CN114539367A - CEY17_ RS11900 gene mutant and application thereof in preparation of L-valine - Google Patents

CEY17_ RS11900 gene mutant and application thereof in preparation of L-valine Download PDF

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CN114539367A
CN114539367A CN202210137842.6A CN202210137842A CN114539367A CN 114539367 A CN114539367 A CN 114539367A CN 202210137842 A CN202210137842 A CN 202210137842A CN 114539367 A CN114539367 A CN 114539367A
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贾慧萍
孟刚
魏爱英
赵春光
杨立鹏
蔡卫卫
毕国东
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Ningxia Eppen Biotech Co ltd
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Abstract

The invention discloses a CEY17_ RS11900 gene mutant and application thereof in preparing L-valine. In particular discloses a CEY17_ RS11900 gene and a mutant CEY17_ RS11900 thereofC260T，GC326‑327TA(SEQ ID No.3) in the construction of L-valine-producing genetic engineering bacteria and the regulation of microbial L-valine yield. Experiments show that the CEY17_ RS11900 gene or mutant gene thereof is subjected to mutation transformation on the coding region of the CEY17_ RS11900 gene or is over-expressed in production bacteria,contributes to the improvement of the yield and the conversion rate of the L-valine. The gene engineering bacterium for producing L-valine is constructed by using the CEY17_ RS11900 gene and the variant thereof, and a high-yield and high-quality strain which meets the industrial production can be cultivated.

Description

CEY17_ RS11900 gene mutant and application thereof in preparation of L-valine
Technical Field
The invention belongs to the technical field of microbial mutation or genetic engineering, and particularly relates to a CEY17_ RS11900 gene mutant and application thereof in preparation of L-valine.
Background
L-Valine (L-Valine), also known as L-2-Amino-3-methylbutyric Acid (L-2-Amino-3-methylbutanoic Acid), is one of the Branched Chain Amino Acids (BCAA), which cannot be synthesized by humans and animals themselves. L-valine is one of eight essential amino acids, has effects of promoting protein synthesis, inhibiting protein decomposition, enhancing immunity, and correcting negative nitrogen balance caused by operation, wound, infection, etc. In addition, L-valine also has the effects of resisting central fatigue, resisting peripheral fatigue, delaying exercise-induced fatigue and accelerating the repair of an organism after exercise, so that the L-valine has wide application and commercial value in the food and medicine industries. L-valine has a large market demand because it has a specific physiological function, so that the production of L-valine is of great interest.
At present, the production method of L-valine mainly comprises an extraction method, a chemical synthesis method and a fermentation method. The extraction method and the chemical synthesis method have the defects of limited raw material sources, high production cost, low yield and serious pollution, and are difficult to realize industrial production. The method for producing L-valine by the direct microbial fermentation method has the advantages of wide raw material source, low cost, mild reaction conditions, easy realization of large-scale production and the like, and is a very economic and efficient production method. The strain with high yield obtained in industrial fermentation is important for the fermentation production of L-valine, is the core of the whole L-valine fermentation industry, and is an important factor for determining the industrial value of a fermentation product. With the increasing market demand of L-valine, breeding high-yield and stable production strains, promoting the accumulation of L-valine in microorganisms, and further improving the yield of L-valine are always hot spots of the technical development and fermentation engineering research of the L-valine fermentation industry, and have important significance for promoting the industrialization process of L-valine.
Disclosure of Invention
The technical problem to be solved by the present invention is how to increase the production of L-valine by a microorganism by genetic modification of a gene, and the technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein will be clearly understood by those skilled in the art from the following description.
To solve the above technical problems, the present invention provides a protein, named CEY17_ RS11900A87V，G109VThe protein may be any one of the following:
A1) a protein having an amino acid sequence of SEQ ID No. 4;
A2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.4, has more than 80% of identity with the protein shown in A1), and has the same function;
A3) a fusion protein with the same function obtained by connecting labels at the N end and/or the C end of A1) or A2).
The invention also provides a gene encoding the protein CEY17_ RS11900A87V，G109VNucleic acid molecule of (4) under the name CEY17_ RS11900C260T，GC326-327TA。
Further, the nucleic acid molecule CEY17_ RS11900C260T，GC326-327TACan be a DNA molecule with a coding sequence shown in SEQ ID No.3 or a DNA molecule with a nucleotide sequence shown in SEQ ID No. 3.
The DNA molecule shown in SEQ ID No.3 is also CEY17_ RS11900 described in the inventionC260T，GC326-327TAA gene.
DNA molecule shown as SEQ ID No.3 (CEY17_ RS11900)C260T，GC326-327TA) Encoding the protein CEY17_ RS11900 shown in SEQ ID No.4A87V，G109V。
The protein CEY17_ RS11900A87V，G109VValine (V) at position 87 in the amino acid sequence (SEQ ID No.4) is mutated from alanine (A) and valine (V) at position 109 is mutated from glycine (G).
The invention also provides a biomaterial, which can be any one of the following:
C1) containing the nucleic acid molecule of claim CEY17_ RS11900C260T，GC326-327TAThe expression cassette of (1);
C2) containing the nucleic acid molecule CEY17_ RS11900C260T，GC326-327TAOr a recombinant vector containing the expression cassette of C1);
C3) containing the nucleic acid molecule CEY17_ RS11900C260T，GC326-327TAOr a recombinant microorganism containing C1) the expression cassette or a recombinant microorganism containing C2) the recombinant vector.
The invention also provides an application of any one of D1) -D8) in constructing a genetically engineered bacterium for producing L-valine, and/or an application in preparing the L-valine, and/or an application in regulating the yield of the L-valine of a microorganism, wherein the D1) -D8) is as follows:
D1) the protein CEY17_ RS11900A87V，G109V；
D2) The nucleic acid molecule CEY17_ RS11900C260T，GC326-327TA；
D3) The biomaterial of claim 4;
D4) a DNA molecule with a nucleotide sequence of SEQ ID No. 1;
D5) a DNA molecule which is obtained by modifying and/or substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID No.1, has more than 90 percent of identity with the DNA molecule shown in SEQ ID No.1 and has the same function;
D6) an expression cassette comprising the DNA molecule of 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_ RS11900 gene.
The DNA molecule shown in SEQ ID No.1 (CEY17_ RS11900 gene) encodes the protein shown in SEQ ID No. 2.
Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost and the Lambda ratio are set to 11, 1 and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of the amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 80% or greater identity can 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 90% or greater identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
The regulation of the production of L-valine by the microorganism as described herein may be carried out by increasing or decreasing the accumulation of L-valine in the microorganism (i.e., by promoting or inhibiting the biosynthesis of L-valine).
The present invention also provides a method for increasing the production of L-valine by a microorganism, which comprises any one of:
E1) increasing the nucleic acid molecule CEY17_ RS11900 in a microorganism of interestC260T，GC326-327TAThe expression amount or content of (a), a microorganism having a higher L-valine yield than the target microorganism is obtained;
E2) increasing the expression level or the content of the DNA molecule D4) or D5) in the target microorganism to obtain a microorganism with higher L-valine yield than the target microorganism;
E3) and (2) carrying out mutation (such as base substitution, base insertion or base deletion) on the DNA molecule with the nucleotide sequence of SEQ ID No.1 in the target microorganism to obtain the microorganism with higher L-valine yield than the target microorganism.
In the above method, the mutation may be a mutation of alanine residue at position 87 to valine residue and a mutation of glycine residue at position 109 to valine residue of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1.
In the above method, the mutation can be that the nucleotide C at the 260 th site in the DNA molecule shown in SEQ ID No.1 is mutated into T and the nucleotide GC at the 326 th and 327 th sites is mutated into TA by a site-directed mutagenesis method.
The mutation is to change one or several bases in the gene by site-directed mutation, which results in the change of the amino acid composition of the corresponding protein, the generation of new protein or the generation of new function of the original protein, i.e., the site-directed mutation of the gene. Techniques for site-directed mutagenesis of genes, 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.
Vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), or viral vectors. Specifically, the compound may be pK18mobsacB or pXMJ 19.
Herein, the microorganism may be yeast, bacteria, algae or fungi. The bacteria may be derived from Brevibacterium (Brevibacterium), Corynebacterium (Corynebacterium), Escherichia (Escherichia), Aerobacter (Aerobacter), Micrococcus (Micrococcus), Flavobacterium (Flavobacterium), or Bacillus (Bacillus), among others.
Specifically, the microorganism may be, but is not limited to, Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Micrococcus glutamicum (Micrococcus glutamicum), Brevibacterium ammoniagenes (Brevibacterium ammoniagenes), Escherichia coli (Escherichia coli), or Aerobacter aerogenes (Aerogenes).
Specifically, the microorganism may be Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260, or Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 14067.
Herein, the recombinant vector may specifically be the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TA、pK18-CEY17_RS11900OE、pK18-CEY17_RS11900C260T，GC326-327TAOE, pXMJ19-CEY17_ RS11900 and/or pXMJ19-CEY17_ RS11900C260T，GC326-327TA。
The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAThe recombinant vector is obtained by replacing a fragment (small fragment) between Xbal I recognition sites and BamH I recognition sites of a pK18mobsacB vector with a DNA fragment shown in the 37 th to 1326 th sites of SEQ ID No.5 in a sequence table and keeping other sequences of the pK18mobsacB vector unchanged. The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAContains mutant gene CEY17_ RS11900 shown in SEQ ID No.3C260T ，GC326-327TAThe DNA molecule shown in positions 1 to 474 of (1).
The recombinant vector pK18-CEY17_ RS11900OE is used for integrating a foreign gene CEY17_ RS11900 into a host chromosome and overexpressing a wild-type CEY17_ RS11900 gene in a production strain.
The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAOE for use of exogenous gene CEY17_ RS11900C260T，GC326-327TAIntegration into host chromosome, overexpression of mutant gene CEY17_ RS11900 in production bacteriaC260T，GC326-327TA。
The recombinant vector pXMJ19-CEY17_ RS11900 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of a pXMJ19 vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.13 in a sequence table and keeping other sequences of the pXMJ19 vector unchanged. The recombinant vector pXMJ19-CEY17_ RS11900 is used for extrachromosomal expression of the foreign gene CEY17_ RS11900 by a plasmid, and further overexpression of the wild-type CEY17_ RS11900 gene in a production strain.
The recombinant vector pXMJ19-CEY17_ RS11900C260T，GC326-327TAThe recombinant expression vector is obtained by replacing a fragment (small fragment) between EcoRI recognition sites and KpnI recognition sites of the pXMJ19 vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.14 in a sequence table and keeping other sequences of the pXMJ19 vector unchanged. The recombinant vector pXMJ19-CEY17_ RS11900C260T，GC326-327TAFor introducing exogenous gene CEY17_ RS11900C260T，GC326-327TAThe mutant CEY17_ RS11900 is overexpressed in a producer by extrachromosomal expression of a plasmidC260T，GC326-327TAA gene.
The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TA、pK18-CEY17_RS11900OE、pK18-CEY17_RS11900C260T，GC326-327TAOE, pXMJ19-CEY17_ RS11900 and pXMJ19-CEY17_ RS11900C260T ，GC326-327TAAre within the scope of the invention.
Herein, the recombinant microorganism may be specifically recombinant bacteria YPV-079, YPV-080, YPV-081, YPV-082 or YPV-083.
The recombinant bacterium YPV-079 is prepared by introducing the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAThe recombinant bacterium obtained by transforming Corynebacterium glutamicum (CGMCC No. 21260) contains a mutated gene CEY17_ RS11900 shown in SEQ ID No.3 in YPV-079C260T，GC326-327TA。
The recombinant strain YPV-080 contains double copies of a CEY17_ RS11900 gene shown in SEQ ID No. 1; specifically, the recombinant bacterium YPV-080 is obtained by replacing the spacer of the upper homologous arm CEY17_02570 and the lower homologous arm CEY17_02575 in the genome of Corynebacterium glutamicum CGMCC No.21260 with a CEY17_ RS11900 gene and keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged. The recombinant strain containing the double-copy CEY17_ RS11900 gene can obviously and stably improve the expression quantity of the CEY17_ RS11900 gene. The recombinant strain YPV-080 is an engineering strain for over-expressing a wild-type CEY17_ RS11900 gene on a genome, and is obtained by introducing the recombinant vector pK18-CEY17_ RS11900OE into escherichia coli DH5 alpha.
The recombinant bacterium YPV-081 contains mutant CEY17_ RS11900 shown in SEQ ID No.3C260T，GC326-327TAA gene; specifically, the recombinant bacterium YPV-081 is obtained by replacing the spacer of the upper homologous arm CEY17_02570 and the lower homologous arm CEY17_02575 in the genome of Corynebacterium glutamicum CGMCC No.21260 with the spacer of CEY17_ RS11900C260T，GC326-327TAGene, recombinant bacterium obtained by keeping other nucleotide in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged. The recombinant bacterium YPV-081 is a mutant CEY17_ RS11900 overexpressed on genomeC260T，GC326-327TAThe genetic engineering bacteria is obtained by using the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAAnd OE is introduced into a recombinant strain obtained by introducing Escherichia coli DH5 alpha.
The recombinant strain YPV-082 contains a CEY17_ RS11900 gene shown in SEQ ID No.1, and the recombinant strain YPV-082 is an engineering strain for over-expressing a wild-type CEY17_ RS11900 gene on a plasmid, namely, the plasmid pXMJ19-CEY17_ RS11900 is over-expressed outside a chromosome.
The recombinant bacterium YPV-083 contains mutant CEY17_ RS11900 shown in SEQ ID No.3C260T，GC326-327TAThe gene, recombinant bacterium YPV-083 is over-expressed mutant CEY17_ RS11900 on plasmidC260T，GC326-327TAGenetically engineered bacteria, i.e. plasmid pXMJ19-CEY17_ RS11900C260T，GC326-327TAThe overexpression is carried out extrachromosomally.
The recombinant bacteria YPV-079, YPV-080, YPV-081, YPV-082 and YPV-083 are all in the protection scope of the invention.
The present invention also provides a method for producing L-valine, which comprises producing L-valine using any of the recombinant microorganisms described herein.
In the above method, the method may be a fermentation method for producing L-valine, and the recombinant microorganism may be Corynebacterium (Corynebacterium), specifically, Corynebacterium glutamicum (Corynebacterium glutamicum) and variants thereof.
The present invention also provides a method for constructing the recombinant microorganism, the method comprising at least any one of:
F1) subjecting said nucleic acid molecule to CEY17_ RS11900C260T，GC326-327TAIntroducing a target microorganism to obtain the recombinant microorganism;
F2) introducing a DNA molecule shown in SEQ ID No.1 into a target microorganism to obtain the recombinant microorganism;
F3) the DNA molecule shown in SEQ ID No.1 is edited by a gene editing means, so that the target microorganism contains the DNA molecule shown in SEQ ID No. 3.
The introduction may be carried out by transforming the host bacterium with the vector carrying the DNA molecule of the present invention by any known transformation method such as chemical transformation or electroporation. The introduced DNA molecule may be in single or multiple copies. The introduction may be the integration of the foreign gene into the host chromosome or the extrachromosomal expression from a plasmid.
The invention firstly introduces mutation into a CEY17_ RS11900 gene coding region (SEQ ID No.1) of Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 (sequence confirmation confirms that a wild-type CEY17_ RS11900 gene is reserved on a strain chromosome) in an allelic gene replacement mode, and constructs genetically engineered bacteria YPV-079 containing the mutation (C-T, GC-TA). For further research, the wild-type CEY17_ RS11900 gene or the mutant gene CEY17_ RS11900 gene is over-expressed in the production bacteriaC260T，GC326-327TACan increase the yield of L-valine, integrate a foreign gene into a host chromosome or express the foreign gene outside the host chromosome by a plasmid, and construct a gene which overexpresses CEY17_ RS11900 or CEY17_ RS11900 on the genome and the plasmidC260T，GC326-327TAGenetically engineered bacteria YPV-080, YPV-081, YPV-082 and YPV-083. Experiments show that the CEY17_ RS11900 gene and the variant thereof are involved in the biosynthesis of L-valine, and the accumulation of L-valine in microorganisms can be regulated by overexpression or knockout or site-directed mutation of the CEY17_ RS11900 gene. Mutation of the coding region of the CEY17_ RS11900 gene or overexpression of the CEY17_ RS11900 gene or its mutated gene CEY17_ RS11900 in a producerC260T，GC326-327TAThe gene is beneficial to improving the yield and the conversion rate of the L-valine, and the knockout or the weakening of the CEY17_ RS11900 gene is not beneficial to the accumulation of the L-valine. The CEY17_ RS11900 gene and variants thereof (e.g., CEY17_ RS11900) can be utilizedC260T，GC326-327TAGene) to construct a genetically engineered strain for producing L-valine to promote the increase of the yield of L-valine and to cultivate the strain meeting the industrial requirementsHigh-yield and high-quality strains are produced in a chemical way.
Deposit description
The strain name is as follows: corynebacterium glutamicum
Latin name: corynebacterium glutamicum
And (3) classification and naming: corynebacterium glutamicum (Corynebacterium glutamicum)
The strain number is as follows: YPFV1
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No.3 of Beijing market facing Yang district
The preservation date is as follows: year 2020, 11 and 30
Registration number of the preservation center: CGMCC No.21260
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.
Corynebacterium glutamicum (Corynebacterium glutamicum) YPFV1 CGMCC No.21260 in the following examples was obtained by mutagenesis of Corynebacterium glutamicum ATCC15168, and was deposited in China general microbiological culture Collection center (CGMCC, address: Sai Lu No.3, institute of microbiology, Ministry of China, GmbH, Ind., Tokyo, N.O.P.C.) on 11/30/2020, and the accession number of CGMCC No. 21260. Corynebacterium glutamicum YPFV1 (also called Corynebacterium glutamicum CGMCC No. 21260).
Example 1 construction of a recombinant vector comprising a fragment of the coding region of the mutated CEY17_ RS11900 Gene
According to a Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC14067 genome sequence published by NCBI, two pairs of primers for amplifying CEY17_ RS11900 gene coding region are designed and synthesized, and mutation is introduced in a mode of allele replacement in a CEY17_ RS11900 gene coding region (SEQ ID No.1) of the Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 (after sequencing confirmation, a wild-type CEY17_ RS11900 gene is remained on the chromosome of the strain), wherein the mutation is that the 260 th cytosine (C) in the nucleotide sequence (SEQ ID No.1) of the CEY17_ RS11900 gene is mutated into thymine (GC), the 326 th guanine cytosine (119327) is mutated into Thymine Adenine (TA), and a DNA molecule (mutated CEY17_ RS 00 gene and the CEY17_ RS 00 gene) shown in SEQ ID No.3 are obtainedC260T ，GC326-327TA)。
Wherein, the DNA molecule shown in SEQ ID No.1 encodes the protein (the protein is named as protein CEY17_ RS11900) with the amino acid sequence of SEQ ID No. 2.
The DNA molecule shown in SEQ ID No.3 encodes the mutant protein with the amino acid sequence of SEQ ID No.4 (the name of the mutant protein is CEY17_ RS11900A87V，G109V. The mutant protein CEY17_ RS11900A87V，G109VValine (V) at position 87 in the amino acid sequence (SEQ ID No.4) was mutated from alanine (A) and valine (V) at position 109 was mutated from glycine (G).
Vector construction is carried out by adopting NEBuilder recombination technology, the CEY17_ RS11900 gene is subjected to site-directed mutagenesis, primers are designed as follows (synthesized by Shanghai invitrogen company), and bases in bold type are mutation positions:
P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTCGCAACAGATGAAACTCC-3'，
Figure BDA0003505070650000081
Figure BDA0003505070650000082
P4:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGCGGGCTAATCCTGGATCT-3'。
the construction method comprises the following steps: using Corynebacterium glutamicum ATCC14067 as a template, primers P1 and P2, and P3 and P4, respectively, were subjected to PCR amplification to obtain two DNA fragments (CEY17_ RS11900 Up and CEY17_ RS11900 Down) having mutated bases and coding regions of the CEY17_ RS11900 gene of 730bp and 706bp, respectively.
The PCR amplification system is as follows: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg2+4. mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L;
the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5min, (denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 40s, 30 cycles), and over-extension at 72 ℃ for 10 min.
The two DNA fragments (CEY17_ RS11900 Up and CEY17_ RS11900 Down) were separated and purified by agarose gel electrophoresis, and then subjected to DNA Assembly reaction with a plasmid pK18mobsacB (available from Addgene, Inc., containing a kanamycin resistance marker) purified by digestion (Xbal I/BamH I) with NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, available from NEB, Inc.): the ligation was carried out at 50 ℃ for 30min, and the single clone grown after transformation of DH5 alpha (purchased from TAKARA) with the ligation product was identified by PCR using M13 primer (M13F:5 'TGTAAAACGACGGCCAGT 3', M13R:5 'CAGGAAACAGCTATGACC 3') to obtain pK18-CEY17_ RS11900C260T ，GC326-327TA. The recombinant vector pK18-CEY17_ RS11900 with correct enzyme digestion is usedC260T，GC326-327TASequencing and identifying by a sequencing company, and carrying out sequencing and sequencing on a recombinant vector pK18-CEY17_ RS11900 containing a correct mutation (C-T, GC-TA)C260T，GC326-327TAAnd (5) storing for later use.
Through sequencing identification, the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAThe DNA fragment containing integrated DNA fragments CEY17_ RS11900 Up and CEY17_ RS11900 Down is named CEY17_ RS11900 Up-Down, the DNA fragment of CEY17_ RS11900 Up-Down has the size of 1364bp, the sequence is shown as SEQ ID No.5, and the DNA fragment containsThe mutation site (C-T, GC-TA) is used for introducing nucleic acid modification into a CEY17_ RS11900 gene coding region (SEQ ID No.1) in a strain Corynebacterium glutamicum CGMCC No.21260, wherein the nucleic acid modification specifically comprises the step of mutating cytosine (C) at the 260 th position of SEQ ID No.1 into thymine (T), and Guanine Cytosine (GC) at the 326 th and 327 th positions into Thymine Adenine (TA), and finally leading both alanine (A) at the 87 th position and glycine (G) at the 109 th position of the encoded protein to be mutated into valine (V).
The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAThe recombinant vector is obtained by replacing a fragment (small fragment) between Xbal I recognition sites and BamH I recognition sites of a pK18mobsacB vector with a DNA fragment shown in the 37 th to 1326 th sites of SEQ ID No.5 in a sequence table and keeping other sequences of the pK18mobsacB vector unchanged.
The recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAContains mutant gene CEY17_ RS11900 shown in SEQ ID No.3C260T，GC326-327TAThe DNA molecule shown in positions 1 to 474 of (1).
Example 2 construction of a plasmid containing the Gene CEY17_ RS11900C260T，GC326-327TAOf (4) an engineered strain
The construction method comprises the following steps: the allele of example 1 was replaced with the plasmid (pK18-CEY17_ RS11900)C260T，GC326-327TA) After the strain is transformed into Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 by electric shock, the culture is carried out in a culture medium, the components of the culture medium and the culture conditions are shown in Table 1, and the single colony generated by the culture is respectively identified by the primer P1 and the universal primer M13R (5'-CAGGAAACAGCTATGACC-3') in example 1, and the strain which can amplify a 1371bp (shown as SEQ ID No. 6) size band is a positive strain. The positive strain was cultured on a medium containing 15% sucrose, the single colonies resulting from the culture were cultured on a medium containing kanamycin and a medium not containing kanamycin, respectively, and strains that grew on a medium not containing kanamycin and did not grow on a medium containing kanamycin were further subjected to PCR identification using the following primers (synthesized by Shanghai Invitrogen Co.):
P5:5'-TGCTCCATTCCTGCTCGAGG-3'，
P6:5'-AAGCAGCTAGCCGCACCGTA-3'。
the resulting PCR amplification product (246bp) was subjected to SSCP (Single-Strand transformation Polymorphis) electrophoresis (plasmid pK18-CEY17_ RS11900) after denaturation at 95 ℃ for 10min and ice-cooling for 5minC260T ，GC326-327TAThe amplified fragment is a positive control, the amplified fragment of corynebacterium glutamicum ATCC14067 is a negative control, and water is used as a blank control), the preparation of PAGE of SSCP electrophoresis and electrophoresis conditions are shown in Table 2, and due to different fragment structures and different electrophoresis positions, the strain with the fragment electrophoresis position inconsistent with the negative control fragment position and the positive control fragment position is a strain with successful allelic replacement. The positive strain CEY17_ RS11900 gene segment is amplified by PCR again through a primer P5/P6 and is connected to a PMD19-T vector for sequencing, and the strain with mutation of the base sequence (C-T, GC-TA) is a positive strain with successful allelic replacement through sequence alignment, and is named as YPV-079.
The recombinant strain YPV-079 is prepared by introducing the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAThe recombinant bacterium obtained by transforming Corynebacterium glutamicum (CGMCC No. 21260) contains a mutated gene CEY17_ RS11900 shown in SEQ ID No.3 in YPV-079C260T，GC326-327TA。
TABLE 1 composition of culture Medium and culture conditions
Figure BDA0003505070650000101
TABLE 2 preparation of SSCP electrophoretic PAGE and electrophoresis conditions
Figure BDA0003505070650000102
Example 3 construction of genome overexpression of CEY17_ RS11900 Gene and CEY17_ RS11900C260T，GC326-327TAEngineered strains of genes
Constructing a vector by adopting a NEBuilder recombination technology, designing and synthesizing three pairs of amplified upstream and downstream homologous sequences according to a Corynebacterium glutamicum ATCC14067 genome sequence published by NCBIArm segment and CEY17_ RS11900 or CEY17_ R S11900C260T，GC326-327TAPrimers of gene coding region and promoter region are introduced into Corynebacterium glutamicum CGMCC No.21260 in homologous recombination mode to obtain CEY17_ RS11900 or CEY17_ RS11900C260T，GC326-327TAA gene.
The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):
P7:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTAGTGCCGTGCGTACCCCA-3'，
P8:5'-CTGTCGTTCCTAACGAATAACCCAACCCCAATCGCAATGT-3'，
P9:5'-ACATTGCGATTGGGGTTGGGTTATTCGTTAGGAACGACAG-3'，
P10:5'-GTGCGGGTTGGGGTTTTTGACAGATACGGAAGTGTCCTAC-3'，
P11:5'-GTAGGACACTTCCGTATCTGTCAAAAACCCCAACCCGCAC-3'，
P12:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTTGGTTTAGCGGAGCTGCA-3'。
the construction method comprises the following steps: respectively taking Corynebacterium glutamicum ATCC14067 or YPV-079 as a template, respectively taking primers P7/P8, P9/P10 and P11/P12 to carry out PCR amplification to obtain an upstream homologous arm fragment 795bp (corresponding to a Corynebacterium glutamicum CGMCC No.21260CEY17_ RS02570 gene and a spacer region of CEY17_ RS02575, the sequence of which is shown as SEQ ID No. 7), a CEY17_ RS11900 gene and a promoter fragment 658bp (the sequence of which is shown as SEQ ID No. 8) or CEY17_ RS11900C260T，GC326-327TAThe gene and the promoter fragment 658bp (the sequence is shown as SEQ ID No. 9) and the downstream homologous arm fragment 769bp (corresponding to the Corynebacterium glutamicum CGMCC No.21260CEY17_ RS02575 gene and the spacer of CEY17_ RS02570, the sequence is shown as SEQ ID No. 10).
After the PCR reaction is finished, 3 fragments obtained by amplifying each template are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. The 3 fragments recovered were subjected to DNA Assembly reaction with pK18mobs acB plasmid (purchased from Addgene, containing kanamycin resistance as a selection marker) purified by Xbal I/BamH I digestion with NEBuil der enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB) under the following conditions: connecting at 50 deg.C for 30min, and transferring the connecting productThe single clone that grows after DH5 alpha is identified by PCR with M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain positive integration plasmid (recombinant vector), pK18-CEY17_ RS11900OE, pK18-CEY17_ RS11900C260T，GC326-327TAOE, the positive integration plasmid contains a kanamycin resistance marker, and recombinants with plasmid integrated into the genome can be obtained by kanamycin selection.
The recombinant vector pK18-CEY17_ RS11900OE is used for integrating the exogenous gene CEY17_ RS11900 into the host chromosome and overexpressing the wild-type CEY17_ RS11900 gene in the producer.
Recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAOE for use of exogenous gene CEY17_ RS11900C260T，GC326-327TAIntegration into host chromosome, overexpression of mutant gene CEY17_ RS11900 in production bacteriaC260T，GC326-327TA。
The PCR reaction system is as follows: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg2+mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.
The PCR reaction program is: pre-denaturation at 94 ℃ for 5min and denaturation at 94 ℃ for 30 s; annealing at 52 ℃ for 30 s; extension at 72 ℃ for 60s (30 cycles) and over-extension at 72 ℃ for 10 min.
The correctly sequenced integrative plasmids (pK18-CEY17_ RS11900OE, pK18-CEY17_ RS11900)C260T ，GC326-327TAOE) are respectively electrically transformed into Corynebacterium glutamicum CGMCC No.21260, the Corynebacterium glutamicum CGMCC No.21260 is cultured in a culture medium, the components of the culture medium and culture conditions are shown in Table 1, a single colony generated by the culture is identified by PCR through a P13/P14 primer, a fragment containing 1361bp (a sequence without mutation is shown as SEQ ID No.11, a GC at position 936 and 937 containing the mutation is mutated into TA, a G at position 1003 is mutated into A, and the rest sequences are shown as SEQ ID No.11) is amplified by PCR, the strain is positive, and the strain without amplified fragment is original bacteria. Culturing the positive strain in a culture medium containing 15% sucrose, and performing PCR identification on the single colony generated by culture by using a P15/P16 primer to amplify the size of 1333bp (the sequence without mutation is shown as SEQ ID No.12, the GC of 65-66 th position of the sequence with mutation is TA, and the G of 132 th position of the sequence with mutation is mutated into TA)A, the rest of the bacteria having the sequence of SEQ ID No.12) is CEY17_ RS11900 or CEY17_ RS11900C260T，GC326-327TAPositive strains with genes integrated into the spacer region of the homology arm CEY17_02570 and the lower homology arm CEY17_02575 of Corynebacterium glutamicum CGMCC No.21260 genome were designated YPV-080 (without mutation points) and YPV-081 (with mutation points), respectively.
The recombinant strain YPV-080 contains double copies of a CEY17_ RS11900 gene shown in SEQ ID No. 1; specifically, the recombinant bacterium YPV-080 is obtained by replacing the spacer of the upper homologous arm CEY17_02570 and the lower homologous arm CEY17_02575 in the genome of Corynebacterium glutamicum CGMCC No.21260 with a CEY17_ RS11900 gene and keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged. The recombinant strain containing the double-copy CEY17_ RS11900 gene can obviously and stably improve the expression quantity of the CEY17_ RS11900 gene. The recombinant strain YPV-080 is an engineering strain for over-expressing a wild-type CEY17_ RS11900 gene on a genome, and is obtained by introducing the recombinant vector pK18-CEY17_ RS11900OE into escherichia coli DH5 alpha.
Recombinant strain YPV-081 contains mutant CEY17_ RS11900 shown in SEQ ID No.3C260T，GC326-327TAA gene; specifically, the recombinant bacterium YPV-081 is obtained by replacing the spacer of the upper homologous arm CEY17_02570 and the lower homologous arm CEY17_02575 in the genome of Corynebacterium glutamicum CGMCC No.21260 with the spacer of CEY17_ RS11900C260T，GC326-327TAGene, recombinant bacterium obtained by keeping other nucleotide in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged. The recombinant bacterium YPV-081 is a mutant CEY17_ RS11900 overexpressed on genomeC260T，GC326-327TAThe genetic engineering bacteria is obtained by using the recombinant vector pK18-CEY17_ RS11900C260T，GC326-327TAAnd OE is introduced into a recombinant strain obtained by introducing Escherichia coli DH5 alpha.
The PCR identifying primers are shown below:
p13:5'-CGGTTAGATTTTTTGGCCCC-3' (corresponding to the outside of the upper homology arm CEY17_ RS 02570),
p14:5'-CAAGATTGGGTCGAGCTATT-3' (corresponding to CEY17_ RS11900 promoter region),
p15:5'-ACACAAAATGGGCGGTTAAG-3' (corresponding to the CEY17_ RS11900 coding region),
p16:5'-TCTGGACTGGGTGTTGCGCT-3' (corresponding to the outside of the lower homology arm CEY17_ RS 02575).
Example 4 construction of plasmids overexpressing the CEY17_ RS11900 Gene or CEY17_ RS11900C260T，GC326-327TAEngineered strains of genes
Constructing a vector by adopting a NEBuilder recombinant technology, designing and synthesizing a pair of amplified CEY17_ RS11900 and CEY17_ RS11900 according to a genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBIC260T，GC326-327TAPrimers for the gene coding region and promoter region were designed as follows (synthesized by Shanghai Invitrogen corporation):
P17:5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTATTCGTTAGGAACGACAG-3' (the underlined nucleotide sequence is that on pXMJ 19),
P18:5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCAGATACGGAAGTGTCCTAC-3' (the underlined nucleotide sequence is that on pXMJ 19).
The construction method comprises the following steps: respectively taking Corynebacterium glutamicum ATCC14067 and YPV-079 as templates, and carrying out PCR amplification by using primers P17/P18 to obtain a CEY17_ RS11900 gene and a promoter fragment (the sequence is shown as SEQ ID No. 13) and a CEY17_ RS11900 geneC260T，GC326-327TAGene and its promoter fragment 688bp (sequence shown as SEQ ID No. 14), electrophoresis of the amplified product and column DNA gel recovery kit for purification and recovery, the recovered DNA fragment and the shuttle plasmid pXMJ19 (from Addgene, containing chloramphenicol resistance as a selection marker) recovered by EcoR I/KpnI enzyme digestion, with NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, from NEB) for DNA Assembly reaction under the following conditions: ligation was carried out at 50 ℃ for 30min, and the single clone grown after transformation of DH 5. alpha. with the ligation product was M13R (-48) (5'AGCGGATAAC AATTTCACAC AGGA 3')the/P18 primer is identified by PCR (the sequence without mutation is shown as SEQ ID No.15, the GC position 223-224 position containing mutation is TA, the G position 290 position containing mutation is A, and the rest sequences are shown as SEQ ID No.15) to obtain positive over-expression vectors pXMJ19-CEY17_ RS11900 (containing CEY17_ RS11900 gene) and pXMJ19-CEY17_ RS11900C260T，GC326-327TA(containing CEY17_ RS11900C260T，GC326-327TAGene), willThe plasmid was sent for sequencing. Since the plasmid contains a chloramphenicol resistance marker, whether the plasmid is transformed into a strain or not can be screened by chloramphenicol.
The recombinant vector pXMJ19-CEY17_ RS11900 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoR I and KpnI recognition sites of the pXMJ19 vector with a DNA fragment having a nucleotide sequence of SEQ ID No.13 in the sequence Listing, and leaving the other sequences of the pXM J19 vector unchanged. The recombinant vector pXMJ19-CEY17_ RS11900 is used for extrachromosomal expression of the foreign gene CEY17_ RS11900 by a plasmid, and further overexpression of the wild-type CEY17_ RS11900 gene in a production strain.
Recombinant vector pXMJ19-CEY17_ RS11900C260T，GC326-327TAThe recombinant expression vector is obtained by replacing a fragment (small fragment) between EcoR I and KpnI recognition sites of a pXMJ19 vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.14 in a sequence table and keeping other sequences of the pXMJ19 vector unchanged. The recombinant vector pXMJ19-CEY17_ RS11900C260T，GC326-327TAFor introducing exogenous gene CEY17_ RS11900C260T，GC326-327TAThe mutant CEY17_ RS11900 is overexpressed in a producer by extrachromosomal expression of a plasmidC260T，GC326-327TAA gene.
The PCR reaction system is as follows: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg2+mu.L (25mM), 2. mu.L each of primers (10pM), 0.25. mu.L of Ex Taq (5U/. mu.L), and a total volume of 50. mu.L.
The PCR reaction program is: pre-denaturation at 94 ℃ for 5min and denaturation at 94 ℃ for 30 s; annealing at 52 ℃ for 30 s; extension at 72 ℃ for 60s (30 cycles) and over-extension at 72 ℃ for 10 min.
The correctly sequenced pXMJ19-CEY17_ RS11900 and pXMJ19-CEY17_ RS11900 will be sequencedC260T，GC326-327TAThe plasmids were separately transformed into Corynebacterium glutamicum CGMCC No.21260, cultured in a medium whose composition and culture conditions are shown in Table 1, and single colonies generated by the culture were isolated by primers M13R (-48) (5' -AGCGGATAACAATTTC)ACACAGGA- 3')Performing PCR identification on the/P18 (the sequence without mutation is shown as SEQ ID No.15, the GC mutation at the 223 rd-224 th site containing the mutation is TA, the G mutation at the 290 th site containing the mutation is A, and the rest sequence is shown as SEQ ID No.15), and performing PCR amplification to obtain a fragment with the size of 727bpThe positive strains are named YPV-082 (without mutation points) and YPV-083 (with mutation points).
The recombinant strain YPV-082 contains a CEY17_ RS11900 gene shown in SEQ ID No.1, and the recombinant strain YPV-082 is an engineering strain for over-expressing a wild-type CEY17_ RS11900 gene on a plasmid, namely, the plasmid pXMJ19-CEY17_ RS11900 is over-expressed outside a chromosome.
Recombinant bacterium YPV-083 contains mutant CEY17_ RS11900 shown in SEQ ID No.3C260T，GC326-327TAThe gene, recombinant bacterium YPV-083 is over-expressed mutant CEY17_ RS11900 on plasmidC260T，GC326-327TAGenetically engineered bacteria, i.e. plasmid pXMJ19-CEY17_ RS11900C260T，GC326-327TAThe overexpression is carried out extrachromosomally.
Example 5 construction of an engineered Strain with deletion of the CEY17_ RS11900 Gene on the genome
The vector construction is carried out by adopting a NEBuilder recombination technology, and two pairs of primers for amplifying fragments at two ends of a coding region of a CEY17_ RS11900 gene are synthesized to be used as upstream and downstream homologous arm fragments according to a genome sequence of Corynebacterium glutamicum ATCC14067 published by NCBI. The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):
P19:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATCTGCACTAGAGGGTCTGC-3'，
P20:5'-CCTGCAGTAAACTGACTTCCACAATTAAAAATGGGCGGTT-3'，
P21:5'-AACCGCCCATTTTTAATTGTGGAAGTCAGTTTACTGCAGG-3'，
P22:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCGCTCGTTAGGTATGGAGT-3'。
the construction method comprises the following steps: PCR amplification is carried out by taking Corynebacterium glutamicum ATCC14067 as a template and primers P19/P20 and P21/P22 respectively to obtain an upstream homologous arm fragment 693bp of CEY17_ RS11900 and a downstream homologous arm fragment 697bp of CEY17_ RS 11900.
The amplified product was electrophoresed and purified using a column DNA gel recovery kit, and the recovered DNA fragment was subjected to DNA Assembly reaction with a plasmid pK18mobsacB (purchased from Addgene, containing kanamycin resistance as a selection marker) purified by Xbal I/BamH I digestion using NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB) under the following conditions: the DNA fragment is ligated for 30min at 50 ℃, and a single clone which grows after the ligation product transforms DH5 alpha is identified by PCR using M13 primer to obtain a positive knockout vector pK 18-delta CEY17_ RS11900, and the recombinant plasmid pK 18-delta CEY17_ RS11900 comprises Up-Down DNA 1350bp (the sequence is shown as SEQ ID No. 16) named as delta CEY17_ RS 11900.
The plasmid is sent for sequencing, a correctly sequenced knock-out plasmid pK 18-delta CEY17_ RS11900 is electrically transformed into Corynebacterium glutamicum CGMCC No.21260, the Corynebacterium glutamicum CGMCC No.21260 is cultured in a culture medium, the components and culture conditions of the culture medium are shown in Table 1, and a single colony generated by the culture is identified by PCR through the following primers (synthesized by Shanghai Invitrogen company):
p23:5'-ATCTGCACTAGAGGGTCTGC-3' (corresponding to the coding region of Corynebacterium glutamicum CGMCC No.21260CEY17_ RS 11895),
p24:5'-GCGCTCGTTAGGTATGGAGT-3' (corresponding to the coding region of Corynebacterium glutamicum CGMCC No.21260CEY17_ RS 11905).
The bacterial strain which is simultaneously amplified by the PCR to obtain bands with sizes of 1276bp and 1750bp is a positive bacterial strain, and the bacterial strain which is only amplified to obtain the bands with sizes of 1750bp is a protobacteria. The positive strains are screened on a 15% sucrose medium, then are respectively cultured on a kanamycin-containing medium and a kanamycin-free medium, strains which grow on the kanamycin-free medium and do not grow on the kanamycin-containing medium are selected, PCR identification is further carried out by adopting a P23/P24 primer, and the strain which is amplified to form a 1276bp band is the positive strain CEY17_ RS11900 with the coding region of the CEY17_ RS11900 gene being knocked out. The positive strain CEY17_ RS11900 fragment was PCR amplified again by P23/P24 primer and ligated to pMD19-T vector for sequencing, and the correctly sequenced strain was named YPV-084 (CEY17_ RS11900 gene on the genome of Corynebacterium glutamicum CGMCC No.21260 was knocked out).
EXAMPLE 6L-valine fermentation experiment
The strains constructed in the above examples and the original strain Corynebacterium glutamicum CGMCC No.21260 were subjected to fermentation experiments in a BLBIO-5GC-4-H model fermenter (purchased from Bailan Biotech Co., Ltd., Shanghai) using the media shown in Table 3 and the control process shown in Table 4. Each strain was replicated three times, and the results are shown in Table 5.
As a result, the coding region of the CEY17_ RS11900 gene was site-directed mutated (e.g., to CEY17_ RS11900) in C.glutamicum as shown in Table 5C260T，GC326-327TA) And overexpression, which is beneficial to the improvement of the yield and the transformation rate of the L-valine, and the gene is knocked out or weakened, which is not beneficial to the accumulation of the L-valine.
TABLE 3 fermentation Medium formulation (balance water)
Composition (I) Formulation(s)
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
Antifoam (CB-442) antifoaming agent) 0.5mL/L
70% glucose (base candy) 40g/L
TABLE 4 fermentation control Process
Figure BDA0003505070650000161
TABLE 5L-valine fermentation test results
Bacterial strains OD610 L-valine yield (g/L)
Corynebacterium glutamicum CGMCC No.21260 98.2 84.1
YPV-079 99.2 84.5
YPV-080 98.7 84.9
YPV-081 99.3 85.1
YPV-082 99.2 85.9
YPV-083 99.3 86.2
YPV-084 96.7 82.4
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 examples, it will be appreciated that the invention may 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.
SEQUENCE LISTING
<110> Ningxia Yipin Biotechnology Ltd
<120> CEY17_ RS11900 gene mutant and application thereof in preparation of L-valine
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 474
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 1
atgcgcgtat accttggagc agaccacgct ggtttcgaaa ctaaaaatgc aatcgcagaa 60
caccttaagg cccacggcca cgaagtgatc gactgcggag cccacaccta tgatgcagaa 120
gatgactacc cagccttctg catcgaagca gctagccgca ccgtaaacga cccaggctca 180
ctcggcatcg tcctgggtgg atccggaaac ggcgagcaga tcgccgccaa caaggtcaag 240
ggtgcacgtt gtgcacttgc ttggtctgtt gaaactgcac gcctcgcccg cgagcacaac 300
aatgcgaacc tcatcggcat cggcggccgc atgcactcag aggaagaggc attggcaatt 360
gtcgacgcct tcctcgagca ggaatggagc aacgccgagc gccaccagcg tcgtatcgac 420
atcctcgctg attacgagcg cactggaatc gcacctgtcg ttcctaacga ataa 474
<210> 2
<211> 157
<212> PRT
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 2
Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Phe Glu Thr Lys Asn
1 5 10 15
Ala Ile Ala Glu His Leu Lys Ala His Gly His Glu Val Ile Asp Cys
20 25 30
Gly Ala His Thr Tyr Asp Ala Glu Asp Asp Tyr Pro Ala Phe Cys Ile
35 40 45
Glu Ala Ala Ser Arg Thr Val Asn Asp Pro Gly Ser Leu Gly Ile Val
50 55 60
Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn Lys Val Lys
65 70 75 80
Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Glu Thr Ala Arg Leu Ala
85 90 95
Arg Glu His Asn Asn Ala Asn Leu Ile Gly Ile Gly Gly Arg Met His
100 105 110
Ser Glu Glu Glu Ala Leu Ala Ile Val Asp Ala Phe Leu Glu Gln Glu
115 120 125
Trp Ser Asn Ala Glu Arg His Gln Arg Arg Ile Asp Ile Leu Ala Asp
130 135 140
Tyr Glu Arg Thr Gly Ile Ala Pro Val Val Pro Asn Glu
145 150 155
<210> 3
<211> 474
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
atgcgcgtat accttggagc agaccacgct ggtttcgaaa ctaaaaatgc aatcgcagaa 60
caccttaagg cccacggcca cgaagtgatc gactgcggag cccacaccta tgatgcagaa 120
gatgactacc cagccttctg catcgaagca gctagccgca ccgtaaacga cccaggctca 180
ctcggcatcg tcctgggtgg atccggaaac ggcgagcaga tcgccgccaa caaggtcaag 240
ggtgcacgtt gtgcacttgt ttggtctgtt gaaactgcac gcctcgcccg cgagcacaac 300
aatgcgaacc tcatcggcat cggcgtacgc atgcactcag aggaagaggc attggcaatt 360
gtcgacgcct tcctcgagca ggaatggagc aacgccgagc gccaccagcg tcgtatcgac 420
atcctcgctg attacgagcg cactggaatc gcacctgtcg ttcctaacga ataa 474
<210> 4
<211> 157
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 4
Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Phe Glu Thr Lys Asn
1 5 10 15
Ala Ile Ala Glu His Leu Lys Ala His Gly His Glu Val Ile Asp Cys
20 25 30
Gly Ala His Thr Tyr Asp Ala Glu Asp Asp Tyr Pro Ala Phe Cys Ile
35 40 45
Glu Ala Ala Ser Arg Thr Val Asn Asp Pro Gly Ser Leu Gly Ile Val
50 55 60
Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn Lys Val Lys
65 70 75 80
Gly Ala Arg Cys Ala Leu Val Trp Ser Val Glu Thr Ala Arg Leu Ala
85 90 95
Arg Glu His Asn Asn Ala Asn Leu Ile Gly Ile Gly Val Arg Met His
100 105 110
Ser Glu Glu Glu Ala Leu Ala Ile Val Asp Ala Phe Leu Glu Gln Glu
115 120 125
Trp Ser Asn Ala Glu Arg His Gln Arg Arg Ile Asp Ile Leu Ala Asp
130 135 140
Tyr Glu Arg Thr Gly Ile Ala Pro Val Val Pro Asn Glu
145 150 155
<210> 5
<211> 1364
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctaggtcg caacagatga aactccgaac 60
tactaccctg gcggtaacgt tgcgggtcgc ccagtccctg ctggttggta ctccgagcct 120
tggtgggcaa gcgcattgcg ttccggtctg tggactgcag gttcggtcat gatgttctca 180
gcaatgttta acggcatggc tggtgtcggc tactccgctg cagcctttga aaatggctat 240
ggcgagggct accaagatgg cctggacgca gctggcggag acatgggcga tgctggcgat 300
atgggtgacg ccggagctga catgggcgat gttggtggtg atgctggcgg agacggtggc 360
ggattcttcg acggactctt tggtggtgga gacggcggcg atggcggcgg attcgatttt 420
gacttcgatt tctagcccaa aataatcccc acataaacac aaaatgggcg gttaagttct 480
tggaaaagaa cttaaccgcc catttttaat tgtttattcg ttaggaacga caggtgcgat 540
tccagtgcgc tcgtaatcag cgaggatgtc gatacgacgc tggtggcgct cggcgttgct 600
ccattcctgc tcgaggaagg cgtcgacaat tgccaatgcc tcttcctctg agtgcatgcg 660
tacgccgatg ccgatgaggt tcgcattgtt gtgctcgcgg gcgaggcgtg cagtttcaac 720
agaccaaaca agtgcacaac gtgcaccctt gaccttgttg gcggcgatct gctcgccgtt 780
tccggatcca cccaggacga tgccgagtga gcctgggtcg tttacggtgc ggctagctgc 840
ttcgatgcag aaggctgggt agtcatcttc tgcatcatag gtgtgggctc cgcagtcgat 900
cacttcgtgg ccgtgggcct taaggtgttc tgcgattgca tttttagttt cgaaaccagc 960
gtggtctgct ccaaggtata cgcgcatgga agtcagttta ctgcaggcat tttcacaacg 1020
gcggataccc ggacttttcc cctatttggg ggttcacaaa tcgcaaaata gctcgaccca 1080
atcttggtaa agcggatcgg ctctgactcg ggtaggacac ttccgtatct gtcatgatgc 1140
ggaacgaata aggaaggggg aacggcttcc ccaagtcacc caacgccata gatactcaag 1200
tggcccctgt gtgtagtagc gcaaccataa agcagaaagc agctcttgaa taatgagaat 1260
acccgcagct aggagcaccg tagctgtcca tgatgtgctg tgtggaagat ccaggattag 1320
cccgccgggt accgagctcg aattcgtaat catggtcata gctg 1364
<210> 6
<211> 1371
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctaggtcg caacagatga aactccgaac 60
tactaccctg gcggtaacgt tgcgggtcgc ccagtccctg ctggttggta ctccgagcct 120
tggtgggcaa gcgcattgcg ttccggtctg tggactgcag gttcggtcat gatgttctca 180
gcaatgttta acggcatggc tggtgtcggc tactccgctg cagcctttga aaatggctat 240
ggcgagggct accaagatgg cctggacgca gctggcggag acatgggcga tgctggcgat 300
atgggtgacg ccggagctga catgggcgat gttggtggtg atgctggcgg agacggtggc 360
ggattcttcg acggactctt tggtggtgga gacggcggcg atggcggcgg attcgatttt 420
gacttcgatt tctagcccaa aataatcccc acataaacac aaaatgggcg gttaagttct 480
tggaaaagaa cttaaccgcc catttttaat tgtttattcg ttaggaacga caggtgcgat 540
tccagtgcgc tcgtaatcag cgaggatgtc gatacgacgc tggtggcgct cggcgttgct 600
ccattcctgc tcgaggaagg cgtcgacaat tgccaatgcc tcttcctctg agtgcatgcg 660
tacgccgatg ccgatgaggt tcgcattgtt gtgctcgcgg gcgaggcgtg cagtttcaac 720
agaccaaaca agtgcacaac gtgcaccctt gaccttgttg gcggcgatct gctcgccgtt 780
tccggatcca cccaggacga tgccgagtga gcctgggtcg tttacggtgc ggctagctgc 840
ttcgatgcag aaggctgggt agtcatcttc tgcatcatag gtgtgggctc cgcagtcgat 900
cacttcgtgg ccgtgggcct taaggtgttc tgcgattgca tttttagttt cgaaaccagc 960
gtggtctgct ccaaggtata cgcgcatgga agtcagttta ctgcaggcat tttcacaacg 1020
gcggataccc ggacttttcc cctatttggg ggttcacaaa tcgcaaaata gctcgaccca 1080
atcttggtaa agcggatcgg ctctgactcg ggtaggacac ttccgtatct gtcatgatgc 1140
ggaacgaata aggaaggggg aacggcttcc ccaagtcacc caacgccata gatactcaag 1200
tggcccctgt gtgtagtagc gcaaccataa agcagaaagc agctcttgaa taatgagaat 1260
acccgcagct aggagcaccg tagctgtcca tgatgtgctg tgtggaagat ccaggattag 1320
cccgccgggt accgagctcg aattcgtaat catggtcata gctgtttcct g 1371
<210> 7
<211> 795
<212> DNA
<213> Artificial sequence (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 ttgggttatt 780
cgttaggaac gacag 795
<210> 8
<211> 658
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
acattgcgat tggggttggg ttattcgtta ggaacgacag gtgcgattcc agtgcgctcg 60
taatcagcga ggatgtcgat acgacgctgg tggcgctcgg cgttgctcca ttcctgctcg 120
aggaaggcgt cgacaattgc caatgcctct tcctctgagt gcatgcggcc gccgatgccg 180
atgaggttcg cattgttgtg ctcgcgggcg aggcgtgcag tttcaacaga ccaagcaagt 240
gcacaacgtg cacccttgac cttgttggcg gcgatctgct cgccgtttcc ggatccaccc 300
aggacgatgc cgagtgagcc tgggtcgttt acggtgcggc tagctgcttc gatgcagaag 360
gctgggtagt catcttctgc atcataggtg tgggctccgc agtcgatcac ttcgtggccg 420
tgggccttaa ggtgttctgc gattgcattt ttagtttcga aaccagcgtg gtctgctcca 480
aggtatacgc gcatggaagt cagtttactg caggcatttt cacaacggcg gatacccgga 540
cttttcccct atttgggggt tcacaaatcg caaaatagct cgacccaatc ttggtaaagc 600
ggatcggctc tgactcgggt aggacacttc cgtatctgtc aaaaacccca acccgcac 658
<210> 9
<211> 658
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
acattgcgat tggggttggg ttattcgtta ggaacgacag gtgcgattcc agtgcgctcg 60
taatcagcga ggatgtcgat acgacgctgg tggcgctcgg cgttgctcca ttcctgctcg 120
aggaaggcgt cgacaattgc caatgcctct tcctctgagt gcatgcgtac gccgatgccg 180
atgaggttcg cattgttgtg ctcgcgggcg aggcgtgcag tttcaacaga ccaaacaagt 240
gcacaacgtg cacccttgac cttgttggcg gcgatctgct cgccgtttcc ggatccaccc 300
aggacgatgc cgagtgagcc tgggtcgttt acggtgcggc tagctgcttc gatgcagaag 360
gctgggtagt catcttctgc atcataggtg tgggctccgc agtcgatcac ttcgtggccg 420
tgggccttaa ggtgttctgc gattgcattt ttagtttcga aaccagcgtg gtctgctcca 480
aggtatacgc gcatggaagt cagtttactg caggcatttt cacaacggcg gatacccgga 540
cttttcccct atttgggggt tcacaaatcg caaaatagct cgacccaatc ttggtaaagc 600
ggatcggctc tgactcgggt aggacacttc cgtatctgtc aaaaacccca acccgcac 658
<210> 10
<211> 769
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
gtaggacact tccgtatctg 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> 1361
<212> DNA
<213> Artificial sequence (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
gggttgggtt attcgttagg aacgacaggt gcgattccag tgcgctcgta atcagcgagg 840
atgtcgatac gacgctggtg gcgctcggcg ttgctccatt cctgctcgag gaaggcgtcg 900
acaattgcca atgcctcttc ctctgagtgc atgcggccgc cgatgccgat gaggttcgca 960
ttgttgtgct cgcgggcgag gcgtgcagtt tcaacagacc aagcaagtgc acaacgtgca 1020
cccttgacct tgttggcggc gatctgctcg ccgtttccgg atccacccag gacgatgccg 1080
agtgagcctg ggtcgtttac ggtgcggcta gctgcttcga tgcagaaggc tgggtagtca 1140
tcttctgcat cataggtgtg ggctccgcag tcgatcactt cgtggccgtg ggccttaagg 1200
tgttctgcga ttgcattttt agtttcgaaa ccagcgtggt ctgctccaag gtatacgcgc 1260
atggaagtca gtttactgca ggcattttca caacggcgga tacccggact tttcccctat 1320
ttgggggttc acaaatcgca aaatagctcg acccaatctt g 1361
<210> 12
<211> 1333
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
tgctccattc ctgctcgagg aaggcgtcga caattgccaa tgcctcttcc tctgagtgca 60
tgcggccgcc gatgccgatg aggttcgcat tgttgtgctc gcgggcgagg cgtgcagttt 120
caacagacca agcaagtgca caacgtgcac ccttgacctt gttggcggcg atctgctcgc 180
cgtttccgga tccacccagg acgatgccga gtgagcctgg gtcgtttacg gtgcggctag 240
ctgcttcgat gcagaaggct gggtagtcat cttctgcatc ataggtgtgg gctccgcagt 300
cgatcacttc gtggccgtgg gccttaaggt gttctgcgat tgcattttta gtttcgaaac 360
cagcgtggtc tgctccaagg tatacgcgca tggaagtcag tttactgcag gcattttcac 420
aacggcggat acccggactt ttcccctatt tgggggttca caaatcgcaa aatagctcga 480
cccaatcttg gtaaagcgga tcggctctga ctcgggtagg acacttccgt atctgtcaaa 540
aaccccaacc cgcacatttt tagatttcta ttttgtgtac atagggttcg gaacaaagct 600
taaaccatcc ccaattgaaa tgtcgttaca cacccacatg tttgaagtgg agcaaaccga 660
aaaccagttt tccccaacgg cagccgcccc ccacgttgaa ccttcgaaat agtaggcaac 720
accatcaagc ggatcttcat caagcgaaat agtgattgac tcttcaccgt tccgcttaca 780
aactgcgtta gtgtcgctat tttccaccca cttgtcacac tcgtacccgt tttcatttag 840
ccatttttcg gcatgtccta ttttctcgaa ccgggcagga gcgtcagggc ttccgcagcc 900
cgctagtagt agtccggctg caatgatgct taatgttttt ttcatgaatt aaacatagta 960
ctttgctggt aaaaatattg gagaacccca ctggcctaca tggtcagtgg gggcattttt 1020
gcgtttcacc cctcaaaaat catcaccaca cttgcgggat ttccccctga tttcccccac 1080
tcccacacca ttcccagtgg acagtgtgga cgtattggac acattaaaca cattgcgacc 1140
aggtaaaacg tcatgaccag gtatcgtcaa tgttcttgat gaatttccgc accgcaggat 1200
tatcattcga ggtggaataa atagcctgca gctccgctaa accaacaggt agatcataaa 1260
aatggcgata ctcaacaccg ctgtaattga gttttttcgc ggactccgga accagcgcaa 1320
cacccagtcc aga 1333
<210> 13
<211> 688
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
gcttgcatgc ctgcaggtcg actctagagg atccccttat tcgttaggaa cgacaggtgc 60
gattccagtg cgctcgtaat cagcgaggat gtcgatacga cgctggtggc gctcggcgtt 120
gctccattcc tgctcgagga aggcgtcgac aattgccaat gcctcttcct ctgagtgcat 180
gcggccgccg atgccgatga ggttcgcatt gttgtgctcg cgggcgaggc gtgcagtttc 240
aacagaccaa gcaagtgcac aacgtgcacc cttgaccttg ttggcggcga tctgctcgcc 300
gtttccggat ccacccagga cgatgccgag tgagcctggg tcgtttacgg tgcggctagc 360
tgcttcgatg cagaaggctg ggtagtcatc ttctgcatca taggtgtggg ctccgcagtc 420
gatcacttcg tggccgtggg ccttaaggtg ttctgcgatt gcatttttag tttcgaaacc 480
agcgtggtct gctccaaggt atacgcgcat ggaagtcagt ttactgcagg cattttcaca 540
acggcggata cccggacttt tcccctattt gggggttcac aaatcgcaaa atagctcgac 600
ccaatcttgg taaagcggat cggctctgac tcgggtagga cacttccgta tctggttttg 660
gcggatgaga gaagattttc agcctgat 688
<210> 14
<211> 688
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
gcttgcatgc ctgcaggtcg actctagagg atccccttat tcgttaggaa cgacaggtgc 60
gattccagtg cgctcgtaat cagcgaggat gtcgatacga cgctggtggc gctcggcgtt 120
gctccattcc tgctcgagga aggcgtcgac aattgccaat gcctcttcct ctgagtgcat 180
gcgtacgccg atgccgatga ggttcgcatt gttgtgctcg cgggcgaggc gtgcagtttc 240
aacagaccaa acaagtgcac aacgtgcacc cttgaccttg ttggcggcga tctgctcgcc 300
gtttccggat ccacccagga cgatgccgag tgagcctggg tcgtttacgg tgcggctagc 360
tgcttcgatg cagaaggctg ggtagtcatc ttctgcatca taggtgtggg ctccgcagtc 420
gatcacttcg tggccgtggg ccttaaggtg ttctgcgatt gcatttttag tttcgaaacc 480
agcgtggtct gctccaaggt atacgcgcat ggaagtcagt ttactgcagg cattttcaca 540
acggcggata cccggacttt tcccctattt gggggttcac aaatcgcaaa atagctcgac 600
ccaatcttgg taaagcggat cggctctgac tcgggtagga cacttccgta tctggttttg 660
gcggatgaga gaagattttc agcctgat 688
<210> 15
<211> 727
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
agcggataac aatttcacac aggaaacaga attaattaag cttgcatgcc tgcaggtcga 60
ctctagagga tccccttatt cgttaggaac gacaggtgcg attccagtgc gctcgtaatc 120
agcgaggatg tcgatacgac gctggtggcg ctcggcgttg ctccattcct gctcgaggaa 180
ggcgtcgaca attgccaatg cctcttcctc tgagtgcatg cggccgccga tgccgatgag 240
gttcgcattg ttgtgctcgc gggcgaggcg tgcagtttca acagaccaag caagtgcaca 300
acgtgcaccc ttgaccttgt tggcggcgat ctgctcgccg tttccggatc cacccaggac 360
gatgccgagt gagcctgggt cgtttacggt gcggctagct gcttcgatgc agaaggctgg 420
gtagtcatct tctgcatcat aggtgtgggc tccgcagtcg atcacttcgt ggccgtgggc 480
cttaaggtgt tctgcgattg catttttagt ttcgaaacca gcgtggtctg ctccaaggta 540
tacgcgcatg gaagtcagtt tactgcaggc attttcacaa cggcggatac ccggactttt 600
cccctatttg ggggttcaca aatcgcaaaa tagctcgacc caatcttggt aaagcggatc 660
ggctctgact cgggtaggac acttccgtat ctggttttgg cggatgagag aagattttca 720
gcctgat 727
<210> 16
<211> 1350
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
cagtgccaag cttgcatgcc tgcaggtcga ctctagatct gcactagagg gtctgcatta 60
tatgaacgcg gctcgtgaga tcatgggtat gactgctggc cctgagctgc ctcctctgga 120
aggtcagcgc aatgctggtc gcgttacaga aaagcgcacc attgagcagg agggtcgcca 180
gatcactgct tcccccgtcg caacagatga aactccgaac tactaccctg gcggtaacgt 240
tgcgggtcgc ccagtccctg ctggttggta ctccgagcct tggtgggcaa gcgcattgcg 300
ttccggtctg tggactgcag gttcggtcat gatgttctca gcaatgttta acggcatggc 360
tggtgtcggc tactccgctg cagcctttga aaatggctat ggcgagggct accaagatgg 420
cctggacgca gctggcggag acatgggcga tgctggcgat atgggtgacg ccggagctga 480
catgggcgat gttggtggtg atgctggcgg agacggtggc ggattcttcg acggactctt 540
tggtggtgga gacggcggcg atggcggcgg attcgatttt gacttcgatt tctagcccaa 600
aataatcccc acataaacac aaaatgggcg gttaagttct tggaaaagaa cttaaccgcc 660
catttttaat tgtggaagtc agtttactgc aggcattttc acaacggcgg atacccggac 720
ttttccccta tttgggggtt cacaaatcgc aaaatagctc gacccaatct tggtaaagcg 780
gatcggctct gactcgggta ggacacttcc gtatctgtca tgatgcggaa cgaataagga 840
agggggaacg gcttccccaa gtcacccaac gccatagata ctcaagtggc ccctgtgtgt 900
agtagcgcaa ccataaagca gaaagcagct cttgaataat gagaataccc gcagctagga 960
gcaccgtagc tgtccatgat gtgctgtgtg gaagatccag gattagcccg ccaatgagca 1020
tgaggatcgt tgcaccaatg tagttggtta gcgccatacg ccctaaaggt gcgaaaactg 1080
cagcgagtgc accgcgaatc ggtgtatgca aggccagcaa cactaaagaa atgtagacgc 1140
cgccaagcgc gagtcccgcc actgtggaaa cgatcgagaa gcctgaagaa gtgatgtcac 1200
gtgcctggat aatcagcgta ggtatagcga ttgctgcgct tacggcgaaa aagactccca 1260
tagcccgcgg tgagttctct gcccgatcga ctactccata cctaacgagc gcgggtaccg 1320
agctcgaatt cgtaatcatg gtcatagctg 1350

Claims (10)

1. A protein, wherein the protein is any one of:
A1) a protein having an amino acid sequence of SEQ ID No. 4;
A2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.4, has more than 80% of identity with the protein shown in A1), and has the same function;
A3) a fusion protein with the same function obtained by connecting labels at the N end and/or the C end of A1) or A2).
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule according to claim 2, wherein the nucleic acid molecule is a DNA molecule whose coding sequence is represented by SEQ ID No.3 or a DNA molecule whose nucleotide sequence is represented by SEQ ID No. 3.
4. A biomaterial, characterized in that the biomaterial is any one of the following:
C1) an expression cassette comprising the nucleic acid molecule of any one of claims 2 or 3;
C2) a recombinant vector comprising any one of the nucleic acid molecules of claim 2 or 3, or a recombinant vector comprising any one of the expression cassettes of C1);
C3) a recombinant microorganism comprising any one of the nucleic acid molecules of claim 2 or 3, or a recombinant microorganism comprising any one of the expression cassettes of C1), or a recombinant microorganism comprising any one of the recombinant vectors of C2).
5, D1) -D8) in the construction of a genetically engineered bacterium producing L-valine, and/or in the preparation of L-valine, and/or in the regulation of the yield of L-valine in a microorganism, wherein the D1) -D8) are as follows:
D1) the protein of any one of claim 1;
D2) the nucleic acid molecule of any one of claims 2 or 3;
D3) the biomaterial of any one of claim 4;
D4) a DNA molecule having the nucleotide sequence of SEQ ID No. 1;
D5) a DNA molecule which is obtained by modifying and/or substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID No.1, has more than 90 percent of identity with the DNA molecule shown in SEQ ID No.1 and has the same function;
D6) an expression cassette comprising the DNA molecule of 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 for increasing the production of L-valine by a microorganism, comprising any one of:
E1) increasing the expression level or the content of the nucleic acid molecule of any one of claims 2 or 3 in a target microorganism to obtain a microorganism having a higher L-valine productivity than the target microorganism;
E2) increasing the expression level or the content of the DNA molecule of claim 5D 4) or D5) in a target microorganism to obtain a microorganism having a higher L-valine yield than the target microorganism;
E3) and (2) mutating the DNA molecule with the nucleotide sequence of SEQ ID No.1 in the target microorganism to obtain the microorganism with the L-valine yield higher than that of the target microorganism.
7. The method of claim 6, wherein the mutation is a mutation of alanine residue 87 to valine residue and a mutation of glycine residue 109 to valine residue of the amino acid sequence encoded by the DNA molecule of SEQ ID No. 1.
8. The method as claimed in claim 7, wherein the mutation is the mutation of nucleotide C at position 260 to T and nucleotide GC at positions 326 and 327 to TA in the DNA molecule shown in SEQ ID No.1 by site-directed mutagenesis.
9. A method for producing L-valine, which comprises producing L-valine by using the recombinant microorganism according to claim 4 or 5.
10. A method for constructing the recombinant microorganism according to claim 4 or 5, wherein the method comprises at least any one of:
F1) introducing any one of the nucleic acid molecules of claim 2 or 3 into a microorganism of interest to obtain said recombinant microorganism;
F2) introducing a DNA molecule shown in SEQ ID No.1 into a target microorganism to obtain the recombinant microorganism;
F3) the DNA molecule shown in SEQ ID No.1 is edited by a gene editing means, so that the target microorganism contains the DNA molecule shown in SEQ ID No. 3.
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Citations (2)

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CN101646687A (en) * 2007-02-19 2010-02-10 赢创德固赛有限责任公司 Method of producing methionine in corynebacteria by over-expressing enzymes of the pentose phosphate pathway
CN109423504A (en) * 2017-08-24 2019-03-05 廊坊梅花生物技术开发有限公司 Produce the bacterial strain and purposes of L-Trp

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CN101646687A (en) * 2007-02-19 2010-02-10 赢创德固赛有限责任公司 Method of producing methionine in corynebacteria by over-expressing enzymes of the pentose phosphate pathway
CN109423504A (en) * 2017-08-24 2019-03-05 廊坊梅花生物技术开发有限公司 Produce the bacterial strain and purposes of L-Trp

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