CN117106042A - YH66-RS07020 mutant protein and application of related biological material in preparation of valine - Google Patents
YH66-RS07020 mutant protein and application of related biological material in preparation of valine Download PDFInfo
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- CN117106042A CN117106042A CN202311178534.9A CN202311178534A CN117106042A CN 117106042 A CN117106042 A CN 117106042A CN 202311178534 A CN202311178534 A CN 202311178534A CN 117106042 A CN117106042 A CN 117106042A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application discloses YH66-RS07020 mutant protein and application of related biological materials thereof in preparation of valine. The YH66-RS07020 mutant protein is obtained by mutating the 84 th amino acid residue of the YH66-RS07020 protein from A to other amino acid residues. The application discovers YH66-RS07020 mutant protein, and a coding gene and application thereof. The application of the mutein and the related biological material thereof is specifically the application in the preparation of valine. The application has great application value for industrial production of valine.
Description
The application relates to engineering bacteria obtained by modifying YH66-RS07020 genes and application thereof in valine preparation, which are classified as '202110967402.9' and 2021, 8 and 23.
Technical Field
The application belongs to the technical field of biology, and relates to application of YH66-RS07020 mutant proteins and related biological materials thereof in preparation of valine.
Background
Valine is one of the 20 amino acids constituting a protein, 8 amino acids and a glycogenic amino acid essential for the human body, and it works together with the other two high concentration amino acids (isoleucine and leucine) to promote normal growth of the body, repair tissues, regulate blood sugar, and supply necessary energy. Valine can provide additional energy to muscles to produce glucose when engaged in intense physical activity to prevent muscle weakness. Valine also helps to scavenge excess nitrogen (potential toxins) from the liver and transport the body's desired nitrogen to various sites.
Valine is an essential amino acid, which means that the body itself cannot produce and must be supplemented by dietary sources. Its natural food sources include cereals, dairy products, mushrooms, peanuts, soy proteins and meats. Although most people can obtain sufficient amounts from their diets, valine deficiency is also common. When valine is deficient, dysfunction of the central nervous system of the brain occurs, and ataxia occurs and limb tremors occur. Through dissecting brain tissue, it is found that erythrocyte degeneration phenomenon is caused, hyperinsulinemia is easy to form in patients with advanced liver cirrhosis due to liver function damage, the ratio of branched chain amino acid to aromatic amino acid in blood is reduced from 3.0-3.5 of normal people to 1.0-1.5, so that injections of branched chain amino acid such as valine are commonly used for treating liver failure and damage caused by alcoholism and drug absorption to the organs. In addition, valine is also useful as a therapeutic agent for accelerating wound healing. L-valine, with the name 2-amino-3-methylbutanoic acid, CAS number 72-18-4, MDL number MFCD00064220, EINECS number 200-773-6. The current preparation of L-valine is mainly a chemical synthesis. Limitations of chemical synthesis: high production cost, complex reaction, multiple steps and a plurality of byproducts.
Disclosure of Invention
The application aims to provide application of YH66-RS07020 mutant proteins and related biological materials thereof in preparation of valine.
The application provides an application of a substance for inhibiting YH66-RS07020 gene expression or a substance for reducing YH66-RS07020 protein abundance or a substance for reducing YH66-RS07020 protein activity;
the application is as follows (I) or (II) or (III):
use of (i) to increase valine production in a bacterium;
(II) use in the production of valine;
(III) use in increasing the bacterial load.
The YH66-RS07020 gene is a gene for encoding YH66-RS07020 protein.
The YH66-RS07020 protein is (a 1) or (a 2) or (a 3) as follows:
(a1) A protein shown in a sequence 3 of a sequence table;
(a2) A protein derived from a bacterium and having an identity of 95% or more to (a 1) and being related to valine production by the bacterium;
(a3) And (b) a protein derived from (a 1) obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein shown in (a 1) and related to valine production of bacteria.
The term "identity" as used herein refers to sequence similarity to a native amino acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The identity of 95% or more may specifically be 96% or more, 97% or more, 98% or more, or 99% or more.
Specifically, the YH66-RS07020 gene is as follows (b 1) or (b 2) or (b 3):
(b1) A DNA molecule with a coding region shown as a sequence 4 of a sequence table;
(b2) A DNA molecule derived from bacteria and having more than 95% identity to (b 1) and encoding said protein;
(b3) A DNA molecule which hybridizes under stringent conditions to (b 1) and which encodes said protein.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The identity of 95% or more may specifically be 96% or more, 97% or more, 98% or more, or 99% or more.
The stringent conditions may be hybridization and washing of the membrane in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.
The inhibition of YH66-RS07020 gene expression can be knockout of YH66-RS07020 gene or mutation of YH66-RS07020 gene.
The substance for inhibiting the expression of the YH66-RS07020 gene can be a DNA molecule shown in a sequence 5 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 5 of the sequence table.
The substance for inhibiting the expression of the YH66-RS07020 gene can be a DNA molecule shown in a sequence 8 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 8 of the sequence table.
Exemplary embodimentsThe substance for inhibiting YH66-RS07020 gene expression may specifically be the recombinant plasmid pK18-YH66-RS07020 in the examples C251T Or recombinant plasmid pK 18-DeltaYH 66-RS07020.
The application also provides a recombinant bacterium which is obtained by inhibiting YH66-RS07020 gene expression in the bacterium.
The inhibition of YH66-RS07020 gene expression in bacteria can be knockout of YH66-RS07020 gene in bacteria or mutant bacteria.
The knockout may be a partial segment of the knockout gene or may be the entire coding box of the knockout gene.
Illustratively, the YH66-RS07020 gene in the knocked-out bacteria can be specifically: the DNA molecule shown in the sequence 4 of the sequence table is deleted in the bacterial genome DNA.
For the YH66-RS07020 gene in mutant bacteria, a person of ordinary skill in the art can easily employ known methods such as directed mutation or gene editing, etc.
Illustratively, the YH66-RS07020 gene in a mutant bacterium can be specifically: the codon of the 84 th amino acid residue of YH66-RS07020 protein coded in bacterial genome DNA is mutated from the codon of A to the codon of other amino acid residues. Specifically, the other amino acid residue is V.
Illustratively, the YH66-RS07020 gene in a mutant bacterium can be specifically: the YH66-RS07020 gene in the bacterial genome DNA is subjected to the following point mutation: the 251 st nucleotide is mutated from C to other nucleotides (which may be T in particular).
Illustratively, the inhibition of YH66-RS07020 gene expression in bacteria may be achieved by: a substance for inhibiting YH66-RS07020 gene expression is introduced into a bacterium.
The substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in a sequence 5 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 5 of the sequence table.
The substance for inhibiting the expression of the YH66-RS07020 gene can be a DNA molecule shown in a sequence 8 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 8 of the sequence table.
The substance for inhibiting YH66-RS07020 gene expression may be exemplified by recombinant plasmid pK18-YH66-RS07020 in the examples C251T Or recombinant plasmid pK 18-DeltaYH 66-RS07020.
The application also protects application of the recombinant bacterium in valine preparation.
The application also provides a method for preparing valine, which comprises the following steps: fermenting the recombinant bacteria.
The person skilled in the art can carry out the fermentation using fermentation methods known in the art. Optimization and improvement of the fermentation process can also be carried out by routine experimentation. The fermentation of the bacteria may be performed in a suitable medium under fermentation conditions known in the art. The medium may comprise: carbon source, nitrogen source, trace elements, and combinations thereof. During the culture, the pH of the culture may be adjusted. In addition, the culture may include prevention of bubble generation, for example, by using an antifoaming agent. In addition, the culturing may include injecting a gas into the culture. The gas may comprise any gas capable of maintaining aerobic conditions of the culture. In the cultivation, the temperature of the culture may be 20 to 45 ℃.
The method may further comprise the steps of: valine was obtained from the culture. Valine can be obtained from culture in a variety of ways including, but not limited to: the culture is treated with sulfuric acid or hydrochloric acid or the like, followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.
In the fermentation, the formula of an exemplary fermentation medium is shown in Table 3, and the balance is water.
An exemplary fermentation control process in the fermentation is shown in table 4.
Illustratively, in the fermentation, the OD value of the system may be 0.3-0.5 at the initial time of completion of inoculation.
Illustratively, during the fermentation process of the fermentation: ammonia water is used for regulating the pH value; when foam exists in the fermentation system, adding a proper amount of defoamer antiiufoam (CB-442); the sugar content (residual sugar) of the system was controlled by supplementing 70% glucose aqueous solution.
The application also provides a method for improving valine yield of bacteria, comprising the following steps: inhibiting YH66-RS07020 gene expression in bacteria or reducing YH66-RS07020 protein abundance in bacteria or reducing YH66-RS07020 protein activity in bacteria.
The inhibition of YH66-RS07020 gene expression in bacteria can be knockout of YH66-RS07020 gene in bacteria or mutant bacteria.
The knockout may be a partial segment of the knockout gene or may be the entire coding box of the knockout gene.
Illustratively, the YH66-RS07020 gene in the knocked-out bacteria can be specifically: the DNA molecule shown in the sequence 4 of the sequence table is deleted in the bacterial genome DNA.
For the YH66-RS07020 gene in mutant bacteria, a person of ordinary skill in the art can easily employ known methods such as directed mutation or gene editing, etc.
Illustratively, the YH66-RS07020 gene in a mutant bacterium can be specifically: the codon of the 84 th amino acid residue of YH66-RS07020 protein coded in bacterial genome DNA is mutated from the codon of A to the codon of other amino acid residues. Specifically, the other amino acid residue is V.
Illustratively, the YH66-RS07020 gene in a mutant bacterium can be specifically: the YH66-RS07020 gene in the bacterial genome DNA is subjected to the following point mutation: the 251 st nucleotide is mutated from C to other nucleotides (which may be T in particular).
Illustratively, the inhibition of YH66-RS07020 gene expression in bacteria may be achieved by: a substance for inhibiting YH66-RS07020 gene expression is introduced into a bacterium.
The substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in a sequence 5 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 5 of the sequence table.
The substance for inhibiting the expression of the YH66-RS07020 gene can be a DNA molecule shown in a sequence 8 of a sequence table or a recombinant plasmid with the DNA molecule shown in the sequence 8 of the sequence table.
The substance for inhibiting YH66-RS07020 gene expression may be exemplified by recombinant plasmid pK18-YH66-RS07020 in the examples C251T Or recombinant plasmid pK 18-DeltaYH 66-RS07020.
The application also protects the application of the YH66-RS07020 protein in regulating and controlling the valine yield of bacteria.
The regulation is negative, namely the YH66-RS07020 protein content is increased, and the valine yield is reduced.
The regulation is negative, namely the YH66-RS07020 protein content is reduced, and the valine yield is increased.
The application also protects the application of the YH66-RS07020 protein in regulating and controlling the bacterial load of bacteria.
The regulation is negative, namely the YH66-RS07020 protein content is increased, and the bacterial load is reduced.
The regulation is negative, namely the YH66-RS07020 protein content is reduced, and the bacterial amount is increased.
The application also protects a mutein named YH66-RS07020 C251T The protein is obtained by mutating the 84 th amino acid residue of YH66-RS07020 protein from A to other amino acid residues.
Specifically, the other amino acid residue is V.
The mutant protein is shown as a sequence 1 in a sequence table.
The application also protects YH66-RS07020 C251T Protein coding gene (named YH66-RS 07020) C251T Genes).
The application also protects the battery with YH66-RS07020 C251T Gene expression cassette or with YH66-RS07020 C251T Recombinant vector of gene or gene with YH66-RS07020 C251T Recombinant bacteria of the gene.
Specifically, YH66-RS07020 C251T The genes are (c 1) or (c 2) or (c 3) as follows:
(c1) A DNA molecule with a coding region shown as a sequence 2 of a sequence table;
(c2) A DNA molecule derived from bacteria and having more than 95% identity to (c 1) and encoding said protein;
(c3) A DNA molecule which hybridizes under stringent conditions to (c 1) and which encodes said protein.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The identity of 95% or more may specifically be 96% or more, 97% or more, 98% or more, or 99% or more.
The stringent conditions may be hybridization and washing of the membrane in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.
The application also protects YH66-RS07020 C251T Protein, YH66-RS07020 C251T Gene, with YH66-RS07020 C251T Gene expression cassette or with YH66-RS07020 C251T Or a recombinant vector having YH66-RS07020 C251T The recombinant strain of (2) is applied to the preparation of valine.
The application also provides a method for improving valine yield of bacteria, comprising the following steps: the codon of the 84 th amino acid residue of YH66-RS07020 protein coded in bacterial genome DNA is mutated from the codon of A to the codon of other amino acid residues.
Specifically, the other amino acid residue is V.
The method specifically comprises the following steps: the YH66-RS07020 gene in the bacterial genome DNA is subjected to the following point mutation: the 251 st nucleotide is mutated from C to other nucleotides (which may be T in particular).
The method specifically comprises the following steps: a DNA molecule shown in a sequence 5 of a sequence table or a recombinant plasmid having the DNA molecule shown in the sequence 5 of the sequence table is introduced into bacteria.
Any of the above bacteria include, but are not limited to, the following: corynebacterium genus bacteria, preferably Corynebacterium acetoacidophilus (Corynebacterium acetoacidophilus), corynebacterium aceti (Corynebacterium acetoglutamicum), corynebacterium maydis (Corynebacterium calune), corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium lactofermentum (Brevibacterium lactofermentum), corynebacterium ammoniagenes (Corynebacterium ammoniagenes), corynebacterium beijing (Corynebacterium pekinense), brevibacterium saccharolyticum (Brevibacterium saccharolyticum), brevibacterium roseum (Brevibacterium roseum), brevibacterium thiogenum (Brevibacterium thiogenitalis).
Any of the above-mentioned bacteria is a bacterium having an ability to produce valine.
"bacterium having an ability to produce valine" means that the bacterium has the following ability: ability to produce and accumulate valine in the medium and/or cells of the bacterium. Thus, valine can be collected when the bacteria are cultured in the medium.
The bacteria may be naturally harvested wild-type bacteria or modified bacteria.
"modified bacteria" refers to engineered bacteria obtained by artificial mutation and/or mutagenesis of naturally acquired wild-type bacteria.
Specifically, the corynebacterium glutamicum can be corynebacterium glutamicum CGMCC21260.
Corynebacterium glutamicum (Corynebacterium glutamicum) YPF 1 was deposited at China general microbiological culture Collection center (CGMCC) at 11 and 30 months in 2020, and the deposit registration number is CGMCC No.21260. Corynebacterium glutamicum (Corynebacterium glutamicum) YPF 1, also known as Corynebacterium glutamicum CGMCC21260.
Valine in any one of the foregoing is meant to be valine in a broad sense and includes valine in free form, salts of valine or mixtures of both.
Specifically, the valine is L-valine.
Any of the above methods or applications can also be used for the preparation of a downstream product of valine.
YH66-RS07 in Corynebacterium glutamicumThe 020 protein is shown as a sequence 3 in a sequence table, and the coding gene is shown as a sequence 4 in the sequence table. In the application, by introducing point mutation, YH66-RS07020 shown in sequence 1 of a sequence table is obtained C251T Protein YH66-RS07020 C251T The coding gene of the protein is shown as a sequence 2 in a sequence table. Compared with YH66-RS07020 gene, YH66-RS07020 C251T The difference between the genes is that nucleotide 251 is mutated from C to T. Compared with YH66-RS07020 protein, YH66-RS07020 C251T The difference between proteins is the mutation of amino acid residue 84 from a to V.
The application discovers that the YH66-RS07020 protein has negative regulation on the valine yield of bacteria, namely the content of the YH66-RS07020 protein is increased, the valine yield is reduced, the content of the YH66-RS07020 protein is reduced, and the valine yield is increased. Inhibiting YH66-RS07020 gene expression can improve valine yield, and over-expressing YH66-RS07020 gene can reduce valine yield. Further, the present application found that YH66-RS07020 C251T Protein and its coding gene and application. The application has great application value for industrial production of valine.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The pK18mobsacB plasmid: addgene company; the pK18mobsacB plasmid has the kanamycin resistance gene as a selectable marker. pXMJ19 plasmid: biovector plasmid vector strain cell gene collection center; the pXMJ19 plasmid has a chloramphenicol resistance gene as a selectable marker. NEBuilder enzyme: NEB corporation. Unless otherwise specified, the medium in the examples was the medium of the formulation of Table 1 (balance water, pH 7.0). The medium shown in Table 1 was not containing kanamycin. The kanamycin-containing medium consisted of the medium shown in Table 1 and kanamycin, the content of which was 50. Mu.g/ml. Unless otherwise indicated, the culture in the examples refers to stationary culture at 32 ℃. Single-strand conformational polymorphism polyacrylamide gel electrophoresis (sscp-PAGE) in the examples: the gel concentration used was 8% and the composition of the electrophoretic gel is shown in table 2; the electrophoresis conditions were: electrophoresis time was 10h using 1 XTBE buffer, 120V voltage.
Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.
TABLE 1
| Component (A) | Concentration in the Medium |
| Sucrose | 10g/L |
| Polypeptone | 10g/L |
| Beef extract | 10g/L |
| Yeast powder | 5g/L |
| Urea | 2g/L |
| Sodium chloride | 2.5g/L |
| Agar powder | 20g/L |
TABLE 2
| Component (A) | Addition amount of |
| 40% acrylamide | 8mL |
| ddH 2 O | 26mL |
| Glycerol | 4mL |
| 10×TBE | 2mL |
| TEMED | 40μL |
| 10％AP | 600μL |
Example 1 obtaining Corynebacterium glutamicum CGMCC21260
Corynebacterium glutamicum ATCC15168: corynebacterium glutamicum (Corynebacterium glutamicum) having accession number 15168 in ATCC.
Corynebacterium glutamicum ATCC15168 was subjected to mutagenesis to obtain Corynebacterium glutamicum (Corynebacterium glutamicum) YPF 1.
Corynebacterium glutamicum (Corynebacterium glutamicum) YPF 1 was deposited at China general microbiological culture Collection center (CGMCC) at 11 and 30 months in 2020, and the deposit registration number is CGMCC No.21260. Corynebacterium glutamicum (Corynebacterium glutamicum) YPF 1, also known as Corynebacterium glutamicum CGMCC21260.
EXAMPLE 2 construction of recombinant bacteria YPV-013
P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAACGCCCGCATCGAAGACCT-3'；
P2：5'-GCCGTAGTCCATGAGTACCCAGACGGCGTGCTCG-3'；
P3：5'-CGAGCACGCCGTCTGGGTACTCATGGACTACGGC-3'；
P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATCCTAGAGGGGCACTTTTC-3'。
P5：5'-CGGACTGTTTTCCAACTGGC-3'；
P6：5'-CCGGGGTTTGTTCCATGAGC-3'。
1. Construction of recombinant plasmids
1. The amplified product (674 bp) was recovered by PCR amplification using Corynebacterium glutamicum ATCC15168 as a template and a primer set consisting of primer P1 and primer P2.
2. The amplified product (674 bp) was recovered by PCR amplification using Corynebacterium glutamicum ATCC15168 as a template and a primer set consisting of primer P3 and primer P4.
3. And simultaneously, taking the amplification product recovered in the step 1 and the amplification product recovered in the step 2 as templates, and adopting a primer pair consisting of a primer P1 and a primer P4 to carry out PCR (overlay PCR) amplification, thereby recovering the amplification product (1314 bp). And sequencing, wherein an amplified product is shown as a sequence 5 in a sequence table.
4. The pK18mobsacB plasmid was taken and subjected to single cleavage with restriction enzyme Xba I to recover the linearized plasmid.
5. Incubating the amplified product recovered in step 3 with the linearized plasmid recovered in step 4 (usingNEBuilder enzyme, incubating for 30min at 50 ℃ to obtain recombinant plasmid pK18-YH66-RS07020 C251T . Sequencing shows that the recombinant plasmid pK18-YH66-RS07020 C251T The DNA molecule shown in the sequence 5 in the sequence table.
2. Construction of recombinant bacterium YPV-013
1. Recombinant plasmid pK18-YH66-RS07020 is adopted C251T The corynebacterium glutamicum CGMCC21260 is subjected to electric shock transformation and then is cultured.
2. The strain in the step 1 is selected, the culture medium containing 15% of sucrose is adopted for culture, then single colonies are selected, the culture medium containing kanamycin and the culture medium not containing kanamycin are adopted for culture respectively, and the strain which can not grow on the culture medium containing kanamycin and can grow on the culture medium not containing kanamycin is selected.
3. And (3) taking the strain screened in the step (2), carrying out PCR amplification by adopting a primer pair consisting of a primer P5 and a primer P6, and then recovering an amplified product (278 bp).
4. Taking the amplified product of the step 3, firstly denaturing at 95 ℃ for 10min, then ice-bathing for 5min, and then performing sscp-PAGE. During electrophoresis, recombinant plasmid pK18-YH66-RS07020 is adopted C251T Is amplified by the recombinant plasmid pK18-YH66-RS07020 C251T As a template, an amplification product obtained by PCR amplification by using a primer pair consisting of the primer P5 and the primer P6 is used as a positive control, an amplification fragment of the corynebacterium glutamicum CGMCC21260 (namely, an amplification product obtained by PCR amplification by using the corynebacterium glutamicum CGMCC21260 as a template and a primer pair consisting of the primer P5 and the primer P6) is used as a negative control, and water is used as a blank control. Due to the different fragment structures, the electrophoresis positions are different, and the strains with the electrophoresis positions inconsistent with the negative control and consistent with the positive control are selected target strains (recombinant strains with successful allelic replacement).
5. And (3) according to the result of the step (4), sequencing and verifying the amplified product of the step (3) of the strain obtained by screening to obtain recombinant bacteria YPV-013. Compared with the corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-013 only has the difference that the YH66-RS07020 gene shown in the sequence 4 of the sequence table in the genome of the corynebacterium glutamicum CGMCC21260 is replaced by the YH66-RS070 shown in the sequence 2 of the sequence table20 C251T And (3) a gene. Sequence 2 and sequence 4 differ by only one nucleotide and are located at position 251. Recombinant YPV-013 is an engineering strain obtained by carrying out mutation (single-point mutation) on YH66-RS07020 genes in corynebacterium glutamicum CGMCC21260.
EXAMPLE 2 construction of recombinant bacteria YPV-015 and YPV-014
P7：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGGACTC-3'；
P8：5'-TGAAATGTAAGATTCAAAGAAATCGGACTCCTTAAATGGG-3'；
P9：5'-CCCATTTAAGGAGTCCGATTTCTTTGAATCTTACATTTCA-3'；
P10：5'-TGGGTGGTAAATTTTTCCATGGAACTCACCGTCCTTACAG-3'；
P11：5'-CTGTAAGGACGGTGAGTTCCATGGAAAAATTTACCACCCA-3'；
P12：5'-CTATGTGAGTAGTCGATTTATTAAGCGTTAGTGCGTGGCT-3'；
P13：5'-AGCCACGCACTAACGCTTAATAAATCGACTACTCACATAG-3'；
P14：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAACCACTT-3'。
P15：5'-GTCCGCTCTGTTGGTGTTCA-3'；
P16：5'-AGAAGTTCGATGTCGGACTG-3'。
P17：5'-CCAACGTGGACACCGACCAG-3'；
P18：5'-TGGAGGAATATTCGGCCCAG-3'。
1. Construction of recombinant bacterium YPV-015
1. The recombinant YPV-013 is used as a template, and a primer pair consisting of a primer P7 and a primer P8 is used for PCR amplification, and an amplification product (806 bp) is recovered.
2. The recombinant YPV-013 is used as a template, and a primer pair consisting of a primer P9 and a primer P10 is used for PCR amplification, and an amplification product (293 bp) is recovered.
3. And (3) taking recombinant bacteria YPV-013 as templates, adopting a primer pair consisting of a primer P11 and a primer P12 to carry out PCR amplification, and recovering an amplification product (634 bp).
4. The recombinant YPV-013 is used as a template, and a primer pair consisting of a primer P13 and a primer P14 is used for PCR amplification, and an amplified product (783 bp) is recovered.
5. The pK18mobsacB plasmid was taken and subjected to single cleavage with restriction enzyme Xba I to recover the linearized plasmid.
6. Incubating the amplification product recovered in the step 1, the amplification product recovered in the step 2, the amplification product recovered in the step 3 and the amplification product recovered in the step 4 with the linearization plasmid recovered in the step 5 (using NEBuilder enzyme, incubating at 50 ℃ for 30 min) to obtain a recombinant plasmid 015. Sequencing shows that the recombinant plasmid 015 has DNA molecule shown in the sequence 6 in the sequence list.
7. The recombinant plasmid 015 is adopted to carry out electric shock transformation on the corynebacterium glutamicum CGMCC21260, then the culture is carried out, and then each single colony is respectively subjected to PCR identification (a primer pair consisting of a primer P15 and a primer P16 is adopted), so that the strain capable of amplifying 1454bp bands is a positive strain.
8. Selecting the positive strain in the step 7, culturing by adopting a culture medium containing 15% of sucrose, then selecting single colonies, culturing by adopting a culture medium containing kanamycin and a culture medium not containing kanamycin respectively, and screening strains which cannot grow on the culture medium containing kanamycin and can grow on the culture medium not containing kanamycin.
9. Taking the strain screened in the step 8, and carrying out PCR amplification by adopting a primer pair consisting of a primer P17 and a primer P18, wherein the strain with 1335bp of amplified band is YH66-RS07020 C251T The positive strain with the gene integrated on the genome of the corynebacterium glutamicum CGMCC21260 is named as recombinant strain YPV-015. Recombinant YPV-015 is the over-expression YH66-RS07020 on genome C251T Engineering strain of gene.
2. Construction of recombinant bacterium YPV-014
The templates were replaced by "recombinant YPV-013" by "Corynebacterium glutamicum ATCC15168", all other steps.
The positive strain of YH66-RS07020 gene integrated on the genome of corynebacterium glutamicum CGMCC21260 is obtained and is named as recombinant strain YPV-014. Recombinant YPV-014 is an engineering strain for over-expressing YH66-RS07020 genes on genome. Recombinant bacteria YPV-014 differ from recombinant bacteria YPV-015 only in: the sequence 4 is integrated into the sequence of the exogenous DNA of the genome of the corynebacterium glutamicum CGMCC21260, and replaces the sequence 2.
EXAMPLE 3 construction of recombinant bacteria YPV-017 and recombinant bacteria YPV-016
1. Construction of recombinant bacterium YPV-017
1. The recombinant YPV-013 is used as a template, and a primer pair consisting of a primer P19 and a primer P20 is used for PCR amplification, and an amplification product (917 bp) is recovered. And sequencing, wherein an amplified product is shown as a sequence 7 in a sequence table.
P19：5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTCTTTGAATCTTACATTTCA-3'；
P20：5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAAGCGTTAGTGCGTGGCT-3'。
2. Taking pXMJ19 plasmid, adopting restriction enzyme EcoRI for single enzyme digestion, and recovering linearization plasmid.
3. Incubating the amplified product recovered in step 1 with the linearized plasmid recovered in step 2 (using NEBuilder enzyme, incubating at 50deg.C for 30 min) to obtain recombinant plasmid pXMJ19-YH66-RS07020 C251T . Sequencing verifies that the recombinant plasmid pXMJ19-YH66-RS07020 C251T The DNA molecule shown in the sequence 7 in the sequence table.
4. Recombinant plasmid pXMJ19-YH66-RS07020 C251T Electric transduction is conducted into corynebacterium glutamicum CGMCC21260 to obtain recombinant bacteria YPV-017. Recombinant YPV-017 is the overexpression of pXMJ19-YH66-RS07020 by plasmid C251T Engineering strain of gene.
2. Construction of recombinant YPV-016
The template was replaced by "recombinant YPV-013" by "Corynebacterium glutamicum ATCC15168", otherwise identical to step one.
Recombinant YPV-016 was obtained. Recombinant YPV-016 is engineering strain through plasmid over-expressing YH66-RS07020 gene. Compared with recombinant bacteria YPV-017, recombinant bacteria YPV-016 differ only in that: sequence 4 replaces sequence 2 in the sequence of the foreign DNA over-expressed by the plasmid.
Example 4 construction of an engineering Strain with deletion of YH66-RS07020 Gene on genome
P21：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGTGCCGGCATGATCGCCCC-3'；
P22：5'-TTTCGCTATCAGACTGAAACTCTTTTTCTAGCCTTCCTTA-3'；
P23：5'-TAAGGAAGGCTAGAAAAAGAGTTTCAGTCTGATAGCGAAA-3'；
P24：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTTGCCCTTCAAACCCACCG-3'。
P25：5'-CGTGCCGGCATGATCGCCCC-3'；
P26：5'-GTTGCCCTTCAAACCCACCG-3'。
1. Construction of recombinant plasmids
1. The amplified product (upstream homology arm fragment, 787 bp) was recovered by PCR amplification using Corynebacterium glutamicum ATCC15168 as a template and a primer pair consisting of primer P21 and primer P22.
2. The Corynebacterium glutamicum ATCC15168 was used as a template, and a primer pair consisting of the primer P23 and the primer P24 was used for PCR amplification to collect an amplified product (a downstream homology arm fragment, 773 bp).
3. Simultaneously, the amplification product recovered in the step 1 and the amplification product recovered in the step 2 are used as templates, and a primer pair consisting of a primer P21 and a primer P24 is adopted for PCR amplification (overlay PCR) to recover the amplification product (1520 bp). And sequencing, wherein an amplified product is shown as a sequence 8 in a sequence table.
4. The pK18mobsacB plasmid was taken and subjected to single cleavage with restriction enzyme Xba I to recover the linearized plasmid.
5. And (3) incubating the amplification product recovered in the step (3) with the linearized plasmid recovered in the step (4) (incubating for 30min at 50 ℃ with NEBuilder enzyme) to obtain the recombinant plasmid pK 18-delta YH66-RS07020. Sequencing verifies that the recombinant plasmid pK 18-delta YH66-RS07020 has the DNA molecule shown in the sequence 8 of the sequence table.
2. Construction of recombinant bacterium YPV-018
1. The recombinant plasmid pK 18-delta YH66-RS07020 is adopted to carry out electric shock transformation on the corynebacterium glutamicum CGMCC21260, then the corynebacterium glutamicum is cultured, and then each single colony is respectively subjected to PCR identification (a primer pair consisting of the primers P25 and P26 is adopted). The strain capable of amplifying 1446bp and 2040bp bands simultaneously is a positive strain. The strain only amplified with 2040bp band is the starting strain with transformation failure, wherein the 2040bp fragment is shown as a sequence 9 of a sequence table.
2. Selecting the positive strain in the step 1, culturing by adopting a culture medium containing 15% of sucrose, then selecting single colonies, culturing by adopting a culture medium containing kanamycin and a culture medium not containing kanamycin respectively, and screening strains which cannot grow on the culture medium containing kanamycin and can grow on the culture medium not containing kanamycin.
3. And 2, taking the strain screened in the step 2, and carrying out PCR amplification by adopting a primer pair consisting of a primer P25 and a primer P26, wherein the amplified product is only one strain with 1446bp, and the strain is a positive strain with the YH66-RS07020 gene coding region knocked out.
4. And (3) carrying out PCR amplification and sequencing on the strain obtained by screening in the step (3) again by adopting a primer pair consisting of a primer P25 and a primer P26, and naming the strain with correct sequencing as recombinant bacteria YPV-018. Compared with the genome DNA of the corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-018 only has the difference that the DNA molecule shown in the sequence 4 of the sequence table is deleted.
Example 5 fermentative preparation of L-valine
The test strains were respectively: corynebacterium glutamicum CGMCC21260, recombinant bacterium YPV-013, recombinant bacterium YPV-014, recombinant bacterium YPV-015, recombinant bacterium YPV-016, recombinant bacterium YPV-017 and recombinant bacterium YPV-018.
A fermentation tank: a BLBIO-5GC-4-H model fermenter (Shanghai Bai Zhi Ku Bio-technology Co., ltd.).
The formulation of the fermentation medium is shown in Table 3, the balance being water.
TABLE 3 fermentation Medium formulation
The fermentation control process is shown in Table 4. At the initial time of completing inoculation, the OD value of the system is 0.3-0.5.
During the fermentation process: ammonia water is used for regulating the pH value; when foam exists in the fermentation system, adding a proper amount of defoamer antiiufoam (CB-442); the sugar content (residual sugar) of the system was controlled by supplementing 70% glucose aqueous solution.
TABLE 4 fermentation control process
After completion of fermentation, the supernatant was collected, and the L-valine yield in the supernatant was measured by HPLC.
The results are shown in Table 5. The L-valine yield of the recombinant bacteria YPV-013 and YPV-018 is obviously higher than that of the corynebacterium glutamicum CGMCC21260. The result shows that the L-valine yield can be improved by inhibiting the expression of the YH66-RS07020 gene, and the L-valine yield can be reduced by over-expressing the YH66-RS07020 gene.
TABLE 5 fermentation test results of L-valine
| Strain | OD 610 | L-valine yield (g/L) |
| Corynebacterium glutamicum CGMCC21260 | 98.1 | 82.4 |
| Recombinant bacterium YPV-013 | 98.7 | 85.9 |
| Recombinant bacterium YPV-014 | 97.9 | 80.9 |
| Recombinant bacterium YPV-015 | 97.4 | 80.6 |
| Recombinant bacterium YPV-016 | 97.3 | 79.7 |
| Recombinant bacterium YPV-017 | 97.2 | 80.8 |
| Recombinant bacterium YPV-018 | 99.7 | 85.3 |
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (7)
1. The mutant protein is obtained by mutating the 84 th amino acid residue of YH66-RS07020 protein from A to other amino acid residues; the YH66-RS07020 protein is the YH66-RS07020 protein in claim 1;
the YH66-RS07020 protein is (a 1) or (a 2) or (a 3) as follows:
(a1) A protein shown in a sequence 3 of a sequence table;
(a2) A protein derived from a bacterium and having an identity of 95% or more to (a 1) and being related to valine production by the bacterium;
(a3) And (b) a protein derived from (a 1) obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein shown in (a 1) and related to valine production of bacteria.
2. A mutant protein encoding gene according to claim 1.
3. An expression cassette comprising a gene encoding the mutein of claim 1.
4. A recombinant vector comprising a gene encoding the mutein of claim 1.
5. A recombinant bacterium having a gene encoding the mutein of claim 1.
6. Use of a mutein according to claim 1, a coding gene according to claim 2, an expression cassette according to claim 3, a recombinant vector according to claim 4 or a recombinant bacterium according to claim 5 for the preparation of valine.
7. A method for increasing valine production of a bacterium comprising the steps of: mutating the codon of the 84 th amino acid residue of YH66-RS07020 protein coded in bacterial genome DNA from the codon of A to the codon of other amino acid residues; the YH66-RS07020 protein is the YH66-RS07020 protein in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311178534.9A CN117106042A (en) | 2021-08-23 | 2021-08-23 | YH66-RS07020 mutant protein and application of related biological material in preparation of valine |
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| CN202110967402.9A CN113683666A (en) | 2021-08-23 | 2021-08-23 | Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine |
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| CN114315999A (en) * | 2022-03-17 | 2022-04-12 | 北京中科伊品生物科技有限公司 | LeuD protein mutant and related biological material and application thereof in preparation of L-valine |
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| CN101082050A (en) * | 1999-06-25 | 2007-12-05 | Basf公司 | Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport |
| DE19951708A1 (en) * | 1999-10-27 | 2001-05-03 | Degussa | Nicleotide sequences coding for the export of branched-chain amino acids, methods for their isolation and their use |
| RU2243260C2 (en) * | 2002-06-25 | 2004-12-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" | Method for preparing l-leucine (variants), strain escherichia coli 505/pacyc-tyr b as producer of l-leucine |
| DE102005045301A1 (en) * | 2005-09-22 | 2007-04-05 | Degussa Ag | Process for the preparation of organochemical compounds using coryneform bacteria |
| US9347048B2 (en) * | 2012-04-27 | 2016-05-24 | Evonik Technochemie Gmbh | Feedback-resistant alpha-isopropylmalate synthases |
| ES2623161T3 (en) * | 2013-06-03 | 2017-07-10 | Evonik Degussa Gmbh | Procedure for the preparation of L-valine using recombinant corinebacteria containing the propionate inducible ilvBN operon |
| KR101747542B1 (en) * | 2015-10-23 | 2017-06-15 | 씨제이제일제당 (주) | Microorganisms having l-isoleucine productivity and process for producing l-isoleucine using the same |
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