Engineering bacterium obtained by YH66-RS07015 gene modification and application thereof in preparation of valine
阅读说明：本技术 Yh66-rs07015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-RS07015 gene modification and application thereof in preparation of valine ) 是由 赵春光 魏爱英 付丽霞 贾慧萍 周晓群 苏厚波 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种YH66-RS07015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-RS07015基因表达的物质或降低YH66-RS07015蛋白丰度的物质或降低YH66-RS07015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-RS07015蛋白对细菌的缬氨酸产量存在负调控,即YH66-RS07015蛋白含量增高、缬氨酸产量降低,YH66-RS07015蛋白含量降低、缬氨酸产量增高。抑制YH66-RS07015基因表达可以提高缬氨酸产量,过表达YH66-RS07015基因降低缬氨酸产量。进一步,本发明发现了YH66-RS07015～(C1187T)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-RS07015 gene modification and application thereof in valine preparation. The invention provides an application of a substance for inhibiting YH66-RS07015 gene expression or a substance for reducing YH66-RS07015 protein abundance or a substance for reducing YH66-RS07015 protein activity in improving the yield of bacterial valine. The invention discovers that YH66-RS07015 protein has negative control on the yield of valine of bacteria, namely YH66-RS07015 protein content is increasedHigh, reduced valine yield, reduced YH66-RS07015 protein content, increased valine yield. The suppression of YH66-RS07015 gene expression can improve valine yield, and the overexpression of YH66-RS07015 gene can reduce valine yield. Further, YH66-RS07015 was found in the invention C1187T 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-RS07015 gene expression or a substance for reducing YH66-RS07015 protein abundance or a substance for reducing YH66-RS07015 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-RS07015 gene is a gene for coding YH66-RS07015 protein;
the YH66-RS07015 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-RS07015 gene expression in bacteria; the YH66-RS07015 gene is the YH66-RS07015 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-RS07015 gene expression in bacteria or reduce YH66-RS07015 protein abundance in bacteria or reduce YH66-RS07015 protein activity in bacteria;
the YH66-RS07015 gene is the YH66-RS07015 gene described in claim 1;
the YH66-RS07015 protein is the YH66-RS07015 protein of claim 1.
The application of the YH66-RS07015 protein in regulating the valine yield of the bacteria or the YH66-RS07015 protein in regulating the bacterial quantity of the bacteria; the YH66-RS07015 protein is the YH66-RS07015 protein of claim 1.
7. The mutant protein is obtained by mutating the 282 th amino acid residue of YH66-RS07015 protein from N to other amino acid residues; the YH66-RS07015 protein is the YH66-RS07015 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 the 396 th amino acid residue of YH66-RS07015 protein encoded in bacterial genome DNA from the codon encoding T to the codon encoding other amino acid residues; the YH66-RS07015 protein is the YH66-RS07015 protein of claim 1.
Technical Field
The invention belongs to the technical field of biology, and relates to an engineering bacterium obtained by YH66-RS07015 gene modification and application thereof in valine preparation, wherein the modification is C1187T.
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-RS07015 gene modification and application thereof in valine preparation.
The invention provides application of a substance for inhibiting YH66-RS07015 gene expression or a substance for reducing YH66-RS07015 protein abundance or a substance for reducing YH66-RS07015 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-RS07015 gene is a gene encoding the YH66-RS07015 protein.
The YH66-RS07015 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-RS07015 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 gene expression for inhibiting YH66-RS07015 can be knock-out of YH66-RS07015 gene, and can also be mutation YH66-RS07015 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-RS07015 gene expression can be specifically a DNA molecule shown in sequence 5 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07015 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-RS07015 gene expression can be exemplified by recombinant plasmid pK18-YH66-RS07015C1187TOr the recombinant plasmid pK 18-delta YH66-RS 07015.
The invention also provides a recombinant bacterium, which is obtained by inhibiting YH66-RS07015 gene expression in bacteria.
The YH66-RS07015 gene expression in the bacteria can be knocked out from YH66-RS07015 genes in the bacteria, and can also be YH66-RS07015 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-RS07015 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-RS07015 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-RS07015 gene in the mutant bacterium can be specifically: the codon of amino acid residue number 396 of YH66-RS07015 protein encoded in bacterial genomic DNA was mutated from the codon encoding T to the codon encoding other amino acid residues. In particular, the other amino acid residue is I.
Illustratively, the YH66-RS07015 gene in the mutant bacterium can be specifically: the YH66-RS07015 gene in bacterial genomic DNA is subjected to the following point mutations: the 1187 th nucleotide is mutated from C to other nucleotides (specifically T).
Illustratively, the manner of inhibiting YH66-RS07015 gene expression in bacteria may be: a substance for inhibiting YH66-RS07015 gene expression was introduced into the bacterium.
Illustratively, the substance for inhibiting YH66-RS07015 gene expression can be specifically a DNA molecule shown in sequence 5 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07015 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-RS07015 gene expression can be specifically a recombinant plasmid pK18-YH66-RS07015 in the embodimentC1187TOr the recombinant plasmid pK 18-delta YH66-RS 07015.
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-RS07015 gene expression in bacteria or reduce YH66-RS07015 protein abundance in bacteria or reduce YH66-RS07015 protein activity in bacteria.
The YH66-RS07015 gene expression in the bacteria can be knocked out from YH66-RS07015 genes in the bacteria, and can also be YH66-RS07015 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-RS07015 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-RS07015 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-RS07015 gene in the mutant bacterium can be specifically: the codon of amino acid residue number 396 of YH66-RS07015 protein encoded in bacterial genomic DNA was mutated from the codon encoding T to the codon encoding other amino acid residues. In particular, the other amino acid residue is I.
Illustratively, the YH66-RS07015 gene in the mutant bacterium can be specifically: the YH66-RS07015 gene in bacterial genomic DNA is subjected to the following point mutations: the 1187 th nucleotide is mutated from C to other nucleotides (specifically T).
Illustratively, the manner of inhibiting YH66-RS07015 gene expression in bacteria may be: a substance for inhibiting YH66-RS07015 gene expression was introduced into the bacterium.
Illustratively, the substance for inhibiting YH66-RS07015 gene expression can be specifically a DNA molecule shown in sequence 5 of the sequence table or a recombinant plasmid with the DNA molecule shown in sequence 5 of the sequence table.
Illustratively, the substance for inhibiting YH66-RS07015 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-RS07015 gene expression can be specifically a recombinant plasmid pK18-YH66-RS07015 in the embodimentC1187TOr the recombinant plasmid pK 18-delta YH66-RS 07015.
The invention also protects the application of the YH66-RS07015 protein in regulating the valine yield of bacteria.
The regulation is negative regulation, namely YH66-RS07015 protein content is increased, and valine yield is reduced.
The regulation is negative regulation, namely YH66-RS07015 protein content is reduced, and valine yield is increased.
The invention also protects the application of the YH66-RS07015 protein in regulating and controlling the bacterial count of bacteria.
The regulation is negative regulation, namely YH66-RS07015 protein content is increased, and bacterial count is reduced.
The regulation is negative regulation, namely YH66-RS07015 protein content is reduced, and bacterial count is increased.
The invention also discloses a mutant protein named YH66-RS07015C1187TThe protein is obtained by mutating YH66-RS07015 protein 396 amino acid residue from T to other amino acid residue.
In particular, the other amino acid residue is I.
Exemplary, the YH66-RS07015C1187TThe protein is shown as a sequence 1 in a sequence table.
The invention also protects YH66-RS07015C1187TEncoding gene of protein or gene with YH66-RS07015C1187TExpression cassette of protein coding gene or gene with YH66-RS07015C1187TRecombinant vector of protein coding gene or recombinant vector with YH66-RS07015C1187TRecombinant bacteria of coding genes of proteins.
YH66-RS07015C1187TThe coding gene of the protein is named YH66-RS07015C1187TA gene.
Specifically, the YH66-RS07015C1187TThe 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-RS07015C1187TProtein YH66-RS07015C1187TGene, gene YH66-RS07015C1187TExpression cassette for gene or gene with YH66-RS07015C1187TRecombinant vector of gene or gene with YH66-RS07015C1187TApplication 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 residue number 396 of YH66-RS07015 protein encoded in bacterial genomic DNA was mutated from the codon encoding T to the codon encoding other amino acid residues.
In particular, the other amino acid residue is I.
The method specifically comprises the following steps: the YH66-RS07015 gene in bacterial genomic DNA is subjected to the following point mutations: the 1187 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 glutamicum (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 thiogens (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 (Corynebacterium glutamicum) also called 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-RS07015 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-RS07015 shown in sequence 1 of a sequence table is obtained by introducing point mutationC1187TProtein, YH66-RS07015C1187TThe coding gene of the protein is shown as a sequence 2 in a sequence table. Compared with YH66-RS07015 gene, YH66-RS07015C1187TThe difference of the genes is that the 1187 th nucleotide is mutated from C to T. Compared with YH66-RS07015 protein, YH66-RS07015C1187TThe difference of the protein is that the 396 th amino acid residue is mutated from T to I.
The invention finds that the YH66-RS07015 protein has negative regulation and control on the valine yield of bacteria, namely the YH66-RS07015 protein content is increased, the valine yield is reduced, the YH66-RS07015 protein content is reduced, and the valine yield is increased. The suppression of YH66-RS07015 gene expression can improve valine yield, and the overexpression of YH66-RS07015 gene can reduce valine yield. Further, YH66-RS07015 was found in the inventionC1187TProtein 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-007
P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGCCAAGATCGGCACCGGTGG-3'；
P2：5'-CCTCTTGCTTGATCCAGATGGAGGAAGGCACGAC-3'；
P3：5'-GTCGTGCCTTCCTCCATCTGGATCAAGCAAGAGG-3'；
P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTGAGGAGAAGACAGCGCGG-3'。
P5：5'-CGACATCAAGATCGACACCG-3'；
P6：5'-GCAGAGCGCTCGCCTGGCTT-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 (628 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 (620 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 P1 and a primer P4, so that the amplification product (1214bp) 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-RS07015C1187T. Sequencing verification shows that the recombinant plasmid pK18-YH66-RS07015C1187THas a DNA molecule shown in a sequence 5 of a sequence table.
Secondly, constructing recombinant bacteria YPV-007
1. Adopts a recombinant plasmid pK18-YH66-RS07015C1187TCorynebacterium 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 (270 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-RS07015 is adoptedC1187TThe amplified fragment of (i.e., the recombinant plasmid pK18-YH66-RS 07015)C1187TThe 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), carrying out sequencing verification on the amplification product obtained in the step (3) of the screened strain to obtain the recombinant bacterium YPV-007. Compared with Corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-007 is different only in that Corynebacterium glutamicum CGMCC21260 gene is usedYH66-RS07015 gene shown in sequence 4 of the sequence table in the group is replaced by YH66-RS07015 gene shown in sequence 2 of the sequence tableC1187TA mutant gene. The sequence 2 and the sequence 4 have only one nucleotide difference and are positioned at the 1187 th position. The recombinant strain YPV-007 is an engineering strain obtained by mutating (single-point mutation) YH66-RS07015 gene in Corynebacterium glutamicum CGMCC 21260.
Example 2 construction of recombinant bacterium YPV-009 and recombinant bacterium YPV-008
P7：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGGACTC-3'；
P8：5'-TGAAATGTAAGATTCAAAGAAATCGGACTCCTTAAATGGG-3'；
P9：5'-CCCATTTAAGGAGTCCGATTTCTTTGAATCTTACATTTCA-3'；
P10：5'-CTATGTGAGTAGTCGATTTATTAGATATCTGCAGGTGAGG-3'；
P11：5'-CCTCACCTGCAGATATCTAATAAATCGACTACTCACATAG-3'；
P12：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAACCACTT-3'。
P13：5'-GTCCGCTCTGTTGGTGTTCA-3'；
P14：5'-GATTGCTTCGCCGCGGTATT-3'。
P15：5'-TCCCACACCTCTACTCACGG-3'；
P16：5'-TGGAGGAATATTCGGCCCAG-3'。
Firstly, constructing recombinant bacteria YPV-009
1. The recombinant bacterium YPV-007 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-007 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 (1739bp) is recovered.
3. The recombinant bacterium YPV-007 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.
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 (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) (by adopting NEBuilder enzyme, incubating at 50 ℃ for 30min) to obtain a recombinant plasmid 009. Through sequencing verification, the recombinant plasmid 009 has a DNA molecule shown as sequence 6 in the sequence table.
6. The corynebacterium glutamicum CGMCC21260 is subjected to electric shock transformation by adopting the recombinant plasmid 009, then cultured, and then each single colony is respectively subjected to PCR identification (a primer pair consisting of a primer P13 and a primer P14 is adopted), so that the strain capable of amplifying a 1755bp 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, wherein the strain with the 1804bp band amplified is YH66-RS07015C1187TThe positive strain with gene integrated into the genome of Corynebacterium glutamicum CGMCC21260 was named recombinant strain YPV-009. The recombinant bacterium YPV-009 is overexpressed YH66-RS07015 on genomeC1187TEngineering strain of gene.
Secondly, constructing recombinant bacteria YPV-008
The templates are all replaced by the recombinant bacterium YPV-007 to the Corynebacterium glutamicum ATCC15168, and the steps are the same.
The positive strain integrating the YH66-RS07015 gene into the genome of Corynebacterium glutamicum CGMCC21260 is obtained and named as recombinant strain YPV-008. The recombinant bacterium YPV-008 is an engineering strain with YH66-RS07015 gene overexpression on genome. Compared with the recombinant bacterium YPV-009, the recombinant bacterium YPV-008 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-011 and recombinant bacterium YPV-010
Firstly, constructing recombinant bacteria YPV-011
1. The recombinant bacterium YPV-007 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 (1769bp) is recovered. And after sequencing, the amplification product is shown as a sequence 7 in the sequence table.
P17：5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTCTTTGAATCTTACATTTCA-3'；
P18：5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAGATATCTGCAGGTGAGG-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-RS07015C1187T. Sequencing verification shows that the recombinant plasmid pXMJ19-YH66-RS07015C1187THas a DNA molecule shown in a sequence 7 of a sequence table.
4. Recombinant plasmid pXMJ19-YH66-RS07015C1187TThe corynebacterium glutamicum CGMCC21260 is electrically transduced to obtain the recombinant bacterium YPV-011. The recombinant bacterium YPV-011 is subjected to plasmid overexpression YH66-RS07015C1187TEngineering strain of gene.
Secondly, constructing recombinant bacteria YPV-010
The template is replaced by the recombinant bacterium YPV-007 to the Corynebacterium glutamicum ATCC15168, and the steps are the same as the steps.
The recombinant strain YPV-010 is obtained. The recombinant strain YPV-010 is an engineering strain for overexpressing YH66-RS07015 gene by plasmid. Compared with the recombinant bacterium YPV-011, the recombinant bacterium YPV-010 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-RS07015 Gene on genome
P19：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTAGGCGGCAAAAACGCGCGC-3'；
P20：5'-CATTCTTTTTCTAGCCTTCCGGAACTCACCGTCCTTACAG-3'；
P21：5'-CTGTAAGGACGGTGAGTTCCGGAAGGCTAGAAAAAGAATG-3'；
P22：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAAATGCACCCCGCGACAATG-3'。
P23：5'-TAGGCGGCAAAAACGCGCGC-3'；
P24：5'-AAATGCACCCCGCGACAATG-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, 764bp) was recovered.
2. The Corynebacterium glutamicum ATCC15168 was used as a template, and a primer pair consisting of a primer P21 and a primer P22 was used for PCR amplification to recover an amplification product (downstream homology arm fragment, 724 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 P19 and a primer P22, and the amplification product (1448bp) 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 07015. Through sequencing, the recombinant plasmid pK 18-delta YH66-RS07015 has a DNA molecule shown in a sequence 8 in a sequence table.
Secondly, constructing recombinant bacteria YPV-012
1. The recombinant plasmid pK 18-delta YH66-RS07015 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 (a primer pair consisting of a primer P23 and a primer P24 is adopted). The strain capable of simultaneously amplifying 1374bp and 2820bp bands is a positive strain. The strain only amplifying the 2820bp band is a failed-transformation starter, wherein the 2820bp 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 which only shows one amplification product and has the size of 1374bp is a positive strain with the YH66-RS07015 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 P23 and a primer P24 again, and naming the strains with correct sequencing as recombinant bacteria YPV-012. Compared with the genome DNA of Corynebacterium glutamicum CGMCC21260, the recombinant bacterium YPV-012 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-007, recombinant bacteria YPV-008, recombinant bacteria YPV-009, recombinant bacteria YPV-010, recombinant bacteria YPV-011 and recombinant bacteria YPV-012.
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-007 and YPV-012 is obviously higher than that of Corynebacterium glutamicum CGMCC 21260. The results show that the suppression of YH66-RS07015 gene expression can improve L-valine yield, and the overexpression of YH66-RS07015 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
98.2
82.1
Recombinant bacterium YPV-007
98.9
85.8
Recombinant bacterium YPV-008
97.6
80.7
Recombinant bacterium YPV-009
97.8
80.4
Recombinant bacterium YPV-010
97.2
79.5
Recombinant bacterium YPV-011
97.4
80.5
Recombinant bacterium YPV-012
99.5
85.2
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 gene modification of YH66-RS07015 and application of engineering bacterium in preparation of valine
<130> GNCYX211993
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 481
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu
1 5 10 15
Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly
20 25 30
Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr
35 40 45
Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg
50 55 60
His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu
65 70 75 80
Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg
85 90 95
Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg
100 105 110
Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly
115 120 125
Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp
130 135 140
Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile
145 150 155 160
Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu
165 170 175
Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro
180 185 190
Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly
195 200 205
Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile
210 215 220
Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile
225 230 235 240
Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe
245 250 255
Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp
260 265 270
Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr
275 280 285
Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile
290 295 300
Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val
305 310 315 320
Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu
325 330 335
Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp
340 345 350
Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile
355 360 365
Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala
370 375 380
Asp Gly Met Arg Met Met Val Val Pro Ser Ser Ile Trp Ile Lys Gln
385 390 395 400
Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala
405 410 415
Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp
420 425 430
Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe
435 440 445
Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala
450 455 460
Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp
465 470 475 480
Ile
<210> 2
<211> 1446
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60
tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120
ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180
cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240
ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300
accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360
cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420
gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480
ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540
accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600
ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660
ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720
gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780
ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840
ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900
accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960
ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020
tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080
ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140
cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccatctg gatcaagcaa 1200
gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260
gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320
gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380
gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440
atctaa 1446
<210> 3
<211> 481
<212> PRT
<213> Corynebacterium glutamicum
<400> 3
Met Thr Ser Pro Val Glu Asn Ser Thr Ser Thr Glu Lys Leu Thr Leu
1 5 10 15
Ala Glu Lys Val Trp Arg Asp His Val Val Ser Lys Gly Glu Asn Gly
20 25 30
Glu Pro Asp Leu Leu Tyr Ile Asp Leu Gln Leu Leu His Glu Val Thr
35 40 45
Ser Pro Gln Ala Phe Asp Gly Leu Arg Met Thr Gly Arg Lys Leu Arg
50 55 60
His Pro Glu Leu His Leu Ala Thr Glu Asp His Asn Val Pro Thr Glu
65 70 75 80
Gly Ile Lys Thr Gly Ser Leu Leu Glu Ile Asn Asp Gln Ile Ser Arg
85 90 95
Leu Gln Val Ser Thr Leu Arg Asp Asn Cys Glu Glu Phe Gly Val Arg
100 105 110
Leu His Pro Met Gly Asp Val Arg Gln Gly Ile Val His Thr Val Gly
115 120 125
Pro Gln Leu Gly Ala Thr Gln Pro Gly Met Thr Ile Val Cys Gly Asp
130 135 140
Ser His Thr Ser Thr His Gly Ala Phe Gly Ser Met Ala Phe Gly Ile
145 150 155 160
Gly Thr Ser Glu Val Glu His Val Met Ala Thr Gln Thr Leu Pro Leu
165 170 175
Lys Pro Phe Lys Thr Met Ala Ile Glu Val Thr Gly Glu Leu Gln Pro
180 185 190
Gly Val Ser Ser Lys Asp Leu Ile Leu Ala Ile Ile Ala Lys Ile Gly
195 200 205
Thr Gly Gly Gly Gln Gly Tyr Val Leu Glu Tyr Arg Gly Glu Ala Ile
210 215 220
Arg Lys Met Ser Met Asp Ala Arg Met Thr Met Cys Asn Met Ser Ile
225 230 235 240
Glu Ala Gly Ala Arg Ala Gly Met Ile Ala Pro Asp Gln Thr Thr Phe
245 250 255
Asp Tyr Val Glu Gly Arg Glu Met Ala Pro Lys Gly Ala Asp Trp Asp
260 265 270
Glu Ala Val Ala Tyr Trp Lys Thr Leu Pro Thr Asp Glu Gly Ala Thr
275 280 285
Phe Asp Lys Val Val Glu Ile Asp Gly Ser Ala Leu Thr Pro Phe Ile
290 295 300
Thr Trp Gly Thr Asn Pro Gly Gln Gly Leu Pro Leu Ser Glu Thr Val
305 310 315 320
Pro Asn Pro Glu Asp Phe Thr Asn Asp Asn Asp Lys Ala Ala Ala Glu
325 330 335
Lys Ala Leu Gln Tyr Met Asp Leu Val Pro Gly Thr Pro Leu Arg Asp
340 345 350
Ile Lys Ile Asp Thr Val Phe Leu Gly Ser Cys Thr Asn Ala Arg Ile
355 360 365
Glu Asp Leu Gln Ile Ala Ala Asp Ile Leu Lys Gly His Lys Ile Ala
370 375 380
Asp Gly Met Arg Met Met Val Val Pro Ser Ser Thr Trp Ile Lys Gln
385 390 395 400
Glu Ala Glu Ala Leu Gly Leu Asp Lys Ile Phe Thr Asp Ala Gly Ala
405 410 415
Glu Trp Arg Thr Ala Gly Cys Ser Met Cys Leu Gly Met Asn Pro Asp
420 425 430
Gln Leu Lys Pro Gly Glu Arg Ser Ala Ser Thr Ser Asn Arg Asn Phe
435 440 445
Glu Gly Arg Gln Gly Pro Gly Gly Arg Thr His Leu Val Ser Pro Ala
450 455 460
Val Ala Ala Ala Thr Ala Ile Arg Gly Thr Leu Ser Ser Pro Ala Asp
465 470 475 480
Ile
<210> 4
<211> 1446
<212> DNA
<213> Corynebacterium glutamicum
<400> 4
atgaccagcc ccgtggagaa cagcacctca actgagaagc tgaccctggc agagaaggtg 60
tggcgcgacc atgtcgtgtc caagggagaa aacggcgagc ccgacctcct ctacatcgac 120
ctgcagctgc tgcatgaagt gacctcacca caggcattcg acggcctgcg catgactggc 180
cgcaaactgc gccacccaga actgcacctg gccaccgaag accacaacgt gccaaccgaa 240
ggcatcaaga ctggctcact gctggaaatc aacgaccaga tttcccgcct gcaggtatcc 300
accctgcgcg acaactgtga agagttcggt gttcgcctgc acccaatggg tgatgtccgc 360
cagggcatcg tgcacaccgt tggcccacag ctgggcgcaa ctcagccggg catgaccatt 420
gtgtgcggtg actcccacac ctctactcac ggcgcgtttg gctccatggc attcggtatc 480
ggtacctctg aggttgagca cgtcatggcc actcagaccc tgccattgaa gcctttcaag 540
accatggcca ttgaagttac tggcgaactg cagccaggtg tttcctccaa ggacctgatc 600
ctggcgatca ttgccaagat cggcaccggt ggtggacaag gctacgttct ggaataccgc 660
ggcgaagcaa tccgcaagat gtccatggat gcacgcatga ccatgtgcaa catgtccatc 720
gaagctggcg cacgtgccgg catgatcgcc ccagaccaaa ccaccttcga ctacgttgaa 780
ggccgcgaaa tggcaccaaa gggcgccgac tgggacgaag cagttgctta ctggaagacc 840
ctgccaaccg acgaaggcgc aacctttgac aaggtcgtag aaatcgatgg ctccgcactg 900
accccattca tcacctgggg caccaaccca ggccaaggtc tgccactgag cgaaaccgtg 960
ccaaacccag aagacttcac caacgacaac gacaaggcag cagccgaaaa ggcactgcag 1020
tacatggacc tggtaccagg aaccccactg cgcgacatca agatcgacac cgtcttcctg 1080
ggatcctgca ccaacgcccg catcgaagac ctgcagatcg ccgctgacat cctcaagggc 1140
cacaaaatcg ccgacggcat gcgcatgatg gtcgtgcctt cctccacctg gatcaagcaa 1200
gaggccgaag cactcggact ggacaaaatc ttcaccgacg ctggcgctga atggcgtacc 1260
gcaggctgct ccatgtgcct gggcatgaac ccagaccaac tgaagccagg cgagcgctct 1320
gcatccacct ccaaccgaaa cttcgaagga cgccaaggac caggaggccg cacccacctg 1380
gtatccccag cagtcgcagc cgccaccgca atccgcggca ccctgtcctc acctgcagat 1440
atctaa 1446
<210> 5
<211> 1214
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctaggcca agatcggcac cggtggtgga 60
caaggctacg ttctggaata ccgcggcgaa gcaatccgca agatgtccat ggatgcacgc 120
atgaccatgt gcaacatgtc catcgaagct ggcgcacgtg ccggcatgat cgccccagac 180
caaaccacct tcgactacgt tgaaggccgc gaaatggcac caaagggcgc cgactgggac 240
gaagcagttg cttactggaa gaccctgcca accgacgaag gcgcaacctt tgacaaggtc 300
gtagaaatcg atggctccgc actgacccca ttcatcacct ggggcaccaa cccaggccaa 360
ggtctgccac tgagcgaaac cgtgccaaac ccagaagact tcaccaacga caacgacaag 420
gcagcagccg aaaaggcact gcagtacatg gacctggtac caggaacccc actgcgcgac 480
atcaagatcg acaccgtctt cctgggatcc tgcaccaacg cccgcatcga agacctgcag 540
atcgccgctg acatcctcaa gggccacaaa atcgccgacg gcatgcgcat gatggtcgtg 600
ccttcctcca tctggatcaa gcaagaggcc gaagcactcg gactggacaa aatcttcacc 660
gacgctggcg ctgaatggcg taccgcaggc tgctccatgt gcctgggcat gaacccagac 720
caactgaagc caggcgagcg ctctgcatcc acctccaacc gaaacttcga aggacgccaa 780
ggaccaggag gccgcaccca cctggtatcc ccagcagtcg cagccgccac cgcaatccgc 840
ggcaccctgt cctcacctgc agatatctaa ggaaggctag aaaaagaatg gaaaaattta 900
ccacccacac cggcgttggc gttccactgc agcgatccaa cgtggacacc gaccagatca 960
tccccgccgt ctacctcaag cgcgtcaccc gcacaggctt cgaagacgga ctgttttcca 1020
actggcgcca aaacgacccc aactttgtcc tcaacaccga cacctacaag aacggctccg 1080
ttctcgtagc aggccctgac tttggcaccg gctcctcccg cgagcacgcc gtctgggcac 1140
tcatggacta cggcttccgc gctgtcttct cctcacgggt accgagctcg aattcgtaat 1200
catggtcata gctg 1214
<210> 6
<211> 3248
<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 tgaccagccc cgtggagaac agcacctcaa ctgagaagct 1080
gaccctggca gagaaggtgt ggcgcgacca tgtcgtgtcc aagggagaaa acggcgagcc 1140
cgacctcctc tacatcgacc tgcagctgct gcatgaagtg acctcaccac aggcattcga 1200
cggcctgcgc atgactggcc gcaaactgcg ccacccagaa ctgcacctgg ccaccgaaga 1260
ccacaacgtg ccaaccgaag gcatcaagac tggctcactg ctggaaatca acgaccagat 1320
ttcccgcctg caggtatcca ccctgcgcga caactgtgaa gagttcggtg ttcgcctgca 1380
cccaatgggt gatgtccgcc agggcatcgt gcacaccgtt ggcccacagc tgggcgcaac 1440
tcagccgggc atgaccattg tgtgcggtga ctcccacacc tctactcacg gcgcgtttgg 1500
ctccatggca ttcggtatcg gtacctctga ggttgagcac gtcatggcca ctcagaccct 1560
gccattgaag cctttcaaga ccatggccat tgaagttact ggcgaactgc agccaggtgt 1620
ttcctccaag gacctgatcc tggcgatcat tgccaagatc ggcaccggtg gtggacaagg 1680
ctacgttctg gaataccgcg gcgaagcaat ccgcaagatg tccatggatg cacgcatgac 1740
catgtgcaac atgtccatcg aagctggcgc acgtgccggc atgatcgccc cagaccaaac 1800
caccttcgac tacgttgaag gccgcgaaat ggcaccaaag ggcgccgact gggacgaagc 1860
agttgcttac tggaagaccc tgccaaccga cgaaggcgca acctttgaca aggtcgtaga 1920
aatcgatggc tccgcactga ccccattcat cacctggggc accaacccag gccaaggtct 1980
gccactgagc gaaaccgtgc caaacccaga agacttcacc aacgacaacg acaaggcagc 2040
agccgaaaag gcactgcagt acatggacct ggtaccagga accccactgc gcgacatcaa 2100
gatcgacacc gtcttcctgg gatcctgcac caacgcccgc atcgaagacc tgcagatcgc 2160
cgctgacatc ctcaagggcc acaaaatcgc cgacggcatg cgcatgatgg tcgtgccttc 2220
ctccatctgg atcaagcaag aggccgaagc actcggactg gacaaaatct tcaccgacgc 2280
tggcgctgaa tggcgtaccg caggctgctc catgtgcctg ggcatgaacc cagaccaact 2340
gaagccaggc gagcgctctg catccacctc caaccgaaac ttcgaaggac gccaaggacc 2400
aggaggccgc acccacctgg tatccccagc agtcgcagcc gccaccgcaa tccgcggcac 2460
cctgtcctca cctgcagata tctaataaat cgactactca catagggtcg ggctagtcat 2520
tctgatcagc gaattccacg ttcacatcgc caattccaga gttcacaacc agattcagca 2580
ttggaccttc tagatcagca ttgtgggcgg tgagatctcc aacatcacag cgcgctgtgc 2640
ccacaccggc ggtacaactt aggctcacgg gcacatcatc gggcagggtg accatgactt 2700
cgccgatccc tgaggtgatt tggatgtttt gttcctgatc caattgggtg aggtggctga 2760
aatcgaggtt catttcaccc acgccagagg tgtagctgct gaggagttca tcgttggtgg 2820
ggatgagatt gacatcgccg attccagggt cgtcttcaaa gtagatggga tcgatatttg 2880
aaataaacag gcctgcgagg gcgctcatga caactccggt accaactaca ccgccgacaa 2940
tccatggcca cacatggcgc tttttctgag gcttttgtgg agggacttgt acatcccagg 3000
tgttgtattg gttttgggca agtggatccc aatgaggcgc ttcgggggtt tgttgcgcga 3060
agggtgcata gtagccctca acgggggtga tagtgcttag atctggttgg ggttgtgggt 3120
agagatcttc gtttttcatg gtggcatcct cagaaacagt gaattcagtg gtgagtagtc 3180
cgcggggtgg aagtggttgt ttcttatgca gggtaccgag ctcgaattcg taatcatggt 3240
catagctg 3248
<210> 7
<211> 1769
<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 tgaccagccc 300
cgtggagaac agcacctcaa ctgagaagct gaccctggca gagaaggtgt ggcgcgacca 360
tgtcgtgtcc aagggagaaa acggcgagcc cgacctcctc tacatcgacc tgcagctgct 420
gcatgaagtg acctcaccac aggcattcga cggcctgcgc atgactggcc gcaaactgcg 480
ccacccagaa ctgcacctgg ccaccgaaga ccacaacgtg ccaaccgaag gcatcaagac 540
tggctcactg ctggaaatca acgaccagat ttcccgcctg caggtatcca ccctgcgcga 600
caactgtgaa gagttcggtg ttcgcctgca cccaatgggt gatgtccgcc agggcatcgt 660
gcacaccgtt ggcccacagc tgggcgcaac tcagccgggc atgaccattg tgtgcggtga 720
ctcccacacc tctactcacg gcgcgtttgg ctccatggca ttcggtatcg gtacctctga 780
ggttgagcac gtcatggcca ctcagaccct gccattgaag cctttcaaga ccatggccat 840
tgaagttact ggcgaactgc agccaggtgt ttcctccaag gacctgatcc tggcgatcat 900
tgccaagatc ggcaccggtg gtggacaagg ctacgttctg gaataccgcg gcgaagcaat 960
ccgcaagatg tccatggatg cacgcatgac catgtgcaac atgtccatcg aagctggcgc 1020
acgtgccggc atgatcgccc cagaccaaac caccttcgac tacgttgaag gccgcgaaat 1080
ggcaccaaag ggcgccgact gggacgaagc agttgcttac tggaagaccc tgccaaccga 1140
cgaaggcgca acctttgaca aggtcgtaga aatcgatggc tccgcactga ccccattcat 1200
cacctggggc accaacccag gccaaggtct gccactgagc gaaaccgtgc caaacccaga 1260
agacttcacc aacgacaacg acaaggcagc agccgaaaag gcactgcagt acatggacct 1320
ggtaccagga accccactgc gcgacatcaa gatcgacacc gtcttcctgg gatcctgcac 1380
caacgcccgc atcgaagacc tgcagatcgc cgctgacatc ctcaagggcc acaaaatcgc 1440
cgacggcatg cgcatgatgg tcgtgccttc ctccatctgg atcaagcaag aggccgaagc 1500
actcggactg gacaaaatct tcaccgacgc tggcgctgaa tggcgtaccg caggctgctc 1560
catgtgcctg ggcatgaacc cagaccaact gaagccaggc gagcgctctg catccacctc 1620
caaccgaaac ttcgaaggac gccaaggacc aggaggccgc acccacctgg tatccccagc 1680
agtcgcagcc gccaccgcaa tccgcggcac cctgtcctca cctgcagata tctaagtttt 1740
ggcggatgag agaagatttt cagcctgat 1769
<210> 8
<211> 1448
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cagtgccaag cttgcatgcc tgcaggtcga ctctagtagg cggcaaaaac gcgcgctgct 60
gaccctgcat ttaaaggcat gcgagtgccc acgggaacca cgttttttag cccggagctg 120
ggctcttggc tggccacaca cgtgcgggtg gtgccggtga ggcgataaag ctgaacggat 180
tcgccggtgc gctccataag gtcggccata attggtacgg ccgtatcgat gagggtgtca 240
gcgccgcgtg cacccaatga ggcaagccgt gcgccgatgg tccatctatt atcgcgggag 300
cgtgccaaca tgccgtgtac ctcaagcgct gaggcgaggc ggtgggctgt agccctgggc 360
agatcggtgg cagctgcgag ctctgccaac gatcgaggct gttctgcgat gacattgagg 420
attaatacag tgcggtctaa aaccttaata ccgctctcgg tggagtcctc gataatttct 480
tgctgtccca ttctttgaat cttacatttc atagagtgag acgcttgcag gttggggttt 540
aaacgttgtg gatatcgatt ccctgcaggg gagctgtata aagtgtgagg taaatctaaa 600
acgcaggacg tgacattttt ggcgcgtttt aggttatact gtctcagaca acgaaactct 660
tgtcccacat tgtgagattt gcttgctaga atgtgggcta gaaattcctg aaaattttta 720
cgcactgtaa ggacggtgag ttccggaagg ctagaaaaag aatggaaaaa tttaccaccc 780
acaccggcgt tggcgttcca ctgcagcgat ccaacgtgga caccgaccag atcatccccg 840
ccgtctacct caagcgcgtc acccgcacag gcttcgaaga cggactgttt tccaactggc 900
gccaaaacga ccccaacttt gtcctcaaca ccgacaccta caagaacggc tccgttctcg 960
tagcaggccc tgactttggc accggctcct cccgcgagca cgccgtctgg gcactcatgg 1020
actacggctt ccgcgctgtc ttctcctcac gattcgccga catcttccgc ggcaactccg 1080
gaaaagctgg catgctcgcc ggcatcatgg aacagtccga catcgaactt ctgtggaagc 1140
tcatggaaca aaccccgggc ctcgaactga ccgtgaacct ggaaaagcag atcgtcactg 1200
caggcgacgt agtgatcagc ttcgaagttg acccttacat tcgctggcgt ttgatggaag 1260
gcctcgacga cgctggcctg accctgcgca agctcgatga aattgaagac tacgaggcta 1320
agcgccctgc gtttaagcca cgcactaacg cttaagtttc agtctgatag cgaaagcacc 1380
ccgcaacctt cattgtcgcg gggtgcattt gggtaccgag ctcgaattcg taatcatggt 1440
catagctg 1448
<210> 9
<211> 2820
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
taggcggcaa aaacgcgcgc tgctgaccct gcatttaaag gcatgcgagt gcccacggga 60
accacgtttt ttagcccgga gctgggctct tggctggcca cacacgtgcg ggtggtgccg 120
gtgaggcgat aaagctgaac ggattcgccg gtgcgctcca taaggtcggc cataattggt 180
acggccgtat cgatgagggt gtcagcgccg cgtgcaccca atgaggcaag ccgtgcgccg 240
atggtccatc tattatcgcg ggagcgtgcc aacatgccgt gtacctcaag cgctgaggcg 300
aggcggtggg ctgtagccct gggcagatcg gtggcagctg cgagctctgc caacgatcga 360
ggctgttctg cgatgacatt gaggattaat acagtgcggt ctaaaacctt aataccgctc 420
tcggtggagt cctcgataat ttcttgctgt cccattcttt gaatcttaca tttcatagag 480
tgagacgctt gcaggttggg gtttaaacgt tgtggatatc gattccctgc aggggagctg 540
tataaagtgt gaggtaaatc taaaacgcag gacgtgacat ttttggcgcg ttttaggtta 600
tactgtctca gacaacgaaa ctcttgtccc acattgtgag atttgcttgc tagaatgtgg 660
gctagaaatt cctgaaaatt tttacgcact gtaaggacgg tgagttccat gaccagcccc 720
gtggagaaca gcacctcaac tgagaagctg accctggcag agaaggtgtg gcgcgaccat 780
gtcgtgtcca agggagaaaa cggcgagccc gacctcctct acatcgacct gcagctgctg 840
catgaagtga cctcaccaca ggcattcgac ggcctgcgca tgactggccg caaactgcgc 900
cacccagaac tgcacctggc caccgaagac cacaacgtgc caaccgaagg catcaagact 960
ggctcactgc tggaaatcaa cgaccagatt tcccgcctgc aggtatccac cctgcgcgac 1020
aactgtgaag agttcggtgt tcgcctgcac ccaatgggtg atgtccgcca gggcatcgtg 1080
cacaccgttg gcccacagct gggcgcaact cagccgggca tgaccattgt gtgcggtgac 1140
tcccacacct ctactcacgg cgcgtttggc tccatggcat tcggtatcgg tacctctgag 1200
gttgagcacg tcatggccac tcagaccctg ccattgaagc ctttcaagac catggccatt 1260
gaagttactg gcgaactgca gccaggtgtt tcctccaagg acctgatcct ggcgatcatt 1320
gccaagatcg gcaccggtgg tggacaaggc tacgttctgg aataccgcgg cgaagcaatc 1380
cgcaagatgt ccatggatgc acgcatgacc atgtgcaaca tgtccatcga agctggcgca 1440
cgtgccggca tgatcgcccc agaccaaacc accttcgact acgttgaagg ccgcgaaatg 1500
gcaccaaagg gcgccgactg ggacgaagca gttgcttact ggaagaccct gccaaccgac 1560
gaaggcgcaa cctttgacaa ggtcgtagaa atcgatggct ccgcactgac cccattcatc 1620
acctggggca ccaacccagg ccaaggtctg ccactgagcg aaaccgtgcc aaacccagaa 1680
gacttcacca acgacaacga caaggcagca gccgaaaagg cactgcagta catggacctg 1740
gtaccaggaa ccccactgcg cgacatcaag atcgacaccg tcttcctggg atcctgcacc 1800
aacgcccgca tcgaagacct gcagatcgcc gctgacatcc tcaagggcca caaaatcgcc 1860
gacggcatgc gcatgatggt cgtgccttcc tccacctgga tcaagcaaga ggccgaagca 1920
ctcggactgg acaaaatctt caccgacgct ggcgctgaat ggcgtaccgc aggctgctcc 1980
atgtgcctgg gcatgaaccc agaccaactg aagccaggcg agcgctctgc atccacctcc 2040
aaccgaaact tcgaaggacg ccaaggacca ggaggccgca cccacctggt atccccagca 2100
gtcgcagccg ccaccgcaat ccgcggcacc ctgtcctcac ctgcagatat ctaaggaagg 2160
ctagaaaaag aatggaaaaa tttaccaccc acaccggcgt tggcgttcca ctgcagcgat 2220
ccaacgtgga caccgaccag atcatccccg ccgtctacct caagcgcgtc acccgcacag 2280
gcttcgaaga cggactgttt tccaactggc gccaaaacga ccccaacttt gtcctcaaca 2340
ccgacaccta caagaacggc tccgttctcg tagcaggccc tgactttggc accggctcct 2400
cccgcgagca cgccgtctgg gcactcatgg actacggctt ccgcgctgtc ttctcctcac 2460
gattcgccga catcttccgc ggcaactccg gaaaagctgg catgctcgcc ggcatcatgg 2520
aacagtccga catcgaactt ctgtggaagc tcatggaaca aaccccgggc ctcgaactga 2580
ccgtgaacct ggaaaagcag atcgtcactg caggcgacgt agtgatcagc ttcgaagttg 2640
acccttacat tcgctggcgt ttgatggaag gcctcgacga cgctggcctg accctgcgca 2700
agctcgatga aattgaagac tacgaggcta agcgccctgc gtttaagcca cgcactaacg 2760
cttaagtttc agtctgatag cgaaagcacc ccgcaacctt cattgtcgcg gggtgcattt 2820
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