0068] (a) regulating the transcription level of a target gene, or preparing a reagent or kit for regulating the transcription level of a target gene;

[0069] (b) producing basic amino acids, or preparing reagents or kits for producing basic amino acids;

[0070] (c) screening a strain with high basic amino acid production, or preparing a reagent, a kit or a biosensor for screening a strain with high basic amino acid production;

[0071] (d) screening proteins or protein encoding genes related to basic amino acid synthesis, or preparing reagents, kits or biosensors for screening proteins or protein encoding genes related to basic amino acid synthesis;

[0072] Optionally, the basic amino acid is histidine, lysine or arginine.

[0073] (14) A method for regulating the expression of a target gene, wherein the method comprises the step of operably linking the polynucleotide according to (7) to a transcription initiation sequence and a target gene; preferably, the transcription initiation sequence is a polynucleotide having LysG-regulated promoter activity. Optionally, the target gene comprises at least one of a gene encoding a protein related to basic amino acid synthesis, a gene encoding a gene expression regulating protein, and a gene encoding a protein related to membrane transport.

[0074] (15) A method for producing a basic amino acid, wherein the method comprises the step of producing the basic amino acid in the presence of the mutant of the transcriptional regulatory factor LysG according to any one of (1) to (6), the polynucleotide according to (7), the nucleic acid construct according to (8), or the recombinant expression vector according to (9); or the method comprises the step of producing the basic amino acid using the recombinant host cell according to any one of (10) to (11);

[0075] Optionally, the method further comprises the step of isolating or purifying the basic amino acid.

[0076] (16) A method for screening strains with high production of basic amino acids, wherein the method comprises the step of screening strains with high production of basic amino acids using a mutant of the transcriptional regulatory factor LysG according to any one of (1) to (6), a polynucleotide according to (7), a nucleic acid construct according to (8), a recombinant expression vector according to (9), a recombinant host cell according to any one of (10) to (11), or a biosensor according to (12).

[0077] (17) A method for screening proteins or protein encoding genes related to basic amino acid synthesis, wherein the method comprises the step of using a mutant of the transcriptional regulatory factor LysG according to any one of (1) to (6), a polynucleotide according to (7), a nucleic acid construct according to (8), a recombinant expression vector according to (9), a recombinant host cell according to any one of (10) to (11), or a biosensor according to (12) to screen proteins or protein encoding genes related to basic amino acid synthesis.

[0078] Effects of the Invention

[0079] In some embodiments, the mutant of the transcriptional regulatory factor LysG provided in the present disclosure is a mutant obtained by mutation of the Linker Helix region of the transcriptional regulatory factor LysG. Compared with the wild-type transcriptional regulatory factor LysG, the intensity of the mutant's response to the induction of basic amino acids is significantly improved; the mutant is used to screen for high-yield strains of basic amino acids and mutations of proteins and protein-coding genes related to basic amino acid synthesis, which can effectively improve the screening sensitivity of high-yield strains of basic amino acids or mutants of proteins or protein-coding genes related to basic amino acid synthesis, and is of great significance for the construction of high-yield strains of basic amino acids and high-throughput screening of strains.

[0080] In some embodiments, the mutant of the transcriptional regulatory factor LysG provided by the present disclosure has an enhanced intensity and sensitivity in responding to basic amino acids, and the mutant has an enhanced transcriptional activation effect on the LysG-regulated promoter. The mutant of LysG and the promoter regulated by it can be used to activate the expression of key genes in the synthesis pathway, construct a high-yield amino acid strain, and effectively increase the yield of the target amino acid.

[0081] In some embodiments, the polynucleotides, nucleic acid constructs, and recombinant expression vectors provided by the present disclosure can express mutants of the transcriptional regulatory factor LysG with increased basic amino acid response intensity, which can be used for high-throughput screening of basic amino acid high-producing strains, or for increasing the production of amino acids.

[0082] In some embodiments, the recombinant host cells provided by the present disclosure, comprising mutants of the transcriptional regulatory factor LysG, or polynucleotides, nucleic acid constructs, and recombinant expression vectors encoding the mutants, can effectively increase the yield and conversion rate of the target amino acid, and realize large-scale industrial production of the target amino acid.

[0083] In some embodiments, the biosensor provided by the present disclosure has a mutant of the transcriptional regulatory factor LysG with improved alkaline amino acid response intensity. On the one hand, the biosensor can screen for high-yield strains of alkaline amino acids or improve the intensity of protein encoding genes related to alkaline amino acid synthesis, thereby enabling high-throughput industrial screening. On the other hand, it can also be used for expression regulation of key genes in the alkaline amino acid synthesis metabolic pathway to construct efficient high-yield strains of alkaline amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Shown is the dimer crystal structure of LysG and a schematic diagram of its Linker Helix region.

[0085] Figure 2 The results of flow cytometry analysis of the LysG Linker Helix library are shown, wherein A: control strain 13032 (pTRCmob); B: control strain 13032 (pLysWT); C: library strain 13032 (pLysWT mut ).

[0086] Figure 3 The comparison results of the induced response fluorescence values ​​of LysG and its mutants to three basic amino acid dipeptides are shown, wherein A: comparison of the induced response fluorescence values ​​of LysG and its mutants to lysine-alanine dipeptide; B: comparison of the induced response fluorescence values ​​of LysG and its mutants to arginine-alanine dipeptide; C: comparison of the induced response fluorescence values ​​of LysG and its mutants to histidine-alanine dipeptide.

[0087] Figure 4The comparison of fluorescence values ​​of pLysWT and pLysJL48 lysine biosensors in response to different lysine production strains is shown. A: Control: ZPCgL1 (pTRCmob + pXMJ19); pXMJ19: ZPCgL1 (pLysWT + pXMJ19); PEPC-WT: ZPCgL1 (pLysWT + pZPW54); PEPC-D299N: ZPCgL1 (pLysWT + pZPW55); PEPC-N917G: ZPCgL1 (pLysWT + pZPW56); B: Control: ZPCgL1 (pTRCmob + pXMJ19); pXMJ19: ZPCgL1 (pLysWT + pXMJ19); PEPC-WT: ZPCgL1 (pLysWT + pZPW54); PEPC-D299N: ZPCgL1 (pLysWT + pZPW55); PEPC-N917G: ZPCgL1 (pLysWT + pZPW56); mob+pXMJ19); pXMJ19: ZPCgL1(pLysJL48+pXMJ19); PEPC-WT: ZPCgL1(pLysJL48+pZPW54); PEPC-D299N: ZPCgL1(pLysJL48+pZPW55); PEPC-N917G: ZPCgL1(pLysWT+pZPW56); C: Extracellular lysine production of different strains after 36h of fermentation; D: Average fluorescence signal of different strains in response. DETAILED DESCRIPTION

[0088] definition

[0089] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0090] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0091] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0092] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or the alternatives are mutually exclusive.

[0093] When used in the claims or specification, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0094] As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0095] 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 is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms.

[0096] As used in the present disclosure, the term "transcriptional regulatory factor" is a class of proteins that can control the transcription of DNA into RNA, i.e., the first stage of gene expression. It regulates the transcription process of the target gene by specifically binding to the DNA sequence of the regulatory region. In some embodiments, the transcriptional regulatory factor is the transcriptional regulatory factor LysG, which belongs to the typical LysR family of transcriptional regulatory factors, and has a Helix-turn-helix motif (HTH) that binds to DNA at its N-terminus, and an effector binding region (Effector binding domain, EBD) at its C-terminus, connected in the middle by a flexible Linker Helix. Exemplarily, the DNA sequence of the regulatory region bound to LysG is the promoter sequence of the gene lysE encoding the alkaline amino acid transporter LysE, or any other type of promoter sequence whose transcription process is regulated by LysG.

[0097] As used in the present disclosure, the term "response intensity" refers to the level of transcription of a transcriptional regulatory factor initiating transcription of a target gene under the induction of an amino acid. In some embodiments, by connecting a fluorescent reporter gene downstream of a transcriptional regulatory factor and detecting the intensity of the fluorescent signal under the induction of a specific amino acid, the response intensity of the transcriptional regulatory factor induced by the specific amino acid is characterized.

[0098] As used in the present disclosure, the term "LysG regulated promoter" refers to a promoter that can bind to LysG and whose transcriptional activity is regulated by LysG. In some embodiments, the LysG regulated promoter is not specifically limited, as long as its activity is regulated by LysG. Exemplarily, the LysG regulated promoter can be a wild-type lysE promoter, also includes a promoter that is still regulated by LysG after the lysE promoter is modified, and also includes an unknown or unidentified promoter that is regulated by LysG.

[0099] The transcriptional regulatory factor LysG in the present disclosure is not specifically limited, as long as it has corresponding transcriptional regulatory activity. In some embodiments, LysG derives from the microorganism of Corynebacterium, and further, LysG derives from Corynebacterium glutamicum. Exemplary, the LysG deriving from Corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO:1, or compared with the amino acid sequence shown in SEQ ID NO:1, there is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (including all ranges and percentages between these numerical values) sequence.

[0100] As used in the present disclosure, the term "mutant" refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying a position.

[0101] As used in the present disclosure, the term "mutated amino acid" includes "substitution, duplication, deletion or addition of one or more amino acids". In the present disclosure, the term "mutation" refers to a change in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0102] The mutant of the transcriptional regulatory factor LysG in the present disclosure is a mutant obtained by mutating the Linker Helix region of the transcriptional regulatory factor LysG, and the mutant has an increased response intensity to basic amino acids compared to the wild-type transcriptional regulatory factor LysG. Further, the mutant of the transcriptional regulatory factor LysG has a mutated amino acid at one or more positions corresponding to positions 57-89 of the sequence shown in SEQ ID NO: 1, and the above positions belong to the Linker Helix region of the transcriptional regulatory factor LysG. After mutating one or more amino acids therein, the mutant has the transcriptional regulatory activity of the transcriptional regulatory factor LysG, and the intensity of the induced response to basic amino acids (lysine, histidine or arginine) is significantly improved. In some embodiments, the mutant has a substituted amino acid at any one or more positions corresponding to positions 57-89 of the sequence shown in SEQ ID NO: 1.

[0103] In the present disclosure, "mutation" may also include the addition, deletion or substitution of amino acids that do not affect the activity of the transcriptional regulator at one or several positions corresponding to the sequence shown in SEQ ID NO: 1. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-important regions, will not substantially change the biological activity. For example, the sequence obtained by appropriately replacing, adding or deleting certain amino acids will not affect its activity. Exemplarily, the "mutation" of the present disclosure includes the deletion or addition of at least one amino acid residue at at least one of the C-terminus and the N-terminus of the polypeptide corresponding to the sequence shown in SEQ ID NO: 1, and the polypeptide has the activity of a transcriptional regulator. In some embodiments, the "mutation" of the present disclosure corresponds to the deletion or addition of 1-15 amino acids, preferably 1-10, more preferably 1-6, from the N-terminus or C-terminus of the polypeptide of the sequence shown in SEQ ID NO: 1, and has the activity of a transcriptional regulator.

[0104] In some embodiments, the "mutation" of the present disclosure may be selected from "conservative mutations". In the present disclosure, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0105] As used in the present disclosure, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged 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).

[0106] As used in the present disclosure, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions may be conservative. Exemplary substitutions that are considered conservative substitutions include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, substitutions of Asp to Asn, Glu or Gln, substitutions of Cys to Ser or Ala, substitutions of Gln to Asn, Glu, Lys, His, Asp or Arg, substitutions of Glu to Gly, Asn, Gln, Lys or Asp, substitutions of Gly to Pro, substitutions of His to Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0107] As used in the present disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0108] As used in the present disclosure, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0109] In some embodiments, two or more sequences or subsequences have a "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (e.g., polynucleotides).

[0110] The term "corresponding to" as used herein has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, with respect to "the amino acid residue corresponding to position 150 of the amino acid sequence shown in SEQ ID NO. 1", if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO. 1, then the position corresponding to position 150 of the amino acid sequence shown in SEQ ID NO. 1 in the resulting mutant may be position 156.

[0111] As used in this disclosure, the term "expression" includes any step involved in RNA production and protein production including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0112] As used in the present disclosure, the term "nucleic acid construct" is a recombinant expression element comprising a polynucleotide with a coding transcription factor mutant polypeptide. In some embodiments, the promoter of a lysE gene is also included in the nucleic acid construct. The polynucleotide of the coding transcription factor mutant polypeptide and the promoter of the lysE gene are "operably connected", refer to the polynucleotide of the coding transcription factor mutant polypeptide and the promoter of the lysE gene are functionally connected, to start and mediate the transcription of the target gene, and the mode operably connected can adopt any mode described by those skilled in the art.

[0113] As used in the present disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence to express a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding 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 that is transcribed into mRNA and translated into a protein; and iii) a transcription subunit that is suitable for transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. In the present disclosure, "recombinant expression vector" and "recombinant vector" can be used interchangeably.

[0114] The term "protein coding gene" in this disclosure refers to a DNA molecule that can guide the synthesis of proteins through certain rules. The process by which protein coding genes guide protein synthesis generally includes a transcription process using double-stranded DNA as a template and a translation process using mRNA as a template. Protein coding genes contain a CDS sequence (Coding Sequence) that can guide the production of mRNA encoding proteins.

[0115] Exemplarily, the protein encoding gene is a gene encoding a protein related to basic amino acid synthesis.

[0116] Exemplarily, the protein encoding gene can be a pyruvate carboxylase gene, a phosphoenolpyruvate carboxylase gene, a γ-glutamyl kinase gene, a glutamate semialdehyde dehydrogenase gene, a pyrroline-5-carboxylic acid reductase gene, an amino acid transport protein gene, a ptsG system-related gene, a pyruvate dehydrogenase gene, a homoserine dehydrogenase gene, an oxaloacetate decarboxylase gene, a gluconate repressor protein gene, a glucose dehydrogenase gene, an aspartate kinase gene, an aspartate semialdehyde dehydrogenase gene, an aspartate ammonia lyase gene, a dihydrodipicolinate synthetase gene, a dihydropicolinate reductase gene, a succinyldiaminopimelate aminotransferase gene, a tetrahydrodipicolinate succinylase gene, a succinyldiamino Pimelate deacylase gene, diaminopimelate epimerase gene, diaminopimelate deacylase gene, glyceraldehyde-3-phosphate dehydrogenase gene, transketolase gene, diaminopimelate dehydrogenase gene, N-acetylglutamyl phosphate reductase gene, ornithine acetyltransferase gene, N-acetylglutamate kinase gene, acetylornithine transaminase gene, ornithine carbamoyltransferase gene, argininosuccinate synthetase gene, argininosuccinate lyase gene and carbamoyl phosphate synthetase gene, glyceraldehyde-3-phosphate dehydrogenase gene, isocitrate dehydrogenase gene, malate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, glucose-6-phosphate isomerase gene and pyridine nucleotide transhydrogenase gene.

[0117] The term "host cell" in the present disclosure means any cell type that is susceptible to the use of a mutant of the transcriptional regulatory factor LysG disclosed herein, or a polynucleotide, a nucleic acid construct or a recombinant expression vector encoding the mutant. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the introduction of a polynucleotide, a nucleic acid construct or a recombinant expression vector, and the recombinant host cell is specifically achieved by transformation.

[0118] The term "transformation" in this disclosure has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl 2 ) precipitation method, microinjection method, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

[0119] Host cell of the present disclosure can be a prokaryotic cell, as long as it can comprise the mutant of the transcriptional regulatory factor LysG of the present disclosure, or the cell of the polynucleotide, nucleic acid construct or recombinant expression vector comprising the encoding mutant. In some embodiments, host cell refers to the microorganism derived from the suitable fermentation production of amino acid, including but not limited to the bacterial strain that can include Escherichia (Escherichia), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providencia), Enterobacteria (Enterobacteria), Salmonella (Salmonella), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Brevibacterium (Brevibacterium), Corynebacterium (Corynebacterium) etc. In some embodiments, host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067 bacterial strain or any of the above-mentioned derivative strains. In some embodiments, the host cell in the present disclosure may also be other types of strains capable of producing amino acids, including wild-type strains and recombinant strains.

[0120] For example, the host cell is a host cell for producing Methionin. In some embodiments, for the host cell for producing Methionin, it can be a bacterial strain expressing the aspartokinase that releases feedback inhibition on the basis of Corynebacterium glutamicum ATCC 13032. In addition, the host cell for producing Methionin can also be a bacterial strain of other types with Methionin production capacity.

[0121] In some embodiments, the lysine-producing host cell may also include, but is not limited to, one or more genes selected from the following being attenuated or having reduced expression:

[0122] a. adhE gene encoding alcohol dehydrogenase;

[0123] b. ackA gene encoding acetate kinase;

[0124] c. pta gene encoding phosphate acetyltransferase;

[0125] d. ldhA gene encoding lactate dehydrogenase;

[0126] e. focA gene encoding formate transporter;

[0127] f. pflB gene encoding pyruvate formate lyase;

[0128] g. poxB gene encoding pyruvate oxidase;

[0129] h. thrA gene encoding the bifunctional enzyme aspartate kinase I / homoserine dehydrogenase I;

[0130] i. thrB gene encoding homoserine kinase;

[0131] j. ldcC gene encoding lysine decarboxylase; and

[0132] h. cadA gene encoding lysine decarboxylase.

[0133] In some embodiments, the lysine-producing host cell may also include, but is not limited to, one or more genes selected from the following being enhanced or overexpressed:

[0134] a. dapA gene encoding dihydrodipyridine synthase that relieves lysine feedback inhibition;

[0135] b. dapB gene encoding dihydrodipicolinate reductase;

[0136] c. ddh gene encoding diaminopimelate dehydrogenase;

[0137] d. dapD encoding tetrahydropicolinate succinylase and dapE encoding succinyl diaminopimelate deacylase;

[0138] e. asd gene encoding aspartate-semialdehyde dehydrogenase;

[0139] f. ppc gene encoding phosphoenolpyruvate carboxylase;

[0140] g. pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase;

[0141] i. The lysE gene encodes the lysine transport protein.

[0142] In some embodiments, the host cell producing arginine may include, but is not limited to, one or more genes selected from the following being enhanced or overexpressed:

[0143] a) gene encoding N-acetylglutamyl phosphate reductase argC;

[0144] b) the gene encoding ornithine acetyltransferase argJ;

[0145] c) gene encoding N-acetylglutamate kinase argB;

[0146] d) gene encoding argD, an acetyl-ornithine aminotransferase;

[0147] e) the gene encoding ornithine carbamoyltransferase argF;

[0148] f) a gene encoding argininosuccinate synthetase argG;

[0149] g) a gene encoding argininosuccinate lyase argH;

[0150] h) A gene encoding carbamoyl-phosphate synthetase.

[0151] In some embodiments, the arginine-producing host cell may further include inactivation of a gene encoding an arginine repressor regulating argR.

[0152] In some embodiments, the host cell producing histidine may include, but is not limited to, one or more genes selected from the following being enhanced or overexpressed:

[0153] a) encoding the hisEG gene and hisDCB gene of the L-histidine biosynthesis operon;

[0154] b) zwf-opcA gene encoding 6-phosphate glucose dehydrogenase;

[0155] c) the gene encoding the PRPP synthase prsA;

[0156] In some embodiments, the host cell for producing histidine may further include reducing the expression of 6-phosphoglucose isomerase pgi gene in the starting bacteria.

[0157] The host cells disclosed herein can be cultured according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0158] The term "amino acid" or "L-amino acid" of the present disclosure refers to the basic building block of a protein in which an amino group and a carboxyl group are bound to the same carbon atom. Exemplarily, the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, 5-aminolevulinic acid, or a derivative of any of the above amino acids. In addition, the amino acid may also be other types of amino acids in the art. In a specific embodiment of the present disclosure, the basic amino acids are lysine, histidine, and arginine.

[0159] The term "culture" disclosed herein can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0160] Unless otherwise defined in the disclosure or clearly indicated by the context, all technical and scientific terms in the disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0161] Mutants of the transcriptional regulator LysG

[0162] The present invention discloses a random mutation of the Linker Helix region of LysG (amino acid 57 to amino acid 89) to obtain a mutant library of the Linker Helix region of LysG, and finds that the mutants can greatly improve the induced response intensity of the biosensor to lysine, histidine and arginine, and the mutants can be used as biosensors for the screening of high-yield strains of basic amino acids; in addition, the mutants and the lysE gene promoter regulated by them can be further used to enhance the expression of key genes in the amino acid synthesis pathway and improve the synthesis and yield of amino acids.

[0163] In some embodiments, the mutant has a mutated amino acid at one or more positions corresponding to the sequence shown in SEQ ID NO: 1, and the mutant has the transcriptional regulatory activity of the transcriptional regulatory factor LysG. The present disclosure found that after the amino acid mutation at the above position, the mutant significantly increased the intensity of response to lysine, histidine and arginine compared with the wild-type polypeptide of the sequence shown in SEQ ID NO: 1, which can improve the sensitivity, dynamic range and operation of the biosensor for screening high-yield strains of basic amino acids or key genes in the basic amino acid synthesis pathway.

[0164] In some more specific embodiments, the mutant has a substituted amino acid at any one or more positions corresponding to 57-89 of the sequence shown in SEQ ID NO: 1, and the amino acid substitution can increase the intensity of the induced response to basic amino acids while maintaining the transcriptional regulatory activity of LysG.

[0165] In some embodiments, the mutant corresponds to the sequence shown in SEQ ID NO: 1, and has a mutated amino acid at any one or more of the following positions: 58, 59, 61, 64, 65, 66, 67, 70, 73, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 88, and 89.

[0166] The present disclosure has found that mutations of the amino acid residues at the above-mentioned positions can increase the induction response strength of LysG to basic amino acids to varying degrees.

[0167] In some more specific embodiments, the mutant has a substituted amino acid at one or more of the above positions. The present disclosure screened the mutant library and found that the substitution of the amino acid at the above position has a positive effect on improving the induction responsiveness to basic amino acids.

[0168] In some preferred embodiments, the mutant has a mutated amino acid at at least one, at least two or at least three positions corresponding to the sequence shown in SEQ ID NO: 1. Exemplarily, the mutant comprises a mutation at one, two, three, four, five, etc. positions corresponding to positions 58, 59, 61, 64, 65, 66, 67, 70, 73, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 88 and 89 of the sequence shown in SEQ ID NO: 1.

[0169] In some specific embodiments, the mutant has an amino acid substitution at any one of positions 58, 59, 61, 64, 65, 66, 67, 70, 73, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 88 and 89 of the sequence shown in SEQ ID NO: 1.

[0170] In some specific embodiments, the mutant has amino acid substitutions at both positions 59 and 80 of the sequence shown in SEQ ID NO:1.

[0171] In some specific embodiments, the mutant has an amino acid substitution at positions 61 and 78 corresponding to the sequence shown in SEQ ID NO:1.

[0172] In some specific embodiments, the mutant has an amino acid substitution at positions 64 and 87 corresponding to the sequence shown in SEQ ID NO:1.

[0173] In some specific embodiments, the mutant has an amino acid substitution at positions 67 and 79 corresponding to the sequence shown in SEQ ID NO:1.

[0174] In some specific embodiments, the mutant has an amino acid substitution at position 67 and position 77 corresponding to the sequence shown in SEQ ID NO:1.

[0175] In some specific embodiments, the mutant has amino acid substitutions at positions 77, 78 and 83 corresponding to the sequence shown in SEQ ID NO:1.

[0176] In some specific embodiments, the mutant has amino acid substitutions at positions 77, 79 and 83 corresponding to the sequence shown in SEQ ID NO:1.

[0177] In some specific embodiments, the mutant has amino acid substitutions at positions 77, 80 and 84 corresponding to the sequence shown in SEQ ID NO:1.

[0178] In some preferred embodiments, the mutant corresponds to the sequence shown in SEQ ID NO: 1 and has the following characteristics: 1 )-(a 22 ) at any one position in the group consisting of, or at any two or more positions:

[0179] (a 1 )E58V;

[0180] (a 2 )A59S;

[0181] (a 3 )E61K or E61V;

[0182] (a 4 )V64A;

[0183] (a 5 )Q65R;

[0184] (a 6 )A66T;

[0185] (a 7 )A67T or A67S;

[0186] (a 8 )A70I;

[0187] (a 9 )L73I;

[0188] (a 10 )A75G;

[0189] (a 11 )E76D, E76C, E76K or E76W;

[0190] (a 12 )T77A;

[0191] (a 13 )K78E or K78R;

[0192] (a 14 )A79V, A79T or A79R;

[0193] (a 15 )Q80R or Q80K;

[0194] (a 16 )G83R;

[0195] (a 17 )R84H;

[0196] (a 18 ) L85P, L85A, L85D, L85G, L85A, L85S, L85T or L85Y;

[0197] (a 19 )A86R or A86D;

[0198] (a 20 )E87G, E87D, E87R, E87N, E87H, E87K or E87S;

[0199] (a 21 )I88V, I88T, I88A, I88N, I88G, I88H, I88K, I88P or I88S;

[0200] (a 22 )P89Q.

[0201] In some preferred embodiments, the mutant corresponds to the sequence shown in SEQ ID NO: 1, having (a 1 )-(a 22 ) at any one position, at any two positions, or at any three positions in the group consisting of ). After the above-mentioned amino acid mutation occurs, the mutant of the transcriptional regulatory factor LysG can be used to construct a biosensor with improved detection sensitivity to achieve high-throughput screening of strains with high production of basic amino acids; or to increase the expression of key genes in the basic amino acid synthesis pathway to improve the synthesis efficiency and yield of basic amino acids.

[0202] In some specific embodiments, the mutant corresponds to the sequence shown in SEQ ID NO: 1 and has the following characteristics (m 1 )-(m 51 ) in any one of the mutated amino acids:

[0203] (m 1 )E58V;(m 2 )A59S and Q80R; (m 3 )E61V;(m 4 )E61K and K78E;

[0204] (m5 )V64A and E87D; (m 6 )Q65R;(m 7 )A66T;(m 8 )A67T and A79V

[0205] (m 9 )A67T;(m 10 )A67S and T77A; (m 11 )M70I;(m 12 )L73I;(m 13 )L73V;

[0206] (m 14 )A75G;(m 15 )E76D;(m 16 )E76C;(m 17 )E76K;(m 18 )E76W

[0207] (m 19 )T77A, K78R and G83R; (m 20 )T77A;(m 21 )A79V;(m 22 )A79R;

[0208] (m 23 )A79T and G83R; (m 24 )Q80R;(m 25 )Q80K and R84H; (m 26 )L85P;

[0209] (m 27 )L85A;(m 28 )L85D;(m 29 )L85G;(m 30 )L85M;(m 31 )L85S;

[0210] (m 32 )L85T;(m 33 )L85Y;(m 34 )A86R;(m 35 )A86D;(m 36 )E87G;

[0211] (m 37 )E87R;(m 38 )E87N;(m 39 )E87H;(m 40 )E87K;(m 41 )E87S;

[0212] (m 42 )I88V;(m 43 )I88T;(m 44 )I88A;(m 45 )I88N;(m 46 )I88G;

[0213] (m 47 )I88H;(m 48 )I88K;(m 49 )I88P;(m 50 )I88S;(m 51 )P89Q.

[0214] After the above-mentioned amino acid mutation occurs, the mutant of the transcriptional regulatory factor LysG can be used to construct a biosensor with improved detection sensitivity to achieve high-throughput screening of high-yield strains of basic amino acids; or to increase the expression of key genes in the basic amino acid synthesis pathway to improve the synthesis efficiency and yield of basic amino acids.

[0215] In some embodiments, after at least one of the N-terminus and the C-terminus of the mutant of the transcription regulatory factor LysG is deleted or added with at least one amino acid, preferably 1-15 amino acids, preferably 1-10, and more preferably 1-6 amino acids, it has the transcription regulatory factor activity of the transcription regulatory factor LysG and an increased response intensity to basic amino acids.

[0216] In some embodiments, the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with any of the above-mentioned mutants of the transcriptional regulatory factor LysG, and the amino acid sequence is not the sequence shown in SEQ ID NO: 1, and has the transcriptional regulatory factor activity of the transcriptional regulatory factor LysG, and an increased response intensity to basic amino acids.

[0217] In some embodiments, the mutants of the transcriptional regulatory factor LysG disclosed herein have 4.58 times, 2.68 times, 2.74 times, 3.15 times, 2.65 times, 3.17 times, 1.94 times, 1.85 times, 1.89 times, 2.22 times, 2.43 times, 2.8 times, 2.85 times, 3.62 times, 3.28 times, 2.07 times, 2.47 times, 3.02 times, 2.51 times, 2.55 times, 2.91 times, 3.04 times, 2.13 times, 2.57 times, 2.41 times, 2.8 times, 2.08 times, 1.78 times, compared to the polypeptide of the sequence shown in SEQ ID NO: 1. , 1.4 times, 1.97 times, 1.51 times, 3.14 times, 2.01 times, 3.1 times, 2.52 times, 3.04 times, 3.07 times, 3.18 times, 2.89 times, 3.06 times, 2.91 times, 1.87 times, 2.81 times, 3.19 times, 2.95 times, 3.18 times, 2.87 times, 2.8 times, 2.79 times, 2.63 times, 2.15 times, 2.1 times, 2.41 times, 2.03 times, 2.28 times, and 1.89 times the induced response intensity of basic amino acids.

[0218] In some embodiments, the mutant of the transcriptional regulatory factor LysG is encoded by a polynucleotide. The present disclosure does not specifically limit the sequence of the polynucleotide, as long as it can encode any of the above-mentioned polypeptides having transcriptional regulatory factor activity.

[0219] Construction of mutant library and mutant screening

[0220] In some embodiments, the present invention uses an artificially synthesized eyfp gene (GeneBank No.AM774338.1) as a template, and uses eyfp-F and eyfp-R as primers to amplify the eyfp gene fragment; uses the genome of Corynebacterium glutamicum 13032 as a template, uses lysGE-F / lysGE-R as primers, and PCR amplifies fragments of the promoter regions of lysG and lysE genes; uses pTRCmob as a template, and uses pTRCmob-rev-F / R as primers to PCR amplify to obtain a plasmid backbone; and connects the above fragments by homologous recombination to obtain a recombinant expression vector pLysWT.

[0221] In some embodiments, the present invention uses an expression vector pLysWT containing a lysine biosensor as a template, and uses random mutation primers LysG-mut-F and LysG-mut-R to obtain coding gene fragments of Linker Helix regions with different mutation rates through error-prone PCR amplification; using pLysWT as a template, the Linker Helix region of LysG is removed by PCR reverse amplification to obtain a linearized vector fragment; and the above fragments are homologously recombined to obtain a recombinant expression vector containing gene fragments with different mutation rates.

[0222] In some embodiments, the recombinant expression vector is transformed into a host cell to obtain a mutant strain library. In some more specific embodiments, the host cell is a competent cell of C. glutamicum ATCC 13032. In other embodiments, the host cell may also be other cell types.

[0223] In some embodiments, the present disclosure transforms the vectors pTRCmob and pLysWT into ATCC 13032 strains, respectively, to obtain control recombinant host cells 13032 (pTRCmob) and 13032 (pLysWT). The recombinant host cells are cultured in the presence of basic amino acids (e.g., lysine), and then the strain library is fluorescently sorted to obtain target recombinant host cells with higher fluorescence intensity than the control recombinant host cells, which contain mutants of the target transcriptional regulatory factor LysG.

[0224] In some embodiments, the present disclosure selects some sites in the Linker Helix region for saturation mutation, designs saturation mutation library primers, and uses specific amino acid site primers NNS (N represents four bases A / T / C / G, and S represents two bases C / G) on the primers. Using the vector pLysWT containing the lysine biosensor as a template, reverse amplification is performed using the saturation mutation library primers to obtain a saturation mutant library plasmid.

[0225] In some embodiments, the saturation mutant library plasmid is transformed into a host cell to obtain a saturation mutant strain library. In some more specific embodiments, the host cell is a competent cell of C. glutamicum ATCC 13032. In other embodiments, the host cell may also be other cell types.

[0226] In some embodiments, the present disclosure transforms the vectors pTRCmob and pLysWT into ATCC 13032 strains, respectively, to obtain control recombinant host cells 13032 (pTRCmob) and 13032 (pLysWT). In the presence of basic amino acids (e.g., lysine), the saturated mutant recombinant host cells are cultured, and then the strain library is fluorescently sorted to obtain target recombinant host cells with higher fluorescence intensity than the control recombinant host cells, which contain mutants of the target transcriptional regulatory factor LysG.

[0227] Biosensors

[0228] The principle of the biosensor is as follows: when the concentration of the basic amino acid (e.g., lysine) as an inducer is low, the transcriptional regulatory factor LysG cannot bind to the amino acid. When the basic amino acid reaches a certain concentration, the transcriptional regulatory factor LysG binds to the basic amino acid (e.g., lysine) as an inducer and can regulate the lysE gene promoter. Therefore, if a reporter gene such as a fluorescent protein is connected downstream of the lysE gene promoter, the expression of the downstream reporter gene can be activated, and the concentration of the basic amino acid can be evaluated by the intensity of the fluorescent signal of the reporter gene, thereby screening for amino acid high-yield strains.

[0229] In some embodiments, the present disclosure utilizes mutants of the transcriptional regulatory factor LysG to construct biosensors. The mutants have greatly improved induced response intensity. Therefore, the fluorescence intensity of the reporter gene expression is significantly improved, which greatly improves the detection sensitivity of the biosensor.

[0230] In some embodiments, the biosensor is used to screen strains with high production of basic amino acids, or to screen protein coding genes related to the synthesis of basic amino acids. Wherein, the strains with high production of basic amino acids are strains with high production of lysine, arginine or histidine. The biosensor in the present disclosure can achieve high-throughput screening of strains with high production of basic amino acids.

[0231] In some preferred embodiments, the basic amino acid high-producing strain is a lysine high-producing strain.

[0232] In some embodiments, the biosensor is cultured under the following culture medium conditions: CGXII culture medium, composed of: 50 g / L glucose, 2 g / L yeast extract, 16.5 g / L NH 4 Cl, 5g / L urea, 1g / L KH 2 PO 4 , 1g / LK 2 HPO 4 , 42g / L MOPS, 0.25g / L MgSO4 , 0.01g / L FeSO 4 ·2H 2 O, 0.01 g / L MnSO 4 ·H 2 O, 0.001g / L ZnSO 4 7H 2 O, 0.2mg / L CuSO 4 , 0.02mg / L NiCl 2 6H 2 O, 0.01 g / L CaCl 2 , 0.03g / Lprotocatechuic acid, 0.2mg / L biotin, 0.1mg / L vitamin B1. The culture medium contained 25μg / mL kanamycin and different concentrations of lysine-alanine, arginine-alanine or histidine-alanine dipeptides (0mM, 0.5mM, 1.0mM, 1.5mM, 2mM, 4mM, 6mM and 8mM). The culture was carried out at 30℃ and 800rpm for 10h. After dilution to a certain multiple, the fluorescence value and OD of the bacterial solution were detected using an enzyme-linked labeling instrument (SpectraMax M5, Molecular Devices, λexcitation=488nm, λemission=520nm). 600 .

[0233] Production process of target amino acids

[0234] (1) A gene encoding a mutant of the transcriptional regulatory factor LysG is operably linked to a LysG-regulated promoter and a gene encoding a protein related to the synthesis of a target amino acid to obtain a recombinant expression vector capable of expressing a protein related to the synthesis of the target amino acid, and a host cell is transformed with the recombinant expression vector to obtain a recombinant host cell.

[0235] (2) Fermenting the recombinant host cells, collecting the target compound from the recombinant host cells or the culture fluid of the recombinant host cells, and completing the production process of the target compound.

[0236] In the above production process, since the mutant of the transcription regulatory factor LysG has an increased induction intensity, its transcriptional regulatory effect on the LysG-regulated promoter is enhanced, thereby increasing the expression level of protein-coding genes related to the synthesis of the target amino acid, thereby significantly increasing the yield of the target compound.

[0237] In some embodiments, the LysG-regulated promoter is a lysE gene promoter; in other embodiments, the gene encoding a mutant of the transcriptional regulatory factor LysG can also be connected to other types of promoters that initiate transcription under the transcriptional regulation of LysG.

[0238] In some embodiments, the target amino acid is an L-amino acid, and the protein encoding gene related to the synthesis of the amino acid refers to a protein encoding gene related to the synthesis of the L-amino acid.

[0239] Exemplarily, the target amino acid includes one or a combination of two or more of the following: lysine, arginine, histidine or a derivative of any of the above amino acids.

[0240] Exemplarily, the protein encoding genes related to the synthesis of amino acids include genes encoding one or a combination of two or more of the following enzymes: pyruvate carboxylase, phosphoenolpyruvate carboxylase, γ-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylic acid reductase, amino acid transport protein, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconate repressor, glucose dehydrogenase, aspartate kinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthetase, dihydropicolinate reductase, succinyl diaminopimelate aminotransferase, tetrahydrodipicolinate succinyltransferase, enzyme, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, diaminopimelate dehydrogenase, N-acetylglutamyl phosphate reductase, ornithine acetyltransferase, N-acetylglutamate kinase, acetylornithine aminotransferase, ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase and carbamoyl phosphate synthetase, glyceraldehyde-3-phosphate dehydrogenase gene, isocitrate dehydrogenase gene, malate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, glucose-6-phosphate isomerase gene and pyridine nucleotide transhydrogenase gene.

[0241] In some preferred embodiments, the target amino acid is lysine.

[0242] In some specific embodiments, the host cell is Corynebacterium glutamicum, which is an important strain for producing target compounds such as amino acids and organic acids.

[0243] In some specific embodiments, the host cell is a modified Corynebacterium glutamicum as follows: the threonine at position 311 of aspartate kinase (encoded by lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 is mutated to isoleucine.

[0244] In some specific embodiments, the target amino acid is lysine, and the lysine fermentation medium composition is: 80 g / L glucose, 8 g / L yeast powder, 9 g / L urea, 1.5 g / L K 2 HPO 4 , 0.01g / L MnSO 4 , 0.6 g / L MgSO 4 , 0.01g / LFeSO 4 , 42 g / L MOPS, pH 7.2.

[0245] In some specific embodiments, the target compound can be recovered from the recombinant host cell or the culture medium of the recombinant cell by methods commonly used in the art, including but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.

[0246] In the art, methods for manipulating microorganisms are known, as explained in publications such as Current Methods in Molecular Biology (Online ISBN: 9780471142720, John Wiley and Sons, Inc.), Microbial Metabolic Engineering: Methods and Procedures (Qiong Cheng Ed., Springer), and Systems Metabolic Engineering: Methods and Procedures (Hal S. Alper Ed., Springer).

[0247] Example

[0248] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

[0249] The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0250] Example 1. Construction of Linker Helix Random Mutation Library of LysG and Flow Cytometry Sorting

[0251] According to the crystal structure reported in the literature (Della Corte, D., et al. (2020) Engineering and application of a biosensor with focused ligand specificity. Nature Communications, 11.), we first found the Linker Helix region, which is located at positions 57-89 of the LysG protein amino acid sequence (such as Figure 1 shown).

[0252] (1) Construction of lysine biosensor expression vector pLysWT

[0253] According to the reported eyfp gene sequence information (GeneBank No.AM774338.1), the whole gene was synthesized, and the eyfp gene fragment was obtained by PCR amplification using eyfp-F and eyfp-R primers. The genome of Corynebacterium glutamicum 13032 was used as a template, and lysGE-F / lysGE-R was used as primers to PCR amplify the fragments of the promoter regions of lysG and lysE genes. Using pTRCmob as a template and pTRCmob-rev-F / R as primers, PCR amplification was used to obtain the plasmid backbone. After purification and recovery, the above fragments were recombined and ligated to obtain the recombinant vector pLysWT.

[0254] (2) Construction of the LysG Linker Helix Random Mutation Library

[0255] According to the protein crystal structure of LysG (PDB: 6XTU), its Linker Helix region (from amino acid 57 to amino acid 89) was locked, and the random mutation primer LysG-mut-F / R was designed for this region. The vector pLysWT containing lysine biosensor was used as a template, and 0.05mM to 0.5mM MnCl was added. 2, error-prone PCR amplification to obtain linker helix fragments with different mutation rates; at the same time, primers LysG-rev-F / R were used, and the lysine biosensor vector pLysWT was used as a template to reversely amplify the linker helix to remove LysG. After Dpn I treatment to remove the template plasmid, the above two fragments were connected using recombinase, chemically transformed into E. coli DH5α, and cultured at 37°C. The next day, the colonies were scraped from the plate, and plasmids with different mutation rates were extracted. After the plasmids with different mutation rates were mixed in an equimolar ratio, they were transformed into competent cells of strain C. glutamicum ATCC13032 by electric pulse. After rescreening at 30°C for 2h, the resuscitation solution was transferred to TSB liquid culture medium containing 25μg / mL kanamycin. After culturing at 30°C for 16h, the library strain 13032 (pLysWT mut ).

[0256] Example 2. LysG mutant library flow cytometric sorting

[0257] According to the response principle of the lysine biosensor, when the intracellular lysine concentration is low, the regulatory protein LysG cannot bind to lysine, and thus cannot initiate the transcription of the fluorescent protein gene by regulating the lysE promoter; when the intracellular lysine reaches a certain concentration, the regulatory protein LysG binds to lysine, causing the conformation of LysG to change, and then binds to the promoter of LysE to initiate the expression of the downstream reporter gene yellow fluorescent protein. Different LysG mutants have different induction response intensities to lysine, and thus different expression intensities of fluorescent proteins. Based on this principle, we screened the random mutation library of the LinkerHelix region of LysG. First, the negative control strains 13032 (pTRCmob), 13032 (pLysWT), and the library strains 13032 (pLysWT mut ) were inoculated with TSB liquid medium containing 25 μg / mL kanamycin and cultured at 30°C for 8 h. Then, the cells were transferred to CGXⅡY medium (50 g / L glucose, 2 g / L yeast extract, 16.5 g / L NH 4 Cl,5g / L urea,1g / L KH 2 PO 4 ,1g / LK 2 HPO 4 ,42g / L MOPS,0.25g / L MgSO 4 ,0.01g / L FeSO 4 ·2H 2 O,0.01g / L MnSO 4 ·H 2O,0.001g / L ZnSO 4 7H 2 O,0.2mg / L CuSO 4 ,0.02mg / LNiCl 2 6H 2 O,0.01g / L CaCl 2 , 0.03g / L protocatechuic acid, 0.2mg / L biotin, and 0.1mg / L vitamin B1), cultured at 30℃ for 6h, and then diluted to 0.1OD using PBS buffer 600 The fluorescence intensity of the above strains was analyzed by flow cytometry. The analysis results are shown in Figure 2. Taking 13032 (pLysWT) as a reference, the flow cytometer sorting area of ​​the library strain was selected. The library strain 13032 (pLysWT mut ) contained 0.12% of cells with enhanced fluorescence. This region may contain mutants with enhanced response to lysine induction, so we sorted the cells in this region.

[0258] Example 3. LysG mutant library rescreening

[0259] 88 single clones were randomly picked from the single-cell colonies obtained by flow cytometry screening in Example 2, and inoculated into a 96-well plate containing 200 μL TSB liquid culture medium (containing 25 μg / mL kanamycin) using a toothpick. Control strains 13032 (pTRCmob) and 13032 (pLysWT) were also inoculated and cultured in a well plate shaker at 30°C and 800 rpm for 8 h. The seed solution was transferred to a 96-well plate containing 200 μL CGXⅡY medium (added with 25 μg / mL kanamycin) at 5% (V / V), and 0 M or 0.8 M lysine was added at the same time. After culturing at 30°C and 800 rpm for 12 h in a well plate shaker, the bacterial solution was diluted 20 times with PBS buffer, and the fluorescence intensity and OD of eYFP were detected using an enzyme-linked label reader (SpectraMax M5, Molecular Devices, λexcitation=488nm, λemission=520nm). 600The mutants with stronger fluorescence values ​​than the control strain 13032 (pLysWT) were sequenced and analyzed for mutation sites. The results are shown in Table 1. The amino acid residues from E58 to P89 are mutated, and the mutations can increase the induction response intensity of LysG to lysine to varying degrees. Among them, E58 mutated to V, which increased the induction response intensity the most, and the mutation sites basically covered the entire Linker Helix region (Figure 1). This result shows that mutations in the Linker Helix region of LysG can greatly increase the induction response intensity of the lysine biosensor to lysine.

[0260] Table 1 Summary of mutant information of Linker Helix region of LysG

[0261]

[0262]

[0263] a = fluorescence value of LysG mutant sensor upon induction / fluorescence value of LysG wild-type biosensor upon induction;

[0264] b = fluorescence value of the LysG mutant sensor when induced / fluorescence value of the LysG mutant sensor when not induced.

[0265] Example 4 Construction and screening of amino acid saturation mutant library of LysG Linker Helix region

[0266] (1) Construction of amino acid saturation mutant library of LysG linker helix region

[0267] Some sites were selected for saturation mutation to verify that mutation of the Linker Helix region to different amino acids also has an enhanced effect. According to the sequence information of pLysWT, the saturation mutation library primers L73-F / L73-R, A75-F / A75-R, E76-F / E76-R, A79-F / A79-R, L85-F / L85-R, A86-F / A86-R, E87-F / E87-R and I88-F / I88-R were designed for amino acid residues at positions L73, A75, E76, A79, L85, A86, E87 and I88, respectively. The primer NNS (N represents four bases A / T / C / G, and S represents two bases C / G) at specific amino acid sites on the primers was used. The full-length plasmid, Dpn I treated the vector fragment PCR product to remove the template plasmid, and after recovery, the saturation mutant library PCR products of different sites were mixed in an equimolar ratio and transformed into E. coli DH5α competent cells. The next day, the library plasmid was extracted and electroporated into C. glutamicum 13032 to obtain the saturation mutant library strain C. glutamicum 13032 (pLysWT Sat-mut ).

[0268] (2) Flow cytometric sorting of the mutant library of amino acid saturation mutants in the linker helix region of LysG

[0269] Using the same screening principle as in Example 2, negative control strains 13032 (pTRCmob), 13032 (pLysWT) and library strains 13032 (pLysWT) were firstly isolated. Sat-mut ) were inoculated with TSB liquid medium containing 25 μg / mL kanamycin and cultured at 30°C for 8 h. Then, the cells were transferred to CGXⅡY medium (50 g / L glucose, 2 g / L yeast extract, 16.5 g / L NH 4 Cl,5g / L urea,1g / L KH 2 PO 4 ,1g / LK 2 HPO 4 ,42g / L MOPS,0.25g / LMgSO 4 ,0.01g / LFeSO 4 ·2H 2 O,0.01g / L MnSO 4 ·H 2 O,0.001g / L ZnSO 4 7H 2 O,0.2mg / LCuSO4 ,0.02mg / LNiCl 2 6H 2 O,0.01g / L CaCl 2 , 0.03g / L protocatechuic acid, 0.2mg / L biotin, and 0.1mg / L vitamin B1), cultured at 30℃ for 6h, and then diluted to 0.1OD using PBS buffer 600 The fluorescence intensity of the above strains was analyzed by flow cytometry, and the part of the library with a stronger fluorescence value than the control strain 13032 (pLysWT) was sorted.

[0270] (3) LysG mutation library rescreening

[0271] 94 single clones were randomly picked from the single-cell colonies obtained by flow cytometry screening, and inoculated into a 96-well plate containing 200 μL TSB liquid culture medium (containing 25 μg / mL kanamycin) using a toothpick. Control strains 13032 (pTRCmob) and 13032 (pLysWT) were also inoculated and cultured in a well plate shaker at 30°C and 800 rpm for 8 h. The seed solution was transferred to a 96-well plate containing 200 μL CGXⅡY medium (added with 25 μg / mL kanamycin) at 5% (V / V), and 0 M or 0.8 M lysine was added at the same time. After culturing at 30°C and 800 rpm for 12 h in a well plate shaker, the bacterial solution was diluted 20 times with PBS buffer, and the fluorescence intensity and OD of eYFP were detected using an enzyme-linked label reader (SpectraMax M5, Molecular Devices, λexcitation=488nm, λemission=520nm). 600 The mutants with stronger fluorescence values ​​than the control strain 13032 (pLysWT) were sequenced and analyzed for mutation sites. The results are shown in Table 2. Except for the L75 amino acid, which was screened to a mutant, the other L73, E76, A79, L85, A86, E87 and I88 amino acid residues were all screened to have mutants mutated into different amino acids, and all of them helped to improve the fluorescence intensity of the response, especially the L85 and I88 amino acids were mutated into 8 and 9 different amino acids, respectively.

[0272] Table 2 Summary of LysG mutant information

[0273]

[0274]

[0275] a = fluorescence value of LysG mutant sensor upon induction / fluorescence value of LysG wild-type biosensor upon induction;

[0276] b = fluorescence value of the LysG mutant sensor when induced / fluorescence value of the LysG mutant sensor when not induced.

[0277] Example 5. Response of LysG mutants to histidine and arginine

[0278] To test the response of LysG LH mutants to the induction of three basic amino acids, strains 13032 (pTRCmob), 13032 (pLysWT), and 13032 (pLysWT) were firstly isolated. Glu58Val ) and 13032(pLysWT Gln80Arg ) were inoculated into a 24-well plate containing 1 mL TSB (containing 25 μg / mL kanamycin) and cultured at 30°C and 800 rpm for 8 h. The seed solution was transferred to a 96-well plate containing 200 μL CGXⅡ (containing 25 μg / mL kanamycin) according to the initial OD 0.5, and 0 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2 mM, 4 mM, 6 mM and 8 mM lysine-alanine, arginine-alanine or histidine-alanine dipeptide were added, respectively. After culture at 30°C and 800 rpm for 10 h, the solution was diluted to a certain multiple and the fluorescence value and OD of the bacterial solution were detected using an enzyme-linked label reader (SpectraMax M5, Molecular Devices, λexcitation=488 nm, λemission=520 nm). 600 The test results are shown in Figure 3. It can be seen that for mutants with increased lysine-induced response intensity, the induced response to arginine and histidine is also significantly increased.

[0279] Example 6. Application of LysG Linker Helix mutant biosensor in screening of amino acid high-producing bacteria

[0280] (1) Construction of pepc gene expression vector

[0281] To detect LysG Linker Helix mutations, the biosensor pLysJL48 (pLysWT E58V) Whether the pLysWT biosensor has screening advantages over the wild-type biosensor. First, we constructed several strains with different lysine production. It has been reported that overexpression of phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum that relieves feedback inhibition of L-aspartate and malate can increase lysine production. Through literature mining, we found two mutants of PEPC, namely D299N (Yokota, A., et al. (2017) Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum. Advances in biochemical engineering / biotechnology, 159, 181-198.) and N917G (Chen, Z., et al. (2014) Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.Applied and environmentalmicrobiology,80,1388-1393.), overexpression of these two mutants increased lysine production by 15% and 37%, respectively.

[0282] According to the published genome sequence of Corynebacterium glutamicum ATCC13032, primers pepc-tac-F / R were designed, and pepc gene was obtained by PCR amplification using ATCC13032 genome as template. In addition, primers pXMJ19-rev-2F / 2R were designed, and plasmid pXMJ19 was used as template to obtain linearized vector fragment by reverse PCR amplification. The PCR product of vector fragment was treated with Dpn I to remove the template plasmid. After recovery, the above two fragments were connected by recombinase and chemically transformed into E.coli DH5α competent cells to obtain the expression vector pXMJ19-pepc of pepc gene, which was named pZPW54.

[0283] (2) Construction of pepc gene mutation vector

[0284] pepc D299N or pepc N917G Expression vector construction, design primers D299N-F / R (or N917G-F / R), use plasmid pZPW54 as template, reverse amplify the full-length plasmid, treat the vector fragment PCR product with Dpn I to remove the template plasmid, and then transfer it into E.coli DH5α competent cells to obtain pepc D299N or pepc N917G The expression vectors were named pZPW55 and pZPW56, respectively.

[0285] (3) Construction of genomic pepc gene knockout strain

[0286] According to the method for constructing lysine strains disclosed in the literature (Becker, J., Zelder, O., S., H.and Wittmann, C. (2011) From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng., 13, 159-168.), using homologous recombination technology based on pK18mobsacB, the threonine at position 311 of aspartate kinase (lysC gene encoding) on ​​the genome of Corynebacterium glutamicum ATCC13032 was mutated to isoleucine, and a strain SCgL30 with a certain lysine synthesis ability was constructed. At the same time, in order to exclude the mutual interference between the genomic expressed PEPC and the overexpressed PEPC mutant on the vector, we used the CRISPR-Cas9 knockout method reported in the literature (Liu, J., et al. (2017) Development of a CRISPR / Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbial cell factories,16,205.), the pepc gene of SCgL30 was knocked out, and the knockout process was as follows: first, the knockout plasmid pgRNA-Δpepc was constructed, and primers pepc-up-F / R and pepc-down-F / R were designed according to the published genome sequence of Corynebacterium glutamicum ATCC13032, and the upstream and downstream homologous arm fragments for the pepc gene were obtained by PCR amplification using the ATCC13032 genome as a template; at the same time, pgRNA2 was used as a template, and primers pepc-pgRNA-2 / pepc-pgRNA-3 were used to PCR amplify backbone fragment 1; pgRNA2 was used as a template, and gRNA-pepc / pepc-pgRNA-1 was used as primers to amplify backbone fragment 2 with pepc-sgRNA (N20, 5′-CGTGCGGGATCAGCGGACGA-3′). After the above fragments were purified and recovered, the above four fragments were connected using a recombinase to obtain the pepc gene knockout plasmid pgRNA-Δpepc.

[0287] The plasmids pCas9 and pgRNA-Δpepc were co-transformed into competent cells of strain SCgL30 by the electric pulse method, and 1 mL of TSB medium preheated at 46°C was added. The cells were incubated at 46°C for 6 min and 30°C for 3 h. TSB solid medium containing 50 μM IPTG + 25 μg / mL kanamycin + 5 μg / mL chloramphenicol was applied and cultured at 30°C for 2 days to obtain recombinant transformants. The transformants were verified using the verification primers pepc-TF / TR to confirm that the target gene on the chromosome of the strain was successfully knocked out. Then, the plasmid was lost by high temperature at 37°C to obtain the gene knockout strain SCgL30 (Δpepc), named ZPCgL1.

[0288] (4) Application of LysG mutants in screening of high-lysine-producing strains

[0289] First, pLysWT or pLysJL48 (pLysWT E58V ) were used to transform the ZPCgL1 strain to obtain the recombinant strains ZPCgL1(pLysWT) and ZPCgL1(pLysJL48). Then, the expression vectors pXMJ19, pZPW54, pZPW55 and pZPW56 were used to transform the above-mentioned recombinant strains, respectively, to obtain the test strains ZPCgL1(pTRCmob+pXMJ19), ZPCgL1(pLysWT+pXMJ19), ZPCgL1(pLysWT+pZPW54), ZPCgL1(pLysWT+pZPW55) and ZPCgL1(pLysWT+pZPW56) as well as ZPCgL1(pLysJL48+pXMJ19), ZPCgL1(pLysJL48+pZPW54), ZPCgL1(pLysJL48+pZPW55) and ZPCgL1(pLysJL48+pZPW56).

[0290] In order to test the induction response of the LysG Linker Helix mutant biosensor pLysJL48 to different lysine production strains relative to the wild-type biosensor pLysWT, the above test strains were first inoculated into a 24-well plate containing 1 mL TSB (added with 25 μg / mL kanamycin and 5 μg / mL chloramphenicol), and cultured in a well plate shaker at 30°C and 800 rpm for 8 h. 600 0.5, transferred to a 24-well plate containing 1 mL of CGXⅡ medium, and cultured in a well plate shaker at 30°C and 800 rpm. The composition of CGXⅡY medium is: 80 g / L glucose, 16.5 g / L NH 4 Cl,5g / L urea,1g / L KH 2 PO 4 ,1g / LK2 HPO 4 ,42g / L MOPS,0.25g / L MgSO 4 ,0.01g / L FeSO 4 ·2H 2 O,0.01g / LMnSO 4 ·H 2 O,0.001g / LZnSO 4 7H 2 O,0.2mg / L CuSO 4 ,0.02mg / L NiCl 2 6H 2 O,0.01g / LCaCl 2 ,0.03g / Lprotocatechuic acid,0.2mg / L biotin,and 0.1mg / L vitamin B1,and add 25μMIPTG,25μg / mL kanamycin and 5μg / mL chloramphenicol. After 6h of culture, detect the OD of the bacterial solution 600 PBS buffer was used to dilute the bacterial cell concentration to 0.1OD 600 The response fluorescence intensity of these strains was analyzed by flow cytometry. The analysis results are as follows: Figure 4 As shown. Figure A shows the flow cytometry analysis results of ZPCgL1 (pLysWT) strains transformed with empty plasmid pXMJ19, pepc-WT and pepc mutant expression vectors; Figure B shows the flow cytometry analysis results of ZPCgL1 (pLysJL48) strains transformed with empty plasmid pXMJ19, pepc-WT and pepc mutant expression vectors; Figure C shows the extracellular lysine production of different strains, and Figure D shows the fluorescence data of different strains. It can be seen from the data that there are certain differences in the lysine production of different strains. As the lysine production increases, the intracellular fluorescence signal gradually increases. Compared with the wild-type LysG, the mutated response system not only enhances the fluorescence signal intensity in the same strain, but also amplifies the signal difference intensity of different lysine-producing strains, which is more conducive to the rapid screening of high-yield strains.

[0291] Example 7. Construction of a lysG gene mutation vector for the chromosome of Corynebacterium glutamicum

[0292] According to the published genome sequence of Corynebacterium glutamicum ATCC13032, primers lysG-up-F, lysG-A66T / L73I / E76D-F, lysG-A66T / L73I / E76D-R, lysG-down-R were designed respectively, and the ATCC13032 genome was used as a template to obtain upstream and downstream homology arm fragments for lysG mutation (wherein the lysG gene A66T / L73I / E76D mutants were respectively) by PCR amplification. In addition, primers lysG-up-F / R and lysG-down-F / R were designed, and the upstream and downstream homology arm fragments for lysG mutation were obtained by PCR amplification using plasmid JL-32 (i.e., pLysWT Glu61Val ) was used as a template to amplify the upstream and downstream homology arm fragments of the E61V mutant, and the plasmid JL-33 (i.e., pLysWT GLn80Arg ) was used as a template to amplify the upstream and downstream homology arm fragments of the Q80R mutant, and the plasmid C2-80 (i.e., pLysWT Leu85Ser ) was used as a template to amplify the upstream and downstream homology arm fragments of the L85S mutant, and the plasmid JL-47 (i.e., pLysWT GLu87Gly ) was used as a template to amplify the upstream and downstream homology arm fragments of the E87G mutant, and the plasmid C1-57 (pLysWT Ile88Ala ) was used as a template to amplify the upstream and downstream homology arm fragments of the I88A mutant. After the above PCR fragment was recovered, it was recombined with the pK18mobsacB vector treated with EcoR I and BamH I to obtain the mutant vector pK18-lysG-A66T / L73I / E76D / E61V / Q80R / L85S / E87G / I88A of the lysG gene.

[0293] Example 8. Construction of lysG gene mutant strain of Corynebacterium glutamicum

[0294] Because wild-type Corynebacterium glutamicum cannot produce lysine, and after Corynebacterium glutamicum introduces point mutations of lysC, pyc and hom, lysine can be produced. The present embodiment takes ATCC 13032 as the starting strain, first constructs a strain producing lysine, namely, introduces T311I point mutation (releasing feedback inhibition of enzyme) into the aspartate kinase gene lysC of Corynebacterium glutamicum ATCC 13032 strain, introduces P458S point mutation (releasing feedback inhibition of enzyme) into the pyruvate carboxylation kinase gene pyc, introduces V59A point mutation (weakening enzyme activity) into the homoserine dehydrogenase gene hom, obtains lysine high-yielding strain AHP-3 (Becker, J., Zelder, O., S., H. and Wittmann, C. (2011) From zero to hero--design-based systems metabolic engineering of Corynebacteriumglutamicum for L-lysine production. Metab. Eng., 13, 159-168..). The competent cells were prepared by the method reported in the literature (Ruan Y, Zhu L, Li Q. Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett. 2015; 37: 2445-2452.), and the competent cells of Corynebacterium glutamicum AHP-3 were obtained. 1 μg of the above-mentioned mutation vector was electrotransformed into the AHP-3 competent cells prepared above, 1 mL of TSB medium preheated at 46° C. was added, incubated at 46° C. for 6 min, incubated at 30° C. for 3 h, coated with TSB solid medium containing 25 μg / mL kanamycin, and cultured at 30° C. for 2 days to obtain the first recombinant transformant. (TSB medium composition (g / L): glucose, 5g / L; yeast powder, 5g / L; soy peptone, 9g / L; urea, 3g / L; succinic acid, 0.5g / L; K 2 HPO 4 ·3H 2 O, 1 g / L; MgSO 4 7H 2 O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L) correct transformants were transferred to TSB medium for overnight culture, and then transferred to TSB medium containing 100 g / L sucrose. After culture at 30°C for 6 h, the strains were spread on TSB medium supplemented with 100 g / L sucrose for screening, and the lysG mutant strains SCgL55-SCgL62 were obtained. The corresponding LysG mutants were E61V, A66T, L73I, E76D, Q80R, L85S, E87G and I88A, respectively.

[0295] Example 9. Effect of lysG gene mutant of Corynebacterium glutamicum on L-lysine synthesis

[0296] In order to test the effect of lysG gene mutant of Corynebacterium glutamicum on lysine production, fermentation tests were carried out on SCgL55-SCgL62 strains. The wild-type AHP-3 strain was used as a control, and the fermentation medium composition was: glucose 80g / L, yeast powder 8g / L, urea 9g / L, K 2 HPO 4 1.5g / L, MOPS 42g / L, FeSO 4 0.01g / L, MnSO 4 0.01g / L, MgSO 4 0.6 g / L, pH 7.2. The strain was first inoculated into TSB liquid medium and cultured for 8 h. The culture was inoculated as seeds into a 24-well plate containing 800 μL of fermentation medium per well. The initial OD 600 The control was about 0.1, cultured at 30°C for 21 hours, the rotation speed of the well plate shaker was 800 rpm, and each strain was replicated three times. After the fermentation, the L-lysine production and glucose consumption were detected, and the sugar-acid conversion rate from glucose to lysine was calculated.

[0297] The results are shown in Table 2. The L-lysine yields were higher than those of the wild-type control strain, and the sugar-acid conversion rates were improved. It can be seen that these amino acid site mutations have good application prospects in the production of L-lysine and its derivatives.

[0298] Table 2

[0299] strain Mutants <![CDATA[OD 600 ]]> L-Lysine(g / L) Sugar-acid conversion rate (g / g, %) AHP-3 WT 12.38±0.65 1.78±0.02 2.70±0.04 SXWh E61V 12.10±0.37 2.38±0.08 3.64±0.09 SXWh A66T 12.12±0.36 1.93±0.04 2.97±0.05 SXWh L73I 12.02±0.39 2.03±0.03 3.09±0.04 SX8 E76D 12.66±0.22 2.01±0.05 3.12±0.02 SXWh Q80R 12.16±0.31 2.05±0.05 3.19±0.08 SCgL60 L85S 12.35±0.17 2.00±0.00 3.15±0.00 SXWh E87G 12.63±0.22 2.03±0.03 3.16±0.00 SXWh I88A 12.45±0.35 2.08±0.03 3.34±0.07

[0300] The primers used in the examples of this disclosure are shown in Table 3 below:

[0301]

[0302]

[0303]

[0304] All technical features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can also be replaced by other features with the same, equal or similar functions. Therefore, unless otherwise specified, each feature disclosed is only an example of a series of equal or similar features.

[0305] In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from this disclosure, and many modifications can be made to the invention to adapt to various different purposes and conditions of use without departing from the spirit and scope of the present disclosure. Therefore, such modifications are also intended to fall within the scope of the appended claims. Sequence Listing <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A mutant of transcriptional regulatory factor LysG and its application <130> 6A17-2173364I <160> 55 <170> SIPOSequenceListing 1.0 <210> 1 <211> 290 <212> PRT <213> Corynebacterium glutamicum <400> 1 Met Asn Pro Ile Gln Leu Asp Thr Leu Leu Ser Ile Ile Asp Glu Gly 1 5 10 15 Ser Phe Glu Gly Ala Ser Leu Ala Leu Ser Ile Ser Pro Ser Ala Val 20 25 30 Ser Gln Arg Val Lys Ala Leu Glu His His Val Gly Arg Val Leu Val 35 40 45 Ser Arg Thr Gln Pro Ala Lys Ala Thr Glu Ala Gly Glu Val Leu Val 50 55 60 Gln Ala Ala Arg Lys Met Val Leu Leu Gln Ala Glu Thr Lys Ala Gln 65 70 75 80 Leu Ser Gly Arg Leu Ala Glu Ile Pro Leu Thr Ile Ala Ile Asn Ala 85 90 95 Asp Ser Leu Ser Thr Trp Phe Pro Pro Val Phe Asn Glu Val Ala Ser 100 105 110 Trp Gly Gly Ala Thr Leu Thr Leu Arg Leu Glu Asp Glu Ala His Thr 115 120 125 Leu Ser Leu Leu Arg Arg Gly Asp Val Leu Gly Ala Val Thr Arg Glu 130 135 140 Ala Asn Pro Val Ala Gly Cys Glu Val Val Glu Leu Gly Thr Met Arg 145 150 155 160 His Leu Ala Ile Ala Thr Pro Ser Leu Arg Asp Ala Tyr Met Val Asp 165 170 175 Gly Lys Leu Asp Trp Ala Ala Met Pro Val Leu Arg Phe Gly Pro Lys 180 185 190 Asp Val Leu Gln Asp Arg Asp Leu Asp Gly Arg Val Asp Gly Pro Val 195 200 205 Gly Arg Arg Arg Val Ser Ile Val Pro Ser Ala Glu Gly Phe Gly Glu 210 215 220 Ala Ile Arg Arg Gly Leu Gly Trp Gly Leu Leu Pro Glu Thr Gln Ala 225 230 235 240 Ala Pro Met Leu Lys Ala Gly Glu Val Ile Leu Leu Asp Glu Ile Pro 245 250 255 Ile Asp Thr Pro Met Tyr Trp Gln Arg Trp Arg Leu Glu Ser Arg Ser 260 265 270 Leu Ala Arg Leu Thr Asp Ala Val Val Asp Ala Ala Ile Glu Gly Leu 275 280 285 Arg Pro 290 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 2 gcccttagtg actcgaattc ctaaggccgc aatccctc 38 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 3 tcttaaagtt catctattac ggtccgatgg acagtaaaag a 41 <210> 4 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 4 gtaatagatg aactttaaga aggagatata catatggtga gcaagggcga g 51 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 5 cagcggccgc tactagttta ttacttgtac agctcgtcca tg 42 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 6 taaactagta gcggccgctg 20 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 7 gaattcgagt cactaagggc ta 22 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 8 cccttagtga ctcgaattcc ta 22 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 9 taaagctctc gagcatcacg tg 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 10 cgtgatgctc gagagcttta 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 11 ggaattcgag tcactaaggg 20 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 12 tttctgcttg snncaacacc attttccgcg ctgctt 36 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 13 aatggtgttg nnscaagcag aaactaaagc gcaactatct gga 43 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 14 ctttagtttc snnttgcagc aacaccattt tccgc 35 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 15 gttgctgcaa nnsgaaacta aagcgcaact atctggacgc c 41 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 16 gcgctttagt snntgcttgc agcaacacca ttttcc 36 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 17 gctgcaagca nnsactaaag cgcaactatc tggacgcc 38 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 18 cagatagttg snntttagtt tctgcttgca gcaacacc 38 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 19 agaaactaaa nnscaactat ctggacgcct tgctgaaat 39 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 20 ggatttcagc snngcgtcca gatagttgcg ctttag 36 <210> twenty one <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> twenty one atctggacgc nnsgctgaaa tcccgttaac catcg 35 <210> twenty two <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> twenty two acgggatttc snnaaggcgt ccagatagtt gcgct 35 <210> twenty three <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> twenty three tggacgcctt nnsgaaatcc cgttaaccat cgccatc 37 <210> twenty four <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> twenty four ttaacggggat snnagcaagg cgtccagata gttgcg 36 <210> 25 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 25 acgccttgct nnsatcccgt taaccatcgc catcaac 37 <210> 26 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 26 tggttaacgg snnttcagca aggcgtccag atagttgc 38 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 27 ccttgctgaa nnsccgttaa ccatcgccat caacg 35 <210> 28 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 28 aggtaacgac cacgtagctt gc 22 <210> 29 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 29 aaagagtgtt taaagtagtt tccagccggc tgggtagt 38 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 30 aactacttta aacactcttt cacattgagg 30 <210> 31 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 31 cagttattgg tgcccttcga gaccagagtc cgtcaagaac ttc 43 <210> 32 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 32 cgtgcgggat cagcggacga gttttagagc tagaaatagc aag 43 <210> 33 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 33 cgcttccttt agcagccctt g 21 <210> 34 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 34 gtagaaagcc agtccgcaga aacg 24 <210> 35 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 35 gattccaaac cgagcaccac tgaattacac tgtacctgtt gcgtc 45 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 36 cgagcatgaa gcccatcatt 20 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 37 gacgcagaga agcagatcgt t 21 <210> 38 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 38 actctagagg atccccgggt ac 22 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 39 agtcatatgt atatctcctt cctgcaggca tgcaagctt 39 <210> 40 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 40 aaggagatat acatatgact gattttttac gcgatgac 38 <210> 41 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 41 acccggggat cctctagagt cctttgccgt ttgagaaccc 40 <210> 42 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 42 tgcgctgcgc ggctccggct agtccagccg g 31 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 43 actagccgga gccgcgcagc gcagtggaaa ga 32 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 44 cagcctgtcg aaccgcatga ataaggtcac ccc 33 <210> 45 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 45 tattcatgcg gttcgacagg ctgagctcat gct 33 <210> 46 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 46 gagctcggta cccggggatc ccaacagcct tgattcactc aacaac 46 <210> 47 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 47 atgctcgaga gctttaacgc gctgactcac cgccga 36 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 48 ttccgcgctg tttgcacaaggacttcacc 29 <210> 49 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 49 tctgcttgga tcaacaccat tttccgcgc 29 <210> 50 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 50 gctttagtgt ctgcttgcag caacaccat 29 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 51 ttgtgcaaac agcgcggaaa atggtgttg 29 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 52 tggtgttgat ccaagcagaa actaaagcg 29 <210> 53 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 53 tgcaagcaga cactaaagcg caactatct 29 <210> 54 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 54 tcggcggtga gtcagcgcgt taaagctctc gagcatcacg t 41 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer sequences <400> 55 cgacggccag tgccaagctt ctaaggccgc aatccctcg 39

Claims

1. A mutant of a transcriptional regulatory factor LysG, wherein the mutant has one or more mutated amino acids located in the Linker Helix region; and compared with the wild-type transcriptional regulatory factor LysG, the mutant has an increased lysine response intensity; the mutant is subjected to the following steps (m1)-(m2) corresponding to the sequence shown in SEQ ID NO: 51 ) in any one of the mutated amino acids: (m1) E58V; (m2) A59S and Q80R; (m3) E61V; (m4) E61K and K78E; (m5) V64A and E87D; (m6) Q65R; (m7) A66T; (m8) A67T and A79V (m9)A67T; (m 10 )A67S and T77A; (m 11 )M70I;(m 12 )L73I;(m 13 )L73V; (m 14 )A75G;(m 15 )E76D;(m 16 )E76C;(m 17 )E76K;(m 18 )E76W; (m 19 )T77A, K78R and G83R; (m 20 )T77A;(m 21 )A79V;(m 22 )A79R; (m 23 )A79T and G83R; (m 24 )Q80R;(m 25 )Q80K and R84H; (m 26 )L85P; (m 27 )L85A;(m 28 )L85D;(m 29 )L85G;(m 30 )L85M;(m 31 )L85S; (m 32 )L85T;(m 33 )L85Y;(m 34 )A86R;(m 35 )A86D;(m 36 )E87G; (m 37 )E87R;(m 38 )E87N;(m 39 )E87H;(m 40 )E87K;(m 41 )E87S; (m 42 )I88V;(m 43 )I88T;(m 44 )I88A;(m 45 )I88N;(m 46 )I88G; (m 47 )I88H;(m 48 )I88K;(m 49 )I88P;(m 50 )I88S;(m 51 )P89Q。 2. The mutant of the transcriptional regulatory factor LysG according to claim 1, wherein Compared with the wild-type transcriptional regulatory factor LysG, the sequence shown in SEQ ID NO: 1 was modified by (m1) E58V or (m 24 ) The mutants with the mutated amino acid shown in Q80R also have improved histidine and arginine response strengths.

3. An isolated polynucleotide, wherein: The polynucleotide encodes the mutant of the transcriptional regulatory factor LysG as described in claim 1 or 2.

4. A nucleic acid construct, wherein: The nucleic acid construct comprises the polynucleotide of claim 3, wherein the polynucleotide is operably linked to a LysG-regulated promoter, and the LysG-regulated promoter directs the expression of the polypeptide.

5. A recombinant expression vector, wherein: The recombinant expression vector comprises the polynucleotide according to claim 3, or the nucleic acid construct according to claim 4.

6. A recombinant host cell, wherein The recombinant host cell comprises the mutant of the transcriptional regulatory factor LysG according to claim 1 or 2, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, or the recombinant expression vector according to claim 5.

7. The recombinant host cell according to claim 6, wherein The host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium or Corynebacterium.

8. The recombinant host cell according to claim 7, wherein The host cell is Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869 or Corynebacterium glutamicum ATCC14067 strain.

9. A biosensor, wherein: The biosensor comprises a mutant of the transcriptional regulatory factor LysG as described in claim 1 or 2, or a polynucleotide as described in claim 3, a nucleic acid construct as described in claim 4, a recombinant expression vector as described in claim 5, or a polypeptide expressed by a recombinant host cell as described in any one of claims 6-8.

10. The mutant of the transcriptional regulatory factor LysG according to claim 1 or 2, Use of the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the recombinant expression vector according to claim 5, or the recombinant host cell according to any one of claims 6 to 8 in at least one of the following (a) to (d): (a) regulating the transcription level of a target gene, or preparing a reagent or kit for regulating the transcription level of a target gene; (b) producing lysine, or preparing a reagent or kit for producing lysine; (c) screening for high-lysine-producing strains, or preparing reagents, kits or biosensors for screening high-lysine-producing strains; (d) Screening for proteins or protein encoding genes related to lysine synthesis, or preparing reagents, kits or biosensors for screening for proteins or protein encoding genes related to lysine synthesis.

11. A method for regulating the expression of a target gene, wherein: The method comprises the step of operably linking the polynucleotide according to claim 3 to a transcription initiation sequence and a target gene.

12. The method according to claim 11, wherein: The transcription initiation sequence is a polynucleotide having LysG-regulated promoter activity.

13. The method according to claim 11, wherein: The target gene includes at least one of a gene encoding a protein related to lysine synthesis, a gene encoding a gene expression regulatory protein, and a gene encoding a protein related to membrane transport.

14. A method for producing lysine, wherein: The method comprises the following steps: The step of producing lysine in the presence of the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, or the recombinant expression vector according to claim 5; or the method comprises the step of producing lysine using the recombinant host cell according to any one of claims 6 to 8.

15. The method according to claim 14, wherein: The method further comprises the step of isolating or purifying the lysine.

16. A method for screening a lysine high-yielding strain, wherein: The method comprises using the mutant of the transcriptional regulatory factor LysG according to claim 1 or 2, The polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to any one of claims 6 to 8, or the step of using the biosensor to screen a strain with high lysine production according to claim 9.

17. A method for screening proteins or protein encoding genes related to lysine synthesis, wherein: The method comprises using the mutant of the transcriptional regulatory factor LysG according to claim 1 or 2, The polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to any one of claims 6 to 8, or the biosensor according to claim 9, comprising the step of screening proteins or protein encoding genes related to lysine synthesis.

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