Use of pncgl1969(C200T) promoter and biomaterial related thereto in construction of high-yield l-lysine engineered strain

Abstract

Disclosed is the use of a PNcgl1969(C200T) promoter and a biomaterial related thereto in the construction of a high-yield L-lysine engineered strain. The promoter is a DNA molecule obtained by means of mutating cytosine at position 200 in the PNcgl1969 promoter to thymine. By means of performing L-lysine fermentation experiments on the constructed engineered strains L-PNcgl1969 and Y-PNcgl1969 containing the PNcgl1969(C200T) promoter, it is found that a point mutation of the PNcgl1969 promoter region in Corynebacterium glutamicum is conducive to improving the yield of L-lysine. The present invention is of great significance for increasing the yield of L-lysine produced by means of bacterial fermentation.

Description

PNcgl1969(C200T) promoter and related biological materials thereof in constructing high-yield L-lysine engineering bacteria

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410728034.6, filed on June 6, 2024, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biotechnology, and specifically relates to the application of PNcgl1969(C200T) promoter and related biological materials thereof in constructing high-yield L-lysine engineering bacteria. BACKGROUND

[0004] The English name of lysine is Lysine, which includes two optical isomers, L-type and D-type, respectively. Among them, D-lysine has no biological activity, and L-lysine can be absorbed by the human body, but L-lysine is an essential amino acid and can only be obtained by external supplementation.

[0005] L-lysine has a wide range of applications, including medicine, food, feed and other aspects, of which L-lysine used in feed additives accounts for 90% of the total.

[0006] Due to the wide application of L-lysine, its demand is increasing every year worldwide, so various studies are being conducted to develop high-efficiency microbial strains and fermentation process technologies for producing L-lysine. For example, replacing the promoter of a key gene affecting L-lysine synthesis in a 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.

[0007] SUMMARY

[0008] The technical problem to be solved by the present application is how to improve the yield of microbial fermentation for producing L-lysine. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0009] To solve the above technical problem, the present application first provides a DNA molecule.

[0010] The DNA molecule provided by the present application is any one of the following A1)-A5):

[0011] A1) a DNA molecule comprising (or whose nucleotide sequence is) the nucleotide sequence shown as Seq ID No. 2;

[0012] A2) a DNA molecule complementary to the nucleotide sequence shown as Seq ID No. 2;

[0013] A3) a DNA molecule capable of hybridizing to the nucleotide sequence of A1) or A2) under high stringency conditions;

[0014] A4) a DNA molecule obtained by modifying the nucleotide sequence of A1) or A2) by substitution and / or deletion and / or addition of one or more nucleotides;

[0015] A5) a DNA molecule having at least 90% identity to the nucleotide sequence of A1) or A2).

[0016] The above-mentioned DNA molecule is PNcgl1969(C200T) promoter in the present application.

[0017] Those skilled in the art can easily mutate the nucleotide sequence of the DNA molecule of the present application by using known methods, such as methods of directed evolution and point mutation. Those nucleotides artificially modified, having 90% or more identity to the nucleotide sequence of the DNA molecule provided by the present application, as long as having the function of a promoter, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0018] In the DNA molecule of A3), the high stringency conditions are hybridization in a solution of 2xSSC, 0.1% SDS at 68°C and washing the membrane twice, each for 5 min; or hybridization in a solution of 0.5xSSC, 0.1% SDS at 68°C and washing the membrane twice, each for 15 min; or hybridization in a solution of 0.1xSSPE (or 0.1xSSC), 0.1% SDS at 65°C.

[0019] In the DNA molecule of A4) above, the substitution and / or deletion and / or addition of the plurality of nucleotides can specifically be a substitution and / or deletion and / or addition of no more than 10 nucleotides, or a substitution and / or deletion and / or addition of no more than 9 nucleotides, or a substitution and / or deletion and / or addition of no more than 8 nucleotides, or a substitution and / or deletion and / or addition of no more than 7 nucleotides, or a substitution and / or deletion and / or addition of no more than 6 nucleotides, or a substitution and / or deletion and / or addition of no more than 5 nucleotides, or a substitution and / or deletion and / or addition of no more than 4 nucleotides, or a substitution and / or deletion and / or addition of no more than 3 nucleotides, or a substitution and / or deletion and / or addition of no more than 2 nucleotides, or a substitution and / or deletion and / or addition of no more than 1 nucleotide.

[0020] In the DNA molecule of A5) above, the identity refers to sequence similarity to the sequence of the natural nucleic acid sequence, including a nucleotide sequence having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 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 the DNA molecule of the present application. The identity can be evaluated by the naked eye 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.

[0021] Any of the DNA molecules described above has promoter activity.

[0022] To solve the above technical problems, the present application further provides a biological material related to the DNA molecule.

[0023] The biological material related to the DNA molecule provided by the present application is any of the following B1) to B5):

[0024] B1) a nucleic acid construct containing the DNA molecule described above;

[0025] B2) an expression cassette containing the DNA molecule described above;

[0026] B3) a recombinant vector containing the DNA molecule described above;

[0027] B4) a recombinant microorganism containing the DNA molecule described above;

[0028] B5) a whole-cell catalyst containing the DNA molecule described above.

[0029] In the above biological material, the nucleic acid construct of B1) comprises the above-mentioned DNA molecule and a gene sequence (such as the Ncgl 1969 gene sequence) operably linked to the DNA molecule. The DNA molecule and the gene sequence can be from the same source or different sources.

[0030] In the above biological material, the expression cassette of B2) can comprise (5' to 3') a promoter region (consisting of the above-mentioned DNA molecule), a transcription initiation region, a gene of interest region, a transcription termination region, and an optional translation termination region. The promoter region and the gene of interest region can be native / analogous to the host cell, or the promoter region and the gene of interest region can be native / analogous to each other, or the promoter region and / or the gene of interest region can be heterologous to the host or to each other. "Heterologous" means a sequence that is foreign to the species, or, if from the same species, has been substantially modified from the native form by deliberate human intervention in terms of components and / or genomic site. The optional transcription termination region can be homologous to the transcription initiation region, to the operably linked gene of interest region, to the host; or the gene of interest region, the host are foreign or heterologous. Further, the expression cassette can further comprise a 5' leader sequence. The 5' leader sequence can enhance translation.

[0031] In preparing the expression cassette, adapters can be used to ligate DNA fragments, or other manipulations can be involved to provide appropriate restriction sites, removal of excess DNA, removal of restriction sites, etc. To achieve this, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, re-substitution, such as transversion and transversion, can be performed.

[0032] The expression cassette can further comprise a selectable marker gene for screening the transformed cells. The selectable marker gene can be used to screen the transformed cells or tissues. The marker gene includes a gene encoding antibiotic resistance. Other selectable markers include phenotypic markers such as fluorescent proteins. The above listed selectable markers are not limiting. Any selectable marker gene can be used in the present application.

[0033] In the above biological material, the vector of B3) includes but is not limited to plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e. cos plasmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0034] The recombinant vector can be a vector containing the above-mentioned DNA molecule or the above-mentioned expression cassette constructed using existing expression vectors.

[0035] In some embodiments, the recombinant vector is a recombinant vector pK18- PNcgl1969(C200T). The recombinant vector pK18-PNcgl1969(C200T) is a recombinant vector obtained by replacing a fragment (a small fragment) between Xbal I and BamH I recognition sites of a pK18mobsacB vector with a DNA fragment shown in SEQ ID No. 3, while maintaining other sequences of the pK18mobsacB vector unchanged.

[0036] Among the above-mentioned biomaterials, the microorganism of B4) can be a bacterium, a fungus, an actinomycete, an alga, or a virus. Among them, the bacterium can be from Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium, or Bacillus, but is not limited thereto. The fungus can be a yeast, which can be from Saccharomyces (e.g., Saccharomyces cerevisiae), Kluyveromyces (e.g., Kluyveromyces lactis), Pichia (e.g., Pichia pastoris), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Hansenula (e.g., Hansenula polymorpha), or the like, but is not limited thereto. The fungus can also be from Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., or the like, but is not limited thereto. The actinomycete can be from Streptomyces sp., Nocardia sp., Micromonospora sp., Streptosporangium sp., Actinoplanes sp., Thermoactinomyces sp., or the like, but is not limited thereto. The alga can be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., or the like, but is not limited thereto. The virus can be a rotavirus, a herpes virus, an influenza virus, an adenovirus, or the like, but is not limited thereto.

[0037] Further, the bacteria include, but are not limited to, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Micrococcus glutamicus, Brevibacterium ammoniagenes, Escherichia coli, Aerobacter aerogenes.

[0038] Further, the bacteria are Corynebacterium glutamicum or a mutant thereof.

[0039] In some embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 strain. The Corynebacterium glutamicum YP097158 strain has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on August 16, 2016, and has the accession number of CGMCC No. 12856.

[0040] In other embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032 strain.

[0041] The recombinant microorganism can be a microorganism containing the nucleic acid construct described above or the expression cassette described above or the recombinant vector described above.

[0042] In some embodiments, the recombinant microorganism is recombinant bacteria L-PNcgl1969. The recombinant bacteria L-PNcgl1969 is a strain obtained by replacing the PNcgl1969 promoter sequence in the genome sequence of Corynebacterium glutamicum YP097158 with the PNcgl1969(C200T) promoter sequence shown in SEQ ID No. 2, and keeping other sequences unchanged.

[0043] In some embodiments, the recombinant microorganism is a recombinant strain Y-PNcgl1969. The recombinant strain Y-PNcgl1969 is a strain obtained by replacing the PNcgl1969 promoter sequence in the genomic sequence of Corynebacterium glutamicum ATCC 13032 with the PNcgl1969 (C200T) promoter sequence shown in SEQ ID No. 2, while keeping other sequences unchanged.

[0044] To solve the above technical problems, the present application also provides a new use of the above DNA molecule or the above biological material.

[0045] The present application provides an application of the above DNA molecule as a promoter.

[0046] The present application also provides an application of the above DNA molecule in promoting expression of a target gene as a promoter.

[0047] The present application also provides an application of the above DNA molecule or the above biological material in constructing a biomolecule engineering strain.

[0048] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for constructing a biomolecule engineering strain.

[0049] The present application also provides an application of the above DNA molecule or the above biological material in improving biomolecule yield.

[0050] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for improving biomolecule yield.

[0051] The present application also provides an application of the above DNA molecule or the above biological material in producing biomolecules.

[0052] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for producing biomolecules.

[0053] The present application also provides an application of the above DNA molecule or the above biological material in regulating expression of a target gene in a microorganism or in microbial breeding.

[0054] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for regulating expression of a target gene in a microorganism or for microbial breeding.

[0055] In any of the above-mentioned applications, the biomolecule is at least one of lysine, valine, glycine, glutamic acid, 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.

[0056] In some embodiments, the biomolecule is L-lysine.

[0057] In actual applications, when the above-mentioned biomolecule is produced, the above-mentioned DNA molecule can be placed upstream of a target gene related to the synthesis pathway of the biomolecule and the DNA molecule drives the expression of the target gene to achieve the production of the target biomolecule or the increase of the yield of the target biomolecule.

[0058] The purpose of the above-mentioned microbial breeding is to breed microorganisms with increased yield of target biomolecule (e.g., microorganisms with increased yield of L-lysine).

[0059] To solve the above-mentioned technical problems, the present application further provides a method for constructing an L-lysine-producing engineered bacterium or a method for increasing the yield of L-lysine.

[0060] The method for constructing an L-lysine-producing engineered bacterium or the method for increasing the yield of L-lysine provided by the present application comprises the step of mutating the base C corresponding to the 200th base in the DNA molecule shown in SEQ ID No. 1 to base T in a microorganism with the ability to produce L-lysine.

[0061] Further, the method for mutating the base C corresponding to the 200th base in the DNA molecule shown in SEQ ID No. 1 to base T in the microorganism with the ability to produce L-lysine can be replacing the DNA molecule shown in SEQ ID No. 1 in the microorganism with the ability to produce L-lysine with the DNA molecule shown in SEQ ID No. 2.

[0062] Still further, the method for replacing the DNA molecule shown in SEQ ID No. 1 in the microorganism with the ability to produce L-lysine with the DNA molecule shown in SEQ ID No. 2 can be introducing a substance that replaces the DNA molecule shown in SEQ ID No. 1 with the DNA molecule shown in SEQ ID No. 2 into the microorganism with the ability to produce L-lysine.

[0063] In some embodiments, the substance that replaces the DNA molecule shown in SEQ ID No. 1 with the DNA molecule shown in SEQ ID No. 2 is the above-mentioned recombinant vector pK18-PNcgl1969(C200T).

[0064] The L-lysine-producing engineering bacteria constructed according to the above method also belong to the protection scope of the present application.

[0065] To solve the above technical problems, the present application finally provides a method for producing L-lysine.

[0066] The method for producing L-lysine provided by the present application comprises the step of fermenting and culturing the L-lysine-producing engineering bacteria constructed according to the above method.

[0067] The fermentation and culturing method can be performed according to the conventional test method in the prior art. The conventional test method can also be optimized and improved.

[0068] In some embodiments, the fermentation medium formula is shown in Table 2 in the following examples.

[0069] In some embodiments, the fermentation culturing conditions are shown in Table 3 in the following examples.

[0070] In any of the above methods, the genome sequence of the microorganism having the ability to produce L-lysine contains the DNA molecule shown in SEQ ID No. 1.

[0071] In any of the above methods, the microorganism having the ability to produce L-lysine can be any bacteria having the ability to produce L-lysine. The bacteria having the ability to produce L-lysine have the ability to produce and accumulate L-lysine in the bacteria body by using external substances (such as culture medium), and further can include the ability to secrete L-lysine into the culture system, so that L-lysine can be collected when the bacteria are cultured in the culture medium.

[0072] Further, the bacteria having the ability to produce L-lysine include but are not limited to bacteria of Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium or Bacillus.

[0073] Further, the bacteria having the ability to produce L-lysine include, but are not limited to, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Micrococcus glutamicus, Brevibacterium ammoniagenes, Escherichia coli, Aerobacter aerogenes.

[0074] Further, the bacteria having the ability to produce L-lysine is Corynebacterium glutamicum or a mutant thereof.

[0075] In some embodiments, the bacteria having the ability to produce L-lysine is Corynebacterium glutamicum YP097158 strain. The Corynebacterium glutamicum YP097158 strain has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on August 16, 2016, and has the accession number of CGMCC No. 12856.

[0076] In other embodiments, the bacteria having the ability to produce L-lysine is Corynebacterium glutamicum ATCC13032 strain.

[0077] Any of the above-mentioned target genes can be an endogenous gene (i.e., a gene in the genome of the organism itself), such as the Ncgl1969 gene associated with L-lysine synthesis in Corynebacterium glutamicum, or an exogenous gene or a heterologous gene, such as the visualization marker gene mCherry.

[0078] The present application provides a PNcgl1969(C200T) promoter, which is a DNA molecule obtained by mutating a cytosine (C) at position 200 in a PNcgl1969 promoter to thymine (T), and by mutating the cytosine (C) at position 200 in the PNcgl1969 promoter region of Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 to thymine (T), two engineering strains L-PNcgl1969 and Y-PNcgl1969 containing the PNcgl1969(C200T) promoter are constructed. Through L-lysine fermentation experiments on the engineering strains L-PNcgl1969 and Y-PNcgl1969, it is found that the point mutation (C200T) of the PNcgl1969 promoter region (PNcgl1969 promoter region) in Corynebacterium glutamicum helps to improve the yield of L-lysine.

[0079] DEPOSIT DESCRIPTION

[0080] Strain name: Corynebacterium glutamicum

[0081] Latin name: Corynebacterium glutamicum

[0082] Strain number: YP097158

[0083] Preservation agency: China General Microbiological Culture Collection Center

[0084] Preservation agency abbreviation: CGMCC

[0085] Address: No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing

[0086] Preservation date: August 16, 2016

[0087] Preservation center registration number: CGMCC No. 12856 Embodiment of the present application

[0088] The present application will be further described in detail below with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0089] In the following examples, the experimental methods are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0090] The Corynebacterium glutamicum YP097158 strain in the following examples was deposited with the China General Microbiological Culture Collection Center (CGMCC) on August 16, 2016, and the deposit number is CGMCC No. 12856.

[0091] The Corynebacterium glutamicum ATCC13032 in the following examples was obtained from the American Type Culture Collection (ATCC), and the deposit number is 13032.

[0092] The plasmid pEC-XK99E in the following examples is described in the literature "Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum Front Bioeng Biotechnol. 2023; 11: 1194511."

[0093] The corn steep liquor in the following examples has a concentration of 18-20°Be and a solvent of water, and is a product of Ningxia Yipin Biotechnology Co., Ltd.

[0094] Example 1, construction of a recombinant vector containing a PNcgl1969 promoter with a point mutation

[0095] According to the Corynebacterium glutamicum ATCC13032 genome sequence published by NCBI, two pairs of primers for amplifying the PNcgl1969 promoter region were designed and synthesized. A point mutation was introduced into the PNcgl1969 promoter region of Corynebacterium glutamicum YP097158 by allelic replacement, which was a mutation of cytosine (C) at position 200 in the PNcgl1969 promoter nucleotide sequence (SEQ ID No. 1) to thymine (T), to obtain the mutated PNcgl1969 promoter. The mutated PNcgl1969 promoter was named PNcgl1969(C200T), and its nucleotide sequence is shown in SEQ ID No. 2. The NEBuilder assembly technology was used to construct a recombinant vector containing the PNcgl1969(C200T) promoter. The specific steps are as follows:

[0096] 1. Design the following primer sequences:

[0097] P1: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGGTGTCTTTGTGCTCTTC-3' (the underlined nucleotide sequence is the sequence on the pK18mobsacB plasmid, SEQ ID No. 5).

[0098] P2: 5'-CGTGGGGCAAGTGTGAGTTTTCCTACTTTTCGG-3' (the underlined bases are the mutation positions, SEQ ID No. 6).

[0099] P3: 5'-CGAAAAGTAGGAAAACTCACACTTGCCCCACG-3' (the underlined bases are the mutation positions, SEQ ID No. 7).

[0100] P4: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGACCTACTGGTATGGAACTTC-3' (the underlined nucleotide sequence is the sequence on the pK18mobsacB plasmid, SEQ ID No. 8).

[0101] The above primer sequences were synthesized by Shanghai Invitrogen Company.

[0102] 2. Taking Corynebacterium glutamicum ATCC13032 as the template, PCR amplification was performed with primers P1 / P2 and P3 / P4 respectively, and two homologous DNA fragments (PNcgl1969Up and PNcgl1969Down) with mutation bases and sizes of 591 bp and 514 bp respectively were obtained.

[0103] The PCR amplification system was as follows: 5×HiFi with Mg Buffer 10 μL, dNTP Mixture (10 mM) 1.5 μL, primers (10 pM) 1.6 μL each, KAPA HiFi HotStart (1 U / μL) 0.5 μL, and ddH2O was added to a total volume of 50 μL. 2+ The PCR amplification system was as follows: 5×HiFi with Mg Buffer 10 μL, dNTP Mixture (10 mM) 1.5 μL, primers (10 pM) 1.6 μL each, KAPA HiFi HotStart (1 U / μL) 0.5 μL, and ddH2O was added to a total volume of 50 μL.

[0104] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 98°C for 20 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, 30 cycles, and over-extension at 72°C for 5 min.

[0105] 3. The two DNA fragments (PNcgl1969Up and PNcgl1969Down) obtained in step 2 were separated and purified by agarose gel electrophoresis. They were then ligated with the pK18mobsacB plasmid (Addgene) purified after enzyme digestion (Xbal I / BamHI) using NEBuilder enzyme (NEB) at 50°C for 30 min to obtain the ligation product. The single clones grown after transformation of the ligation product were identified by PCR using primers P1 / P4. The positive recombinant vector that amplified a 1073 bp fragment (the nucleotide sequence of the 1073 bp fragment is shown in SEQ ID No. 3) was named pK18-PNcgl1969(C200T). This recombinant vector pK18-PNcgl1969(C200T) contained a kanamycin resistance marker. The recombinant vector pK18-PNcgl1969(C200T) was identified by enzyme digestion. The correctly digested recombinant vector pK18-PNcgl1969(C200T) was sent to a sequencing company for sequencing identification. The recombinant vector pK18-PNcgl1969(C200T) containing the correct point mutation (C200T) was stored for future use.

[0106] The recombinant vector pK18-PNcgl1969(C200T) contains the mutation site (C200T) shown in SEQ ID No.2, which will cause the cytosine (C) at position 200 of the promoter region of PNcgl1969 in Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 to be mutated to thymine (T).

[0107] The recombinant vector pK18-PNcgl1969(C200T) is a recombinant vector obtained by replacing the fragment (small fragment) between the Xbal I and BamH I recognition sites of the pK18mobsacB vector with the DNA fragment shown in SEQ ID No. 3, while keeping the other sequences of the pK18mobsacB vector unchanged.

[0108] Example 2: Activity detection of promoter PNcgl1969 (C200T)

[0109] The nucleotide sequence of the mCherry (red fluorescent protein) gene was downloaded and synthesized on the NCBI website (the nucleotide sequence is shown as SEQ ID No. 4); the plasmid backbone was amplified from the plasmid pEC-XK99E using primers pEC-F / pEC-R, and the recombinant vector pEC-PNcgl1969-mCherry was constructed using the NEBuilder assembly technique; the recombinant vector pEC-PNcgl1969-mCherry was transformed into C. glutamicum YP097158 to verify whether PNcgl1969 (C200T) has promoter activity. The specific steps are as follows:

[0110] 1. The following primer sequences were designed:

[0111] pEC-F: 5'-CTAGAGTCGACCTGCAGGCATGCAAGCTTG-3' (SEQ ID No. 9).

[0112] pEC-R: 5'-CTAGAGGATCCCCGGGTACCGAGCTCGAATTC-3' (SEQ ID No. 10).

[0113] P5: 5'-GAATTCGAGCTCGGTACCCGGGGATCCTCTAGCAGGTGTCAAAATCGCCCATTAC-3' (the underlined nucleotide sequence is the sequence on the pEC-XK99E plasmid, SEQ ID No. 11).

[0114] P6: 5'-GTTATCTTCTTCTCCTTTACGCATCCAGCTTTCGATTGTGGCATC-3' (SEQ ID No. 12).

[0115] P7: 5'-GATGCCACAATCGAAAGCTGGATGCGTAAAGGAGAAGAAGATAAC-3' (SEQ ID No. 13).

[0116] P8: 5'-CAAGCTTGCATGCCTGCAGGTCGACTCTAGTTTGTATAGTTCATCCATGCCAC-3' (the underlined nucleotide sequence is the sequence on the pEC-XK99E plasmid, SEQ ID No. 14).

[0117] The above-mentioned primer sequences were synthesized by Shanghai invitrogen company.

[0118] 2. Using the plasmid pk18-PNcgl1969(C200T) and mCherry gene fragment from Example 1 as templates, PCR amplification was performed using primers P5 / P6 and P7 / P8, respectively, to obtain the 330bp promoter PNcgl1969(C200T) and the 705bp mCherry gene fragment.

[0119] 3. After separating and purifying the two DNA fragments obtained in step 2 and the pEC-XK99E plasmid backbone by agarose gel electrophoresis, the fragments were ligated using NEBuilder enzyme (NEB) at 50℃ for 30 min to obtain the ligation product. The ligation product was transformed into E. coli DH5α competent cells, and the resulting single clones were identified by PCR using primers P5 / P8. The correctly sequenced positive recombinant vector was named pEC-PNcgl1969-mCherry, which contains a kanamycin resistance marker.

[0120] 4. The recombinant vector pEC-PNcgl1969-mCherry was transformed into Corynebacterium glutamicum YP097158 by electroporation. The culture was carried out at 30°C for 40 h on a solid medium plate containing kanamycin (50 mg / L) (the composition of the medium is shown in Table 1). The single colonies that grew out of the culture were pink in appearance, which proved that mCherry (red fluorescent protein) was correctly expressed, indicating that PNcgl1969 (C200T) of the present invention has promoter activity.

[0121] Example 3: Construction of an engineered strain containing a point mutation PNcgl1969 promoter

[0122] The allelic substitution plasmid pK18-PNcgl1969(C200T) constructed in Example 1 was transformed into *Corynebacterium glutamicum* YP097158 and *Corynebacterium glutamicum* strain ATCC13032 to construct an engineered strain containing the point mutation PNcgl1969 promoter. The specific steps are as follows:

[0123] 1. The allelic substitution plasmid pK18-PNcgl1969(C200T) constructed in Example 1 was transformed into *Corynebacterium glutamicum* YP097158 and *Corynebacterium glutamicum* ATCC13032 by electroporation (sequencing confirmed that the wild-type PNcgl1969 promoter was retained on the chromosomes of both *Corynebacterium glutamicum* YP097158 and *Corynebacterium glutamicum* ATCC13032). Then, it was cultured at 30°C for 40 h on a solid medium plate containing kanamycin (50 mg / L) (the composition of the medium is shown in Table 1).

[0124] 2. Single colonies produced by culture were identified by PCR using primers P1 / P4 from Example 1. Strains that could amplify a band of 1073 bp were considered positive strains.

[0125] 3. Positive strains were streaked onto solid medium containing 15% sucrose (this medium was obtained by increasing the sucrose concentration in the medium shown in Table 1 to 150 g / L) for 40 h. Single colonies produced were cultured on solid medium plates containing and without kanamycin. Strains that grew on the kanamycin-free medium but not on the kanamycin-containing medium were further identified by PCR using primers P1 / P4 and sequenced. Through sequence alignment, strains with a base sequence mutation (C200T) were identified as positive strains with successful allelic substitution. The positive strains obtained from *Corynebacterium glutamicum* YP097158 and *Corynebacterium glutamicum* ATCC13032 were named L-PNcgl1969 and Y-PNcgl1969, respectively.

[0126] The only difference between recombinant strain L-PNcgl1969 and Corynebacterium glutamicum YP097158 is that L-PNcgl1969 is obtained by replacing the PNcgl1969 promoter sequence in the genome sequence of Corynebacterium glutamicum YP097158 with the sequence shown in SEQ ID No.2, while keeping other sequences unchanged.

[0127] The only difference between recombinant strain Y-PNcgl1969 and Corynebacterium glutamicum ATCC13032 is that Y-PNcgl1969 is a strain obtained by replacing the PNcgl1969 promoter sequence in the genome sequence of Corynebacterium glutamicum ATCC13032 with the sequence shown in SEQ ID No.2, while keeping other sequences unchanged.

[0128] Table 1. Composition of Corynebacterium glutamicum culture medium (solvent is water)

[0129] Example 4: L-Lysine fermentation experiment

[0130] The recombinant bacteria L-PNcgl1969 and Y-PNcgl1969 constructed in Example 3, as well as Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032, were fermented in a BLBIO-5GC-4-H fermenter (Shanghai Bailun Biotechnology Co., Ltd.) using the culture medium shown in Table 2 and the control process shown in Table 3. After fermentation, the L-lysine yield was detected by the ninhydrin colorimetric method. Each strain was repeated three times, and the results are shown in Table 4.

[0131] Table 2. Fermentation medium formulation (solvent is water)

[0132] Table 3, fermentation control process

[0133] Table 4, L-lysine production of PNcgl1969 engineering strain and significance analysis

[0134] The results are shown in Table 4, and the results show that: in Corynebacterium glutamicum, point mutation (C200T, base C at position 200 is mutated to base T) of the Ncgl1969 gene promoter region (PNcgl1969 promoter region, Seq ID No. 1) helps to improve the yield of L-lysine. Industrial applicability

[0135] The application provides a PNcgl1969 (C200T) promoter. Experiments prove that point mutation (C200T, base C at position 200 is mutated to base T) of the PNcgl1969 promoter region (Seq ID No. 1) in Corynebacterium glutamicum helps to improve the yield of L-lysine. The PNcgl1969 (C200T) promoter (Seq ID No. 2) provided by the application is of great significance for constructing L-lysine engineering bacteria and improving the yield of L-lysine produced by bacterial fermentation, and has a good application prospect in L-lysine production.

Claims

1. A DNA molecule, which is any one of the following A1) to A5): A1) a DNA molecule whose nucleotide sequence comprises the nucleotide sequence shown in Seq ID No. 2; A2) a DNA molecule complementary to the nucleotide sequence shown in Seq ID No. 2; A3) a DNA molecule capable of hybridizing to the nucleotide sequence of A1) or A2) under high stringency conditions; A4) a DNA molecule obtained by modifying the nucleotide sequence of A1) or A2) by substitution and / or deletion and / or addition of one or more nucleotides; and A5) a DNA molecule having at least 90% identity to the nucleotide sequence of A1) or A2).

2. A biological material associated with the DNA molecule of claim 1; the biological material is any one of the following B1) to B5): B1) a nucleic acid construct containing the DNA molecule of claim 1; B2) an expression cassette containing the DNA molecule of claim 1; B3) a recombinant vector containing the DNA molecule of claim 1; B4) a recombinant microorganism containing the DNA molecule of claim 1; and B5) a whole-cell catalyst containing the DNA molecule of claim 1.

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

4. Use of the DNA molecule of claim 1 to initiate expression of a target gene as a promoter.

5. Use of the DNA molecule of claim 1 or the biological material of claim 2 in constructing a biomolecule-producing engineered microorganism; or, use of the DNA molecule of claim 1 or the biological material of claim 2 in the preparation of a product for constructing a biomolecule-producing engineered microorganism.

6. Use of the DNA molecule of claim 1 or the biological material of claim 2 in the production of a biomolecule; or, use of the DNA molecule of claim 1 or the biological material of claim 2 in the preparation of a product for producing a biomolecule.

7. Use of the DNA molecule of claim 1 or the biological material of claim 2 in the improvement of the yield of a biomolecule; or, use of the DNA molecule of claim 1 or the biological material of claim 2 in the preparation of a product for improving the yield of a biomolecule. The biomolecule is at least one of the following: lysine, valine, glycine, glutamic acid, 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. The biomolecule is L-lysine.

10. Use of the DNA molecule of claim 1 or the biological material of claim 2 in the regulation of the expression of a target gene in a microorganism or in the breeding of a microorganism; or, use of the DNA molecule of claim 1 or the biological material of claim 2 in the preparation of a product for regulating the expression of a target gene in a microorganism or for the breeding of a microorganism. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8. Use according to any one of claims 5 to 7, characterized in that: ​ 9. Use according to any one of claims 5 to 8, characterized in that: ​ ​ ​ 11. A method for constructing an L-lysine-producing engineered bacterium, comprising the step of mutating a base C at position 200 in a DNA molecule shown in SEQ ID No. 1 to a base T in a microorganism having an L-lysine-producing ability.

12. The method of claim 11, wherein: The microorganism having an L-lysine-producing ability is a bacterium having an L-lysine-producing ability.

13. The method of claim 12, wherein: The bacterium is a bacterium of the genus Corynebacterium.

14. The method of claim 13, wherein: The bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

15. An L-lysine-producing engineered bacterium constructed by the method according to any one of claims 11 to 14.

16. A method for increasing the production of L-lysine, comprising the step of mutating a base C at position 200 in a DNA molecule shown in SEQ ID No. 1 to a base T in a microorganism having an L-lysine-producing ability.

17. The method of claim 16, wherein: The microorganism having an L-lysine-producing ability is a bacterium having an L-lysine-producing ability.

18. The method of claim 17, wherein: The bacterium is a bacterium of the genus Corynebacterium.

19. The method of claim 18, wherein: The bacterium of the genus Corynebacterium is Corynebacterium glutamicum.

20. A method for producing L-lysine, comprising the step of subjecting an L-lysine-producing engineered bacterium constructed by the method according to any one of claims 11 to 14 to fermentation culture.

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