A histidine kinase mutant and recombinant microorganism containing the same and its application

Abstract

The present invention relates to the field of genetic engineering technology, and specifically discloses a histidine kinase mutant, a recombinant microorganism containing the same, and applications thereof. The histidine kinase mutant provided by the present invention, using the amino acid sequence of the wild-type histidine kinase as a reference sequence, contains a mutation in which glutamic acid at position 53 is replaced by aspartic acid, or a mutation in which glutamic acid at position 58 is replaced by another amino acid. Specifically, the mutation contains a substitution of glutamic acid at position 58 by aspartic acid or arginine. Recombinant microorganisms containing the mutants of the present invention have a stronger ability to produce amino acids, especially lysine, and provide a new method for increasing amino acid yield through fermentation.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a histidine kinase mutant and a recombinant microorganism containing the same and applications thereof. Background Art

[0002] Histidine kinases are a family of signaling enzymes that phosphorylate conserved histidine residues. Together with their downstream target proteins, histidine kinases form a two-component signaling system. Histidine kinases are ubiquitous in various microorganisms and often exist as protein families. In Corynebacterium glutamicum, numerous histidine kinases exist. Some have been extensively studied, while others have had their functions less well-documented. As signaling proteins, histidine kinases promote histidine phosphorylation. Phosphorylated histidine acts as a signaling factor, triggering a series of downstream cascades, ultimately affecting cellular metabolism.

[0003] Corynebacterium glutamicum is a Gram-positive microorganism with a fast growth rate, non-pathogenic, and clear genetic background and mature metabolic modification method. It is an excellent industrial microorganism chassis bacteria. In recent years, Corynebacterium glutamicum has been widely used in the production of various amino acids, nucleotides and other organic acids. The history of Corynebacterium glutamicum for the production of amino acid can be traced back to the 1960s. Corynebacterium glutamicum can produce glutamic acid under natural environment, and the induced Corynebacterium glutamicum can also produce various amino acids such as lysine and valine. Along with the fast development of metabolic engineering technology and genome sequencing technology, the metabolic pathway research of Corynebacterium glutamicum is more and more clear, and researchers have successively identified a lot of high-yield mechanisms on genetics, and have achieved the efficient production of various metabolites. There are more reports about the research of Corynebacterium glutamicum, but relatively less research about the effect of its histidine kinase, and also rarely research and report on the sudden change of this enzyme. Summary of the Invention

[0004] The object of the present invention is to provide a novel mutant capable of increasing the production of amino acids and a recombinant strain containing the mutant.

[0005] The technical solutions of the present invention are as follows:

[0006] A histidine kinase mutant, which uses the amino acid sequence of a wild-type histidine kinase as a reference sequence and contains a mutation in which glutamic acid at position 53 is replaced by aspartic acid, or a mutation in which glutamic acid at position 58 is replaced by other amino acids.

[0007] Preferably, the mutation contains a substitution of glutamic acid at position 58 by aspartic acid or arginine.

[0008] In the present invention, the amino acid sequence of the histidine kinase mutant is shown in any one of SEQ ID NOs. 7, 9, and 10.

[0009] The gene encoding the wild-type histidine kinase is Cgl2964, and its corresponding gene number in NCBI is NCgl2862.

[0010] The inventors have devoted many years to the study of the metabolic mechanism and amino acid production of Corynebacterium glutamicum and unexpectedly discovered that some specific mutations can lead to subtle changes in the global metabolism of cells and promote better production results, thus completing the present invention.

[0011] Specifically, the present invention provides a mutant histidine kinase, wherein the amino acid at position 53 of its protein sequence is mutated from glutamic acid to other amino acids, preferably to aspartic acid or lysine, or the amino acid at position 58 of its protein sequence is mutated from glutamic acid to other amino acids, preferably to aspartic acid or arginine. The wild-type protein sequence is shown in SEQ ID NO: 6, the protein sequence in which the amino acid at position 53 is mutated to aspartic acid is shown in SEQ ID NO: 7, the protein sequence in which the amino acid at position 53 is mutated to lysine is shown in SEQ ID NO: 8, the protein sequence in which the amino acid at position 58 is mutated to aspartic acid is shown in SEQ ID NO: 9, and the protein sequence in which the amino acid at position 58 is mutated to arginine is shown in SEQ ID NO: 10.

[0012] The acquisition of the histidine kinase mutant of the present invention includes the construction of a tool plasmid carrying the target mutation, a genetic manipulation method for introducing the target mutation into the genome of Corynebacterium glutamicum, screening of the target mutant, and a method for testing the effects of the above mutants.

[0013] The present invention uses two model strains of Corynebacterium glutamicum as implementation objects to test the impact of this mutation on Corynebacterium glutamicum. The strain numbers of the two Corynebacterium glutamicum model strains are ATCC 13032 and ATCC 13869, respectively. The results show that Corynebacterium glutamicum carrying the above-mentioned histidine kinase mutant has a faster sugar consumption rate and can secrete more amino acids, particularly lysine, in the culture medium.

[0014] The present invention continues to test two high-lysine-producing Corynebacterium glutamicum strains, the strain numbers of which are CGMCC No. 11942 and CGMCC No. 13407. The results show that the strain carrying the mutant histidine kinase has better lysine production capacity than the strain carrying the wild-type histidine kinase.

[0015] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown in any one of SEQ ID NOs. 2-5.

[0016] The present invention also provides nucleotide sequences of genes encoding histidine kinases. The nucleotide sequence of the gene encoding the wild-type histidine kinase is shown in SEQ ID NO: 1. The nucleotide sequence of the gene encoding the histidine kinase mutant carrying the E53D mutation (amino acid at position 53 is mutated from glutamic acid to aspartic acid) is shown in SEQ ID NO: 2. The nucleotide sequence of the gene encoding the histidine kinase mutant carrying the E53K mutation (amino acid at position 53 is mutated from glutamic acid to lysine) is shown in SEQ ID NO: 3. The nucleotide sequence of the gene encoding the histidine kinase mutant carrying the E58D mutation (amino acid at position 58 is mutated from glutamic acid to aspartic acid) is shown in SEQ ID NO: 4. The nucleotide sequence of the gene encoding the histidine kinase mutant carrying the E58R mutation (amino acid at position 58 is mutated from glutamic acid to arginine) is shown in SEQ ID NO: 5.

[0017] The present invention further provides a recombinant microorganism that expresses the above-mentioned histidine kinase mutant containing a mutation in which glutamic acid at position 53 is replaced by aspartic acid, or a mutation in which glutamic acid at position 58 is replaced by other amino acids.

[0018] The starting strain of the recombinant microorganism of the present invention is Corynebacterium, preferably, Corynebacterium glutamicum.

[0019] The present invention has found that Corynebacterium glutamicum carrying a histidine kinase mutant has the effect of improving the production efficiency of amino acids when applied to the fermentation production of amino acids.

[0020] The present invention also provides any of the following applications of the recombinant microorganism:

[0021] (1) Application in fermentation production of amino acids;

[0022] (2) Application in genetic breeding of microorganisms for amino acid production;

[0023] (3) Application in increasing the yield of amino acids in fermentation production;

[0024] The recombinant microorganism is as described above, or the recombinant microorganism expresses a histidine kinase mutant containing a mutation in which glutamic acid at position 53 is replaced by lysine, with the amino acid sequence of the wild-type histidine kinase as a reference sequence.

[0025] Preferably, the amino acid is lysine.

[0026] The present invention further provides a method for producing amino acids, comprising the step of fermentation culture using a recombinant microorganism, wherein the recombinant microorganism is as described above.

[0027] The beneficial effects of the present invention are at least:

[0028] The present invention provides a histidine kinase mutant. A recombinant microorganism containing the histidine kinase mutant is beneficial to the synthesis of amino acids, especially lysine, and provides a new method for improving amino acid production efficiency. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Specifically, if the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased through regular channels.

[0031] In the present invention, the model strains of Corynebacterium glutamicum ATCC 13032 and ATCC 13869 can be purchased from public channels. Their genome sequences have been made public and can be searched from the NCBI website.

[0032] Lysine-producing bacteria CGMCC No. 11942 is a strain of Corynebacterium glutamicum, which was deposited on December 25, 2015, at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified as Corynebacterium glutamicum and has been disclosed in patent CN105734004B.

[0033] Lysine-producing bacteria CGMCC No. 13407 is a strain of Corynebacterium glutamicum, which was deposited on November 30, 2016, at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified as Corynebacterium glutamicum. The strain has been disclosed in patent application CN106635944A.

[0034] The amino acid sequence of the wild-type histidine kinase encoding gene Cgl2964 is shown in SEQ ID NO: 6, and the corresponding nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence carrying the E53D mutation is shown in SEQ ID NO: 7, and the corresponding nucleotide sequence is shown in SEQ ID NO: 2. The amino acid sequence carrying the E53K mutation is shown in SEQ ID NO: 8, and the corresponding nucleotide sequence is shown in SEQ ID NO: 3. The amino acid sequence carrying the E58D mutation is shown in SEQ ID NO: 9, and the corresponding nucleotide sequence is shown in SEQ ID NO: 4. The amino acid sequence carrying the E58R mutation is shown in SEQ ID NO: 10, and the corresponding nucleotide sequence is shown in SEQ ID NO: 5.

[0035] The four mutant nucleotide sequences can be obtained by designing special primers and performing fusion PCR, or by whole gene synthesis, the latter of which is more convenient. The four mutant nucleotide sequences of the present invention (Sequence 2, Sequence 3, Sequence 4, and Sequence 5) were synthesized by a gene synthesis company (Jiangsu Jinweizhi).

[0036] The primer sequence information used in the examples of the present invention is shown in Table 1. The examples of the present invention are only used to illustrate the effects of the present invention, but are not intended to limit the scope of the present invention.

[0037] Table 1 Primer sequences (SEQ ID No. 11-16)

[0038]

[0039]

[0040] Example 1 Construction of an engineered plasmid carrying a target mutation

[0041] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification with the primer pair Cgl2964-1f / Cgl2964-1r yielded the upstream homology arm fragment Cgl2964-up. PCR amplification with the primer pair Cgl2964-2f / Cgl2964-2r, also from the ATCC 13032 genome, yielded the downstream homology arm fragment Cgl2964-dn. PCR amplification with the primer pair Cgl2964-f / Cgl2964-r, using the fully synthesized sequence 2 as a template, yielded the amplified product of sequence 2, which carries the E53D mutation and is labeled Cgl2964-E53D. The upstream homology arm fragments Cgl2964-up and Cgl2964-E53D, and the downstream homology arm fragment Cgl2964-dn were subjected to fusion PCR using primers Cgl2964-1f / Cgl2964-2r to obtain a full-length fusion fragment of the three fragments. This full-length fragment was double-digested with XbaI and NheI, and the product was recovered using a gel recovery kit. The plasmid vector pK18mobsacB (GenBank: FJ1287239.1; kindly provided by Professor Yang Sheng of the Shanghai Institutes for Biological Studies, Chinese Academy of Sciences, and also available from public sources) was double-digested with the same restriction endonucleases and gel-recovered. The fragments and vector after digestion and gel recovery were ligated with T4 DNA Ligase and transformed into Trans1 T1 competent cells. The resulting transformants were subjected to plasmid extraction and sequencing confirmation. The correct plasmid was designated pK18-Cgl2964-E53D.

[0042] In the same manner as above, the amplified products of sequence 3, sequence 4, and sequence 5 were fused with the upstream homology arm Cgl2964-up and the downstream homology arm Cgl2964-dn, respectively, and subjected to enzyme digestion, ligation, and transformation. Finally, three other tool plasmids carrying the target mutations were obtained, which were named pK18-Cgl2964-E53K, pK18-Cgl2964-E58D, and pK18-Cgl2964-E58R, respectively.

[0043] Example 2: Mutation modification of Cgl2964 in the Corynebacterium glutamicum model strain ATCC 13032

[0044] Competent cells of the Corynebacterium glutamicum model bacterium ATCC13032 were prepared and gene recombination was performed according to the method in the C. glutamicum Handbook (Chapter 23).

[0045] The recombinant plasmid pK18-Cgl2964-E53D was transformed into ATCC13032 competent cells by electroporation, and transformants were selected on BHI selective medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in ordinary BHI liquid medium at 33°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange and simultaneously introducing the target mutation. The culture was serially diluted (10 -2 Serial dilution to 10 -4 ), spread the dilution onto standard BHI solid medium containing 10% sucrose and incubate at 33°C for 48 hours. Transformants should harbor the desired mutation and lack the inserted vector sequence. The desired sequence was amplified by PCR and analyzed by nucleotide sequencing. The resulting transformed strain was designated 32-Cgl2964-E53D.

[0046] In the same manner as above, the above genetic manipulations were performed on the model strain ATCC 13032 of Corynebacterium glutamicum using the other three plasmids constructed in Example 1. The target transformed strains finally obtained were named 32-Cgl2964-E53K, 32-Cgl2964-E58D and 32-Cgl2964-E58R, respectively.

[0047] Example 3: Mutation modification of Cgl2964 in the Corynebacterium glutamicum model strain ATCC 13869

[0048] Competent cells of the Corynebacterium glutamicum model bacterium ATCC 13869 were prepared according to the method in the C. glutamicum Handbook (Chapter 23), and ATCC 13869 was genetically manipulated according to the method of Example 2. Specifically, all four tool plasmids constructed in Example 1 were used to genetically modify ATCC 13869 strains, and target modified strains carrying four histidine kinase mutants were obtained. The four modified strains were named 69-Cgl2964-E53D, 69-Cgl2964-E53K, 69-Cgl2964-E58D, and 69-Cgl2964-E58R, respectively.

[0049] Example 4 Fermentation performance test of histidine kinase mutants

[0050] The culture medium used for the fermentation performance test is as follows:

[0051] Plate activation medium: BHI 37g / L, 18g / L agar powder.

[0052] Seed culture medium: sucrose 20 g / L, yeast powder 5 g / L, peptone 10 g / L, urea 5 g / L, magnesium sulfate heptahydrate 0.4 g / L, adjust pH to 7.0.

[0053] Fermentation medium: glucose 30 g / L, ammonium sulfate 10 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, soybean meal hydrolyzate 10 g / L, calcium carbonate 30 g / L, adjust pH to 7.0.

[0054] Fermentation method:

[0055] 1. Seed activation: Take the strain to be verified from the cryopreserved tube, streak it on the seed activation medium, and culture it at 33℃ for 24 hours;

[0056] 2. Seed culture: Pick 1 activated seed from the plate and transfer it to a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Culture at 33°C, 220 rpm, and shake for 6 h.

[0057] 3. Fermentation culture: inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium. Culture at 33°C and 220 rpm for 15 h. Make three replicates for each strain.

[0058] 4. OD 562 Assay: Dilute the fermentation broth 100-fold with 0.01 M dilute hydrochloric acid and measure absorbance at 562 nm using a spectrophotometer. Perform three replicates for each strain and calculate the average. The results are shown in Table 2, which represents the average of the three replicates.

[0059] 5. Glucose Concentration Determination: Centrifuge 2 mL of fermentation broth (12,000 rpm, 2 min), collect the supernatant, dilute 10-fold with distilled water, and analyze using a biosensor analyzer. Three replicates were performed for each strain, and the average was calculated. The results are shown in Table 2, where the data are the average of the three replicates.

[0060] 6. Determination of Amino Acid Concentration: 2 mL of fermentation broth was centrifuged (12,000 rpm, 2 min), and the supernatant was collected and analyzed using Agilent high-performance liquid chromatography (HPLC). Three replicates were performed for each strain, and the average was calculated. The results are shown in Table 2, where the data are the average of the three replicates.

[0061] Table 2 Fermentation performance of shake flask test

[0062]

[0063] Testing of the control strain and its four modified strains from the same batch revealed consistent results: the modified strains carrying the histidine kinase mutation showed increased production of alanine, glutamate, and lysine, with a particularly significant increase in lysine. The other amino acids were present at lower levels and showed less significant changes, so the data are not presented in the table.

[0064] Example 5: Mutation modification of Cgl2964 in a lysine-producing strain CGMCC No. 11942

[0065] Competent cells of CGMCC No. 11942 were prepared according to the method in the C. glutamicum Handbook (Chapter 23), and genetic manipulation of CGMCC No. 11942 was performed according to the method of Example 2. Specifically, the two tool plasmids (pK18-Cgl2964-E53D and pK18-Cgl2964-E58R) constructed in Example 1 were used to genetically modify CGMCC No. 11942, and target modified strains carrying two histidine kinase mutants were obtained. The two modified strains were named 42-Cg12964-E53D and 42-Cg12964-E58R, respectively.

[0066] Example 6: Mutation modification of Cgl2964 in a lysine-producing strain CGMCC No. 13407

[0067] Competent cells of CGMCC No. 13407 were prepared according to the method in the C. glutamicum Handbook (Chapter 23), and genetic manipulation of CGMCC No. 13407 was performed according to the method of Example 2. Specifically, the two tool plasmids (pK18-Cgl2964-E53D and pK18-Cgl2964-E58R) constructed in Example 1 were used to genetically modify CGMCC No. 13407, and the target modified strains carrying two histidine kinase mutants were obtained. The two modified strains were named 07-Cgl2964-E53D and 07-Cgl2964-E58R, respectively.

[0068] Example 7 Fermentation performance test of histidine kinase mutants

[0069] The culture medium used for the fermentation performance test is as follows:

[0070] Plate activation medium: BHI 37g / L, 18g / L agar powder.

[0071] Seed culture medium: sucrose 20 g / L, yeast powder 5 g / L, peptone 10 g / L, urea 5 g / L, magnesium sulfate heptahydrate 0.4 g / L, adjust pH to 7.0.

[0072] Fermentation medium: glucose 60 g / L, ammonium sulfate 25 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, soybean meal hydrolyzate 10 g / L, calcium carbonate 30 g / L, adjust pH to 7.0.

[0073] Fermentation method:

[0074] 1. Seed activation: Take the strain to be verified from the cryopreserved tube, streak it on the seed activation medium, and culture it at 33℃ for 24 hours;

[0075] 2. Seed culture: Pick 1 activated seed from the plate and transfer it to a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Culture at 33°C, 220 rpm, and shake for 6 h.

[0076] 3. Fermentation culture: inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium. Culture at 33°C and 220 rpm for 15 h. Make three replicates for each strain.

[0077] 4. OD 562 Assay: Dilute the fermentation broth 100-fold with 0.01 M dilute hydrochloric acid and measure absorbance at 562 nm using a spectrophotometer. Perform three replicates for each strain and calculate the average. The results are shown in Table 3, which represents the average of the three replicates.

[0078] 5. Glucose Concentration Determination: Centrifuge 2 mL of fermentation broth (12,000 rpm, 2 min). Collect the supernatant, dilute 10-fold with distilled water, and analyze using a biosensor analyzer. Three replicates were performed for each strain, and the average was calculated. The results are shown in Table 3, which represents the average of the three replicates.

[0079] 6. Determination of Amino Acid Concentration: 2 mL of fermentation broth was centrifuged (12,000 rpm, 2 min), and the supernatant was collected and analyzed using Agilent high-performance liquid chromatography (HPLC). Three replicates were performed for each strain, and the average was calculated. The results are shown in Table 3, where the data are the average of the three replicates.

[0080] Table 3 Fermentation performance of shake flask test

[0081]

[0082]

[0083] In a test of two lysine-producing strains and two modified strains with histidine kinase mutations from the same batch, the modified strains carrying the histidine kinase mutants showed significant improvements in both lysine production and yield. This result demonstrates that introducing the histidine kinase mutants of the present invention into Corynebacterium glutamicum facilitates amino acid synthesis, particularly lysine synthesis.

[0084] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A histidine kinase mutant, characterized in that: The amino acid sequence of the histidine kinase mutant is shown in any one of SEQ ID NOs. 7, 9, and 10.

2. A DNA molecule, the nucleotide sequence of which is shown in any one of SEQ ID NOs. 2-5.

3. A recombinant microorganism, characterized in that The starting strain of the recombinant microorganism is Corynebacterium glutamicum, in which the wild-type histidine kinase is replaced by the histidine kinase mutant according to claim 1.

4. Any of the following applications of the recombinant microorganism: (1) Application in fermentation production of amino acids; (2) Application in genetic breeding of microorganisms for amino acid production; (3) Application in increasing the yield of amino acids in fermentation production; The recombinant microorganism is as described in claim 3, wherein the amino acid is lysine.

5. A method for producing lysine, characterized in that: The method comprises the step of fermenting and culturing a recombinant microorganism, wherein the recombinant microorganism is as described in claim 4.

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