Use of pncgl1855 (A244C) promoter and biomaterial related thereto in preparation of l-lysine

Abstract

Disclosed is the use of a PNcgl1855 (A244C) promoter and a biomaterial related thereto in preparation of L-lysine. The promoter is a DNA molecule obtained by mutating adenine at position 244 of a PNcgl1855 promoter into cytosine. L-lysine fermentation experiments performed on constructed engineered strains, i.e. L-PNcgl1855 and Y-PNcgl1855, containing the PNcgl1855 (A244C) promoter show that introducing a point mutation into the promoter region of the Ncgl1855 gene in Corynebacterium glutamicum contributes to an increase in the yield of L-lysine. The present invention is of great significance for increasing the yield of L-lysine in bacterial fermentation.

Description

Application of PNcgl1855(A244C) promoter and related biological materials thereof in preparation of L-lysine

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410728429.6, 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 application of PNcgl1855(A244C) promoter and related biological materials thereof in preparation of L-lysine. BACKGROUND

[0004] Lysine, as an essential amino acid, has a wide range of applications in food, feed, medicine and other fields. China is a major producer of lysine, with an annual output of over 1 million tons, ranking first in the world. However, the current domestic lysine production enterprises have problems such as slow growth of production strains, poor stress resistance, low acid production rate and low conversion rate, and high production cost, which seriously restricts the development of China's lysine industry. Therefore, various researches are being carried out to develop high-efficiency microbial strains and fermentation process technology for the production of L-lysine. For example, the promoters of key genes affecting the synthesis of L-lysine in microbial strains are replaced to increase or weaken their activity; or some genes (byproducts or toxins affecting cell growth, etc.) that do not need to be expressed are deleted in the microbial strains. However, with the increasing demand for L-lysine 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.

[0005] SUMMARY

[0006] The technical problem to be solved by the present application is how to improve the yield of microbial fermentation production of 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.

[0007] To solve the above technical problem, the present application first provides a DNA molecule.

[0008] The DNA molecule provided by the present application is any one of the following A1)-A5):

[0009] A1) a DNA molecule whose nucleotide sequence comprises or is the nucleotide sequence shown in Seq ID No. 2;

[0010] A2) a DNA molecule complementary to the nucleotide sequence shown in Seq ID No. 2;

[0011] A3) a DNA molecule capable of hybridizing to the nucleotide sequence of A1) or A2) under high stringency conditions;

[0012] 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;

[0013] A5) a DNA molecule having at least 90% identity to the nucleotide sequence of A1) or A2).

[0014] The above-mentioned DNA molecule is the PNcgl1855(A244C) promoter in the present application.

[0015] 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 in 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.

[0016] 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.

[0017] In the DNA molecule of A4), the substitution and / or deletion and / or addition of the plurality of nucleotides can be specifically substitution and / or deletion and / or addition of no more than 10 nucleotides, or no more than 9 nucleotides, or no more than 8 nucleotides, or no more than 7 nucleotides, or no more than 6 nucleotides, or no more than 5 nucleotides, or no more than 4 nucleotides, or no more than 3 nucleotides, or no more than 2 nucleotides, or no more than 1 nucleotide.

[0018] The DNA molecule of A5) above has the same origin as the natural nucleic acid sequence, and includes 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 with the nucleotide sequence of the DNA molecule 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.

[0019] The DNA molecule of any one of the above has promoter activity.

[0020] To solve the above technical problem, the present application further provides a biological material related to the above DNA molecule.

[0021] The biological material related to the above DNA molecule provided by the present application is a nucleic acid construct, an expression cassette, a recombinant vector, a recombinant microorganism, or a whole cell catalyst containing the above DNA molecule.

[0022] In the above biological material, the nucleic acid construct contains the above DNA molecule and a gene sequence (such as the Ncgl1855 gene sequence) operably linked to the DNA molecule. The DNA molecule and the gene sequence can be of the same origin or different origin.

[0023] In the above biological material, the expression cassette (5' to 3') can include a promoter region (consisting of the above DNA molecule), a transcription initiation region, a target gene region, a transcription termination region, and an optional translation termination region. The promoter region and the target gene region can be native / analogous to the host cell, or the promoter region and the target gene region can be native / analogous to each other, or the promoter region and / or the target gene region can be heterologous to the host or to each other. "Heterologous" means a sequence derived from a foreign species, or, if from the same species, has been substantially modified through deliberate human intervention in the components and / or genomic locus. The optional transcription termination region can be homologous to the transcription initiation region, homologous to the operably linked target gene region, homologous to the host; or the target gene region, the host are exogenous or heterologous. Further, the expression cassette can further include a 5' leader sequence. The 5' leader sequence can enhance translation.

[0024] In preparing the expression cassette, adapters can be used to ligate DNA fragments, or other manipulations can be involved to provide appropriate restriction enzyme sites, remove excess DNA, remove restriction enzyme sites, etc. To achieve this purpose, in vitro mutation, primer repair, restriction enzyme digestion, annealing, re-substitution, such as conversion and transversion, can be performed.

[0025] The expression cassette can also include a selectable marker gene for screening transformed cells. Selectable marker genes can be used to screen transformed cells or tissues. Marker genes include genes that encode 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 with the present application.

[0026] The above-mentioned biological material, the vector includes but is not limited to plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cos plasmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0027] The recombinant vector can be a vector containing the above-mentioned DNA molecule or the above-mentioned expression cassette constructed using an existing expression vector.

[0028] In some embodiments, the recombinant vector is a recombinant vector pK18-PNcgl1855(A244C). The recombinant vector pK18-PNcgl1855(A244C) 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 other sequences of the pK18mobsacB vector unchanged.

[0029] In the above-mentioned biomaterials, the microorganism 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.

[0030] Further, the bacteria include, but are not limited to, bacteria of the genus Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium, or Bacillus.

[0031] Still further, the bacteria include, but are not limited to, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Micrococcus glutamicus, Brevibacterium ammoniagenes, Escherichia coli, Aerobacter aerogenes.

[0032] Still further, the bacteria is Corynebacterium glutamicum or a mutant thereof.

[0033] In some embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 strain.

[0034] The Corynebacterium glutamicum YP097158 strain has been deposited with the China General Microbiological Culture Collection Center (CGMCC, address: No. 1, Huayuancun, Beijing, China, 100101, phone: 010-64807355) on August 16, 2016, and has the accession number of CGMCC No. 12856.

[0035] In other embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032 strain.

[0036] 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.

[0037] In some embodiments, the recombinant microorganism is a recombinant bacterium L-PNcgl1855. The recombinant bacterium L-PNcgl1855 is a strain obtained by replacing the PNcgl1855 promoter sequence in the Corynebacterium glutamicum YP097158 genomic sequence with the PNcgl1855(A244C) promoter sequence shown in SEQ ID No. 2, while keeping other sequences unchanged.

[0038] In other embodiments, the recombinant microorganism is a recombinant bacterium Y-PNcgl1855. The recombinant bacterium Y-PNcgl1855 is a strain obtained by replacing the PNcgl1855 promoter sequence in the Corynebacterium glutamicum ATCC13032 genomic sequence with the PNcgl1855(A244C) promoter sequence shown in SEQ ID No. 2, while keeping other sequences unchanged.

[0039] To solve the above technical problems, the present application also provides a new use of the above DNA molecule or the above biological material.

[0040] The present application provides an application of the above DNA molecule as a promoter.

[0041] The present application also provides an application of the above DNA molecule in promoting expression of a target gene as a promoter.

[0042] The present application also provides an application of the above DNA molecule or the above biological material in constructing a biomolecule engineering bacterium.

[0043] 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 bacterium.

[0044] The present application also provides an application of the above DNA molecule or the above biological material in producing a biomolecule.

[0045] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for producing a biomolecule.

[0046] The present application also provides an application of the above DNA molecule or the above biological material in improving the yield of a biomolecule.

[0047] The present application also provides an application of the above DNA molecule or the above biological material in preparing a product for improving the yield of a biomolecule.

[0048] 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.

[0049] The present application also provides the use of the above-mentioned DNA molecule or the above-mentioned biological material in the preparation of a product for regulating the expression of a target gene in a microorganism or for breeding a microorganism.

[0050] In any of the above-mentioned uses, the biological molecule 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.

[0051] In some embodiments, the biological molecule is L-lysine.

[0052] In practical applications, the above-mentioned DNA molecule can be placed upstream of a target gene related to the synthesis pathway of the above-mentioned biological molecule and the DNA molecule can drive the expression of the target gene to achieve the production of the target biological molecule or to increase the yield of the target biological molecule.

[0053] The purpose of the above-mentioned breeding of a microorganism is to breed a microorganism with increased yield of a target biological molecule (e.g., a microorganism with increased yield of L-lysine).

[0054] To solve the above-mentioned technical problems, the present application finally provides any one of the following B1) or B2) or B3) methods:

[0055] B1) A method for constructing an L-lysine-producing engineered bacterium, comprising the step of mutating the base A at position 244 in the DNA molecule shown in SEQ ID No. 1 to base C in a microorganism having the ability to produce L-lysine;

[0056] B2) A method for increasing the yield of L-lysine, comprising the step of mutating the base A at position 244 in the DNA molecule shown in SEQ ID No. 1 to base C in a microorganism having the ability to produce L-lysine;

[0057] B3) A method for producing L-lysine, comprising the step of fermenting and culturing the L-lysine-producing engineered bacterium constructed according to the method of B1).

[0058] In any of the above-mentioned methods, the genome sequence of the microorganism having the ability to produce L-lysine contains the DNA molecule shown in SEQ ID No. 1.

[0059] In the above-mentioned method of B1) or B2), the method of mutating the base A at position 244 in the DNA molecule shown in SEQ ID No. 1 to base C in the microorganism having the ability to produce L-lysine can be replacing the DNA molecule shown in SEQ ID No. 1 in the microorganism having the ability to produce L-lysine with the DNA molecule shown in SEQ ID No. 2.

[0060] Further, the method of replacing the DNA molecule shown in SEQ ID No. 1 with the DNA molecule shown in SEQ ID No. 2 in the microorganism having the ability to produce L-lysine 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 having the ability to produce L-lysine.

[0061] Still further, the substance that replaces the DNA molecule shown in SEQ ID No. 1 with the DNA molecule shown in SEQ ID No. 2 can be the recombinant vector pK18-PNcgl1855(A244C). The recombinant vector pK18-PNcgl1855(A244C) 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.

[0062] In the above-mentioned method of B3), the fermentation culture method can be performed according to the conventional test method in the prior art. Alternatively, the fermentation culture method can be performed using an optimized and improved conventional test method.

[0063] In some embodiments, the fermentation culture medium formula is shown in Table 2 in the following examples.

[0064] In some embodiments, the fermentation culture conditions are shown in Table 3 in the following examples.

[0065] In any of the above-mentioned methods, the microorganism having the ability to produce L-lysine can be any microorganism having the ability to produce L-lysine. The microorganism having the ability to produce L-lysine has the ability to utilize external substances (such as culture medium) to produce and accumulate L-lysine in the bacterial body, 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.

[0066] Further, the bacteria having the ability to produce L-lysine include, but are not limited to, bacteria of the genus Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium, or Bacillus.

[0067] Further, the bacteria having the ability to produce L-lysine include, but are not limited to, bacteria of the genus Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium, or Bacillus.

[0068] Further, the bacteria having the ability to produce L-lysine include, but are not limited to, bacteria of the genus Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium, or Bacillus.

[0069] In some embodiments, the bacteria having the ability to produce L-lysine is the Corynebacterium glutamicum YP097158 strain.

[0070] The Corynebacterium glutamicum YP097158 strain has been deposited with the China General Microbiological Culture Collection Center (CGMCC, address: No. 1, Huayuancun, Beijing, China, postcode: 100101, telephone: 010-64807355) on August 16, 2016, and has the accession number CGMCC No. 12856.

[0071] In other embodiments, the bacteria having the ability to produce L-lysine is the Corynebacterium glutamicum ATCC13032 strain.

[0072] The present application provides a PNcgl1855(A244C) promoter, which is a DNA molecule obtained by mutating an adenine (A) at position 244 in a PNcgl1855 promoter to a cytosine (C), and an engineering strain L-PNcgl1855 and Y-PNcgl1855 containing the PNcgl1855(A244C) promoter are constructed by mutating the adenine (A) at position 244 in the PNcgl1855 promoter region of Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 to a cytosine (C). Through L-lysine fermentation experiments on the engineering strains L-PNcgl1855 and Y-PNcgl1855, it is found that point mutation (A244C) of the PNcgl1855 promoter region (PNcgl1855 promoter region) in Corynebacterium glutamicum is helpful to improve the yield of L-lysine.

[0073] DEPOSIT DESCRIPTION

[0074] Strain name: Corynebacterium glutamicum

[0075] Latin name: Corynebacterium glutamicum

[0076] Strain number: YP097158

[0077] Preservation agency: China General Microbiological Culture Collection Center

[0078] Preservation agency abbreviation: CGMCC

[0079] Address: No. 1, Michen West Road, Beijing City, Chaoyang District, No. 3

[0080] Preservation date: August 16, 2016

[0081] Preservation center registration number: CGMCC No. 12856 Embodiment of the present application

[0082] The present application is further described in detail below with specific reference to the embodiments. The examples provided are only to illustrate the present application, and are not intended to limit 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.

[0083] The experimental methods in the following examples 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 commercially, unless otherwise specified.

[0084] 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 was assigned accession number CGMCC No. 12856.

[0085] The Corynebacterium glutamicum ATCC13032 in the following examples was obtained from the American Type Culture Collection (ATCC) and was assigned accession number 13032.

[0086] 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.”

[0087] 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.

[0088] Example 1, Construction of a Recombinant Vector Containing a PNcgl1855 Promoter with a Point Mutation

[0089] According to the Corynebacterium glutamicum ATCC13032 genome sequence published by NCBI, two pairs of primers for amplifying the PNcgl1855 promoter region were designed and synthesized. A point mutation was introduced into the PNcgl1855 promoter region of Corynebacterium glutamicum YP097158 in an allelic replacement manner, which was a mutation of adenine (A) at position 244 in the PNcgl1855 promoter nucleotide sequence (SEQ ID No. 1) to cytosine (C), to obtain a mutated PNcgl1855 promoter. The mutated PNcgl1855 promoter was named PNcgl1855(A244C), and its nucleotide sequence is shown in SEQ ID No. 2. The NEBuilder assembly technology was used to construct a recombinant vector containing the PNcgl1855(A244C) promoter. The specific steps are as follows:

[0090] 1. The following primer sequences were designed:

[0091] P1: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTAACCAGCCCAAACAGTCC-3' (The underlined nucleotide sequence is the sequence on the pK18mobsacB plasmid, SEQ ID No. 5).

[0092] P2: 5'-CTTCCTCTCATTGTGTGCCCGTTAAAG-3' (The underlined bases are the mutation positions, SEQ ID No. 6).

[0093] P3: 5'-CTTTAACGGGCACACAATGAGAGGAAG-3' (underlined bases indicate mutation positions, SEQ ID No. 7).

[0094] P4: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCCAGATGAATCCTTGTG-3' (The underlined nucleotide sequence is the sequence on the pK18mobsacB plasmid, SEQ ID No. 8).

[0095] The above primer sequences were synthesized by Shanghai Invitrogen Company.

[0096] 2. Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed with primers P1 / P2 and P3 / P4, respectively, to obtain two homologous DNA fragments (PNcgl1855Up and PNcgl1855Down) with mutant bases, respectively.

[0097] The PCR amplification system is as follows: 5×HiFi with Mg 2+ Buffer 10μL, dNTP Mixture (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, add ddH2O to a total volume of 50μL.

[0098] The PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 20 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, 30 cycles; 72℃ over-extension for 5 min.

[0099] 3. The two DNA fragments (PNcgl1855Up and PNcgl1855Down) 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 1408 bp fragment (the nucleotide sequence of the 1408 bp fragment is shown in SEQ ID No. 3) was named pK18-PNcgl1855(A244C). This recombinant vector pK18-PNcgl1855(A244C) contains a kanamycin resistance marker. The recombinant vector pK18-PNcgl1855(A244C) was identified by enzyme digestion. Then, the recombinant vector pK18-PNcgl1855(A244C) with the correct enzyme digestion was sent to a sequencing company for sequencing identification. The recombinant vector pK18-PNcgl1855(A244C) containing the correct point mutation (A244C) was stored for future use.

[0100] The recombinant vector pK18-PNcgl1855(A244C) contains the mutation site (A244C) shown in SEQ ID No.2, which will cause the adenine (A) at position 244 of the promoter region of PNcgl1855 in Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 to be mutated to cytosine (C).

[0101] The recombinant vector pK18-PNcgl1855(A244C) 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.

[0102] Example 2: Activity detection of promoter PNcgl1855 (A244C)

[0103] 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 pEC-XK99E plasmid using primers pEC-F / pEC-R, and the NEBuilder assembly technique was used to construct the recombinant vector pEC-PNcgl1855-mCherry; the recombinant vector pEC-PNcgl1855-mCherry was transformed into C. glutamicum YP097158 to verify whether PNcgl1855 (A244C) has promoter activity. The specific steps are as follows:

[0104] 1. The following primer sequences were designed:

[0105] pEC-F: 5'-CTAGAGTCGACCTGCAGGCATGCAAGCTTG-3' (SEQ ID No. 9).

[0106] pEC-R: 5'-CTAGAGGATCCCCGGGTACCGAGCTCGAATTC-3' (SEQ ID No. 10).

[0107] P5: 5'-GAATTCGAGCTCGGTACCCGGGGATCCTCTAGGTTCACATGTTCGACTAAG-3' (the underlined nucleotide sequence is the sequence on the pEC-XK99E plasmid, SEQ ID No. 11).

[0108] P6: 5'-GTTATCTTCTTCTCCTTTACGCATCTTCCGTTCCCTTCCTCTC-3' (SEQ ID No. 12).

[0109] P7: 5'-GAGAGGAAGGGAACGGAAGATGCGTAAAGGAGAAGAAGATAAC-3' (SEQ ID No. 13).

[0110] P8: 5'-CAAGCTTGCATGCCTGCAGGTCGACTCTAGTTTGTATAGTTCATCCATGCCAC-3' (the underlined nucleotide sequence is the sequence on the pEC-XK99E plasmid, SEQ ID No. 14).

[0111] The above-mentioned primer sequences were synthesized by Shanghai invitrogen company.

[0112] 2. Using the plasmid pK18-PNcgl1855(A244C) and mCherry gene fragment from Example 1 as templates, PCR amplification was performed using primers P5 / P6 and P7 / P8, respectively, to obtain the promoter PNcgl1855(A244C) with a size of 268 bp and the mCherry gene fragment with a size of 705 bp.

[0113] 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-PNcgl1855-mCherry, which contains a kanamycin resistance marker.

[0114] 4. The vector pEC-PNcgl1855-mCherry was transformed into Corynebacterium glutamicum YP097158 by electroporation. 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). 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 PNcgl1855 (A244C) of the present invention has promoter activity.

[0115] Example 3: Constructing an engineered strain containing a point mutation PNcgl1855 promoter

[0116] The allelic substitution plasmid pK18-PNcgl1855(A244C) constructed in Example 1 was transformed into *Corynebacterium glutamicum* YP097158 and *Corynebacterium glutamicum* ATCC13032 to construct an engineered strain containing the point mutation PNcgl1855 promoter. The specific steps are as follows:

[0117] 1. The allelic substitution plasmid pK18-PNcgl1855 (A244C) constructed in Example 1 was transformed into Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 by electroporation (sequencing confirmed that both Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032 retained the wild-type PNcgl1855 promoter on their chromosomes). The plasmid was then cultured at 30°C for 40 h on solid medium containing kanamycin (50 mg / L) (the composition of the medium is shown in Table 1).

[0118] 2、The single colonies produced in the culture were respectively identified by PCR using primers P1 / P4 in Example 1, and the strain with a 1408 bp band amplified was a positive strain.

[0119] 3、The positive strain was streaked on a solid medium containing 15% sucrose (the medium was obtained by increasing the concentration of sucrose in the medium shown in Table 1 to 150 g / L) for 40 h, and the single colonies produced in the culture were respectively cultured on solid medium containing kanamycin and solid medium without kanamycin, and the strain growing on the medium without kanamycin but not growing on the medium containing kanamycin was further identified by PCR using primers P1 / P4 and sequencing. Through sequence alignment, the strain with a base sequence mutation (A244C) was a positive strain of successful allelic replacement. The positive strains obtained from C. glutamicum YP097158 and C. glutamicum ATCC13032 were named as L-PNcgl1855 and Y-PNcgl1855, respectively.

[0120] The recombinant strain L-PNcgl1855 differed from C. glutamicum YP097158 only in that the PNcgl1855 promoter sequence in the genome sequence of C. glutamicum YP097158 was replaced with the PNcgl1855(A244C) promoter sequence shown in SEQ ID No. 2, and other sequences remained unchanged.

[0121] The recombinant strain Y-PNcgl1855 differed from C. glutamicum ATCC13032 only in that the PNcgl1855 promoter sequence in the genome sequence of C. glutamicum ATCC13032 was replaced with the PNcgl1855(A244C) promoter sequence shown in SEQ ID No. 2, and other sequences remained unchanged.

[0122] Table 1, Components of C. glutamicum medium (solvent is water)

[0123] Example 4, L-lysine fermentation experiment

[0124] The recombinant strains L-PNcgl1855 and Y-PNcgl1855 constructed in Example 3 and C. glutamicum YP097158 and C. glutamicum ATCC13032 were respectively subjected to fermentation experiment in a fermentation tank (BLBIO-5GC-4-H model, Shanghai Bailong Biological Science and Technology Co., Ltd.) with the medium shown in Table 2 and the control process shown in Table 3. After the fermentation was completed, 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.

[0125] Table 2, fermentation medium formula (solvent is water)

[0126] Table 3, fermentation control process

[0127] Table 4, L-lysine production of different engineering strains and significance analysis

[0128] The results are shown in Table 4, and the results show that the L-lysine production of Y-PNcgl1855 is significantly improved compared with ATCC13032, and the L-lysine production of L-PNcgl1855 is significantly improved compared with YP097158. It is shown that the point mutation (A244C, the base A at position 244 is mutated to base C) of the Ncgl1855 gene promoter region (PNcgl1855 promoter region, Seq ID No. 1) in Corynebacterium glutamicum helps to improve the L-lysine production. Industrial applicability

[0129] The application provides a PNcgl1855 (A244C) promoter and application. Experiments prove that the point mutation (A244C, the base A at position 244 is mutated to base C) of the PNcgl1855 promoter region (Seq ID No. 1) in Corynebacterium glutamicum helps to improve the L-lysine production. The PNcgl1855 (A244C) promoter (Seq ID No. 2) provided by the application is of great significance for constructing L-lysine engineering bacteria and improving the L-lysine production of bacterial fermentation, and has a good application prospect in the production of L-lysine.

Claims

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

2. A biological material related to the DNA molecule of claim 1; the biological material is a nucleic acid construct, an expression cassette, a recombinant vector, a recombinant microorganism, a whole-cell catalyst containing the DNA molecule of claim 1.

3. The biomaterial of claim 2, wherein: The nucleic acid construct comprises the DNA molecule of claim 1 and a gene sequence operably linked to the DNA molecule.

4. Use of the DNA molecule of claim 1 as a promoter; or, 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 or 3 in constructing a biomolecule engineering strain; or, use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in preparing a product for constructing a biomolecule engineering strain.

6. Use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in producing a biomolecule; or, use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in preparing a product for producing a biomolecule.

7. Use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in increasing the yield of a biomolecule; or, use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in preparing a product for increasing the yield of a biomolecule.

8. Use according to any one of claims 5 to 7, characterized in that: 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.

9. Use according to any one of claims 5 to 8, characterized in that: The biomolecule is L-lysine.

10. Use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in regulating the expression of a target gene in a microorganism or in microbial breeding; or, use of the DNA molecule of claim 1 or the biological material of claim 2 or 3 in preparing a product for regulating the expression of a target gene in a microorganism or for microbial breeding.

11. A method for constructing an L-lysine-producing engineered bacterium, comprising the step of mutating a base A at position 244 in a DNA molecule shown in SEQ ID No. 1 to a base C 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. The L-lysine-producing engineered bacterium obtained 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 A at position 244 in a DNA molecule shown in SEQ ID No. 1 to a base C 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 the L-lysine-producing engineered bacterium obtained by the method according to any one of claims 11 to 14 to fermentation culture.

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