阅读说明：本技术 Yh66-rs07020基因改造得到的工程菌及其在制备缬氨酸中的应用 (Engineering bacterium obtained by YH66-RS07020 gene modification and application thereof in preparation of valine ) 是由 田斌 孟刚 魏爱英 赵春光 贾慧萍 杨立鹏 于 2021-08-23 设计创作，主要内容包括：本发明公开了YH66-RS07020基因改造得到的工程菌及其在制备缬氨酸中的应用。本发明提供了用于抑制YH66-RS07020基因表达的物质或降低YH66-RS07020蛋白丰度的物质或降低YH66-RS07020蛋白活性的物质在提高细菌缬氨酸产量中的应用。本发明发现YH66-RS07020蛋白对细菌的缬氨酸产量存在负调控,即YH66-RS07020蛋白含量增高、缬氨酸产量降低,YH66-RS07020蛋白含量降低、缬氨酸产量增高。抑制YH66-RS07020基因表达可以提高缬氨酸产量,过表达YH66-RS07020基因降低缬氨酸产量。进一步,本发明发现了YH66-RS07020～(C251T)蛋白及其编码基因和应用。本发明对于缬氨酸工业化生产,具有重大的应用价值。(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 invention C251T Protein and its coding gene and application. The invention has great application value for industrial production of valine.)

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.

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 embodiment^C251TOr 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-RS07020^C251TOr 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-RS07020^C251TOr 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-RS07020^C251TThe 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-RS07020^C251TProtein coding gene (named YH66-RS 07020)^C251TA gene).

The invention also protects the polypeptide with YH66-RS07020^C251TExpression cassette for gene or gene with YH66-RS07020^C251TRecombinant vector of gene or gene with YH66-RS07020^C251TRecombinant bacteria of genes.

In particular, YH66-RS07020^C251TThe 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-RS07020^C251TProtein YH66-RS07020^C251TGene, gene YH66-RS07020^C251TExpression cassette for gene or gene with YH66-RS07020^C251THas YH66-RS07020^C251TThe 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 mutation^C251TProtein, YH66-RS07020^C251TThe coding gene of the protein is shown as a sequence 2 in a sequence table. Compared with YH66-RS07020 gene, YH66-RS07020^C251TThe difference of the genes is that the 251 th nucleotide is mutated from C to T. Compared with YH66-RS07020 protein, YH66-RS07020^C251TThe 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 invention^C251TProtein 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-RS07020^C251T. Sequencing verification shows that the recombinant plasmid pK18-YH66-RS07020^C251THas 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-RS07020^C251TCorynebacterium 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 adopted^C251TThe 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 table^C251TA 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 band^C251TThe 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 genome^C251TEngineering 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-RS07020^C251T. Sequencing verification shows that the recombinant plasmid pXMJ19-YH66-RS07020^C251THas a DNA molecule shown in a sequence 7 of a sequence table.

4. Recombinant plasmid pXMJ19-YH66-RS07020^C251TElectrically 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 plasmid^C251TEngineering 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