CN113683666A - Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine - Google Patents
Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine Download PDFInfo
- Publication number
- CN113683666A CN113683666A CN202110967402.9A CN202110967402A CN113683666A CN 113683666 A CN113683666 A CN 113683666A CN 202110967402 A CN202110967402 A CN 202110967402A CN 113683666 A CN113683666 A CN 113683666A
- Authority
- CN
- China
- Prior art keywords
- protein
- valine
- gene
- bacteria
- bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- 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 invention discloses an engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in valine preparation. The invention 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 in improving the yield of bacterial valine. The invention discovers that YH66-RS07020 protein has negative regulation and control on the yield of valine of bacteria, namely YH66-RS07020 protein content is increased,The yield of valine is reduced, the content of YH66-RS07020 protein is reduced, and the yield of valine is increased. The suppression of YH66-RS07020 gene expression can improve valine yield, and the overexpression of YH66-RS07020 gene can reduce valine yield. Further, YH66-RS07020 was found in the inventionC251TProtein and its coding gene and application. The invention has great application value for industrial production of valine.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in valine preparation, wherein the modification is C251T.
Background
Valine, which is one of 20 amino acids constituting proteins, is 8 amino acids and glycogenic amino acid essential to the human body, and works together with other two high-concentration amino acids (isoleucine and leucine) to promote the normal growth of the body, repair tissues, regulate blood glucose, and supply required energy. Valine can provide additional energy to the muscle to produce glucose when engaged in strenuous physical activity to prevent muscle weakness. Valine also helps to clear excess nitrogen (a potential toxin) from the liver and to transport the body's required nitrogen to various sites.
Valine is an essential amino acid, which means that the body cannot produce itself and must be supplemented by dietary sources. Its natural food sources include grains, dairy products, mushrooms, peanuts, soy protein, and meats. Although most people can obtain sufficient quantities from their diets, cases of valine deficiency are also rare. When valine is insufficient, the central nervous system of the brain is disturbed, and limb tremor occurs due to ataxia. When the brain tissue is dissected and sliced, the degeneration phenomenon of the red nucleus cells is found, hyperinsulinemia is easy to form due to the liver function damage of a patient with late cirrhosis, so that the branched chain amino acid in the blood is reduced, the ratio of the branched chain amino acid to the aromatic amino acid is reduced from 3.0-3.5 of a normal person to 1.0-1.5, and therefore, the injection of the branched chain amino acid such as valine is commonly used for treating liver failure and the damage to the organs caused by alcoholism and drug absorption. In addition, valine can also be used as a therapeutic agent for accelerating wound healing. L-valine, also known as 2-amino-3-methylbutyric acid, CAS number 72-18-4, MDL number MFCD00064220, EINECS number 200-773-6. The current method for preparing L-valine is mainly a chemical synthesis method. Limitations of chemical synthesis: the production cost is high, the reaction is complex, the steps are multiple, and a plurality of byproducts exist.
Disclosure of Invention
The invention aims to provide an engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in valine preparation.
The invention provides 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), (II) or (III):
the application of (I) in improving the yield of the bacterial valine;
(II) use in the production of valine;
(III) application in improving bacterial quantity.
The YH66-RS07020 gene is a gene encoding the YH66-RS07020 protein.
The YH66-RS07020 protein is (a1) or (a2) or (a 3):
(a1) protein shown in a sequence 3 in a sequence table;
(a2) a protein derived from a bacterium, having 95% or more identity to (a1), and being associated with valine production by the bacterium;
(a3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in (a1) and is related to the production of valine by bacteria and derived from (a 1).
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 assess the identity between related sequences.
The identity of 95% or more may be 96% or more, 97% or more, 98% or more, or 99% or more.
Specifically, the YH66-RS07020 gene is (b1) or (b2) or (b 3):
(b1) the coding region is a DNA molecule shown as a sequence 4 in the sequence table;
(b2) a DNA molecule derived from a bacterium and having 95% or more identity to (b1) and encoding said protein;
(b3) a DNA molecule that hybridizes under stringent conditions to (b1) and 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 assess the identity between related sequences.
The identity of 95% or more may be 96% or more, 97% or more, 98% or more, or 99% or more.
The stringent conditions may be hybridization and membrane washing at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.
The YH66-RS07020 gene expression inhibition can be knock-out of the YH66-RS07020 gene, and can also be mutation of the YH66-RS07020 gene.
Illustratively, the substance for inhibiting YH66-RS07020 gene expression may be specifically a DNA molecule shown in sequence 5 of the sequence table or a recombinant plasmid having the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in sequence 8 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 8 of the sequence table.
The substance for inhibiting YH66-RS07020 gene expression can be specifically a recombinant plasmid pK18-YH66-RS07020 in the embodimentC251TOr the recombinant plasmid pK 18-delta YH66-RS 07020.
The invention also provides a recombinant bacterium, which is obtained by inhibiting YH66-RS07020 gene expression in bacteria.
The YH66-RS07020 gene expression in the bacteria can be knocked out from YH66-RS07020 genes in the bacteria, and can also be YH66-RS07020 genes in mutant bacteria.
The knockout may be a partial segment of the knockout gene or may be the entire coding frame of the knockout gene.
Illustratively, the YH66-RS07020 gene in the knockout bacterium can be specifically: and (3) deleting the DNA molecule shown in the sequence 4 in the sequence table in the bacterial genome DNA.
For the YH66-RS07020 gene in mutant bacteria, a person of ordinary skill in the art can easily adopt known methods, such as directed mutation or gene editing, and the like.
Illustratively, the YH66-RS07020 gene in the mutant bacterium can be specifically: the codon of amino acid residue 84 of YH66-RS07020 protein encoded in bacterial genomic DNA was mutated from the codon encoding A to the codon encoding other amino acid residues. In particular, the other amino acid residue is V.
Illustratively, the YH66-RS07020 gene in the mutant bacterium can be specifically: the YH66-RS07020 gene in bacterial genomic DNA is subjected to the following point mutations: the 251 th nucleotide is mutated from C to other nucleotides (specifically T).
Illustratively, the manner of inhibiting YH66-RS07020 gene expression in bacteria may be: a substance for inhibiting YH66-RS07020 gene expression was introduced into the bacterium.
The substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in sequence 5 of a sequence table or a recombinant plasmid with the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in sequence 8 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 8 of the sequence table.
Exemplary, the substance for inhibiting YH66-RS07020 gene expression can be specifically the recombinant plasmid pK18-YH66-RS07020C251TOr the recombinant plasmid pK 18-delta YH66-RS 07020.
The invention also protects the application of the recombinant bacterium in the preparation of valine.
The invention also provides a method for preparing valine, which comprises the following steps: and fermenting the recombinant strain.
The fermentation can be carried out by a person skilled in the art using fermentation methods known in the art. Optimization and modification of the fermentation process can also be carried out by routine experimentation. Fermentation of the bacteria may be carried out in a suitable medium under fermentation conditions known in the art. The culture medium may comprise: carbon sources, nitrogen sources, trace elements, and combinations thereof. In the culture, the pH of the culture may be adjusted. Further, prevention of bubble generation, for example, by using an antifoaming agent, may be included in the culture. In addition, the culturing may include injecting a gas into the culture. The gas may include any gas capable of maintaining aerobic conditions of the culture. In the culture, the temperature of the culture may be 20 to 45 ℃.
The method may further comprise the steps of: valine was obtained from the culture. Obtaining valine from the culture can be accomplished 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, an exemplary fermentation medium formulation is shown in table 3, with the balance being water.
An exemplary fermentation control process for the fermentation is shown in table 4.
In the fermentation, the OD value of the system can be 0.3-0.5 at the initial moment of completing the inoculation.
Illustratively, the fermentation process of the fermentation is: ammonia water is used for adjusting the pH value; when foam exists in the fermentation system, adding a proper amount of antifoaming agent anifoam (CB-442); the sugar content (residual sugar) of the system is controlled by supplementing 70% glucose aqueous solution.
The invention also provides a method for improving the valine yield of bacteria, which comprises the following steps: inhibit YH66-RS07020 gene expression in bacteria or reduce YH66-RS07020 protein abundance in bacteria or reduce YH66-RS07020 protein activity in bacteria.
The YH66-RS07020 gene expression in the bacteria can be knocked out from YH66-RS07020 genes in the bacteria, and can also be YH66-RS07020 genes in mutant bacteria.
The knockout may be a partial segment of the knockout gene or may be the entire coding frame of the knockout gene.
Illustratively, the YH66-RS07020 gene in the knockout bacterium can be specifically: and (3) deleting the DNA molecule shown in the sequence 4 in the sequence table in the bacterial genome DNA.
For the YH66-RS07020 gene in mutant bacteria, a person of ordinary skill in the art can easily adopt known methods, such as directed mutation or gene editing, and the like.
Illustratively, the YH66-RS07020 gene in the mutant bacterium can be specifically: the codon of amino acid residue 84 of YH66-RS07020 protein encoded in bacterial genomic DNA was mutated from the codon encoding A to the codon encoding other amino acid residues. In particular, the other amino acid residue is V.
Illustratively, the YH66-RS07020 gene in the mutant bacterium can be specifically: the YH66-RS07020 gene in bacterial genomic DNA is subjected to the following point mutations: the 251 th nucleotide is mutated from C to other nucleotides (specifically T).
Illustratively, the manner of inhibiting YH66-RS07020 gene expression in bacteria may be: a substance for inhibiting YH66-RS07020 gene expression was introduced into the bacterium.
The substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in sequence 5 of a sequence table or a recombinant plasmid with the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07020 gene expression can be specifically a DNA molecule shown in sequence 8 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 8 of the sequence table.
The substance for inhibiting YH66-RS07020 gene expression can be specifically the recombinant plasmid pK18-YH in the embodiment66-RS07020C251TOr the recombinant plasmid pK 18-delta YH66-RS 07020.
The invention also protects the application of YH66-RS07020 protein in regulating and controlling the valine yield of bacteria.
The regulation is negative regulation, namely YH66-RS07020 protein content is increased, and valine yield is reduced.
The regulation is negative regulation, namely YH66-RS07020 protein content is reduced, and valine yield is increased.
The invention also protects the application of the YH66-RS07020 protein in regulating and controlling the bacterial count of bacteria.
The regulation is negative regulation, namely YH66-RS07020 protein content is increased, and bacterial count is reduced.
The regulation is negative regulation, namely YH66-RS07020 protein content is reduced, and bacterial count is increased.
The invention also discloses a mutant protein named YH66-RS07020C251TThe protein is obtained by mutating YH66-RS07020 protein 84 amino acid residue from A to other amino acid residue.
In particular, the other amino acid residue is V.
Illustratively, the mutant protein is shown as a sequence 1 in a sequence table.
The invention also protects YH66-RS07020C251TProtein coding gene (named YH66-RS 07020)C251TA gene).
The invention also protects the polypeptide with YH66-RS07020C251TExpression cassette for gene or gene with YH66-RS07020C251TRecombinant vector of gene or gene with YH66-RS07020C251TRecombinant bacteria of genes.
In particular, YH66-RS07020C251TThe genes are (c1) or (c2) or (c3) as follows:
(c1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;
(c2) a DNA molecule derived from a bacterium and having 95% or more identity to (c1) and encoding said protein;
(c3) a DNA molecule that hybridizes under stringent conditions to (c1) and 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 assess the identity between related sequences.
The identity of 95% or more may be 96% or more, 97% or more, 98% or more, or 99% or more.
The stringent conditions may be hybridization and membrane washing at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.
The invention also protects YH66-RS07020C251TProtein YH66-RS07020C251TGene, gene YH66-RS07020C251TExpression cassette for gene or gene with YH66-RS07020C251THas YH66-RS07020C251TThe recombinant strain is applied to the preparation of valine.
The invention also provides a method for improving the valine yield of bacteria, which comprises the following steps: the codon of amino acid residue 84 of YH66-RS07020 protein encoded in bacterial genomic DNA was mutated from the codon encoding A to the codon encoding other amino acid residues.
In particular, the other amino acid residue is V.
The method specifically comprises the following steps: the YH66-RS07020 gene in bacterial genomic DNA is subjected to the following point mutations: the 251 th nucleotide is mutated from C to other nucleotides (specifically T).
The method specifically comprises the following steps: introducing 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 into bacteria.
Any of the above bacteria include, but are not limited to, the following: corynebacterium bacteria, preferably Corynebacterium acetoacidophilum (Corynebacterium acetoacidophilum), Corynebacterium acetoglutamicum (Corynebacterium acetoglutamicum), Corynebacterium melallum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium pekinense (Corynebacterium pekinense), Brevibacterium saccharolyticum (Brevibacterium saccharolyticum), Brevibacterium roseum (Brevibacterium roseum), Brevibacterium thiolyticum (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: the ability to produce and accumulate valine in the culture medium and/or in the cells of the bacterium. Thus, valine can be collected when the bacterium is cultured in a medium.
The bacteria may be naturally collected wild-type bacteria or modified bacteria.
"modified bacterium" refers to an engineered bacterium obtained by artificially mutating and/or mutagenizing a naturally collected wild-type bacterium.
Specifically, the corynebacterium glutamicum can be corynebacterium glutamicum CGMCC 21260.
Corynebacterium glutamicum YPFV1, which has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3, institute of microbiology, China academy of sciences, North West Lu 1, Kyoho, Beijing, Inc.) at 11 months and 30 days of 2020, with the deposition registration number of CGMCC No. 21260. Corynebacterium glutamicum YPFV1 (also called Corynebacterium glutamicum CGMCC 21260).
Any valine mentioned above is meant to be taken in the broad sense of valine including valine in free form, a salt of valine or a mixture of both.
Specifically, the valine is L-valine.
Any of the above methods or applications may also be used for the production of downstream products of valine.
YH66-RS07020 protein in Corynebacterium glutamicum is shown as sequence 3 in the sequence table, and coding gene thereof is shown as sequence 4 in the sequence table. In the invention, YH66-RS07020 shown in sequence 1 of a sequence table is obtained by introducing point mutationC251TProtein, YH66-RS07020C251TThe coding gene of the protein is shown as a sequence 2 in a sequence table. Compared with YH66-RS07020 gene, YH66-RS07020C251TThe difference of the genes is that the 251 th nucleotide is mutated from C to T. Compared with YH66-RS07020 protein, YH66-RS07020C251TThe difference between the proteins is that the 84 th amino acid residue is mutated from A to V.
The invention finds that the YH66-RS07020 protein has negative regulation and control on the valine yield of bacteria, namely the YH66-RS07020 protein content is increased, the valine yield is reduced, the YH66-RS07020 protein content is reduced, and the valine yield is increased. The suppression of YH66-RS07020 gene expression can improve valine yield, and the overexpression of YH66-RS07020 gene can reduce valine yield. Further, YH66-RS07020 was found in the inventionC251TProtein and its coding gene and application. The invention has great application value for industrial production of valine.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. pK18mobsacB plasmid: addgene, Inc.; the plasmid pK18mobsacB has the kanamycin resistance gene as a selection marker. plasmid pXMJ 19: biovector plasmid vector strain cell gene collection center; the plasmid pXMJ19 has a chloramphenicol resistance gene as a selection marker. NEBuilder enzyme: NEB corporation. Unless otherwise specified, the medium in the examples was a medium having the formulation shown in Table 1 (balance water, pH 7.0). The kanamycin-free medium was the medium shown in Table 1. The kanamycin-containing medium consisted of the medium shown in Table 1 and kanamycin at a content of 50. mu.g/ml. Unless otherwise specified, the culture in the examples refers to a static culture at 32 ℃. Single-stranded conformational polymorphic polyacrylamide gel electrophoresis (sscp-PAGE) in the examples: the concentration of the gel used was 8%, and the composition of the electrophoretic gel is shown in table 2; the electrophoresis conditions are as follows: 1 XTBE buffer, 120V voltage, electrophoresis time 10 h.
Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
TABLE 1
| Components | Concentration in the culture 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
| Components | Amount of addition |
| 40% acrylamide | 8mL |
| ddH2O | 26mL |
| Glycerol | 4mL |
| 10×TBE | 2mL |
| TEMED | 40μL |
| 10％AP | 600μL |
Example 1 obtaining of Corynebacterium glutamicum CGMCC21260
Corynebacterium glutamicum ATCC 15168: corynebacterium glutamicum (Corynebacterium glutamicum) No. 15168 in ATCC.
Corynebacterium glutamicum ATCC15168 was subjected to mutagenesis to obtain Corynebacterium glutamicum (Corynebacterium glutamicum) YPFV 1.
Corynebacterium glutamicum YPFV1, which has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3, institute of microbiology, China academy of sciences, North West Lu 1, Kyoho, Beijing, Inc.) at 11 months and 30 days of 2020, with the deposition registration number of CGMCC No. 21260. Corynebacterium glutamicum YPFV1 (also called Corynebacterium glutamicum CGMCC 21260).
Example 2 construction of recombinant bacterium 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'。
Construction of recombinant plasmid
1. The Corynebacterium glutamicum ATCC15168 was used as a template, and a primer pair consisting of primer P1 and primer P2 was used for PCR amplification to recover an amplification product (674 bp).
2. The Corynebacterium glutamicum ATCC15168 was used as a template, and a primer pair consisting of primer P3 and primer P4 was used for PCR amplification to recover an amplification product (674 bp).
3. Meanwhile, the amplification product recovered in the step 1 and the amplification product recovered in the step 2 are used as templates, and PCR amplification (Overlap PCR) is carried out by adopting a primer pair consisting of a primer P1 and a primer P4, so that the amplification product (1314bp) is recovered. And after sequencing, the amplification product is shown as a sequence 5 in the sequence table.
4. The pK18mobsacB plasmid was digested with restriction enzyme Xba I, and the linearized plasmid was recovered.
5. Co-incubating the amplification product recovered in the step 3 with the linearized plasmid recovered in the step 4 (with NEBuilder enzyme, incubating at 50 ℃ for 30min) to obtain the recombinant plasmid pK18-YH66-RS07020C251T. Sequencing verification shows that the recombinant plasmid pK18-YH66-RS07020C251THas a DNA molecule shown in a sequence 5 of a sequence table.
Secondly, constructing recombinant bacteria YPV-013
1. Adopts a recombinant plasmid pK18-YH66-RS07020C251TCorynebacterium glutamicum CGMCC21260 was transformed by electric shock and then cultured.
2. The strain in step 1 is picked up and cultured by using a culture medium containing 15% of sucrose, then a single colony is picked up and cultured by using a culture medium containing kanamycin and a culture medium not containing kanamycin respectively, and strains which cannot grow on the culture medium containing kanamycin and can grow on the culture medium not containing kanamycin are screened.
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 amplification product (278 bp).
4. Taking the amplification product in the step 3, firstly, denaturing at 95 ℃ for 10min, then, carrying out ice bath for 5min, and then, carrying out sscp-PAGE. During electrophoresis, the recombinant plasmid pK18-YH66-RS07020 is adoptedC251TThe amplified fragment of (i.e., the recombinant plasmid pK18-YH66-RS 07020)C251TThe primer pair consisting of the primer P5 and the primer P6 is used as a template for PCR amplification, the amplification product) is used as a positive control, the amplification fragment of the Corynebacterium glutamicum CGMCC21260 (namely, the amplification product of the Corynebacterium glutamicum CGMCC21260 is used as a template and the primer pair consisting of the primer P5 and the primer P6 is used for PCR amplification) is used as a negative control, and water is used as a blank control. Due to different fragment structures and different electrophoresis positions, the strains with the electrophoresis positions inconsistent with the negative control and consistent with the positive control are the target strains for screening (recombinant strains with successful allelic replacement).
5. And (4) according to the result of the step (4), sequencing and verifying the amplified product of the step (3) of the screened strain to obtain the recombinant bacterium YPV-013. Compared with Corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-013 only has the difference that YH66-RS07020 gene shown in sequence 4 of a sequence table in the genome of Corynebacterium glutamicum CGMCC21260 is replaced by YH66-RS07020 gene shown in sequence 2 of the sequence tableC251TA gene. The sequence 2 and the sequence 4 have only one nucleotide difference and are positioned at the 251 th position. The recombinant bacterium YPV-013 is obtained by mutating (single-point mutation) YH66-RS07020 gene in Corynebacterium glutamicum CGMCC21260The engineered strain of (1).
Example 2 construction of recombinant bacterium YPV-015 and recombinant bacterium 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'。
Firstly, constructing recombinant bacteria YPV-015
1. The recombinant bacterium YPV-013 is used as a template, a primer pair consisting of a primer P7 and a primer P8 is adopted for PCR amplification, and an amplification product (806bp) is recovered.
2. The recombinant bacterium YPV-013 is used as a template, a primer pair consisting of a primer P9 and a primer P10 is adopted for PCR amplification, and an amplification product (293bp) is recovered.
3. The recombinant bacterium YPV-013 is used as a template, a primer pair consisting of a primer P11 and a primer P12 is adopted for PCR amplification, and an amplification product (634bp) is recovered.
4. The recombinant bacterium YPV-013 is used as a template, a primer pair consisting of a primer P13 and a primer P14 is adopted for PCR amplification, and an amplification product (783bp) is recovered.
5. The pK18mobsacB plasmid was digested with restriction enzyme Xba I, and the linearized plasmid was recovered.
6. And (3) co-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), the amplification product recovered in the step (4) and the linearized plasmid recovered in the step (5) (incubating for 30min at 50 ℃ by using NEBuilder enzyme) to obtain a recombinant plasmid 015. Through sequencing verification, the recombinant plasmid 015 has a DNA molecule shown in sequence 6 of the sequence table.
7. The corynebacterium glutamicum CGMCC21260 is subjected to electric shock transformation by adopting the recombinant plasmid 015, then cultured, and then PCR identification is respectively carried out on each single colony (a primer pair consisting of a primer P15 and a primer P16 is adopted), so that the strain capable of amplifying a 1454bp strip is a positive strain.
8. And (3) selecting the positive strain in the step 7, culturing the positive strain by using a culture medium containing 15% of sucrose, then selecting a single colony, culturing the single colony by using a culture medium containing kanamycin and a culture medium not containing kanamycin respectively, and screening the strain which can not 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, carrying out PCR amplification by adopting a primer pair consisting of a primer P17 and a primer P18, and obtaining the strain YH66-RS07020 with a 1335bp bandC251TThe positive strain with gene integrated into the genome of Corynebacterium glutamicum CGMCC21260 is named recombinant strain YPV-015. The recombinant bacterium YPV-015 is used for overexpressing YH66-RS07020 on genomeC251TEngineering strain of gene.
Secondly, constructing a recombinant bacterium YPV-014
The templates are all replaced by the recombinant bacteria YPV-013 'to the corynebacterium glutamicum ATCC 15168', and the steps are the same.
The positive strain integrating the YH66-RS07020 gene into the genome of Corynebacterium glutamicum CGMCC21260 was obtained and named recombinant strain YPV-014. The recombinant strain YPV-014 is an engineering strain with YH66-RS07020 gene overexpression on genome. Compared with the recombinant bacterium YPV-015, the recombinant bacterium YPV-014 only has the following differences: the sequence 4 replaces the sequence 2 in the sequence of the foreign DNA integrated into the genome of Corynebacterium glutamicum CGMCC 21260.
Example 3 construction of recombinant bacterium YPV-017 and recombinant bacterium YPV-016
Firstly, constructing recombinant bacteria YPV-017
1. The recombinant bacterium YPV-013 is used as a template, a primer pair consisting of a primer P19 and a primer P20 is adopted for PCR amplification, and an amplification product (917bp) is recovered. And after sequencing, the amplification product is shown as a sequence 7 in the sequence table.
P19：5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTCTTTGAATCTTACATTTCA-3'；
P20：5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAAGCGTTAGTGCGTGGCT-3'。
2. Taking pXMJ19 plasmid, adopting restriction enzyme EcoR I to perform single enzyme digestion, and recovering the linearized plasmid.
3. Co-incubating the amplification product recovered in step 1 with the linearized plasmid recovered in step 2 (with NEBuilder enzyme, incubation at 50 ℃ for 30min) to obtain recombinant plasmid pXMJ19-YH66-RS07020C251T. Sequencing verification shows that the recombinant plasmid pXMJ19-YH66-RS07020C251THas a DNA molecule shown in a sequence 7 of a sequence table.
4. Recombinant plasmid pXMJ19-YH66-RS07020C251TElectrically transduced into Corynebacterium glutamicum CGMCC21260 to obtain recombinant bacterium YPV-017. The recombinant bacterium YPV-017 is prepared by overexpressing pXMJ19-YH66-RS07020 by virtue of plasmidC251TEngineering strain of gene.
Secondly, constructing recombinant bacteria YPV-016
The template is replaced by 'recombinant bacterium YPV-013' to 'corynebacterium glutamicum ATCC 15168', and the steps are the same.
The recombinant strain YPV-016 is obtained. The recombinant strain YPV-016 is an engineering strain for overexpressing YH66-RS07020 gene by plasmid. Compared with the recombinant bacterium YPV-017, the recombinant bacterium YPV-016 has the following differences: sequence 4 replaces sequence 2 in the sequence of the foreign DNA over-expressed by the plasmid.
Example 4 construction of engineered 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'。
Construction of recombinant plasmid
1. Using Corynebacterium glutamicum ATCC15168 as a template, PCR amplification was carried out using a primer pair consisting of primer P21 and primer P22, and an amplification product (upstream homology arm fragment, 787bp) was recovered.
2. The Corynebacterium glutamicum ATCC15168 was used as a template, and a primer pair consisting of a primer P23 and a primer P24 was used for PCR amplification to recover an amplification product (a downstream homology arm fragment, 773 bp).
3. Meanwhile, the amplification product recovered in the step 1 and the amplification product recovered in the step 2 are used as templates, and PCR amplification (Overlap PCR) is carried out by using a primer pair consisting of a primer P21 and a primer P24, so that the amplification product (1520bp) is recovered. And after sequencing, the amplification product is shown as a sequence 8 in the sequence table.
4. The pK18mobsacB plasmid was digested with restriction enzyme Xba I, and the linearized plasmid was recovered.
5. And (3) co-incubating the amplification product recovered in the step (3) with the linearized plasmid recovered in the step (4) (by adopting NEBuilder enzyme, incubating for 30min at 50 ℃) to obtain a recombinant plasmid pK 18-delta YH66-RS 07020. Through sequencing verification, the recombinant plasmid pK 18-delta YH66-RS07020 has a DNA molecule shown as a sequence 8 in a sequence table.
Secondly, constructing recombinant bacteria 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 culture is carried out, and PCR identification is carried out on each single colony (by adopting a primer pair consisting of primers P25 and P26). The strain capable of amplifying bands of 1446bp and 2040bp simultaneously is a positive strain. The strain only amplifying the 2040bp band is a failed-transformation spawn, wherein the 2040bp fragment is shown as a sequence 9 in a sequence table.
2. Selecting the positive strain in the step 1, culturing the positive strain by using a culture medium containing 15% of sucrose, then selecting a single colony, culturing the single colony by using a culture medium containing kanamycin and a culture medium not containing kanamycin respectively, and screening the strain which can not grow on the culture medium containing kanamycin and can grow on the culture medium not containing kanamycin.
3. And (3) taking the strains screened in the step (2), carrying out PCR amplification by adopting a primer pair consisting of a primer P25 and a primer P26, wherein the amplification product is only one and 1446bp strain which is a positive strain with a YH66-RS07020 gene coding region knocked out.
4. And (3) performing PCR amplification and sequencing on the strains obtained by screening in the step (3) by using a primer pair consisting of a primer P25 and a primer P26 again, and naming the strains with correct sequencing as recombinant bacteria YPV-018. Compared with the genome DNA of Corynebacterium glutamicum CGMCC21260, the recombinant bacteria YPV-018 only have the difference that the DNA molecule shown in sequence 4 of the sequence table is deleted.
Example 5 fermentative preparation of L-valine
The test strains are respectively as follows: corynebacterium glutamicum CGMCC21260, recombinant bacteria YPV-013, recombinant bacteria YPV-014, recombinant bacteria YPV-015, recombinant bacteria YPV-016, recombinant bacteria YPV-017 and recombinant bacteria YPV-018.
Fermentation tank: BLBIO-5GC-4-H model fermenter (Shanghai Bailun Biotech Co., Ltd.).
The formulation of the fermentation medium is shown in Table 3, with the balance being water.
TABLE 3 fermentation Medium formulation
| Composition (I) | Content (wt.) |
| Ammonium sulfate | 14g/L |
| Potassium dihydrogen phosphate | 1g/L |
| Dipotassium hydrogen phosphate | 1g/L |
| Magnesium sulfate | 0.5g/L |
| Yeast powder | 2g/L |
| Ferrous sulfate | 18mg/L |
| Manganese sulfate | 4.2mg/L |
| Biotin | 0.02mg/L |
| Vitamin B1 | 2mg/L |
| Antifoaming agent antidioam (CB-442) | 0.5mL/L |
| Glucose (base candy) | 40g/L |
The fermentation control process is shown in Table 4.
At the initial moment of completion of inoculation, the OD value of the system is 0.3-0.5.
In the fermentation process: ammonia water is used for adjusting the pH value; when foam exists in the fermentation system, adding a proper amount of antifoaming agent anifoam (CB-442); the sugar content (residual sugar) of the system is controlled by supplementing 70% glucose aqueous solution.
TABLE 4 fermentation control Process
After completion of the fermentation, the supernatant was collected, and the L-valine production in the supernatant was measured by HPLC.
The results are shown in Table 5. The yield of L-valine of the recombinant bacteria YPV-013 and YPV-018 is obviously higher than that of Corynebacterium glutamicum CGMCC 21260. The results show that the suppression of YH66-RS07020 gene expression can improve L-valine yield, and the overexpression of YH66-RS07020 gene can reduce L-valine yield.
TABLE 5 results of L-valine fermentation experiments
| Bacterial strains | OD610 | 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 invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Heilongjiang Yipin Biotechnology Ltd
Engineering bacterium obtained by gene modification of YH66-RS07020 and application of engineering bacterium in preparation of valine
<130> GNCYX211994
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 197
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Val Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
<210> 2
<211> 594
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctggg tactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 3
<211> 197
<212> PRT
<213> Corynebacterium glutamicum
<400> 3
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Ala Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
<210> 4
<211> 594
<212> DNA
<213> Corynebacterium glutamicum
<400> 4
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctggg cactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 5
<211> 1314
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctagaacg cccgcatcga agacctgcag 60
atcgccgctg acatcctcaa gggccacaaa atcgccgacg gcatgcgcat gatggtcgtg 120
ccttcctcca cctggatcaa gcaagaggcc gaagcactcg gactggacaa aatcttcacc 180
gacgctggcg ctgaatggcg taccgcaggc tgctccatgt gcctgggcat gaacccagac 240
caactgaagc caggcgagcg ctctgcatcc acctccaacc gaaacttcga aggacgccaa 300
ggaccaggag gccgcaccca cctggtatcc ccagcagtcg cagccgccac cgcaatccgc 360
ggcaccctgt cctcacctgc agatatctaa ggaaggctag aaaaagaatg gaaaaattta 420
ccacccacac cggcgttggc gttccactgc agcgatccaa cgtggacacc gaccagatca 480
tccccgccgt ctacctcaag cgcgtcaccc gcacaggctt cgaagacgga ctgttttcca 540
actggcgcca aaacgacccc aactttgtcc tcaacaccga cacctacaag aacggctccg 600
ttctcgtagc aggccctgac tttggcaccg gctcctcccg cgagcacgcc gtctgggtac 660
tcatggacta cggcttccgc gctgtcttct cctcacgatt cgccgacatc ttccgcggca 720
actccggaaa agctggcatg ctcgccggca tcatggaaca gtccgacatc gaacttctgt 780
ggaagctcat ggaacaaacc ccgggcctcg aactgaccgt gaacctggaa aagcagatcg 840
tcactgcagg cgacgtagtg atcagcttcg aagttgaccc ttacattcgc tggcgtttga 900
tggaaggcct cgacgacgct ggcctgaccc tgcgcaagct cgatgaaatt gaagactacg 960
aggctaagcg ccctgcgttt aagccacgca ctaacgctta agtttcagtc tgatagcgaa 1020
agcaccccgc aaccttcatt gtcgcggggt gcatttgtgc gtcttggtgg gcgagtggag 1080
tgggcatgtc tggaatagac caagaggccc tggattccct taaggtcttg gtctttttcg 1140
tacgttttga ggcctgggaa gtgcatatcc agaccaaggg accccggcaa gccaagaccc 1200
ttggtttaat atgacgttcc gccccgagca agccgaaacc attacctaga acgcatgaaa 1260
agtgcccctc taggatgggt accgagctcg aattcgtaat catggtcata gctg 1314
<210> 6
<211> 2396
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctagcatg acggctgact ggactcgact 60
tccatacgag gttctggaga agatctccac ccgcatcacc aacgaagttc cagatgtgaa 120
ccgcgtggtt ttggacgtaa cctccaagcc accaggaacc atcgaatggg agtaggcctt 180
aaatgagcct tcgttaagcg gcaatcacct tattggagat tgtcgctttt cccatttctc 240
cgggttttct ggaacttttt gggcgtatgc tgggaatgat tctattattg ccaaatcaga 300
aagcaggaga gacccgatga gcgaaatcct agaaacctat tgggcacccc actttggaaa 360
aaccgaagaa gccacagcac tcgtttcata cctggcacaa gcttccggcg atcccattga 420
ggttcacacc ctgttcgggg atttaggttt agacggactc tcgggaaact acaccgacac 480
tgagattgac ggctacggcg acgcattcct gctggttgca gcgctatccg tgttgatggc 540
tgaaaacaaa gcaacaggtg gcgtgaatct gggtgagctt gggggagctg ataaatcgat 600
ccggctgcat gttgaatcca aggagaacac ccaaatcaac accgcattga agtattttgc 660
gctctcccca gaagaccacg cagcagcaga tcgcttcgat gaggatgacc tgtctgagct 720
tgccaacttg agtgaagagc tgcgcggaca gctggactaa ttgtctccca tttaaggagt 780
ccgatttctt tgaatcttac atttcataga gtgagacgct tgcaggttgg ggtttaaacg 840
ttgtggatat cgattccctg caggggagct gtataaagtg tgaggtaaat ctaaaacgca 900
ggacgtgaca tttttggcgc gttttaggtt atactgtctc agacaacgaa actcttgtcc 960
cacattgtga gatttgcttg ctagaatgtg ggctagaaat tcctgaaaat ttttacgcac 1020
tgtaaggacg gtgagttcca tggaaaaatt taccacccac accggcgttg gcgttccact 1080
gcagcgatcc aacgtggaca ccgaccagat catccccgcc gtctacctca agcgcgtcac 1140
ccgcacaggc ttcgaagacg gactgttttc caactggcgc caaaacgacc ccaactttgt 1200
cctcaacacc gacacctaca agaacggctc cgttctcgta gcaggccctg actttggcac 1260
cggctcctcc cgcgagcacg ccgtctgggt actcatggac tacggcttcc gcgctgtctt 1320
ctcctcacga ttcgccgaca tcttccgcgg caactccgga aaagctggca tgctcgccgg 1380
catcatggaa cagtccgaca tcgaacttct gtggaagctc atggaacaaa ccccgggcct 1440
cgaactgacc gtgaacctgg aaaagcagat cgtcactgca ggcgacgtag tgatcagctt 1500
cgaagttgac ccttacattc gctggcgttt gatggaaggc ctcgacgacg ctggcctgac 1560
cctgcgcaag ctcgatgaaa ttgaagacta cgaggctaag cgccctgcgt ttaagccacg 1620
cactaacgct taataaatcg actactcaca tagggtcggg ctagtcattc tgatcagcga 1680
attccacgtt cacatcgcca attccagagt tcacaaccag attcagcatt ggaccttcta 1740
gatcagcatt gtgggcggtg agatctccaa catcacagcg cgctgtgccc acaccggcgg 1800
tacaacttag gctcacgggc acatcatcgg gcagggtgac catgacttcg ccgatccctg 1860
aggtgatttg gatgttttgt tcctgatcca attgggtgag gtggctgaaa tcgaggttca 1920
tttcacccac gccagaggtg tagctgctga ggagttcatc gttggtgggg atgagattga 1980
catcgccgat tccagggtcg tcttcaaagt agatgggatc gatatttgaa ataaacaggc 2040
ctgcgagggc gctcatgaca actccggtac caactacacc gccgacaatc catggccaca 2100
catggcgctt tttctgaggc ttttgtggag ggacttgtac atcccaggtg ttgtattggt 2160
tttgggcaag tggatcccaa tgaggcgctt cgggggtttg ttgcgcgaag ggtgcatagt 2220
agccctcaac gggggtgata gtgcttagat ctggttgggg ttgtgggtag agatcttcgt 2280
ttttcatggt ggcatcctca gaaacagtga attcagtggt gagtagtccg cggggtggaa 2340
gtggttgttt cttatgcagg gtaccgagct cgaattcgta atcatggtca tagctg 2396
<210> 7
<211> 917
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcttgcatgc ctgcaggtcg actctagagg atcccctctt tgaatcttac atttcataga 60
gtgagacgct tgcaggttgg ggtttaaacg ttgtggatat cgattccctg caggggagct 120
gtataaagtg tgaggtaaat ctaaaacgca ggacgtgaca tttttggcgc gttttaggtt 180
atactgtctc agacaacgaa actcttgtcc cacattgtga gatttgcttg ctagaatgtg 240
ggctagaaat tcctgaaaat ttttacgcac tgtaaggacg gtgagttcca tggaaaaatt 300
taccacccac accggcgttg gcgttccact gcagcgatcc aacgtggaca ccgaccagat 360
catccccgcc gtctacctca agcgcgtcac ccgcacaggc ttcgaagacg gactgttttc 420
caactggcgc caaaacgacc ccaactttgt cctcaacacc gacacctaca agaacggctc 480
cgttctcgta gcaggccctg actttggcac cggctcctcc cgcgagcacg ccgtctgggt 540
actcatggac tacggcttcc gcgctgtctt ctcctcacga ttcgccgaca tcttccgcgg 600
caactccgga aaagctggca tgctcgccgg catcatggaa cagtccgaca tcgaacttct 660
gtggaagctc atggaacaaa ccccgggcct cgaactgacc gtgaacctgg aaaagcagat 720
cgtcactgca ggcgacgtag tgatcagctt cgaagttgac ccttacattc gctggcgttt 780
gatggaaggc ctcgacgacg ctggcctgac cctgcgcaag ctcgatgaaa ttgaagacta 840
cgaggctaag cgccctgcgt ttaagccacg cactaacgct taagttttgg cggatgagag 900
aagattttca gcctgat 917
<210> 8
<211> 1520
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cagtgccaag cttgcatgcc tgcaggtcga ctctagcgtg ccggcatgat cgccccagac 60
caaaccacct tcgactacgt tgaaggccgc gaaatggcac caaagggcgc cgactgggac 120
gaagcagttg cttactggaa gaccctgcca accgacgaag gcgcaacctt tgacaaggtc 180
gtagaaatcg atggctccgc actgacccca ttcatcacct ggggcaccaa cccaggccaa 240
ggtctgccac tgagcgaaac cgtgccaaac ccagaagact tcaccaacga caacgacaag 300
gcagcagccg aaaaggcact gcagtacatg gacctggtac caggaacccc actgcgcgac 360
atcaagatcg acaccgtctt cctgggatcc tgcaccaacg cccgcatcga agacctgcag 420
atcgccgctg acatcctcaa gggccacaaa atcgccgacg gcatgcgcat gatggtcgtg 480
ccttcctcca cctggatcaa gcaagaggcc gaagcactcg gactggacaa aatcttcacc 540
gacgctggcg ctgaatggcg taccgcaggc tgctccatgt gcctgggcat gaacccagac 600
caactgaagc caggcgagcg ctctgcatcc acctccaacc gaaacttcga aggacgccaa 660
ggaccaggag gccgcaccca cctggtatcc ccagcagtcg cagccgccac cgcaatccgc 720
ggcaccctgt cctcacctgc agatatctaa ggaaggctag aaaaagagtt tcagtctgat 780
agcgaaagca ccccgcaacc ttcattgtcg cggggtgcat ttgtgcgtct tggtgggcga 840
gtggagtggg catgtctgga atagaccaag aggccctgga ttcccttaag gtcttggtct 900
ttttcgtacg ttttgaggcc tgggaagtgc atatccagac caagggaccc cggcaagcca 960
agacccttgg tttaatatga cgttccgccc cgagcaagcc gaaaccatta cctagaacgc 1020
atgaaaagtg cccctctagg atgggttcta agcccctaac aggctcaaac ccaagcccat 1080
gcccgctcgc caaaccggag gccttaaacg cgctcctatt taaccggcag ggaactcgcc 1140
aggtaatcag cgccggtgaa cacaccgtcg tgaaagctca acacccacac gctgcccttt 1200
ttcgccttga tcttctcatc gatagggagg gtgccgttct cggagaacca tttgatcatt 1260
tccggaatga tgtcgccctg cccaacgatc atcggcacgc caccttgtgc aaccacgtcg 1320
gtgaagcgct tcttgcaggc ctcgggatcg gtttcccagg cgtcgtcgcc gaacagtcgg 1380
ttgacggaca cgtcgaggcc gagctcatcg gcaaggggga gcgcggtggc ttggcagcga 1440
tcgggcaccg ccgagtaaat tgcggtgggt ttgaagggca acgggtaccg agctcgaatt 1500
cgtaatcatg gtcatagctg 1520
<210> 9
<211> 2040
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgtgccggca tgatcgcccc agaccaaacc accttcgact acgttgaagg ccgcgaaatg 60
gcaccaaagg gcgccgactg ggacgaagca gttgcttact ggaagaccct gccaaccgac 120
gaaggcgcaa cctttgacaa ggtcgtagaa atcgatggct ccgcactgac cccattcatc 180
acctggggca ccaacccagg ccaaggtctg ccactgagcg aaaccgtgcc aaacccagaa 240
gacttcacca acgacaacga caaggcagca gccgaaaagg cactgcagta catggacctg 300
gtaccaggaa ccccactgcg cgacatcaag atcgacaccg tcttcctggg atcctgcacc 360
aacgcccgca tcgaagacct gcagatcgcc gctgacatcc tcaagggcca caaaatcgcc 420
gacggcatgc gcatgatggt cgtgccttcc tccacctgga tcaagcaaga ggccgaagca 480
ctcggactgg acaaaatctt caccgacgct ggcgctgaat ggcgtaccgc aggctgctcc 540
atgtgcctgg gcatgaaccc agaccaactg aagccaggcg agcgctctgc atccacctcc 600
aaccgaaact tcgaaggacg ccaaggacca ggaggccgca cccacctggt atccccagca 660
gtcgcagccg ccaccgcaat ccgcggcacc ctgtcctcac ctgcagatat ctaaggaagg 720
ctagaaaaag aatggaaaaa tttaccaccc acaccggcgt tggcgttcca ctgcagcgat 780
ccaacgtgga caccgaccag atcatccccg ccgtctacct caagcgcgtc acccgcacag 840
gcttcgaaga cggactgttt tccaactggc gccaaaacga ccccaacttt gtcctcaaca 900
ccgacaccta caagaacggc tccgttctcg tagcaggccc tgactttggc accggctcct 960
cccgcgagca cgccgtctgg gcactcatgg actacggctt ccgcgctgtc ttctcctcac 1020
gattcgccga catcttccgc ggcaactccg gaaaagctgg catgctcgcc ggcatcatgg 1080
aacagtccga catcgaactt ctgtggaagc tcatggaaca aaccccgggc ctcgaactga 1140
ccgtgaacct ggaaaagcag atcgtcactg caggcgacgt agtgatcagc ttcgaagttg 1200
acccttacat tcgctggcgt ttgatggaag gcctcgacga cgctggcctg accctgcgca 1260
agctcgatga aattgaagac tacgaggcta agcgccctgc gtttaagcca cgcactaacg 1320
cttaagtttc agtctgatag cgaaagcacc ccgcaacctt cattgtcgcg gggtgcattt 1380
gtgcgtcttg gtgggcgagt ggagtgggca tgtctggaat agaccaagag gccctggatt 1440
cccttaaggt cttggtcttt ttcgtacgtt ttgaggcctg ggaagtgcat atccagacca 1500
agggaccccg gcaagccaag acccttggtt taatatgacg ttccgccccg agcaagccga 1560
aaccattacc tagaacgcat gaaaagtgcc cctctaggat gggttctaag cccctaacag 1620
gctcaaaccc aagcccatgc ccgctcgcca aaccggaggc cttaaacgcg ctcctattta 1680
accggcaggg aactcgccag gtaatcagcg ccggtgaaca caccgtcgtg aaagctcaac 1740
acccacacgc tgcccttttt cgccttgatc ttctcatcga tagggagggt gccgttctcg 1800
gagaaccatt tgatcatttc cggaatgatg tcgccctgcc caacgatcat cggcacgcca 1860
ccttgtgcaa ccacgtcggt gaagcgcttc ttgcaggcct cgggatcggt ttcccaggcg 1920
tcgtcgccga acagtcggtt gacggacacg tcgaggccga gctcatcggc aagggggagc 1980
gcggtggctt ggcagcgatc gggcaccgcc gagtaaattg cggtgggttt gaagggcaac 2040
Claims (10)
1. The 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), (II) or (III):
the application of (I) in improving the yield of the bacterial valine;
(II) use in the production of valine;
(III) use in increasing bacterial load;
the YH66-RS07020 gene is a gene for coding YH66-RS07020 protein;
the YH66-RS07020 protein is (a1) or (a2) or (a 3):
(a1) protein shown in a sequence 3 in a sequence table;
(a2) a protein derived from a bacterium, having 95% or more identity to (a1), and being associated with valine production by the bacterium;
(a3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in (a1) and is related to the production of valine by bacteria and derived from (a 1).
2. A recombinant bacterium is obtained by inhibiting YH66-RS07020 gene expression in bacteria; the YH66-RS07020 gene is the YH66-RS07020 gene described in claim 1.
3. Use of the recombinant bacterium of claim 2 for producing valine.
4. A process for producing valine, comprising the steps of: fermenting the recombinant bacterium of claim 2.
5. A method for increasing valine production in a bacterium, comprising the steps of: inhibit YH66-RS07020 gene expression in bacteria or reduce YH66-RS07020 protein abundance in bacteria or reduce YH66-RS07020 protein activity in bacteria;
the YH66-RS07020 gene is the YH66-RS07020 gene described in claim 1;
the YH66-RS07020 protein is the YH66-RS07020 protein of claim 1.
The application of the YH66-RS07020 protein in regulating the valine yield of the bacteria or the YH66-RS07020 protein in regulating the bacterial quantity of the bacteria; the YH66-RS07020 protein is the YH66-RS07020 protein of claim 1.
7. 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 of claim 1.
8. The mutant protein coding gene or claim 7 with the mutant protein coding gene expression cassettes or claim 7 with the mutant protein coding gene recombinant vector or claim 7 with the mutant protein coding gene recombinant bacteria.
9. Use of the mutein of claim 7, the coding gene of claim 8, the expression cassette of claim 8, the recombinant vector of claim 8 or the recombinant bacterium of claim 8 for producing valine.
10. A method for increasing valine production in a bacterium, comprising the steps of: mutating the codon of amino acid residue 84 of YH66-RS07020 protein encoded in bacterial genomic DNA from the codon encoding A to the codon encoding other amino acid residues; the YH66-RS07020 protein is the YH66-RS07020 protein of claim 1.
Priority Applications (2)
| 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 |
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311178534.9A Division CN117106042A (en) | 2021-08-23 | 2021-08-23 | YH66-RS07020 mutant protein and application of related biological material in preparation of valine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113683666A true CN113683666A (en) | 2021-11-23 |
Family
ID=78581405
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311178534.9A Pending CN117106042A (en) | 2021-08-23 | 2021-08-23 | YH66-RS07020 mutant protein and application of related biological material in preparation of valine |
| CN202110967402.9A Pending CN113683666A (en) | 2021-08-23 | 2021-08-23 | Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311178534.9A Pending CN117106042A (en) | 2021-08-23 | 2021-08-23 | YH66-RS07020 mutant protein and application of related biological material in preparation of valine |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN117106042A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114540399A (en) * | 2022-02-15 | 2022-05-27 | 宁夏伊品生物科技股份有限公司 | Method for preparing L-valine and gene mutant and biological material used by same |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302873A (en) * | 1999-10-27 | 2001-07-11 | 德古萨－于尔斯股份公司 | Nucleotide sequence of output protein of encode side chain amino acid and separation method and application thereof |
| US20040091980A1 (en) * | 2002-06-25 | 2004-05-13 | Ajinomoto Co., Inc. | Method for producing L-leucine |
| CN1715413A (en) * | 1999-06-25 | 2006-01-04 | Basf公司 | Corynebacterium glutamicum genes encoding metabolic pathway proteins |
| EP1767616A2 (en) * | 2005-09-22 | 2007-03-28 | Degussa GmbH | Method for producing L-amino acids or ketoacids employing coryneform bacteria with attenuated aceE (pdhA) expression |
| CN104302765A (en) * | 2012-04-27 | 2015-01-21 | 赢创工业集团股份有限公司 | Feedback-resistant alpha-isopropylmalate synthases |
| CN105492616A (en) * | 2013-06-03 | 2016-04-13 | 赢创德固赛有限公司 | Method for producing l-leucine, l-valine, l-isoleucine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, or alpha-ketoisocaproate using recombinant corynebacteria that contain the ilvbn operon which can be induced by propionate |
| CN108368514A (en) * | 2015-10-23 | 2018-08-03 | Cj第制糖株式会社 | Certain microorganism of corynebacterium with l-Isoleucine production capacity and the method for producing l-Isoleucine using it |
| CN110551670A (en) * | 2019-09-19 | 2019-12-10 | 天津科技大学 | Genetically engineered bacterium for producing L-leucine and application thereof |
-
2021
- 2021-08-23 CN CN202311178534.9A patent/CN117106042A/en active Pending
- 2021-08-23 CN CN202110967402.9A patent/CN113683666A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1715413A (en) * | 1999-06-25 | 2006-01-04 | Basf公司 | Corynebacterium glutamicum genes encoding metabolic pathway proteins |
| CN1302873A (en) * | 1999-10-27 | 2001-07-11 | 德古萨－于尔斯股份公司 | Nucleotide sequence of output protein of encode side chain amino acid and separation method and application thereof |
| US20040091980A1 (en) * | 2002-06-25 | 2004-05-13 | Ajinomoto Co., Inc. | Method for producing L-leucine |
| EP1767616A2 (en) * | 2005-09-22 | 2007-03-28 | Degussa GmbH | Method for producing L-amino acids or ketoacids employing coryneform bacteria with attenuated aceE (pdhA) expression |
| CN104302765A (en) * | 2012-04-27 | 2015-01-21 | 赢创工业集团股份有限公司 | Feedback-resistant alpha-isopropylmalate synthases |
| CN105492616A (en) * | 2013-06-03 | 2016-04-13 | 赢创德固赛有限公司 | Method for producing l-leucine, l-valine, l-isoleucine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, or alpha-ketoisocaproate using recombinant corynebacteria that contain the ilvbn operon which can be induced by propionate |
| CN108368514A (en) * | 2015-10-23 | 2018-08-03 | Cj第制糖株式会社 | Certain microorganism of corynebacterium with l-Isoleucine production capacity and the method for producing l-Isoleucine using it |
| CN110551670A (en) * | 2019-09-19 | 2019-12-10 | 天津科技大学 | Genetically engineered bacterium for producing L-leucine and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHENG CHEN ET AL.: "Metabolic engineering of Corynebacterium glutamicum ATCC13869 for l-valine production", 《METABOLIC ENGINEERING》, pages 66 - 75 * |
| GENBANK: "WP_003861502.1", 《GENBANK》 * |
| YUECHAO MA ET AL.: "Comparative Genomic and Genetic Functional Analysis of Industrial L-Leucin- and L-Valine-Producing Corynebacterium glutamicum Strains", 《J. MICROBIOL. BIOTECHNOL.》, vol. 28, no. 11, pages 1916 - 1927, XP055874919, DOI: 10.4014/jmb.1805.05013 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540399A (en) * | 2022-02-15 | 2022-05-27 | 宁夏伊品生物科技股份有限公司 | Method for preparing L-valine and gene mutant and biological material used by same |
| CN114540399B (en) * | 2022-02-15 | 2024-05-03 | 宁夏伊品生物科技股份有限公司 | Method for preparing L-valine, and gene mutant and biological material used by same |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117106042A (en) | 2023-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113666991B (en) | Engineering bacterium obtained by YH66-RS07015 gene modification and application thereof in valine preparation | |
| CN113683667A (en) | Engineering bacterium obtained by YH66-RS10865 gene transformation and application thereof in preparation of valine | |
| CN113683666A (en) | Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine | |
| CN113667682A (en) | YH66-RS11190 gene mutant and application thereof in preparation of L-valine | |
| EP4273228A1 (en) | Strain having enhanced l-glutamic acid productivity, construction method therefor and application thereof | |
| CN111961635B (en) | Recombinant strain for producing L-lysine and construction method and application thereof | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| CN112646766A (en) | Recombinant strain for producing L-glutamic acid by modifying gene BBD 29-04920 and construction method and application thereof | |
| CN113563436A (en) | Engineering bacterium obtained by YH66-RS03880 gene transformation and application thereof in preparation of valine | |
| CN114540399B (en) | Method for preparing L-valine, and gene mutant and biological material used by same | |
| CN114349831B (en) | aspA gene mutant, recombinant bacterium and method for preparing L-valine | |
| CN112725253B (en) | Recombinant strain for modifying gene BBD29_14900 and construction method and application thereof | |
| CN114835783A (en) | NCgl2747 gene mutant and application thereof in preparation of L-lysine | |
| CN114410615B (en) | YH66_00525 protein and application of encoding gene thereof in regulating and controlling bacterial arginine yield | |
| CN114317583B (en) | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule used in method | |
| CN114249803B (en) | Engineering bacterium for high-yield arginine and construction method and application thereof | |
| CN114277069B (en) | Method for preparing L-valine and biological material used by same | |
| CN114507273B (en) | YH66_07020 protein and application of related biological material thereof in improving arginine yield | |
| CN114315998B (en) | CEY17_RS00300 gene mutant and application thereof in preparation of L-valine | |
| CN114539367B (en) | CEY17_RS11900 gene mutant and application thereof in preparation of L-valine | |
| CN114369559B (en) | Recombinant strain for producing L-amino acid and construction method and application thereof | |
| CN114540262B (en) | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same | |
| CN118147035A (en) | Recombinant microorganism and application thereof in preparation of L-lysine | |
| CN114410614A (en) | Application of YH66_05415 protein or mutant thereof in preparation of L-arginine | |
| CN114315999A (en) | LeuD protein mutant and related biological material and application thereof in preparation of L-valine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |