CN115851802A - Construction method of glutamic acid high-producing strain and application of glutamic acid high-producing strain in glutamic acid production - Google Patents

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a construction method of a DNA ligase mutant for a glutamic acid high-producing strain and application of the DNA ligase mutant in glutamic acid production. The DNA ligase mutant has threonine 442 replaced by isoleucine and leucine 535 replaced by phenylalanine based on the DNA ligase shown in SEQ ID NO. 3 or homologous enzyme thereof. The mutant can obviously improve the yield of the L-glutamic acid and the conversion rate of the saccharic acid, can be used for constructing a high-yield glutamic acid strain, and has a good application prospect in the production of the L-glutamic acid and derivatives thereof, thereby greatly reducing the production cost of the glutamic acid, providing a new choice for large-scale production, and having a great application value.

Description

Construction method of glutamic acid high-producing strain and application of glutamic acid high-producing strain in glutamic acid production

Technical Field

The invention relates to the technical field of microorganisms and biology, in particular to a method for constructing a glutamic acid high-producing strain and application thereof in glutamic acid production.

Background

L-glutamic acid is the first major amino acid product in the world, is a basic substance for forming protein required by animal nutrition, plays an important role in the protein metabolic process in organisms, participates in a plurality of important chemical reactions in animals, plants and microorganisms, is mainly used for producing seasonings such as monosodium glutamate and chicken essence and various foods, and is widely applied in the fields of medicine, chemical industry, animal husbandry and the like. Because of large demand and high yield, the industrial fermentation of the L-glutamic acid is one of the important fermentation industries in China.

At present, L-glutamic acid is produced mainly by a microbial fermentation method, and the most predominant production strain is Corynebacterium glutamicum. In corynebacterium glutamicum, the intermediate product alpha-ketoglutarate mainly circulated by TCA produces L-glutamic acid under the action of glutamate dehydrogenase, the synthetic route is short, the target point for improving the strain so as to improve the yield of L-glutamic acid is relatively less, and the performance improvement of the L-glutamic acid producing strain is greatly limited. Therefore, the field needs to further develop new targets for L-glutamic acid modification, so as to further improve the yield and the conversion rate of L-glutamic acid.

Disclosure of Invention

According to the invention, through genome sequence analysis of a glutamic acid high-producing strain SL4 obtained by early screening, the existence of point mutation in a DNA ligase gene coding gene ligA is found, and further research shows that the encoded DNA ligase of which the 442 th threonine is replaced by isoleucine and the 535 th leucine is replaced by phenylalanine can improve the glutamic acid yield of the strain, so that the production efficiency of glutamic acid can be improved, and the production cost is reduced. On the basis of this, the present invention has been completed.

The invention firstly provides a construction method of a high-yield glutamic acid strain, which is characterized in that an original strain is a production strain of L-glutamic acid, and a coding gene of a DNA ligase protein mutant is introduced into the strain, wherein the amino acid sequence of the DNA ligase protein mutant is replaced by isoleucine at the 442 th position and replaced by phenylalanine at the 535 th position corresponding to the amino acid sequence shown in SEQ ID NO. 3.

Preferably, the starting strain is selected from a glutamic acid-producing strain of the genus Corynebacterium (Corynebacterium), pantoea (Pantoea), brevibacterium (Brevibacterium); preferably, it is Corynebacterium glutamicum (Corynebacterium glutamicum), more preferably, it is Corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum B253, corynebacterium glutamicum ATCC14067, and produced by the above strains of mutant strains of glutamic acid.

Further, the starting strain is a strain obtained by modifying the strain based on corynebacterium glutamicum ATCC 13032 and corynebacterium glutamicum ATCC13869, wherein the modification comprises but is not limited to the enhancement or overexpression of one or more genes selected from the following genes:

a. the yggB gene encoding mechanosensitive channel protein (CN 108250278A);

b. the fxpk gene encoding phosphoketolase (WO 2006016705A 1);

c. the pyc gene encoding pyruvate carboxylase (WO 2004069996A 2);

d. the gdh gene encoding glutamate dehydrogenase (CN 103205390 a);

e. a gene encoding carbonic anhydrase (WO 2011024583A 1);

optionally, one or more genes selected from the group consisting of:

a. the odhA gene encoding alpha-ketoglutarate dehydrogenase (WO 2006028298A 2);

b. the amtR gene encoding a transcriptional regulatory gene (EP 2276845A 1);

c. the acnR gene encoding the transcription repressor (CN 111334535A);

furthermore, the glutamic acid-producing strain was obtained by introducing a111V mutation into the gene numbered BBD29_06760 (NCgl 1221 homologous gene or yggB) and a C361Y mutation into citrate synthase GltA.

The invention also provides a DNA ligase mutant which has an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:3 by isoleucine for the threonine at position 442 and phenylalanine for the leucine at position 535.

Further, the amino acid sequence of the DNA ligase mutant is shown as SEQ ID NO:1 is shown.

Among them, the "DNA ligase" and its abbreviation name "LigA" as described herein refer to a protein derived from Corynebacterium glutamicum which catalyzes the formation of a phosphodiesterase bond between a 5 '-phosphate group and a 3' -hydroxyl group in double-stranded DNA and is essential for DNA replication and repair of damaged DNA. As used herein, ligA is not particularly limited as long as it has a corresponding activity, and it may be derived from corynebacterium glutamicum, but is not limited thereto. For example, ligA may be a wild-type sequence as the amino acid sequence of SEQ ID No. 3 or an amino acid sequence having at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the amino acid sequence of SEQ ID No. 3. In addition, if the amino acid sequence has homology with the above-mentioned sequence and has substantially the same or corresponding biological activity as the protein of SEQ ID NO. 3, it is apparent that the amino acid sequence having deletion, modification, substitution or addition should also fall within the scope of the present disclosure. In the present invention, any polynucleotide sequence encoding LigA is within the scope of the present disclosure. For example, the polynucleotide sequence can be a polynucleotide sequence having at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the polynucleotide sequence of SEQ ID No. 4. In addition, a polynucleotide sequence encoding a protein may have various variants on a coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, based on codon degeneracy or considering codons preferred by an organism to express the protein.

The "polypeptides", "peptides" and "proteins" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

By "fragment" is meant a polypeptide or a catalytic or carbohydrate binding module that lacks one or more (e.g., several) amino acids from the amino and/or carboxy terminus of the mature polypeptide or domain. In a specific embodiment, the fragment has DNA ligase activity.

The term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature, and is not intentionally modified by man in the laboratory, is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms. In some embodiments, the wild-type DNA ligase of the present invention refers to a wild-type LigA protein, i.e. the DNA ligase as set forth in SEQ ID NO: 3.

The "mutant" refers to a polynucleotide that comprises alterations (i.e., substitutions, insertions, and/or deletions) at one or more (e.g., several) positions relative to a "wild-type", or "comparable" polynucleotide or polypeptide, wherein a substitution refers to the substitution of a nucleotide occupying a position with a different nucleotide.

The "amino acid mutation" or "nucleotide mutation" includes "substitution, repetition, deletion or addition of one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to a change in nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

In some embodiments, the "mutation" is contained in SEQ ID NO:3 by isoleucine for the threonine at position 442 and phenylalanine for the leucine at position 535. And SEQ ID NO:3, the L-glutamic acid yield of the strain containing the mutant is improved by more than 11 percent compared with that of the strain containing the mutant.

The invention also provides encoding polynucleotides encoding the DNA ligases.

The "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments, or may be a component of a larger nucleotide sequence structure, derived from nucleotide sequences that have been isolated at least once in quantity or concentration, and which are capable of being recognized, manipulated, and recovered in sequence, and their component nucleotide sequences, by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individually or as a whole) or may be an integral part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

Specifically, the polynucleotide encoding the DNA ligase of the present invention includes a polynucleotide represented by SEQ ID NO. 4, and a mutant polynucleotide exists at positions 1325 and 1603 thereof. In addition, the polynucleotide of the present invention also includes any polynucleotide having homology of 75% or more, specifically 80% or more, more specifically 85% or more, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more with the polynucleotide represented by SEQ ID NO. 4.

The term "homology" in the context of the present invention refers to the percentage of identity between two polynucleotide or polypeptide parts. Homology between the sequence of one portion and the other can be determined by techniques known in the art. For example, homology can be determined by directly aligning the sequence information of two polynucleotide molecules or two polypeptide molecules using readily available computer programs. Examples of computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), megAlignTM (DNASTAR Inc.), and the like. In addition, the homology between polynucleotides can be determined by: polynucleotides are hybridized under conditions that form a stable double strand between homologous regions, cleaved with a single strand specific nuclease, and the cleaved fragments are then sized.

The invention further provides a recombinant host cell or a recombinant strain containing the DNA ligase mutant or a coding gene thereof. In particular, the host cell is an L-glutamic acid-producing strain.

L-glutamic acid of the present inventionThe production strain is constructed by transforming a DNA ligase or a gene encoding the DNA ligase into a host cell or by introducing the DNA ligase into the host cell by homologous recombination. "transformation" herein has the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell including, but not limited to, electroporation, calcium phosphate (CaPO) ₄ ) Precipitation method, calcium chloride (CaCl) ₂ ) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.

The "host cell" of the present invention is a cell having a meaning generally understood by those of ordinary skill in the art, i.e., capable of introducing a nucleic acid having promoter activity of the present invention, and the introduction is hereinafter referred to as a recombinant host cell. In other words, the present invention can utilize any host cell as long as the cell contains the nucleic acid having promoter activity of the present invention and is operably linked to a gene to mediate transcription of the gene. The host cell of the present invention may be a prokaryotic or eukaryotic cell, preferably an enterobacterium or a coryneform bacterium, more preferably a Corynebacterium glutamicum including, but not limited to Corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum B253, corynebacterium glutamicum ATCC14067, and L-amino acid-producing mutants or strains prepared from the above strains.

Meanwhile, the invention provides the application of the DNA ligase mutant or the coding gene or the recombinant host cell thereof in improving the yield and the conversion rate of the L-glutamic acid.

Further, the present invention provides a method for producing L-glutamic acid, which comprises culturing the host cell of the third aspect to produce L-glutamic acid, and further comprises, for example, a step of isolating L-glutamic acid from the culture medium or recovering L-glutamic acid. In the present invention, the culture of the host cell may be performed according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a batch culture, a continuous culture, a fed-batch culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to the actual circumstances.

The invention has the beneficial effects that: the DNA ligase mutant provided by the invention can improve the yield and the conversion rate of L-glutamic acid of the strain, and can be used for constructing a glutamic acid high-yield strain, so that the production cost of glutamic acid can be reduced, and a new choice is provided for large-scale production of glutamic acid.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental techniques and experimental procedures used in the examples are, unless otherwise specified, conventional techniques, e.g., those in the following examples in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.

The media used in the examples are as follows:

the TSB plate medium comprises the following components (g/L): glucose, 5g/L; 5g/L of yeast powder; soybean peptone, 9g/L; 3g/L of urea; succinic acid, 0.5g/L; k ₂ HPO ₄ ·3H ₂ O，1g/L；MgSO ₄ ·7H ₂ O,0.1g/L; biotin, 0.01mg/L; vitamin B1,0.1mg/L; MOPS,20g/L; agar powder, 15g/L.

The TSB liquid culture medium comprises the following components (g/L): glucose, 5g/L; 5g/L of yeast powder; soybean peptone, 9g/L; 3g/L of urea; succinic acid, 0.5g/L; k ₂ HPO ₄ ·3H ₂ O，1g/L；MgSO ₄ ·7H ₂ O,0.1g/L; biotin, 0.01mg/L; vitamin B1,0.1mg/L; MOPS,20g/L.

The seed culture medium used in the glutamic acid fermentation experiment comprises the following components: glucose, 25g/L; KH (Perkin Elmer) ₂ PO ₄ ·3H ₂ O,2.2g/L; 3g/L of urea; 33mL of corn steep liquor; mgSO (MgSO) in vitro ₄ ·7H ₂ O,0.9g/L; 22mL of bean cake hydrolysate; MOPS,20g/L; initial pH7.2.

The fermentation medium used in the glutamic acid fermentation experiment comprises the following components: glucose, 80g/L; KH (Perkin Elmer) ₂ PO ₄ 1g/L; 10g/L of urea; 5g/L of corn steep liquor dry powder; mgSO (MgSO) ₄ ·7H ₂ O，0.4g/L；FeSO ₄ ·7H ₂ O，10mg/L；MnSO ₄ ·4H ₂ O，10mg/L；VB ₁ 200 mu g/L; MOPS,40g/L; initial pH7.5.

Example 1 construction of LigA mutant editing plasmid

A mutant strain capable of producing glutamic acid at a high yield was obtained by mutagenesis screening in the past by the inventors and named SL4 (Liu Jiano, et al, mutations in peptide Synthesis Gene punA improved electrophoresis Efficiency of Corynebacterium glutamicum ATCC 13869.Appl. Environ. Microbiol.,2018,84, e02225-02218.). The genome of the SL4 strain was subjected to sequencing analysis, and the DNA ligase of the strain was found to be mutated. Given that DNA ligase is critical for DNA replication and repair of damaged DNA, we speculate that this mutation may play a role in the synthesis of high-producing glutamate by the strain.

According to the published sequence of Corynebacterium glutamicum ATCC13869 genome (GenBank: CP 016335.1), amplification primers ligA-F1/R1, ligA-F2/R2 and ligA-F3/R3 are designed, and the ATCC13869 genome is used as a template to obtain the strain containing LigA through PCR amplification ^T442I,L535F Gene segments of mutation sites and upstream and downstream recombination segments. Designing a primer pK-F/R according to the sequence information of the plasmid pK18mobsacB, and obtaining a linearized vector fragment by PCR reverse amplification by taking the plasmid pK18mobsacB as a template. The primers used in this example are shown in Table 1. The four fragments are recovered and recombined and connected to obtain the recombinant plasmid with LigA ^T442I,L535F Editing plasmid pK18-LigA of mutant ^T44 2I,L535F。

TABLE 1 construction of mutant editing plasmid primers

Primer and method for producing the same	Nucleotide sequence
		pK-F	AAGCTTGGCACTGGCCGTCG
pK-R	GAATTCGTAATCATGTCATAGCTGT
		ligA-F1	tgacatgattacgaattcTGATGAATATCCTGTGCCTG
ligA-R1	caaggtagAtcaaacgcgtggacagctga
		ligA-F2	gcgtttgaTctaccttgctggtcgtggcg
ligA-R2	acctgcaaAggcgcgcgctgcggtggggc
		ligA-F3	cgcgcgccTttgcaggtcgctatcattcc
ligA-R3	cgacggccagtgccaagcttAGAAATGGTCGATGTCATCC

Example 2 construction of glutamic acid producing Strain of mutant LigA

(1) Construction of L-glutamic acid-producing Strain

The introduction of A111V mutation into NCgl1221 homologous gene (BBD 29_06760 or yggB) of Corynebacterium glutamicum ATCC13869 has been reported in the literature, so that the strain has the capability of constitutive synthesis and secretion of L-glutamic acid. To verify the use of the above mutants in the production of L-glutamic acid, the above mutations were first introduced into the genome of Corynebacterium glutamicum ATCC 13869.

Primers A111V-UH-F/R and A111V-DH-F/R were designed based on the published sequence of the Corynebacterium glutamicum ATCC13869 genome (GenBank: CP 016335.1). The gene group ATCC13869 is taken as a template, and the YggB gene is obtained by PCR amplification by using the primers ^A111V A mutated DNA fragment; designing a primer pK-F/R according to the sequence information of the plasmid pK18mobsacB, and obtaining a linearized vector fragment by PCR reverse amplification by taking the plasmid pK18mobsacB as a template; the three fragments are recovered and recombined and connected, and the clone obtained after transformation is collected and extracted to obtain YggB ^A111V Mutant editing vector pK18-YggB ^A111V 。

Preparing competent cells of glutamic acid ATCC13869 (stimulating the electro-transformation of Corynebacterium by fermentation cell wall and stimulating the cytoplastic membrane fluid, biotechnology Letters,2015,37 2445-52.) by a method reported in the literature, and electroporating 1. Mu.g of pK18-YggB into the 13869 competent cells obtained by the above preparation ^A111V The plasmid was added to 1mL of 46 ℃ preheated TSB medium, incubated at 46 ℃ for 6min, incubated at 30 ℃ for 3h, plated with TSB solid medium containing 25. Mu.g/mL kanamycin, and cultured at 30 ℃ for 1 day to obtain a first recombinant transformant. The correct transformant is transferred to a TSB culture medium containing 5g/L glucose for overnight culture, then transferred to a TSB culture medium containing 100g/L sucrose, cultured for 6h at 30 ℃, spread on the TSB culture medium added with 100g/L sucrose and screened to obtain the L-glutamic acid production strain SCgGC5.

Preliminary studies have shown that GltA ^C361Y The mutation can obviously improve the L-glutamic acid yield of the strain, so the mutant is introduced into SCgGC5 to obtain the L-glutamic acid producing strain with higher yield. The specific operation is as follows: primers gltA-F1/R1 and gltA-F2/R2 were designed based on the published sequence of Corynebacterium glutamicum ATCC13869 genome (GenBank: CP 016335.1), and amplification of a strain containing GltA using ATCC13869 genome as a template ^C361Y Upstream and downstream duplication of mutantsGroup fragments. The primer pK-F/R is designed according to the sequence information of the plasmid pK18mobsacB, and the linearized vector fragment is obtained by PCR reverse amplification by taking the plasmid pK18mobsacB as a template. The three fragments are recovered and recombined and connected to obtain the recombinant plasmid with GltA ^C361Y Editing plasmid pK18-GltA of mutant ^C361Y 。

SCgGC5 competent cells were prepared in the same manner, and 1. Mu.g of pK18-GltA was electroporated into SCgGC5 competent cells ^C361Y The plasmid was added to 1mL of 46 ℃ preheated TSB medium, incubated at 46 ℃ for 6min, incubated at 30 ℃ for 3 hours, plated with TSB solid medium containing 25. Mu.g/mL kanamycin, and cultured at 30 ℃ for 24 hours to obtain a first recombinant transformant. The correct transformant is transferred to a TSB culture medium containing 5g/L glucose for overnight culture, then the TSB culture medium containing 100g/L sucrose is transferred, cultured for 4h at 30 ℃, spread on the TSB culture medium added with 100g/L sucrose for screening to obtain the strain with YggB ^A111V And GltA ^C361Y Mutant strain scgcc 7. The primers used in this example are shown in Table 2.

TABLE 2 primers used in this example

Primer and method for producing the same	Nucleotide sequence
		A111V-UH-F	tgacatgattacgaattcATCCACTGGAGTTTTGCCAATTCTC
A111V-UH-R	gtcttggtGTGcagtcgattgttgcg
		A111V-DH-F	atcgactgCACaccaagaccaatggc
A111V-DH-R	cgacggccagtgccaagcttTGGAGGAATAGAGCGGGTCATACAC
		pK-F	AAGCTTGGCACTGGCCGTCG
pK-R	GAATTCGTAATCATGTCATAGCTGT
		gltA-F1	tgacatgattacgaattcTTTGACCCAGGTTATGTGAG
gltA-R1	aatcatcagccagtgcaatttct
		gltA-F2	cactggctgatgattActtcatctcccgcaa
gltA-R2	cgacggccagtgccaagcttCGGAAATCATAGAGCGACAA

(2) Construction of a production Strain containing a LigA mutant

The same method was used to prepare SCgGC7 competent cells of the L-glutamic acid-producing strain, and 1. Mu.g of pK18-LigA was electroporated into the SCgGC7 competent cells obtained by the above preparation ^T442I,L535F The plasmid was added to 1mL of 46 ℃ preheated TSB medium, incubated at 46 ℃ for 6min, incubated at 30 ℃ for 3 hours, plated with TSB solid medium containing 25. Mu.g/mL kanamycin, and cultured at 30 ℃ for 24 hours to obtain a first recombinant transformant. The correct transformant is transferred to a TSB culture medium containing 5g/L glucose for overnight culture, then transferred to a TSB culture medium containing 100g/L sucrose, cultured for 4h at 30 ℃, coated on the TSB culture medium added with 100g/L sucrose for screening to obtain the L-glutamic acid producing strain SCgGC7-LigA with mutant LigA ^T442I,L535F The amino acid sequence of the mutant LigA expressed in the strain is shown as SEQ ID NO:1, and the nucleotide sequence is shown as SEQ ID NO:2, and the corresponding wild type LigA has the amino acid sequence shown as SEQ ID NO:3, respectively.

Wherein, SEQ ID NO:1 is as follows:

MTEDNAQLRRTWNDLAEKVRYHRDRYYNEQPEIPDADFDALFKQLQQLEEDHPELAVPDSPTMVVG

APVAEQSSFDNVEHLERMLSLDNVFDEQELRDWLGRTPAKQYLTELKIDGLSIDLVYRNGQLERAATR

GDGRVGEDITANARVIEDIPHQLQGTDEYPVPAVLEIRGEVFITVEDFPEVNAQRIADGGKPFANPRNA

AAGSLRQKNIEDVKKRRLRMISHGIGFTEGFSPASQHDAYLALAAWGLPTSPYTEAVTDPEDVVKKVS

YWADHRHDALHEMDGLVIKVDDIASQRALGSTSRAPRWAIAYKYPPEEVTTKLLDIQVGVGRTGRVT

PFAVMEPVLVAGSTVSMATLHNQSEVKRKGVLIGDTVVIRKAGEVIPEVLGPVVELRDGTEREYIFPTL

CPECGTRLAPAKADDVDWRCPNMQSCPGQLSTRLIYLAGRGAFDIEALGEKGADDLIRTGILLDESGL

FDLTEDDLLSSNVYTTNAGKVNASGKKLLDNLQKSKQTDLWRVLVALSIRHVGPTAARAFAGRYHSIQ

ALNDAPLEELSETDGVGTIIAQSFKDWFEVDWHKAIVDKWAAAGVTMEEEVGEVAEQTLEGLTIVVT

GGLEGFTRDSVKEAIISRGGKASGSVSKKTDYVVVGENAGSKATKAEELGLRILDEAGFVRLLNTGSA

DE。

SEQ ID NO:2 is as follows:

gtgactgaagataatgctcaactgcgtagaacgtggaacgacttagccgagaaggttcgttatcaccgagatcgttattacaacgaacagccagagatccctgat

gctgattttgatgcgctttttaagcagcttcagcagttggaagaagaccacccggagctcgccgtccctgatagccccacgatggttgtgggcgctccggtggca

gagcaatcaagctttgacaatgttgagcacttggagcgaatgctcagcttggacaatgtttttgatgagcaggagttgcgtgattggttgggcaggacgccagcca

agcagtatttgacggagttgaaaattgatggcttgtccatcgacttggtgtatcgcaatggccagttagagcgtgcggctactcgtggtgatggtcgcgtgggcga

ggacatcacggccaatgctcgcgtgatcgaagatatcccgcaccagcttcagggcactgatgaatatcctgtgcctgctgtgctggagattcgcggtgaggtgtt

catcactgtggaggatttcccagaggtcaacgcgcagcgcattgctgatggtggcaagccgtttgccaacccgcgtaatgctgcggctggttctctgcgtcagaa

aaatattgaggacgtgaagaagcgtcgcctgcggatgatcagccatggcatcggtttcactgaaggctttagccctgcgtctcagcatgatgcgtatctggcattg

gctgcctggggtttgcccacctcgccgtacacagaggctgtgactgatccagaagatgtggtgaaaaaggtcagctactgggctgatcaccgccacgacgcac

tccatgagatggatggcctggtgattaaggtcgatgacatcgcatctcagcgtgctttgggctccaccagccgcgcgcctcgctgggccattgcgtataagtacc

ctccggaggaggtcaccaccaagctgcttgatattcaggttggcgttggtcgcaccggccgtgtcaccccattcgcggtcatggagcctgttcttgttgcaggatc

aacggtgtctatggcgacgctgcataaccagagcgaagtcaagcgtaaaggcgtgctcatcggtgacaccgtggtcatccgcaaggcgggcgaggttatccc

agaggtgcttggccctgtcgtagagcttcgtgacggcacagagcgcgagtacatcttcccaacgctgtgcccagaatgcggtacccgtctggcgcccgcgaag

gccgatgacgtggattggcgttgccccaacatgcaaagctgtccaggtcagctgtccacgcgtttgaTctaccttgctggtcgtggcgcttttgatattgaagcatt

gggcgaaaagggcgctgatgacctcatccgcaccggcattttgcttgacgagtctggcctgttcgacctcacagaggacgatctgctgagctccaatgtctacac

caccaacgccggcaaagtaaatgccagcggcaagaaactgctggacaacctgcaaaaatccaagcagaccgacctctggcgagtcctcgtggcactatctatc

aggcacgtaggccccaccgcagcgcgcgccTttgcaggtcgctatcattccatccaggcgcttaacgacgcccccctcgaggaactctccgaaaccgatgga

gtaggtaccatcattgcccaatccttcaaggactggttcgaggttgattggcacaaggccatcgtggacaagtgggcagccgctggtgtgactatggaggaaga

agtaggggaggtcgctgaacaaacccttgaaggcctaaccatcgtggtcaccggaggattggaaggcttcaccagagattcggtgaaggaagccatcatctcc

cgtggcggaaaagcctctggatctgtctcgaagaaaactgactacgtggtggttggtgaaaacgcaggttccaaggccaccaaggcagaagaactagggctg

cgcattctggatgaggcaggattcgtccgtttgctcaataccggctcagctgacgaatag。

SEQ ID NO:3 is as follows:

MTEDNAQLRRTWNDLAEKVRYHRDRYYNEQPEIPDADFDALFKQLQQLEEDHPELAVPDSPTMVVG

APVAEQSSFDNVEHLERMLSLDNVFDEQELRDWLGRTPAKQYLTELKIDGLSIDLVYRNGQLERAATR

GDGRVGEDITANARVIEDIPHQLQGTDEYPVPAVLEIRGEVFITVEDFPEVNAQRIADGGKPFANPRNA

AAGSLRQKNIEDVKKRRLRMISHGIGFTEGFSPASQHDAYLALAAWGLPTSPYTEAVTDPEDVVKKVS

YWADHRHDALHEMDGLVIKVDDIASQRALGSTSRAPRWAIAYKYPPEEVTTKLLDIQVGVGRTGRVT

PFAVMEPVLVAGSTVSMATLHNQSEVKRKGVLIGDTVVIRKAGEVIPEVLGPVVELRDGTEREYIFPTL

CPECGTRLAPAKADDVDWRCPNMQSCPGQLSTRLTYLAGRGAFDIEALGEKGADDLIRTGILLDESGL

FDLTEDDLLSSNVYTTNAGKVNASGKKLLDNLQKSKQTDLWRVLVALSIRHVGPTAARALAGRYHSI

QALNDAPLEELSETDGVGTIIAQSFKDWFEVDWHKAIVDKWAAAGVTMEEEVGEVAEQTLEGLTIVV

TGGLEGFTRDSVKEAIISRGGKASGSVSKKTDYVVVGENAGSKATKAEELGLRILDEAGFVRLLNTGS

ADE。

example 3 Effect of LigA mutants on L-glutamic acid Synthesis

To verify the effect of the LigA mutant on glutamic acid production, SCgGC7-LigA constructed as described above was subjected ^T442I,L535F The strains were subjected to fermentation tests while using the strain SCgGC7 as a control.

The strains were first inoculated into seed medium for 8h and the cultures were inoculated as seeds into 24-well plates containing 800. Mu.L of fermentation medium per well, starting OD ₆₀₀ Controlling the rotation speed of a pore plate shaker to be about 0.5, paralleling 3 strains of the strain at the temperature of 30 ℃, supplementing 5g/L urea for 21h, finishing fermentation for 25h, detecting the yield and the consumption of the L-glutamic acid, and calculating the saccharic acid conversion rate from the glucose to the L-glutamic acid. The results are shown in Table 3.

TABLE 3L-glutamic acid production

Bacterial strains	L-glutamic acid (g/L)	Conversion of sugar to acid (g/g,%)
			ATCC 13869	0.20±0.00	0.27±0.00
SCgGC5	4.50±0.22	5.86±0.33
			SCgGC7	8.53±0.31	12.03±0.56
SCgGC7-LigA T44 2I,L535F	9.53±0.92	13.14±1.44

As can be seen from the table, the mutant LigA ^T442I,L535F Compared with a control strain SCgGC7, the yield of the L-glutamic acid is improved by about 12 percent, so that the L-glutamic acid can obviously improve the yield and the saccharic acid conversion rate of the L-glutamic acid, can also obviously improve the production level of the L-glutamic acid by combining and transforming with YggB and GltA mutants, and has application value in the production of the L-glutamic acid and derivatives thereof.

Claims (10)

1. A method for constructing a high-producing strain of glutamic acid, wherein the starting strain is a strain producing L-glutamic acid, and a gene encoding a DNA ligase protein mutant having an amino acid sequence in which threonine 442-th position corresponding to the amino acid sequence shown in SEQ ID NO. 3 is substituted with isoleucine and leucine 535-th position is substituted with phenylalanine is introduced.

2. The method of claim 1, wherein the starting strain is selected from the group consisting of Corynebacterium (C.sp.) (C.) (I.)Corynebacterium) Pantoea genus (Pantoea) Brevibacterium (Brevibacterium) (II)Brevibacteriumsp) The glutamic acid-producing strain of (1); preferably, it is Corynebacterium glutamicum (C.glutamicum) (C.glutamicum)Corynebacterium glutamicum). More preferably, it is Corynebacterium glutamicum ATCC13869, corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum B253, corynebacterium glutamicum ATCC14067, and a mutant strain producing glutamic acid prepared from the above strains.

3. The method of claim 2, wherein the starting strain is a strain obtained by modifying the strain based on the strains Corynebacterium glutamicum ATCC 13032 and ATCC13869, wherein the modification includes but is not limited to the enhancement or overexpression of one or more genes selected from the group consisting of:

a. encoding mechanosensitive channel proteinsyggBA gene;

b. encoding phosphoketolasefxpkA gene;

c. encoding pyruvate carboxylasepycA gene;

d. encoding glutamate dehydrogenasegdhA gene;

e. a gene encoding carbonic anhydrase;

optionally, one or more genes selected from the group consisting of:

a. encoding alpha-ketoglutarate dehydrogenaseodhAA gene;

b. encoding a transcriptional regulatory geneamtRA gene;

c. encoding transcription repressorsacnRA gene;

further, the gene numbered BBD29_06760 in said bacterium (II) ((III))NCgl1221Homologous gene oryggB) Introducing mutation of A111V and mutation of C361Y into citrate synthase GltA to obtain glutamic acid producing strain.

4. A DNA ligase protein mutant characterized in that the amino acid sequence thereof has a substitution of threonine at position 442 with isoleucine and a substitution of leucine at position 535 with phenylalanine, which correspond to the amino acid sequence shown in SEQ ID NO. 3.

5. The DNA ligase protein mutant according to claim 4, wherein the amino acid sequence is shown in SEQ ID NO 1.

6. The polynucleotide encoding the mutant DNA ligase protein of claim 4 or 5, preferably the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 4.

7. An expression vector comprising the encoding polynucleotide of claim 6.

8. A recombinant host cell comprising the encoding polynucleotide of claim 6.

9. Use of the mutant DNA ligase protein according to claim 4 or 5, or the encoding polynucleotide according to claim 6, or the expression vector according to claim 7, or the recombinant host cell according to claim 8 for the production of L-glutamic acid.

10. A method for producing L-glutamic acid, comprising the steps of:

culturing the glutamic acid-producing strain obtained by the construction method according to any one of claims 1 to 3 to produce a culture solution containing L-glutamic acid; and optionally separating the produced L-glutamic acid from the culture broth.