CN114540399A - A kind of method for preparing L-valine and its used gene mutant and biological material - Google Patents

Abstract

The invention discloses a method for preparing L-valine and a gene mutant and a biological material used by the method. Specifically disclosed is a mutein CEY17_ RS01410^A245TAnd a gene encoding the same. The invention constructs a genetically engineered bacterium containing a point mutation (G-A) and overexpresses a CEY17_ RS01410 gene or CEY17_ RS01410 gene^G733AEngineering bacteria of genes. Experiments show that the CEY17_ RS01410 gene and variants thereof are involved in L-valine biosynthesis, and the gene is CEY1The point mutation of the 7_ RS01410 gene coding region or the overexpression of the CEY17_ RS01410 gene and/or a mutant thereof in a producing strain is beneficial to the improvement of the yield and the conversion rate of L-valine, can culture high-yield and high-quality strains meeting the industrial production, and has important significance for the industrial production of L-valine.

Description

A kind of method for preparing L-valine and its used gene mutant and biological material

technical field

The invention belongs to the technical field of microbial variation or genetic engineering, and in particular relates to a method for preparing L-valine and a gene mutant and biological material used therefor.

Background technique

The production methods of L-valine mainly include extraction method, chemical synthesis method and fermentation method. The extraction method and chemical synthesis method are difficult to realize industrialized production due to the limited source of raw materials, high production cost, low yield and serious pollution. Microbial direct fermentation to produce L-valine has the advantages of wide source of raw materials, low cost, mild reaction conditions, and easy large-scale production. It is a very economical and efficient production method. The high-yielding strains obtained in industrial fermentation are crucial to the fermentation production of L-valine, the core of the entire L-valine fermentation industry, and an important factor determining the industrial value of fermented products. With the increasing market demand for L-valine, breeding high-yield and stable production strains, promoting the accumulation of L-valine in microorganisms, and further improving the production of L-valine has always been the focus of L-valine. The hot spot of acid fermentation industrial technology development and fermentation engineering research is of great significance to promote the process of L-valine industrialization.

L-Valine (L-Valine), also known as L-2-Amino-3-methylbutyric acid (L-2-Amino-3-methylbutyricacid), is a branched chain amino acid (Branched Chain Amino Acid, BCAA). It is a kind of human and animals that cannot be synthesized by themselves. L-valine is one of the eight essential amino acids in the human body. It can promote protein synthesis, inhibit protein decomposition, enhance the body's immune protection, and help correct negative nitrogen balance caused by surgery, trauma, infection, etc. In addition, L-valine also has the effect of anti-central fatigue and anti-peripheral fatigue, which can delay exercise fatigue and speed up the repair of the body after exercise. The lack of L-valine in the human body will affect the growth and development of the body, causing neurological disorders, ataxia, anemia, etc. The compound branched-chain amino acid infusion prepared by L-valine is used in the treatment of blood-brain barrier, hepatic coma, chronic liver cirrhosis and renal failure, dietary treatment of congenital metabolic defects, treatment of sepsis and postoperative diabetic patients, to speed up It is widely used in the treatment of surgical wound healing and nutritional support for tumor patients. Because of its special structure and function, L-valine occupies a particularly important position in the metabolism of human life. It is mainly used in food industry as food additives, nutritional additives, feed additives, etc. The market demand is large, making L-valine The production of acid is of great concern.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve the yield of microbial L-valine, the technical problem to be solved is not limited to the described technical subject, and those skilled in the art can clearly understand other technologies not mentioned in this paper through the following description theme.

In order to solve the above-mentioned technical problems, the present invention first provides a method for preparing L-valine, the method comprising using a recombinant microorganism expressing protein to produce L-valine;

The protein name is CEY17_RS01410 ^A245T , which can be any of the following:

A1) the amino acid sequence is the protein of SEQ ID No.4;

A2) A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.4;

A3) A fusion protein with the same function obtained by linking a tag to the N-terminus and/or C-terminus of A1) or A2).

In the above method, the method may be fermentation to prepare L-valine, and the recombinant microorganism may be Corynebacterium, specifically Corynebacterium glutamicum and its variants.

Specifically, the recombinant microorganism can be recombinant bacteria YPV-085, YPV-086, YPV-087, YPV-088 and/or YPV-089.

The protein CEY17_RS01410 ^{A245T is} also within the protection scope of the present invention.

The present invention also provides a nucleic acid molecule named CEY17_RS01410 ^G733A , and the nucleic acid molecule can be any of the following:

B1) a nucleic acid molecule encoding the protein CEY17_RS01410 ^A245T ;

B2) the coding sequence is the DNA molecule shown in SEQ ID No.3;

B3) The nucleotide sequence is the DNA molecule shown in SEQ ID No.3.

The DNA molecule shown in SEQ ID No. 3 is also the CEY17_RS01410 ^G733A gene described in the present invention.

The DNA molecule shown in SEQ ID No. 3 ( ^{CEY17_RS01410G733A} ) encodes the protein ^{CEY17_RS01410A245T} shown in SEQ ID No.4.

The threonine (T) at position 245 in the amino acid sequence (SEQ ID No. 4) of the protein CEY17_RS01410 ^A245T is mutated from alanine (A).

The present invention also provides biological materials, and the biological materials can be any of the following:

C1) an expression cassette containing the nucleic acid molecule CEY17_RS01410 ^G733A ;

C2) a recombinant vector containing the nucleic acid molecule CEY17_RS01410 ^G733A or a recombinant vector containing the expression cassette of C1);

C3) a recombinant microorganism containing the nucleic acid molecule CEY17_RS01410 ^G733A , or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2).

The present invention also provides any one of the following applications of any one of D1)-D8):

F1) the application of any one of D1)-D8) in regulating the production of L-valine of microorganisms;

F2) the application of any one of D1)-D8) in the construction of L-valine-producing genetically engineered bacteria;

F3) the application of any one of D1)-D8) in the preparation of L-valine;

Wherein, described D1)-D8) are:

D1) the protein CEY17_RS01410 ^A245T ;

D2) the nucleic acid molecule CEY17_RS01410 ^G733A ;

D3) the biological material;

D4) DNA molecule whose nucleotide sequence is SEQ ID No.1;

D5) The nucleotide sequence shown in SEQ ID No.1 is modified and/or one or several nucleotides are substituted and/or deleted and/or added to obtain the DNA molecule shown in SEQ ID No.1. DNA molecules that are more than 90% identical and have the same function;

D6) an expression cassette comprising the DNA molecule described in D4) or D5);

D7) a recombinant vector containing the DNA molecule described in D4) or D5), or a recombinant vector containing the expression cassette described in D6);

D8) a recombinant microorganism containing the DNA molecule described in D4) or D5), or a recombinant microorganism containing the expression cassette described in D6), or a recombinant microorganism containing the recombinant vector described in D7).

The DNA molecule shown in SEQ ID No. 1 is also the CEY17_RS01410 gene of the present invention.

The DNA molecule shown in SEQ ID No. 1 (CEY17_RS01410 gene) encodes the protein shown in SEQ ID No. 2.

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

Herein, the identity of more than 80% may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the above 90% identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

Modulating the production of L-valine in a microorganism as described herein may increase or decrease the amount of L-valine accumulated in the microorganism (ie, promote or inhibit L-valine biosynthesis).

The present invention also provides a method for improving the output of microorganism L-valine, the method comprising any of the following:

E1) improve the expression amount or content of the nucleic acid molecule CEY17_RS01410 ^G733A in the target microorganism, obtain a microorganism whose output of L-valine is higher than the target microorganism;

E2) improve the expression amount or content of the DNA molecule described in D4) or D5) in the target microorganism, and obtain a microorganism whose output of L-valine is higher than that of the target microorganism;

E3) mutating the DNA molecule whose nucleotide sequence is SEQ ID No. 1 in the target microorganism (such as base substitution, base insertion or base deletion), to obtain a higher yield of L-valine than that of the target microorganism Microorganisms that describe the microorganisms of interest.

In the above method, the mutation may be a point mutation, ie a mutation of a single nucleotide.

In the above method, the point mutation may be to mutate the alanine residue at position 245 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to another amino acid residue.

In the above method, the point mutation may be to mutate the alanine residue at position 245 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to a threonine residue.

In the above method, the point mutation may be nucleic acid modification of the guanine (G) at position 733 of the CEY17_RS01410 gene (SEQ ID No. 1).

In the above method, the point mutation may be a mutation of guanine (G) at position 733 in the DNA molecule shown in SEQ ID No. 1 to adenine (A) by a site-directed mutagenesis method.

The mutation refers to changing one or several bases in a gene through site-directed mutation, resulting in a change in the amino acid composition of the corresponding protein, resulting in a new protein or a new function of the original protein, that is, gene site-directed mutation. Gene site-directed mutagenesis techniques such as oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis or cassette mutagenesis are well known to those skilled in the art.

The point mutation described herein can be a single-base substitution, a single-base insertion or a single-base deletion, specifically a single-base substitution. The single base substitution can be an allelic substitution.

The vectors described herein are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (eg, lambda phage or M13 filamentous phage, etc.), cosmids (ie, cosmids), or viral vectors. Specifically, it can be pK18mobsacB or pXMJ19.

Herein, the microorganism may be yeast, bacteria, algae or fungi. Wherein, the bacteria can be from Brevibacterium, Corynebacterium, Escherichia, Aerobacter, Micrococcus, Flavobacterium or Bacillus Bacillus, etc.

Specifically, the microorganism can be Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Micrococcus glutamicus, Brevibacterium ammoniagenes (Brevibacterum ammoniagenes), Escherichia coli or Aerobacter aerogenes but not limited thereto.

Specifically, the microorganism may be Corynebacterium glutamicum CGMCCNo.21260, or Corynebacterium glutamicum ATCC14067.

Herein, the recombinant vector may specifically be the recombinant vector pK18- ^{CEY17_RS01410G733A} , pK18-CEY17_RS01410OE, ^pK18 -CEY17_RS01410G733AOE, pXMJ19-CEY17_RS01410 and/or pXMJ19- ^{CEY17_RS01410G733A} .

Described recombinant vector pK18-CEY17_RS01410 ^G733A is to replace the fragment (small fragment) between the Xbal I and BamH I recognition sites of pK18mobsacB vector with the DNA fragment shown in the 37th-1266th position of SEQ ID No.5 in the sequence listing, Keep the other sequences of the pK18mobsacB vector unchanged to obtain a recombinant vector. The recombinant vector pK18-CEY17_RS01410 ^G733A contains the DNA molecule shown in positions 128-1281 of the mutant gene CEY17_RS01410 ^G733A shown in SEQ ID No. 3.

The recombinant vector pK18-CEY17_RS01410OE is used to integrate the exogenous gene CEY17_RS01410 into the host chromosome, and overexpress the wild-type CEY17_RS01410 gene in the production bacteria.

The recombinant vector pK18-CEY17_RS01410 ^G733A OE is used to integrate the exogenous gene CEY17_RS01410 ^G733A into the host chromosome, and overexpress the mutant gene CEY17_RS01410 ^G733A in the production bacteria.

The recombinant vector pXMJ19-CEY17_RS01410 is a fragment (small fragment) between the EcoR I and KpnI recognition sites of the pXMJ19 vector is replaced with a DNA fragment whose nucleotide sequence is SEQ ID No. 13 in the sequence listing, and other parts of the pXMJ19 vector are maintained The sequence is unchanged, and the obtained recombinant expression vector. The recombinant vector pXMJ19-CEY17_RS01410 is used to express the exogenous gene CEY17_RS01410 extrachromosomally through a plasmid, and then overexpress the wild-type CEY17_RS01410 gene in the production bacteria.

The recombinant vector pXMJ19-CEY17_RS01410 ^G733A is a fragment (small fragment) between the EcoR I and KpnI recognition sites of the pXMJ19 vector is replaced with a DNA fragment whose nucleotide sequence is SEQ ID No. 14 in the sequence listing, maintaining the pXMJ19 vector. Other sequences remain unchanged, and the obtained recombinant expression vector. The recombinant vector pXMJ19-CEY17_RS01410 ^G733A is used to express the exogenous gene CEY17_RS01410 ^G733A extrachromosomally through a plasmid, and then overexpress the mutant CEY17_RS01410 ^G733A gene in the production bacteria.

The recombinant vectors pK18- ^{CEY17_RS01410G733A} , pK18-CEY17_RS01410OE, ^pK18 -CEY17_RS01410G733AOE, pXMJ19-CEY17_RS01410 and pXMJ19- ^{CEY17_RS01410G733A} are all within the protection scope of the present invention.

Herein, the recombinant microorganism can specifically be recombinant bacteria YPV-085, YPV-086, YPV-087, YPV-088 and/or YPV-089.

The recombinant bacterium YPV-085 is obtained by transforming the recombinant vector pK18-CEY17_RS01410 ^G733A into Corynebacterium glutamicum CGMCC No. 21260, and the recombinant bacterium YPV-085 contains SEQ ID No. 3. The mutated gene CEY17_RS01410 ^G733A .

The recombinant bacteria YPV-086 contains double copies of the CEY17_RS01410 gene shown in SEQ ID No. 1; The spacer of the lower homology arm CEY17_02575 is replaced with the CEY17_RS01410 gene, and the recombinant bacteria obtained by keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 21260 unchanged. Recombinant bacteria containing double copies of CEY17_RS01410 gene can significantly and stably increase the expression of CEY17_RS01410 gene. The recombinant strain YPV-086 is an engineered strain that overexpresses the wild-type CEY17_RS01410 gene on the genome, and is a recombinant strain obtained by introducing the recombinant vector pK18-CEY17_RS01410OE into Escherichia coli DH5α. .

The recombinant bacteria YPV-087 contains the mutated CEY17_RS01410 ^G733A gene shown in SEQ ID No.3; The spacer region of the lower homology arm CEY17_02575 was replaced with the CEY17_RS01410 ^G733A gene, and the recombinant bacteria obtained by keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 21260 unchanged. The recombinant strain YPV-087 is an engineered strain that overexpresses the mutant CEY17_RS01410 ^G733A gene on the genome, and is a recombinant strain obtained by introducing the recombinant vector pK18-CEY17_RS01410 ^G733A OE into Escherichia coli DH5α.

The recombinant bacteria YPV-088 contains the CEY17_RS01410 gene shown in SEQ ID No. 1, and the recombinant bacteria YPV-088 is an engineering bacteria that overexpresses the wild-type CEY17_RS01410 gene on a plasmid, that is, extrachromosomally overexpressed from the plasmid pXMJ19-CEY17_RS01410 .

The recombinant bacteria YPV-089 contains the mutant CEY17_RS01410 ^G733A gene shown in SEQ ID No. 3, and the recombinant bacteria YPV-089 is an engineering bacteria that overexpresses the mutant CEY17_RS01410 ^G733A gene on a plasmid, that is, the plasmid pXMJ19-CEY17_RS01410 ^G733A expresses Extrachromosomal overexpression.

The recombinant bacteria YPV-085, YPV-086, YPV-087, YPV-088 and YPV-089 are all within the protection scope of the present invention.

The present invention also provides a method for constructing the recombinant microorganism, the method comprising at least any of the following:

F1) introducing the nucleic acid molecule CEY17_RS01410 ^G733A into the target microorganism to obtain the recombinant microorganism;

F2) introduce the DNA molecule shown in SEQ ID No.1 into the target microorganism to obtain the recombinant microorganism;

F3) Editing the DNA molecule shown in SEQ ID No. 1 by gene editing means (such as single-base gene editing), so that the target microorganism contains the DNA molecule shown in SEQ ID No. 3.

The introduction can be by transforming the host bacteria with the vector carrying the DNA molecule of the present invention by any known transformation method such as chemical transformation method or electroporation transformation method. The introduced DNA molecule can be single copy or multiple copies. The introduction may be the integration of the exogenous gene into the host chromosome, or the extrachromosomal expression of the plasmid.

In the present invention, the CEY17_RS01410 gene coding region (SEQ ID No. 1) of Corynebacterium glutamicum CGMCC No. 21260 (which is confirmed by sequencing to retain the wild-type CEY17_RS01410 gene on the chromosome) is first replaced by alleles. ) and introduced point mutation to construct a genetically engineered strain YPV-085 containing the mutation (GA). In order to further study and verify that overexpression of the wild-type CEY17_RS01410 gene or its mutant gene CEY17_RS01410 ^G733A in the production bacteria can increase the production of L-valine, the exogenous genes were integrated into the host chromosome or expressed by plasmids extrachromosomally. Engineering bacteria YPV-086, YPV-087, YPV-088 and YPV-089 overexpress CEY17_RS01410 gene or CEY17_RS01410 ^G733A gene on genome and plasmid. Experiments show that CEY17_RS01410 gene and its variants are involved in the biosynthesis of L-valine, and the accumulation of L-valine in microorganisms can be regulated by overexpression or knockout of CEY17_RS01410 gene, or site-directed mutation. Mutating the coding region of the CEY17_RS01410 gene or overexpressing the CEY17_RS01410 gene or its mutant gene CEY17_RS01410 ^G733A in the production strain is helpful for the improvement of L-valine production and conversion rate, while the knockout or weakening of the CEY17_RS01410 gene is not conducive to L-valine accumulation. The CEY17_RS01410 gene and its variants (such as CEY17_RS01410 ^G733A gene) can be used to construct a genetically engineered strain that produces L-valine, so as to promote the increase of L-valine yield and cultivate high-yield, high-quality strains that are in line with industrial production.

Preservation Instructions

Species name: Corynebacterium glutamicum

Latin name: Corynebacterium glutamicum

Taxonomic name: Corynebacterium glutamicum

Strain number: YPFV1

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: November 30, 2020

Depository center registration number: CGMCC No.21260

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Corynebacterium glutamicum (Corynebacterium glutamicum) YPFV1 CGMCCNo.21260 in the following examples is obtained by mutagenizing Corynebacterium glutamicum ATCC15168, and has been deposited in the China Microorganism Culture Collection Management Committee on November 30, 2020 General Microbiology Center (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC No.21260. Corynebacterium glutamicum YPFV1, also known as Corynebacterium glutamicum CGMCC No.21260.

Example 1 Construction of the recombinant vector comprising the coding region fragment of the CEY17_RS01410 gene with point mutation

According to the genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBI, two pairs of primers to amplify the coding region of CEY17_RS01410 gene were designed and synthesized. 21260 (confirmed by sequencing that the CEY17_RS01410 gene of the strain retains the wild-type CEY17_RS01410 gene), a point mutation was introduced into the coding region (SEQ ID No. 1) of the CEY17_RS01410 gene, which was the nucleotide sequence (SEQ ID No. The 733rd guanine (G) in ID No. 1) was mutated to adenine (A) to obtain the DNA molecule shown in SEQ ID No. 3 (mutated CEY17_RS01410 gene, named CEY17_RS01410 ^G733A ).

Wherein, the DNA molecule shown in SEQ ID No. 1 encodes a protein whose amino acid sequence is SEQ ID No. 2 (the protein name is protein CEY17_RS01410).

The DNA molecule shown in SEQ ID No. 3 encodes a mutein whose amino acid sequence is SEQ ID No. 4 (the name of the mutein is CEY17_RS01410 ^A245T ). The 245th threonine (T) in the amino acid sequence (SEQ ID No. 4) of the mutein CEY17_RS01410 ^A245T is mutated from alanine (A).

The NEBuilder recombination technology was used to construct the vector, and the CEY17_RS01410 gene was subjected to site-directed mutation. The primers were designed as follows (synthesized by Shanghai Invitrogen Company), and the bases in bold font are the mutation positions:

P1:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG CGTCGGAGCAGTTGGATTTC-3',

P2: 5'-GTTCGGGTTACCCGTCGCCTCACCAAGCCCAACG-3',

P3:5'-CGTTGGGCTTGGTGAGGCGACGGGTAACCCGAAC-3',

P4: 5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC CGCGCCAACCTGGTGAAACT-3'.

Construction method: Using Corynebacterium glutamicum ATCC14067 as the template, PCR amplification was performed with primers P1 and P2, P3 and P4, respectively, to obtain two CEY17_RS01410 gene coding regions with mutated bases and sizes of 656bp and 682bp respectively. DNA fragments (CEY17_RS01410 Up and CEY17_RS01410 Down).

PCR amplification system: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg ²⁺ (25mM) 4μL, primers (10pM) 2μL each, Ex Taq (5U/μL) 0.25μL, total volume 50μL ;

The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 5 min, (denaturation at 94°C for 30s; annealing at 52°C for 30s; extension at 72°C for 40s; 30 cycles), and overextension at 72°C for 10 min.

After the above-mentioned two DNA fragments (CEY17_RS01410 Up and CEY17_RS01410 Down) were separated and purified by agarose gel electrophoresis, they were separated with the pK18mobsacB plasmid (purchased from Add gene company) and purified by enzyme digestion (Xbal I/BamH I). Anamycin resistance marker) DNA assembly reaction was carried out with NEBuilder enzyme (NEBuilder HiFi DNA AssemblyMaster Mix, purchased from NEB Company), and the reaction conditions were: ligation at 50°C for 30 min, and the ligation product was transformed into DH5α (purchased from TAKARA Company) after the growth. The single clone was identified by primer M13 (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive recombinant vector pK18-CEY17_RS01410 ^G733A , and sent to a sequencing company for sequencing identification, which will contain the correct point mutation ( The recombinant vector pK18-CEY17_RS01410 ^G733A of GA) was kept for future use.

After sequencing identification, the recombinant vector pK18-CEY17_RS01410 ^G733A contains DNA fragments integrating DNA fragments CEY17_RS01410Up and CEY17_RS01410 Down, named CEY17_RS01410 Up-Down, CEY17_RS01410Up-Down DNA fragment size is 1304bp, the sequence is shown in SEQ ID No.5, its Contains a mutation site (GA) for introducing nucleic acid modification at position 733 of the CEY17_RS01410 gene coding region (SEQ ID No.1) in the strain Corynebacterium glutamicum CGMCC No.21260, and the nucleic acid modification can specifically be Guanine (G) at position 733 of SEQ ID No. 1 was mutated to adenine (A), which eventually resulted in the mutation of alanine (A) at position 245 of the encoded protein to threonine (T).

Described recombinant vector pK18-CEY17_RS01410 ^G733A is to replace the fragment (small fragment) between the Xbal I and BamH I recognition sites of pK18mobsacB vector with the DNA fragment shown in the 37th-1266th position of SEQ ID No.5 in the sequence listing, Keep the other sequences of the pK18mobsacB vector unchanged to obtain a recombinant vector.

The recombinant vector pK18-CEY17_RS01410 ^G733A contains the DNA molecule shown in positions 128-1281 of the mutant gene CEY17_RS01410 ^G733A shown in SEQ ID No. 3.

Example 2 Construction of engineering strain comprising gene CEY17_RS01410 ^G733A

The construction method is as follows: after the allelic replacement plasmid (pK18- ^{CEY17_RS01410G733A} ) in Example 1 was transformed into Corynebacterium glutamicum CGMCC No.21260 by electric shock, it was cultured in the medium, and the medium composition and The culture conditions are shown in Table 1. The single colony produced by the culture is identified by the primer P1 in Example 1 and the universal primer M13R (5'CAGGAAACAGCTATGACC3'), and the size of 1311bp (sequence shown in SEQ ID No. 6) can be amplified. The strain in the band is the positive strain. The positive strains were cultured on the medium containing 15% sucrose, the single colonies produced by the culture were cultured on the medium containing kanamycin and without kanamycin, and the culture medium without kanamycin was selected. The strains that grow on the base and do not grow on the medium containing kanamycin were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen Company):

P5:5'-CTGGTTTCACGGTGTCGGAG-3',

P6:5'-CCACTTACGGTTTCTTCGCAG-3'.

The obtained PCR amplification product (260bp) was subjected to SSCP (Single-Strand Conformation Polymorphis) electrophoresis (Single-Strand Conformation Polymorphis) electrophoresis (using the amplified fragment of plasmid pK18-CEY17_RS01410 ^G733A as a positive control, Corynebacterium glutamicum The amplified fragment of ATCC14067 is a negative control, and water is used as a blank control), and the preparation and electrophoresis conditions of PAGE for SSCP electrophoresis are shown in Table 2. Due to the different structure of the fragments and the different electrophoresis positions, the electrophoresis positions of the fragments are inconsistent with those of the negative control fragment and are different from those of the positive control. The strains with the same fragment position are the strains with successful allelic substitution. The positive strain CEY17_RS01410 gene fragment was amplified again by primer P5/P6 PCR, and connected to the PMD19-T vector for sequencing. Through sequence comparison, the strain with mutation (GA) in the base sequence was the positive strain with successful allelic replacement, and Designated as YPV-085.

Recombinant bacterium YPV-085 is a recombinant bacterium obtained by transforming the recombinant vector pK18-CEY17_RS01410 ^G733A into Corynebacterium glutamicum CGMCC No.21260, and the recombinant bacterium YPV-085 contains the mutation shown in SEQID No.3. Gene CEY17_RS01410 ^G733A .

Table 1 Composition and culture conditions of medium

Table 2 Preparation of PAGE for SSCP electrophoresis and electrophoresis conditions

Example 3 Construction of engineering strains overexpressing CEY17_RS01410 gene and CEY17_RS01410 ^G733A gene on genome

The NEBuilder recombination technology was used to construct the vector. According to the genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBI, three pairs of primers for amplifying the upstream and downstream homology arm fragments and the CEY17_RS01410 or CEY17_RS01410 ^G733A gene coding region and promoter region were designed and synthesized. The CEY17_RS01410 or CEY17_RS01410 ^G733A gene was introduced into Corynebacterium glutamicum CGMCC No. 21260 by means of homologous recombination.

The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

P7:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GTAGTGCCGTGCGTACCCCA-3',

P8:5'-CAAGCCACTACTCTAGACGTCCCAACCCCAATCGCAATGT-3',

P9:5'-ACATTGCGATTGGGGTTGGGACGTCTAGAGTAGTGGCTTG-3',

P10: 5'-GTGCGGGTTGGGGTTTTTGACTAGCTAGCGTAGTGCTCCG-3',

P11: 5'-CGGAGCACTACGCTAGCTAGTCAAAAACCCCAACCCGCAC-3',

P12: 5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GTTGGTTTAGCGGAGCTGCA-3'.

The construction method is as follows: take Corynebacterium glutamicum ATCC14067 or YPV-085 as the template, respectively, carry out PCR amplification with primers P7/P8, P9/P10, P11/P12, and obtain the upstream homology arm fragment of 795bp (corresponding to glutamate). Corynebacterium acidophilus CGMCCNo.21260 CEY17_RS02570 gene and the spacer region of CEY17_RS02575, sequence shown in SEQ ID No.7), CEY17_RS01410 gene and its promoter fragment 1413bp (sequence shown in SEQ ID No.8) or CEY17_RS01410 ^G733A gene and Its promoter fragment 1413bp (sequence shown as SEQ ID No.9) and downstream homology arm fragment 769bp (corresponding to Corynebacterium glutamicum CGMCC No.21260 CEY17_RS02575 gene and its spacer region with CEY17_RS02570, sequence as SEQ ID No. .10).

After the PCR reaction, the three fragments amplified from each template were recovered by electrophoresis using a column-type DNA gel recovery kit. The recovered 3 fragments and the pK18mobsacB plasmid purified after Xbal I/BamH I digestion (purchased from Addgene Company, the plasmid contains kanamycin resistance as a selection marker) were treated with NEBuilder enzyme (NEBuilderHiFi DNA Assembly Master Mix, Purchased from NEB Company) for DNA assembly reaction, the reaction conditions are: ligation at 50°C for 30min, the monoclonal grown after the ligation product is transformed into DH5α is used M13 primers (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC- 3') Positive integration plasmids (recombinant vectors) are obtained through PCR identification, which are respectively pK18-CEY17_RS01410OE, pK18- ^{CEY17_RS01410G733A} OE, and the positive integration plasmids contain kanamycin resistance markers, and the plasmids can be obtained by kanamycin screening Recombinants integrated into the genome.

The recombinant vector pK18-CEY17_RS01410OE was used to integrate the exogenous gene CEY17_RS01410 into the host chromosome and overexpress the wild-type CEY17_RS01410 gene in the production strain.

The recombinant vector pK18-CEY17_RS01410 ^G733A OE was used to integrate the exogenous gene CEY17_RS01410 ^G733A into the host chromosome, and the mutant gene CEY17_RS01410 ^G733A was overexpressed in the production strain.

The PCR reaction system was: 10×Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg ²⁺ (25 mM) 4 μL, primers (10 pM) 2 μL each, Ex Taq (5U/μL) 0.25 μL, the total volume was 50 μL.

The PCR reaction program was: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s; annealing at 52 °C for 30 s; extension at 72 °C for 60 s (30 cycles), and overextension at 72 °C for 10 min.

The integration plasmids with correct sequencing (pK18-CEY17_RS01410OE, pK18-CEY17_RS01410 ^G733A OE) were electrotransformed into Corynebacterium glutamicum CGMCC No.21260 respectively, and cultured in the medium. The single colony was identified by P13/P14 primers by PCR, and the PCR amplification contained a sequence of size 1693bp (the sequence without point mutation was shown in SEQ ID No. The fragment of 11) is the positive strain, and the fragment that cannot be amplified is the original strain. The positive strain was cultured on a medium containing 15% sucrose, and the single colony produced by the culture was further identified by PCR using the P15/P16 primer, and the amplified size was 1582bp (the sequence without point mutation is as shown in SEQ ID No.12. It is shown that the 236th position of the sequence containing the point mutation is A, and the other bacteria such as SEQ ID No. 12) are CEY17_RS01410 or CEY17_RS01410 ^G733A gene integrated into the homology arm CEY17_02570 and the lower homology arm CEY17_02575 on the genome of Corynebacterium glutamicum CGMCCNo.21260 The positive strains on the spacer were named YPV-086 (without mutation) and YPV-087 (with mutation).

Recombinant bacterium YPV-086 contains double copies of the CEY17_RS01410 gene shown in SEQ ID No.1; specifically, recombinant bacterium YPV-086 is the homology arm CEY17_02570 in the genome of Corynebacterium glutamicum CGMCC No.21260 and the same below The spacer of the source arm CEY17_02575 was replaced with the CEY17_RS01410 gene, and the recombinant bacteria obtained by keeping other nucleotides in the genome of Corynebacterium glutamicum CG MCCNo.21260 unchanged. Recombinant bacteria containing double copies of CEY17_RS01410 gene can significantly and stably increase the expression of CEY17_RS01410 gene. The recombinant strain YPV-086 is an engineered strain that overexpresses the wild-type CEY17_RS01410 gene on the genome, and is a recombinant strain obtained by introducing the recombinant vector pK18-CEY17_RS01410OE into Escherichia coli DH5α.

Recombinant bacterium YPV-087 contains the mutant CEY17_RS01410 ^G733A gene shown in SEQ ID No.3; specifically, recombinant bacterium YPV-087 is the homology arm CEY17_02570 in the genome of Corynebacterium glutamicum CGMCC No.21260 and the lower homology The spacer of the source arm CEY17_02575 was replaced with the CEY17_RS01410 ^G733A gene, and the recombinant bacteria obtained by keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 21260 unchanged. The recombinant strain YPV-087 is an engineered strain that overexpresses the mutant CEY17_RS01410 ^G733A gene on the genome, and is a recombinant strain obtained by introducing the recombinant vector pK18-CEY17_RS01410 ^G733A OE into Escherichia coli DH5α.

PCR identification primers are as follows:

P13: 5'-CGGTTAGATTTTTTGGCCCC-3' (corresponding to the outer side of the upper homology arm CEY17_RS02570),

P14: 5'-AAGTCATGAAGAAGGACACG-3' (corresponding to the inside of the CEY17_RS01410 gene),

P15: 5'-TGATATGGATGCTGTTTGAGG-3' (corresponding to the inside of CEY17_RS01410 gene),

P16: 5'-TCTGGACTGGGTGTTGCGCT-3' (corresponds to the outer side of the lower homology arm CEY17_RS02575).

Example 4 Construction of an engineered strain that overexpresses the CEY17_RS01410 gene or the CEY17_RS01410 ^G733A gene on a plasmid

NEBuilder recombination technology was used to construct the vector. According to the genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBI, a pair of primers for amplifying the CEY17_RS01410 and CEY17_RS01410 ^G733A gene coding regions and promoter regions were designed and synthesized. The primers were designed as follows (synthesized by Shanghai Invitrogen Company). ):

P17: 5'- GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC ACGTCTAGAGTAGTGGCTTG-3' (the underlined nucleotide sequence is the sequence on pXMJ19),

P18: 5'- ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAAC CTAGCTAGCGTAGTGCTCCG-3' (the underlined nucleotide sequence is the sequence on pXMJ19).

The construction method is as follows: respectively take Corynebacterium glutamicum ATCC14067 and YPV-085 as templates, and carry out PCR amplification with primers P17/P18 to obtain CEY17_RS01410 gene and its promoter fragment (sequence shown in SEQ ID No. 13) and CEY17_RS01410 ^G733A gene and its promoter fragment 1443bp (sequence shown in SEQ ID No. 14), the amplified product was electrophoresed and purified and recovered using a column DNA gel recovery kit. The recovered DNA fragment was combined with EcoR I/KpnI The shuttle plasmid pXMJ19 recovered by enzyme digestion (purchased from Addgene Company, which contains chloramphenicol resistance as a selection marker) was subjected to DNA assembly reaction with NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB Company), and the reaction conditions were: : ligated at 50°C for 30min, the monoclonal grown after the ligation product was transformed into DH5α was identified by PCR with M1R(-48)(5'- AGCGGATAACAATTTCACACAGGA-3') /P18 primer to obtain a positive overexpression vector pXMJ19-CEY17_RS01410 (containing CEY17_RS01410 gene) and pXMJ19-CEY17_RS01410 ^G733A (containing the CEY17_RS01410 ^G733A gene), the plasmid was sent for sequencing. Because the plasmid contains a chloramphenicol resistance marker, chloramphenicol can be used to screen whether the plasmid is transformed into the strain.

The recombinant vector pXMJ19-CEY17_RS01410 replaces the fragment (small fragment) between the EcoR I and KpnI recognition sites of the pXMJ19 vector with a DNA fragment whose nucleotide sequence is SEQ ID No. 13 in the sequence listing, and maintains other sequences of the pXM J19 vector unchanged, the resulting recombinant expression vector. The recombinant vector pXMJ19-CEY17_RS01410 is used to express the exogenous gene CEY17_RS01410 extrachromosomally through a plasmid, and then overexpress the wild-type CEY17_RS01410 gene in the production bacteria.

The recombinant vector pXMJ19-CEY17_RS01410 ^G733A replaces the fragment (small fragment) between the EcoR I and KpnI recognition sites of the pXMJ19 vector with a DNA fragment whose nucleotide sequence is SEQ ID No. 14 in the sequence listing, and maintains other sequences of the pXMJ19 vector unchanged, the resulting recombinant expression vector. The recombinant vector pXMJ19-CEY17_RS01410 ^G733A is used to express the exogenous gene CEY17_RS01410 ^G733A extrachromosomally through a plasmid, and then overexpress the mutant CEY17_RS01410 ^G733A gene in the production bacteria.

The PCR reaction system was: 10×Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg ²⁺ (25 mM) 4 μL, primers (10 pM) 2 μL each, Ex Taq (5U/μL) 0.25 μL, the total volume was 50 μL.

The PCR reaction program was: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s; annealing at 52 °C for 30 s; extension at 72 °C for 60 s (30 cycles), and overextension at 72 °C for 10 min.

The correctly sequenced pXMJ19-CEY17_RS01410 and pXMJ19-CEY17_RS01410 ^G733A plasmids were electro-transformed into Corynebacterium glutamicum CGMCC No.21260, respectively, and cultured in the medium. See Table 1 for medium components and culture conditions. The primers M13R(-48)/P18 were used for PCR identification, and the PCR amplified sequence containing a size of 1482 bp (the sequence without point mutation is shown in SEQ ID No. 15, the 900th position of the sequence containing point mutation is A, and the rest are shown in SEQ ID No. .15) fragments were positive strains, which were named as YPV-088 (without mutation point) and YPV-089 (with mutation point).

The recombinant strain YPV-088 contains the CEY17 RS01410 gene shown in SEQ ID No. 1, and the recombinant strain YPV-088 is an engineered strain that overexpresses the wild-type CEY17_RS01410 gene on a plasmid, that is, extrachromosomal overexpression from the plasmid pXMJ19-CEY17_RS01410.

Recombinant bacterium YPV-089 contains the mutant CEY17_RS01410 ^G733A gene shown in SEQ ID No. 3, and recombinant bacterium YPV-089 is an engineering bacterium that overexpresses the mutant CEY17_RS01410 ^G733A gene on a plasmid, that is, extrachromosomally derived from plasmid pXMJ19-CEY17_RS01410 ^G733A overexpressed.

Example 5 Construction of an engineered strain that lacks the CEY17_RS01410 gene on the genome

NEBuilder recombination technology was used to construct the vector. According to the genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBI, two pairs of primers for amplifying the fragments at both ends of the CEY17_RS01410 gene coding region were synthesized as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

P19:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG AGAATTACCGGCGATGGGGC-3',

P20:5'-CAGTTTCGCGGTATTCACTAACTGCGTACCTCCGCATGT-3',

P21:5'-ACATGCGGAGGGTACGCAGTTAGTGAATACCGCGGAAACTG-3',

P22: 5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GTACGCATGGTTGATGCGCC-3'.

The construction method is as follows: using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification is carried out with primers P19/P20 and P21/P22, respectively, to obtain the upstream homology arm fragment of CEY17_RS01410 709bp and the downstream homology arm fragment of CEY17_RS01410 824bp.

The amplified product was electrophoresed and purified using a column DNA gel recovery kit. The recovered DNA fragment was the same as the pK18mobsacB plasmid (purchased from Addgene, which was purified by Xbal I/BamH I digestion) Mycin resistance was used as a selection marker) DNA assembly reaction was performed with NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB Company), and the reaction conditions were: ligation at 50 °C for 30 min, and M13 was used for the monoclonal grown after the ligation product was transformed into DH5α. Primers (M13F: 5'TGTAAAACGACGGCCAGT 3', M13R: 5'CAGGAAACAGCTATGACC 3') were identified by PCR to obtain a positive knockout vector pK18-ΔCEY17_RS01410. This recombinant plasmid pK18-ΔCEY17_RS01410 contains the Up-Down DNA 1493 bp of ΔCEY17_RS01410 (sequence as shown in SEQ ID). No. 16).

The plasmid was sent for sequencing, and the correctly sequenced knockout plasmid pK18-ΔCEY17_RS01410 was electrotransformed into Corynebacterium glutamicum CGMCC No. 21260, and cultured in the medium. See Table 1 for medium components and culture conditions. Colonies were identified by PCR with the following primers (synthesized by Shanghai Invitrogen Company):

P23: 5'-AGAATTACCGGCGATGGGGC-3' (corresponding to the spacer region of C. glutamicum CGMCC No. 21260 CEY17_RS01395 and CEY17_RS01400),

P24: 5'-GTACGCATGGTTGATGCGCC-3' (corresponding to the coding region of Corynebacterium glutamicum CGMCC No. 21260CEY17_RS01420).

The strains that amplified the bands of 1419bp and 2700bp simultaneously by the above PCR were positive strains, and the strains that only amplified the 2700bp band were the original strains. Positive strains were selected on 15% sucrose medium and cultured on kanamycin-containing and kanamycin-free medium, respectively. The strains that did not grow on the medium of mycin were further identified by PCR using P23/P24 primers, and the strain with a size of 1419 bp amplified was the positive strain CEY17_RS01410 whose coding region of the CEY17_RS01410 gene was knocked out. The positive strain CEY17_RS01410 fragment was amplified by P23/P24 primer PCR again, and connected to the pMD19-T vector for sequencing, and the correctly sequenced strain was named YPV-090 (CEY17_RS01410 on the genome of Corynebacterium glutamicum CGMCC No. 21260). gene was knocked out).

Example 6L-valine fermentation experiment

The bacterial strain and the original strain Corynebacterium glutamicum CGMCC No.21260 constructed in the above example were cultivated in the fermentor of the BLBIO-5GC-4-H model (purchased from Shanghai Bailun Biotechnology Co., Ltd.) as shown in Table 3 Fermentation experiments were carried out based on the control process shown in Table 4. Each strain was repeated three times and the results are shown in Table 5.

The results are shown in Table 5. In Corynebacterium glutamicum, the CEY17_RS01410 gene coding region is subjected to point mutation (such as mutation to CEY17_RS01410 ^G733A gene) and overexpression, which is conducive to the improvement of L-valine production and transformation rate, while Knockout or weakening of the CEY17_RS01410 gene is not conducive to the accumulation of L-valine.

Table 3 fermentation medium formula (the rest are water)

Element formula Ammonium sulfate 14g/L Potassium dihydrogen phosphate 1g/L Dipotassium phosphate 1g/L Magnesium sulfate 0.5g/L yeast 2g/L Ferrous sulfate 18mg/L Manganese sulfate 4.2mg/L Biotin 0.02mg/L Vitamin B1 2mg/L antifoam (CB-442) defoamer) 0.5mL/L 70% glucose (bottom sugar) 40g/L

Table 4 fermentation control process

Table 5L-valine fermentation test results

strain OD₆₁₀ L-valine yield (g/L) Corynebacterium glutamicum CGMCC No.21260 98.2 84.1 YPV-085 99.4 85.6 YPV-086 99.9 85.5 YPV-087 99.7 85.9 YPV-088 100.5 86.2 YPV-089 100.3 86.9 YPV-090 96.9 82.6

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the present invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application.

SEQUENCE LISTING

<110> Ningxia Yipin Biotechnology Co., Ltd.

<120> A method for preparing L-valine and gene mutants and biological materials used therefor

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 1281

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

atgagttcag tttcgctgca ggattttgat gcagagcgaa ttggtctgtt ccacgaggac 60

attaaacgca agtttgatga gctcaagtca aaaaatctga agctggatct tactcgcggt 120

aagccttcgt cggagcagtt ggatttcgct gatgagctgt tggcgttgcc tggtaagggc 180

gatttcaagg ctgcggatgg tactgatgtc cgtaactatg gcgggctgga tggcattgtt 240

gatattcgtc agatttgggc ggatttgctg ggtgttcctg tggagcaggt cttggcgggg 300

gatgcttcga gcttgaacat catgtttgat gtgatcagct ggtcgtacat tttcggtaac 360

aatgattcgg ttcagccttg gtcgaaggaa gagaccgtta agtggatttg ccctgttccg 420

ggctatgatc gccatttctc catcacggag cgtttcggct ttgagatgat ttctgtgcca 480

atgaatgaag acggccctga tatggatgct gttgaggaat tggtgaagga tcctcaggtt 540

aagggcatgt gggttgtgcc ggtattttct aacccgactg gtttcacggt gtcggaggac 600

gtcgcaaagc gtctgagcac gatggaaact gcggcgccgg acttccgcgt ggtgtgggat 660

aacgcttacg ccgttcatac gttgactgat gagttccctg aggtcatcga catcgttggg 720

cttggtgagg cggcgggtaa cccgaaccgt ttctgggcgt tcacttctac ttcgaagatc 780

actctcgcgg gtgcgggcgt gtccttcttc atgacttctg cgaagaaccg taagtggtac 840

tccggtcatg cgggtatccg tggcattggc cctaacaagg tcaatcagtt ggctcatgcg 900

cgttactttg gcgatgctga gggagtgcgc gcggtgatgc gtaagcatgc tgcgtcgttg 960

gctccgaagt tcaacaaggt tctggagatt ctggattctc gccttgctga gtacggtgtc 1020

gcgcagtgga ctgtccctgc gggcggctac ttcatttccc ttgatgtggt tcctggtacg 1080

gcgtctcgcg tggctgagtt ggctaaggaa gccggcatcg cgttgacggg tgcgggttcg 1140

tccttcccat tgcatcagga tccagagaac aaaaatctcc gtttggcgcc ttctctgcct 1200

cctgttgagg aacttgaggt tgccatggat ggcgtggcta cgtgtgtttt gctggcagct 1260

gcggagcact acgctagcta g 1281

<210> 2

<211> 426

<212> PRT

<213> Corynebacterium glutamicum

<400> 2

Met Ser Ser Val Ser Leu Gln Asp Phe Asp Ala Glu Arg Ile Gly Leu

1 5 10 15

Phe His Glu Asp Ile Lys Arg Lys Phe Asp Glu Leu Lys Ser Lys Asn

20 25 30

Leu Lys Leu Asp Leu Thr Arg Gly Lys Pro Ser Ser Glu Gln Leu Asp

35 40 45

Phe Ala Asp Glu Leu Leu Ala Leu Pro Gly Lys Gly Asp Phe Lys Ala

50 55 60

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

65 70 75 80

Asp Ile Arg Gln Ile Trp Ala Asp Leu Leu Gly Val Pro Val Glu Gln

85 90 95

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

100 105 110

Ser Trp Ser Tyr Ile Phe Gly Asn Asn Asp Ser Val Gln Pro Trp Ser

115 120 125

Lys Glu Glu Thr Val Lys Trp Ile Cys Pro Val Pro Gly Tyr Asp Arg

130 135 140

His Phe Ser Ile Thr Glu Arg Phe Gly Phe Glu Met Ile Ser Val Pro

145 150 155 160

Met Asn Glu Asp Gly Pro Asp Met Asp Ala Val Glu Glu Leu Val Lys

165 170 175

Asp Pro Gln Val Lys Gly Met Trp Val Val Pro Val Phe Ser Asn Pro

180 185 190

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

195 200 205

Glu Thr Ala Ala Pro Asp Phe Arg Val Val Trp Asp Asn Ala Tyr Ala

210 215 220

Val His Thr Leu Thr Asp Glu Phe Pro Glu Val Ile Asp Ile Val Gly

225 230 235 240

Leu Gly Glu Ala Ala Gly Asn Pro Asn Arg Phe Trp Ala Phe Thr Ser

245 250 255

Thr Ser Lys Ile Thr Leu Ala Gly Ala Gly Val Ser Phe Phe Met Thr

260 265 270

Ser Ala Lys Asn Arg Lys Trp Tyr Ser Gly His Ala Gly Ile Arg Gly

275 280 285

Ile Gly Pro Asn Lys Val Asn Gln Leu Ala His Ala Arg Tyr Phe Gly

290 295 300

Asp Ala Glu Gly Val Arg Ala Val Met Arg Lys His Ala Ala Ser Leu

305 310 315 320

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

325 330 335

Glu Tyr Gly Val Ala Gln Trp Thr Val Pro Ala Gly Gly Tyr Phe Ile

340 345 350

Ser Leu Asp Val Val Pro Gly Thr Ala Ser Arg Val Ala Glu Leu Ala

355 360 365

Lys Glu Ala Gly Ile Ala Leu Thr Gly Ala Gly Ser Ser Phe Pro Leu

370 375 380

His Gln Asp Pro Glu Asn Lys Asn Leu Arg Leu Ala Pro Ser Leu Pro

385 390 395 400

Pro Val Glu Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr Cys Val

405 410 415

Leu Leu Ala Ala Ala Glu His Tyr Ala Ser

420 425

<210> 3

<211> 1281

<212> DNA

<213> Artificial sequence

<400> 3

atgagttcag tttcgctgca ggattttgat gcagagcgaa ttggtctgtt ccacgaggac 60

attaaacgca agtttgatga gctcaagtca aaaaatctga agctggatct tactcgcggt 120

aagccttcgt cggagcagtt ggatttcgct gatgagctgt tggcgttgcc tggtaagggc 180

gatttcaagg ctgcggatgg tactgatgtc cgtaactatg gcgggctgga tggcattgtt 240

gatattcgtc agatttgggc ggatttgctg ggtgttcctg tggagcaggt cttggcgggg 300

gatgcttcga gcttgaacat catgtttgat gtgatcagct ggtcgtacat tttcggtaac 360

aatgattcgg ttcagccttg gtcgaaggaa gagaccgtta agtggatttg ccctgttccg 420

ggctatgatc gccatttctc catcacggag cgtttcggct ttgagatgat ttctgtgcca 480

atgaatgaag acggccctga tatggatgct gttgaggaat tggtgaagga tcctcaggtt 540

aagggcatgt gggttgtgcc ggtattttct aacccgactg gtttcacggt gtcggaggac 600

gtcgcaaagc gtctgagcac gatggaaact gcggcgccgg acttccgcgt ggtgtgggat 660

aacgcttacg ccgttcatac gttgactgat gagttccctg aggtcatcga catcgttggg 720

cttggtgagg cgacgggtaa cccgaaccgt ttctgggcgt tcacttctac ttcgaagatc 780

actctcgcgg gtgcgggcgt gtccttcttc atgacttctg cgaagaaccg taagtggtac 840

tccggtcatg cgggtatccg tggcattggc cctaacaagg tcaatcagtt ggctcatgcg 900

cgttactttg gcgatgctga gggagtgcgc gcggtgatgc gtaagcatgc tgcgtcgttg 960

gctccgaagt tcaacaaggt tctggagatt ctggattctc gccttgctga gtacggtgtc 1020

gcgcagtgga ctgtccctgc gggcggctac ttcatttccc ttgatgtggt tcctggtacg 1080

gcgtctcgcg tggctgagtt ggctaaggaa gccggcatcg cgttgacggg tgcgggttcg 1140

tccttcccat tgcatcagga tccagagaac aaaaatctcc gtttggcgcc ttctctgcct 1200

cctgttgagg aacttgaggt tgccatggat ggcgtggcta cgtgtgtttt gctggcagct 1260

gcggagcact acgctagcta g 1281

<210> 4

<211> 426

<212> PRT

<213> Artificial sequence

<400> 4

Met Ser Ser Val Ser Leu Gln Asp Phe Asp Ala Glu Arg Ile Gly Leu

1 5 10 15

Phe His Glu Asp Ile Lys Arg Lys Phe Asp Glu Leu Lys Ser Lys Asn

20 25 30

Leu Lys Leu Asp Leu Thr Arg Gly Lys Pro Ser Ser Glu Gln Leu Asp

35 40 45

Phe Ala Asp Glu Leu Leu Ala Leu Pro Gly Lys Gly Asp Phe Lys Ala

50 55 60

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

65 70 75 80

Asp Ile Arg Gln Ile Trp Ala Asp Leu Leu Gly Val Pro Val Glu Gln

85 90 95

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

100 105 110

Ser Trp Ser Tyr Ile Phe Gly Asn Asn Asp Ser Val Gln Pro Trp Ser

115 120 125

Lys Glu Glu Thr Val Lys Trp Ile Cys Pro Val Pro Gly Tyr Asp Arg

130 135 140

His Phe Ser Ile Thr Glu Arg Phe Gly Phe Glu Met Ile Ser Val Pro

145 150 155 160

Met Asn Glu Asp Gly Pro Asp Met Asp Ala Val Glu Glu Leu Val Lys

165 170 175

Asp Pro Gln Val Lys Gly Met Trp Val Val Pro Val Phe Ser Asn Pro

180 185 190

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

195 200 205

Glu Thr Ala Ala Pro Asp Phe Arg Val Val Trp Asp Asn Ala Tyr Ala

210 215 220

Val His Thr Leu Thr Asp Glu Phe Pro Glu Val Ile Asp Ile Val Gly

225 230 235 240

Leu Gly Glu Ala Thr Gly Asn Pro Asn Arg Phe Trp Ala Phe Thr Ser

245 250 255

Thr Ser Lys Ile Thr Leu Ala Gly Ala Gly Val Ser Phe Phe Met Thr

260 265 270

Ser Ala Lys Asn Arg Lys Trp Tyr Ser Gly His Ala Gly Ile Arg Gly

275 280 285

Ile Gly Pro Asn Lys Val Asn Gln Leu Ala His Ala Arg Tyr Phe Gly

290 295 300

Asp Ala Glu Gly Val Arg Ala Val Met Arg Lys His Ala Ala Ser Leu

305 310 315 320

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

325 330 335

Glu Tyr Gly Val Ala Gln Trp Thr Val Pro Ala Gly Gly Tyr Phe Ile

340 345 350

Ser Leu Asp Val Val Pro Gly Thr Ala Ser Arg Val Ala Glu Leu Ala

355 360 365

Lys Glu Ala Gly Ile Ala Leu Thr Gly Ala Gly Ser Ser Phe Pro Leu

370 375 380

His Gln Asp Pro Glu Asn Lys Asn Leu Arg Leu Ala Pro Ser Leu Pro

385 390 395 400

Pro Val Glu Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr Cys Val

405 410 415

Leu Leu Ala Ala Ala Glu His Tyr Ala Ser

420 425

<210> 5

<211> 1304

<212> DNA

<213> Artificial sequence

<400> 5

cagtgccaag cttgcatgcc tgcaggtcga ctctagcgtc ggagcagttg gatttcgctg 60

atgagctgtt ggcgttgcct ggtaagggcg atttcaaggc tgcggatggt actgatgtcc 120

gtaactatgg cgggctggat ggcattgttg atattcgtca gatttgggcg gatttgctgg 180

gtgttcctgt ggagcaggtc ttggcggggg atgcttcgag cttgaacatc atgtttgatg 240

tgatcagctg gtcgtacatt ttcggtaaca atgattcggt tcagccttgg tcgaaggaag 300

agaccgttaa gtggatttgc cctgttccgg gctatgatcg ccatttctcc atcacggagc 360

gtttcggctt tgagatgatt tctgtgccaa tgaatgaaga cggccctgat atggatgctg 420

ttgaggaatt ggtgaaggat cctcaggtta agggcatgtg ggttgtgccg gtattttcta 480

acccgactgg tttcacggtg tcggaggacg tcgcaaagcg tctgagcacg atggaaactg 540

cggcgccgga cttccgcgtg gtgtgggata acgcttacgc cgttcatacg ttgactgatg 600

agttccctga ggtcatcgac atcgttgggc ttggtgaggc gacgggtaac ccgaaccgtt 660

tctgggcgtt cacttctact tcgaagatca ctctcgcggg tgcgggcgtg tccttcttca 720

tgacttctgc gaagaaccgt aagtggtact ccggtcatgc gggtatccgt ggcattggcc 780

ctaacaaggt caatcagttg gctcatgcgc gttactttgg cgatgctgag ggagtgcgcg 840

cggtgatgcg taagcatgct gcgtcgttgg ctccgaagtt caacaaggtt ctggagattc 900

tggattctcg ccttgctgag tacggtgtcg cgcagtggac tgtccctgcg ggcggctact 960

tcatttccct tgatgtggtt cctggtacgg cgtctcgcgt ggctgagttg gctaaggaag 1020

ccggcatcgc gttgacgggt gcgggttcgt ccttcccatt gcatcaggat ccagagaaca 1080

aaaatctccg tttggcgcct tctctgcctc ctgttgagga acttgaggtt gccatggatg 1140

gcgtggctac gtgtgttttg ctggcagctg cggagcacta cgctagctag agtgaatacc 1200

gcggaaactg cacattggat taaccgtttg ctgccgggtc agccggagtt tcaccaggtt 1260

ggcgcggggt accgagctcg aattcgtaat catggtcata gctg 1304

<210> 6

<211> 1311

<212> DNA

<213> Artificial sequence

<400> 6

cagtgccaag cttgcatgcc tgcaggtcga ctctagcgtc ggagcagttg gatttcgctg 60

atgagctgtt ggcgttgcct ggtaagggcg atttcaaggc tgcggatggt actgatgtcc 120

gtaactatgg cgggctggat ggcattgttg atattcgtca gatttgggcg gatttgctgg 180

gtgttcctgt ggagcaggtc ttggcggggg atgcttcgag cttgaacatc atgtttgatg 240

tgatcagctg gtcgtacatt ttcggtaaca atgattcggt tcagccttgg tcgaaggaag 300

agaccgttaa gtggatttgc cctgttccgg gctatgatcg ccatttctcc atcacggagc 360

gtttcggctt tgagatgatt tctgtgccaa tgaatgaaga cggccctgat atggatgctg 420

ttgaggaatt ggtgaaggat cctcaggtta agggcatgtg ggttgtgccg gtattttcta 480

acccgactgg tttcacggtg tcggaggacg tcgcaaagcg tctgagcacg atggaaactg 540

cggcgccgga cttccgcgtg gtgtgggata acgcttacgc cgttcatacg ttgactgatg 600

agttccctga ggtcatcgac atcgttgggc ttggtgaggc gacgggtaac ccgaaccgtt 660

tctgggcgtt cacttctact tcgaagatca ctctcgcggg tgcgggcgtg tccttcttca 720

tgacttctgc gaagaaccgt aagtggtact ccggtcatgc gggtatccgt ggcattggcc 780

ctaacaaggt caatcagttg gctcatgcgc gttactttgg cgatgctgag ggagtgcgcg 840

cggtgatgcg taagcatgct gcgtcgttgg ctccgaagtt caacaaggtt ctggagattc 900

tggattctcg ccttgctgag tacggtgtcg cgcagtggac tgtccctgcg ggcggctact 960

tcatttccct tgatgtggtt cctggtacgg cgtctcgcgt ggctgagttg gctaaggaag 1020

ccggcatcgc gttgacgggt gcgggttcgt ccttcccatt gcatcaggat ccagagaaca 1080

aaaatctccg tttggcgcct tctctgcctc ctgttgagga acttgaggtt gccatggatg 1140

gcgtggctac gtgtgttttg ctggcagctg cggagcacta cgctagctag agtgaatacc 1200

gcggaaactg cacattggat taaccgtttg ctgccgggtc agccggagtt tcaccaggtt 1260

ggcgcggggt accgagctcg aattcgtaat catggtcata gctgtttcct g 1311

<210> 7

<211> 795

<212> DNA

<213> Artificial sequence

<400> 7

cagtgccaag cttgcatgcc tgcaggtcga ctctaggtag tgccgtgcgt accccattag 60

aaagtgaaaa ttcactgatt ctagccagtc acgctgggaa tcattacatg ggccttcttc 120

gatcattcca tgatcgacaa gaaaagcctc acgttcatca ggttgtaaat aggggacagt 180

agacattaat tacacctaaa aagaaaaggg cccccatgag gcgcatcgtt gagaggcgtt 240

gggggtgctg ttggcttcta cgatatatct aattttgcct gatgtgtcag tagctcgaac 300

gtcactttca cttgtcgtct gaagtttcga tgtttctgca ccataaacgg tgtttatgaa 360

ttatcccccc ctctaccccc cgggggtgag gttttcgctg agaaggctgg cttcaaacgg 420

gggctggaca cgtacgcgga gatggcgacg cgttctgtca cgaatcgtgc gttgcgtgct 480

ggccattccg ccacccaagc cagatccagg tcatgagggc taccaggcca cacagaagca 540

gcgctaccta gaacgccaga tcagggcgtc gaaacggatg gaagctgcag ccatcgaccc 600

tagagacatt gacaccgcaa aacagcgcat acgggcatac caggcaaaac tacgcgacca 660

catcaaacag cacgacctgc caaggcgcag acaccgagaa cagattaaaa tgcgctaaag 720

aagttaacat catgctgcca ccgcccaagc gggaaacatt gcgattgggg ttgggacgtc 780

tagagtagtg gcttg 795

<210> 8

<211> 1413

<212> DNA

<213> Artificial sequence

<400> 8

acattgcgat tggggttggg acgtctagag tagtggcttg aggtcactac tctttttttg 60

tgcccttttt ttggtccgtc tattttgcca ccacatgcgg aggtacgcag ttatgagttc 120

agtttcgctg caggattttg atgcagagcg aattggtctg ttccacgagg acattaaacg 180

caagtttgat gagctcaagt caaaaaatct gaagctggat cttactcgcg gtaagccttc 240

gtcggagcag ttggatttcg ctgatgagct gttggcgttg cctggtaagg gcgatttcaa 300

ggctgcggat ggtactgatg tccgtaacta tggcgggctg gatggcattg ttgatattcg 360

tcagatttgg gcggatttgc tgggtgttcc tgtggagcag gtcttggcgg gggatgcttc 420

gagcttgaac atcatgtttg atgtgatcag ctggtcgtac attttcggta acaatgattc 480

ggttcagcct tggtcgaagg aagagaccgt taagtggatt tgccctgttc cgggctatga 540

tcgccatttc tccatcacgg agcgtttcgg ctttgagatg atttctgtgc caatgaatga 600

agacggccct gatatggatg ctgttgagga attggtgaag gatcctcagg ttaagggcat 660

gtgggttgtg ccggtatttt ctaacccgac tggtttcacg gtgtcggagg acgtcgcaaa 720

gcgtctgagc acgatggaaa ctgcggcgcc ggacttccgc gtggtgtggg ataacgctta 780

cgccgttcat acgttgactg atgagttccc tgaggtcatc gacatcgttg ggcttggtga 840

ggcggcgggt aacccgaacc gtttctgggc gttcacttct acttcgaaga tcactctcgc 900

gggtgcgggc gtgtccttct tcatgacttc tgcgaagaac cgtaagtggt actccggtca 960

tgcgggtatc cgtggcattg gccctaacaa ggtcaatcag ttggctcatg cgcgttactt 1020

tggcgatgct gagggagtgc gcgcggtgat gcgtaagcat gctgcgtcgt tggctccgaa 1080

gttcaacaag gttctggaga ttctggattc tcgccttgct gagtacggtg tcgcgcagtg 1140

gactgtccct gcgggcggct acttcatttc ccttgatgtg gttcctggta cggcgtctcg 1200

cgtggctgag ttggctaagg aagccggcat cgcgttgacg ggtgcgggtt cgtccttccc 1260

attgcatcag gatccagaga acaaaaatct ccgtttggcg ccttctctgc ctcctgttga 1320

ggaacttgag gttgccatgg atggcgtggc tacgtgtgtt ttgctggcag ctgcggagca 1380

ctacgctagc tagtcaaaaa ccccaacccg cac 1413

<210> 9

<211> 1413

<212> DNA

<213> Artificial sequence

<400> 9

acattgcgat tggggttggg acgtctagag tagtggcttg aggtcactac tctttttttg 60

tgcccttttt ttggtccgtc tattttgcca ccacatgcgg aggtacgcag ttatgagttc 120

agtttcgctg caggattttg atgcagagcg aattggtctg ttccacgagg acattaaacg 180

caagtttgat gagctcaagt caaaaaatct gaagctggat cttactcgcg gtaagccttc 240

gtcggagcag ttggatttcg ctgatgagct gttggcgttg cctggtaagg gcgatttcaa 300

ggctgcggat ggtactgatg tccgtaacta tggcgggctg gatggcattg ttgatattcg 360

tcagatttgg gcggatttgc tgggtgttcc tgtggagcag gtcttggcgg gggatgcttc 420

gagcttgaac atcatgtttg atgtgatcag ctggtcgtac attttcggta acaatgattc 480

ggttcagcct tggtcgaagg aagagaccgt taagtggatt tgccctgttc cgggctatga 540

tcgccatttc tccatcacgg agcgtttcgg ctttgagatg atttctgtgc caatgaatga 600

agacggccct gatatggatg ctgttgagga attggtgaag gatcctcagg ttaagggcat 660

gtgggttgtg ccggtatttt ctaacccgac tggtttcacg gtgtcggagg acgtcgcaaa 720

gcgtctgagc acgatggaaa ctgcggcgcc ggacttccgc gtggtgtggg ataacgctta 780

cgccgttcat acgttgactg atgagttccc tgaggtcatc gacatcgttg ggcttggtga 840

ggcgacgggt aacccgaacc gtttctgggc gttcacttct acttcgaaga tcactctcgc 900

gggtgcgggc gtgtccttct tcatgacttc tgcgaagaac cgtaagtggt actccggtca 960

tgcgggtatc cgtggcattg gccctaacaa ggtcaatcag ttggctcatg cgcgttactt 1020

tggcgatgct gagggagtgc gcgcggtgat gcgtaagcat gctgcgtcgt tggctccgaa 1080

gttcaacaag gttctggaga ttctggattc tcgccttgct gagtacggtg tcgcgcagtg 1140

gactgtccct gcgggcggct acttcatttc ccttgatgtg gttcctggta cggcgtctcg 1200

cgtggctgag ttggctaagg aagccggcat cgcgttgacg ggtgcgggtt cgtccttccc 1260

attgcatcag gatccagaga acaaaaatct ccgtttggcg ccttctctgc ctcctgttga 1320

ggaacttgag gttgccatgg atggcgtggc tacgtgtgtt ttgctggcag ctgcggagca 1380

ctacgctagc tagtcaaaaa ccccaacccg cac 1413

<210> 10

<211> 769

<212> DNA

<213> Artificial sequence

<400> 10

cggagcacta cgctagctag tcaaaaaccc caacccgcac atttttagat ttctattttg 60

tgtacatagg gttcggaaca aagcttaaac catccccaat tgaaatgtcg ttacacaccc 120

acatgtttga agtggagcaa accgaaaacc agttttcccc aacggcagcc gccccccacg 180

ttgaaccttc gaaatagtag gcaacaccat caagcggatc ttcatcaagc gaaatagtga 240

ttgactcttc accgttccgc ttacaaactg cgttagtgtc gctattttcc acccacttgt 300

cacactcgta cccgttttca tttagccatt tttcggcatg tcctattttc tcgaaccggg 360

caggagcgtc agggcttccg cagcccgcta gtagtagtcc ggctgcaatg atgcttaatg 420

tttttttcat gaattaaaca tagtactttg ctggtaaaaa tattggagaa ccccactggc 480

ctacatggtc agtgggggca tttttgcgtt tcacccctca aaaatcatca ccacacttgc 540

gggatttccc cctgatttcc cccactccca caccattccc agtggacagt gtggacgtat 600

tggacacatt aaacacattg cgaccaggta aaacgtcatg accaggtatc gtcaatgttc 660

ttgatgaatt tccgcaccgc aggattatca ttcgaggtgg aataaatagc ctgcagctcc 720

gctaaaccaa cgggtaccga gctcgaattc gtaatcatgg tcatagctg 769

<210> 11

<211> 1697

<212> DNA

<213> Artificial sequence

<400> 11

cggttagatt ttttggcccc tcccaatggg actcattaat gagatttcgg tagtgccgtg 60

cgtaccccat tagaaagtga aaattcactg attctagcca gtcacgctgg gaatcattac 120

atgggccttc ttcgatcatt ccatgatcga caagaaaagc ctcacgttca tcaggttgta 180

aataggggac agtagacatt aattacacct aaaaagaaaa gggcccccat gaggcgcatc 240

gttgagaggc gttgggggtg ctgttggctt ctacgatata tctaattttg cctgatgtgt 300

cagtagctcg aacgtcactt tcacttgtcg tctgaagttt cgatgtttct gcaccataaa 360

cggtgtttat gaattatccc cccctctacc ccccgggggt gaggttttcg ctgagaaggc 420

tggcttcaaa cgggggctgg acacgtacgc ggagatggcg acgcgttctg tcacgaatcg 480

tgcgttgcgt gctggccatt ccgccaccca agccagatcc aggtcatgag ggctaccagg 540

ccacacagaa gcagcgctac ctagaacgcc agatcagggc gtcgaaacgg atggaagctg 600

cagccatcga ccctagagac attgacaccg caaaacagcg catacgggca taccaggcaa 660

aactacgcga ccacatcaaa cagcacgacc tgccaaggcg cagacaccga gaacagatta 720

aaatgcgcta aagaagttaa catcatgctg ccaccgccca agcgggaaac attgcgattg 780

gggttgggac gtctagagta gtggcttgag gtcactactc ttttttttgtg cccttttttt 840

ggtccgtcta ttttgccacc acatgcggag gtacgcagtt atgagttcag tttcgctgca 900

ggattttgat gcagagcgaa ttggtctgtt ccacgaggac attaaacgca agtttgatga 960

gctcaagtca aaaaatctga agctggatct tactcgcggt aagccttcgt cggagcagtt 1020

ggatttcgct gatgagctgt tggcgttgcc tggtaagggc gatttcaagg ctgcggatgg 1080

tactgatgtc cgtaactatg gcgggctgga tggcattgtt gatattcgtc agatttgggc 1140

ggatttgctg ggtgttcctg tggagcaggt cttggcgggg gatgcttcga gcttgaacat 1200

catgtttgat gtgatcagct ggtcgtacat tttcggtaac aatgattcgg ttcagccttg 1260

gtcgaaggaa gagaccgtta agtggatttg ccctgttccg ggctatgatc gccatttctc 1320

catcacggag cgtttcggct ttgagatgat ttctgtgcca atgaatgaag acggccctga 1380

tatggatgct gttgaggaat tggtgaagga tcctcaggtt aagggcatgt gggttgtgcc 1440

ggtattttct aacccgactg gtttcacggt gtcggaggac gtcgcaaagc gtctgagcac 1500

gatggaaact gcggcgccgg acttccgcgt ggtgtgggat aacgcttacg ccgttcatac 1560

gttgactgat gagttccctg aggtcatcga catcgttggg cttggtgagg cggcgggtaa 1620

cccgaaccgt ttctgggcgt tcacttctac ttcgaagatc actctcgcgg gtgcgggcgt 1680

gtccttcttc atgactt 1697

<210> 12

<211> 1582

<212> DNA

<213> Artificial sequence

<400> 12

tgatatggat gctgttgagg aattggtgaa ggatcctcag gttaagggca tgtgggttgt 60

gccggtattt tctaacccga ctggtttcac ggtgtcggag gacgtcgcaa agcgtctgag 120

cacgatggaa actgcggcgc cggacttccg cgtggtgtgg gataacgctt acgccgttca 180

tacgttgact gatgagttcc ctgaggtcat cgacatcgtt gggcttggtg aggcggcggg 240

taacccgaac cgtttctggg cgttcacttc tacttcgaag atcactctcg cgggtgcggg 300

cgtgtccttc ttcatgactt ctgcgaagaa ccgtaagtgg tactccggtc atgcgggtat 360

ccgtggcatt ggccctaaca aggtcaatca gttggctcat gcgcgttact ttggcgatgc 420

tgagggagtg cgcgcggtga tgcgtaagca tgctgcgtcg ttggctccga agttcaacaa 480

ggttctggag attctggatt ctcgccttgc tgagtacggt gtcgcgcagt ggactgtccc 540

tgcgggcggc tacttcattt cccttgatgt ggttcctggt acggcgtctc gcgtggctga 600

gttggctaag gaagccggca tcgcgttgac gggtgcgggt tcgtccttcc cattgcatca 660

ggatccagag aacaaaaatc tccgtttggc gccttctctg cctcctgttg aggaacttga 720

ggttgccatg gatggcgtgg ctacgtgtgt tttgctggca gctgcggagc actacgctag 780

ctagtcaaaa accccaaccc gcacattttt agatttctat tttgtgtaca tagggttcgg 840

aacaaagctt aaaccatccc caattgaaat gtcgttacac acccacatgt ttgaagtgga 900

gcaaaccgaa aaccagtttt ccccaacggc agccgccccc cacgttgaac cttcgaaata 960

gtaggcaaca ccatcaagcg gatcttcatc aagcgaaata gtgattgact cttcaccgtt 1020

ccgcttacaa actgcgttag tgtcgctatt ttccacccac ttgtcacact cgtacccgtt 1080

ttcatttagc catttttcgg catgtcctat tttctcgaac cgggcaggag cgtcagggct 1140

tccgcagccc gctagtagta gtccggctgc aatgatgctt aatgtttttt tcatgaatta 1200

aacatagtac tttgctggta aaaatattgg agaaccccac tggcctacat ggtcagtgggg 1260

ggcatttttg cgtttcaccc ctcaaaaatc atcaccacac ttgcgggatt tccccctgat 1320

ttcccccact cccacaccat tcccagtgga cagtgtggac gtattggaca cattaaacac 1380

attgcgacca ggtaaaacgt catgaccagg tatcgtcaat gttcttgatg aatttccgca 1440

ccgcaggatt atcattcgag gtggaataaa tagcctgcag ctccgctaaa ccaacaggta 1500

gatcataaaa atggcgatac tcaacaccgc tgtaattgag ttttttcgcg gactccggaa 1560

ccagcgcaac acccagtcca ga 1582

<210> 13

<211> 1443

<212> DNA

<213> Artificial sequence

<400> 13

gcttgcatgc ctgcaggtcg actctagagg atccccacgt ctagagtagt ggcttgaggt 60

cactactctt ttttttgtgcc ctttttttgg tccgtctatt ttgccaccac atgcggaggt 120

acgcagttat gagttcagtt tcgctgcagg attttgatgc agagcgaatt ggtctgttcc 180

acgaggacat taaacgcaag tttgatgagc tcaagtcaaa aaatctgaag ctggatctta 240

ctcgcggtaa gccttcgtcg gagcagttgg atttcgctga tgagctgttg gcgttgcctg 300

gtaagggcga tttcaaggct gcggatggta ctgatgtccg taactatggc gggctggatg 360

gcattgttga tattcgtcag atttgggcgg atttgctggg tgttcctgtg gagcaggtct 420

tggcggggga tgcttcgagc ttgaacatca tgtttgatgt gatcagctgg tcgtacattt 480

tcggtaacaa tgattcggtt cagccttggt cgaaggaaga gaccgttaag tggatttgcc 540

ctgttccggg ctatgatcgc catttctcca tcacggagcg tttcggcttt gagatgattt 600

ctgtgccaat gaatgaagac ggccctgata tggatgctgt tgaggaattg gtgaaggatc 660

ctcaggttaa gggcatgtgg gttgtgccgg tattttctaa cccgactggt ttcacggtgt 720

cggaggacgt cgcaaagcgt ctgagcacga tggaaactgc ggcgccggac ttccgcgtgg 780

tgtgggataa cgcttacgcc gttcatacgt tgactgatga gttccctgag gtcatcgaca 840

tcgttgggct tggtgaggcg gcgggtaacc cgaaccgttt ctgggcgttc acttctactt 900

cgaagatcac tctcgcgggt gcgggcgtgt ccttcttcat gacttctgcg aagaaccgta 960

agtggtactc cggtcatgcg ggtatccgtg gcattggccc taacaaggtc aatcagttgg 1020

ctcatgcgcg ttactttggc gatgctgagg gagtgcgcgc ggtgatgcgt aagcatgctg 1080

cgtcgttggc tccgaagttc aacaaggttc tggagattct ggattctcgc cttgctgagt 1140

acggtgtcgc gcagtggact gtccctgcgg gcggctactt catttccctt gatgtggttc 1200

ctggtacggc gtctcgcgtg gctgagttgg ctaaggaagc cggcatcgcg ttgacgggtg 1260

cgggttcgtc cttcccattg catcaggatc cagagaacaa aaatctccgt ttggcgcctt 1320

ctctgcctcc tgttgaggaa cttgaggttg ccatggatgg cgtggctacg tgtgttttgc 1380

tggcagctgc ggagcactac gctagctagg ttttggcgga tgagagaaga ttttcagcct 1440

gat 1443

<210> 14

<211> 1443

<212> DNA

<213> Artificial sequence

<400> 14

gcttgcatgc ctgcaggtcg actctagagg atccccacgt ctagagtagt ggcttgaggt 60

cactactctt ttttttgtgcc ctttttttgg tccgtctatt ttgccaccac atgcggaggt 120

acgcagttat gagttcagtt tcgctgcagg attttgatgc agagcgaatt ggtctgttcc 180

acgaggacat taaacgcaag tttgatgagc tcaagtcaaa aaatctgaag ctggatctta 240

ctcgcggtaa gccttcgtcg gagcagttgg atttcgctga tgagctgttg gcgttgcctg 300

gtaagggcga tttcaaggct gcggatggta ctgatgtccg taactatggc gggctggatg 360

gcattgttga tattcgtcag atttgggcgg atttgctggg tgttcctgtg gagcaggtct 420

tggcggggga tgcttcgagc ttgaacatca tgtttgatgt gatcagctgg tcgtacattt 480

tcggtaacaa tgattcggtt cagccttggt cgaaggaaga gaccgttaag tggatttgcc 540

ctgttccggg ctatgatcgc catttctcca tcacggagcg tttcggcttt gagatgattt 600

ctgtgccaat gaatgaagac ggccctgata tggatgctgt tgaggaattg gtgaaggatc 660

ctcaggttaa gggcatgtgg gttgtgccgg tattttctaa cccgactggt ttcacggtgt 720

cggaggacgt cgcaaagcgt ctgagcacga tggaaactgc ggcgccggac ttccgcgtgg 780

tgtgggataa cgcttacgcc gttcatacgt tgactgatga gttccctgag gtcatcgaca 840

tcgttgggct tggtgaggcg acgggtaacc cgaaccgttt ctgggcgttc acttctactt 900

cgaagatcac tctcgcgggt gcgggcgtgt ccttcttcat gacttctgcg aagaaccgta 960

agtggtactc cggtcatgcg ggtatccgtg gcattggccc taacaaggtc aatcagttgg 1020

ctcatgcgcg ttactttggc gatgctgagg gagtgcgcgc ggtgatgcgt aagcatgctg 1080

cgtcgttggc tccgaagttc aacaaggttc tggagattct ggattctcgc cttgctgagt 1140

acggtgtcgc gcagtggact gtccctgcgg gcggctactt catttccctt gatgtggttc 1200

ctggtacggc gtctcgcgtg gctgagttgg ctaaggaagc cggcatcgcg ttgacgggtg 1260

cgggttcgtc cttcccattg catcaggatc cagagaacaa aaatctccgt ttggcgcctt 1320

ctctgcctcc tgttgaggaa cttgaggttg ccatggatgg cgtggctacg tgtgttttgc 1380

tggcagctgc ggagcactac gctagctagg ttttggcgga tgagagaaga ttttcagcct 1440

gat 1443

<210> 15

<211> 1482

<212> DNA

<213> Artificial sequence

<400> 15

agcggataac aatttcacac aggaaacaga attaattaag cttgcatgcc tgcaggtcga 60

ctctagagga tccccacgtc tagagtagtg gcttgaggtc actactcttt ttttgtgccc 120

ttttttttggt ccgtctattt tgccaccaca tgcggaggta cgcagttatg agttcagttt 180

cgctgcagga ttttgatgca gagcgaattg gtctgttcca cgaggacatt aaacgcaagt 240

ttgatgagct caagtcaaaa aatctgaagc tggatcttac tcgcggtaag ccttcgtcgg 300

agcagttgga tttcgctgat gagctgttgg cgttgcctgg taagggcgat ttcaaggctg 360

cggatggtac tgatgtccgt aactatggcg ggctggatgg cattgttgat attcgtcaga 420

tttgggcgga tttgctgggt gttcctgtgg agcaggtctt ggcgggggat gcttcgagct 480

tgaacatcat gtttgatgtg atcagctggt cgtacatttt cggtaacaat gattcggttc 540

agccttggtc gaaggaagag accgttaagt ggatttgccc tgttccgggc tatgatcgcc 600

atttctccat cacggagcgt ttcggctttg agatgatttc tgtgccaatg aatgaagacg 660

gccctgatat ggatgctgtt gaggaattgg tgaaggatcc tcaggttaag ggcatgtggg 720

ttgtgccggt attttctaac ccgactggtt tcacggtgtc ggaggacgtc gcaaagcgtc 780

tgagcacgat ggaaactgcg gcgccggact tccgcgtggt gtgggataac gcttacgccg 840

ttcatacgtt gactgatgag ttccctgagg tcatcgacat cgttggggctt ggtgaggcgg 900

cgggtaaccc gaaccgtttc tgggcgttca cttctacttc gaagatcact ctcgcgggtg 960

cgggcgtgtc cttcttcatg acttctgcga agaaccgtaa gtggtactcc ggtcatgcgg 1020

gtatccgtgg cattggccct aacaaggtca atcagttggc tcatgcgcgt tactttggcg 1080

atgctgaggg agtgcgcgcg gtgatgcgta agcatgctgc gtcgttggct ccgaagttca 1140

acaaggttct ggagattctg gattctcgcc ttgctgagta cggtgtcgcg cagtggactg 1200

tccctgcggg cggctacttc atttcccttg atgtggttcc tggtacggcg tctcgcgtgg 1260

ctgagttggc taaggaagcc ggcatcgcgt tgacgggtgc gggttcgtcc ttcccattgc 1320

atcaggatcc agagaacaaa aatctccgtt tggcgccttc tctgcctcct gttgaggaac 1380

ttgaggttgc catggatggc gtggctacgt gtgttttgct ggcagctgcg gagcactacg 1440

ctagctaggt tttggcggat gagagaagat tttcagcctg at 1482

<210> 16

<211> 1493

<212> DNA

<213> Artificial sequence

<400> 16

cagtgccaag cttgcatgcc tgcaggtcga ctctagagaa ttaccggcga tggggcaaac 60

caagtggccg aagagcccct aactgagaga ttctcggtca tatgggacgg tttccagacc 120

tcaaaaccat cccatatgac cgacggcagt gaaattcggc gttcattggt cacctggttt 180

gggcttgcag ggactcacca gagaaaagca cacatttttgt cccttaacca ccaaagagaa 240

tccccaatac atgaaaaaac cagcttccca ccgaagtgag aagctggttt ggtttgcgga 300

ggatagggga tttgaacccc tgagggattg ctcccaaccc gcgttccagg cgagcgacat 360

aggccgctag tcgaatcctc cagctagaac ggctgcaaca catggctgct ttgttctggg 420

gattagatta cacaaaagtc gtttagaaac tcaaatccgc tcgcagttgg cgttttctgg 480

ggcggttcag ctagagttat gcgaaggatc ccgtgcggcg tttatcttgt gaactccccc 540

agggcaggaa tgcagcaagg gtcagcgagc tctgacgggt gcgcggggtc ccctaaaacg 600

tctagagtag tggcttgagg tcactactct ttttttgtgc ccttttttttg gtccgtctat 660

tttgccacca catgcggagg tacgcagtta gtgaataccg cggaaactgc acattggatt 720

aaccgtttgc tgccgggtca gccggagttt caccaggttg gcgcgtttaa agtggcgggt 780

tacacgcttg atgatgagtc aattgcgtgt tccgtcgact tcgggcgcgt caacacgggc 840

ctggtcaccg agacaggcgc ggaaaccgtc gatgtgcgaa gtgagatttt gagcctggcc 900

agggccgacg tgtccgtgcc tgggcgcgcc gtcggcgctg ctgcaacaat gcttctcgac 960

gcctctctct ccttcaaatc cgcccccgat tccagtgtca ctcccatgca tgcccaacca 1020

ggtcagatcc tgcctggtgt gggtatcatg gcgaatcttc cccaagaggg tttttctgtg 1080

gtgcacggaa ttttggcgga tccccgcatt tggggcccgg agattccgta tgttcgtgaa 1140

gaagcaggtc aagtaaatgt ggaagcaccc gacgaggctg gcgatctagc ccgattgacg 1200

ttgcctctgc agttgatcct gctcacggag gaagagtttg ccatcgctct caacgagggc 1260

agcgacgtca tgttcgaacg aatggcggag caagaagtcg atcttctgga tctgaaacgt 1320

taagacggta ggctcaattc cgtggctttg tatagcaagt atcgaccggc aagttttggt 1380

gaactagttg ggcagtcgca agtgactgac cctctgtccg cagctttgga tagcgggcgc 1440

atcaaccatg cgtacgggta ccgagctcga attcgtaatc atggtcatag ctg 1493

Claims (10)

1. a method for preparing L-valine, is characterized in that, described method comprises utilizing the recombinant microorganism of expressing protein to produce L-valine; The protein is any of the following: A1) the amino acid sequence is the protein of SEQ ID No.4; A2) A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.4; A3) A fusion protein with the same function obtained by linking a tag to the N-terminus and/or C-terminus of A1) or A2). 2. Any protein of claim 1. 3. nucleic acid molecule, it is characterised in that the nucleic acid molecule is any of the following: B1) a nucleic acid molecule encoding any one of the proteins of claim 1 or 2; B2) the coding sequence is the DNA molecule shown in SEQ ID No.3; B3) The nucleotide sequence is the DNA molecule shown in SEQ ID No.3. 4. biological material, it is characterised in that the biological material is any of the following: C1) an expression cassette containing any one of the nucleic acid molecules of claim 3; C2) a recombinant vector containing any one of the nucleic acid molecules described in claim 3, or a recombinant vector containing any one of the expression cassettes described in C1); C3) a recombinant microorganism comprising any one of the nucleic acid molecules described in claim 3, or a recombinant microorganism comprising any one of the expression cassettes described in C1), or a recombinant microorganism comprising any one of the recombinant vectors described in C2). 5. Any of the following applications of any one of D1)-D8): F1) the application of any one of D1)-D8) in regulating the production of L-valine of microorganisms; F2) the application of any one of D1)-D8) in the construction of L-valine-producing genetically engineered bacteria; F3) the application of any one of D1)-D8) in the preparation of L-valine; Wherein, described D1)-D8) are: D1) any protein described in claim 1 or 2; D2) any nucleic acid molecule of claim 3; D3) any biological material described in claim 4; D4) DNA molecule whose nucleotide sequence is SEQ ID No.1; D5) The nucleotide sequence shown in SEQ ID No.1 is modified and/or one or several nucleotides are substituted and/or deleted and/or added to obtain the DNA molecule shown in SEQ ID No.1. DNA molecules that are more than 90% identical and have the same function; D6) an expression cassette comprising the DNA molecule described in D4) or D5); D7) a recombinant vector containing the DNA molecule described in D4) or D5), or a recombinant vector containing the expression cassette described in D6); D8) a recombinant microorganism containing the DNA molecule described in D4) or D5), or a recombinant microorganism containing the expression cassette described in D6), or a recombinant microorganism containing the recombinant vector described in D7). 6. a method that improves the output of microorganism L-valine, it is characterised in that the method comprises any of the following: E1) improve the expression amount or the content of any nucleic acid molecule described in claim 3 in the target microorganism, obtain the microorganism whose output of L-valine is higher than the target microorganism; E2) improve the expression amount or content of the DNA molecule described in D4) or D5) in claim 5 in the target microorganism, and obtain a microorganism whose output of L-valine is higher than that of the target microorganism; E3) mutating the DNA molecule whose nucleotide sequence is SEQ ID No. 1 in the target microorganism to obtain a microorganism whose output of L-valine is higher than that of the target microorganism. 7. The method of claim 6, wherein the mutation is a point mutation. 8. The method according to claim 7, wherein the point mutation is to mutate the alanine residue at position 245 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to another amino acid Residues. The method according to claim 7 or 8, wherein the point mutation is to mutate the alanine residue at position 245 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to threonine acid residue. 10. a method for constructing a recombinant microorganism described in claim 4 or 5, wherein the method comprises at least any of the following: F1) any nucleic acid molecule described in claim 3 is introduced into the microorganism of interest, obtains described recombinant microorganism; F2) introduce the DNA molecule shown in SEQ ID No.1 into the target microorganism to obtain the recombinant microorganism; F3) Editing the DNA molecule shown in SEQ ID No. 1 by means of gene editing, so that the target microorganism contains the DNA molecule shown in SEQ ID No. 3.