YH66-RS11190 gene mutant and application thereof in preparation of L-valine

文档序号：1856603 发布日期：2021-11-19 浏览：30次中文

阅读说明：本技术 Yh66-rs11190基因突变体及其在制备l-缬氨酸中的应用 (YH66-RS11190 gene mutant and application thereof in preparation of L-valine ) 是由孟刚魏爱英贾慧萍赵春光毕国栋于 2021-09-10 设计创作，主要内容包括：本发明公开了YH66-RS11190基因突变体及其在制备L-缬氨酸中的应用。具体地公开了SEQ ID No.1所示的YH66-RS11190基因在调控微生物的L-缬氨酸的产量中的应用。本发明通过对YH66-RS11190基因进行过表达或敲除、或定点突变(SEQ ID No.3)可以调控L-缬氨酸的产量。对YH66-RS11190基因编码区进行点突变及过表达,有助于L-缬氨酸产量及转化率的提高,而对基因进行敲除或弱化,不利于L-缬氨酸的积累。可利用YH66-RS11190基因来构建符合工业化生产的L-缬氨酸高产菌种,对L-缬氨酸的工业化生产具有广泛的应用价值和重要的经济意义。(The invention discloses a YH66-RS11190 gene mutant and application thereof in preparation of L-valine. Specifically discloses the application of YH66-RS11190 gene shown in SEQ ID No.1 in regulating and controlling the yield of L-valine of microorganisms. The invention can regulate the yield of L-valine by carrying out overexpression or knockout or site-directed mutagenesis (SEQ ID No.3) on YH66-RS11190 gene. The YH66-RS11190 gene coding region is subjected to point mutation and overexpression, which is beneficial to the improvement of L-valine yield and transformation rate, and the gene is knocked out or weakened, which is not beneficial to the accumulation of L-valine. The YH66-RS11190 gene can be used for constructing an L-valine high-yield strain which accords with industrial production, and the strain has wide application value and important economic significance for the industrial production of the L-valine.)

1. Use of a nucleic acid molecule, wherein said use is any one of:

A1) the use of said nucleic acid molecule for regulating the production of L-valine by a microorganism;

A2) the application of the nucleic acid molecule in constructing genetic engineering bacteria for producing L-valine;

A3) the use of said nucleic acid molecule for the preparation of L-valine;

the nucleic acid molecule is B1) or B2) as follows:

B1) a DNA molecule with a nucleotide sequence of SEQ ID No. 1;

B2) the 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 B1) and has the same function.

2. Use of a biological material associated with a nucleic acid molecule as claimed in claim 1, wherein the use is any one of:

C1) use of a biological material related to the nucleic acid molecule of claim 1 for regulating the production of L-valine by a microorganism;

C2) use of a biological material related to the nucleic acid molecule of claim 1 for constructing a genetically engineered bacterium that produces L-valine;

C3) use of a biological material related to the nucleic acid molecule of claim 1 for the preparation of L-valine;

the biomaterial is any one of the following D1) to D3):

D1) an expression cassette comprising the nucleic acid molecule of claim 1;

D2) a recombinant vector comprising the nucleic acid molecule of claim 1, or a recombinant vector comprising the expression cassette of D1);

D3) a recombinant microorganism comprising the nucleic acid molecule of claim 1, or a recombinant microorganism comprising the expression cassette of D1), or a recombinant microorganism comprising the recombinant vector of D2).

3. A method for increasing the production of L-valine in a microorganism, which comprises increasing the expression level or amount of the nucleic acid molecule of claim 1 in a target microorganism, and/or mutating the nucleic acid molecule of claim 1 in the target microorganism to obtain a microorganism having a higher L-valine production than the target microorganism.

4. The method of claim 3, wherein the mutation is a point mutation.

5. The method as claimed in claim 4, wherein the point mutation is a mutation of alanine at position 101 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 to threonine, resulting in a mutein having the amino acid sequence SEQ ID No. 4.

6. A biomaterial, characterized in that the biomaterial is any one of the following:

E1) protein with an amino acid sequence of SEQ ID No. 4;

E2) a nucleic acid molecule encoding the protein of E1);

E3) a DNA molecule shown as SEQ ID No.3 and encoding the protein E1);

E4) an expression cassette comprising the DNA molecule of E2) or E3);

E5) a recombinant vector containing the DNA molecule of E2) or E3), or a recombinant vector containing the expression cassette of E4);

E6) a recombinant microorganism containing the DNA molecule of E2) or E3), or a recombinant microorganism containing the expression cassette of E4), or a recombinant microorganism containing the recombinant vector of E5).

7. A method for producing L-valine, which comprises producing L-valine using the recombinant microorganism as claimed in claim 2 or 6.

8. The method according to claim 7, wherein the method is a fermentation method for producing L-valine and the recombinant microorganism is Corynebacterium.

9. A method for constructing the recombinant microorganism according to claim 2 or 6, comprising at least any one of:

F1) introducing the nucleic acid molecule of claim 1 into a microorganism of interest to obtain said recombinant microorganism;

F2) introducing a DNA molecule shown in SEQ ID No.3 into a target microorganism to obtain the recombinant microorganism;

F3) editing the nucleic acid molecule of claim 1 by gene editing means so that the desired microorganism contains the DNA molecule represented by SEQ ID No. 3.

10. Use of the biomaterial of claim 6 for the production of L-valine.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a YH66-RS11190 gene mutant and application thereof in preparation of L-valine.

Background

L-valine (L-valine), also known as 2-amino-3-methylbutyric acid, belongs to branched chain amino acid, is one of eight essential amino acids of a human body, and has the effects of promoting normal growth of the body, regulating blood sugar, enhancing the immune protection effect of the body, resisting central fatigue, resisting peripheral fatigue, accelerating the repair of the body after exercise and the like, so the L-valine has wide application and commercial value in the industries of food, medicine and cosmetics. And the L-valine gel has a positively charged end group, is a novel low molecular weight gel, can be prepared into hydrogel, and is widely applied to the fields of tissue engineering, photochemistry, electrochemistry and the like. Since L-valine has a special physiological function and cannot be synthesized by a human body, the market demand is large, so that the production of L-valine is concerned. At present, L-valine is mostly produced by a direct fermentation method, the valine produced by the fermentation method is L-type, optical resolution is not needed, and the method has the advantages of low raw material cost, mild reaction conditions, large-scale production and the like, and is a very economic 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 hot spots of technical development and fermentation engineering research of the L-valine fermentation industry, and have important significance for promoting the industrialization process of L-valine along with the development of the L-valine fermentation industry.

Disclosure of Invention

The technical problem to be solved by the invention is how to construct a recombinant plasmid and a recombinant bacterium by using YH66-RS11190 gene and/or improve the yield of L-valine. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the above technical problems, the present invention provides the application of the nucleic acid molecule (named YH66-RS11190) in the first place, wherein the application can be any one of the following:

A1) the application of the nucleic acid molecule YH66-RS11190 in regulating and controlling the yield of L-valine of the microorganism;

A2) the application of the nucleic acid molecule YH66-RS11190 in constructing a genetic engineering bacterium for producing L-valine;

A3) the application of the nucleic acid molecule YH66-RS11190 in preparing L-valine;

the nucleic acid molecule YH66-RS11190 may be B1) or B2) as follows:

B1) a DNA molecule with a nucleotide sequence of SEQ ID No. 1;

The DNA molecule shown in SEQ ID No.1 is the YH66-RS11190 gene.

The DNA molecule shown as SEQ ID No.1 (YH66-RS11190 gene) encodes the protein shown as SEQ ID No. 2.

The invention also provides an application of biological materials related to the nucleic acid molecule YH66-RS11190, wherein the application can be any one of the following:

C1) the application of the biological material related to the nucleic acid molecule YH66-RS11190 in regulating and controlling the yield of L-valine of microorganisms;

C2) the application of the biological material related to the nucleic acid molecule YH66-RS11190 in constructing the genetic engineering bacteria for producing L-valine;

C3) the application of the biological material related to the nucleic acid molecule YH66-RS11190 in the preparation of L-valine;

the biomaterial may be any one of the following D1) to D3):

D1) an expression cassette comprising the nucleic acid molecule YH66-RS 11190;

D2) a recombinant vector containing the nucleic acid molecule YH66-RS11190 or a recombinant vector containing the expression cassette described in D1);

D3) the recombinant microorganism containing the nucleic acid molecule YH66-RS11190, or the recombinant microorganism containing the expression cassette D1), or the recombinant microorganism containing the recombinant vector D2).

Further, the application can be realized by modifying the nucleic acid molecule YH66-RS11190 so as to improve the yield of L-valine of the microorganism.

Further, the application can also be used for obtaining the microorganism with higher L-valine yield than the target microorganism by increasing the expression amount or the content of the nucleic acid molecule YH66-RS11190 in the target microorganism, so as to improve the L-valine yield of the microorganism.

Such modifications include, but are not limited to: base substitution, base insertion, base deletion, or base modification.

Further, the alteration may be a point mutation.

Further, the point mutation may be mutation (alteration) of guanine (G) at position 301 of YH66-RS11190 gene (SEQ ID No. 1).

Further, the point mutation can be that guanine (G) at position 301 of YH66-RS11190 gene (SEQ ID No.1) is mutated into adenine (A), so as to obtain the DNA molecule shown in SEQ ID No. 3.

Herein, identity refers to the identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in BLAST2.1, the identity (%) can be obtained by searching using blastn as a program and calculating the identity of nucleotide sequences.

Herein, the 90% or greater identity can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

The regulation of the production of L-valine by the microorganism as described herein may be an increase or decrease in the production of L-valine by the microorganism (may be an enhancement or inhibition of L-valine biosynthesis).

The invention also provides a method for improving the yield of L-valine in a microorganism, which comprises the step of improving the expression quantity or the content of the nucleic acid molecule YH66-RS11190 in a target microorganism, and/or the step of mutating (base substitution, base insertion or base deletion) the nucleic acid molecule YH66-RS11190 in the target microorganism to obtain the microorganism with the yield of L-valine higher than that of the target microorganism.

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.

In the above method, the mutation may be a point mutation (point mutation), i.e., a mutation of a single nucleotide.

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

Specifically, the point mutation can be that guanine (G) at position 301 of YH66-RS11190 gene (SEQ ID No.1) is mutated into adenine (A), so as to obtain the DNA molecule shown in SEQ ID No. 3.

In the above method, the point mutation may be to mutate alanine at position 101 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 into threonine to obtain the mutein having the amino acid sequence of SEQ ID No. 4.

The invention also provides a biomaterial, which can be any one of the following:

E1) protein with an amino acid sequence of SEQ ID No. 4;

E2) a nucleic acid molecule encoding the protein of E1);

E3) a DNA molecule shown as SEQ ID No.3 and encoding the protein E1);

E4) an expression cassette comprising the DNA molecule of E2) or E3);

E5) a recombinant vector containing the DNA molecule of E2) or E3), or a recombinant vector containing the expression cassette of E4);

Herein, the vector may be a plasmid, cosmid, phage, or viral vector.

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), Bacillus (Bacillus), etc.

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 Brevibacterium flavum (Brevibacterium flavum) ATCC 15168.

The present invention also provides a method for producing L-valine, which comprises producing L-valine using the recombinant microorganism described in any one of the above.

In the above method, the method may be a fermentation method for producing L-valine, and the recombinant microorganism may be Corynebacterium (Corynebacterium glutamicum), and specifically Corynebacterium glutamicum (Corynebacterium glutamicum).

In one embodiment of the present invention, the recombinant microorganism is Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No. 21260.

The present invention also provides a method of constructing a recombinant microorganism as described herein, the method comprising at least any one of:

F1) introducing the nucleic acid molecule YH66-RS11190 into a target microorganism to obtain the recombinant microorganism;

F2) introducing a DNA molecule shown in SEQ ID No.3 into a target microorganism to obtain the recombinant microorganism;

F3) the nucleic acid molecule YH66-RS11190 was edited by a gene editing means (e.g., single-nucleotide gene editing) so that the objective microorganism contained the DNA molecule represented by SEQ ID No. 3.

The invention also provides application of the biological material in preparation of L-valine.

The present inventors have extensively and intensively studied and found for the first time that YH66-RS11190 gene is involved in L-valine biosynthesis, and that the production of L-valine in a microorganism can be controlled by overexpressing or knocking out or by site-directed mutagenesis (e.g., point mutagenesis) of YH66-RS11190 gene. Experiments show that point mutation and overexpression of the YH66-RS11190 gene coding region are beneficial to the improvement of L-valine yield and transformation rate, and the gene is knocked out or weakened to be not beneficial to the accumulation of L-valine. The YH66-RS11190 gene can be used for constructing a genetic engineering strain for producing L-valine so as to promote the yield increase of the L-valine, and the strain with high yield and high quality which meets the industrial production is cultivated, thereby having wide application value and important economic significance for the industrial production of the L-valine.

Deposit description

The strain name is as follows: corynebacterium glutamicum

Latin name: 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 recombinant vector containing Point-mutated YH66-RS11190 Gene coding region fragment

According to the genome sequence of Brevibacterium flavum (Brevibacterium flavum) ATCC15168 published by NCBI, two pairs of primers for amplifying YH66-RS11190 gene coding region are designed and synthesized, and YH66-RS of Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21260 (sequence determination confirms that wild type YH66-RS11190 gene is reserved on the chromosome of the strain) is subjected to allelic gene replacementThe 11190 gene coding region (SEQ ID No.1) is introduced with point mutation, and the point mutation is to mutate guanine (G) at position 301 in the nucleotide sequence (SEQ ID No.1) of YH66-RS11190 gene into adenine (A) to obtain a DNA molecule (mutant YH66-RS11190 gene, named YH66-RS11190) shown in SEQ ID No.3^G301A)。

Wherein, the DNA molecule shown in SEQ ID No.1 encodes a protein (the protein is named as protein YH66-RS11190) 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 mutant protein is named YH66-RS11190^A101T). The mutant protein YH66-RS11190^A101TThreonine 101 (T) in the amino acid sequence (SEQ ID No.4) is mutated from alanine (A).

The site-directed mutagenesis of the gene was performed by the overlap PCR (overlap PCR) technique, the primers were designed as follows (synthesized by Shanghai Invitrogen), and the base in bold font is the position of the mutation:

P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGGACAACCGTGACTTTGCGG 3'，

P2:5'AGCCCGAATAAAGCGTATTATCGACGCCAACCGC 3'，

P3:5'GCGGTTGGCGTCGATAATACGCTTTATTCGGGCT 3'，

P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATCAGCGCGAAAACAGTGGC 3'。

the construction method comprises the following steps: the Brevibacterium flavum ATCC15168 was used as a template, and PCR amplification was carried out with primers P1 and P2, and P3 and P4, respectively, to obtain two DNA fragments (YH66-RS11190 Up and YH66-RS11190 Down) having mutant bases and sizes of 651bp and 658bp, respectively, of the coding region of the YH66-RS11190 gene.

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 (YH66-RS11190 Up and YH66-RS11190 Down) were separated and purified by agarose gel electrophoresis, the desired band was recovered, and the two DNA fragments were used as templates and P1 and P4 were used as primers to amplify by Overlap PCR to obtain a DNA fragment of 1274bp (named YH66-RS11190Up-Down, the sequence of which is shown in SEQ ID No. 5). In the DNA fragment shown in SEQ ID No.5, the 335-1236 th (902bp) position is YH66-RS11190 containing a mutation site^G301AGene fragment (i.e. positions 1-902 of SEQ ID No. 3).

The Overlap PCR amplification reaction 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 procedure of the Overlap PCR amplification reaction is as follows: pre-denaturation at 94 ℃ for 5min, (denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 60s, 30 cycles), and over-extension at 72 ℃ for 10 min.

This DNA fragment (YH66-RS11190 Up-Down, SEQ ID No.5) contains a mutation site which leads to a change of guanine (G) at position 301 to adenine (A) and finally to a change of alanine (A) at position 101 to threonine (T) in the coding region of the YH66-RS11190 gene of the strain Corynebacterium glutamicum CGMCC No. 21260. The DNA fragment (YH66-RS11190 Up-Down) was separated by agarose gel electrophoresis, purified, ligated with pK18mobsacB plasmid (obtained from Addgene, digested with XbaI/BamHI) purified by digestion with enzyme (XbaI/BamHI) with NEBuilder enzyme (obtained from NEB) at 50 ℃ for 30min, and the single clone grown after transformation of the ligation product was identified by PCR to obtain the positive recombinant vector pK18-YH66-RS11190^G301AThe recombinant vector contains a kanamycin resistance marker. The recombinant vector pK18-YH66-RS11190 with correct enzyme digestion is used^G301ASequencing and identifying by a sequencing company, and carrying out sequencing and identification on the recombinant vector pK18-YH66-RS11190 containing the correct point mutation (G-A)^G301AAnd (5) storing for later use.

The recombinant vector pK18-YH66-RS11190^G301AThe recombinant vector is obtained by replacing a fragment (small fragment) between XbaI and/BamHI recognition sites of the pK18mobsacB vector with a DNA fragment shown in the 37 th to 1236 th positions of SEQ ID No.5 in a sequence table and keeping other sequences of the pK18mobsacB vector unchanged.

The recombinant vector pK18-YH66-RS11190^G301AGene YH66-RS11190 containing mutation shown in SEQ ID No.3^G301AThe DNA molecule shown in positions 1-902 of (1).

Example 2 construction of a plasmid containing Gene YH66-RS11190^G301AOf (4) an engineered strain

The construction method comprises the following steps: the allelic replacement plasmid (pK18-YH66-RS 11190) in example 1 was used^G301A) 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 in the example 1, so that the strain which can amplify a 1244bp 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'TATCGCGGTG GTGTTGTCGG 3'，

P6:5'GCAACGCTGG CGTGAGCCAC 3'。

the resulting PCR amplification product (278bp) was subjected to SSCP (Single-Strand transformation Polymorphis) electrophoresis (using plasmid pK18-YH66-RS 11190) after denaturation at 95 ℃ for 10min and ice-cooling for 5min^G301AThe amplified fragment is a positive control, the amplified fragment of Brevibacterium flavum ATCC15168 is a negative control, water is used as a blank control), the preparation of the PAGE of SSCP electrophoresis and the electrophoresis conditions are shown in Table 2, and because the fragment structure is different and the electrophoresis position is different, the strain with the fragment electrophoresis position different from that of the negative control and the position identical with that of the positive control is a strain with successful allelic replacement. The positive strain YH66-RS11190 gene fragment was PCR-amplified again by primers P5/P6 and ligated to PMD19-T vector for sequencing, and the strain having a mutation in base sequence (G-A) was a positive strain with successful allelic substitution by sequence alignment and was named YPV-025.

RecombinationStrain YPV-025 has mutant gene YH66-RS11190 shown in SEQ ID No.3^G301A。

TABLE 1 composition of culture Medium and culture conditions

TABLE 2 preparation of SSCP electrophoretic PAGE and electrophoresis conditions

Example 3 construction of overexpression of YH66-RS11190 Gene or YH66-RS11190 Gene on genome^G301AEngineered strains of genes

Three pairs of amplified upstream and downstream homologous arm fragments YH66-RS11190 or YH66-RS11190 were designed and synthesized based on the genomic sequence of Brevibacterium flavum ATCC15168 published by NCBI^G301APrimer of gene coding region and promoter region, introducing YH66-RS11190 or YH66-RS11190 into Corynebacterium glutamicum CGMCC No.21260 by homologous recombination^G301AA gene.

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

P7:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG CATGACGGCT GACTGGACTC3'，

P8:5'ACAGACTTTT TACTCATTAT AATCGGACTC CTTAAATGGG 3'，

P9:5'CCCATTTAAG GAGTCCGATT ATAATGAGTA AAAAGTCTGT 3'，

P10:5'CTATGTGAGT AGTCGATTTA TTAGACGGAA ATGGATTCCT 3'，

P11:5'AGGAATCCAT TTCCGTCTAA TAAATCGACT ACTCACATAG 3'，

P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC TGCATAAGAA ACAACCACTT 3'。

the construction method comprises the following steps: are respectively in yellowBrevibacterium ATCC15168 or YPV-025 as template, respectively using primers P7/P8, P9/P10 and P11/P12 to perform PCR amplification to obtain upstream homologous arm fragment 806bp (corresponding to Corynebacterium glutamicum CGMCC No.21260YH66_ RS03355 gene and promoter region thereof or spacer region of the last gene, sequence shown as SEQ ID No. 6), YH66-RS11190 gene and promoter fragment 1324bp (sequence shown as SEQ ID No. 7) or YH66-RS11190^G301AThe gene and the promoter fragment 1324bp (the sequence is shown as SEQ ID No. 8) and the downstream homologous arm fragment 783bp (corresponding to the Corynebacterium glutamicum CGMCC No.21260YH66_ RS03360 gene and a partial spacer region of the gene YH66_ RS03355, the sequence is shown as SEQ ID No. 9). 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 recovered 3 fragments were ligated with pK18mobsacB plasmid (purchased from Addgene) purified by Xbal I/BamH I digestion at 50 ℃ for 30min, and the single clone grown after ligation was identified by PCR using M13 primer to obtain positive integration plasmid (recombinant vector) as pK18-YH66-RS11190OE, pK18-YH66-RS11190^G301AOE, the positive integration plasmid contains a kanamycin resistance marker, and recombinants with plasmid integrated into the genome can be obtained by kanamycin selection.

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-YH66-RS11190OE, pK18-YH66-RS 11190)^G301AOE) are respectively electrically transformed into Corynebacterium glutamicum CGMCC No.21260, and cultured in a culture medium, the components and culture conditions of the culture medium are shown in Table 1, a single colony generated by culture is identified by PCR through a P13/P14 primer, a strain which is positive and contains a 1488bp fragment is amplified by PCR, and a strain which is original is not amplified. Culturing the positive strain in a medium containing 15% sucrose, and culturing the produced monomerThe colony is further identified by PCR using P15/P16 primer, and the strain with the amplified size of 1536bp is YH66-RS11190 or YH66-RS11190^G301APositive strains genetically integrated to the spacer regions of the homology arm YH66_ RS03355 and the lower homology arm YH66_ RS03360 of the genome of Corynebacterium glutamicum CGMCC No.21260 were designated YPV-026 (without mutation points) and YPV-027 (with mutation points), respectively.

The recombinant bacterium YPV-026 contains double copies of YH66-RS11190 gene shown in SEQ ID No. 1; specifically, the recombinant bacterium YPV-026 is a recombinant bacterium obtained by replacing the spacer region of the upper homologous arm YH66_ RS03355 and the lower homologous arm YH66_ RS03360 in the genome of Corynebacterium glutamicum CGMCC No.21260 with YH66-RS11190 gene and keeping other nucleotides in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged. The recombinant bacterium containing the double copies of the YH66-RS11190 gene can obviously and stably improve the expression level of the YH66-RS11190 gene.

Recombinant bacterium YPV-027 contains mutant YH66-RS11190 shown in SEQ ID No.3^G301AA gene; specifically, the recombinant bacterium YPV-027 is obtained by replacing the spacer region of the upper homologous arm YH66_ RS03355 and the lower homologous arm YH66_ RS03360 in the genome of Corynebacterium glutamicum CGMCC No.21260 with YH66-RS11190^G301AGene, recombinant bacterium obtained by keeping other nucleotide in the genome of Corynebacterium glutamicum CGMCC No.21260 unchanged.

The PCR identifying primers are shown below:

p13:5'GTCCGCTCTG TTGGTGTTCA 3' (corresponding to the outside of the upper homology arm YH66_ RS 03355),

p14:5'CAGTTGCCGT GCGGACCTTG 3' (corresponding to the interior of YH66-RS11190 gene),

p15:5'CGGGGGCGGG TTTGCTTGAG 3' (corresponding to the interior of YH66-RS11190 gene),

p16:5'TGGAGGAATA TTCGGCCCAG3' (corresponding to the outside of the lower homology arm YH66_ RS 03360).

Example 4 construction of plasmids overexpressing YH66-RS11190 Gene or YH66-RS11190^G301AEngineered strains of genes

According to the genome sequence of Brevibacterium flavum ATCC15168 published by NCBI, a pair of amplifications YH66-RS11190 or YH66-RS11190 is designed and synthesized^G301AGene coding region andprimers for the promoter region were designed as follows (synthesized by Shanghai Invitrogen corporation):

P17:5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCATAATGAGTA AAAAGTCTGT 3' (the underlined nucleotide sequence is that on pXMJ 19),

P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAGACGGAA ATGGATTCCT 3' (the underlined nucleotide sequence is that on pXMJ 19).

The construction method comprises the following steps: YPV-025 and YPV-026 are respectively taken as templates, PCR amplification is carried out by using primers P17/P18, and YH66-RS11190 gene and promoter fragment (shown as SEQ ID No. 10) and YH66-RS11190 are obtained^G301AGene and its promoter fragment 1354bp (sequence is shown in SEQ ID No. 11), electrophoresing the amplified product and purifying and recovering it by column type DNA gel recovery kit, connecting the recovered DNA fragment with shuttle plasmid pXMJ19 recovered by EcoR I enzyme digestion by NEBuilder enzyme (purchased from NEB company) at 50 deg.C for 30min, PCR identifying the single clone grown after the transformation of the connection product by M13 primer to obtain positive over-expression plasmid pXMJ19-YH66-RS11190 (containing YH66-RS11190 gene) and pXMJ19-YH66-RS11190^G301A(containing YH66-RS11190^G301AGene), the 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 correctly sequenced pXMJ19-YH66-RS11190 and pXMJ19-YH66-RS11190 will be^G301AThe plasmids are respectively electrically transformed into Corynebacterium glutamicum CGMCC No.21260, cultured in a culture medium, the components of the culture medium and culture conditions are shown in Table 1, a single colony generated by culture is subjected to PCR identification through a primer M13R (-48)/P18, and a positive strain containing a 1394bp fragment is obtained through PCR amplification, and the positive strain is named as YPV-028 (without a mutation point) and YPV-029 (with the mutation point))。

The recombinant bacterium YPV-028 contains YH66-RS11190 gene shown in SEQ ID No. 1;

recombinant bacterium YPV-029 contains mutant YH66-RS11190 shown in SEQ ID No.3^G301AA gene.

Example 5 construction of engineered Strain lacking YH66-RS11190 Gene on genome

According to the genome sequence of Brevibacterium flavum ATCC15168 published by NCBI, two pairs of primers for amplifying fragments at both ends of the coding region of YH66-RS11190 gene were synthesized as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):

P19:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GCTCACCGAT ATTGCGGCGA 3'，

P20:5'AGACTCCTCG CAATTAGCGA CAAAGAAGTA ATCAGGACAG 3'，

P21:5'CTGTCCTGAT TACTTCTTTG TCGCTAATTG CGAGGAGTCT 3'，

P22:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCTCAGGATG CTGCTCTGAA 3'。

the construction method comprises the following steps: PCR amplification was performed using Brevibacterium flavum ATCC15168 as template and primers P19/P20 and P21/P22, respectively, to obtain 684bp upstream homology arm fragment of YH66-RS11190 and 722bp downstream homology arm fragment of YH66-RS 11190. Then, the primer P19/P22 is used for carrying out Overlap PCR to obtain a whole homologous arm fragment of 1366bp (the sequence is shown as SEQ ID No. 12). And (2) carrying out electrophoresis on the amplified product and purifying by adopting a column type DNA gel recovery kit, connecting the recovered DNA fragment with a pK18mobsacB plasmid (purchased from Addgene company) which is purified after XbaI/BamHI enzyme digestion for 30min at 50 ℃ by NEBuilder enzyme (purchased from NEB company), carrying out PCR identification on a single clone which grows out after the transformation of a connection product by using an M13 primer to obtain a positive knockout vector pK 18-delta YH66-RS11190, and sequencing the plasmid. This plasmid contains kanamycin resistance as a selection marker.

The Overlap PCR amplification 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 procedure of the Overlap PCR amplification reaction is as follows: pre-denaturation at 94 ℃ for 5min and denaturation at 94 ℃ for 30 s; annealing at 52 ℃ for 30 s; extension at 72 ℃ for 90s (30 cycles) and over-extension at 72 ℃ for 10 min.

The correctly sequenced knockout plasmid pK 18-delta YH66-RS11190 was electrically transformed into Corynebacterium glutamicum CGMCC No.21260, cultured in a medium whose composition and culture conditions are shown in Table 1, and the single colonies generated by the culture were identified by PCR using the following primers (synthesized by Shanghai Invitrogen):

p23:5'GCTCACCGAT ATTGCGGCGA 3' (corresponding to the interior of the Corynebacterium glutamicum CGMCC No.21260YH66-RS11185 gene),

p24:5'GCTCAGGATG CTGCTCTGAA 3' (corresponding to the interior of the Corynebacterium glutamicum CGMCC No.21260YH66_ RS11195 gene).

The bacterial strain which has the bands with the sizes of 1292bp and 2540bp and is simultaneously amplified by the PCR is a positive bacterial strain, and the bacterial strain which has the band with the size of 2540bp and is only amplified by the PCR 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, the 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 with a 1292bp band is amplified to be the positive strain YH66-RS11190 with the coding region of the YH66-RS11190 gene knocked out. The positive strain YH66-RS11190 fragment was amplified again by PCR with P23/P24 primer and ligated to pMD19-T vector for sequencing, and the correctly sequenced strain was named YPV-030 (YH66-RS11190 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 medium shown in Table 3 and the control process shown in Table 4, and the valine content was measured by high performance liquid chromatography. Each strain was replicated three times, and the results are shown in Table 5.

As a result, the coding region of YH66-RS11190 gene was point-mutated in Corynebacterium glutamicum YH66-RS11190^G301AAnd the expression of the protein is carried out,the gene knockout or weakening of the gene is beneficial to the improvement of the yield and the conversion rate of the L-valine, and is not beneficial to the accumulation of the L-valine.

TABLE 3 fermentation Medium formulation (balance water)

Composition (I)	Formulation of
		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

TABLE 5 results of L-valine fermentation experiments

Bacterial strains	OD₆₁₀	L-valine yield (g/L)
			Corynebacterium glutamicum CGMCC No.21260	98.3	83.8
YPV-025	98.2	85.7
			YPV-026	97.4	84.9
YPV-027	97.2	85.6
			YPV-028	98.9	85.4
YPV-029	99.2	85.8
			YPV-030	97.7	82.3

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> Ningxia Yipin Biotechnology Ltd

<120> YH66-RS11190 gene mutant and application thereof in preparation of L-valine

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 1248

<212> DNA

<213> Brevibacterium flavum (Brevibacterium flavum)

<400> 1

atgctgtttt ccatgttctt cggagctgga aacctcatct tcccgccgat gcttggattg 60

tcggcaggaa ccaactatct accagctatc ttaggatttc tagcaacgag tgttctgctc 120

ccggtgctgg cgattatcgc ggtggtgttg tcgggagaaa atgtcaagga catggcttct 180

cgtggcggta agatctttgg cctggtgttt cctattgctg cctatttgtc catcggtgcg 240

ttttacgcgc tgccgaggac tggggcggtg agctattcga cggcggttgg cgtcgataat 300

gcgctttatt cgggcttgtt taactttgtg ttttttgcgg tggcactggc gttgtcgtgg 360

aatccgaatg gcattgcaga caagttgggt aagtggctca cgccagcgtt gctcacgttg 420

attgtggtgc tggtggtgtt gtcggtagcc aagttggatg gcacgccagg tgagccaagt 480

agtgcgtatg cgcagcagcc tgcgggggcg ggtttgcttg agggctacat gacgatggat 540

gcgattgctg cgttggcgtt tggcatcgtg gtgatttctg cgttcaagta ccaaaaggtt 600

aacaaggtcc gcacggcaac tgtcgtgtcg gcgttcattg ccggaatttt gttggcgctg 660

gtttatcttg gtttgggctc aatcggtcaa gtagtaaacg gtgagttcgc tgatggcacc 720

gcaattttga actacgctgc actgtccacg atgggtcagg ctggtcgcat catgttcgtg 780

gccattttga tccttgcatg tatgaccacc gcagttggtc tgatcagtgc gacgtctgag 840

tttttcaatt cgctgctgcc aggtgtcaag taccacgtct gggccactgt tttcgcgctg 900

atttcctttg gcgttgccac gatgggattg gatacggtgt tggccgttgc ggctccagtg 960

attagtttca tttacccatc ggccatcacc ttggtgttct tgtcgctcat cgagcccctg 1020

ctgttccgtc tcaagtggac ctacctattc ggcatttgga ctgcagttgt gtgggcgctg 1080

ttcatgtcta tccctgcgct gaatccattc atcgaatggg cgccgctgca cagcatgtct 1140

ttgggttggg ttgtcccagt tctcgtggcc tctgccatcg gtttggctat tgattggaac 1200

aagaaaggtg cccagtctgt tgcagagaag gaatccattt ccgtctaa 1248

<210> 2

<211> 415

<212> PRT

<213> Brevibacterium flavum (Brevibacterium flavum)

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Met Leu Phe Ser Met Phe Phe Gly Ala Gly Asn Leu Ile Phe Pro Pro

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Met Leu Gly Leu Ser Ala Gly Thr Asn Tyr Leu Pro Ala Ile Leu Gly

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Phe Leu Ala Thr Ser Val Leu Leu Pro Val Leu Ala Ile Ile Ala Val

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

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Ile Phe Gly Leu Val Phe Pro Ile Ala Ala Tyr Leu Ser Ile Gly Ala

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Phe Tyr Ala Leu Pro Arg Thr Gly Ala Val Ser Tyr Ser Thr Ala Val

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Gly Val Asp Asn Ala Leu Tyr Ser Gly Leu Phe Asn Phe Val Phe Phe

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Ala Val Ala Leu Ala Leu Ser Trp Asn Pro Asn Gly Ile Ala Asp Lys

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Leu Gly Lys Trp Leu Thr Pro Ala Leu Leu Thr Leu Ile Val Val Leu

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

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Ser Ala Tyr Ala Gln Gln Pro Ala Gly Ala Gly Leu Leu Glu Gly Tyr

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Met Thr Met Asp Ala Ile Ala Ala Leu Ala Phe Gly Ile Val Val Ile

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Ser Ala Phe Lys Tyr Gln Lys Val Asn Lys Val Arg Thr Ala Thr Val

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Val Ser Ala Phe Ile Ala Gly Ile Leu Leu Ala Leu Val Tyr Leu Gly

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Leu Gly Ser Ile Gly Gln Val Val Asn Gly Glu Phe Ala Asp Gly Thr

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Ala Ile Leu Asn Tyr Ala Ala Leu Ser Thr Met Gly Gln Ala Gly Arg

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Ile Met Phe Val Ala Ile Leu Ile Leu Ala Cys Met Thr Thr Ala Val

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

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Val Lys Tyr His Val Trp Ala Thr Val Phe Ala Leu Ile Ser Phe Gly

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Pro Phe Ile Glu Trp Ala Pro Leu His Ser Met Ser Leu Gly Trp Val

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Val Pro Val Leu Val Ala Ser Ala Ile Gly Leu Ala Ile Asp Trp Asn

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