CN113683667A

CN113683667A - Engineering bacterium obtained by YH66-RS10865 gene transformation and application thereof in preparation of valine

Info

Publication number: CN113683667A
Application number: CN202110980059.1A
Authority: CN
Inventors: 付丽霞; 贾慧萍; 孟刚; 赵春光; 魏爱英; 田斌
Original assignee: Ningxia Eppen Biotech Co ltd
Current assignee: Ningxia Eppen Biotech Co ltd
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-11-23
Anticipated expiration: 2041-08-25
Also published as: CN113683667B

Abstract

The invention discloses an engineering bacterium obtained by modifying YH66-RS10865 gene and application thereof in preparation of valine. The invention provides an application of a substance for inhibiting YH66-RS10865 gene expression or a substance for reducing YH66-RS10865 protein abundance or a substance for reducing YH66-RS10865 protein activity in improving the yield of bacterial valine. The invention provides a recombinant bacterium, which is obtained by inhibiting YH66-RS10865 gene expression in bacteria. The invention also protects the preparationValine method: and fermenting the recombinant strain. The invention finds that the YH66-RS10865 protein negatively regulates the valine yield of bacteria, inhibits the expression of the YH66-RS10865 gene to improve the valine yield, and overexpresses the YH66-RS10865 gene to reduce the valine yield. Further, YH66-RS10865 was found in the present invention^A844C,C846AProtein and its coding gene and application. The invention has great application value for industrial production of valine.

Description

Engineering bacterium obtained by YH66-RS10865 gene transformation and application thereof in preparation of valine

Technical Field

The invention belongs to the technical field of biology, and relates to an engineering bacterium obtained by YH66-RS10865 gene modification and application thereof in valine preparation, wherein the modification specifically comprises A844C and C846A.

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-RS10865 gene modification and application thereof in valine preparation.

The invention provides application of a substance for inhibiting YH66-RS10865 gene expression or a substance for reducing YH66-RS10865 protein abundance or a substance for reducing YH66-RS10865 protein activity;

the application is as follows (I) or (II):

the application of (I) in improving the yield of the bacterial valine;

(II) the application in the production of valine.

The YH66-RS10865 gene is a gene encoding YH66-RS10865 protein;

the YH66-RS10865 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-RS10865 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 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 suppression of the YH66-RS10865 gene expression can be realized by knocking out the YH66-RS10865 gene or mutating the YH66-RS10865 gene.

The knockout may be a partial segment of the knockout gene or may be the entire coding frame of the knockout gene.

Illustratively, the substance for inhibiting YH66-RS10865 gene expression may specifically be a DNA molecule represented by sequence 5 of the sequence table or a recombinant plasmid having a DNA molecule represented by sequence 5 of the sequence table.

Illustratively, the substance for inhibiting YH66-RS10865 gene expression may specifically be a DNA molecule represented by sequence 8 of the sequence table or a recombinant plasmid having a DNA molecule represented by sequence 8 of the sequence table.

The substance for inhibiting YH66-RS10865 gene expression can be exemplified by recombinant plasmid pK18-YH66-RS10865^A844C,C846AOr recombinantPlasmid pK18- Δ YH66-RS 10865.

The invention also provides a recombinant bacterium obtained by inhibiting YH66-RS10865 gene expression in bacteria.

The YH66-RS10865 gene expression in the bacteria can be knocked out from YH66-RS10865 gene in the bacteria, and can also be YH66-RS10865 gene in mutant bacteria.

Illustratively, the YH66-RS10865 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 YH66-RS10865 gene in mutant bacteria, a person of ordinary skill in the art can easily adopt known methods such as directed mutation or gene editing, etc.

Illustratively, the YH66-RS10865 gene in the mutant bacterium specifically can be: the codon of amino acid 282 of YH66-RS10865 protein encoded in bacterial genomic DNA was mutated from the codon encoding N to the codon encoding other amino acid residues. In particular, the other amino acid residue is Q.

Illustratively, the YH66-RS10865 gene in the mutant bacterium specifically can be: the YH66-RS10865 gene in the bacterial genomic DNA was subjected to the following two point mutations: the 844 th nucleotide is mutated from A to other nucleotides (specifically C), and the 846 th nucleotide is mutated from C to other nucleotides (specifically A).

Illustratively, the manner of inhibiting YH66-RS10865 gene expression in bacteria may be: a substance for inhibiting YH66-RS10865 gene expression was introduced into the bacterium.

The substance for inhibiting YH66-RS10865 gene expression can be exemplified by recombinant plasmid pK18-YH66-RS10865^A844C,C846AOr recombinant plasmid pK 18-delta YH66-RS 10865.

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-RS10865 gene expression or reduce YH66-RS10865 protein abundance or reduce YH66-RS10865 protein activity in bacteria.

The invention also protects the application of YH66-RS10865 protein in regulating the valine yield of bacteria.

The regulation is negative regulation, namely YH66-RS10865 protein content is increased, and valine yield is reduced.

The regulation is negative regulation, namely YH66-RS10865 protein content is reduced, and valine yield is increased.

The invention also discloses a mutant protein named YH66-RS10865^A844C,C846AThe protein is obtained by mutating YH66-RS10865 protein 282 amino acid residue from N to other amino acid residue.

In particular, the other amino acid residue is Q.

Illustratively, the mutant protein is shown as a sequence 1 in a sequence table.

The invention also protects YH66-RS10865^A844C,C846AThe coding gene of the protein is named YH66-RS10865^A844C ^,C846AA gene.

Specifically, YH66-RS10865^A844C,C846AThe 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 invention also protects the product with YH66-RS10865^A844C,C846AExpression cassette for Gene or Gene having YH66-RS10865^A844C ^,C846ARecombinant vector of gene or gene with YH66-RS10865^A844C,C846ARecombinant bacteria of genes.

The invention also protects YH66-RS10865^A844C,C846AProtein YH66-RS10865^A844C,C846AGene, having YH66-RS10865^A844C,C846AExpression cassette for Gene or Gene having YH66-RS10865^A844C,C846ARecombinant vector of gene or gene with YH66-RS10865^A844C,C846AApplication of recombinant bacteria of the gene in 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 282 of YH66-RS10865 protein encoded in bacterial genomic DNA was mutated from the codon encoding N to the codon encoding other amino acid residues. In particular, the other amino acid residue is Q.

The method specifically comprises the following steps: the YH66-RS10865 gene in the bacterial genomic DNA was subjected to the following two point mutations: the 844 th nucleotide is mutated from A to other nucleotides (specifically C), and the 846 th nucleotide is mutated from C to other nucleotides (specifically A).

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 lactofermentum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium pekinense (Corynebacterium pekinense), Brevibacterium saccharolyticum (Brevibacterium saccharolyticum), Brevibacterium roseum (Brevibacterium roseum), Brevibacterium thiolyticum (Brevibacterium thiogenitum), Brevibacterium thiogenitalis (Brevibacterium thiogeniticum), and Brevibacterium thiogenitalis (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-RS10865 protein in Corynebacterium glutamicum is shown as sequence 3 in a sequence table, and coding genes thereof are shown as sequence 4 in the sequence table. In the invention, YH66-RS1 shown in sequence 1 of a sequence table is obtained by introducing point mutation0865^A844C,C846AProtein, YH66-RS10865^A844C,C846AThe coding gene of the protein is shown as a sequence 2 in a sequence table. Compared with YH66-RS10865 gene, YH66-RS10865^A844C,C846AThe difference of the genes is that the 844 th nucleotide is mutated from A to C and the 846 th nucleotide is mutated from C to A. Compared with YH66-RS10865 protein, YH66-RS10865^A844C,C846AThe difference between the proteins is that the 282 th amino acid residue is mutated from N to Q.

The invention finds that YH66-RS10865 protein has negative control on the valine yield of bacteria, namely YH66-RS10865 protein content is increased, valine yield is reduced, YH66-RS10865 protein content is reduced, and valine yield is increased. The suppression of the YH66-RS10865 gene expression can improve the valine yield, and the overexpression of the YH66-RS10865 gene can reduce the valine yield. Further, YH66-RS10865 was found in the present invention^A844C,C846AProtein 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.

Example 2 construction of recombinant bacterium YPV-031

P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGCTTCGTTTGCTTGAGCGTG-3'；

P2：5'-CGCTGCAGGTTGCATTGAACCACGAAGGTCAGGT-3'；

P3：5'-ACCTGACCTTCGTGGTTCAATGCAACCTGCAGCG-3'；

P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTAGCCACCGAGCGCTGCGCG-3'。

P5：5'-ACGTTTCAACCGCTACCTCG-3'；

P6：5'-ATCCCACTCGCGACCCCAAAC-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 (720 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 (678 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, and the amplification product (1364bp) 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 step 3 with the linearized plasmid recovered in step 4 (with NEBuilder enzyme, incubation at 50 ℃ for 30min) to obtain recombinant plasmid pK18-YH66-RS10865^A844C,C846A. Measured bySequence verification, recombinant plasmid pK18-YH66-RS10865^A844C,C846AHas a DNA molecule shown in a sequence 5 of a sequence table.

Secondly, constructing recombinant bacteria YPV-031

1. Adopts a recombinant plasmid pK18-YH66-RS10865^A844C,C846ACorynebacterium 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 (283 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-RS10865 is adopted^A844C,C846AThe amplified fragment of (1) (i.e., the recombinant plasmid pK18-YH66-RS 10865)^A844C,C846AThe 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), performing sequencing verification on the amplified product of the step (3) of the screened strain to obtain the recombinant bacterium YPV-031. Compared with Corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-031 has the difference that: the YH66-RS10865 gene shown in sequence 4 of the sequence table in the Corynebacterium glutamicum CGMCC21260 genome is replaced by YH66-RS10865 gene shown in sequence 2 of the sequence table^A844C,C846AA gene. Sequence 2 and sequence 4 present only two nucleiA nucleotide difference between 844 th and 846 th. The recombinant strain YPV-031 is an engineering strain obtained by mutating (mutating at two sites) YH66-RS10865 gene in Corynebacterium glutamicum CGMCC 21260.

Example 2 construction of recombinant bacterium YPV-032 and recombinant bacterium YPV-033

P7：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGGACTC-3'；

P8：5'-ACTGGGATTTCAGGTATCCAAATCGGACTCCTTAAATGGG-3'；

P9：5'-CCCATTTAAGGAGTCCGATTTGGATACCTGAAATCCCAGT-3'；

P10：5'-CTATGTGAGTAGTCGATTTATTATTCCTCAGGAGCGTTTG-3'；

P11：5'-CAAACGCTCCTGAGGAATAATAAATCGACTACTCACATAG-3'；

P12：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAACCACTT-3'。

P13：5'-GTCCGCTCTGTTGGTGTTCA-3'；

P14：5'-TGTTACCGCGGACAGGTCCG-3'。

P15：5'-GCAAGCAGCAGATCGCTACC-3'；

P16：5'-TGGAGGAATATTCGGCCCAG-3'。

Firstly, constructing recombinant bacteria YPV-033

1. The recombinant bacterium YPV-031 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-031 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 (3014bp) is recovered.

3. The recombinant bacterium YPV-031 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 (783bp) is recovered.

5. 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) and the linearized plasmid recovered in the step (4) (incubating for 30min at 50 ℃ by using NEBuilder enzyme) to obtain a recombinant plasmid 033. Through sequencing verification, the recombinant plasmid 033 has a DNA molecule shown as a sequence 6 in a sequence table.

6. The corynebacterium glutamicum CGMCC21260 is subjected to electric shock transformation by adopting the recombinant plasmid 033, then cultured, and then each single colony is subjected to PCR identification (by adopting a primer pair consisting of a primer P13 and a primer P14), so that the strain capable of amplifying a 1921bp band is a positive strain.

7. And (3) selecting the positive strain in the step 6, 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.

8. Taking the strain screened in the step 7, carrying out PCR amplification by adopting a primer pair consisting of a primer P15 and a primer P16, and obtaining the strain YH66-RS10865 with 2013bp bands^A844C,C846AThe positive strain with gene integrated into the genome of Corynebacterium glutamicum CGMCC21260 was named recombinant strain YPV-033. Recombinant bacterium YPV-033 is overexpressed YH66-RS10865 on genome^A844C,C846AEngineering strain of gene.

Secondly, constructing recombinant bacteria YPV-032

Replacing the templates with recombinant bacteria YPV-031 to Corynebacterium glutamicum ATCC15168, and carrying out the same steps.

The positive strain integrating the YH66-RS10865 gene into the Corynebacterium glutamicum CGMCC21260 genome is obtained and named recombinant strain YPV-032. The recombinant strain YPV-032 is an engineering strain with YH66-RS10865 gene overexpression on genome. Compared with the recombinant bacterium YPV-033, the recombinant bacterium YPV-032 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-035 and recombinant bacterium YPV-034

Firstly, constructing recombinant bacteria YPV-035

1. The recombinant bacterium YPV-031 is used as a template, a primer pair consisting of a primer P17 and a primer P18 is adopted for PCR amplification, and an amplification product (3044bp) is recovered. And after sequencing, the amplification product is shown as a sequence 7 in the sequence table.

P17：5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTGGATACCTGAAATCCCAGT-3'；

P18：5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTATTCCTCAGGAGCGTTTG-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-RS10865^A844C,C846A. Sequencing verification shows that the recombinant plasmid pXMJ19-YH66-RS10865^A844C,C846AHas a DNA molecule shown in a sequence 7 of a sequence table.

4. The recombinant plasmid pXMJ19-YH66-RS10865^A844C,C846AThe Corynebacterium glutamicum CGMCC21260 is electrically transduced to obtain recombinant bacteria YPV-035. The recombinant bacterium YPV-035 is prepared by over-expressing pXMJ19-YH66-RS10865 by a plasmid^A844C,C846AEngineering strain of gene.

Secondly, constructing recombinant bacteria YPV-034

The template is replaced by the recombinant bacterium YPV-031 to the Corynebacterium glutamicum ATCC15168, and the steps are the same as the first step.

The recombinant strain YPV-034 is obtained. The recombinant strain YPV-034 is an engineering strain for overexpressing YH66-RS10865 gene by a plasmid. Compared with the recombinant bacteria YPV-035, the recombinant bacteria YPV-034 only have 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-RS10865 Gene on genome

P19：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCGGCCACTTTAATAATCCT-3'；

P20：5'-TATCTGTCCCTTGAGGTGATTTCCACACCTCCTGTTGGAA-3'；

P21：5'-TTCCAACAGGAGGTGTGGAAATCACCTCAAGGGACAGATA-3'；

P22：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAACAATGTAAACAAGATGTC-3'。

P23：5'-TCGGCCACTTTAATAATCCT-3'；

P24：5'-AACAATGTAAACAAGATGTC-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 P19 and primer P20, and an amplification product (upstream homology arm fragment, 730bp) was recovered.

2. 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 (downstream homology arm fragment, 725bp) was recovered.

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 P19 and a primer P22, so that the amplification product is recovered. And after sequencing, the amplification product is shown as a sequence 8 in the sequence table.

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 at 50 ℃ for 30min) to obtain the recombinant plasmid pK 18-delta YH66-RS 10865. Through sequencing, the recombinant plasmid pK 18-delta YH66-RS10865 has a DNA molecule shown as a sequence 8 in a sequence table.

Secondly, constructing recombinant bacteria YPV-036

1. The Corynebacterium glutamicum CGMCC21260 is subjected to electric shock transformation by adopting a recombinant plasmid pK 18-delta YH66-RS10865, then cultured, and then PCR identification is respectively carried out on each single colony (by adopting a primer pair consisting of a primer P23 and a primer P24). The bacterial strain capable of amplifying bands of 1341bp and 4110bp simultaneously is a positive bacterial strain. The strain only amplifying the 4110bp band is a starting strain which fails in transformation, wherein the 4110bp 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 strain screened in the step (2), carrying out PCR amplification by adopting a primer pair consisting of a primer P23 and a primer P24, wherein the strain only shows a single amplification product and has the size of 1341bp and is a positive strain with the YH66-RS10865 gene coding region knocked out.

4. And (3) taking the strain obtained by screening in the step (3), performing PCR amplification and sequencing by using a primer pair consisting of a primer P23 and a primer P24 again, and naming the strain with correct sequencing as recombinant bacteria YPV-036. Compared with the genome DNA of Corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-036 is different only in that the DNA molecule shown in sequence 4 in 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-031, recombinant bacteria YPV-032, recombinant bacteria YPV-033, recombinant bacteria YPV-034, recombinant bacteria YPV-035 and recombinant bacteria YPV-036.

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 L-valine yield of the recombinant bacteria YPV-031 and YPV-036 is obviously higher than that of Corynebacterium glutamicum CGMCC 21260. The results show that the suppression of YH66-RS10865 gene expression can increase L-valine production, and the overexpression of YH66-RS10865 gene can decrease L-valine production.

TABLE 5 results of L-valine fermentation experiments

Bacterial strains	OD₆₁₀	L-valine yield (g/L)
			Corynebacterium glutamicum CGMCC21260	97.8	83.2
Recombinant bacterium YPV-031	98.1	85.6
			Recombinant bacterium YPV-032	98.4	82.3
Recombinant bacterium YPV-033	98.2	83.1
			Recombinant bacterium YPV-034	98.6	82.4
Recombinant bacterium YPV-035	98.3	82.6
			Recombinant bacterium YPV-036	97.9	84.3

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> Ningxia Yipin Biotechnology Ltd

Engineering bacterium obtained by YH66-RS10865 gene modification and application thereof in preparation of valine

<130> GNCYX211995

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 922

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Gln Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 2

<211> 2769

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60

cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120

gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180

cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240

tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300

aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360

ccaggcatcg gcgtcggcgg gcacatttcc acttacgcag gcgcagcccc tctgtacgaa 420

gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480

ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540

gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc 600

taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660

ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720

aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780

gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840

gttcaatgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900

gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960

gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020

acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080

ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140

tggaagctgc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200

cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260

ggccacaatt tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320

ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380

ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440

gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500

caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560

gctaccacca tggcgactgt tcgcaccttc aaggaactga tgcgcgataa gggcttggct 1620

gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680

ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740

atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800

ggttccatgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860

attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920

gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980

ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040

gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100

ggcatcgacc gcatgtacgg cccaggcaag ggcgaagatg ttatctacta catcaccatc 2160

tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220

aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280

tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340

gttcgtgcaa acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400

cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460

cagctgaagc agacctctgg cccatacgtc gcagtgtctg acttctccac tgatctgcca 2520

aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580

ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640

gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700

gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760

gaggaataa 2769

<210> 3

<211> 922

<212> PRT

<213> Corynebacterium glutamicum

<400> 3

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 4

<211> 2769

<212> DNA

<213> Corynebacterium glutamicum

<400> 4

atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60

cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120

gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180

cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240

tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300

aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360

ccaggcatcg gcgtcggcgg gcacatttcc acttacgcag gcgcagcccc tctgtacgaa 420

gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480

ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540

gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc 600

taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660

ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720

aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780

gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840

gttaactgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900

gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960

gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020

acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080

ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140

tggaagctgc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200

cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260

ggccacaatt tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320

ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380

ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440

gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500

caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560

gctaccacca tggcgactgt tcgcaccttc aaggaactga tgcgcgataa gggcttggct 1620

gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680

ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740

atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800

ggttccatgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860

attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920

gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980

ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040

gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100

ggcatcgacc gcatgtacgg cccaggcaag ggcgaagatg ttatctacta catcaccatc 2160

tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220

aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280

tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340

gttcgtgcaa acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400

cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460

cagctgaagc agacctctgg cccatacgtc gcagtgtctg acttctccac tgatctgcca 2520

aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580

ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640

gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700

gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760

gaggaataa 2769

<210> 5

<211> 1364

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cagtgccaag cttgcatgcc tgcaggtcga ctctaggctt cgtttgcttg agcgtgcatc 60

tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac tacgtcaaca ccattccaac 120

ctctatggaa cctgaattcc caggcgatga ggaaatggag aagcgttacc gtcgttggat 180

tcgctggaac gcagccatca tggttcaccg cgctcagcga ccaggcatcg gcgtcggcgg 240

gcacatttcc acttacgcag gcgcagcccc tctgtacgaa gttggcttca accacttctt 300

ccgcggcaag gatcacccag gcggcggcga ccagatcttc ttccagggcc acgcatcacc 360

aggtatgtac gcacgtgcat tcatggaggg tcgcctttct gaagacgatc tcgatggctt 420

ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc taccctcacc cacacggtat 480

gaaggacttc tgggagttcc caactgtgtc catgggtctt ggcccaatgg atgccattta 540

ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc aaggacacct ctgaccagca 600

cgtctgggcc ttccttggcg acggcgaaat ggacgagcca gaatcacgtg gtctcatcca 660

gcaggctgca ctgaacaacc tggacaacct gaccttcgtg gttcaatgca acctgcagcg 720

tctcgacgga cctgtccgcg gtaacaccaa gatcatccag gaactcgagt ccttcttccg 780

tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc gagtgggatg aacttctgga 840

gaaggaccag gatggtgcac ttgttgagat catgaacaac acctccgatg gtgactacca 900

gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac ttcttcggac gtgacccacg 960

caccgcaaag ctcgttgaga acatgaccga cgaagaaatc tggaagctgc cacgtggcgg 1020

ccacgattac cgcaaggttt acgcagccta caagcgagct cttgagacca aggatcgccc 1080

aaccgtcatc cttgctcaca ccattaaggg ctacggactc ggccacaatt tcgaaggccg 1140

taacgcaacc caccagatga agaagctgac gcttgatgat ctgaagttgt tccgcgacaa 1200

gcagggcatc ccaatcaccg atgagcagct ggagaaggat ccttaccttc ctccttacta 1260

ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag gaacgtcgcg cagcgctcgg 1320

tggctagggt accgagctcg aattcgtaat catggtcata gctg 1364

<210> 6

<211> 4523

<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

ccgatttgga tacctgaaat cccagtgagc gcaccactcc ccttacgtca cagtctgtaa 840

aacaaatctt cggtgttgcg tatccttgtt aataacttat gcgttgactc attcgtgcac 900

ttcggcgtgt cacaattagg tacgaccaag aatgggaccg ggaaaccggg acgtataaac 960

gaaataaaac attccaacag gaggtgtgga aatggccgat caagcaaaac ttggtggcaa 1020

gccctcggat gactctaact tcgcgatgat ccgcgatggc gtggcatctt atttgaacga 1080

ctcagatccg gaggagacca acgagtggat ggattcactc gacggattac tccaggagtc 1140

ttctccagaa cgtgctcgtt acctcatgct tcgtttgctt gagcgtgcat ctgcaaagcg 1200

cgtatctctt cccccaatga cgtcaaccga ctacgtcaac accattccaa cctctatgga 1260

acctgaattc ccaggcgatg aggaaatgga gaagcgttac cgtcgttgga ttcgctggaa 1320

cgcagccatc atggttcacc gcgctcagcg accaggcatc ggcgtcggcg ggcacatttc 1380

cacttacgca ggcgcagccc ctctgtacga agttggcttc aaccacttct tccgcggcaa 1440

ggatcaccca ggcggcggcg accagatctt cttccagggc cacgcatcac caggtatgta 1500

cgcacgtgca ttcatggagg gtcgcctttc tgaagacgat ctcgatggct tccgtcagga 1560

agtttcccgt gagcagggtg gtattccgtc ctaccctcac ccacacggta tgaaggactt 1620

ctgggagttc ccaactgtgt ccatgggtct tggcccaatg gatgccattt accaggcacg 1680

tttcaaccgc tacctcgaaa accgtggcat caaggacacc tctgaccagc acgtctgggc 1740

cttccttggc gacggcgaaa tggacgagcc agaatcacgt ggtctcatcc agcaggctgc 1800

actgaacaac ctggacaacc tgaccttcgt ggttcaatgc aacctgcagc gtctcgacgg 1860

acctgtccgc ggtaacacca agatcatcca ggaactcgag tccttcttcc gtggcgcagg 1920

ctggtctgtg atcaaggttg tttggggtcg cgagtgggat gaacttctgg agaaggacca 1980

ggatggtgca cttgttgaga tcatgaacaa cacctccgat ggtgactacc agaccttcaa 2040

ggctaacgac ggcgcatatg ttcgtgagca cttcttcgga cgtgacccac gcaccgcaaa 2100

gctcgttgag aacatgaccg acgaagaaat ctggaagctg ccacgtggcg gccacgatta 2160

ccgcaaggtt tacgcagcct acaagcgagc tcttgagacc aaggatcgcc caaccgtcat 2220

ccttgctcac accattaagg gctacggact cggccacaat ttcgaaggcc gtaacgcaac 2280

ccaccagatg aagaagctga cgcttgatga tctgaagttg ttccgcgaca agcagggcat 2340

cccaatcacc gatgagcagc tggagaagga tccttacctt cctccttact accacccagg 2400

tgaagacgct cctgaaatca agtacatgaa ggaacgtcgc gcagcgctcg gtggctacct 2460

gccagagcgt cgtgagaact acgatccaat tcaggttcca ccactggata agcttcgctc 2520

tgtccgtaag ggctccggca agcagcagat cgctaccacc atggcgactg ttcgcacctt 2580

caaggaactg atgcgcgata agggcttggc tgatcgcctt gtcccaatca ttcctgatga 2640

ggcacgtacc ttcggtcttg actcttggtt cccaaccttg aagatctaca acccgcacgg 2700

tcagaactac gtgcctgttg accacgacct gatgctctcc taccgtgagg cacctgaagg 2760

acagatcctg cacgaaggca tcaacgaggc tggttccatg gcatcgttca tcgctgcggg 2820

tacctcctac gccacccacg gcaaggccat gattccgctg tacatcttct actcgatgtt 2880

cggattccag cgcaccggtg actccatctg ggcagcagcc gatcagatgg cacgtggctt 2940

cctcttgggc gctaccgcag gtcgcaccac cctgaccggt gaaggcctcc agcacatgga 3000

tggacactcc cctgtcttgg cttccaccaa cgagggtgtc gagacctacg acccatcctt 3060

tgcgtacgag atcgcacacc tggttcaccg tggcatcgac cgcatgtacg gcccaggcaa 3120

gggcgaagat gttatctact acatcaccat ctacaacgag ccaaccccac agccagctga 3180

gccagaagga ctggacgtag aaggcctgca caagggcatc tacctctact cccgcggtga 3240

aggcaccggc catgaggcaa acatcttggc ttccggtgtt ggtatgcagt gggctctcaa 3300

ggctgcatcc atccttgagg ctgactacgg agttcgtgca aacatttact ccgctacttc 3360

ttgggttaac ttggctcgcg atggcgctgc tcgtaacaag gcacagctgc gcaacccagg 3420

tgcagatgct ggcgaggcat tcgtaaccac ccagctgaag cagacctctg gcccatacgt 3480

cgcagtgtct gacttctcca ctgatctgcc aaaccagatc cgtgaatggg tcccaggcga 3540

ctacaccgtt ctcggtgcag atggcttcgg tttctctgat acccgcccag ctgctcgtcg 3600

cttcttcaac atcgacgctg agtccattgt tgttgcagtg ctgaactccc tggcacgcga 3660

aggcaagatc gacgtctccg ttgctgctca ggctgctgag aagttcaagt tggatgatcc 3720

tacgagtgtt tccgtagatc caaacgctcc tgaggaataa taaatcgact actcacatag 3780

ggtcgggcta gtcattctga tcagcgaatt ccacgttcac atcgccaatt ccagagttca 3840

caaccagatt cagcattgga ccttctagat cagcattgtg ggcggtgaga tctccaacat 3900

cacagcgcgc tgtgcccaca ccggcggtac aacttaggct cacgggcaca tcatcgggca 3960

gggtgaccat gacttcgccg atccctgagg tgatttggat gttttgttcc tgatccaatt 4020

gggtgaggtg gctgaaatcg aggttcattt cacccacgcc agaggtgtag ctgctgagga 4080

gttcatcgtt ggtggggatg agattgacat cgccgattcc agggtcgtct tcaaagtaga 4140

tgggatcgat atttgaaata aacaggcctg cgagggcgct catgacaact ccggtaccaa 4200

ctacaccgcc gacaatccat ggccacacat ggcgcttttt ctgaggcttt tgtggaggga 4260

cttgtacatc ccaggtgttg tattggtttt gggcaagtgg atcccaatga ggcgcttcgg 4320

gggtttgttg cgcgaagggt gcatagtagc cctcaacggg ggtgatagtg cttagatctg 4380

gttggggttg tgggtagaga tcttcgtttt tcatggtggc atcctcagaa acagtgaatt 4440

cagtggtgag tagtccgcgg ggtggaagtg gttgtttctt atgcagggta ccgagctcga 4500

attcgtaatc atggtcatag ctg 4523

<210> 7

<211> 3044

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcttgcatgc ctgcaggtcg actctagagg atcccctgga tacctgaaat cccagtgagc 60

gcaccactcc ccttacgtca cagtctgtaa aacaaatctt cggtgttgcg tatccttgtt 120

aataacttat gcgttgactc attcgtgcac ttcggcgtgt cacaattagg tacgaccaag 180

aatgggaccg ggaaaccggg acgtataaac gaaataaaac attccaacag gaggtgtgga 240

aatggccgat caagcaaaac ttggtggcaa gccctcggat gactctaact tcgcgatgat 300

ccgcgatggc gtggcatctt atttgaacga ctcagatccg gaggagacca acgagtggat 360

ggattcactc gacggattac tccaggagtc ttctccagaa cgtgctcgtt acctcatgct 420

tcgtttgctt gagcgtgcat ctgcaaagcg cgtatctctt cccccaatga cgtcaaccga 480

ctacgtcaac accattccaa cctctatgga acctgaattc ccaggcgatg aggaaatgga 540

gaagcgttac cgtcgttgga ttcgctggaa cgcagccatc atggttcacc gcgctcagcg 600

accaggcatc ggcgtcggcg ggcacatttc cacttacgca ggcgcagccc ctctgtacga 660

agttggcttc aaccacttct tccgcggcaa ggatcaccca ggcggcggcg accagatctt 720

cttccagggc cacgcatcac caggtatgta cgcacgtgca ttcatggagg gtcgcctttc 780

tgaagacgat ctcgatggct tccgtcagga agtttcccgt gagcagggtg gtattccgtc 840

ctaccctcac ccacacggta tgaaggactt ctgggagttc ccaactgtgt ccatgggtct 900

tggcccaatg gatgccattt accaggcacg tttcaaccgc tacctcgaaa accgtggcat 960

caaggacacc tctgaccagc acgtctgggc cttccttggc gacggcgaaa tggacgagcc 1020

agaatcacgt ggtctcatcc agcaggctgc actgaacaac ctggacaacc tgaccttcgt 1080

ggttcaatgc aacctgcagc gtctcgacgg acctgtccgc ggtaacacca agatcatcca 1140

ggaactcgag tccttcttcc gtggcgcagg ctggtctgtg atcaaggttg tttggggtcg 1200

cgagtgggat gaacttctgg agaaggacca ggatggtgca cttgttgaga tcatgaacaa 1260

cacctccgat ggtgactacc agaccttcaa ggctaacgac ggcgcatatg ttcgtgagca 1320

cttcttcgga cgtgacccac gcaccgcaaa gctcgttgag aacatgaccg acgaagaaat 1380

ctggaagctg ccacgtggcg gccacgatta ccgcaaggtt tacgcagcct acaagcgagc 1440

tcttgagacc aaggatcgcc caaccgtcat ccttgctcac accattaagg gctacggact 1500

cggccacaat ttcgaaggcc gtaacgcaac ccaccagatg aagaagctga cgcttgatga 1560

tctgaagttg ttccgcgaca agcagggcat cccaatcacc gatgagcagc tggagaagga 1620

tccttacctt cctccttact accacccagg tgaagacgct cctgaaatca agtacatgaa 1680

ggaacgtcgc gcagcgctcg gtggctacct gccagagcgt cgtgagaact acgatccaat 1740

tcaggttcca ccactggata agcttcgctc tgtccgtaag ggctccggca agcagcagat 1800

cgctaccacc atggcgactg ttcgcacctt caaggaactg atgcgcgata agggcttggc 1860

tgatcgcctt gtcccaatca ttcctgatga ggcacgtacc ttcggtcttg actcttggtt 1920

cccaaccttg aagatctaca acccgcacgg tcagaactac gtgcctgttg accacgacct 1980

gatgctctcc taccgtgagg cacctgaagg acagatcctg cacgaaggca tcaacgaggc 2040

tggttccatg gcatcgttca tcgctgcggg tacctcctac gccacccacg gcaaggccat 2100

gattccgctg tacatcttct actcgatgtt cggattccag cgcaccggtg actccatctg 2160

ggcagcagcc gatcagatgg cacgtggctt cctcttgggc gctaccgcag gtcgcaccac 2220

cctgaccggt gaaggcctcc agcacatgga tggacactcc cctgtcttgg cttccaccaa 2280

cgagggtgtc gagacctacg acccatcctt tgcgtacgag atcgcacacc tggttcaccg 2340

tggcatcgac cgcatgtacg gcccaggcaa gggcgaagat gttatctact acatcaccat 2400

ctacaacgag ccaaccccac agccagctga gccagaagga ctggacgtag aaggcctgca 2460

caagggcatc tacctctact cccgcggtga aggcaccggc catgaggcaa acatcttggc 2520

ttccggtgtt ggtatgcagt gggctctcaa ggctgcatcc atccttgagg ctgactacgg 2580

agttcgtgca aacatttact ccgctacttc ttgggttaac ttggctcgcg atggcgctgc 2640

tcgtaacaag gcacagctgc gcaacccagg tgcagatgct ggcgaggcat tcgtaaccac 2700

ccagctgaag cagacctctg gcccatacgt cgcagtgtct gacttctcca ctgatctgcc 2760

aaaccagatc cgtgaatggg tcccaggcga ctacaccgtt ctcggtgcag atggcttcgg 2820

tttctctgat acccgcccag ctgctcgtcg cttcttcaac atcgacgctg agtccattgt 2880

tgttgcagtg ctgaactccc tggcacgcga aggcaagatc gacgtctccg ttgctgctca 2940

ggctgctgag aagttcaagt tggatgatcc tacgagtgtt tccgtagatc caaacgctcc 3000

tgaggaataa gttttggcgg atgagagaag attttcagcc tgat 3044

<210> 8

<211> 1415

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagtgccaag cttgcatgcc tgcaggtcga ctctagtcgg ccactttaat aatcctcctc 60

gtgtgggccc cgatgtgttt ttcgattaca tggattcaac atgaaaccgc ggggctattg 120

atatatccga attgcacatt accgtccaac cggtactttg aaccaccttt ccctggaatt 180

ttttcctttt cctccccctt tacgctcaag aatcaatgaa ttcaatcact ggccagcgat 240

taacttttcg agttttcagt cttggatttc cacaattctc ttcaaaataa tggtggctag 300

atttttcatc aaaccttcac caaaaggaca tcagacctgt agttttatgc gattcgcgtc 360

aaacgtgaga gaaacatcac atctcgcggg aaactacccg ataattcttt gcaaaacttt 420

gcaaagcgga atgaacatgc agctagtttc cgtagaaatg ttctttaaaa aatccacaac 480

aattgccagg aagcacaccg attgatggat acctgaaatc ccagtgagcg caccactccc 540

cttacgtcac agtctgtaaa acaaatcttc ggtgttgcgt atccttgtta ataacttatg 600

cgttgactca ttcgtgcact tcggcgtgtc acaattaggt acgaccaaga atgggaccgg 660

gaaaccggga cgtataaacg aaataaaaca ttccaacagg aggtgtggaa atcacctcaa 720

gggacagata aatcccgccg ccagacgtta gtctggcggc gggattcgtc gtaaagcaag 780

ctctttttag ccgaggaacg ccttgtcaga caatgttgcg cccttgatgt tggcgaactc 840

ctgcagcaaa tcgcgcacag tcaacttcga cttggtagcc tgatctgcct ggtagacaat 900

ctggccttca tgcatcatga tcaggcgatt gcccaggcga attgcctgtt ccatgttgtg 960

cgtgaccata agcgtagtca gagttccatc tgccacgatc ttttcggtca aggtggtcac 1020

aagctctgca cgctgtggat caagtgctgc ggtgtgctca tccaacagca tgattttagg 1080

ttgagtaaaa ccagccatca gcagggacaa tgcctgacgc tgaccgccag agagcaaacc 1140

aactttggca gtgagcctgt tttccagacc cagctcgagg cgctcaagtt cctgcttgaa 1200

ttgctcacgg cgcttcgagg tcagtgcaaa gcccaatcca cggcgcttgc cgcgcagcaa 1260

cgcgatggcc agattctctt caatggtgag attcggcgcg gtgccggcca gaggatcctg 1320

gaaaacgcgg ccgatgtagc gggcacgctt gtgctctgac atcttgttta cattgttggg 1380

taccgagctc gaattcgtaa tcatggtcat agctg 1415

<210> 9

<211> 4110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcggccactt taataatcct cctcgtgtgg gccccgatgt gtttttcgat tacatggatt 60

caacatgaaa ccgcggggct attgatatat ccgaattgca cattaccgtc caaccggtac 120

tttgaaccac ctttccctgg aattttttcc ttttcctccc cctttacgct caagaatcaa 180

tgaattcaat cactggccag cgattaactt ttcgagtttt cagtcttgga tttccacaat 240

tctcttcaaa ataatggtgg ctagattttt catcaaacct tcaccaaaag gacatcagac 300

ctgtagtttt atgcgattcg cgtcaaacgt gagagaaaca tcacatctcg cgggaaacta 360

cccgataatt ctttgcaaaa ctttgcaaag cggaatgaac atgcagctag tttccgtaga 420

aatgttcttt aaaaaatcca caacaattgc caggaagcac accgattgat ggatacctga 480

aatcccagtg agcgcaccac tccccttacg tcacagtctg taaaacaaat cttcggtgtt 540

gcgtatcctt gttaataact tatgcgttga ctcattcgtg cacttcggcg tgtcacaatt 600

aggtacgacc aagaatggga ccgggaaacc gggacgtata aacgaaataa aacattccaa 660

caggaggtgt ggaaatggcc gatcaagcaa aacttggtgg caagccctcg gatgactcta 720

acttcgcgat gatccgcgat ggcgtggcat cttatttgaa cgactcagat ccggaggaga 780

ccaacgagtg gatggattca ctcgacggat tactccagga gtcttctcca gaacgtgctc 840

gttacctcat gcttcgtttg cttgagcgtg catctgcaaa gcgcgtatct cttcccccaa 900

tgacgtcaac cgactacgtc aacaccattc caacctctat ggaacctgaa ttcccaggcg 960

atgaggaaat ggagaagcgt taccgtcgtt ggattcgctg gaacgcagcc atcatggttc 1020

accgcgctca gcgaccaggc atcggcgtcg gcgggcacat ttccacttac gcaggcgcag 1080

cccctctgta cgaagttggc ttcaaccact tcttccgcgg caaggatcac ccaggcggcg 1140

gcgaccagat cttcttccag ggccacgcat caccaggtat gtacgcacgt gcattcatgg 1200

agggtcgcct ttctgaagac gatctcgatg gcttccgtca ggaagtttcc cgtgagcagg 1260

gtggtattcc gtcctaccct cacccacacg gtatgaagga cttctgggag ttcccaactg 1320

tgtccatggg tcttggccca atggatgcca tttaccaggc acgtttcaac cgctacctcg 1380

aaaaccgtgg catcaaggac acctctgacc agcacgtctg ggccttcctt ggcgacggcg 1440

aaatggacga gccagaatca cgtggtctca tccagcaggc tgcactgaac aacctggaca 1500

acctgacctt cgtggttaac tgcaacctgc agcgtctcga cggacctgtc cgcggtaaca 1560

ccaagatcat ccaggaactc gagtccttct tccgtggcgc aggctggtct gtgatcaagg 1620

ttgtttgggg tcgcgagtgg gatgaacttc tggagaagga ccaggatggt gcacttgttg 1680

agatcatgaa caacacctcc gatggtgact accagacctt caaggctaac gacggcgcat 1740

atgttcgtga gcacttcttc ggacgtgacc cacgcaccgc aaagctcgtt gagaacatga 1800

ccgacgaaga aatctggaag ctgccacgtg gcggccacga ttaccgcaag gtttacgcag 1860

cctacaagcg agctcttgag accaaggatc gcccaaccgt catccttgct cacaccatta 1920

agggctacgg actcggccac aatttcgaag gccgtaacgc aacccaccag atgaagaagc 1980

tgacgcttga tgatctgaag ttgttccgcg acaagcaggg catcccaatc accgatgagc 2040

agctggagaa ggatccttac cttcctcctt actaccaccc aggtgaagac gctcctgaaa 2100

tcaagtacat gaaggaacgt cgcgcagcgc tcggtggcta cctgccagag cgtcgtgaga 2160

actacgatcc aattcaggtt ccaccactgg ataagcttcg ctctgtccgt aagggctccg 2220

gcaagcagca gatcgctacc accatggcga ctgttcgcac cttcaaggaa ctgatgcgcg 2280

ataagggctt ggctgatcgc cttgtcccaa tcattcctga tgaggcacgt accttcggtc 2340

ttgactcttg gttcccaacc ttgaagatct acaacccgca cggtcagaac tacgtgcctg 2400

ttgaccacga cctgatgctc tcctaccgtg aggcacctga aggacagatc ctgcacgaag 2460

gcatcaacga ggctggttcc atggcatcgt tcatcgctgc gggtacctcc tacgccaccc 2520

acggcaaggc catgattccg ctgtacatct tctactcgat gttcggattc cagcgcaccg 2580

gtgactccat ctgggcagca gccgatcaga tggcacgtgg cttcctcttg ggcgctaccg 2640

caggtcgcac caccctgacc ggtgaaggcc tccagcacat ggatggacac tcccctgtct 2700

tggcttccac caacgagggt gtcgagacct acgacccatc ctttgcgtac gagatcgcac 2760

acctggttca ccgtggcatc gaccgcatgt acggcccagg caagggcgaa gatgttatct 2820

actacatcac catctacaac gagccaaccc cacagccagc tgagccagaa ggactggacg 2880

tagaaggcct gcacaagggc atctacctct actcccgcgg tgaaggcacc ggccatgagg 2940

caaacatctt ggcttccggt gttggtatgc agtgggctct caaggctgca tccatccttg 3000

aggctgacta cggagttcgt gcaaacattt actccgctac ttcttgggtt aacttggctc 3060

gcgatggcgc tgctcgtaac aaggcacagc tgcgcaaccc aggtgcagat gctggcgagg 3120

cattcgtaac cacccagctg aagcagacct ctggcccata cgtcgcagtg tctgacttct 3180

ccactgatct gccaaaccag atccgtgaat gggtcccagg cgactacacc gttctcggtg 3240

cagatggctt cggtttctct gatacccgcc cagctgctcg tcgcttcttc aacatcgacg 3300

ctgagtccat tgttgttgca gtgctgaact ccctggcacg cgaaggcaag atcgacgtct 3360

ccgttgctgc tcaggctgct gagaagttca agttggatga tcctacgagt gtttccgtag 3420

atccaaacgc tcctgaggaa taaatcacct caagggacag ataaatcccg ccgccagacg 3480

ttagtctggc ggcgggattc gtcgtaaagc aagctctttt tagccgagga acgccttgtc 3540

agacaatgtt gcgcccttga tgttggcgaa ctcctgcagc aaatcgcgca cagtcaactt 3600

cgacttggta gcctgatctg cctggtagac aatctggcct tcatgcatca tgatcaggcg 3660

attgcccagg cgaattgcct gttccatgtt gtgcgtgacc ataagcgtag tcagagttcc 3720

atctgccacg atcttttcgg tcaaggtggt cacaagctct gcacgctgtg gatcaagtgc 3780

tgcggtgtgc tcatccaaca gcatgatttt aggttgagta aaaccagcca tcagcaggga 3840

caatgcctga cgctgaccgc cagagagcaa accaactttg gcagtgagcc tgttttccag 3900

acccagctcg aggcgctcaa gttcctgctt gaattgctca cggcgcttcg aggtcagtgc 3960

aaagcccaat ccacggcgct tgccgcgcag caacgcgatg gccagattct cttcaatggt 4020

gagattcggc gcggtgccgg ccagaggatc ctggaaaacg cggccgatgt agcgggcacg 4080

cttgtgctct gacatcttgt ttacattgtt 4110

Claims

1. The application of a substance for inhibiting YH66-RS10865 gene expression or reducing YH66-RS10865 protein abundance or reducing YH66-RS10865 protein activity;

the application is as follows (I) or (II):

the application of (I) in improving the yield of the bacterial valine;

(II) use in the production of valine;

the YH66-RS10865 gene is a gene encoding YH66-RS10865 protein;

the YH66-RS10865 protein is (a1) or (a2) or (a 3):

(a1) protein shown in a sequence 3 in a sequence table;

2. A recombinant bacterium is obtained by inhibiting YH66-RS10865 gene expression in bacteria; the YH66-RS10865 gene is the YH66-RS10865 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: inhibiting YH66-RS10865 gene expression or reducing YH66-RS10865 protein abundance or reducing YH66-RS10865 protein activity in bacteria;

the YH66-RS10865 gene described in claim 1;

the YH66-RS10865 protein as set forth in claim 1, which is YH66-RS10865 protein.

Use of the YH66-RS10865 protein for regulating valine production in a bacterium; the YH66-RS10865 protein as set forth in claim 1, which is YH66-RS10865 protein.

7. The mutant protein is obtained by mutating the 282 th amino acid residue of YH66-RS10865 protein from N to other amino acid residues; the YH66-RS10865 protein as set forth in claim 1, which is YH66-RS10865 protein.

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 282 th amino acid residue of YH66-RS10865 protein encoded in bacterial genomic DNA from the codon encoding N to the codon encoding other amino acid residue; the YH66-RS10865 protein as set forth in claim 1, which is YH66-RS10865 protein.