CN114540262B - Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same - Google Patents
Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same Download PDFInfo
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- CN114540262B CN114540262B CN202210137750.8A CN202210137750A CN114540262B CN 114540262 B CN114540262 B CN 114540262B CN 202210137750 A CN202210137750 A CN 202210137750A CN 114540262 B CN114540262 B CN 114540262B
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- cey17
- gene
- valine
- corynebacterium glutamicum
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- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims abstract description 107
- 229960004295 valine Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 32
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 23
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 23
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 23
- 239000012620 biological material Substances 0.000 title claims abstract description 9
- 244000005700 microbiome Species 0.000 title abstract description 41
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- 150000001413 amino acids Chemical group 0.000 claims description 13
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- 241000894006 Bacteria Species 0.000 abstract description 49
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- 238000006243 chemical reaction Methods 0.000 abstract description 19
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The invention discloses a method for constructing recombinant microorganism producing L-valine, and nucleic acid molecules and biological materials used by the method. Specifically disclosed are mutant proteins cey17_rs14810 G397Y and genes encoding them. The invention constructs genetic engineering bacteria containing mutation (GG-TA) and engineering bacteria over-expressing CEY17_RS14810 gene or mutant gene thereof. Experiments show that the CEY17_RS14810 gene and variants thereof participate in biosynthesis of L-valine, and mutation of the CEY17_RS14810 gene coding region or overexpression of the CEY17_RS14810 gene and/or mutants thereof in production bacteria are beneficial to improvement of the yield and conversion rate of the L-valine, can cultivate high-yield and high-quality strains conforming to industrial production, and has important significance for industrial production of the L-valine.
Description
Technical Field
The invention belongs to the technical field of microbial variation or genetic engineering, and particularly relates to a method for constructing recombinant microorganisms producing L-valine, and nucleic acid molecules and biological materials used by the method.
Background
L-valine (L-valine), known by the chemical name α -aminoisovaleric acid, is one of the branched-chain amino acids, and cannot be synthesized by humans and animals themselves. L-valine is one of eight essential amino acids of human body, has the effects of promoting protein synthesis, inhibiting protein decomposition, enhancing immunity, and is helpful for correcting negative nitrogen balance caused by operation, wound, infection, etc. In addition, the L-valine also has the effects of resisting central fatigue, resisting peripheral fatigue, delaying exercise fatigue and accelerating the restoration of a body after exercise, so that the L-valine has wide application and commercial value in the food and pharmaceutical industries. The compound branched-chain amino acid transfusion prepared from the L-valine has wide application in the treatment of blood brain barrier, hepatic coma, chronic liver cirrhosis and renal failure, the dietary treatment of congenital metabolic defect, the treatment of septicemia and postoperative diabetes patients, the treatment for accelerating the healing of surgical wounds and the nutritional support treatment of tumor patients. L-valine is mainly used as a food additive, a nutrient supplement liquid, a flavoring agent and the like in the food industry. The L-valine gel has positively charged terminal groups, is a novel low molecular weight gel, can be prepared to form hydrogel, and has been widely applied in the fields of biological medicine, tissue engineering, photochemistry, electrochemistry, food industry, cosmetics and the like.
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 advantages of limited sources of raw materials, high production cost, low yield, serious pollution and difficult realization of industrial production. The microorganism direct fermentation method for producing L-valine has the advantages of wide raw material source, low cost, mild reaction condition, easy realization of large-scale production and the like, and is a very economical and efficient production method. The strain with high yield obtained in industrial fermentation is of great importance for the fermentation production of L-valine, and is a core of the whole L-valine fermentation industry and an important factor for determining the industrial value of fermentation products. Along with the continuous development of genetic engineering breeding technology, the production bacteria are modified from the molecular level, and the functions of related genes are researched and excavated, thus providing a wide prospect for the industrialized fermentation production of L-valine. The breeding of high-yield and stable production strains promotes the accumulation of L-valine in microorganisms, and further improves the yield of L-valine, which always accompanies the development of the L-valine fermentation industry, and has important significance for promoting the progress of L-valine industrialization.
Disclosure of Invention
The technical problem to be solved by the present invention is how to increase the yield of microbial L-valine, and the technical problem to be solved is not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be apparent to those skilled in the art from the following description.
To solve the above technical problems, the present invention provides a method for constructing a recombinant microorganism, the method comprising at least any one of the following:
F1 Introducing a nucleic acid molecule encoding a protein, named cey17_rs14810 G397Y, which may be any of the following, into a microorganism of interest to obtain said recombinant microorganism:
A1 A protein whose amino acid sequence is SEQ ID No.4,
A2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residues in the amino acid sequence shown in SEQ ID No.4, has more than 80 percent of identity with the protein shown in A1) and has the same function,
A3 Fusion proteins with the same function obtained by connecting labels to the N end and/or the C end of A1) or A2);
F2 Introducing the DNA molecule shown in SEQ ID No.1 into a microorganism of interest to obtain the recombinant microorganism;
F3 Editing the DNA molecule shown in SEQ ID No.1 by using a gene editing means (such as single base gene editing) to make the target microorganism contain the DNA molecule shown in SEQ ID No. 3.
The introduction may be by transforming a host bacterium with a vector carrying the DNA molecule of the present invention by any known transformation method such as chemical transformation or electric shock transformation. The DNA molecules to be introduced may be either single or multiple copies. The introduction may be by integrating the exogenous gene into the host chromosome or by extrachromosomal expression from a plasmid.
The protein CEY17_RS14810 G397Y is also within the scope of the invention.
The invention also provides a nucleic acid molecule, named cey17_rs14810 GG1189-1190TA, which may be any of the following:
B1 A nucleic acid molecule encoding said protein cey17_rs14810 G397Y;
B2 A DNA molecule with a coding sequence shown in SEQ ID No. 3;
b3 The nucleotide sequence is a DNA molecule shown as SEQ ID No. 3.
The DNA molecule shown in SEQ ID No.3 is also the CEY17_RS14810 GG1189-1190TA gene according to the invention.
The DNA molecule shown in SEQ ID No.3 (CEY17_RS 14810 GG1189-1190TA) encodes the protein CEY17_RS14810 G397Y shown in SEQ ID No. 4.
The protein CEY17_RS14810 G397Y has its amino acid sequence (SEQ ID No. 4) mutated from glycine (G) at tyrosine (Y) 397.
The present invention also provides a biomaterial which may be any one of the following:
C1 An expression cassette containing said nucleic acid molecule cey17_rs14810 GG1189-1190TA;
c2 A recombinant vector comprising said nucleic acid molecule cey17_rs14810 GG1189-1190TA or a recombinant vector comprising said expression cassette of C1);
C3 A recombinant microorganism containing said nucleic acid molecule cey17_rs14810 GG1189-1190TA, or a recombinant microorganism containing said expression cassette of C1), or a recombinant microorganism containing said recombinant vector of C2).
The invention also provides any one of the following applications of any one of D1) to D8):
g1 Use of any one of D1) to D8) for regulating the production of L-valine by a microorganism;
g2 Application of any one of D1) to D8) in construction of L-valine-producing genetically engineered bacteria;
G3 Use of any one of D1) to D8) for the preparation of L-valine;
Wherein, D1) -D8) are:
D1 The protein cey17_rs14810 G397Y;
D2 The nucleic acid molecule cey17_rs14810 GG1189-1190TA;
D3 A) the biological material;
D4 A DNA molecule with a nucleotide sequence of SEQ ID No. 1;
D5 A DNA molecule which has more than 90 percent of identity with the DNA molecule shown in SEQ ID No.1 and has the same function after the nucleotide sequence shown in SEQ ID No.1 is modified and/or one or more nucleotide substitutions and/or deletions and/or additions;
d6 An expression cassette comprising the DNA molecule described under D4) or D5);
d7 A recombinant vector comprising the DNA molecule described in D4) or D5), or a recombinant vector comprising the expression cassette described in D6);
d8 A recombinant microorganism comprising the DNA molecule described in D4) or D5), or a recombinant microorganism comprising the expression cassette described in D6), or a recombinant microorganism comprising the recombinant vector described in D7).
The DNA molecule shown in SEQ ID No.1 is also the CEY17_RS14810 gene according to the invention.
The DNA molecule shown in SEQ ID No.1 (CEY17_RS 14810 gene) encodes the protein shown in SEQ ID No. 2.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.
Herein, the 80% identity or more 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 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The regulation of L-valine production by a microorganism as described herein can be an increase or decrease in the accumulation of L-valine in the microorganism (i.e., promotion or inhibition of L-valine biosynthesis).
The present invention also provides a method for increasing the production of L-valine in a microorganism, the method comprising any one of the following:
E1 Increasing the expression level or the content of the nucleic acid molecule cey17_rs14810 GG1189-1190TA in the microorganism of interest, to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
E2 Increasing the expression level or the content of the DNA molecule of D4) or D5) in the microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
E3 The DNA molecule with the nucleotide sequence of SEQ ID No.1 in the target microorganism is subjected to mutation (such as base substitution, base insertion or base deletion) to obtain a microorganism with higher L-valine yield than the target microorganism.
In the above method, the mutation may be a mutation of glycine residue at position 397 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 mutation may be a mutation of glycine residue at position 397 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 to tyrosine residue.
In the above method, the mutation may be a site-directed mutagenesis method of mutating nucleotide GG at 1189-1190 in the DNA molecule shown in SEQ ID No.1 to TA.
The mutation refers to a site-directed mutation in which a certain or a few bases in a gene are changed to change the amino acid composition of the corresponding protein, so that a new protein is produced or a new function is produced in the original protein, namely, the gene site-directed mutation. Site-directed mutagenesis techniques for genes, such as oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis or cassette mutagenesis, and the like, are well known to those skilled in the art.
Vectors described herein are well known to those of skill 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, pK18mobsacB or pXMJ19 is used.
Herein, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria may be derived from Brevibacterium (Brevibacterium), corynebacterium (Corynebacterium), escherichia (Escherichia), aerobacter (Aerobacter), micrococcus (Micrococcus), flavobacterium (Flavobacterium), bacillus (Bacillus), etc.
Specifically, the microorganism may be Corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium lactofermentum (Brevibacterium lactofermentum), micrococcus glutamicum (Micrococcus glutamicus), brevibacterium ammoniagenes (Brevibacterum ammoniagenes), escherichia coli (ESCHERICHIA COLI) or Aerobacter aerogenes (Aerobacter aerogenes), but is not limited thereto.
Specifically, the microorganism may be Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260, or Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC14067.
Herein, the recombinant vector may specifically be the recombinant vector pK18-cey17_rs14810 GG1189-1190TA、pK18-CEY17_RS14810OE、pK18-CEY17_RS14810GG1189-1190TA OE, pXMJ19-cey17_rs14810 and/or pXMJ-cey17_rs 14810 GG1189-1190TA.
The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA is obtained by replacing a fragment (small fragment) between Xbal I and BamH I recognition sites of a pK18mobsacB plasmid with a DNA fragment shown at 37-1266 th site of SEQ ID No.5 in a sequence table, and keeping other sequences of the pK18mobsacB vector unchanged. The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA contains a DNA molecule shown in 597-1269 of a mutant gene CEY17_RS14810 GG1189-1190TA shown in SEQ ID No. 3.
The recombinant vector pK18-CEY17_RS14810OE is used for integrating the exogenous gene CEY17_RS14810 into the host chromosome and overexpressing the wild-type CEY17_RS14810 gene in the producer.
The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA OE is used for integrating the exogenous gene CEY17_RS14810 GG1189-1190TA into host chromosome and over-expressing the mutant gene CEY17_RS14810 GG1189-1190TA in the producer.
The recombinant vector pXMJ-CEY17_RS 14810 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of the pXMJ vector with a DNA fragment with a nucleotide sequence of SEQ ID No.13 in a sequence table, and keeping other sequences of the pXMJ vector unchanged. Recombinant vector pXMJ-CEY17_RS 14810 is used to express exogenous gene CEY17_RS14810 extrachromosomally by plasmid, and then over-express wild CEY17_RS14810 gene in producer.
The recombinant vector pXMJ-CEY17_RS 14810 GG1189-1190TA is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of the pXMJ vector with a DNA fragment with a nucleotide sequence of SEQ ID No.14 in a sequence table, and keeping other sequences of the pXMJ vector unchanged. The recombinant vector pXMJ-CEY17_RS 14810 GG1189-1190TA is used for expressing the exogenous gene CEY17_RS14810 GG1189-1190TA extrachromosomally through a plasmid, and further over-expressing the mutant CEY17_RS14810 GG1189-1190TA gene in a production strain.
The recombinant vectors pK18-CEY17_RS14810 GG1189-1190TA、pK18-CEY17_RS14810OE、pK18-CEY17_RS14810GG1189-1190TA OE, pXMJ19-CEY17_RS14810 and pXMJ19-CEY17_RS14810 GG1189-1190TA are within the scope of the invention.
Herein, the recombinant microorganism may specifically be recombinant bacteria YPV-091, YPV-092, YPV-093, YPV-094, and/or YPV-095.
The recombinant bacterium YPV-091 is obtained by transforming the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA into corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260, and the recombinant bacterium YPV-091 contains a mutated gene CEY17_RS14810 GG1189-1190TA shown in SEQ ID No. 3.
The recombinant bacterium YPV-092 contains a CEY17_RS14810 gene shown in double-copy SEQ ID No. 1; specifically, the recombinant bacterium YPV-092 is obtained by replacing the spacer region of the upper homology arm CEY17_02570 and the lower homology arm CEY17_02575 in the genome of the corynebacterium glutamicum CGMCC No.21260 with the CEY17_RS14810 gene and keeping other nucleotides in the genome of the corynebacterium glutamicum CGMCC No.21260 unchanged. Recombinant bacteria containing double copies of the CEY17_RS14810 gene can significantly and stably increase the expression level of the CEY17_RS14810 gene. Recombinant bacterium YPV-092 is an engineering bacterium for over-expressing wild CEY17_RS14810 gene on genome, and is obtained by introducing the recombinant vector pK18-CEY17_RS14810OE into Escherichia coli DH5 alpha.
The recombinant bacterium YPV-093 contains a mutant CEY17_RS14810 GG1189-1190TA gene shown in SEQ ID No. 3; specifically, the recombinant bacterium YPV-093 is obtained by replacing the spacer region of the upper homology arm CEY17_02570 and the lower homology arm CEY17_02575 in the genome of the corynebacterium glutamicum CGMCC No.21260 with the CEY17_RS14810 GG1189-1190TA gene and keeping other nucleotides in the genome of the corynebacterium glutamicum CGMCC No.21260 unchanged. Recombinant bacterium YPV-093 is an engineering bacterium for over-expressing mutant CEY17_RS14810 GG1189-1190TA gene on genome, and is obtained by introducing the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA OE into Escherichia coli DH5 alpha.
The recombinant bacterium YPV-094 contains CEY17_RS14810 gene shown in SEQ ID No.1, and the recombinant bacterium YPV-094 is engineering bacterium for over-expressing wild CEY17_RS14810 gene on plasmid, namely over-expressing the recombinant bacterium outside chromosome by plasmid pXMJ-CEY17_RS14810.
The recombinant bacterium YPV-095 contains the mutated CEY17_RS14810 GG1189-1190TA gene shown in SEQ ID No.3, and the recombinant bacterium YPV-095 is engineering bacterium for over-expressing the mutated CEY17_RS14810 GG1189-1190TA gene on a plasmid, namely over-expressing the mutated CEY17_RS14810 GG1189-1190TA gene outside a chromosome by the plasmid pXMJ-CEY17_RS 14810 GG1189-1190TA.
The recombinant bacteria YPV-091, YPV-092, YPV-093, YPV-094 and YPV-095 are all within the scope of the invention.
The present invention also provides a method for producing L-valine, which comprises producing L-valine using the recombinant microorganism 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), and particularly Corynebacterium glutamicum (Corynebacterium glutamicum) and variants thereof.
In one embodiment of the invention, the recombinant microorganism is recombinant strain YPV-091, YPV-092, YPV-093, YPV-094, or YPV-095.
The invention firstly introduces mutation in a coding region (SEQ ID No. 1) of CEY17_RS14810 gene of corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 (the chromosome of the strain is confirmed to be reserved with wild CEY17_RS14810 by sequencing) in an allele replacement mode, and constructs genetically engineered bacterium YPV-091 containing mutation (GG-TA). To further study and verify that overexpression of the wild-type cey17_rs14810 gene or its mutant gene cey17_rs14810 GG1189-1190TA in producer bacteria can increase the yield of L-valine, engineering bacteria YPV-092, YPV-093, YPV-094 and YPV-095 were constructed that overexpress the cey17_rs14810 gene or cey17_rs14810 GG1189-1190TA gene on the genome and on the plasmid, respectively, by integrating the exogenous gene into the host chromosome or by extrachromosomal expression from the plasmid. Experiments show that the CEY17_RS14810 gene and variants thereof are involved in biosynthesis of L-valine, and the accumulation of L-valine in microorganisms can be regulated by over-expression or knockout or site-directed mutation of the CEY17_RS14810 gene. Mutation of the CEY17_RS14810 gene coding region or overexpression of the CEY17_RS14810 gene or the mutant gene CEY17_RS14810 GG1189-1190TA in the producer is beneficial to the improvement of the yield and conversion rate of L-valine, and knocking out or weakening of the CEY17_RS14810 gene is not beneficial to the accumulation of L-valine. The CEY17_RS14810 gene and variants thereof (such as CEY17_RS14810 GG1189-1190TA gene) can be utilized to construct genetic engineering strains for producing L-valine so as to promote the yield improvement of the L-valine, and cultivate high-yield and high-quality strains which accord with industrial production, thus having wide application value and important economic significance for the industrial production of the L-valine.
Preservation description
Strain name: corynebacterium glutamicum
Latin name: corynebacterium glutamicum A
Classification naming: corynebacterium glutamicum (Corynebacterium glutamicum)
Strain number: YPFV 1A 1
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing, chaoyang district North Star, west Lu No. 1, 3
Preservation date: 11/30/2020
Accession numbers of the preservation center: CGMCC No.21260
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below 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 with China general microbiological culture Collection center (CGMCC, address: no. 3 of West Song 1, national academy of sciences of China) at 11 and 30 of 2020, and the accession number was CGMCC No.21260. Corynebacterium glutamicum (Corynebacterium glutamicum) YPFV1, also known as Corynebacterium glutamicum CGMCC No.21260.
EXAMPLE 1 construction of recombinant vector comprising mutated CEY17_RS14810 Gene coding region fragment
Two pairs of primers for amplifying the coding region of the CEY17_RS14810 gene were designed and synthesized based on the genomic sequence of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC14067 published by NCBI, and a mutation was introduced into the coding region (SEQ ID No. 1) of the CEY17_RS14810 gene of Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 (the wild-type CEY17_RS14810 gene was confirmed to remain on the chromosome of the strain) in an allelic substitution manner, which was a mutation of guanine (GG) to Thymine Adenine (TA) at positions 1189-1190 in the nucleotide sequence (SEQ ID No. 1) of the CEY17_RS14810 gene, to give a DNA molecule (mutated CEY17_RS14810 gene, named CEY17_RS14810 GG1189-1190TA) shown in SEQ ID No. 3.
Wherein the DNA molecule shown in SEQ ID No.1 encodes a protein with the amino acid sequence of SEQ ID No.2 (the name of the protein is protein CEY17_RS 14810).
The DNA molecule shown in SEQ ID No.3 encodes a mutein of the amino acid sequence SEQ ID No.4 (said mutein is named CEY17_RS14810 G397Y). The 397 th tyrosine (Y) in the amino acid sequence (SEQ ID No. 4) of the mutant protein CEY17_RS14810 G397Y is mutated from glycine (G).
Vector construction is carried out by adopting NEBuilder recombinant technology, site-directed mutagenesis is carried out on the CEY17_RS14810 gene, the primer design is as follows (synthesized by Shanghai in vitro), and the base in bold font is the mutation position:
P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGGTGCCATCACCTTCGCCC-3'，
P2:
P3:
P4:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCGAGCTGAATCTCGGCGAG-3'。
The construction method comprises the following steps: the Corynebacterium glutamicum ATCC14067 was used as a template, and primers P1 and P2, P3 and P4 were used for PCR amplification, respectively, to obtain two DNA fragments (CEY17_RS14810Up and CEY17_RS14810Down) each having a mutated base and a CEY17_RS14810 gene coding region of 646bp and 692bp, respectively.
The PCR amplification system is as follows: 10 XEx Taq Buffer 5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, mg 2+ (25 mM) 4. Mu.L, primer (10 pM) 2. Mu.L each, ex Taq (5U/. Mu.L) 0.25. Mu.L, and total volume 50. Mu.L;
The PCR amplification reaction procedure was: pre-denaturation at 94℃for 5min, (denaturation at 94℃for 30s; annealing at 52℃for 30s; extension at 72℃for 40s;30 cycles), over-extension at 72℃for 10min.
The two DNA fragments (CEY17_RS 14810Up and CEY17_RS 14810Down) were separated and purified by agarose gel electrophoresis, and then subjected to DNA assembly reaction with the pK18mobsacB plasmid (available from Add gene, which contains kanamycin resistance marker) purified by cleavage (Xbal I/BamH I) using NEBuilder enzyme (NEBuilder HiFi DNA A ssembly Master Mix, available from NEB) under the following reaction conditions: the resultant was ligated at 50℃for 30min, and the single clone grown after DH 5. Alpha. Transformation (purchased from TAKARA) was identified by primer M13 (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive recombinant vector pK18-CEY17_RS14810 GG1189 -1190TA. The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA with correct mutation (GG-TA) was sequenced and identified by sequencing company, and the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA with correct mutation was stored for use.
Sequencing identified that the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA contains DNA fragments of integrated DNA fragments CEY17_RS14810 Up and CEY17_RS14810 Down, named CEY17_RS14810 Up-Down, the CEY17_RS14810 Up-Down DNA fragment being 1304bp in size and having the sequence shown in SEQ ID No.5, contains a mutation site (GG-TA) for introducing nucleic acid modification into the coding region (SEQ ID No. 1) of the CEY17_RS14810 gene in the strain Corynebacterium glutamicum CGMCC No.21260, in particular mutation of guanine (G) at 1189 th position of SEQ ID No.1 into thymine (T), mutation of guanine (G) at 1189-0 th position of SEQ ID No. 1191 into adenine (A), finally mutation of glycine (G) at 397 th position of the encoded protein into tyrosine (Y).
The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA is obtained by replacing a fragment (small fragment) between Xbal I and BamH I recognition sites of a pK18mobsacB plasmid with a DNA fragment shown at 37-1266 th site of SEQ ID No.5 in a sequence table, and keeping other sequences of the pK18mobsacB vector unchanged.
The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA contains a DNA molecule shown in 597-1269 of a mutant gene CEY17_RS14810 GG1189-1190TA shown in SEQ ID No. 3.
EXAMPLE 2 construction of an engineering Strain comprising the Gene CEY17_RS14810 GG1189-1190TA
The construction method comprises the following steps: the allelic substitution plasmid (pK 18-CEY17_RS14810 GG1189-1190TA) in example 1 was transformed into Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 by electric shock, and then cultured in a medium, the composition of the medium and the culture conditions are shown in Table 1, and single colonies generated by the culture were identified by the primer P1 and the universal primer M13R (5'-CAGGAAACAGCTATGACC-3') in example 1, respectively, so that 1311bp (the sequence of which is shown in SEQ ID No. 6) of the size band was amplified as positive strains. Positive strains were cultured on a medium containing 15% sucrose, single colonies generated by the culture were cultured on a medium containing kanamycin and a medium not containing kanamycin, respectively, strains grown on a medium not containing kanamycin were selected, and strains not grown on a medium containing kanamycin were further identified by PCR using the following primers (synthesized by shanghai invitrogen corporation):
P5:5'-CGACGGTGTCATCACCGCTG-3'，
P6:5'-CAGAATCGCCTCTGAGGGAT-3'。
The PCR amplified product (270 bp) obtained was subjected to SSCP (Single-Strand Conformation Polymorphis) electrophoresis (positive control with plasmid pK18-CEY17_RS14810 GG1189 -1190TA amplified fragment, negative control with Corynebacterium glutamicum ATCC14067 amplified fragment and water as blank) after denaturing at a high temperature of 95℃for 10min and ice bath for 5min, and the preparation of PAGE and electrophoresis conditions of SSCP electrophoresis are shown in Table 2, and the strain with fragment electrophoresis position inconsistent with that of the negative control fragment and consistent with that of the positive control fragment was the strain with successful allelic replacement due to different fragment structures and different electrophoresis positions. The positive strain CEY17_RS14810 gene fragment was amplified again by primer P5/P6 PCR and ligated to PMD19-T vector for sequencing, and the strain with mutation in the base sequence (GG-TA) was the positive strain with successful allelic replacement by sequence alignment and designated YPV-091.
Recombinant bacterium YPV-091 is obtained by transforming the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA into Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260, and recombinant bacterium YPV-091 contains mutated gene CEY17_RS14810 GG1189-1190TA shown in SEQ ID No. 3.
TABLE 1 composition of the culture medium and culture conditions
TABLE 2 preparation of PAGE for SSCP electrophoresis and electrophoresis conditions
EXAMPLE 3 construction of engineering strains that overexpress the CEY17_RS14810 Gene and the CEY17_RS14810 GG1189-1190TA Gene on the genome
The vector construction is carried out by adopting NEBuilder recombination technology, three pairs of primers for amplifying an upstream and downstream homologous arm fragment and a CEY17_RS14810 or a CEY17_RS14810 GG1189-1190TA gene coding region and a promoter region are designed and synthesized according to the genome sequence of the corynebacterium glutamicum ATCC14067 published by NCBI, and the CEY17_RS14810 or the CEY17_RS14810 GG1189-1190TA gene is introduced into the corynebacterium glutamicum CGMCC No.21260 in a homologous recombination mode.
Primers were designed as follows (synthesized by the company epivitrogen, shanghai):
P7:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTAGTGCCGTGCGTACCCCA-3'，
P8:5'-AAAAATGTGCAGAATCGCTTCCCAACCCCAATCGCAATGT-3'，
P9:5'-ACATTGCGATTGGGGTTGGGAAGCGATTCTGCACATTTTT-3'，
P10:5'-GTGCGGGTTGGGGTTTTTGATTAGTTTGATGGAGCGCCAG-3'，
P11:5'-CTGGCGCTCCATCAAACTAATCAAAAACCCCAACCCGCAC-3'，
P12:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTTGGTTTAGCGGAGCTGCA-3'。
The construction method comprises the following steps: the upstream homology arm fragment 795bp (corresponding to the spacer region of the Corynebacterium glutamicum CGMCC No.21260 CEY17_RS02570 gene and CEY17_RS02575 thereof, the sequence shown as SEQ ID No. 7), the CEY17_RS14810 gene and the promoter fragment 1489bp (sequence shown as SEQ ID No. 8) thereof, or the CEY17_RS14810 GG1189-1190TA gene and the promoter fragment 1489bp (sequence shown as SEQ ID No. 9) thereof, and the downstream homology arm fragment 769bp (corresponding to the Corynebacterium glutamicum CGMCC No.21260 CEY17_RS02575 gene and the spacer region of the CEY17_RS02570 thereof, the sequence shown as SEQ ID No. 10) are obtained by PCR amplification with primers P7/P8, P9/P10 and P11/P12, respectively, using Corynebacterium glutamicum ATCC14067 or YPV-091 as templates.
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 the pK18mobsacB plasmid (from Addgene, which contains kanamycin resistance as a selection marker) purified by Xbal I/BamH I cleavage using NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, from NEB), under the following reaction conditions: the resulting ligation was ligated at 50℃for 30min, and the M13 primer (M13F: 5'TGTAAAACGACGGCCAGT 3', M13R:5'CAGGAAACAGCTATGACC 3') used for the monoclonal after DH 5. Alpha. Transformation was used to obtain positive integration plasmids (recombinant vectors) containing kanamycin resistance markers, pK18-CEY17_RS14810OE and pK18-CEY17_RS14810 GG1189-1190TA OE, respectively, by PCR, and recombinants were obtained by screening with kanamycin to integrate the plasmids into the genome.
The recombinant vector pK18-CEY17_RS14810OE is used to integrate the exogenous gene CEY17_RS14810 into the host chromosome and to overexpress the wild-type CEY17_RS14810 gene in the producer.
The recombinant vector pK18-CEY17_RS14810 GG1189-1190TA OE was used to integrate the exogenous gene CEY17_RS14810 GG1189 -1190TA into the host chromosome and to overexpress the mutant gene CEY17_RS14810 GG1189-1190TA in the producer.
The PCR reaction system is as follows: 10 XEx Taq Buffer 5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, mg 2+ (25 mM) 4. Mu.L, primer (10 pM) 2. Mu.L each, ex Taq (5U/. Mu.L) 0.25. Mu.L, and total volume 50. Mu.L.
The PCR reaction procedure was: pre-denaturing for 5min at 94℃and denaturing for 30s at 94 ℃; annealing at 52 ℃ for 30s; extending at 72℃for 60s (30 cycles), and over-extending at 72℃for 10min.
The integrated plasmids (pK 18-CEY17_RS14810 OE, pK18-CEY17_RS14810 GG1189 -1190TA OE) with correct sequence are respectively electrically transformed into Corynebacterium glutamicum CGMCC No.21260, cultured in a culture medium, the components of the culture medium and the culture conditions are shown in Table 1, single colonies generated by the culture are identified by PCR through P13/P14 primers, the PCR amplified contains 2214bp (the sequence without mutation is shown as SEQ ID No.11, the 2157-2158 position of the sequence with mutation is TA, the rest fragments are positive strains of the fragments shown as SEQ ID No. 11), and the fragments cannot be amplified to primordia. The positive strain is cultivated on a culture medium containing 15% of sucrose, and single colonies generated by cultivation are further subjected to PCR identification by using a P15/P16 primer, so that positive strains with the size of 1571bp (the sequence without mutation is shown as SEQ ID No.12, the 693-694 th position of the sequence with mutation is TA, and the rest of the bacteria with the sequence as SEQ ID No.12 are CEY17_RS14810 or CEY17_RS14810 GG1189-1190TA genes integrated on a spacer region of a homology arm CEY17_02570 and a lower homology arm CEY17_02575 on a genome of the corynebacterium glutamicum CGMCC No.21260 are respectively named YPV-092 (without mutation point) and YPV-093 (with mutation point).
Recombinant bacterium YPV-092 contains double copies of CEY17_RS14810 gene shown in SEQ ID No. 1; specifically, the recombinant bacterium YPV-092 is obtained by replacing the spacer region of the upper homology arm CEY17_02570 and the lower homology arm CEY17_02575 in the genome of the corynebacterium glutamicum CGMCC No.21260 with the CEY17_RS14810 gene and keeping other nucleotides in the genome of the corynebacterium glutamicum CGMCC No.21260 unchanged. Recombinant bacteria containing double copies of the CEY17_RS14810 gene can significantly and stably increase the expression level of the CEY17_RS14810 gene. Recombinant bacterium YPV-092 is an engineering bacterium for over-expressing wild CEY17_RS14810 gene on genome, and is obtained by introducing the recombinant vector pK18-CEY17_RS14810OE into Escherichia coli DH5 alpha.
Recombinant bacterium YPV-093 contains mutated CEY17_RS14810 GG1189-1190TA gene shown in SEQ ID No. 3; specifically, the recombinant bacterium YPV-093 is obtained by replacing the spacer region of the upper homology arm CEY17_02570 and the lower homology arm CEY17_02575 in the genome of the corynebacterium glutamicum CGMCC No.21260 with the CEY17_RS14810 GG1189-1190TA gene and keeping other nucleotides in the genome of the corynebacterium glutamicum CGMCC No.21260 unchanged. Recombinant bacterium YPV-093 is an engineering bacterium for over-expressing mutant CEY17_RS14810 GG1189-1190TA gene on genome, and is obtained by introducing the recombinant vector pK18-CEY17_RS14810 GG1189-1190TA OE into Escherichia coli DH5 alpha.
The PCR identification primers are shown below:
P13:5'-CGGTTAGATTTTTTGGCCCC-3' (corresponding to the outside of the upper homology arm CEY17_RS02570),
P14:5'-GCATTTCACCAGTAGGCATG-3' (corresponding to CEY17_RS14810 coding region),
P15:5'-GCCAGTACTACATCGCATTC-3' (corresponding to CEY17_RS14810 coding region),
P16:5'-TCTGGACTGGGTGTTGCGCT-3' (corresponding to the outer side of the lower homology arm CEY17_RS 02575).
EXAMPLE 4 construction of engineering strains over-expressing the CEY17_RS14810 Gene or the CEY17_RS14810 GG1189-1190TA Gene on plasmids
Vector construction was performed by NEBuilder recombinant technology, and a pair of primers for amplifying the coding region and the promoter region of the CEY17_RS14810 and CEY17_RS14810 GG1189-1190TA genes were designed and synthesized according to the genomic sequence of Corynebacterium glutamicum ATCC14067 published by NCBI, and the primers were designed as follows (synthesized by Shanghai in vitro company):
p17:5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCAAGCGATTCTGCACATTTTT-3' (underlined nucleotide sequence is the sequence on pXMJ),
P18:5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAGTTTGATGGAGCGCCAG-3' (underlined nucleotide sequence is the sequence on pXMJ).
The construction method comprises the following steps: the amplified products were subjected to PCR amplification using Corynebacterium glutamicum ATCC14067 and YPV-091 as templates and primers P17/P18, respectively, to obtain the CEY17_RS14810 gene and its promoter fragment (sequence shown in SEQ ID No. 13) and the CEY17_RS14810 GG1189-1190TA gene and its promoter fragment 1519bp (sequence shown in SEQ ID No. 14), and the amplified products were subjected to DNA assembly reaction using a column type DNA gel recovery kit, and the recovered DNA fragments were subjected to DNA assembly reaction with a shuttle plasmid pXMJ (purchased from Addgene, which contains chloramphenicol resistance as a selection marker) recovered by EcoRI/KpnI cleavage using NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB, under the following conditions: the resulting single clone was ligated at 50℃for 30min, and after DH 5. Alpha. Transformation of the ligation product, positive overexpression vectors pXMJ-CEY17_RS 14810 (containing the CEY17_RS14810 gene) and pXMJ-CEY17_RS 14810 GG1189-1190TA (containing the CEY17_RS14810 GG1189-1190TA gene) were obtained by PCR identification using M13R (-48) (5'AGCGGATAAC AATTTCACAC AGGA3')/P18 primers, and the plasmids were sequenced. Since the plasmid contains a chloramphenicol resistance marker, it is possible to select whether the plasmid is transformed into a strain by chloramphenicol.
The recombinant vector pXMJ-CEY17_RS14810 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of the pXMJ 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 pXM J19 vector unchanged. Recombinant vector pXMJ-CEY17_RS 14810 is used to express exogenous gene CEY17_RS14810 extrachromosomally by plasmid, and then over-express wild CEY17_RS14810 gene in producer.
Recombinant vector pXMJ-CEY17_RS 14810 GG1189-1190TA is a recombinant expression vector obtained by replacing the fragment (small fragment) between EcoRI and KpnI recognition sites of the pXMJ vector with a DNA fragment whose nucleotide sequence is SEQ ID No.14 in the sequence Listing, and keeping the other sequences of the pXMJ vector unchanged. The recombinant vector pXMJ-CEY17_RS 14810 GG1189-1190TA is used for expressing the exogenous gene CEY17_RS14810 GG1189-1190TA extrachromosomally through a plasmid, and further over-expressing the mutant CEY17_RS14810 GG1189-1190TA gene in a production strain.
The PCR reaction system is as follows: 10 XEx Taq Buffer 5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, mg 2+ (25 mM) 4. Mu.L, primer (10 pM) 2. Mu.L each, ex Taq (5U/. Mu.L) 0.25. Mu.L, and total volume 50. Mu.L.
The PCR reaction procedure was: pre-denaturing for 5min at 94℃and denaturing for 30s at 94 ℃; annealing at 52 ℃ for 30s; extending at 72℃for 60s (30 cycles), and over-extending at 72℃for 10min.
The correctly sequenced pXMJ-CEY17_RS 14810 and pXMJ-CEY17_RS 14810 GG1189-1190TA plasmids were respectively electrotransformed into Corynebacterium glutamicum CGMCC No.21260, cultured in medium, the medium composition and culture conditions are shown in Table 1, single colonies generated by the culture were identified by PCR through primers M13R (-48)/P18, PCR amplified to contain fragments of 1558bp (the sequence without mutation is shown as SEQ ID No.15, the position 1444-1445 of the sequence with mutation is TA, and the rest is shown as SEQ ID No. 15) as positive strains, which were named YPV-094 (without mutation point) and YPV-095 (with mutation point).
Recombinant bacterium YPV-094 contains CEY17_RS14810 gene shown in SEQ ID No.1, and recombinant bacterium YPV-094 is engineering bacterium for over-expressing wild CEY17_RS14810 gene on plasmid, namely over-expressing plasmid pXMJ-CEY17_RS 14810 outside chromosome.
Recombinant bacterium YPV-095 contains mutated CEY17_RS14810 GG1189-1190TA gene shown in SEQ ID No.3, recombinant bacterium YPV-095 is engineering bacterium of over-expressing mutated CEY17_RS14810 GG1189-1190TA gene on plasmid, namely over-expressing by plasmid pXMJ-CEY17_RS 14810 GG1189-1190TA outside chromosome.
EXAMPLE 5 construction of an engineering Strain with the deletion of the CEY17_RS14810 Gene on the genome
Vector construction is carried out by adopting NEBuilder recombinant technology, and two pairs of primers for amplifying fragments at two ends of a CEY17_RS14810 gene coding region are synthesized to serve as upstream and downstream homology arm fragments according to the genome sequence of corynebacterium glutamicum ATCC14067 published by NCBI. Primers were designed as follows (synthesized by the company epivitrogen, shanghai):
P19:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTGCCGAGAAATTTTCATTGG-3'，
P20:5'-GTTCGGCCTCTTAAATGGCGGAGAACTAATATAAAAGTAA-3'，
P21:5'-TTACTTTTATATTAGTTCTCCGCCATTTAAGAGGCCGAAC-3'，
P22:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCGGCAAGGCTCACATCCGTT-3'。
The construction method comprises the following steps: the upstream homology arm fragment 720bp of CEY17_RS14810 and the downstream homology arm fragment 695bp of CEY17_RS14810 are obtained by PCR amplification with Corynebacterium glutamicum ATCC14067 as a template and with primers P19/P20 and P21/P22, respectively.
The amplified product was electrophoresed and purified using a column type DNA gel recovery kit, and the recovered DNA fragment was subjected to DNA assembly reaction with the pK18mobsacB plasmid (purchased from Addgene, inc. and containing kanamycin resistance as a selection marker) purified by Xbal I/BamH I cleavage using NEBuilder enzyme (NEBuilder HiFi DNA Assembly Master Mix, purchased from NEB, inc.), under the following reaction conditions: the ligation was performed at 50℃for 30min, and the M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') for the monoclonal antibody obtained after DH 5. Alpha. Transformation was used for PCR identification to obtain a positive knockout vector pK 18-. DELTA.CEY17-RS 14810, wherein the recombinant plasmid pK 18-. DELTA.CEY17-RS 14810 contains the Up-Down DNA 1375bp (the sequence is shown in SEQ ID No. 16) named as DeltaCEY17-RS 14810.
The plasmid was sequenced, the correctly sequenced knockout plasmid pK 18-. DELTA.CEY17_RS 14810 was electrotransformed into Corynebacterium glutamicum CGMCC No.21260, cultured in medium, medium composition and culture conditions are shown in Table 1, and single colonies resulting from the culture were identified by PCR using the following primers (synthesized by Shanghai Invitrogen):
P23:5'-TGCCGAGAAATTTTCATTGG-3' (corresponding to the inside of the Corynebacterium glutamicum CGMCC No.21260CEY17_RS11895 gene),
P24:5'-CGGCAAGGCTCACATCCGTT-3' (corresponding to the inside of the Corynebacterium glutamicum CGMCC No.21260CEY17_RS11905 gene).
The strain amplified by the PCR to obtain bands of 1301bp and 2570bp simultaneously is a positive strain, and the strain amplified to obtain bands of 2570bp only is a primary strain. Positive strains are respectively cultured on a medium containing kanamycin and a medium not containing kanamycin after being screened on a 15% sucrose medium, the strains which do not grow on the medium not containing kanamycin are selected to grow on the medium not containing kanamycin, and the strains which do not grow on the medium containing kanamycin are further identified by PCR (polymerase chain reaction) by using a P23/P24 primer, and the strains amplified into the 1301bp band are positive strains CEY17_RS14810 with the CEY17_RS14810 gene coding region knocked out. The positive strain CEY17_RS14810 fragment was amplified again by PCR with the P23/P24 primer and ligated into the pMD19-T vector for sequencing, the correctly sequenced strain was designated YPV-096 (the CEY17_RS14810 gene on the genome on 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 fermentation tank (purchased from Shanghai Biotechnology Co., ltd.) of BLBIO-5GC-4-H type with the medium shown in Table 3 and the control process shown in Table 4. Each strain was repeated three times and the results are shown in table 5.
As shown in Table 5, site-directed mutagenesis (e.g., mutation to CEY17_RS14810 GG1189-1190TA) and overexpression of the CEY17_RS14810 gene coding region in Corynebacterium glutamicum contributes to improvement of L-valine yield and conversion rate, while knockout or attenuation of the CEY17_RS14810 gene is detrimental to accumulation of L-valine.
TABLE 3 fermentation Medium formulation (balance water)
| Composition of the components | Formulation of |
| Ammonium sulfate | 14g/L |
| Monopotassium 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) defoamer | 0.5mL/L |
| 70% Glucose (bottom candy) | 40g/L |
TABLE 4 fermentation control process
TABLE 5L results of valine fermentation experiments
| Strain | OD610 | L-valine yield (g/L) |
| Corynebacterium glutamicum CGMCC No.21260 | 98.2 | 84.1 |
| YPV-091 | 100.2 | 85.2 |
| YPV-092 | 100.1 | 84.5 |
| YPV-093 | 99.4 | 85.7 |
| YPV-094 | 100.6 | 85.3 |
| YPV-095 | 101.3 | 86.1 |
| YPV-096 | 97.7 | 83.1 |
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
SEQUENCE LISTING
<110> Ningxia Yipin biotechnology Co., ltd
<120> Method for constructing L-valine-producing recombinant microorganism, nucleic acid molecule and biological material therefor
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 1269
<212> DNA
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 1
atgcgcctca acaaacgact cccagcggca ctctccggac tgctgctctc tgctgccctt 60
cttgccggat gctccacttc tggaaccgcc gagaccacga caacaaccgt ttcatctgct 120
gcggcatcaa caaccacttc cacctcctcc gattcctctt cctcctcttc ctccgactca 180
agcaccaccg ccgaaaccat ctccaacacc gcggaagctg cccaagcttt cttgtccacc 240
ctgtccaccg aagaacaaga cgccgtactc tacgactacg acgctgaaga aaagtccacc 300
ggctggtcta acttcccagt caccttcgtg cagcgttccg gcgtgaacct caccgacctc 360
actgaggaac agcaagcagc tgccctcaac gtgctgaaga acctgctcaa cgacgacgcc 420
taccaaatga tcgaagacat catggctagc gatcagtacc tcaacgacga aagcaacacc 480
accgaggatt ccctcggcca gtactacatc gcattcttcg gcgatccaag cagcgactcc 540
gactggtcca tccaattcgg cggacaccac atcggcatca acgccacctt ctccgacggt 600
gccatcacct tcgccccaac ccaccttggc acccagcctt ccgagtggac caaccaggac 660
ggcgaaaccg ttgcagcact aagcaacatg tacgaaaccg ccttcgcctt ctacgacagc 720
ctcaccgaag agcagcaagc acagctctac cagggtgaag agttggattc catggtctgc 780
gcaccgggca gcacctgcga ctacccaacc ggcaccggct tgaaaggctc cgacctcacc 840
gacgagcaaa aggaattgct tctcgacgtg atcgccaact gggttggtct agccgatgag 900
gaaaccaccg aaactgaact cgatgccatc cgcgaaaccc tggatgacac ctacatcaac 960
tggtccggag ccaccgagta cgacacctcc accggcgacg gcatctactt ccagatcagt 1020
ggcccaaagg tctacattga gttcgctaac cagcaaggtt ctgcaggtgc cgacatcgac 1080
ggtgtcatca ccgctggatg gggccacatt cacaccatct accgcgaccc aaccaatgat 1140
tacgctaact ccgtaactca ggaagcagcc agcggaatga tgggcggcgg ccctggtggt 1200
aatggtggcg agatgcctag cggtgacatg cctactggtg aaatgccttc tggcgctcca 1260
tcaaactaa 1269
<210> 2
<211> 422
<212> PRT
<213> Corynebacterium glutamicum (Corynebacterium glutamicum)
<400> 2
Met Arg Leu Asn Lys Arg Leu Pro Ala Ala Leu Ser Gly Leu Leu Leu
1 5 10 15
Ser Ala Ala Leu Leu Ala Gly Cys Ser Thr Ser Gly Thr Ala Glu Thr
20 25 30
Thr Thr Thr Thr Val Ser Ser Ala Ala Ala Ser Thr Thr Thr Ser Thr
35 40 45
Ser Ser Asp Ser Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Thr Ala
50 55 60
Glu Thr Ile Ser Asn Thr Ala Glu Ala Ala Gln Ala Phe Leu Ser Thr
65 70 75 80
Leu Ser Thr Glu Glu Gln Asp Ala Val Leu Tyr Asp Tyr Asp Ala Glu
85 90 95
Glu Lys Ser Thr Gly Trp Ser Asn Phe Pro Val Thr Phe Val Gln Arg
100 105 110
Ser Gly Val Asn Leu Thr Asp Leu Thr Glu Glu Gln Gln Ala Ala Ala
115 120 125
Leu Asn Val Leu Lys Asn Leu Leu Asn Asp Asp Ala Tyr Gln Met Ile
130 135 140
Glu Asp Ile Met Ala Ser Asp Gln Tyr Leu Asn Asp Glu Ser Asn Thr
145 150 155 160
Thr Glu Asp Ser Leu Gly Gln Tyr Tyr Ile Ala Phe Phe Gly Asp Pro
165 170 175
Ser Ser Asp Ser Asp Trp Ser Ile Gln Phe Gly Gly His His Ile Gly
180 185 190
Ile Asn Ala Thr Phe Ser Asp Gly Ala Ile Thr Phe Ala Pro Thr His
195 200 205
Leu Gly Thr Gln Pro Ser Glu Trp Thr Asn Gln Asp Gly Glu Thr Val
210 215 220
Ala Ala Leu Ser Asn Met Tyr Glu Thr Ala Phe Ala Phe Tyr Asp Ser
225 230 235 240
Leu Thr Glu Glu Gln Gln Ala Gln Leu Tyr Gln Gly Glu Glu Leu Asp
245 250 255
Ser Met Val Cys Ala Pro Gly Ser Thr Cys Asp Tyr Pro Thr Gly Thr
260 265 270
Gly Leu Lys Gly Ser Asp Leu Thr Asp Glu Gln Lys Glu Leu Leu Leu
275 280 285
Asp Val Ile Ala Asn Trp Val Gly Leu Ala Asp Glu Glu Thr Thr Glu
290 295 300
Thr Glu Leu Asp Ala Ile Arg Glu Thr Leu Asp Asp Thr Tyr Ile Asn
305 310 315 320
Trp Ser Gly Ala Thr Glu Tyr Asp Thr Ser Thr Gly Asp Gly Ile Tyr
325 330 335
Phe Gln Ile Ser Gly Pro Lys Val Tyr Ile Glu Phe Ala Asn Gln Gln
340 345 350
Gly Ser Ala Gly Ala Asp Ile Asp Gly Val Ile Thr Ala Gly Trp Gly
355 360 365
His Ile His Thr Ile Tyr Arg Asp Pro Thr Asn Asp Tyr Ala Asn Ser
370 375 380
Val Thr Gln Glu Ala Ala Ser Gly Met Met Gly Gly Gly Pro Gly Gly
385 390 395 400
Asn Gly Gly Glu Met Pro Ser Gly Asp Met Pro Thr Gly Glu Met Pro
405 410 415
Ser Gly Ala Pro Ser Asn
420
<210> 3
<211> 1269
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
atgcgcctca acaaacgact cccagcggca ctctccggac tgctgctctc tgctgccctt 60
cttgccggat gctccacttc tggaaccgcc gagaccacga caacaaccgt ttcatctgct 120
gcggcatcaa caaccacttc cacctcctcc gattcctctt cctcctcttc ctccgactca 180
agcaccaccg ccgaaaccat ctccaacacc gcggaagctg cccaagcttt cttgtccacc 240
ctgtccaccg aagaacaaga cgccgtactc tacgactacg acgctgaaga aaagtccacc 300
ggctggtcta acttcccagt caccttcgtg cagcgttccg gcgtgaacct caccgacctc 360
actgaggaac agcaagcagc tgccctcaac gtgctgaaga acctgctcaa cgacgacgcc 420
taccaaatga tcgaagacat catggctagc gatcagtacc tcaacgacga aagcaacacc 480
accgaggatt ccctcggcca gtactacatc gcattcttcg gcgatccaag cagcgactcc 540
gactggtcca tccaattcgg cggacaccac atcggcatca acgccacctt ctccgacggt 600
gccatcacct tcgccccaac ccaccttggc acccagcctt ccgagtggac caaccaggac 660
ggcgaaaccg ttgcagcact aagcaacatg tacgaaaccg ccttcgcctt ctacgacagc 720
ctcaccgaag agcagcaagc acagctctac cagggtgaag agttggattc catggtctgc 780
gcaccgggca gcacctgcga ctacccaacc ggcaccggct tgaaaggctc cgacctcacc 840
gacgagcaaa aggaattgct tctcgacgtg atcgccaact gggttggtct agccgatgag 900
gaaaccaccg aaactgaact cgatgccatc cgcgaaaccc tggatgacac ctacatcaac 960
tggtccggag ccaccgagta cgacacctcc accggcgacg gcatctactt ccagatcagt 1020
ggcccaaagg tctacattga gttcgctaac cagcaaggtt ctgcaggtgc cgacatcgac 1080
ggtgtcatca ccgctggatg gggccacatt cacaccatct accgcgaccc aaccaatgat 1140
tacgctaact ccgtaactca ggaagcagcc agcggaatga tgggcggcta ccctggtggt 1200
aatggtggcg agatgcctag cggtgacatg cctactggtg aaatgccttc tggcgctcca 1260
tcaaactaa 1269
<210> 4
<211> 422
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
Met Arg Leu Asn Lys Arg Leu Pro Ala Ala Leu Ser Gly Leu Leu Leu
1 5 10 15
Ser Ala Ala Leu Leu Ala Gly Cys Ser Thr Ser Gly Thr Ala Glu Thr
20 25 30
Thr Thr Thr Thr Val Ser Ser Ala Ala Ala Ser Thr Thr Thr Ser Thr
35 40 45
Ser Ser Asp Ser Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Thr Ala
50 55 60
Glu Thr Ile Ser Asn Thr Ala Glu Ala Ala Gln Ala Phe Leu Ser Thr
65 70 75 80
Leu Ser Thr Glu Glu Gln Asp Ala Val Leu Tyr Asp Tyr Asp Ala Glu
85 90 95
Glu Lys Ser Thr Gly Trp Ser Asn Phe Pro Val Thr Phe Val Gln Arg
100 105 110
Ser Gly Val Asn Leu Thr Asp Leu Thr Glu Glu Gln Gln Ala Ala Ala
115 120 125
Leu Asn Val Leu Lys Asn Leu Leu Asn Asp Asp Ala Tyr Gln Met Ile
130 135 140
Glu Asp Ile Met Ala Ser Asp Gln Tyr Leu Asn Asp Glu Ser Asn Thr
145 150 155 160
Thr Glu Asp Ser Leu Gly Gln Tyr Tyr Ile Ala Phe Phe Gly Asp Pro
165 170 175
Ser Ser Asp Ser Asp Trp Ser Ile Gln Phe Gly Gly His His Ile Gly
180 185 190
Ile Asn Ala Thr Phe Ser Asp Gly Ala Ile Thr Phe Ala Pro Thr His
195 200 205
Leu Gly Thr Gln Pro Ser Glu Trp Thr Asn Gln Asp Gly Glu Thr Val
210 215 220
Ala Ala Leu Ser Asn Met Tyr Glu Thr Ala Phe Ala Phe Tyr Asp Ser
225 230 235 240
Leu Thr Glu Glu Gln Gln Ala Gln Leu Tyr Gln Gly Glu Glu Leu Asp
245 250 255
Ser Met Val Cys Ala Pro Gly Ser Thr Cys Asp Tyr Pro Thr Gly Thr
260 265 270
Gly Leu Lys Gly Ser Asp Leu Thr Asp Glu Gln Lys Glu Leu Leu Leu
275 280 285
Asp Val Ile Ala Asn Trp Val Gly Leu Ala Asp Glu Glu Thr Thr Glu
290 295 300
Thr Glu Leu Asp Ala Ile Arg Glu Thr Leu Asp Asp Thr Tyr Ile Asn
305 310 315 320
Trp Ser Gly Ala Thr Glu Tyr Asp Thr Ser Thr Gly Asp Gly Ile Tyr
325 330 335
Phe Gln Ile Ser Gly Pro Lys Val Tyr Ile Glu Phe Ala Asn Gln Gln
340 345 350
Gly Ser Ala Gly Ala Asp Ile Asp Gly Val Ile Thr Ala Gly Trp Gly
355 360 365
His Ile His Thr Ile Tyr Arg Asp Pro Thr Asn Asp Tyr Ala Asn Ser
370 375 380
Val Thr Gln Glu Ala Ala Ser Gly Met Met Gly Gly Tyr Pro Gly Gly
385 390 395 400
Asn Gly Gly Glu Met Pro Ser Gly Asp Met Pro Thr Gly Glu Met Pro
405 410 415
Ser Gly Ala Pro Ser Asn
420
<210> 5
<211> 1304
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctagcggt gccatcacct tcgccccaac 60
ccaccttggc acccagcctt ccgagtggac caaccaggac ggcgaaaccg ttgcagcact 120
aagcaacatg tacgaaaccg ccttcgcctt ctacgacagc ctcaccgaag agcagcaagc 180
acagctctac cagggtgaag agttggattc catggtctgc gcaccgggca gcacctgcga 240
ctacccaacc ggcaccggct tgaaaggctc cgacctcacc gacgagcaaa aggaattgct 300
tctcgacgtg atcgccaact gggttggtct agccgatgag gaaaccaccg aaactgaact 360
cgatgccatc cgcgaaaccc tggatgacac ctacatcaac tggtccggag ccaccgagta 420
cgacacctcc accggcgacg gcatctactt ccagatcagt ggcccaaagg tctacattga 480
gttcgctaac cagcaaggtt ctgcaggtgc cgacatcgac ggtgtcatca ccgctggatg 540
gggccacatt cacaccatct accgcgaccc aaccaatgat tacgctaact ccgtaactca 600
ggaagcagcc agcggaatga tgggcggcta ccctggtggt aatggtggcg agatgcctag 660
cggtgacatg cctactggtg aaatgccttc tggcgctcca tcaaactaac gccatttaag 720
aggccgaacc cgtgagacga gcatttacat cacctcacgg gtttggatcc ctcagaggcg 780
attctgtgaa gtcggtttct gctgggccca ggtcagtttc tccaggcggt tagcattgga 840
gaggcgtttc agaccaaaag cgctcggatt cttccgatcc ttggcctcga atgacagttt 900
cctcgcctac aaagtgctgt ttcagaccaa gaaccccact tttcgactgg ccctttggtc 960
tctttttcaa gtccacccac aaagcgcgtg tccctcgtca aagaagggca cgcgcttgaa 1020
gtgtttttaa gagattttag cggatggtca cctggcggga cttgatgttc tccagctgac 1080
ggcgctcgtc ggcgttgagc tgtgcatcgt tatccagttc agcgacgatc ttctcgttga 1140
gtgcaaccaa aacatcggcg taatcggtgg atggacgctc aggatcgaga tcccacactg 1200
ggacgacgat gccgtgggtg cggaaagcgc cagcgaactt ggtttcctcg ccgagattca 1260
gctcgcgggt accgagctcg aattcgtaat catggtcata gctg 1304
<210> 6
<211> 1311
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctagcggt gccatcacct tcgccccaac 60
ccaccttggc acccagcctt ccgagtggac caaccaggac ggcgaaaccg ttgcagcact 120
aagcaacatg tacgaaaccg ccttcgcctt ctacgacagc ctcaccgaag agcagcaagc 180
acagctctac cagggtgaag agttggattc catggtctgc gcaccgggca gcacctgcga 240
ctacccaacc ggcaccggct tgaaaggctc cgacctcacc gacgagcaaa aggaattgct 300
tctcgacgtg atcgccaact gggttggtct agccgatgag gaaaccaccg aaactgaact 360
cgatgccatc cgcgaaaccc tggatgacac ctacatcaac tggtccggag ccaccgagta 420
cgacacctcc accggcgacg gcatctactt ccagatcagt ggcccaaagg tctacattga 480
gttcgctaac cagcaaggtt ctgcaggtgc cgacatcgac ggtgtcatca ccgctggatg 540
gggccacatt cacaccatct accgcgaccc aaccaatgat tacgctaact ccgtaactca 600
ggaagcagcc agcggaatga tgggcggcta ccctggtggt aatggtggcg agatgcctag 660
cggtgacatg cctactggtg aaatgccttc tggcgctcca tcaaactaac gccatttaag 720
aggccgaacc cgtgagacga gcatttacat cacctcacgg gtttggatcc ctcagaggcg 780
attctgtgaa gtcggtttct gctgggccca ggtcagtttc tccaggcggt tagcattgga 840
gaggcgtttc agaccaaaag cgctcggatt cttccgatcc ttggcctcga atgacagttt 900
cctcgcctac aaagtgctgt ttcagaccaa gaaccccact tttcgactgg ccctttggtc 960
tctttttcaa gtccacccac aaagcgcgtg tccctcgtca aagaagggca cgcgcttgaa 1020
gtgtttttaa gagattttag cggatggtca cctggcggga cttgatgttc tccagctgac 1080
ggcgctcgtc ggcgttgagc tgtgcatcgt tatccagttc agcgacgatc ttctcgttga 1140
gtgcaaccaa aacatcggcg taatcggtgg atggacgctc aggatcgaga tcccacactg 1200
ggacgacgat gccgtgggtg cggaaagcgc cagcgaactt ggtttcctcg ccgagattca 1260
gctcgcgggt accgagctcg aattcgtaat catggtcata gctgtttcct g 1311
<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 ttgggaagcg 780
attctgcaca ttttt 795
<210> 8
<211> 1489
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
acattgcgat tggggttggg aagcgattct gcacattttt taacatcccc aaggcgtgat 60
ttcgattttc ggaatcacgc ctttcccatt ttcgcgttaa aataccaggt caacacacac 120
aggaaccgtt cagaaacctt ccagattgct cactttttga tttcactttt ttgagaagtt 180
ttacttttat attagttctc atgcgcctca acaaacgact cccagcggca ctctccggac 240
tgctgctctc tgctgccctt cttgccggat gctccacttc tggaaccgcc gagaccacga 300
caacaaccgt ttcatctgct gcggcatcaa caaccacttc cacctcctcc gattcctctt 360
cctcctcttc ctccgactca agcaccaccg ccgaaaccat ctccaacacc gcggaagctg 420
cccaagcttt cttgtccacc ctgtccaccg aagaacaaga cgccgtactc tacgactacg 480
acgctgaaga aaagtccacc ggctggtcta acttcccagt caccttcgtg cagcgttccg 540
gcgtgaacct caccgacctc actgaggaac agcaagcagc tgccctcaac gtgctgaaga 600
acctgctcaa cgacgacgcc taccaaatga tcgaagacat catggctagc gatcagtacc 660
tcaacgacga aagcaacacc accgaggatt ccctcggcca gtactacatc gcattcttcg 720
gcgatccaag cagcgactcc gactggtcca tccaattcgg cggacaccac atcggcatca 780
acgccacctt ctccgacggt gccatcacct tcgccccaac ccaccttggc acccagcctt 840
ccgagtggac caaccaggac ggcgaaaccg ttgcagcact aagcaacatg tacgaaaccg 900
ccttcgcctt ctacgacagc ctcaccgaag agcagcaagc acagctctac cagggtgaag 960
agttggattc catggtctgc gcaccgggca gcacctgcga ctacccaacc ggcaccggct 1020
tgaaaggctc cgacctcacc gacgagcaaa aggaattgct tctcgacgtg atcgccaact 1080
gggttggtct agccgatgag gaaaccaccg aaactgaact cgatgccatc cgcgaaaccc 1140
tggatgacac ctacatcaac tggtccggag ccaccgagta cgacacctcc accggcgacg 1200
gcatctactt ccagatcagt ggcccaaagg tctacattga gttcgctaac cagcaaggtt 1260
ctgcaggtgc cgacatcgac ggtgtcatca ccgctggatg gggccacatt cacaccatct 1320
accgcgaccc aaccaatgat tacgctaact ccgtaactca ggaagcagcc agcggaatga 1380
tgggcggcgg ccctggtggt aatggtggcg agatgcctag cggtgacatg cctactggtg 1440
aaatgccttc tggcgctcca tcaaactaat caaaaacccc aacccgcac 1489
<210> 9
<211> 1489
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 9
acattgcgat tggggttggg aagcgattct gcacattttt taacatcccc aaggcgtgat 60
ttcgattttc ggaatcacgc ctttcccatt ttcgcgttaa aataccaggt caacacacac 120
aggaaccgtt cagaaacctt ccagattgct cactttttga tttcactttt ttgagaagtt 180
ttacttttat attagttctc atgcgcctca acaaacgact cccagcggca ctctccggac 240
tgctgctctc tgctgccctt cttgccggat gctccacttc tggaaccgcc gagaccacga 300
caacaaccgt ttcatctgct gcggcatcaa caaccacttc cacctcctcc gattcctctt 360
cctcctcttc ctccgactca agcaccaccg ccgaaaccat ctccaacacc gcggaagctg 420
cccaagcttt cttgtccacc ctgtccaccg aagaacaaga cgccgtactc tacgactacg 480
acgctgaaga aaagtccacc ggctggtcta acttcccagt caccttcgtg cagcgttccg 540
gcgtgaacct caccgacctc actgaggaac agcaagcagc tgccctcaac gtgctgaaga 600
acctgctcaa cgacgacgcc taccaaatga tcgaagacat catggctagc gatcagtacc 660
tcaacgacga aagcaacacc accgaggatt ccctcggcca gtactacatc gcattcttcg 720
gcgatccaag cagcgactcc gactggtcca tccaattcgg cggacaccac atcggcatca 780
acgccacctt ctccgacggt gccatcacct tcgccccaac ccaccttggc acccagcctt 840
ccgagtggac caaccaggac ggcgaaaccg ttgcagcact aagcaacatg tacgaaaccg 900
ccttcgcctt ctacgacagc ctcaccgaag agcagcaagc acagctctac cagggtgaag 960
agttggattc catggtctgc gcaccgggca gcacctgcga ctacccaacc ggcaccggct 1020
tgaaaggctc cgacctcacc gacgagcaaa aggaattgct tctcgacgtg atcgccaact 1080
gggttggtct agccgatgag gaaaccaccg aaactgaact cgatgccatc cgcgaaaccc 1140
tggatgacac ctacatcaac tggtccggag ccaccgagta cgacacctcc accggcgacg 1200
gcatctactt ccagatcagt ggcccaaagg tctacattga gttcgctaac cagcaaggtt 1260
ctgcaggtgc cgacatcgac ggtgtcatca ccgctggatg gggccacatt cacaccatct 1320
accgcgaccc aaccaatgat tacgctaact ccgtaactca ggaagcagcc agcggaatga 1380
tgggcggcta ccctggtggt aatggtggcg agatgcctag cggtgacatg cctactggtg 1440
aaatgccttc tggcgctcca tcaaactaat caaaaacccc aacccgcac 1489
<210> 10
<211> 769
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 10
ctggcgctcc atcaaactaa 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> 2214
<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
gggttgggaa gcgattctgc acatttttta acatccccaa ggcgtgattt cgattttcgg 840
aatcacgcct ttcccatttt cgcgttaaaa taccaggtca acacacacag gaaccgttca 900
gaaaccttcc agattgctca ctttttgatt tcactttttt gagaagtttt acttttatat 960
tagttctcat gcgcctcaac aaacgactcc cagcggcact ctccggactg ctgctctctg 1020
ctgcccttct tgccggatgc tccacttctg gaaccgccga gaccacgaca acaaccgttt 1080
catctgctgc ggcatcaaca accacttcca cctcctccga ttcctcttcc tcctcttcct 1140
ccgactcaag caccaccgcc gaaaccatct ccaacaccgc ggaagctgcc caagctttct 1200
tgtccaccct gtccaccgaa gaacaagacg ccgtactcta cgactacgac gctgaagaaa 1260
agtccaccgg ctggtctaac ttcccagtca ccttcgtgca gcgttccggc gtgaacctca 1320
ccgacctcac tgaggaacag caagcagctg ccctcaacgt gctgaagaac ctgctcaacg 1380
acgacgccta ccaaatgatc gaagacatca tggctagcga tcagtacctc aacgacgaaa 1440
gcaacaccac cgaggattcc ctcggccagt actacatcgc attcttcggc gatccaagca 1500
gcgactccga ctggtccatc caattcggcg gacaccacat cggcatcaac gccaccttct 1560
ccgacggtgc catcaccttc gccccaaccc accttggcac ccagccttcc gagtggacca 1620
accaggacgg cgaaaccgtt gcagcactaa gcaacatgta cgaaaccgcc ttcgccttct 1680
acgacagcct caccgaagag cagcaagcac agctctacca gggtgaagag ttggattcca 1740
tggtctgcgc accgggcagc acctgcgact acccaaccgg caccggcttg aaaggctccg 1800
acctcaccga cgagcaaaag gaattgcttc tcgacgtgat cgccaactgg gttggtctag 1860
ccgatgagga aaccaccgaa actgaactcg atgccatccg cgaaaccctg gatgacacct 1920
acatcaactg gtccggagcc accgagtacg acacctccac cggcgacggc atctacttcc 1980
agatcagtgg cccaaaggtc tacattgagt tcgctaacca gcaaggttct gcaggtgccg 2040
acatcgacgg tgtcatcacc gctggatggg gccacattca caccatctac cgcgacccaa 2100
ccaatgatta cgctaactcc gtaactcagg aagcagccag cggaatgatg ggcggcggcc 2160
ctggtggtaa tggtggcgag atgcctagcg gtgacatgcc tactggtgaa atgc 2214
<210> 12
<211> 1571
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 12
gccagtacta catcgcattc ttcggcgatc caagcagcga ctccgactgg tccatccaat 60
tcggcggaca ccacatcggc atcaacgcca ccttctccga cggtgccatc accttcgccc 120
caacccacct tggcacccag ccttccgagt ggaccaacca ggacggcgaa accgttgcag 180
cactaagcaa catgtacgaa accgccttcg ccttctacga cagcctcacc gaagagcagc 240
aagcacagct ctaccagggt gaagagttgg attccatggt ctgcgcaccg ggcagcacct 300
gcgactaccc aaccggcacc ggcttgaaag gctccgacct caccgacgag caaaaggaat 360
tgcttctcga cgtgatcgcc aactgggttg gtctagccga tgaggaaacc accgaaactg 420
aactcgatgc catccgcgaa accctggatg acacctacat caactggtcc ggagccaccg 480
agtacgacac ctccaccggc gacggcatct acttccagat cagtggccca aaggtctaca 540
ttgagttcgc taaccagcaa ggttctgcag gtgccgacat cgacggtgtc atcaccgctg 600
gatggggcca cattcacacc atctaccgcg acccaaccaa tgattacgct aactccgtaa 660
ctcaggaagc agccagcgga atgatgggcg gcggccctgg tggtaatggt ggcgagatgc 720
ctagcggtga catgcctact ggtgaaatgc cttctggcgc tccatcaaac taatcaaaaa 780
ccccaacccg cacattttta gatttctatt ttgtgtacat agggttcgga acaaagctta 840
aaccatcccc aattgaaatg tcgttacaca cccacatgtt tgaagtggag caaaccgaaa 900
accagttttc cccaacggca gccgcccccc acgttgaacc ttcgaaatag taggcaacac 960
catcaagcgg atcttcatca agcgaaatag tgattgactc ttcaccgttc cgcttacaaa 1020
ctgcgttagt gtcgctattt tccacccact tgtcacactc gtacccgttt tcatttagcc 1080
atttttcggc atgtcctatt ttctcgaacc gggcaggagc gtcagggctt ccgcagcccg 1140
ctagtagtag tccggctgca atgatgctta atgttttttt catgaattaa acatagtact 1200
ttgctggtaa aaatattgga gaaccccact ggcctacatg gtcagtgggg gcatttttgc 1260
gtttcacccc tcaaaaatca tcaccacact tgcgggattt ccccctgatt tcccccactc 1320
ccacaccatt cccagtggac agtgtggacg tattggacac attaaacaca ttgcgaccag 1380
gtaaaacgtc atgaccaggt atcgtcaatg ttcttgatga atttccgcac cgcaggatta 1440
tcattcgagg tggaataaat agcctgcagc tccgctaaac caacaggtag atcataaaaa 1500
tggcgatact caacaccgct gtaattgagt tttttcgcgg actccggaac cagcgcaaca 1560
cccagtccag a 1571
<210> 13
<211> 1519
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 13
gcttgcatgc ctgcaggtcg actctagagg atccccaagc gattctgcac attttttaac 60
atccccaagg cgtgatttcg attttcggaa tcacgccttt cccattttcg cgttaaaata 120
ccaggtcaac acacacagga accgttcaga aaccttccag attgctcact ttttgatttc 180
acttttttga gaagttttac ttttatatta gttctcatgc gcctcaacaa acgactccca 240
gcggcactct ccggactgct gctctctgct gcccttcttg ccggatgctc cacttctgga 300
accgccgaga ccacgacaac aaccgtttca tctgctgcgg catcaacaac cacttccacc 360
tcctccgatt cctcttcctc ctcttcctcc gactcaagca ccaccgccga aaccatctcc 420
aacaccgcgg aagctgccca agctttcttg tccaccctgt ccaccgaaga acaagacgcc 480
gtactctacg actacgacgc tgaagaaaag tccaccggct ggtctaactt cccagtcacc 540
ttcgtgcagc gttccggcgt gaacctcacc gacctcactg aggaacagca agcagctgcc 600
ctcaacgtgc tgaagaacct gctcaacgac gacgcctacc aaatgatcga agacatcatg 660
gctagcgatc agtacctcaa cgacgaaagc aacaccaccg aggattccct cggccagtac 720
tacatcgcat tcttcggcga tccaagcagc gactccgact ggtccatcca attcggcgga 780
caccacatcg gcatcaacgc caccttctcc gacggtgcca tcaccttcgc cccaacccac 840
cttggcaccc agccttccga gtggaccaac caggacggcg aaaccgttgc agcactaagc 900
aacatgtacg aaaccgcctt cgccttctac gacagcctca ccgaagagca gcaagcacag 960
ctctaccagg gtgaagagtt ggattccatg gtctgcgcac cgggcagcac ctgcgactac 1020
ccaaccggca ccggcttgaa aggctccgac ctcaccgacg agcaaaagga attgcttctc 1080
gacgtgatcg ccaactgggt tggtctagcc gatgaggaaa ccaccgaaac tgaactcgat 1140
gccatccgcg aaaccctgga tgacacctac atcaactggt ccggagccac cgagtacgac 1200
acctccaccg gcgacggcat ctacttccag atcagtggcc caaaggtcta cattgagttc 1260
gctaaccagc aaggttctgc aggtgccgac atcgacggtg tcatcaccgc tggatggggc 1320
cacattcaca ccatctaccg cgacccaacc aatgattacg ctaactccgt aactcaggaa 1380
gcagccagcg gaatgatggg cggcggccct ggtggtaatg gtggcgagat gcctagcggt 1440
gacatgccta ctggtgaaat gccttctggc gctccatcaa actaagtttt ggcggatgag 1500
agaagatttt cagcctgat 1519
<210> 14
<211> 1519
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 14
gcttgcatgc ctgcaggtcg actctagagg atccccaagc gattctgcac attttttaac 60
atccccaagg cgtgatttcg attttcggaa tcacgccttt cccattttcg cgttaaaata 120
ccaggtcaac acacacagga accgttcaga aaccttccag attgctcact ttttgatttc 180
acttttttga gaagttttac ttttatatta gttctcatgc gcctcaacaa acgactccca 240
gcggcactct ccggactgct gctctctgct gcccttcttg ccggatgctc cacttctgga 300
accgccgaga ccacgacaac aaccgtttca tctgctgcgg catcaacaac cacttccacc 360
tcctccgatt cctcttcctc ctcttcctcc gactcaagca ccaccgccga aaccatctcc 420
aacaccgcgg aagctgccca agctttcttg tccaccctgt ccaccgaaga acaagacgcc 480
gtactctacg actacgacgc tgaagaaaag tccaccggct ggtctaactt cccagtcacc 540
ttcgtgcagc gttccggcgt gaacctcacc gacctcactg aggaacagca agcagctgcc 600
ctcaacgtgc tgaagaacct gctcaacgac gacgcctacc aaatgatcga agacatcatg 660
gctagcgatc agtacctcaa cgacgaaagc aacaccaccg aggattccct cggccagtac 720
tacatcgcat tcttcggcga tccaagcagc gactccgact ggtccatcca attcggcgga 780
caccacatcg gcatcaacgc caccttctcc gacggtgcca tcaccttcgc cccaacccac 840
cttggcaccc agccttccga gtggaccaac caggacggcg aaaccgttgc agcactaagc 900
aacatgtacg aaaccgcctt cgccttctac gacagcctca ccgaagagca gcaagcacag 960
ctctaccagg gtgaagagtt ggattccatg gtctgcgcac cgggcagcac ctgcgactac 1020
ccaaccggca ccggcttgaa aggctccgac ctcaccgacg agcaaaagga attgcttctc 1080
gacgtgatcg ccaactgggt tggtctagcc gatgaggaaa ccaccgaaac tgaactcgat 1140
gccatccgcg aaaccctgga tgacacctac atcaactggt ccggagccac cgagtacgac 1200
acctccaccg gcgacggcat ctacttccag atcagtggcc caaaggtcta cattgagttc 1260
gctaaccagc aaggttctgc aggtgccgac atcgacggtg tcatcaccgc tggatggggc 1320
cacattcaca ccatctaccg cgacccaacc aatgattacg ctaactccgt aactcaggaa 1380
gcagccagcg gaatgatggg cggctaccct ggtggtaatg gtggcgagat gcctagcggt 1440
gacatgccta ctggtgaaat gccttctggc gctccatcaa actaagtttt ggcggatgag 1500
agaagatttt cagcctgat 1519
<210> 15
<211> 1558
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 15
agcggataac aatttcacac aggaaacaga attaattaag cttgcatgcc tgcaggtcga 60
ctctagagga tccccaagcg attctgcaca ttttttaaca tccccaaggc gtgatttcga 120
ttttcggaat cacgcctttc ccattttcgc gttaaaatac caggtcaaca cacacaggaa 180
ccgttcagaa accttccaga ttgctcactt tttgatttca cttttttgag aagttttact 240
tttatattag ttctcatgcg cctcaacaaa cgactcccag cggcactctc cggactgctg 300
ctctctgctg cccttcttgc cggatgctcc acttctggaa ccgccgagac cacgacaaca 360
accgtttcat ctgctgcggc atcaacaacc acttccacct cctccgattc ctcttcctcc 420
tcttcctccg actcaagcac caccgccgaa accatctcca acaccgcgga agctgcccaa 480
gctttcttgt ccaccctgtc caccgaagaa caagacgccg tactctacga ctacgacgct 540
gaagaaaagt ccaccggctg gtctaacttc ccagtcacct tcgtgcagcg ttccggcgtg 600
aacctcaccg acctcactga ggaacagcaa gcagctgccc tcaacgtgct gaagaacctg 660
ctcaacgacg acgcctacca aatgatcgaa gacatcatgg ctagcgatca gtacctcaac 720
gacgaaagca acaccaccga ggattccctc ggccagtact acatcgcatt cttcggcgat 780
ccaagcagcg actccgactg gtccatccaa ttcggcggac accacatcgg catcaacgcc 840
accttctccg acggtgccat caccttcgcc ccaacccacc ttggcaccca gccttccgag 900
tggaccaacc aggacggcga aaccgttgca gcactaagca acatgtacga aaccgccttc 960
gccttctacg acagcctcac cgaagagcag caagcacagc tctaccaggg tgaagagttg 1020
gattccatgg tctgcgcacc gggcagcacc tgcgactacc caaccggcac cggcttgaaa 1080
ggctccgacc tcaccgacga gcaaaaggaa ttgcttctcg acgtgatcgc caactgggtt 1140
ggtctagccg atgaggaaac caccgaaact gaactcgatg ccatccgcga aaccctggat 1200
gacacctaca tcaactggtc cggagccacc gagtacgaca cctccaccgg cgacggcatc 1260
tacttccaga tcagtggccc aaaggtctac attgagttcg ctaaccagca aggttctgca 1320
ggtgccgaca tcgacggtgt catcaccgct ggatggggcc acattcacac catctaccgc 1380
gacccaacca atgattacgc taactccgta actcaggaag cagccagcgg aatgatgggc 1440
ggcggccctg gtggtaatgg tggcgagatg cctagcggtg acatgcctac tggtgaaatg 1500
ccttctggcg ctccatcaaa ctaagttttg gcggatgaga gaagattttc agcctgat 1558
<210> 16
<211> 1375
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 16
cagtgccaag cttgcatgcc tgcaggtcga ctctagtgcc gagaaatttt cattgggcct 60
ggatttcatt gaaattcagg cccttgccat tcccgatgct gctaggttcg tgttaagcaa 120
ccacaagttg caccaccatt cttccatcca gagaatacga attcgtttac ctctagaaag 180
gcactttccc atgtcttacc acgatcacag cgacatcgaa tacctcgaga agatcggcgc 240
caactcccct gacgccttca aagcttttgc ccattttgat gaggcagctc tccgcggccc 300
gaacaagaaa atcccacgca actacaccga aatgatcgca cttgcggtcg cattcacaac 360
ccaatgcgcc tactgcatcg acatccacac tgccgctgcg aagaaggaag gtgtcaccac 420
cgaggagctc gctgaggttg cgctcatcgc cgcagcactt cgggcaggcg gcgccatgac 480
gcacggcgca cttgccatga agctttacga cgaaaactag aagcgattct gcacattttt 540
taacatcccc aaggcgtgat ttcgattttc ggaatcacgc ctttcccatt ttcgcgttaa 600
aataccaggt caacacacac aggaaccgtt cagaaacctt ccagattgct cactttttga 660
tttcactttt ttgagaagtt ttacttttat attagttctc cgccatttaa gaggccgaac 720
ccgtgagacg agcatttaca tcacctcacg ggtttggatc cctcagaggc gattctgtga 780
agtcggtttc tgctgggccc aggtcagttt ctccaggcgg ttagcattgg agaggcgttt 840
cagaccaaaa gcgctcggat tcttccgatc cttggcctcg aatgacagtt tcctcgccta 900
caaagtgctg tttcagacca agaaccccac ttttcgactg gccctttggt ctctttttca 960
agtccaccca caaagcgcgt gtccctcgtc aaagaagggc acgcgcttga agtgttttta 1020
agagatttta gcggatggtc acctggcggg acttgatgtt ctccagctga cggcgctcgt 1080
cggcgttgag ctgtgcatcg ttatccagtt cagcgacgat cttctcgttg agtgcaacca 1140
aaacatcggc gtaatcggtg gatggacgct caggatcgag atcccacact gggacgacga 1200
tgccgtgggt gcggaaagcg ccagcgaact tggtttcctc gccgagattc agctcgccac 1260
gtgctgcgat gcgtgccaaa gcgttgaaca gtgcggtttc gttttcggtg cggacccaac 1320
ggatgtgagc cttgccgggg taccgagctc gaattcgtaa tcatggtcat agctg 1375
Claims (9)
1. A method for constructing recombinant corynebacterium glutamicum is characterized by comprising the step of introducing a nucleic acid molecule encoding a protein into corynebacterium glutamicum to obtain the recombinant corynebacterium glutamicum, wherein the amino acid sequence of the protein is shown as SEQ ID No. 4.
2. The protein according to claim 1.
3. A nucleic acid molecule encoding a protein as claimed in claim 1 or 2.
4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID No. 3.
5. A biomaterial characterized in that the biomaterial is any one of the following:
C1 A cassette comprising the nucleic acid molecule of any one of claims 3 or 4;
c2 A recombinant vector comprising the nucleic acid molecule of any one of claims 3 or 4;
C3 A recombinant corynebacterium glutamicum comprising the nucleic acid molecule of any one of claims 3 or 4.
Use of any one of D1) -D6) for increasing the production of L-valine in corynebacterium glutamicum;
wherein, D1) -D6) are:
D1 A protein as claimed in claim 1 or 2;
d2 A nucleic acid molecule according to claim 3 or 4;
D3 An expression cassette or recombinant vector as claimed in claim 5.
D4 A DNA molecule with a nucleotide sequence of SEQ ID No. 1;
d5 An expression cassette containing D4) said DNA molecule;
d6 A recombinant vector comprising D4) said DNA molecule.
7. A method for increasing the production of L-valine in corynebacterium glutamicum, said method comprising any one of the following steps:
E1 Increasing the expression level or the content of the nucleic acid molecule according to claim 3 or 4 in corynebacterium glutamicum;
E2 Increasing the expression level or the content of the DNA molecule according to D4) in claim 6 in Corynebacterium glutamicum.
8. A method for increasing the production of L-valine in coryneform glutamicum, said method comprising mutating nucleotide GG at positions 1189-1190 of the DNA molecule shown in SEQ ID No.1 to TA.
9. A process for producing L-valine, which comprises producing L-valine using the recombinant corynebacterium glutamicum according to claim 1 and/or claim 5.
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|---|---|---|---|
| CN202210137750.8A CN114540262B (en) | 2022-02-15 | 2022-02-15 | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same |
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| CN202210137750.8A CN114540262B (en) | 2022-02-15 | 2022-02-15 | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same |
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| CN114540262B true CN114540262B (en) | 2024-05-03 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667682A (en) * | 2021-09-10 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | YH66-RS11190 gene mutant and application thereof in preparation of L-valine |
| CN113666991A (en) * | 2021-08-20 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | Engineering bacterium obtained by YH66-RS07015 gene modification and application thereof in preparation of valine |
| CN113683667A (en) * | 2021-08-25 | 2021-11-23 | 宁夏伊品生物科技股份有限公司 | Engineering bacterium obtained by YH66-RS10865 gene transformation and application thereof in preparation of valine |
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| CN113666991A (en) * | 2021-08-20 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | Engineering bacterium obtained by YH66-RS07015 gene modification and application thereof in preparation of valine |
| CN113683667A (en) * | 2021-08-25 | 2021-11-23 | 宁夏伊品生物科技股份有限公司 | Engineering bacterium obtained by YH66-RS10865 gene transformation and application thereof in preparation of valine |
| CN113667682A (en) * | 2021-09-10 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | YH66-RS11190 gene mutant and application thereof in preparation of L-valine |
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| MULTISPECIES: DUF3500 domain-containing protein [Corynebacterium];无;《Genabnk Database》;WP_003862594.1 * |
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