CN116904418A - yfdH gene mutant and application thereof in preparation of L-valine - Google Patents
yfdH gene mutant and application thereof in preparation of L-valine Download PDFInfo
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- CN116904418A CN116904418A CN202311058879.0A CN202311058879A CN116904418A CN 116904418 A CN116904418 A CN 116904418A CN 202311058879 A CN202311058879 A CN 202311058879A CN 116904418 A CN116904418 A CN 116904418A
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Abstract
The application discloses a yfdH gene mutant and application thereof in preparation of L-valine, and belongs to the technical field of biology. The technical problem to be solved is how to increase the yield of the L-valine of the escherichia coli. In order to solve the technical problem, the application provides a protein with an amino acid sequence shown as SEQ ID No.4, a coding gene thereof, related biological materials and application thereof in improving the yield of L-valine. The application constructs the over-expression mutant yfdH T743C Recombinant bacteria of the gene or the wild yfdH gene, and point mutation recombinant bacteria, and experimental results show that: overexpression of yfdH Gene or yfdH T743C The gene can obviously improve the yield of L-valine. Point mutation (T-C) of yfdH gene also contributes to the improvement of L-valine production. The genetic engineering strain constructed by the method can obviously promote accumulation of L-valine and improve the yield of L-valine.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a yfdH gene mutant and application thereof in preparation of L-valine.
Background
L-Valine (L-Valine) is a branched nonpolar alpha-amino acid containing five carbon atoms, is an essential amino acid and a glycogenic amino acid for mammals, and cannot be synthesized by humans and animals themselves. L-valine has effects of promoting protein synthesis, inhibiting protein decomposition, enhancing immunity, and 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 industries of animal feed, cosmetics, foods and medicines. 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. Therefore, 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, thereby being beneficial to promoting the industrialization process of L-valine.
Disclosure of Invention
The technical problem to be solved by the present application is how to increase the yield of L-valine in E.coli by genetic engineering of genes, 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 clearly understood by those skilled in the art from the following description.
To solve the technical problems, the application firstly provides protein which can be named YfdH I248T The protein may comprise any one of the following:
a1 Amino acid sequence comprising a protein as shown in SEQ ID No. 4;
a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.4, has more than 90% of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).
Labels described herein include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
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, the Expect value is 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 search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The application also provides nucleic acid molecules, which may be named yfdH T743C The nucleic acid molecule may comprise any one of the following:
b1 Encoding the protein YfdH I248T Is a nucleic acid molecule of (a);
b2 The coding sequence is a DNA molecule shown as SEQ ID No. 3.
The DNA molecule shown in SEQ ID No.3 is the mutant gene yfdH of the application T743C 。
The DNA molecule shown in SEQ ID No.3 (yfdH T743C Gene) encodes a mutant protein YfdH shown in SEQ ID No.4 I248T 。
The yfdH T743C The 743 rd cytosine (C) in the nucleotide sequence (SEQ ID No. 3) of the gene is mutated from thymine (T), the protein YfdH I248T Threonine (T) at position 248 in the amino acid sequence (SEQ ID No. 4) is mutated from isoleucine (I).
The present application also provides a biomaterial that may include any one of the following:
c1 Containing said nucleic acid molecule yfdH T743C Is a gene expression cassette;
c2 Containing said nucleic acid molecule yfdH T743C Or a recombinant vector comprising the expression cassette of C1);
c3 Containing said nucleic acid molecule yfdH T743C Or a recombinant microorganism comprising the expression cassette of C1) or a recombinant microorganism comprising the recombinant vector of C2);
c4 Containing said nucleic acid molecule yfdH T743C Or a recombinant cell comprising the expression cassette of C1) or a recombinant cell comprising the recombinant vector of C2).
Further, C3) the recombinant microorganism may be any one of the following:
d1 A recombinant microorganism obtained by introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 into a microorganism of interest;
d2 A recombinant microorganism obtained by introducing a DNA molecule having a coding sequence represented by SEQ ID No.3 into a microorganism of interest;
d3 A recombinant microorganism obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest to C.
The application also provides the protein YfdH I248T Said nucleic acid molecule yfdH T743C Or any one of the following applications of the biomaterial:
e1 Use in regulating the production of microbial L-valine;
e2 Use in the preparation of L-valine;
e3 The application of the strain in constructing genetically engineered bacteria for producing L-valine.
The modulation described herein may be either an increase (up-regulation) or a decrease (down-regulation).
Further, the modulation of L-valine production by a microorganism as described herein can be an increase (up-regulation) or a decrease (down-regulation) in the accumulation of L-valine in the microorganism (i.e., promotion or inhibition of L-valine biosynthesis).
The application also provides an application of any one of F1) -F4) in regulating and controlling the yield of L-valine of microorganisms, preparing L-valine or constructing genetically engineered bacteria producing L-valine, wherein the F1) -F4) is as follows:
f1 Amino acid sequence comprising a protein as shown in SEQ ID No. 2;
f2 A nucleic acid molecule encoding the protein of F1);
f3 A DNA molecule with a coding sequence shown in SEQ ID No. 1;
f4 An expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell containing F2) said nucleic acid molecule or F3) said DNA molecule.
The DNA molecule shown in SEQ ID No.1 is also the yfdH gene of the present application.
The DNA molecule shown in SEQ ID No.1 (yfdH gene) encodes the protein YfdH shown in SEQ ID No. 2.
The present application also provides a method for increasing the yield of L-valine or producing L-valine of a microorganism of interest, which can comprise any one of the following:
g1 Increasing the nucleic acid molecule yfdH in a microorganism of interest T743C The expression amount or content of (a) to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g2 Increasing the expression level or the content of F2) the nucleic acid molecule or F3) the DNA molecule in the microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g3 A DNA molecule having a nucleotide sequence of SEQ ID No.1 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest, the mutation may comprise: isoleucine at position 248 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 was mutated to threonine.
Further, the G1) may be achieved by any one of the following methods: (1) Increasing yfdH in the microorganism of interest T743C Copy number of Gene (SEQ ID No. 3) (e.g., yfdH to be single-or multiple-copy T743C Gene transfer into the microorganism of interest); (2) Enhancement of yfdH by expression control sequences on the basis of (1) T743C Expression of genes (e.g. modified yfdH T743C Expression control sequences of the genes).
Further, the G2) may be achieved by any one of the following methods: (1) Increasing the copy number of the yfdH gene (SEQ ID No. 1) in the microorganism of interest (e.g., introducing a single copy or multiple copies of the yfdH gene into the microorganism of interest); (2) The expression of the yfdH gene is enhanced by an expression control sequence (e.g., the expression control sequence of the yfdH gene is modified).
The expression control sequence may be a promoter, enhancer or silencer sequence.
Further, the mutation in G3) may be performed by a gene editing technique, a homologous recombination technique, or a site-directed mutagenesis, etc.
In the above method, the microorganism of interest may be Escherichia coli.
In the above method, the method may include any one of the following:
h1 Introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 and/or SEQ ID No.2 into the microorganism of interest;
h2 Introducing a DNA molecule comprising the coding sequence shown as SEQ ID No.3 and/or SEQ ID No.1 into the microorganism of interest;
h3 The nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest is mutated to C.
The application also provides the protein YfdH I248T Said nucleic acid molecule yfdH T743C Use of said biological material, a protein as described in F1) herein, a nucleic acid molecule as described in F2) herein, a DNA molecule as described in F3) herein, an expression cassette as described in F4) herein, a recombinant vector, a recombinant microorganism or a recombinant cell for the preparation of a food, a cosmetic, a pharmaceutical or a feed comprising L-valine.
The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), or viral vectors.
The microorganism described herein may be a yeast, bacterium, algae or fungus. The bacteria may be derived from, but not limited to, escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, alcaligenes sp, pseudomonas sp, bacillus sp, brevibacterium sp, corynebacterium sp, aerobacter sp, enterobacter sp, micrococcus sp, serratia sp, salmonella sp, streptomyces sp, provicum sp, and the like.
Further, the bacterium may be Escherichia coli (Escherichia coli), corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium lactofermentum (Brevibacterium lactofermentum), brevibacterium flavum (Brevibacterium flavum), or Corynebacterium beijing (Corynebacterium pekinense).
The cells described herein may be plant cells or animal cells. The cell may be any biological cell that can synthesize the amino acid of interest.
The recombinant vector described herein may be recombinant vector pSTV28-yfdH and/or recombinant vector pSTV28-yfdH T743C 。
The recombinant vector pSTV28-yfdH contains the DNA molecule shown in SEQ ID No.6 (i.e., the yfdH gene and regulatory element fragment). The DNA molecule shown in SEQ ID No.6 comprises a promoter P J23105 RBS sequence of pTrc99a plasmid, wild-type yfdH gene, terminator Trc. Promoter P J23105 And RBS sequences were designed in primer P1 (SEQ ID No. 8) and primer P6 (SEQ ID No. 13), and terminator Trc was designed in primer P4 (SEQ ID No. 11) and primer P5 (SEQ ID No. 12).
The recombinant vector pSTV28-yfdH T743C Contains the DNA molecule shown in SEQ ID No.7 (i.e., yfdH T743C Gene and regulatory element fragments). The DNA molecule shown in SEQ ID No.7 comprises a promoter P J23105 RBS sequence, yfdH of pTrc99a plasmid T743C Gene, terminator Ttrc. Promoter P J23105 And RBS sequences were designed in primer P1 (SEQ ID No. 8) and primer P6 (SEQ ID No. 13), and terminator Trc was designed in primer P4 (SEQ ID No. 11) and primer P5 (SEQ ID No. 12).
The recombinant microorganism described herein may be VHY (overexpressing the yfdH gene), VHY (overexpressing yfdH) T743C Gene), VHY (yfdH) T743C Point mutation), MG1655-02 (overexpressing the yfdH gene), MG1655-03 (overexpressing the yfdH gene) T743C Gene) and/or MG1655-04 (yfdH) T743C Point mutations).
The recombinant microorganisms VHY and VHY are the recombinant vectors pSTV28-yfdH and pSTV28-yfdH T743C Recombinant microorganisms obtained by introducing E.coli VHY.
The recombinant microorganisms MG1655-02 and MG1655-03 were prepared by subjecting the recombinant vectors pSTV28-yfdH and pSTV28-yfdH T743C Recombinant microorganisms obtained by introducing E.coli MG1655, respectively.
The recombinant microorganism VHY is obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the escherichia coli VHY into C.
The recombinant microorganism MG1655-04 is obtained by mutating a nucleotide T at 743 of a DNA molecule shown in SEQ ID No.1 in a genome of Escherichia coli MG1655 into C.
The introduction may be a transformation of a vector carrying the DNA molecule of the present application into a host bacterium by any known transformation method such as a chemical transformation method (e.g., ca2+ -induced transformation method, polyethylene glycol-mediated transformation method or metal cation-mediated transformation method) or electroporation transformation method; the DNA molecules of the application may also be transduced into host bacteria by phage transduction. 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 method for producing L-valine described herein may be a fermentation method for producing L-valine, and specifically may be a fermentation method for producing L-valine by using the recombinant microorganisms VHY, VHY, VHY24, MG1655-02, MG1655-03 and/or MG 1655-04.
The microorganism of interest described herein may be E.coli (Escherichia coli), and specifically E.coli VHY or E.coli MG1655.
Using the yfdH gene or a variant thereof (e.g., yfdH T743C Genes) can also be used to produce a variety of products including, but not limited to, lysine, glutamic acid, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine and/or citrulline.
The application constructs the over-expression yfdH by taking the escherichia coli MG1655 and the escherichia coli VHY as starting bacteria T743C Recombinant strain of gene (SEQ ID No. 3) or yfdH gene (SEQ ID No. 1), and yfdH gene against Escherichia coli MG1655 and Escherichia coli VHY20The point mutation is carried out, recombinant bacteria with the point mutation are constructed, and fermentation experiments are carried out on the constructed engineering strain, and the result shows that: overexpression of the yfdH gene or yfdH in either the L-valine-producing strain (e.g., VHY) or the wild-type E.coli (e.g., MG 1655) T743C The genes all contribute to the synthesis of L-valine and over-express yfdH T743C The gene can remarkably improve the yield of L-valine. Furthermore, in either L-valine-producing strain or wild-type E.coli, the yfdH gene was subjected to a point mutation, and after the T at position 743 was mutated to C (the mutant protein YfdH was obtained accordingly) I248T Mutation of isoleucine to threonine at position 248 of the amino acid sequence) contributes to a significant increase in L-valine production. Meanwhile, the expression of the deletion yfdH gene is unfavorable for the synthesis of L-valine. Since the sugar supplement amounts are the same, an increase in yield means an increase in conversion. The genetic engineering strain constructed by the application can obviously promote accumulation of L-valine and improve the yield of L-valine. The application cultivates high-yield and high-quality strains which accord with industrial production, and is beneficial to promoting the industrial production process of L-valine.
Drawings
FIG. 1 shows the composition of L-valine fermentation medium in example 7.
FIG. 2 shows the results of fermentation experiments for L-valine in example 7.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application 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 application 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.
The pSTV28 plasmid in the following examples is a Bao Bio Inc., cat: 3331.
coli MG1655 in the following examples was derived from beijing na-invasive alliance biotechnology institute (BNCC), strain number: BNCC363342.
The following examples are described in E.coli VHY: wang Jiachu, wu Faqing, wu Heyun, et al. Engineering strains for the production of valine by oxygen-limited fermentation and optimization of the fermentation process [ J ]. Food and fermentation industry, 2023,49 (1): 8.
The pREDCas9 plasmid and pGRB vector in the examples below are products of the addgene company.
Example 1 construction of a coding region fragment of yfdH Gene and wild-type yfdH Gene comprising Point mutations
According to NCBI published Escherichia coli (Escherichia coli) MG1655 genome sequence, two pairs of primers for amplifying the coding region of the yfdH gene were designed and synthesized, and a point mutation was introduced into the yfdH gene coding region (SEQ ID No. 1) of Escherichia coli MG1655 (it was confirmed by sequencing that the wild-type yfdH gene remained on the chromosome of the strain) in an allelic substitution manner, the point mutation being a mutation of thymine (T) at position 743 in the nucleotide sequence (SEQ ID No. 1) of the yfdH gene into cytosine (C), to give a DNA molecule shown in SEQ ID No.3 (mutated yfdH gene, which is named yfdH) T743C )。
Wherein the DNA molecule shown in SEQ ID No.1 encodes a protein having the amino acid sequence of SEQ ID No.2 (said protein is named protein YfdH).
The DNA molecule shown in SEQ ID No.3 encodes a mutein having the amino acid sequence of SEQ ID No.4 (said mutein is named YfdH I248T ). The mutant protein YfdH I248T Threonine (T) at position 248 in the amino acid sequence (SEQ ID No. 4) is mutated from isoleucine (I).
The mutant fragment was constructed by PCR, and the primer design was as follows (synthesized by Jin Wei, suzhou) with the bold base as the mutation position:
P1:5'-TAGGTACTATGCTAGCAGGAAACAGACCATGAAGATATCTCTTGTAGTTCC-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 8),
P2:
P3:
P4:5'-CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTT GTCATTTTTTGACTCTCTTGATG-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 11).
The construction method comprises the following steps: using Escherichia coli MG1655 as a template, and performing PCR amplification with primers P1 and P2, P3 and P4, respectively, to obtain two DNA fragments (yfdH) each having a coding region of the yfdH gene having a mutation base and a length of 784bp and 230bp, respectively T743C -Up and yfdH T743C Down). The Escherichia coli MG1655 is used as a template, and primers P1 and P4 are used for PCR amplification to obtain a DNA fragment with the length of 988bp and containing the coding region of the yfdH gene.
The PCR amplification system is as follows: 5 XPrimeSTAR Buffer (Mg) 2+ Plus) 10. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, primer (10 pM) 2. Mu.L each, template<200 ng), primeSTAR HS DNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, sterile water was added to a total volume of 50. Mu.L.
The PCR amplification reaction procedure was: pre-denaturation at 98℃for 10sec, (denaturation at 95℃for 5min; annealing at 55℃for 30s; extension at 72℃for 60sec;30 cycles), over-extension at 72℃for 10min.
Two DNA fragments (yfdH T743C -Up and yfdH T743C Down) is separated and purified by agarose gel electrophoresis, and then subjected to yfdH T743C -Up and yfdH T743C Overlapping PCR amplification is carried out by using the primer P1 and the primer P4 by taking Down as a template to obtain the primer containing yfdH with the length of 988bp T743C DNA fragments of the coding region of the gene.
Example 2 construction of overexpressed yfdH Gene or yfdH T743C Recombinant vector of gene
PCR was used to construct pSTV28 plasmid linearized vector fragment, and the primer design was as follows (synthesized by Jin Wei, suzhou):
P5:5'-AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA TGTGAGCGAGGAAGCGGAATA-3' (underlined nucleotide sequence comprising the sequence of the gene regulatory element) (SEQ ID No. 12),
P6:5'-GCTAGCATAGTACCTAGGACTGAGCTAGCCGTAAAGAAGATGCCAGGAAGATACTTAACA-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 13).
The construction method comprises the following steps: the pSTV28 plasmid (containing chloramphenicol resistance marker) is used as a template, and primers P5 and P6 are used for PCR amplification to obtain a pSTV28 plasmid linearization vector fragment with the length of 3027bp, and the pSTV28 plasmid linearization vector fragment is separated and purified by agarose gel electrophoresis for later use. The nucleotide sequence of the pSTV28 plasmid linearized vector fragment is shown in SEQ ID No. 5.
The DNA fragment (988 bp, without point mutation) containing the coding region of the yfdH gene constructed in example 1 and the gene containing yfdH were subjected to T743C The DNA fragment (988 bp, containing point mutation) of the gene coding region is respectively connected with the pSTV28 plasmid linearization vector fragment to obtain recombinant vectors, which are respectively named as: pSTV28-yfdH and pSTV28-yfdH T743C 。
Positive recombinant vector pSTV28-yfdH T743C Sequencing and identification of the recombinant vector pSTV28-yfdH containing the correct Point mutation (T-C) by the company Jin Weizhi, suzhou T743C And (5) storing for standby. Sequencing results showed that thymine (T) at position 743 of the coding region of yfdH gene was mutated to cytosine (C), ultimately resulting in mutation of isoleucine (I) at position 248 of the encoded protein to threonine (T).
The recombinant vector pSTV28-yfdH contains the DNA molecule shown in SEQ ID No.6 (i.e., the yfdH gene and regulatory element fragment). The DNA molecule shown in SEQ ID No.6 comprises a promoter P J23105 RBS sequence of pTrc99a plasmid, wild-type yfdH gene, terminator Trc. Promoter P J23105 And RBS sequences are designed in primer P1 and primer P6, and terminator Trc is designed in primer P4 and primer P5.
Recombinant vector pSTV28-yfdH T743C Contains the DNA molecule shown in SEQ ID No.7 (i.e., yfdH T743C Gene and regulatory element fragments). The DNA molecule shown in SEQ ID No.7 comprises a promoter P J23105 RBS sequence, yfdH of pTrc99a plasmid T743C Gene, terminator Ttrc.Promoter P J23105 And RBS sequences are designed in primer P1 and primer P6, and terminator Trc is designed in primer P4 and primer P5.
EXAMPLE 3 construction of plasmids containing pSTV28, pSTV28-yfdH T743C Is an engineered strain of (2)
The construction method comprises the following steps: plasmids pSTV28 and the plasmids of example 2 (pSTV 28-yfdH, pSTV28-yfdH T743C ) After electric transduction into E.coli VHY, the single colony generated by culture is identified by primer P7 (5'-CAGGGATTGGCTGAGACGAA-3', SEQ ID No. 14) and primer P8 (5'-TGTTAAGTATCTTCCTGGCATCTTC-3', SEQ ID No. 15), positive strains can amplify 1075bp, 1996bp and 1996bp size bands respectively. The positive strains were named VHY, VHY, VHY23, respectively. Recombinant VHY contained plasmid pSTV28; recombinant VHY contained plasmid pSTV28-yfdH; recombinant VHY contains plasmid pSTV28-yfdH T743C 。
EXAMPLE 4 construction of the plasmid containing pSTV28, pSTV28-yfdH in wild type E.coli MG1655 T743C Is an engineered strain of (2)
The construction method comprises the following steps: plasmids pSTV28 and the plasmids of example 2 (pSTV 28-yfdH, pSTV28-yfdH T743C ) After electric transduction into the wild type MG1655 of the escherichia coli respectively, the single colonies generated by culture are respectively identified by a primer P7 (SEQ ID No. 14) and a primer P8 (SEQ ID No. 15), and positive strains can respectively amplify 1075bp, 1996bp and 1996bp size bands. Positive strains were designated MG1655-01, MG1655-02, MG1655-03, respectively. Recombinant MG1655-01 contains plasmid pSTV28; recombinant MG1655-02 contained plasmid pSTV28-yfdH; recombinant MG1655-03 containing plasmid pSTV28-yfdH T743C 。
Example 5, wild-type E.coli MG1655 and valine-producing bacterium VHY yfdH T743C Construction of Point mutations
According to the genomic sequence of Escherichia coli MG1655 published by NCBI, thymine (T) at 743bp in the yfdH gene coding region of MG1655 and L-valine producing bacterium VHY is mutated into cytosine (C) by CRISPR/Cas9 gene editing technique,thus, the mutant yfdH was studied more intensively T473C Influence of the Gene on the amount of L-valine synthesized in wild-type E.coli and L-valine-producing bacterium VHY.
1. Construction of sgRNA plasmids
According to NCBI published Escherichia coli (Escherichia coli) MG1655 genome sequence, using CRISPRRGEN Tools (http:// www.rgenome.net/cas-designer /) to design the sgRNA target sequence, after selecting the appropriate sgRNA target sequence, linearization pGRB cloning vector terminal sequences were added at the 5 'and 3' extreme ends of the target sequence to form the complete sgRNA plasmid by recombination.
The amplification of the DNA fragment containing the sgRNA target sequence is carried out without a template and only by carrying out the PCR annealing process, and the system and the procedure are as follows. PCR reaction system: 10. Mu.L of sgRNA-F and 10. Mu.L of sgRNA-R; PCR reaction procedure: denaturation at 95℃for 5min and annealing at 50℃for 1min. After the completion of annealing, the target fragment (DNA fragment containing the sgRNA target sequence) was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng/. Mu.L.
The pGRB plasmid was digested with SpeI to give a linearized pGRB plasmid, which was then dephosphorylated to prevent the pGRB plasmid from self-ligating. And (3) enzyme cutting system: 10 XBuffer 5. Mu.L, speI 2.5. Mu.L, pGRB plasmid DNA 3000-5000ng, ddH 2 O to 50. Mu.L. Enzyme digestion at 37 ℃ for 3 hours, agarose gel electrophoresis gel cutting recovery, dephosphorylation reaction and dephosphorylation system: 10 XBuffer 5. Mu.L, linearized pGRB plasmid DNA 1000-2000ng, CIAP 2.5. Mu.L, ddH 2 O to 50. Mu.L. After 1h treatment at 37℃the linearized pGRB plasmid was recovered using a DNA purification kit.
The DNA fragment containing the sgRNA target sequence was subjected to homologous recombination ligation with the linearized pGRB plasmid using the Gibson Assemblem kit (New England). Recombination system: NEB assembly enzyme 2.5. Mu.L, linearized pGRB plasmid 2. Mu.L, DNA fragment containing sgRNA target sequence 0.5. Mu.L. After 30min of assembly at 50 ℃, DH5 alpha competent cells are transformed from the product, plasmids are extracted, and sequencing primers sgRNA-PF/sgRNA-PR are used for sequencing and identification. The constructed sgRNA plasmid was designated pGRB-sgRNA-yfdH.
The primers used in this experiment were designed as follows (synthesized by Shanghai Invitrogen) with underlined bases as pGRB cloning vector homology arm sequence and lowercase bases as sgRNA target sequence:
sgRNA-F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTactttggaatttagcacttgGTT TTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3'(SEQ ID No.16)，
sgRNA-R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcaagtgctaaattccaaagtACT AGTATTATACCTAGGACTGAGCTAGCTGTCA-3'(SEQ ID No.17)，
sgRNA-PF：5'-GTCTCATGAGCGGATACATATTTG-3'(SEQ ID No.18)，
sgRNA-PR：5'-ATGAGAAAGCGCCACGCT-3'(SEQ ID No.19)。
2. DNA fragment Up-yfdH of yfdH gene point mutation T743C Acquisition of Down
Two pairs of primers for amplifying the upstream and downstream homology arm sequences are designed and synthesized according to the genome sequence of Escherichia coli MG1655 published by NCBI, and T743C point mutation is introduced into yfdH genes of Escherichia coli MG1655 and valine producer VHY respectively in a CRISPR/Cas9 gene editing mode.
The primers were designed as follows (synthesized by the company epivitrogen, shanghai) with the bolded bases as mutation positions:
P9:5'-CCTGTCTTCAATGAAGAAGAAG-3'(SEQ ID No.20)，
P10:5'-AATATGATAGTATCTAAAGT CATCCACGCCCCATAAATAA-3'(SEQ ID No.21)，
P11:5'-TTATTTATGGGGCGTGGATGACTTTAGATACTATCATATT-3'(SEQ ID No.22)，
P12:5'-ACATTGACAGTGTTGATGAAG-3'(SEQ ID No.23)。
the genome DNA of MG1655 or valine producing bacterium VHY is used as a template, and primers P9/P10, P11/P12 and KAPA HiFi HotStart are used for PCR amplification, so that an upper homology arm fragment 740bp and a lower homology arm fragment 620bp are obtained. After the PCR reaction is finished, agarose gel electrophoresis recovery is respectively carried out by adopting a column type DNA gel recovery kit. The recovered DNA is used as a template, and primers P9 and P12 are used for overlap PCR amplification to obtain a DNA fragment Up-yfdH with yfdH gene point mutation T743C -Down 1320bp。
PCR amplification and overlap PCR amplificationThe system comprises: 5 XHiFi with Mg 2+ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) 1.6. Mu.L, KAPA HiFi HotStart (1U/. Mu.L) 0.5. Mu.L, and ddH were supplemented 2 O to a total volume of 50. Mu.L.
PCR amplification and overlap PCR amplification procedure: pre-denaturation at 95℃for 5min, (denaturation at 98℃for 20s; annealing at 56℃for 15s; extension at 72℃for 150s;30 cycles), over-extension at 72℃for 5min.
3. Preparation and transformation of competent cells
pREDCas9 plasmid (containing spectinomycin resistance gene) is respectively transformed into MG1655 and valine producer VHY competent cells, the cells are spread on a 2-YT agar plate containing spectinomycin (100 MG/L) for culture at 32 ℃, single colony of the spectinomycin (100 MG/L) is selected and PCR identification is carried out by taking pRedCas9-PF/pRedCas9-PR as a primer and r Taq, so that 943bp MG1655-Cas9 and VHY-Cas 9 transformants containing pREDCas9 plasmid are obtained.
Preparation of MG1655-Cas9, VHY20-Cas9 competent cells when the thallus grows to OD 600 =0.1 IPTG was added at a final concentration of 0.1mM to induce λ -Red mediated homologous recombination. When OD is 600 When=0.6, cells were collected to prepare competent cells, and pGRB-sgRNA-yfdH plasmid and Up-yfdH plasmid were transformed, respectively T743C Down fragment, spread onto 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L) and incubated at 32 ℃. And (3) carrying out PCR identification on single colonies generated by culture through a primer P13/P14 by using rTaq, carrying out PCR amplification to obtain a fragment with 660bp, sequencing, and comparing sequencing results to obtain positive bacteria with T743C point mutation.
Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% to eliminate the plasmid pGRB-sgRNA-yfdH, colonies that grew on spectinomycin (100 mg/L) but did not grow on ampicillin (100 mg/L) were selected, these colonies were transferred to 2-YT medium for 42℃cultivation to eliminate pREDCas9 plasmid, colonies that did not grow on spectinomycin (100 mg/L) but grew on nonreactive 2-YT were selected, identified again by primer P13/P14 using rTaq PCR, PCR amplified to contain a 660bp fragment, and sequenced to produce a positive transformant with a T743C point mutation.
Gene editing of MG1655 was performed to obtain a mutant yfdH containing the point mutation T473C Recombinant strain (MG 1655/yfdH) T473C ) Designated MG1655-04, was obtained by gene editing of valine-producing bacterium VHY, and was found to contain the point mutation yfdH T473C Recombinant bacterium (VHY/yfdH) T473C ) Named VHY.
Recombinant MG1655-04 and VHY24 genome each containing point mutated yfdH T743C And (3) a gene.
Primers were designed as follows (synthesized by the company epivitrogen, shanghai):
P13:5'-AAATGGCAAGCAGGTGCTG-3'(SEQ ID No.24)，
P14:5'-CTTCAATTAATGGCTTAATG-3'(SEQ ID No.25)，
pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3'(SEQ ID No.26)，
pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3'(SEQ ID No.27)。
PCR amplification system: 2 XPromix Taq 12.5. Mu.L, 1. Mu.L each of primer (10 pM), and ddH was added 2 The total volume of O was 25. Mu.L.
PCR amplification procedure: pre-denaturing for 5min at 94℃and denaturing for 30s at 94 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 90s (30 cycles), and overextensing at 72 ℃ for 10min.
EXAMPLE 6 construction of genetically engineered bacterium MG1655. Delta. YfdH and VHY. Delta. YfdH
The genome of E.coli MG1655 or VHY was used as a template, and an upstream homology arm primer (UP-yfdH-S, UP-yfdH-A) and a downstream homology arm primer (DN-yfdH-S, DN-yfdH-A) were designed based on the sequence upstream and downstream of the coding region of the yfdH gene. PCR amplification was performed with primers UP-yfdH-S/UP-yfdH-A and DN-yfdH-S/DN-yfdH-A and KAPA HiFi HotStart to obtain an upper homology arm fragment 461bp and a lower homology arm fragment 898bp. After the PCR reaction is finished, agarose gel electrophoresis recovery is respectively carried out by adopting a column type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR amplification with primers UP-yfdH-S and DN-yfdH-A to obtain a DNA fragment of UP-delta yfdH-Down 1359bp from which the yfdH gene was knocked out.
PCR amplification and overlap PCR amplification system: 5 XHiFi with Mg 2+ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) 1.6. Mu.L, KAPA HiFi HotStart (1U/. Mu.L) 0.5. Mu.L eachL, supplement ddH 2 O to a total volume of 50. Mu.L.
PCR amplification and overlap PCR amplification procedure: pre-denaturation at 95℃for 5min, (denaturation at 98℃for 20s; annealing at 56℃for 15s; extension at 72℃for 150s;30 cycles), over-extension at 72℃for 5min.
Construction of pGRB-yfdH: the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yfdH-S and gRNA-yfdH-A.
Preparation of competent cells of MG1655 or valine-producing bacterium VHY, construction of engineering strains MG1655 having reduced expression strength of yfdH Gene. Delta. YfdH and VHY: Δyfdh. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 1359bp, and the length of the PCR amplified fragment of the original bacterium is 1842bp. The resulting MG1655 was designated as MG1655-05; the resulting VHY20: Δyfdh is designated VHY25.MG1655-05 is a strain in which the yfdH gene is deleted in the genome of MG 1655; VHY 25A strain deleted of yfdH gene in the genome of VHY.
The primer sequences are shown below:
primer UP-yfdH-S:5'-TTTGTGTGTATAAGTTTTGTC-3' (SEQ ID No. 28),
primer UP-yfdH-A:5'-ATGATACTTTTATTGCTTTATTTCGCATCCCTAAAGACAATG-3' (SEQ ID No. 29),
primer DN-yfdH-S:5'-CATTGTCTTTAGGGATGCGAAATAAAGCAATAAAAGTATCAT-3' (SEQ ID No. 30),
primer DN-yfdH-A:5'-AAAAGAGTGCATTCGAAAACG-3' (SEQ ID No. 31),
primer gRNA-yfdH-S:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTACTTTGGAATTTAGCACTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 32),
primer gRNA-yfdH-A:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCAAGTGCTAAATTCCAAAGTACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 33).
Example 7L-valine fermentation experiment
Strains VHY (empty vector control), VHY (overexpressing yfdH gene), VHY23 (overexpressing yfdH) constructed in the above examples were used T743C Gene), VHY (yfdH) T743C Point mutation), VHY (deletion of yfdH Gene), MG1655-01 (empty vector control)) MG1655-02 (overexpressing the yfdH gene), MG1655-03 (overexpressing the yfdH gene) T743C Gene), MG1655-04 (yfdH) T743C Point mutations), MG1655-05 (deletion of the yfdH gene) and control strains VHY, MG1655 were subjected to shake flask fermentation test in a shaking incubator model ZQZY-CS8T (available from Shanghai know Chu instruments Co., ltd.). The fermentation test was performed at 37℃and 80rpm using the medium shown in FIG. 1, and each strain was repeated three times, and the results are shown in FIG. 2. Wherein the seed solution is cultured for 12 hours by using LB culture medium, and the fermentation inoculation amount is 10 percent (27 mL fermentation liquid and 3mL seed liquid).
As shown in FIG. 2, the yields of the over-expressed and point mutant genetically engineered bacteria (VHY, VHY, VHY24, MG1655-02, MG1655-03 and MG 1655-04) constructed by the application are obviously improved, and the yields of recombinant bacteria (VHY and MG 1655-05) after gene knockout are reduced, wherein compared with the yield of VHY of a control group, the yield of VHY is 16.23g/L, and the yield is obviously improved by 19.96%. At the same time, the yield (13.53 g/L) of strain VHY (empty vector control) was not significantly different from that of the blank VHY (13.24 g/L).
As can be seen from the above fermentation results, the yfdH gene or yfdH was overexpressed in either the L-valine-producing strain (e.g., VHY) or the wild-type E.coli (e.g., MG 1655) T743C The genes all contribute to the synthesis of L-valine and over-express yfdH T743C The gene can remarkably improve the yield of L-valine. Furthermore, in either L-valine-producing strain or wild-type E.coli, the yfdH gene was subjected to a point mutation, and after the T at position 743 was mutated to C (the mutant protein YfdH was obtained accordingly) I248T Mutation of isoleucine to threonine at position 248 of the amino acid sequence) contributes to a significant increase in L-valine production. Meanwhile, the expression of the deletion yfdH gene is unfavorable for the synthesis of L-valine. Since the sugar supplement amounts are the same, an increase in yield means an increase in conversion.
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.
Claims (10)
1. A protein, characterized in that the protein comprises any one of the following:
a1 Amino acid sequence comprising a protein as shown in SEQ ID No. 4;
a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.4, has more than 90% of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).
2. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises any one of the following:
b1 A nucleic acid molecule encoding the protein of claim 1;
b2 The coding sequence is a DNA molecule shown as SEQ ID No. 3.
3. A biomaterial, characterized in that the biomaterial comprises any one of the following:
c1 An expression cassette comprising the nucleic acid molecule of claim 2;
c2 A recombinant vector comprising the nucleic acid molecule of claim 2, or a recombinant vector comprising the expression cassette of C1);
c3 A recombinant microorganism comprising the nucleic acid molecule of claim 2, or a recombinant microorganism comprising the expression cassette of C1), or a recombinant microorganism comprising the recombinant vector of C2);
c4 A recombinant cell comprising the nucleic acid molecule of claim 2, or a recombinant cell comprising the expression cassette of C1), or a recombinant cell comprising the recombinant vector of C2).
4. A biomaterial according to claim 3 wherein C3) the recombinant microorganism is any one of the following:
d1 A recombinant microorganism obtained by introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 into a microorganism of interest;
d2 A recombinant microorganism obtained by introducing a DNA molecule having a coding sequence represented by SEQ ID No.3 into a microorganism of interest;
d3 A recombinant microorganism obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest to C.
5. Use of the protein of claim 1, the nucleic acid molecule of claim 2 or any of the following of the biological material of claim 3 or 4:
e1 Use in regulating the production of microbial L-valine;
e2 Use in the preparation of L-valine;
e3 The application of the strain in constructing genetically engineered bacteria for producing L-valine.
Use of any one of F1) -F4) in regulating the yield of L-valine in a microorganism, in the preparation of L-valine or in the construction of a genetically engineered bacterium producing L-valine, wherein the F1) -F4) is:
f1 Amino acid sequence comprising a protein as shown in SEQ ID No. 2;
f2 A nucleic acid molecule encoding the protein of F1);
f3 A DNA molecule with a coding sequence shown in SEQ ID No. 1;
f4 An expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell containing F2) said nucleic acid molecule or F3) said DNA molecule.
7. A method for increasing the production of L-valine or producing L-valine of a microorganism of interest, comprising any one of the following:
g1 Increasing the expression level or the content of the nucleic acid molecule according to claim 2 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g2 Increasing the expression level or the content of the nucleic acid molecule F2) or the DNA molecule F3) in the microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g3 A DNA molecule having a nucleotide sequence of SEQ ID No.1 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest, comprising: isoleucine at position 248 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 was mutated to threonine.
8. The method of claim 7, wherein the microorganism of interest is escherichia coli.
9. The method according to claim 7 or 8, characterized in that the method comprises any one of the following:
h1 Introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 and/or SEQ ID No.2 into the microorganism of interest;
h2 Introducing a DNA molecule comprising the coding sequence shown as SEQ ID No.3 and/or SEQ ID No.1 into the microorganism of interest;
h3 The nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest is mutated to C.
10. Use of the protein of claim 1, the nucleic acid molecule of claim 2, the biological material of claim 3 or 4, the protein of claim 6, the nucleic acid molecule, the DNA molecule, the expression cassette, the recombinant vector, the recombinant microorganism or the recombinant cell for the preparation of a food, a cosmetic, a pharmaceutical or a feed comprising L-valine.
Priority Applications (1)
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