Modified strong promoter, strain containing same and application of modified strong promoter and strain in amino acid production

Abstract

The invention discloses a modified strong promoter, a strain containing the same and application of the modified strong promoter and the strain in amino acid production. The invention also discloses a promoter core region of the strong promoter, an expression cassette containing the strong promoter, a recombinant expression vector containing the strong promoter, a construction method of the strain, and a method for enhancing target gene expression and preparing target protein. A sequence of a core region of the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The strong promoter screened by the invention is helpful for modifying a strain and improving the amino acid production capacity of the strain: the activities of the promoters eftu30, eftu5 or eftu24 are respectively 12, 1.3 or 1.1 times of the activities of the original eftu; the arginine yield of a strain replaced by a single eftu30 promoter, a single eftu5 promoter or a single eftu24 promoter is respectively increased by 27.2%, 1.2% or 3.2% compared with that of a strain replaced by an eftu promoter; compared with a strain replaced by a double-eftu promoter, the strain replaced by the double-eftu promoter is improved by 29.0%, 9.38% or 7.31% respectively, and the strain replaced by the double-eftu promoter is improved by 29.0%, 9.38% or 7.31% respectively.

Description

A modified strong promoter, a strain containing the same, and their use in amino acid production Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a modified strong promoter, a strain containing the same, and their use in amino acid production. The present invention also relates to the promoter core region of the strong promoter, an expression cassette and recombinant expression vector containing the same, a method for constructing the strain, and methods for enhancing target gene expression and producing target proteins. Background Art

[0002] L-arginine is a semi-essential amino acid. It is not an essential amino acid for adults and animals, but it is an essential amino acid for infants and young animals. L-arginine has many physiological functions. It participates in the production of urea in the body to prevent ammonia poisoning, acts as a precursor for NO synthesis, thereby lowering blood pressure and enhancing the body's immunity. It can also prevent thrombosis and atherosclerosis. Therefore, arginine is widely used in food, medicine, and animal feed. At present, there are two main methods for producing arginine. One is the hair hydrolysis method, which has abundant raw materials and low prices, but pollutes the environment. The other is the microbial fermentation method, which is green and environmentally friendly and has a relatively simple production process. It is a more popular production method. Germany, Japan, and South Korea were the first to realize large-scale fermentation production of arginine. However, the acid production level of domestic arginine strains is relatively low, which is far behind that of foreign countries.

[0003] The transformation strategies of arginine engineering bacteria mainly include removing feedback regulation, enhancing the synthesis pathway of target products, reducing the metabolic flux of competing pathways, and optimizing the supply of cofactors and intermediates. Among them, enhancing the synthesis pathway of target products is a relatively more common and effective way, by replacing the promoter of key synthases, increasing the copy number of the synthesis pathway gene cluster, etc. For example, the sod promoter P sod Replace the promoter of carbamyl phosphate synthase gene carAB to increase the supply of auxiliary precursor carbamyl phosphate. eftu Replacing the argGH operon promoter increases the activity of argininosuccinate synthase and argininosuccinate lyase, promoting the synthesis of L-arginine from L-citrulline (Park SH, Kim HU, Kim TY, et al. Metabolic engineering of Corynebacterium glutamicum for L-arginine production[J]. Nat Commun, 2014, 5.).

[0004] A promoter is a non-translated sequence that regulates the transcription intensity of a target gene. It can be divided into constitutive promoters and inducible promoters according to whether induction is required. Constitutive promoters do not require induction for expression and can continuously express the target protein as long as the bacteria are in a growing state. Therefore, they have greater advantages in actual production. Currently, more than 50 transcription promoters have been experimentally located in Corynebacterium glutamicum, but there are not many promoters in actual application. The ones that are used and studied more are relatively strong promoters, such as P eftu and P sod The yield of the target product is increased by significantly increasing the expression of key enzyme-encoding genes in the target strain. However, due to the effects of other genetic elements such as the untranslated region (5'UTR) and the transcription initiation region (TIR), the use of strong promoters does not always guarantee higher expression of the target gene. The process of metabolic engineering of bacterial strains still requires appropriate enhancement or weakening of the expression of some genes. Therefore, it is necessary to develop more optional endogenous promoters of different strengths that can be stably expressed in Corynebacterium glutamicum to meet the metabolic engineering requirements of Corynebacterium glutamicum.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the prior art lacks a promoter that can be stably expressed in Corynebacterium glutamicum and has stronger activity, thereby providing a modified strong promoter, a strain containing the same and their use in amino acid production.

[0007] To solve the above technical problems, one of the technical solutions provided by the present invention is: a promoter core region, which comprises replacements at one or more positions 20-25, 27-31, 33-36, 43 and 45-47 of the nucleotide sequence shown in SEQ ID NO:12.

[0008] In a specific embodiment of the present invention, the promoter core region comprises replacements at 2, 3, 4, 5, 10, 14 or 15 positions of the nucleotide sequence shown in SEQ ID NO: 12, 20-25, 27-31, 33-36, 43 and 45-47.

[0009] In a specific embodiment of the present invention, the promoter core region is selected from any one of the following (i) to (iii):

[0010] (i) comprising substitutions at one or more positions 20-21, 23-25, 27-28, 30-31, 33, 35, 43, and 45-46 of the nucleotide sequence shown in SEQ ID NO: 12;

[0011] (ii) comprising substitutions at one or more positions 20-25, 28-29, 31, 33-36, 45, and 47 of the nucleotide sequence as set forth in SEQ ID NO: 12;

[0012] (iii) comprising substitution at one or both of positions 43 and 45 of the sequence as shown in SEQ ID NO: 12.

[0013] In a preferred embodiment of the present invention, the promoter core region has 60% to 98% identity with the nucleotide sequence shown in SEQ ID NO: 12.

[0014] In a more preferred embodiment of the present invention, the promoter core region has at least 68%, at least 70% or at least 95% identity with the nucleotide sequence shown in SEQ ID NO:12.

[0015] As used herein, the term "identity" refers to the matching of sequences between two nucleic acid molecules. When a certain position in the two sequences being compared is occupied by the same base (e.g., a certain position in each of the two DNA molecules is occupied by adenine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100%. For example, if 6 out of 10 positions of the two sequences match, the two sequences have 60% identity. Such an alignment can be performed using disclosed and commercially available alignment algorithms and programs, such as, but not limited to, ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, BLAST, and those skilled in the art can reasonably select and use them. Those skilled in the art can determine the appropriate parameters for aligning sequences, including, for example, any algorithm that achieves a better alignment or optimal comparison over the full length of the compared sequences, and any algorithm that achieves a better alignment or optimal comparison over a portion of the compared sequences.

[0016] In a specific embodiment of the present invention, the promoter comprising the promoter core region has higher promoter activity than the promoter comprising the promoter core region of the sequence shown in SEQ ID NO:12.

[0017] In a specific embodiment of the present invention, the promoter comprising the promoter core region has an enhanced promoter activity greater than 1 times, greater than 1.5 times, greater than 2 times, greater than 5 times, greater than 10 times, and preferably greater than 12 times compared to the promoter comprising the promoter core region of the sequence shown in SEQ ID NO:12.

[0018] In a specific embodiment of the present invention, the promoter core region comprises one or more replacements selected from the following: position 20 of the nucleotide sequence as shown in SEQ ID NO: 12 is replaced by T or G, position 21 is replaced by G or T, position 22 is replaced by G, position 23 is replaced by A, position 24 is replaced by T or C, position 25 is replaced by G or T, position 27 is replaced by T, position 28 is replaced by T or C, position 29 is replaced by A, position 30 is replaced by C, position 31 is replaced by A or C, position 33 is replaced by T, position 34 is replaced by A, position 35 is replaced by C or T, position 36 is replaced by C, position 43 is replaced by C, position 45 is replaced by G or C, position 46 is replaced by C and position 47 is replaced by T.

[0019] In a specific embodiment of the present invention, the promoter core region comprises one or more replacements selected from the following: position 20 of the nucleotide sequence shown in SEQ ID NO: 12 is replaced by T, position 21 is replaced by G, position 23 is replaced by A, position 24 is replaced by T, position 25 is replaced by G, position 27 is replaced by T, position 28 is replaced by T, position 30 is replaced by C, position 31 is replaced by A, position 33 is replaced by T, position 35 is replaced by C, position 43 is replaced by C, position 45 is replaced by G and position 46 is replaced by C.

[0020] In a specific embodiment of the present invention, the promoter core region comprises one or more replacements selected from the following: position 20 of the nucleotide sequence shown in SEQ ID NO: 12 is replaced by G, position 21 is replaced by T, position 22 is replaced by G, position 23 is replaced by A, position 24 is replaced by C, position 25 is replaced by T, position 28 is replaced by C, position 29 is replaced by A, position 31 is replaced by C, position 33 is replaced by T, position 34 is replaced by A, position 35 is replaced by T, position 36 is replaced by C, position 45 is replaced by C and position 47 is replaced by T.

[0021] In a specific embodiment of the present invention, the promoter core region comprises one or two substitutions selected from the following: substitution of position 43 with C and substitution of position 45 with G in the nucleotide sequence shown in SEQ ID NO: 12.

[0022] In a specific embodiment of the present invention, the promoter core region comprises the nucleotide sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0023] In a specific embodiment of the present invention, the nucleotide sequence of the promoter core region is shown as SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0024] In this context, the term "promoter" refers to a nucleic acid molecule typically located upstream of the target gene's coding sequence, providing a recognition site for RNA polymerase and located 5' upstream of the mRNA transcription start site. It is an untranslated nucleic acid sequence that RNA polymerase binds to and initiates transcription of the target gene.

[0025] In the present invention, the "promoter core region" refers to a nucleic acid sequence located on the promoter in prokaryotes, which is the core sequence region that exerts the promoter function, mainly including the ‐35 region, the ‐10 region, the region between the ‐35 region and the ‐10 region, and the transcription start site. The ‐35 region is the recognition site of RNA polymerase, and the ‐10 region is the binding site of RNA polymerase.

[0026] In order to solve the above technical problems, the second technical solution provided by the present invention is: a strong promoter, which comprises the promoter core region as described in one of the technical solutions.

[0027] In a specific embodiment of the present invention, the strong promoter is a variant of the eftu promoter; the eftu promoter is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

[0028] In a specific embodiment of the present invention, the eftu promoter comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0029] In the present invention, the "eftu promoter" refers to the promoter of the gene encoding elongation factor Tu.

[0030] In the present invention, the "variant" of the eftu promoter refers to a nucleotide molecule that contains the promoter core region as described in one of the technical solutions of the present invention and still maintains or even improves the activity of the original eftu promoter. For example, the original eftu promoter can have the promoter core region as described in one of the technical solutions of the present invention through base mutation or direct replacement.

[0031] In a specific embodiment of the present invention, the strong promoter comprises the nucleotide sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0032] In a specific embodiment of the present invention, the nucleotide sequence of the strong promoter is shown as SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0033] Promoters of the present invention can be isolated or prepared using standard molecular biology techniques. For example, suitable primer sequences can be used to isolate promoters of the present invention by PCR. In addition, promoters of the present invention can also be prepared using automatic DNA synthesizers by standard synthesis techniques.

[0034] To solve the above technical problems, the third technical solution provided by the present invention is: an expression cassette, which comprises the promoter core region as described in one of the technical solutions or the strong promoter as described in the second technical solution.

[0035] In the present invention, the term "expression cassette" has the meaning generally understood by those skilled in the art, i.e., an element containing a promoter, a target gene, and capable of expressing the target gene. The target gene is a protein-coding gene operably linked to the strong promoter.

[0036] The strong promoter described in the second technical solution of the present invention can be used to moderately regulate the expression of various target genes to achieve efficient production of target products.

[0037] In a specific embodiment of the present invention, the protein encoding gene is a gene encoding an enzyme for synthesizing amino acids.

[0038] In a specific embodiment of the present invention, the enzymes for synthesizing amino acids include: NAD + kinase (Ppnk), acetylglutamate semialdehyde dehydrogenase (ArgC), ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithide aminotransferase (ArgD), ornithine carbamoyltransferase (ArgF), argininosuccinate synthetase (ArgG), argininosuccinate lyase (ArgH), arginine transporter, carbamyl phosphate synthetase (CarAB), aspartate ammonia lyase (AspA), aspartate aminotransferase (AspB) and glutamate dehydrogenase (GDH). One or a combination of two or more of the following.

[0039] In a specific embodiment of the present invention, the amino acid synthesizing enzyme is derived from Corynebacterium glutamicum, such as Corynebacterium glutamicum H5 with a deposit number of CCTCC NO: M2016609, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

[0040] In a specific embodiment of the present invention, the nucleotide sequence of the gene encoding the carbamyl phosphate synthetase is shown in SEQ ID NO: 19.

[0041] To solve the above technical problems, the fourth technical solution provided by the present invention is: a recombinant expression vector, which comprises the promoter core region as described in one of the technical solutions, the strong promoter as described in the second technical solution, or the expression cassette as described in the third technical solution.

[0042] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pXMJ19 or pK18mobsacB.

[0043] In the present invention, the term "recombinant expression vector" refers to a DNA construct comprising a DNA sequence operably linked to appropriate control sequences, thereby expressing a gene of interest in a suitable host. The vector used in the present invention is not particularly limited and can be any vector known in the art, as long as it is capable of replicating in the host. Specifically, such vectors include, but are not limited to, plasmids or bacteriophages, such as the pXMJ19 or pK18mobsacB plasmids used in specific embodiments of the present invention. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

[0044] To solve the above technical problems, the fifth technical solution provided by the present invention is: a genetically engineered bacterium, which comprises the promoter core region as described in one of the technical solutions, the strong promoter as described in the second technical solution, the expression cassette as described in the third technical solution, or the recombinant expression vector as described in the fourth technical solution.

[0045] In a specific embodiment of the present invention, the starting bacteria of the genetically engineered bacteria are derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably Corynebacterium glutamicum, such as Corynebacterium glutamicum H5 with a deposit number of CCTCC NO: M2016609, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067; and / or, the expression cassette in the genetically engineered bacteria is a single copy, a double copy or a multiple copy.

[0046] The "starting bacteria" of the present invention is a meaning generally understood by those of ordinary skill in the art, that is, a strain capable of importing a nucleic acid molecule with promoter activity of the present invention, which is referred to as a genetically engineered bacterium after the introduction. In other words, the present invention can utilize any starting bacteria, as long as the nucleic acid molecule with promoter activity of the present invention is contained in its cell and operably connected to a certain gene to mediate the transcription of the gene. The starting bacteria of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably an enterobacterium or a corynebacterium, more preferably a Corynebacterium glutamicum, including but not limited to Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067, and a mutant or strain producing L-amino acids prepared from the above-mentioned strains (e.g., Corynebacterium glutamicum H5 with a deposit number of CCTCC NO: M2016609).

[0047] To solve the above technical problems, the sixth technical solution provided by the present invention is: a method for constructing a genetically engineered bacterium as described in the fifth technical solution, the construction method comprising transferring the promoter core region as described in one of the technical solutions, the strong promoter as described in the second technical solution, the expression cassette as described in the third technical solution, or the recombinant expression vector as described in the fourth technical solution into the starting bacteria.

[0048] The "genetically engineered bacteria" described herein are constructed through transformation. "Transformation" as generally understood by those skilled in the art refers to the process of introducing exogenous DNA into a starting bacterium. Transformation methods include any method for introducing nucleic acid into a starting bacterium, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO methods.

[0049] In a specific embodiment of the present invention, the promoter core region is used to replace the original promoter core region of the target gene of the starting bacteria; and / or, the strong promoter is used to replace the original promoter of the target gene of the starting bacteria; and / or, the expression cassette is inserted into the genome of the starting bacteria, and the insertion site is preferably an invalid gene of the starting bacteria, such as the msrA or ddh gene; and / or, the genetically engineered bacteria contains a free recombinant expression vector.

[0050] The "invalid gene" mentioned in the present invention refers to a gene that has no effect on the growth and metabolism of the strain after destruction, such as the msrA or ddh gene sites of Corynebacterium glutamicum.

[0051] In a specific embodiment of the present invention, when the promoter core region is used to replace the original promoter core region of the gene of the starting bacterium, the gene is an elongation factor Tu encoding gene; the elongation factor Tu encoding gene is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

[0052] To solve the above technical problems, the seventh technical solution provided by the present invention is: a method for enhancing the expression of a target gene, the method comprising replacing the original promoter core region of the target gene with the promoter core region described in one of the technical solutions; or the method comprising operably connecting the strong promoter described in the second technical solution to the target gene.

[0053] In a specific embodiment of the invention, the method is for non-diagnostic and / or therapeutic purposes.

[0054] In a specific embodiment of the present invention, when the promoter core region replaces the original promoter core region of the target gene, the target gene is an elongation factor Tu encoding gene; the elongation factor Tu encoding gene is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

[0055] The term "operably linked" in the present invention means that the strong promoter as described in the second technical solution of the present invention is functionally linked to the coding gene to initiate and mediate the transcription of the gene, indicating that the strong promoter as described in the second technical solution of the present invention is operably linked to the coding gene to control the transcriptional activity of the operon gene. The operably linked method can be any method described by those skilled in the art. In the method for enhancing the expression of the target gene described in the present invention, based on the strong promoter as described in the second technical solution of the present invention, it is implemented by methods commonly used by those skilled in the art.

[0056] To solve the above technical problems, the present invention provides an eighth technical solution: a method for preparing a target protein, comprising culturing the genetically engineered bacteria as described in the fifth technical solution.

[0057] In a specific embodiment of the present invention, the method further comprises separating and purifying the target protein.

[0058] In the present invention, the "target protein" refers to a protein whose expression is enhanced by operably linking the gene encoding the target protein to a promoter containing the promoter core region as described in one of the technical solutions or a strong promoter as described in the second technical solution.

[0059] To solve the above technical problems, the present invention provides a ninth technical solution: a method for producing amino acids, comprising culturing the genetically engineered bacteria as described in the fifth technical solution.

[0060] In a specific embodiment of the present invention, the amino acid is L-arginine, L-isoleucine, L-citrulline or L-ornithine; and / or, the method further comprises collecting the amino acid from the fermentation broth after the culturing is completed.

[0061] There are reports in the prior art of enhancing carAB activity to increase arginine, citrulline, and ornithine. For example, KR102269637B1 reported that the use of eftu promoter or sod promoter to enhance carAB activity can increase the production of L-arginine or L-citrulline. Therefore, those skilled in the art can reasonably expect that when the strong promoter as described in the second technical solution of the present invention is used to enhance the expression of genes encoding enzymes for synthesizing amino acids, the production of amino acids in the strain, especially proline, lysine, glutamic acid, hydroxyproline, isoleucine, arginine, citrulline, ornithine, and glutamic acid amide, can be increased.

[0062] In the present invention, the culture of the genetically engineered bacteria 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, and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0063] The amino acid production method of the present invention utilizes the strong promoter described in the second technical solution of the present invention and employs methods commonly used by those skilled in the art, and also includes a step of recovering the amino acids from cells or culture medium. Methods for recovering amino acids from cells or culture medium are well known in the art and include, but are not limited to, filtration, anion exchange chromatography, crystallization, and HPLC.

[0064] To solve the above technical problems, the tenth technical solution provided by the present invention is: the use of the promoter core region as described in the first technical solution, the strong promoter as described in the second technical solution, the expression cassette as described in the third technical solution, the recombinant expression vector as described in the fourth technical solution, or the genetically engineered bacteria as described in the fifth technical solution in the preparation of a reagent or kit for enhancing gene transcription levels, preparing a target protein, or producing amino acids.

[0065] In a specific embodiment of the invention, said use is for non-diagnostic and / or therapeutic purposes.

[0066] In a specific embodiment of the present invention, the amino acid is L-arginine, L-isoleucine, L-citrulline or L-ornithine.

[0067] To solve the above technical problems, the present invention provides an eleventh technical solution: a kit comprising the promoter core region as described in one of the technical solutions, the strong promoter as described in the second technical solution, the expression cassette as described in the third technical solution, the recombinant expression vector as described in the fourth technical solution, or the genetically engineered bacteria as described in the fifth technical solution.

[0068] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0069] The reagents and raw materials used in the present invention are commercially available.

[0070] The positive progress effect of the present invention is:

[0071] 1. The activity of the screened promoter mutant eftu30 is 12 times that of the original eftu; the activity of eftu5 is 1.3 times that of the original eftu; and the activity of eftu24 is 1.1 times that of the original eftu, which can be used to transform bacterial strains and improve their amino acid acid production capacity.

[0072] 2. The arginine yield of the strain modified with a single eftu30 promoter replacement increased by 130% compared to the control (ATCC 13869), and by 27.2% compared to the strain modified with the eftu promoter replacement. The strain modified with dual eftu30 promoter replacement increased arginine yield by 180% compared to the control, and by 29.0% compared to the strain modified with dual eftu promoter replacement. Furthermore, in terms of sugar consumption, the eftu30 promoter-modified strain consumed less sugar, produced more acid, and had an improved sugar-to-acid conversion rate. Furthermore, the production of mixed acids in the fermentation broth was reduced compared to the control.

[0073] 3. The arginine production of the transformed strains after replacing a single eftu5 promoter and a single eftu24 promoter increased by 1.2% and 3.2% respectively compared with the transformed strain replaced by the eftu promoter; the arginine production of the transformed strains after replacing the double eftu promoter and the eftu24 promoter increased by 9.38% and 7.31% respectively compared with the transformed strain replaced by the double eftu promoter. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 shows the TLC test results of shake flask fermentation broths of the original strain (ATCC 13869) and four modified strains (H1-R1, H1-R2, H1-R5, and H1-R6). 0.5% arg and 1% arg are arginine standard solutions, and the fermentation broth samples are loaded after being diluted 10 times. DETAILED DESCRIPTION

[0075] The purpose of the present invention is, firstly, to screen out a more active eftu promoter mutant by constructing a promoter mutant library, and secondly, to apply the promoter mutant to a Corynebacterium glutamicum production strain of amino acids, such as arginine, for example, to replace the natural promoter of the gene carAB encoding carbamoyl phosphate synthetase CPSII, thereby enhancing the supply of arginine precursor carbamoyl phosphate, thereby indirectly increasing arginine production.

[0076] The present invention first randomly mutates the eftu promoter sequence reported in the literature. The mutation regions are mainly distributed in the non-conserved base region between the -35 and -10 regions and the 6bp base downstream of the -10 region. EGFP is used as a reporter gene to detect the promoter strength. Through fluorescence detection, promoter mutants with significantly enhanced fluorescence signals are selected. Sequencing and comparison analysis are used to determine the position and mutation type of the mutated bases, and finally the optimal promoter mutants eftu30, eftu5 and eftu24 are screened.

[0077] Using ATCC 13869, a standard strain of Corynebacterium glutamicum, as the starting strain, the carAB gene was identified in the ATCC 13869 genome. Software analysis predicted its native promoter region. Gene-editing vectors were then constructed to replace the carAB gene promoter with the eftu promoter or the selected eftu30, eftu5, or eftu24 promoters, or to simultaneously overexpress a carAB expression cassette carrying the eftu, eftu30, eftu5, or eftu24 promoters. This modified strain enhanced its arginine acid production.

[0078] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0079] Example 1: Construction and screening of eftu promoter mutation library

[0080] Corynebacterium glutamicum (ATCC 13869 strain) was inoculated into LBG medium (LBG formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose) and shaken at 30°C 220 rpm overnight. The cells were collected and the genome was extracted using a bacterial genome mini kit (Sanggong). Using the genome as a template, primers (eftu-F and eftu-R) were designed to amplify the eftu promoter (SEQ ID NO: 1). EGFP was used as a reporter gene and the pXMJ19 (Miaoling Biotechnology, P0100) backbone vector was used to construct the recombinant vector p19-eftu-EGFP, which served as a control plasmid. Using the p19-eftu-EGFP plasmid as a template, degenerate PCR primers (random-eftu-EGFP-FP and random-eftu-RP) were designed to randomly mutate the eftu promoter. The mutations occurred in the non-conserved region between the -35 and -10 regions of the promoter, as well as in the 6 bp downstream of the -10 region. EGFP was still used as the reporter gene. The primers used in the present invention are shown in Table 1.

[0081] Original eftu promoter sequence 285bp (SEQ ID NO: 1)

[0082] The framed region is the promoter core region (including region ‐35, region ‐10, the region between region ‐35 and region ‐10, and the transcription start site).

[0083] Table 1: Primer sequence list

[0084] The control plasmid and the promoter random mutation plasmid were electrotransformed into Corynebacterium glutamicum. The competent state and electrotransformation steps were prepared according to the method of van der Rest et al. (ME van der Rest, C. Lange, D. Molenaar. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol., 1999, 52: 541-545). The transformants of the promoter mutant strain and the original strain were inoculated into 30 mL LBG medium and cultured at 30 ° C 220 rpm to an OD of about 2. The cells were collected by centrifugation and washed twice with PBS buffer. A preliminary high-throughput screening was performed using the Qpix microbial screening system and the Biomek FXP laboratory automatic workstation to obtain an initial mutation library. The promoter mutation library included 50 promoters. Subsequently, 10 promoters were selected from the 50 promoters obtained and their fluorescence intensity was further accurately measured using a microplate reader. The promoter's activation strength (i.e., activity) was evaluated based on the fluorescence intensity. The intensity span of the promoters in the final library construction results was about 10 times. The three promoters with the strongest fluorescence signals were numbered eftu30, eftu5, and eftu24, as shown in Table 2.

[0085] Sequencing of eftu30, eftu5, and eftu24 (sequences shown in SEQ ID NOs: 16-18, respectively) and comparison with eftu by Balst analysis revealed that eftu30 harbored 14 base mutations, located at positions 161-162, 164-166, 168-169, 171-172, 174, 176, 184, and 186-187; eftu5 harbored 15 base mutations, located at positions 161-166, 169-170, 172, 174-177, 186, and 188; and eftu24 harbored two base mutations, located at positions 184 and 186. All mutations were located in the promoter core region.

[0086] Table 2: Comparison of fluorescence intensity before and after mutation of the promoter core region

[0087] eftu30 promoter sequence 285 bp (SEQ ID NO: 16)

[0088] eftu5 promoter sequence 285 bp (SEQ ID NO: 17)

[0089] eftu24 promoter sequence 285 bp (SEQ ID NO: 18)

[0090] The framed region is the promoter core region (including region ‐35, region ‐10, the region between region ‐35 and region ‐10, and the transcription start site).

[0091] Example 2: Mutated eftu promoter or original eftu promoter replaces the natural promoter of carAB gene

[0092] Based on the genome sequence of Corynebacterium glutamicum published in the NCBI database, the gene encoding CPSII (carbamyl phosphate synthetase) was found. One carAB gene (SEQ ID NO: 19) is present in the genome of ATCC strain 13869. The promoter region of this gene was predicted using promoter prediction software. With the promoter region as the center, 1000 bp were extended upstream and downstream as upstream and downstream homology arms (L and R, respectively). Primers were designed to amplify the upstream and downstream homology arms, respectively. Primers were also designed to amplify the eftu promoter, eftu30 promoter, eftu5 promoter, or eftu24 promoter. The three fragments were assembled into the universal gene editing vector pK18mobsacB (Miaoling Biotechnology, P0100) for Corynebacterium glutamicum using a multi-fragment cloning kit (abclonal) in the order L-promoter-R, constructing the recombinant vectors pK18-eftu301, pK18-eftu1, pK18-eftu51, and pK18-eftu241 (for the first carAB gene). The ligation products were transformed into DH5α chemically competent cells, plated onto LB plates containing kanamycin, and cultured overnight at 37°C. Transformants were selected and verified by PCR using the designed vector sequencing primers (Pk18-cexu-Fp and Pk18-cexu-Rp). After the correct transformants were expanded and cultured, the plasmids were extracted and sent to Jinkairui Biotechnology Co., Ltd. for sequencing verification.

[0093] The correct recombinant vector pK18-eftu301, pK18-eftu1, pK18-eftu51 or pK18-eftu241 was transformed into the competent state of Corynebacterium glutamicum. The preparation of competent state and electroporation steps were referenced to the method of van der Rest et al. (ME van der Rest, C. Lange, D. Molenaar. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol., 1999, 52: 541-545). After 2-3 days of culture at 30 ° C, transformants grew, and colonies were picked and inoculated into LBG medium containing kan. The culture was expanded at 30 ° C 220 rpm overnight. The genome was extracted the next day, and a sequence was selected from the upstream of the left homologous arm of the genome and on the plasmid as primers (L1-Fp and L-Rp) to verify whether homologous recombination occurred and the location of homologous recombination. The PCR product was sent for sequencing for further confirmation. The transformants that were verified to have undergone one round of exchange were inoculated into LBG medium and cultured at 30°C and 90 rpm for 24 h. The bacterial solution was diluted 10 -2 , 10 -1 The strain was plated on a 10% sucrose plate for a second round of exchange. The strain was cultured at 30°C for 3 days until transformants emerged. The transformed strains were streaked simultaneously on 10% sucrose plates and LBG (kan, 50 μg / mL) plates and cultured at 30°C for 1-2 days. Transformants that did not grow on the resistant plate but grew on the sucrose plate were selected and primers were designed for PCR verification (L1-Fp and 301-Rp). The PCR products were further verified by sequencing. The successfully verified transformants were named H1-R1 (natural promoter of the carAB gene replaced by the eftu30 promoter), H1-R2 (natural promoter of the carAB gene replaced by the eftu promoter), H1-R3 (natural promoter of the carAB gene replaced by the eftu5 promoter), and H1-R4 (natural promoter of the carAB gene replaced by the eftu24 promoter).

[0094] CarAB nucleotide sequence (SEQ ID NO: 19)

[0095] Example 3: Overexpression of carAB genes using a mutated eftu promoter or the original eftu promoter

[0096] A carAB expression cassette was overexpressed at the msrA gene locus of Corynebacterium glutamicum ATCC 13869 using the eftu, eftu30, eftu5, or eftu24 promoter. First, vectors pK18-eftu302, pK18-eftu2, pK18-eftu52, and pK18-eftu242 were constructed. The msrA gene was centered, with 1000 bp extending upstream and downstream as upstream and downstream homology arms (L and R, respectively). Primers were designed to amplify the upstream and downstream homology arms, respectively. Primers were also designed to amplify the eftu promoter + carAB, eftu30 promoter + carAB, eftu5 promoter + carAB, or eftu24 promoter + carAB. The three fragments were assembled into the universal gene-editing vector pK18mobsacB for Corynebacterium glutamicum using the multi-fragment cloning kit (abclonal) in the order of L-promoter + carAB-R, creating recombinant vectors pK18-eftu302, pK18-eftu52, pK18-eftu242, and pK18-eftu2. The ligation products were transformed into DH5α chemically competent cells, plated onto LB plates containing kan resistance, and cultured overnight at 37°C. Transformants were selected and verified by PCR using the designed vector sequencing primers (Pk18-cexu-Fp and Pk18-cexu-Rp). After the correct transformants were expanded, plasmids were extracted and sent to Jinkairui Biotechnology Co., Ltd. for sequencing verification.

[0097] The strains H1-R1, H1-R2, H1-R3 or H1-R4 with successful promoter replacement were prepared into electrocompetent cells, and pK18-eftu302, pK18-eftu2, pK18-eftu52, and pK18-eftu242 were transformed into H1-R1, H1-R2, H1-R3 or H1-R4, respectively. After culturing at 30°C for 2-3 days, transformants grew out. Colonies were picked and inoculated into LBG medium containing kan, and cultured overnight at 30°C and 220rpm. The genome was extracted the next day, and a sequence upstream of the left homologous arm of the genome and on the plasmid was selected as primers (L2-Fp and L-Rp) to verify whether homologous recombination occurred and the location of homologous recombination. The PCR product was sent for sequencing for further confirmation. The transformants that were verified to have undergone one round of exchange were inoculated into LBG medium, cultured at 30°C and 90rpm for 24 hours, and the bacterial solution was diluted 10 -2 , 10 -1The cells were plated on 10% sucrose plates for a second round of exchange. The cells were cultured at 30°C for 3 days until transformants emerged. The transformed cells were streaked onto 10% sucrose plates and LBG (kan, 50 μg / mL) plates simultaneously and cultured at 30°C for 1-2 days. Transformants that did not grow on resistant plates but did grow on sucrose plates were selected and primers designed for PCR verification (L2-Fp and 301-Rp). The PCR products were further verified by sequencing. Successfully verified transformants were named H1-R5 (overexpressing the eftu30 promoter and carAB genes based on H1-R1), H1-R6 (overexpressing the eftu promoter and carAB genes based on H1-R2), H1-R7 (overexpressing the eftu5 promoter and carAB genes based on H1-R3), and H1-R8 (overexpressing the eftu24 promoter and carAB genes based on H1-R4).

[0098] Example 4: Validation of acid production in shake flasks using single and dual promoter replacements

[0099] ATCC 13869, H1-R1, H1-R2, H1-R3, H1-R4, H1-R5, H1-R6, H1-R7 and H1-R8 were streaked into three zones on plate 1 (formula: peptone 10 g / L, yeast powder 5 g / L, beef powder 5 g / L, sodium chloride 2.5 g / L, agar 20 g / L, pH 6.8-7.0), and cultured at 30°C for 48 h. A loop of activated bacterial sludge was taken with an inoculation loop and densely streaked on a new plate 1, and cultured at 30°C for 24 h. Use an inoculating loop to take a loop of secondary activated bacterial sludge and streak densely on plate 2 (formula: monohydrate glucose 11g / L, peptone 10g / L, yeast powder 10g / L, sodium chloride 2.5g / L, urea 2g / L, agar 20g / L, pH 6.8-7.0). Streak 3 plates for each strain and incubate at 30℃ for 15-20h. The next morning, scrape the bacterial sludge on plate 2 with a sterilized wooden stick into a sterile EP tube containing 1ml of sterile water, stir evenly, take an appropriate amount of bacterial solution to dilute it to an appropriate multiple, and detect the OD 562 According to the measured OD, inoculate an appropriate amount of bacterial liquid into 30mL shake flask medium (formula: glucose monohydrate 50g / L, peptone 4g / L, yeast extract 10g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate trihydrate 1.31g / L, sodium chloride 2.5g / L, urea 2g / L, biotin 0.1mg / L), making the inoculum amount as consistent as possible. Incubate at 30℃ 220rpm for 1 hour, then take a sample and measure the OD 562 , set as the initial OD, 48h, detect the net increase of OD, pH, sugar consumption, and acid production (HPLC detection and TLC detection are carried out simultaneously).

[0100] Table 3: Shake flask fermentation results

[0101] As can be seen from the results in Table 3, the strains after promoter replacement were compared with the control strain (ATCC 13869) for the same fermentation time: the arginine production of the transformed strain with a single eftu30 promoter replacement increased by 130% compared to the control, and increased by 27.2% relative to the transformed strain with eftu promoter replacement, and the arginine production of the transformed strain with a single eftu promoter replacement increased by 81% compared to the control. The transformed strain with double eftu30 promoter replacement increased by 180% compared to the control, and increased by 29.0% relative to the transformed strain with double eftu promoter replacement, and the arginine production of the transformed strain with double eftu promoter replacement increased by 120% compared to the control. The arginine production of the transformed strains after replacing a single eftu5 promoter and a single eftu24 promoter increased by 83.2% and 86.8% respectively compared with the control, and the arginine production increased by 1.2% and 3.2% respectively compared with the transformed strains replaced with the eftu promoter. The arginine production of the transformed strains replaced with double eftu5 promoters and eftu24 promoters increased by 142% and 138% respectively compared with the control strain, and the arginine production increased by 9.38% and 7.31% respectively compared with the transformed strains replaced with double eftu promoters.

[0102] Comparing the growth of the strains, the growth rate of the modified strain was slightly slower than that of the original strain, but the difference was not obvious. Compared with the sugar consumption, the strain modified with the eftu30 promoter consumed less sugar, produced more acid, and had an improved sugar-acid conversion rate, while the sugar-acid conversion rate of the strain modified with the eftu5 and eftu24 promoters did not increase.

[0103] As can be seen from Figure 1 , H1-R1 and H1-R2 have some reduction in impurities compared to the control, while H1-R6 and H1-R5 have a more obvious reduction in impurities compared to the control.

[0104] Example 5: Overexpression of the ppnk gene using a mutant eftu promoter

[0105] A ppnk expression cassette was overexpressed at the ddh gene locus of Corynebacterium glutamicum H5 (an L-isoleucine-producing strain, deposited with CCTCC NO: M2016609, derived from patent CN106701648A). That is, with ddh as the center, 1000bp were extended upstream and downstream as upstream and downstream homology arms (L and R, respectively). Primers were designed to amplify the upstream and downstream homology arms, respectively, and primers were designed to amplify the eftu30 promoter + ppnk. The above fragments were assembled into the universal gene editing vector pK18mobsacB of Corynebacterium glutamicum to construct the recombinant vector pK18-eftu30-ppnk. The ligation product was transformed into DH5α chemically competent medium, plated onto LB plates containing kan resistance, and cultured overnight at 37°C. Transformants were selected and verified by PCR using the designed vector sequencing primers (Pk18-cexu-Fp and Pk18-cexu-Rp). After the correct transformants were expanded and cultured, the plasmids were extracted and sent to Jinkairui Biotechnology Co., Ltd. for sequencing verification.

[0106] Prepare Corynebacterium glutamicum H5 into electrocompetent state, and transform plasmid pK18-eftu30-ppnk into Corynebacterium glutamicum H5. After culturing at 30℃ for 2-3 days, transformants grow out, pick colonies and inoculate them into LBG medium containing kan, expand and culture at 30℃ 220rpm overnight, extract genome the next day, use a sequence upstream of the left homologous arm of the genome and a sequence on the plasmid as primers to verify whether homologous recombination occurs and the location of homologous recombination. PCR products are sent for sequencing for further confirmation. The transformants that have been verified to have undergone one round of exchange are inoculated into LBG medium, cultured at 30℃ 90rpm for 24h, and the bacterial solution is diluted 10 -2 , 10 -1 The cells were plated on 10% sucrose plates for two rounds of exchange. The cells were cultured at 30°C for 3 days until transformants emerged. The transformed cells were streaked simultaneously on 10% sucrose plates and LBG (kan, 50 μg / mL) plates and cultured at 30°C for 1-2 days. Transformants that did not grow on the resistant plates but grew on the sucrose plates were selected and primers were designed for PCR verification. The PCR products were sent for sequencing for further verification. The successfully verified transformants were named Corynebacterium glutamicum K7 (overexpressing the eftu30 promoter + ppnk gene based on Corynebacterium glutamicum H5).

[0107] Example 6: Isoleucine production by small-tank fermentation of Corynebacterium glutamicum K7

[0108] The activation culture medium formula is: 5% corn steep liquor, 1% tryptone, 0.5% sodium chloride, 0.4% ammonium sulfate, 0.3% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.004% alanine, 2% agar powder, and the balance is deionized water. The pH is 7, all are weight percentages.

[0109] The seed culture medium formula is: 3% glucose, 2.5% ammonium sulfate, 3.5% corn steep liquor, 0.3% yeast extract, 0.3% silk peptide powder, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.00003% vitamin B1, 0.00002% biotin, 0.00001% iron sulfate, 4% calcium carbonate, 0.004% alanine, and the balance is deionized water. The pH is 7. All percentages are by weight.

[0110] The fermentation medium formula is: 16.0% glucose, 0.8% ammonium sulfate, 3.5% corn steep liquor, 0.3% yeast extract, 0.3% silk peptide powder, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.00003% vitamin B1, 0.00002% biotin, 0.00001% iron sulfate, 0.004% alanine, and the balance is deionized water. The pH is 7. All percentages are by weight.

[0111] A 1ml glycerol culture of Corynebacterium glutamicum H5 and K7 was streaked onto solid-state activation medium and incubated at 30°C for 16 hours. A loopful of inoculated bacteria was transferred from the activation medium to a seed culture medium and incubated at 30°C with shaking at 200 rpm for 16 hours. A 10% volume ratio of the seed culture medium was inoculated into a 5L fermentor. The temperature was maintained at 30°C, the pH was controlled at 7.0 with ammonia water, and the dissolved oxygen level was maintained at 30% by adjusting the rotational speed and aeration rate. When the residual sugar content fell below 1.5%, 80% glucose was fed to maintain the residual sugar content between 1.5 and 2.5%. The fermentation was continued for 65 hours. Samples were taken from each strain and analyzed by HPLC to determine the difference in fermentation results between the two strains. Corynebacterium glutamicum H5 produced 35g / L of L-isoleucine, while Corynebacterium glutamicum K7 produced 37.5g / L, representing an approximately 7% increase in L-isoleucine yield compared to H5.

[0112] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A promoter core region, characterized in that, the promoter core region comprises substitutions at one or more positions among the 20th - 25th, 27th - 31st, 33rd - 36th, 43rd, and 45th - 47th positions of the nucleotide sequence shown in SEQ ID NO:

12.

2. The promoter core region according to claim 1, characterized in that, the promoter core region is selected from any one of the following (i) - (iii): (i) Comprises substitutions at one or more positions among the 20th - 21st, 23rd - 25th, 27th - 28th, 30th - 31st, 33rd, 35th, 43rd, and 45th - 46th positions of the nucleotide sequence shown in SEQ ID NO:12; (ii) Comprises substitutions at one or more positions among the 20th - 25th, 28th - 29th, 31st, 33rd - 36th, 45th, and 47th positions of the nucleotide sequence shown in SEQ ID NO:12; (iii) Comprises substitutions at one or two positions among the 43rd and 45th positions of the nucleotide sequence shown in SEQ ID NO:

12.

3. The promoter core region according to claim 1, characterized in that, the promoter core region has 60% - 98% identity compared with the nucleotide sequence shown in SEQ ID NO:12; Preferably, the promoter core region has at least 68%, at least 70%, or at least 95% identity compared with the nucleotide sequence shown in SEQ ID NO:

12.

4. The promoter core region according to any one of claims 1 - 3, characterized in that, the promoter core region comprises substitutions selected from one or more of the following: substituting T or G at the 20th position, substituting G or T at the 21st position, substituting G at the 22nd position, substituting A at the 23rd position, substituting T or C at the 24th position, substituting G or T at the 25th position, substituting T at the 27th position, substituting T or C at the 28th position, substituting A at the 29th position, substituting C at the 30th position, substituting A or C at the 31st position, substituting T at the 33rd position, substituting A at the 34th position, substituting C or T at the 35th position, substituting C at the 36th position, substituting C at the 43rd position, substituting G or C at the 45th position, substituting C at the 46th position, and substituting T at the 47th position of the nucleotide sequence shown in SEQ ID NO:12; Preferably: the promoter core region comprises substitutions selected from one or more of the following: substituting T at the 20th position, substituting G at the 21st position, substituting A at the 23rd position, substituting T at the 24th position, substituting G at the 25th position, substituting T at the 27th position, substituting T at the 28th position, substituting C at the 30th position, substituting A at the 31st position, substituting T at the 33rd position, substituting C at the 35th position, substituting C at the 43rd position, substituting G at the 45th position, and substituting C at the 46th position of the nucleotide sequence shown in SEQ ID NO:12; The promoter core region contains one or more substitutions selected from the following: substitution of G at position 20, substitution of T at position 21, substitution of G at position 22, substitution of A at position 23, substitution of C at position 24, substitution of T at position 25, substitution of C at position 28, substitution of A at position 29, substitution of C at position 31, substitution of T at position 33, substitution of A at position 34, substitution of T at position 35, substitution of C at position 36, substitution of C at position 45 and substitution of T at position 47; or, The promoter core region contains one or two substitutions selected from the following: substitution of C at position 43 and substitution of G at position 45 in the nucleotide sequence shown in SEQ ID NO:

12.

5. The promoter core region according to claim 4, wherein, the promoter core region contains the nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:

15.

6. A strong promoter, wherein, the strong promoter contains the promoter core region according to any one of claims 1 to 5; Preferably, the strong promoter is a variant of the eftu promoter; the eftu promoter is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC13869 or Corynebacterium glutamicum ATCC 14067; More preferably, the eftu promoter contains the nucleotide sequence shown in SEQ ID NO:1; Even more preferably, the strong promoter contains the nucleotide sequence shown in SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

7. An expression cassette, wherein, the expression cassette contains the promoter core region according to any one of claims 1 to 5 or the strong promoter according to claim 6; Preferably, the expression cassette further comprises a protein-coding gene, which is operably linked to the strong promoter; the protein-coding gene is, for example, a gene encoding an enzyme for synthesizing amino acids; the enzyme for synthesizing amino acids preferably comprises: NAD + kinase, acetylglutamate semialdehyde dehydrogenase, ornithine acetyltransferase, acetylglutamate kinase, acetylornithine aminotransferase, ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, arginine transporter, carbamoyl phosphate synthetase, aspartate ammonia-lyase, aspartate aminotransferase, and glutamate dehydrogenase, or a combination of two or more of them; Preferably, the enzyme for synthesizing amino acids is from Corynebacterium glutamicum, such as Corynebacterium glutamicum H5 with the preservation number of CCTCC NO: M2016609, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067; Even more preferably, the nucleotide sequence of the gene encoding the carbamoyl phosphate synthetase is as shown in SEQ ID NO:

19.

8. A recombinant expression vector, wherein, the recombinant expression vector contains the promoter core region according to any one of claims 1 to 5, the strong promoter according to claim 6 or the expression cassette according to claim 7; Preferably, the backbone of the recombinant expression vector is pXMJ19 or pK18mobsacB.

9. A genetically engineered bacterium, wherein, The genetically engineered bacterium contains the promoter core region as described in any one of claims 1 to 5, the strong promoter as described in claim 6, the expression cassette as described in claim 7, or the recombinant expression vector as described in claim 8; Preferably, the starting bacterium of the genetically engineered bacterium is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably Corynebacterium glutamicum, such as Corynebacterium glutamicum H5 with the preservation number of CCTCC NO: M2016609, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067; and / or, the expression cassette in the genetically engineered bacterium is single-copy, double-copy or multi-copy.

10. A method for constructing a genetically engineered bacterium as described in claim 9, characterized in that, the construction method includes transferring the promoter core region as described in any one of claims 1 to 5, the strong promoter as described in claim 6, the expression cassette as described in claim 7, or the recombinant expression vector as described in claim 8 into the starting bacterium; Preferably, the original promoter core region of the target gene of the starting bacterium is replaced with the promoter core region; and / or, the original promoter of the target gene of the starting bacterium is replaced with the strong promoter; and / or, the expression cassette is inserted into the genome of the starting bacterium, and the insertion site is preferably the ineffective gene of the starting bacterium, such as the msrA or ddh gene; and / or, the genetically engineered bacterium contains the free recombinant expression vector; More preferably, when the original promoter core region of the gene of the starting bacterium is replaced with the promoter core region, the gene is the elongation factor Tu encoding gene; the elongation factor Tu encoding gene is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

11. A method for enhancing the expression of a target gene, characterized in that, the method includes replacing the original promoter core region of the target gene with the promoter core region as described in any one of claims 1 to 5; or the method includes operably connecting the strong promoter as described in claim 6 to the target gene; Preferably, when the original promoter core region of the target gene is replaced with the promoter core region, the target gene is the elongation factor Tu encoding gene; the elongation factor Tu encoding gene is preferably from Corynebacterium glutamicum, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

12. A method for preparing a target protein, characterized in that, the method includes culturing the genetically engineered bacterium as described in claim 9; Preferably, the method further includes separating and purifying the target protein.

13. A method for producing an amino acid, characterized in that, the method includes culturing the genetically engineered bacterium as described in claim 9; Preferably, the amino acid is L-arginine, L-isoleucine, L-citrulline or L-ornithine; and / or, the method further comprises collecting the amino acid from the fermentation broth after the completion of cultivation.

14. Use of the promoter core region according to any one of claims 1 to 5, the strong promoter according to claim 6, the expression cassette according to claim 7, the recombinant expression vector according to claim 8, or the genetically engineered bacterium according to claim 9 in the preparation of a reagent or kit for enhancing gene transcription level, the preparation of a target protein or the production of an amino acid; Preferably, the amino acid is L-arginine, L-isoleucine, L-citrulline or L-ornithine.

15. A kit, characterized in that the kit contains the promoter core region according to any one of claims 1 to 5, the strong promoter according to claim 6, the expression cassette according to claim 7, the recombinant expression vector according to claim 8, or the genetically engineered bacterium according to claim 9.

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