Promoter, and related biological material thereof and use thereof

Abstract

Disclosed in the present invention are a promoter, and a related biological material thereof and a use thereof. The present invention provides a DNA molecule, which can be used as a promoter. Also provided are an expression cassette, a recombinant vector, a recombinant host cell, or a recombinant microorganism, each containing the DNA molecule. Also provided is a method for using the DNA molecule to produce an L-amino acid or increase the content of an L-amino acid in bacteria.

Description

Promoter and related biological materials and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a promoter and related biological materials and applications thereof. BACKGROUND

[0002] Lysine, chemical name 2, 6-diaminohexanoic acid. Lysine is an essential amino acid, the body can not synthesize itself, and must be supplemented from food. Lysine mainly exists in animal food and legumes, and the content of lysine in cereal food is very low, and it is easy to be destroyed in the processing process and lack of, so it is called the first limiting amino acid. Only L-type lysine is absorbed by organisms, and the CAS number of L-lysine is 56-87-1.

[0003] L-lysine has positive nutritional significance in promoting human growth and development, enhancing immune function, resisting viruses, promoting fat oxidation, and relieving anxiety, etc. It can also promote the absorption of some nutrients and synergize with some nutrients to better play the physiological functions of various nutrients.

[0004] Due to the important utilization value of L-lysine, its demand is gradually increasing every year, so various researches are being carried out to develop high-efficiency microbial strains and fermentation process technology for producing L-lysine. For example, replacing the promoter of the key gene affecting L-lysine synthesis in the microbial strain to increase or weaken its activity; or deleting some genes (byproducts or toxins affecting cell growth, etc.) that do not need to be expressed in the microbial strain. However, as the demand for L-lysine increases year by year, further research on microbial strains is still needed to find suitable genes or promoters to effectively increase the production capacity of L-lysine. INVENTION

[0005] The purpose of the present application is to provide a new type of promoter and related biological materials and applications thereof, such as applications in the preparation of L-lysine.

[0006] The present application claims a DNA molecule, which is a new type of promoter named EP7 by the present application. The DNA molecule claimed by the present application comprises (or is) any of the following:

[0007] (a1) a DNA molecule with a nucleotide sequence of SEQ ID NO: 1;

[0008] (a2) a DNA molecule with a promoter function obtained by conservatively replacing a plurality of nucleotides in SEQ ID NO: 1;

[0009] (a3) a DNA molecule having 70% or more identity to SEQ ID NO: 1.

[0010] The term "identity" herein refers to the identity of nucleotide sequences. Alignment for purposes of determining percent identity can be achieved in various ways. For instance, a comparison of sequences can be performed by using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including algorithms needed to achieve maximal alignment over the entire length of the sequences being compared.

[0011] In some embodiments, the 70% or more identity is 75% or more identity. In some embodiments, the 75% or more identity is 80% or more identity. In some embodiments, the 75% or more identity is 85% or more identity. In some embodiments, the 75% or more identity is 90% or more identity. In some embodiments, the 75% or more identity is 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity. In some embodiments, the 70% or more identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In some embodiments, the 75% or more identity can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0012] The present application claims the use of the DNA molecule as described above as a promoter.

[0013] The use is the use of the DNA molecule as a promoter to drive the expression of a gene of interest.

[0014] The gene of interest can be a visual marker gene (such as GUS gene) or a functional gene to be expressed.

[0015] In the present application, the gene of interest is specifically a functional gene (such as lysC gene or ddh gene or lysA gene or gnd gene) in a microbial genome for producing an L-amino acid (such as L-lysine). The microorganism can be a yeast, a bacterium, an algae or a fungus. Among them, the bacterium can be Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Ammoniphilus sp., Corynebacterium crenatum or Pantoea. In some embodiments, the bacterium is Corynebacterium glutamicum. In some embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum YP097158.

[0016] The present application claims an expression cassette, a recombinant vector or a recombinant bacterium containing the DNA molecule described above.

[0017] The term "expression cassette" herein refers to DNA capable of initiating expression of a gene of interest in a host cell from the DNA molecule described above. The expression cassette can also include other regulatory sequences. The regulatory sequences are capable, under their inherent conditions, of directing expression of the coding sequence of the gene of interest in a suitable host cell. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum, the expression cassette includes the DNA molecule described above as a promoter, the gene of interest to be transcribed, and a termination signal for transcription, the DNA molecule being functionally linked to the gene of interest, and the gene of interest being linked to the termination signal for transcription. The termination signal for transcription can be a suitable transcription termination sequence, i.e. a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' end of the gene of interest. Any terminator that functions in the host cell of choice can be used in the present application. The leader sequence is an untranslated region of mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the gene of interest. Any leader sequence that functions in the host cell of choice can be used in the present application. The expression cassette can include, in addition to the gene of interest, a marker gene, a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) addition signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly linked or indirectly linked through a linker.

[0018] The term "recombinant vector" as used herein refers to a recombinant DNA molecule constructed by in vitro joining of the DNA molecule described above with a vector, which can be constructed in any suitable way, provided that the recombinant vector thus constructed can carry the DNA molecule described above into a recipient cell and provide the DNA molecule described above with the ability to replicate, integrate and / or be transcribed in the recipient cell.

[0019] In the present application, the recombinant vector can be a cloning vector or an expression vector. In the case of an expression vector, the gene of interest and the DNA molecule described above (promoter) are located in the vector and are operably linked. The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can conveniently be subjected to recombinant DNA procedures and that can bring about the expression of nucleic acid sequences to which it is operably linked. The choice of vector will often depend on the design of the vector, the host cells to be used for expression, and the functions desired of the vector. The vector can be linear or closed circular. The vector can be an autonomously replicating vector (i.e., a vector that exists as an episome in a host cell, replicating independent of the chromosomal DNA), e.g., a plasmid, a non-chromosomal element, a mini-chromosome, a cosmid, a phage or a virus vector. The vector can comprise any mechanism for replication of the vector within a host cell. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the cell chromosome and replicated with the chromosome. The vector system can further comprise one or more selectable marker genes to facilitate selection of host cells into which the vector is introduced. A selectable marker gene is a gene whose product confers antibiotic or viral resistance, resistance to heavy metals, or the like, on a host cell into which the gene is introduced. Examples of bacterial selectable marker genes are the dal genes of Bacillus subtilis or Bacillus licheniformis, or antibiotic resistance genes such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. The vector can comprise an element(s) that permits stable integration of the vector into the host cell genome or alternatively, the vector can comprise an element that permits autonomous replication of the vector in the cell independent of the genome. In the case of autonomous replication, the vector can further comprise a replication origin, enabling the vector to replicate autonomously in a host cell. The replication origin can be provided with a mutation that renders it temperature- sensitive (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA 75:1433). More than one copy of a nucleic acid sequence of the present application can be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the nucleic acid sequence into the host cell genome or by including an amplifiable selectable marker gene on the vector where appropriate. When an amplifiable selectable marker gene is used, the gene can be amplified by growing the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements of the vectors of the present application, to assemble vectors, to ligate nucleic acid fragments into vectors, to ligate vectors into plasmids, and to integrate vectors into the chromosomes of host cells are known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.

[0020] In some embodiments, the recombinant bacteria are recombinant bacteria containing the expression cassette or the recombinant vector.

[0021] In some embodiments, the recombinant bacteria are recombinant bacteria. The bacteria can be from Corynebacterium sp., Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serratia sp., Salmonella sp., Streptomyces sp., Providencia sp., etc., but are not limited thereto. In some embodiments, the bacteria can be Corynebacterium glutamicum, Escherichia coli (also known as E. coli), Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, or Pantoea, but are not limited thereto.

[0022] In some embodiments, the recombinant bacteria are recombinant bacteria A or recombinant bacteria B or recombinant bacteria C or recombinant bacteria D.

[0023] The recombinant bacteria A is a recombinant bacteria obtained by inserting the DNA molecule described above before the start codon of the lysC gene of the bacteria;

[0024] The lysC gene can be any one of the following:

[0025] (b1) a DNA molecule having a nucleotide sequence of SEQ ID NO: 14;

[0026] (b2) a DNA molecule derived from bacteria and having 95% or more identity with (b1) and encoding the same amino acid sequence.

[0027] In some embodiments, the insertion of the aforementioned DNA molecule before the start codon of the lysC gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 2 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 2.

[0028] The recombinant bacteria B is a recombinant bacteria obtained after the insertion of the aforementioned DNA molecule before the start codon of the ddh gene of the bacteria;

[0029] The ddh gene can be any one of the following:

[0030] (c1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 15;

[0031] (c2) a DNA molecule derived from bacteria and having 95% or more identity with (c1) and encoding the same amino acid sequence.

[0032] In some embodiments, the insertion of the aforementioned DNA molecule before the start codon of the ddh gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 5 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 5.

[0033] The recombinant bacteria C is a recombinant bacteria obtained after the insertion of the aforementioned DNA molecule before the start codon of the lysA gene of the bacteria.

[0034] The lysA gene can be any one of the following:

[0035] (d1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 16;

[0036] (d2) a DNA molecule derived from bacteria and having 95% or more identity with (d1) and encoding the same amino acid sequence.

[0037] In some embodiments, the insertion of the aforementioned DNA molecule before the start codon of the lysA gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 8 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 8.

[0038] The recombinant bacteria D is a recombinant bacteria obtained by inserting the DNA molecule described above before the start codon of the gnd gene of the bacteria.

[0039] The gnd gene can be any one of the following:

[0040] (e1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 17;

[0041] (e2) a DNA molecule derived from bacteria and having 95% or more identity to (e1) and encoding the same amino acid sequence.

[0042] In some embodiments, the insertion of the DNA molecule described above before the start codon of the gnd gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 11 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 11.

[0043] In the DNA molecules of (b2), (c2), (d2) and (e2) described above, the term "identity" used herein refers to sequence similarity with the natural nucleotide sequence. The "identity" includes a nucleotide sequence having 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more identity to the nucleotide sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 17 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%) which can be used to evaluate the identity between related sequences. The same applies hereinafter.

[0044] In some embodiments, the bacteria can be a Corynebacterium microorganism, such as Corynebacterium glutamicum. In a specific embodiment of the present application, the Corynebacterium glutamicum is Corynebacterium glutamicum YP097158.

[0045] The present application also provides a recombinant microorganism or a recombinant host cell containing the DNA molecule described above.

[0046] The term "recombinant microorganism" generally refers to a recombinant microorganism obtained by manipulating and modifying the genes of the microorganism of interest, thereby changing the function of the recombinant microorganism. For example, the DNA molecule described above or the recombinant vector described above is introduced into the microorganism of interest, or the DNA molecule described above is directly inserted before the endogenous target gene of the microorganism of interest.

[0047] The term "recombinant host cell" generally refers to a host cell whose genome has been manipulated and modified so that the recombinant host cell has a changed function. The host cell is manipulated and modified by introducing into the host cell a DNA molecule as described hereinbefore or a recombinant vector as described hereinbefore, or by directly inserting the DNA molecule as described hereinbefore in front of an endogenous gene of interest in the microorganism of interest.

[0048] The term "recombinant" refers to a substance (such as a nucleic acid, a protein, a cell, a microorganism, a plant, an animal) that is man-made and that does not normally exist in nature. The term "does not normally exist in nature" means that it does not exist in nature without human introduction.

[0049] The cell can be a microbial cell, an animal cell or a plant cell.

[0050] The present application claims protection for any of the following applications:

[0051] P1. Use of a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector or a recombinant bacterium as described hereinbefore for the production of an L-amino acid.

[0052] P2. Use of a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector for the construction of an L-amino acid-producing engineered strain.

[0053] P3. Use of a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector for the preparation of a food product, a pharmaceutical product, a feed or a cosmetic product containing an L-amino acid.

[0054] The present application also provides a method for producing an L-amino acid.

[0055] The method for producing an L-amino acid provided by the present application is achieved by using a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector or a recombinant bacterium as described hereinbefore.

[0056] The present application also provides a method for constructing an L-amino acid-producing engineered strain.

[0057] The method for constructing an L-amino acid-producing engineered strain provided by the present application is achieved by using a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector.

[0058] The present application also provides a method for preparing a food product, a pharmaceutical product, a feed or a cosmetic product containing an L-amino acid.

[0059] The method for preparing a food product, a pharmaceutical product, a feed or a cosmetic product containing an L-amino acid provided by the present application is achieved by using a DNA molecule as described hereinbefore or an expression cassette as described hereinbefore or a recombinant vector.

[0060] In some embodiments, the L-amino acid can be L-lysine.

[0061] The present application claims a method for improving the production of L-amino acid by bacteria.

[0062] The method for improving the production of L-amino acid by bacteria claimed in the present application can comprise the following step (A) or step (B) or step (C) or step (D):

[0063] (A) inserting the DNA molecule described above before the start codon of the lysC gene of bacteria to improve the production of the L-amino acid by the bacteria.

[0064] The lysC gene can be any of the following:

[0065] (b1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 14;

[0066] (b2) a DNA molecule derived from bacteria and having more than 95% identity with (b1) and encoding the same amino acid sequence.

[0067] In some embodiments, inserting the DNA molecule described above before the start codon of the lysC gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 2 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 2.

[0068] (B) inserting the DNA molecule described above before the start codon of the ddh gene of bacteria to improve the production of the L-amino acid by the bacteria.

[0069] The ddh gene can be any of the following:

[0070] (c1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 15;

[0071] (c2) a DNA molecule derived from bacteria and having more than 95% identity with (c1) and encoding the same amino acid sequence.

[0072] In some embodiments, inserting the DNA molecule described above before the start codon of the ddh gene of the bacteria can be achieved by introducing into the bacteria a doner fragment having the nucleotide sequence of SEQ ID NO: 5 or a doner vector containing the doner fragment having the nucleotide sequence of SEQ ID NO: 5.

[0073] (C) inserting the DNA molecule described above before the start codon of the lysA gene of bacteria to improve the production of the L-amino acid by the bacteria;

[0074] The lysA gene can be any one of the following:

[0075] (d1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 16;

[0076] (d2) a DNA molecule derived from a bacterium and having more than 95% identity with (d1) and encoding the same amino acid sequence.

[0077] In some embodiments, the insertion of the aforementioned DNA molecule before the start codon of the lysA gene of the bacterium can be achieved by introducing into the bacterium a doner fragment having the nucleotide sequence of SEQ ID NO: 8 or a doner vector containing a doner fragment having the nucleotide sequence of SEQ ID NO: 8.

[0078] (D) the insertion of the aforementioned DNA molecule before the start codon of the gnd gene of the bacterium to achieve the improvement of the production of the L-amino acid by the bacterium;

[0079] The gnd gene can be any one of the following:

[0080] (e1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 17;

[0081] (e2) a DNA molecule derived from a bacterium and having more than 95% identity with (e1) and encoding the same amino acid sequence.

[0082] In some embodiments, the insertion of the aforementioned DNA molecule before the start codon of the gnd gene of the bacterium can be achieved by introducing into the bacterium a doner fragment having the nucleotide sequence of SEQ ID NO: 11 or a doner vector containing a doner fragment having the nucleotide sequence of SEQ ID NO: 11.

[0083] In some embodiments, the method further comprises a step of culturing the resulting recombinant bacterium in fermentation after the insertion of the aforementioned DNA molecule before the start codon of the lysC gene (or ddh gene or lysA gene or gnd gene) of the bacterium in the method. The L-amino acid can be obtained from the fermentation culture product.

[0084] In some embodiments, the L-amino acid can be L-lysine.

[0085] In some embodiments, the bacteria can be from the genus Corynebacterium sp., Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serratia sp., Salmonella sp., Streptomyces sp., Providencia sp., etc., but are not limited thereto. In some embodiments, the bacteria can be Corynebacterium glutamicum, Escherichia coli (also known as E. coli), Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, or Pantoea, but are not limited thereto.

[0086] The present application claims a method for producing L-amino acid.

[0087] The method for producing L-amino acid claimed by the present application comprises the following steps: introducing the DNA molecule as described above into a biological cell capable of synthesizing the L-amino acid, allowing the DNA molecule as described above to drive the expression of relevant genes in the L-amino acid synthesis pathway in the biological cell, to obtain a recombinant biological cell; culturing the recombinant biological cell, to obtain the L-amino acid.

[0088] In some embodiments, in the method, the culturing of the recombinant biological cell is specifically fermentation culture of the recombinant biological cell.

[0089] The biological cell can be bacteria, yeast, algae, fungi, plant cells, or animal cells that are capable of synthesizing the L-amino acid. In some embodiments, the bacteria can be from the genus Corynebacterium sp., Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serratia sp., Salmonella sp., Streptomyces sp., Providencia sp., etc., but not limited thereto. In some embodiments, the bacteria can be Corynebacterium glutamicum, Escherichia coli (also known as E. coli), Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, or Pantoea, but not limited thereto.

[0090] In some embodiments, the L-amino acid can be L-lysine. Accordingly, the relevant gene can be the lysC gene described supra, or the ddh gene described supra, or the lysA gene described supra, or the gnd gene described supra.

[0091] In the above-described method, the fermentation culture can be performed by those skilled in the art using the methods in the prior art, or by optimizing and improving the fermentation method through routine experiments. The fermentation culture can be performed in a suitable culture medium under the fermentation conditions known in the art. The culture medium can comprise a carbon source, a nitrogen source, trace elements, and combinations thereof. In the culture, the pH of the culture can be adjusted (e.g., controlled to be 7.0). In the culture, the temperature of the culture can be 30 to 40°C (e.g., 30°C). In the culture, the rotation speed can be controlled (e.g., 220 rpm). In the culture, the fermentation period can be controlled (e.g., 48 h).

[0092] In the specific embodiments of the present application, the formulation of the culture medium used in the fermentation culture is shown in Table 2, and the balance is water. In the fermentation culture, the fermentation control conditions are shown in Table 3.

[0093] In the present application, the above-described bacteria are all bacteria having the ability to produce L-amino acids (e.g., L-lysine).

[0094] The "bacteria having the ability to produce L-amino acids (e.g., L-lysine)" refers to the ability of the bacteria to produce and accumulate L-amino acids (e.g., L-lysine) in the bacterial body using external substances (e.g., culture medium), and further includes the ability to secrete L-amino acids (e.g., L-lysine) into the culture system. Thus, when the bacteria are cultured in the culture medium, L-amino acids (e.g., L-lysine) can be collected.

[0095] The bacteria can be naturally collected wild-type bacteria or modified bacteria.

[0096] The "modified bacteria" refers to the modified bacteria obtained by artificially mutating and / or mutagenizing the naturally collected wild-type bacteria.

[0097] Specifically, the bacteria can be Corynebacterium microorganisms, such as Corynebacterium glutamicum. In one specific embodiment of the present application, the Corynebacterium glutamicum is Corynebacterium glutamicum YP097158.

[0098] Correspondingly, the above-described recombinant bacteria are specifically recombinant bacteria L-lysC or recombinant bacteria L-ddh or recombinant bacteria L-lysA or recombinant bacteria L-gnd.

[0099] The recombinant bacteria L-lysC differs from Corynebacterium glutamicum YP097158 only in that the lysC gene start codon of the recombinant bacteria L-lysC is inserted with the EP7 promoter sequence (SEQ ID NO: 1) in front of the lysC gene start codon of the Corynebacterium glutamicum YP097158 genome, and the other sequences remain unchanged. The recombinant bacteria L-ddh differs from Corynebacterium glutamicum YP097158 only in that the ddh gene start codon of the recombinant bacteria L-ddh is inserted with the EP7 promoter sequence (SEQ ID NO: 1) in front of the ddh gene start codon of the Corynebacterium glutamicum YP097158 genome, and the other sequences remain unchanged. The recombinant bacteria L-lysA differs from Corynebacterium glutamicum YP097158 only in that the lysA gene start codon of the recombinant bacteria L-lysA is inserted with the EP7 promoter sequence (SEQ ID NO: 1) in front of the lysA gene start codon of the Corynebacterium glutamicum YP097158 genome, and the other sequences remain unchanged. The recombinant bacteria L-gnd differs from Corynebacterium glutamicum YP097158 only in that the gnd gene start codon of the recombinant bacteria L-gnd is inserted with the EP7 promoter sequence (SEQ ID NO: 1) in front of the gnd gene start codon of the Corynebacterium glutamicum YP097158 genome, and the other sequences remain unchanged.

[0100] The DNA molecules of the present application can be used to produce a variety of products, including but not limited to lysine in the examples, and the produced products can also be glutamic acid, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, a-ketoglutaric acid, citric acid, ornithine, citrulline, etc. When producing various products, the DNA molecules of the present application are placed upstream of the genes in the synthesis pathway of the target product and the DNA molecules drive the synthesis of the genes in the synthesis pathway of the target product to achieve the production of the target product.

[0101] DEPOSIT INFORMATION

[0102] Reference biological material (Co.): YP097158

[0103] Suggested classification name: Corynebacterium glutamicum

[0104] Preservation unit: China General Microbiological Culture Collection Center

[0105] Abbreviation of preservation unit: CGMCC

[0106] Address: No. 3, Beichen West Road, Haidian District, Beijing

[0107] Preservation date: August 16, 2016

[0108] Preservation center registration number: CGMCC No. 12856 Best mode of the present application

[0109] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified. In the following examples, quantitative tests were set up with three repeated experiments, and the results were averaged.

[0110] In the following examples, Corynebacterium glutamicum ATCC13032 is Corynebacterium glutamicum numbered 13032 in ATCC. Hereinafter, it is referred to as Corynebacterium glutamicum ATCC13032.

[0111] In the following examples, Corynebacterium glutamicum YP097158 is recorded in Chinese patent application CN 110607313 B, with preservation number CGMCC No. 12856, preservation date August 16, 2016, preservation unit China General Microbiological Culture Collection Center, Beijing City, Haidian District, No. 3, Beichen West Road, telephone: 010-64807355. Hereinafter, it is referred to as Corynebacterium glutamicum YP097158.

[0112] Example 1, obtaining of new promoter EP7

[0113] The wild-type promoter of sod gene of Corynebacterium glutamicum ATCC13032 published on NCBI was used as a template, and the promoter was mutated using a random mutagenesis kit (Agilent Technologies, USA), and finally a new type of promoter was obtained, and the nucleotide sequence thereof was SEQ ID NO: 1. The new type of promoter was directly synthesized according to SEQ ID NO: 1 (synthesized by Shanghai Invitrogen Company), and was connected into a pMD19-T vector for sequencing, and the sequencing result was correct, and the sequence was SEQ ID NO: 1. The new type of promoter was named as EP7.

[0114] Example 2, construction of a strain in which the EP7 promoter regulates the expression of the lysC gene

[0115] According to the sequence of the EP7 promoter with the nucleotide sequence of SEQ ID NO: 1 and the genome sequence of Corynebacterium glutamicum ATCC13032 published on NCBI, primers were designed and synthesized for inserting the EP7 promoter into the front end of the start codon GTG of the lysC gene to enhance the expression of the lysC gene, and the specific primer design was as follows (synthesized by Shanghai Invitrogen Company):

[0116] P1: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGGTGAGGATTAGG-3' (SEQ ID NO: 18, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector);

[0117] P2: 5'-CCGGAATAATTGGCAGCTCTTTGTGCACCTTTCGATC-3' (SEQ ID NO: 19);

[0118] P3: 5'-GATCGAAAGGTGCACAAAGAGCTGCCAATTATTCCGG-3' (SEQ ID NO: 20);

[0119] P4: 5'-GGTAAAAAATCCTTTCGTAGGTTTCCGTGTCGAG-3' (SEQ ID NO: 21);

[0120] P5: 5'-GAAACCTACGAAAGGATTTTTTACCCGTGGCCCTGGTCGTACAGAAATATG-3' (SEQ ID NO: 22);

[0121] P6: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGAGCGTATTCAACACTGCG-3' (SEQ ID NO: 23, underlined nucleotide sequence is the sequence on the pK18mobsacB vector).

[0122] Construction method:

[0123] The genomic DNA of C. glutamicum ATCC13032 was used as a template for PCR amplification with primers P1 / P2, P3 / P4, and P5 / P6, respectively, to obtain a 931 bp fragment containing the upstream homologous arm (amplification product of primers P1 / P2, sequence "SEQ ID NO: 24 + positions 1-895 of SEQ ID NO: 2), a 209 bp fragment containing the EP7 promoter (amplification product of primers P3 / P4, sequence positions 859-1067 of SEQ ID NO: 2), and a 725 bp fragment containing the downstream homologous arm (amplification product of primers P5 / P6, sequence "positions 1043-1729 of SEQ ID NO: 2 + SEQ ID NO: 25).

[0124] After the PCR reaction, the three fragments obtained above were electrophoretically recovered using a column-type DNA gel recovery kit. The three recovered fragments and the pK18mobsacB plasmid (product of Addgene) digested with Xbal I and BamH I and purified were ligated with NEBuilder enzyme (product of NEB) at 50 °C for 60 min, and the single colony that grew after transformation of the ligation product was identified by PCR with primers P1 / P6 (amplification product sequence "SEQ ID NO: 24 + SEQ ID NO: 2 + SEQ ID NO: 25") to obtain a positive integration plasmid (recombinant vector). The positive integration plasmid contains a kanamycin resistance marker and can be used to obtain a recombinant in which the plasmid is integrated into the genome by kanamycin selection. The recombinant plasmid pK18-EP7-lysC has been sequenced and verified. Compared with the pK18mobsacB plasmid, the recombinant plasmid pK18-EP7-lysC differs only in that the double-stranded DNA molecule with the nucleotide sequence of SEQ ID NO: 2 replaces "AGGATCCCC" in the pK18mobsacB plasmid. Positions 1-877 of SEQ ID NO: 2 correspond to part of the coding region of the NCgl0246 gene of C. glutamicum ATCC13032, positions 878-1068 are the EP7 promoter sequence, and positions 1069-1729 correspond to part of the coding region of the lysC gene of C. glutamicum ATCC13032.

[0125] The correctly sequenced integration plasmid pK18-EP7-lysC was electroporated into Corynebacterium glutamicum YP097158, and then cultured on solid medium plates (see Table 1 for medium composition) for 40 h. Single colonies produced during the culture were identified by PCR using P7 / P8 primers. A positive strain was identified by PCR amplification of a 908 bp fragment (nucleotide sequence of SEQ ID NO: 3), and a wild type strain was identified by failure to amplify the fragment. The positive strain was streaked on solid medium plates containing 15% sucrose (adjust the sucrose content in the medium of Table 1 to 150 g / L) and cultured for 40 h. Single colonies produced during the culture were further identified by PCR using P9 / P10 primers. A positive strain with EP7 promoter sequence inserted before the start codon of the lysC gene was identified by PCR amplification of an 803 bp fragment (nucleotide sequence of SEQ ID NO: 4), and the strain was named L-lysC.

[0126] P7: 5'-GATTGTTCCAAGTAATGCGG-3' (SEQ ID NO: 26);

[0127] P8: 5'-CCGGAATAATTGGCAGCT-3' (SEQ ID NO: 27);

[0128] P9: 5'-CTCGACACGGAAACCTACG-3' (SEQ ID NO: 28);

[0129] P10: 5'-GGTAAGGACTGCTTCTTCCAC-3' (SEQ ID NO: 29).

[0130]

[0131] The recombinant strain L-lysC differs from Corynebacterium glutamicum YP097158 only in that the recombinant strain L-lysC has EP7 promoter sequence (SEQ ID NO: 1) inserted before the start codon of the lysC gene in the genome of Corynebacterium glutamicum YP097158, and the other sequences remain unchanged.

[0132] Example 3, Construction of a strain with EP7 promoter regulating ddh gene expression

[0133] According to the sequence of the EP7 promoter with the nucleotide sequence of SEQ ID NO: 1 and the genome sequence of Corynebacterium glutamicum ATCC13032 published on NCBI, primers are designed and synthesized for inserting the EP7 promoter in front of the start codon ATG of the ddh gene to enhance the expression of the ddh gene. The specific primer design is as follows (synthesized by Shanghai Invitrogen Company):

[0134] P11: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGATCAGATCGTCCAAGTTCTC-3' (SEQ ID NO: 30, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector);

[0135] P12: 5'-ACGGAAACCTACGAAAGGATTTTTTACCCATGACCAACATCCGCGTAGCTATCGTG-3' (SEQ ID NO: 31);

[0136] P13: 5'-GTAAAAAATCCTTTCGTAGGTTTCCGT-3' (SEQ ID NO: 32);

[0137] P14: 5'-CAATTTTGGAGGATTACAAGAACAGCTGCCAATTATTCCGGG-3' (SEQ ID NO: 33);

[0138] P15: 5'-GAATAATTGGCAGCTGTTCTTGTAATCCTCCAAAATTGTG-3' (SEQ ID NO: 34);

[0139] P16: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCAAAGAACTTCCCAATCTCC-3' (SEQ ID NO: 35, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector).

[0140] Construction method:

[0141] The genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and primers P11 / P12, P13 / P14 and P15 / P16 were used for PCR amplification, respectively, to obtain a 1013 bp fragment containing the upstream homologous arm (the amplification product of primers P11 / P12, the sequence is "SEQ ID NO: 24+SEQ ID NO: 5, 1-977"), a 212 bp fragment containing the EP7 promoter (the amplification product of primers P13 / P14, the sequence is SEQ ID NO: 5, 951-1162), and a 915 bp fragment containing the downstream homologous arm (the amplification product of primers P15 / P16, the sequence is "SEQ ID NO: 5, 1125-2001+SEQ ID NO: 25").

[0142] After the PCR reaction, the three fragments obtained by amplification were recovered by column type DNA gel recovery kit. The three recovered fragments and the pK18mobsacB plasmid (Addgene product) digested by Xbal I and BamH I were ligated at 50°C for 60 min using NEBuilder enzyme (NEB product). The positive integration plasmid (recombinant vector) was obtained by PCR identification of the single clone obtained after transformation of the ligation product using primers P11 / P16 (the sequence of the amplification product is "SEQ ID NO: 24+SEQ ID NO: 5+SEQ ID NO: 25"). The positive integration plasmid contains a kanamycin resistance marker, and the recombinant plasmid can be obtained by kanamycin screening. The recombinant plasmid pK18-EP7-ddh has been sequenced and verified. Compared with the pK18mobsacB plasmid, the difference of the recombinant plasmid pK18-EP7-ddh is only that the double-stranded DNA molecule with the nucleotide sequence of SEQ ID NO: 5 replaces "AGGATCCCC" in the pK18mobsacB plasmid. SEQ ID NO: 5, 1-948 corresponds to the reverse complement sequence of the partial coding region of the ddh gene of Corynebacterium glutamicum ATCC13032, 949-1139 is the reverse complement sequence of the EP7 promoter, and 1140-2001 corresponds to the reverse complement sequence of the partial coding region of the NCgl2529 gene of Corynebacterium glutamicum ATCC13032.

[0143] The correctly sequenced integration plasmid pK18-EP7-ddh was electroporated into Corynebacterium glutamicum YP097158, and then cultured on solid medium plates (see Table 1 for medium composition) for 40 h. The single colonies produced by the culture were identified by PCR using P17 / P18 primers. The positive strain was identified by PCR amplification of a 1399 bp fragment (nucleotide sequence: SEQ ID NO: 6), and the original strain was identified by failure to amplify the fragment. The positive strain was streaked on solid medium plates containing 15% sucrose (the sucrose content in the medium in Table 1 was adjusted to 150 g / L) and cultured for 40 h. The single colonies produced by the culture were further identified by PCR using P19 / P20 primers. The positive strain was identified by PCR amplification of a 1034 bp fragment (nucleotide sequence: SEQ ID NO: 7) containing the EP7 promoter inserted before the start codon of the ddh gene, and the strain was named L-ddh.

[0144] P17: 5'-CCACGAGACCCAATCCTATC-3' (SEQ ID NO: 36);

[0145] P18: 5'-CTCGACACGGAAACCTACG-3' (SEQ ID NO: 37);

[0146] P19: 5'-CCGGAATAATTGGCAGCT-3' (SEQ ID NO: 38);

[0147] P20: 5'-TCTCTCCCTATTTGAGGCG-3' (SEQ ID NO: 39).

[0148] The recombinant strain L-ddh differs from Corynebacterium glutamicum YP097158 only in that the recombinant strain L-ddh has the EP7 promoter sequence (SEQ ID NO: 1) inserted before the start codon of the ddh gene in the genome of Corynebacterium glutamicum YP097158, and the other sequences remain unchanged.

[0149] Example 4, Construction of a strain in which the EP7 promoter regulates the expression of the lysA gene

[0150] According to the sequence of the EP7 promoter with the nucleotide sequence of SEQ ID NO: 1 and the genome sequence of Corynebacterium glutamicum ATCC13032 published on NCBI, primers are designed and synthesized for inserting the EP7 promoter in front of the start codon ATG of the lysA gene to enhance the expression of the lysA gene. The specific primer design is as follows (synthesized by Shanghai Invitrogen Company):

[0151] P21: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGATGTCTACTACCACGAGAAC-3' (SEQ ID NO: 40, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector),

[0152] P22: 5'-AGCCCGGAATAATTGGCAGCTTGTTACATCTTCTCCGGTG-3' (SEQ ID NO: 41);

[0153] P23: 5'-CACCGGAGAAGATGTAACAAGCTGCCAATTATTCCGGGCT-3' (SEQ ID NO: 42);

[0154] P24: 5'-GAAATTTTCAACTGTAGCCATGGGTAAAAAATCCTTTCGTAG-3' (SEQ ID NO: 43);

[0155] P25: 5'-CTACGAAAGGATTTTTTACCCATGGCTACAGTTGAAAATTTC-3' (SEQ ID NO: 44);

[0156] P26: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCGTATTCGGAGCCGTAGAG-3' (SEQ ID NO: 45, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector).

[0157] Construction method:

[0158] The genomic DNA of C. glutamicum ATCC13032 was used as a template for PCR amplification with primers P21 / P22, P23 / P24 and P25 / P26, respectively, to obtain a 966 bp fragment containing the upstream homologous arm (amplification product of primers P21 / P22, sequence: "SEQ ID NO: 24 + SEQ ID NO: 8, positions 1-930"), a 231 bp fragment containing the EP7 promoter (amplification product of primers P23 / P24, sequence: SEQ ID NO: 8, positions 891-1121), and a 1114 bp fragment containing the downstream homologous arm (amplification product of primers P25 / P26, sequence: "SEQ ID NO: 8, positions 1080-2155 + SEQ ID NO: 25").

[0159] After the PCR reaction, the three fragments obtained by amplification were recovered by column type DNA gel recovery kit respectively. The three recovered fragments and the pK18mobsacB plasmid (product of Addgene company) digested by Xbal I and BamH I and purified were ligated by NEBuilder enzyme (product of NEB company) at 50 °C for 60 min, and the single colony grown after transformation of the ligation product was identified by PCR with primers P21 / P26 (amplification product sequence: "SEQ ID NO: 24 + SEQ ID NO: 8 + SEQ ID NO: 25") to obtain a positive integrated plasmid (recombinant vector). The obtained recombinant vector was pK18-EP7-lysA, and the positive integrated plasmid contained a kanamycin resistance marker, which could be used to screen the recombinants in which the plasmid was integrated into the genome by kanamycin. The recombinant plasmid pK18-EP7-lysA has been sequenced and verified. Compared with the pK18mobsacB plasmid, the difference of the recombinant plasmid pK18-EP7-lysA is only that the double-stranded DNA molecule with the nucleotide sequence of SEQ ID NO: 8 replaces "AGGATCCCC" in the pK18mobsacB plasmid. SEQ ID NO: 8, positions 1-909 correspond to part of the coding region of the NCgl1132 gene of C. glutamicum ATCC13032, positions 910-1100 are the EP7 promoter sequence, and positions 1101-2155 correspond to part of the coding region of the lysA gene of C. glutamicum ATCC13032.

[0160] The correctly sequenced integration plasmid pK18-EP7-lysA was electroporated into Corynebacterium glutamicum YP097158, and then cultured on solid medium plates (see Table 1 for medium components) for 40 h. The single colonies produced by the culture were identified by PCR using primers P27 / P28. The positive strain was identified by PCR amplification of a 987 bp fragment (nucleotide sequence: SEQ ID NO: 9), and the original strain was identified by the absence of amplification. The positive strain was streaked on solid medium plates containing 15% sucrose (the sucrose content in the medium in Table 1 was adjusted to 150 g / L) and cultured for 40 h. The single colonies produced by the culture were further identified by PCR using primers P29 / P30. The positive strain was identified by PCR amplification of a 1209 bp fragment (nucleotide sequence: SEQ ID NO: 10) containing the EP7 promoter inserted before the start codon of the lysA gene, and the strain was named L-lysA.

[0161] P27: 5'-AGGCGTGGAGATGATGTTC-3' (SEQ ID NO: 46);

[0162] P28: 5'-CCGGAATAATTGGCAGCT-3' (SEQ ID NO: 47);

[0163] P29: 5'-CTCGACACGGAAACCTACG-3' (SEQ ID NO: 48);

[0164] P30: 5'-TGGTGATGTCAGATGGGTAG-3' (SEQ ID NO: 49).

[0165] The recombinant strain L-lysA differs from Corynebacterium glutamicum YP097158 only in that the recombinant strain L-lysA has the EP7 promoter sequence (SEQ ID NO: 1) inserted before the start codon of the lysA gene in the genome of Corynebacterium glutamicum YP097158, and the other sequences remain unchanged.

[0166] Example 5, Construction of a strain in which the EP7 promoter regulates the expression of the gnd gene

[0167] According to the sequence of EP7 of SEQ ID NO: 1 and the genome sequence of Corynebacterium glutamicum ATCC13032 published on NCBI, primers were designed and synthesized for inserting the EP7 promoter in front of the start codon ATG of the gnd gene to enhance the expression of the gnd gene. The primers are as follows (synthesized by Shanghai Invitrogen Company):

[0168] P31: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTAGCGGAGAAGGTGGTATG-3' (SEQ ID NO: 50, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector);

[0169] P32: 5'-GAAAGGATTTTTTACCCATGACTAATGGAGATAATCTCGCACAG-3' (SEQ ID NO: 51);

[0170] P33: 5'-CATTAGTCATGGGTAAAAAATCCTTTCGTAGGTTTCCGT-3' (SEQ ID NO: 52);

[0171] P34: 5'-GATTTTGCTGACACCGGGCTAGCTGCCAATTATTCCGGGCTC-3' (SEQ ID NO: 53);

[0172] P35: 5'-GAGCCCGGAATAATTGGCAGCTAGCCCGGTGTCAGCAAAATC-3' (SEQ ID NO: 54);

[0173] P36: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTTGGCGTAATCAATCAGG-3' (SEQ ID NO: 55, the underlined nucleotide sequence is the sequence on the pK18mobsacB vector).

[0174] Construction method: The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primers P31 / P32, P33 / P34, and P35 / P36 were used for PCR amplification, respectively, to obtain a 682 bp fragment containing the upstream homologous arm (the primer P31 / P32 amplification product, the sequence is "SEQ ID NO: 24+SEQ ID NO: 11 1-646"), a 221 bp fragment containing the EP7 promoter (the primer P33 / P34 amplification product, the sequence is SEQ ID NO: 11 620-840), and a 910 bp fragment containing the downstream homologous arm (the primer P35 / P36 amplification product, the sequence is "SEQ ID NO: 11 799-1670+ SEQ ID NO: 25").

[0175] After the PCR reaction, the three fragments obtained by amplification were recovered by column type DNA gel recovery kit respectively. The three recovered fragments and the pK18mobsacB plasmid (Addgene product) digested by Xbal I and BamH I were ligated at 50°C for 60 min using NEBuilder enzyme (NEB product). The single colony obtained after transformation of the ligation product was identified by PCR using primers P31 / P36 (amplification product sequence "SEQ ID NO: 24+SEQ ID NO: 11+SEQ ID NO: 25") to obtain a positive integration plasmid (recombinant vector). The obtained recombinant vector was pK18-EP7-gnd, which contained a kanamycin resistance marker on the positive integration plasmid, and the recombinant strain with the plasmid integrated into the genome could be obtained by kanamycin screening. The recombinant plasmid pK18-EP7-gnd has been sequenced and verified. Compared with the pK18mobsacB plasmid, the difference of the recombinant plasmid pK18-EP7-gnd is only that the double-stranded DNA molecule with the nucleotide sequence of SEQ ID NO: 11 replaces "AGGATCCCC" in the pK18mobsacB plasmid. SEQ ID NO: 11 1-629 corresponds to the reverse complementary sequence of part of the coding region of the gnd gene of Corynebacterium glutamicum ATCC13032, 630-820 is the reverse complementary sequence of the EP7 promoter, and 821-1670 corresponds to the reverse complementary sequence of part of the coding region of the NCgl1397 gene of Corynebacterium glutamicum ATCC13032.

[0176] The correctly sequenced integrated plasmid pK18-EP7-gnd was electroporated into Corynebacterium glutamicum YP097158, and then cultured on solid medium plates (see Table 1 for medium composition) for 40 h. Single colonies produced during the culture were identified by PCR using primers P37 / P38. A positive strain was identified by PCR amplification of a 1015 bp fragment (nucleotide sequence: SEQ ID NO: 12) using primers P37 / P38. A strain that did not amplify the fragment was the original strain. The positive strain was streaked on solid medium plates containing 15% sucrose (adjust the sucrose content in the medium in Table 1 to 150 g / L) and cultured for 40 h. Single colonies produced during the culture were further identified by PCR using primers P39 / P40. A positive strain was identified by PCR amplification of a 988 bp fragment (nucleotide sequence: SEQ ID NO: 13) using primers P39 / P40. The strain was named L-gnd.

[0177] P37: 5'-ACGAACTGTGCCTTGTCCAC-3' (SEQ ID NO: 56);

[0178] P38: 5'-CTCGACACGGAAACCTACG-3' (SEQ ID NO: 57);

[0179] P39: 5'-CCGGAATAATTGGCAGCT-3' (SEQ ID NO: 58);

[0180] P40: 5'-ACCATCCTCAGCGAGAAAG-3' (SEQ ID NO: 59).

[0181] The recombinant strain L-gnd differs from Corynebacterium glutamicum YP097158 only in that the recombinant strain L-gnd has an EP7 promoter sequence (SEQ ID NO: 1) inserted before the start codon of the gnd gene in the genome of Corynebacterium glutamicum YP097158, and the other sequences remain unchanged.

[0182] Example 6, L-lysine fermentation experiment

[0183] The strains constructed in Examples 2-5 and the original strain Corynebacterium glutamicum YP097158 were subjected to fermentation experiments in 500 mL baffled shake flasks using the medium in Table 2 and the control conditions in Table 3. After the fermentation, the L-lysine yield was determined using an SBA-biosensor analyzer (Shandong Academy of Sciences). Each strain was repeated three times, and the results are shown in Table 4.

[0184]

[0185]

[0186]

[0187] Note: P<0.01 in the table means that there is a significant difference compared with the L-lysine yield of YP097158.

[0188] As can be seen from Table 4, the expression of the lysC, ddh, lysA and gnd genes in Corynebacterium glutamicum YP097158 is regulated by the EP7 promoter (SEQ ID NO: 1), which helps to improve the yield of L-lysine.

[0189] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In general, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

[0190] Cross-reference of related applications:

[0191] This application claims priority to Chinese patent application (application number 202410996454.2) with a filing date of July 24, 2024, the entire contents of which are hereby incorporated by reference. Industrial applicability

[0192] The present application replaces any promoter of the lysC gene, ddh gene, lysA gene and gnd gene in Corynebacterium glutamicum with the EP7 promoter with a nucleotide sequence of SEQ ID NO: 1, which can significantly improve the yield of L-lysine (p<0.01). The present application shows that replacing the wild-type promoter of other key genes in the L-lysine metabolic pathway on the chromosome of Corynebacterium bacillus with the EP7 promoter can also enhance the expression of the corresponding genes and ultimately improve the yield of L-lysine. In particular, this method does not conflict with the existing chromosome modification sites of a large number of high-yield L-lysine bacteria, and can be superimposed to improve the effect, so it can be used in practice for the fermentation production of L-lysine by various bacteria. The present application has important significance for improving the yield of L-lysine.

Claims

1. A DNA molecule comprising any one of the following: (a1) a DNA molecule having a nucleotide sequence of SEQ ID NO: 1; (a2) a DNA molecule having a promoter function obtained by conservatively replacing some nucleotides in SEQ ID NO:

1.

2. Use of the DNA molecule of claim 1 as a promoter.

3. An expression cassette, a recombinant vector or a recombinant bacterium comprising the DNA molecule of claim 1.

4. The recombinant bacteria of claim 3, wherein: The recombinant bacterium is recombinant bacterium A or recombinant bacterium B or recombinant bacterium C or recombinant bacterium D; The recombinant bacterium A is a recombinant bacterium obtained by inserting the DNA molecule of claim 1 before the start codon of the bacterial lysC gene; The recombinant bacterium B is a recombinant bacterium obtained by inserting the DNA molecule of claim 1 before the start codon of the bacterial ddh gene; The recombinant bacterium C is a recombinant bacterium obtained by inserting the DNA molecule of claim 1 before the start codon of the bacterial lysA gene; The recombinant bacterium D is a recombinant bacterium obtained by inserting the DNA molecule of claim 1 before the start codon of the bacterial gnd gene.

5. The recombinant bacteria of claim 4, wherein: The bacterium is a Corynebacterium microorganism; Further, the Corynebacterium microorganism is Corynebacterium glutamicum.

6. Use of any one of the following: P1, the DNA molecule of claim 1 or the expression cassette or the recombinant vector or the recombinant bacterium of any one of claims 3-5 in the production of L-amino acid; P2, the DNA molecule of claim 1 or the expression cassette or the recombinant vector of any one of claims 3-5 in the construction of an L-amino acid-producing engineering strain; P3, the DNA molecule of claim 1 or the expression cassette or the recombinant vector of any one of claims 3-5 in the preparation of food, medicine, feed or cosmetics containing L-amino acid.

7. Use according to claim 6, characterized in that: The L-amino acid is L-lysine.

8. A method for improving the production of L-amino acid by a bacterium, comprising the following steps (A) or (B) or (C) or (D): (A) inserting the DNA molecule of claim 1 before the start codon of the bacterial lysC gene to improve the production of the L-amino acid by the bacterium; (B) inserting the DNA molecule of claim 1 before the start codon of the bacterial ddh gene to improve the production of the L-amino acid by the bacterium; (C) inserting the DNA molecule of claim 1 before the start codon of the bacterial lysA gene to improve the production of the L-amino acid by the bacterium; (D) inserting the DNA molecule of claim 1 before the start codon of the bacterial gnd gene to improve the production of the L-amino acid by the bacterium.

9. The method of claim 8, wherein: The bacterium is a Corynebacterium microorganism; Further, the Corynebacterium microorganism is Corynebacterium glutamicum.

10. The method according to claim 8 or 9, characterized in that: The L-amino acid is L-lysine.

11. A method for producing an L-amino acid, comprising the steps of: introducing the DNA molecule of claim 1 into a biological cell capable of synthesizing the L-amino acid, allowing the DNA molecule of claim 1 to drive expression of related genes in the L-amino acid synthesis pathway in the biological cell to obtain a recombinant biological cell; culturing the recombinant biological cell to obtain the L-amino acid.

12. The method of claim 11, wherein: The biological cell is a bacterium, a yeast, an alga, a fungus, a plant cell, or an animal cell capable of synthesizing the L-amino acid.

13. The method of claim 12, wherein: The bacterium is a Corynebacterium glutamicum microorganism. Further, the Corynebacterium glutamicum microorganism is Corynebacterium glutamicum.

14. The method of any one of claims 11-13, wherein: The L-amino acid is L-lysine.

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