CN115850406A

CN115850406A - L-tyrosine yield-related protein yedZ and biological material and application thereof

Info

Publication number: CN115850406A
Application number: CN202211622967.4A
Authority: CN
Inventors: 魏爱英; 孟刚; 苏厚波; 赵春光; 贾慧萍; 田斌
Original assignee: Ningxia Eppen Biotech Co ltd
Current assignee: Ningxia Eppen Biotech Co ltd
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2023-03-28

Abstract

The invention discloses an L-tyrosine yield-related protein yedZ, a biological material and application thereof, and belongs to the technical field of biology. The problem to be solved by the present application is to regulate the production of microbial L-tyrosine. The protein is any one of the following proteins: the protein is any one of the following proteins: a1 Protein which is yedZ protein or valine residue at alanine residue 180 of the amino acid sequence of the yedZ protein, the yedZ protein is protein with the amino acid sequence of sequence 2; a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling the yield of the microorganism L-tyrosine; a3 A fusion protein which is obtained by connecting labels at the N end and/or the C end of A1) or A2) and has the function of regulating and controlling the yield of the microorganism L-tyrosine. The substance for regulating the expression of the yedZ protein gene can be used for regulating the L-tyrosine output of microorganisms or the breeding of L-tyrosine output strains.

Description

L-tyrosine yield-related protein yedZ and biological material and application thereof

Technical Field

The application belongs to the field of biotechnology. In particular to a protein yedZ related to the L-tyrosine yield, and a biological material and application thereof.

Background

Tyrosine (L-tyrosine, tyr) is an important nutritional essential amino acid, plays an important role in metabolism, growth and development of human and animals, and is widely applied to industries such as food, feed, medicine, chemical industry and the like. It is often used as nutritional supplement for phenylketonuria patients, and raw material for preparing pharmaceutical and chemical products such as polypeptide hormone, antibiotic, L-dopa, melanin, p-hydroxycinnamic acid, p-hydroxystyrene, etc. With the discovery of more high value-added L-tyrosine derivatives such as tanshinol, resveratrol and hydroxytyrosol in organisms, L-tyrosine is developing towards a platform type compound.

Disclosure of Invention

The application provides an L-tyrosine yield-related protein yedZ and a biological material and application thereof, and solves the technical problem of regulating or improving the yield of L-amino acid of microorganisms, particularly L-tyrosine.

In order to solve the above problems, the present application provides a protein.

The protein provided by the application is any one of the following:

the protein is any one of the following proteins:

a1 Protein) which is a yedZ protein or a yedZ mutein obtained by replacing the alanine residue at position 180 of the amino acid sequence of said yedZ protein with any one of the following amino acid residues:

an arginine residue, a cysteine residue, a phenylalanine residue, a leucine residue, an isoleucine, a methionine residue, a valine residue, a serine residue, a proline residue, a threonine residue, a tyrosine residue, a histidine residue, a glutamine residue, an asparagine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a tryptophan residue, a serine residue, or a glycine residue; the yedZ protein is a protein with an amino acid sequence of sequence 2;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling the yield of the microorganism L-tyrosine;

a3 A fusion protein which is obtained by connecting labels at the N end and/or the C end of A1) or A2) and has the function of regulating and controlling the yield of the microorganism L-tyrosine.

The protein is yedZ protein or the alanine residue at the position 180 of the amino acid sequence of the yedZ protein is replaced by any one of the following amino acid residues;

the amino acid residue is valine residue, tryptophan residue, phenylalanine residue, leucine residue, isoleucine residue or methionine residue; the yedZ protein is a protein with an amino acid sequence of sequence 2.

As described above, the amino acid sequence of the protein obtained by mutating the alanine residue at position 180 of the sequence No.2 to a valine residue is the sequence No. 6.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gapexistencecost, perresidugaPCost, and Lambdatoratio to 11,1 and 0.85 (default values), respectively, and performing a calculation by searching for identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 80% or greater identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above protein, SEQ ID No.2 (SEQ ID No. 2) consists of 211 amino acid residues.

In order to solve the above problems, the present application provides a biomaterial.

The biological material is any one of the following materials:

b1 Nucleic acid molecules encoding the above proteins;

b2 An expression cassette containing the nucleic acid molecule of B1);

b3 A recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 ) an expressed nucleic acid molecule that inhibits or reduces or down-regulates a gene encoding said protein;

b6 Expressing a gene encoding the RNA molecule of B5);

b7 An expression cassette containing the gene according to B6);

b8 A recombinant vector containing the gene of B6) or a recombinant vector containing the expression cassette of B7);

b9 A recombinant microorganism having the gene of B6), or a recombinant microorganism containing the expression cassette of B7), or a recombinant microorganism containing the recombinant vector of B4).

In the above biological material, the nucleic acid molecule according to B1) is any one of the following:

z1) the coding sequence is a DNA molecule shown in SEQ ID No. 1;

z2) the coding sequence is a DNA molecule shown in SEQ ID No. 5;

z3) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with a thymine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue, and 540 th thymine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue;

z4) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with a thymine deoxyribonucleotide residue, and 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z5) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with a cytosine deoxyribonucleotide residue, and 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z6) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue and 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z7) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue, and 540 th thymine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue;

z8) the nucleotide sequence is a DNA molecule shown in SEQ ID No. 1;

z9) the nucleotide sequence is a DNA molecule shown in SEQ ID No. 5;

z10) the nucleotide sequence is a DNA molecule obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with a thymine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue, and 540 th thymine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue;

z11) the nucleotide sequence is a DNA molecule obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with a thymine deoxyribonucleotide residue, and 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z12) the nucleotide sequence is a DNA molecule obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with a cytosine deoxyribonucleotide residue, and 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z13) the nucleotide sequence is a DNA molecule obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue and the 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z14) the nucleotide sequence is a DNA molecule obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue, and 540 th thymine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue.

In order to solve the above problems, the present application also provides the following uses:

the use of any one of the following materials in regulating the L-tyrosine yield of a microorganism, or in preparing a microorganism for regulating the L-tyrosine yield, or in breeding a microorganism:

c1 Protein) which is the above protein;

c2 Substances which regulate the expression of the genes coding for the proteins according to C1);

c3 Substances which regulate the activity or content of the protein according to C1).

In the above use, the substance that regulates expression of a gene encoding the protein is any one of:

d1 Nucleic acid molecules encoding the protein;

d2 An expression cassette containing the nucleic acid molecule according to D1);

d3 A recombinant vector containing the nucleic acid molecule according to D1), or a recombinant vector containing the expression cassette according to D2);

d4 A recombinant microorganism containing the nucleic acid molecule according to D1), or a recombinant microorganism containing the expression cassette according to D2), or a recombinant microorganism containing the recombinant vector according to D3);

d5 ) an expressed nucleic acid molecule that inhibits or reduces or down-regulates a gene encoding the above protein;

d6 Expressing a gene encoding the RNA molecule of D5);

d7 An expression cassette containing the gene according to D6);

d8 A recombinant vector containing the gene according to D6) or a recombinant vector containing the expression cassette according to D7);

d9 A recombinant microorganism having a gene according to D6), or a recombinant microorganism having an expression cassette according to D7), or a recombinant microorganism having a recombinant vector according to D4).

D1 Or D5), the nucleotide sequence of the invention coding for the protein yedZ can be easily mutated by a person skilled in the art using known methods, for example directed evolution or point mutation. Those nucleotides which are artificially modified to have 80% or more of identity with the nucleotide sequence of the protein yedZ isolated in the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as they encode the protein yedZ and have the function of the protein yedZ.

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

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, gapexisteccost, perresigugaptost and Lambdariratio are set to 11,1 and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of amino acid sequences, and then the value (%) of identity can be obtained.

In the above biological material, the nucleic acid molecule according to D1) or D5) may be a gene encoding the protein.

As above, the protein with alanine mutated from 180 th alanine to valine in the sequence 2 is shown in the sequence 6. The coding gene is shown as a sequence 5.

As described above, the 180 th alanine in the sequence 2 is mutated into tryptophan residue, and the coding gene of the protein is the nucleotide obtained by replacing the 538 st guanine deoxyribonucleotide residue in the sequence 1 to thymine deoxyribonucleotide residue, replacing the 539 th cytosine deoxyribonucleotide residue to guanine deoxyribonucleotide residue, and replacing the 540 th thymine deoxyribonucleotide residue to guanine deoxyribonucleotide residue.

As described above, the encoding gene of the protein in which alanine at position 180 of the sequence 2 is mutated into phenylalanine residue is a nucleotide obtained by replacing guanine deoxyribonucleotide residue at position 538 of the sequence 1 with thymine deoxyribonucleotide residue, and replacing cytosine deoxyribonucleotide residue at position 539 with thymine deoxyribonucleotide residue.

As described above, the 180 th alanine in the sequence 2 is mutated into leucine residue, and the coding gene of the protein is obtained by replacing the 538 st guanine deoxyribonucleotide residue in the sequence 1 with a cytosine deoxyribonucleotide residue, and replacing the 539 st cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue.

As described above, the 180 th alanine mutation in SEQ ID No.2 is a nucleotide obtained by replacing the 538 rd guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue and the 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue.

As described above, the 180 th alanine of the sequence 2 is mutated into methionine, the coding gene of the protein is the nucleotide obtained by replacing the 538 st guanine deoxyribonucleotide residue of the sequence 1 to adenine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue to thymine deoxyribonucleotide residue and 540 th thymine deoxyribonucleotide residue to guanine deoxyribonucleotide residue.

Herein, the vectors are well known to those skilled in the art, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. Specifically, the vector can be pEASY-Blunt and/or pCAMBIA-139;

in the above-mentioned biological materials, the expression cassette described in D2) or D7) means a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence.

In the above biological material, the nucleic acid molecule described in D1) or D5) may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

D5 In the nucleic acid molecule, the nucleotide sequence encoding the protein yedZ of the invention can be easily mutated by a person of ordinary skill in the art using known methods, for example directed evolution or point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the protein yedZ isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the protein yedZ and have the function of the protein yedZ.

The above-mentioned identity of 75% or more may be 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, gapexisteccost, perresigugaptost and Lambdariratio are set to 11,1 and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of amino acid sequences, and then the value (%) of identity can be obtained.

In order to solve the above problems, the present application also provides a method for regulating the production of L-tyrosine by a microorganism.

The method for regulating the production of the microorganism L-tyrosine comprises regulating the production of the microorganism L-tyrosine by regulating the expression of the coding gene of the protein in the target microorganism or the activity or content of the protein.

In order to solve the above problems, the present application also provides a method of constructing a recombinant microorganism

The method for constructing the recombinant microorganism comprises the step of regulating and controlling the expression of a coding gene of the protein or the activity or content of the protein in a target microorganism to obtain the recombinant microorganism with the changed amino acid yield, wherein the L-tyrosine yield of the recombinant microorganism is higher or lower than that of the target microorganism.

As above, the modulation may be up-regulation or enhancement or increase, or down-regulation or attenuation or decrease.

Wherein up-regulating or enhancing or increasing the expression of a gene encoding said protein or the activity or content of said protein is capable of up-regulating or enhancing or increasing microbial amino acid production.

Downregulating or attenuating or reducing the expression of a gene encoding said protein or the activity or content of said protein enables the downregulation or attenuation or reduction of microbial amino acid production.

In the above, the expression of the gene encoding the protein (abbreviated as gene) can be regulated by at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above method, the regulation of the expression of the gene encoding the protein in the target microorganism is carried out by any one of the following methods:

e1 An expression cassette for introducing the above-mentioned protein-encoding gene into the target microorganism;

e2 Knock-out or down-regulation or attenuation or reduction of the expression of the gene encoding E1) in the microorganism of interest.

Above, E1)) can be achieved by inserting the encoding gene into a chromosome. The site of insertion into the chromosome may specifically be the yai T coding region. E1 In a microorganism of interest) may also be introduced by expression of a recombinant plasmid encoding the gene, which may be present as an extrachromosomal genetic element. The nucleotide sequence of the coding gene of the recombinant plasmid can be at least one of sequence 1 and sequence 5.

Above, the knockout can be a gene knockout technique. The knockout vector can be a pGRB vector.

In the above method, the regulation of the activity or content of the protein in the target microorganism is achieved by mutating yedZ gene in the genome of the target microorganism, wherein the mutation is to mutate the codon of alanine at position 180 of the amino acid sequence encoded by the yedZ gene into valine codon; the yedZ gene encodes any one of the following proteins:

m1) the amino acid sequence is the protein of sequence 2;

m2) protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of M1), has more than 80% of identity with the protein shown by M1), and has the function of regulating and controlling the yield of microbial amino acids.

As described above, the method for making the change may be to obtain the above-mentioned protein by genetic engineering site-directed mutagenesis. The above proteins can also be obtained by random mutagenesis.

In the above, the nucleotide sequence of the gene encoding the protein whose amino acid sequence is sequence 2 may be sequence 1. The nucleotide sequence of the gene encoding the protein of which the amino acid sequence is the sequence 6 can be obtained by mutating the 539 th cytosine (C) of the sequence 1 into thymine (T) and keeping other nucleotides of the sequence 1 unchanged.

In order to solve the above problems, the present application also provides a method for producing an L-amino acid.

The method for producing an L-amino acid comprises producing an L-amino acid using the above-mentioned protein or the above-mentioned biomaterial or the recombinant microorganism according to claim 3 or 4.

In the above-mentioned biomaterial, the above-mentioned use, or the above-mentioned method, the microorganism is any one of:

c1 Bacteria kingdom);

c2 Enterobacteriaceae;

c3 Escherichia coli;

c4 Coli).

The Escherichia coli can be W3110, CGMCC No.25231.

In the present application, the amino acid may be at least one of L-threonine, L-tryptophan, L-arginine, and L-valine.

The recombinant microorganism can be used to produce a variety of products including, but not limited to, lysine, glutamic acid, and valine in the examples, 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, citrulline, and the like.

The present invention also provides a method for producing an amino acid, the method comprising: introducing the biological material into biological cells capable of synthesizing the target amino acid to obtain recombinant biological cells, and culturing the recombinant biological cells to obtain the target amino acid.

In the above method, the biological cell may be a yeast, a bacterium, an algae, a fungus, a plant cell or an animal cell capable of synthesizing the desired amino acid. The biological cell is any biological cell capable of synthesizing the target amino acid. The bacterium may be Escherichia coli, corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium lactofermentum, brevibacterium flavum, corynebacterium pekinense, brevibacterium ammoniagenes, corynebacterium crenatum, or Pantoea (Pantoea).

In the present application, escherichia coli is preferred, and any type of Escherichia coli can be used, and the strains selected in the examples are only representative strains, and have general practicability of Escherichia coli.

Advantageous effects

The invention discloses an L-tyrosine yield-related protein yedZ, a biological material and application thereof, and belongs to the technical field of biology.

The present application constructs pGRB-sgRNA-1 plasmid and Up-yedZ ^A180V A Down fragment, which is introduced into CGMCC No.25231 and Escherichia coli W3110 to obtain an L-tyrosine-producing bacterium CGMCC No.25231 strain containing a mutant gene yedZ A180V and Escherichia coli W3110 (where cytosine (C) at the 539 th position of the nucleotide sequence of the coding region of the yedZ gene is mutated to thymine (T)), and which are respectively named YPTyr-yedZ-01 and W3110-yedZ-01. The fermentation result shows that the mutant strain has improved L-tyrosine yield.

Meanwhile, pGRB-sgRNA-2 plasmid and Up-yedZ-Down fragment are constructed and are introduced into CGMCC No.25231 and Escherichia coli W3110 to obtain yaiT partial coding region on genome replaced by yedZ ^A180V The gene and its promoter, CGMCC No.25231 strain and colibacillus W3110 with unchanged other nucleotides in its genome are named recombinant YPTyr-yedZ-02 and W3110-yedZ-02.

Meanwhile, pGRB-sgRNA-2 plasmid and Up-yedZ are constructed ^A180V -Down fragment, which was introduced into CGMCC No.25231 and E.coli W3110 to obtain a genomic replacement of yaiT partial coding region with yedZ ^A180V The gene and its promoter, CGMCC No.25231 strain and colibacillus W3110 with unchanged other nucleotides in its genome are named recombinant bacteria YPTyr-yedZ-03 and W3110-yedZ-03. The fermentation results showed that either yaiT partial coding region was double-copied yedZ or yedZ ^A180V The CGMCC No.25231 strain of the coding gene or Escherichia coli W3110 has high L-tyrosine yield.

Meanwhile, pGRB-sgRNA-3 plasmid and delta yedZ-Up-Dwon fragment are constructed and introduced into CGMCCNo.25231 and Escherichia coli W3110 to obtain CGMCCNo.25231 strain of yedZ gene deleted on genome and wild type Escherichia coli W3110, which are named YPTyr-yedZ-04 and W3110-yedZ-04 respectively. The fermentation results show that the CG MCCNo.25231 strain with the yedZ gene deleted or the wild type Escherichia coli W3110 has reduced L-tyrosine production.

Meanwhile, pET28a-yedZ is also constructed ^A180V 、pET28(a)-yedZ ^A180W 、pET28(a)-yedZ ^A180F 、pET28(a)-ye dZ ^A180L 、pET28(a)-yedZ ^A180I 、pET28(a)-yedZ ^A180M Then, it was introduced into wild type E.coli W3110 to obtain W3110-pET28 (a) -yedZ ^A180V 、W3110-pET28(a)-yedZ ^A180W 、W3110-pET28(a)-yedZ ^A180F 、W3110-pET28(a)-yedZ ^A180L 、W3110-pET28(a)-yedZ ^A180I And W3110-pET28 (a) -yedZ ^A180M . The W3110-yedZ mutant strain has the ability to produce a part of L-amino acids. And W3110-pET28 (a) -yedZ ^A180V 、W3110-pET28(a)-yedZ ^A180W 、W3110-pET28(a)-yedZ ^A180F 、W3110-pET28(a)-yedZ ^A180L 、W3110-pET28(a)-yedZ ^A180I And W3110-pET28 (a) -yedZ ^A180M All have the ability to produce L-tyrosine, in particular W3110-pET28 (a) -yedZ ^A180V The L-tyrosine producing ability is strongest.

Meanwhile, pET28a-yedZ plasmid and pET28a-yedZ are constructed ^A180V Plasmid, introducing it into CGMCC No.25231 strains, YPTyr-yedZ-05 overexpressing yedZ encoding gene and yedZ overexpressing yedZ were obtained ^A180V The bacterial strain YPTyr-yedZ-06 of the coding gene.

Meanwhile, pET28a-yedZ plasmid and pET28a-yedZ are constructed ^A180V Plasmid, which is introduced into wild type Escherichia coli W3110 to obtain strain W3110-yedZ-05 overexpressing yedZ-encoding gene and strain W3110-yedZ-05 overexpressing yedZ ^A180V The strain W3110-yedZ-06 encoding the gene. The fermentation results showed that either yedZ encoding gene was overexpressed or yedZ ^A180V The CGMCC No.25231 strain of the coding gene is also wild type colibacillus W3110, and the L-tyrosine yield is improved.

The above results show that, in both of the high L-tyrosine productivity strain CGMCCNo.25231 and the model strain W3110, the substitution of alanine at position 180 in the amino acid sequence of yedZ gene with valine, tryptophan, phenylalanine, leucine, isoleucine or methionine contributes to the improvement of L-tyrosine productivity, especially valine; for the strain with high L-tyrosine yield, wild type yedZ gene and mutant yedZ ^A180V Mutant yedZ ^A180W Mutant yedZ ^A180F Mutant yedZ ^A180L Mutant yedZ ^A180I And mutant yedZ ^A180M The overexpression of (A) contributes to the increase of the production of L-tyrosine, in particular the mutant yedZ ^A180V . While the knockout of the yedZ gene decreases the production of L-tyrosine.

Deposit description

The strain name is as follows: escherichia coli;

latin name: escherichia coli;

the strain number is as follows: YP052-1;

the preservation organization: china general microbiological culture Collection center;

the preservation organization is abbreviated as: CGMCC;

address: xilu No.1 Hospital No.3, beijing, chaoyang, north;

the preservation date is as follows: 7/2021, 04/month;

registration number of the preservation center: CGMCC No.25231.

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.

The following examples were processed using SPSS11.5 statistical software and the results were expressed as mean ± standard deviation, with One-way ANOVA test, P < 0.05 (x) indicating a significant difference, P < 0.01 (x) indicating a very significant difference, and P < 0.001 (x) indicating a very significant difference.

Escherichia coli (Escherichia coli) W3110 in the following examples is Escherichia coli str.K-12substr.W3110 in the following documents: NCBI Reference Sequence NC _007779.1 (17-MAY-2022). The biological material is available to the public from the applicant and is only useful for repeating the experiments of the present invention and is not useful for other purposes. Escherichia coli (Escherichia coli) W3110 was purchased from CGMCC (China general microbiological culture Collection center) with the number of CGMCC1.7052.

Example 1 construction of W3110 Strain comprising a yedZ Gene mutant

1. Construction of plasmid for yedZ Gene mutant

For the convenience of the study, the wild-type yedZ gene (sequence as SEQ ID No. 1) and its promoter were first cloned into expression vector pET28 a. PCR amplification is carried out by taking a genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI as a template and primers yedZ-F/yedZ-R to obtain a wild type yedZ promoter and a coding region fragment (the sequence is shown as SEQ ID No. 3). The fragment is recovered and is connected with an expression vector pET28a (purchased from TaKaRa company and containing kanamycin resistance) recovered by enzyme digestion of EcoR I and Hind III for 30min at 50 ℃ by NEBuilder enzyme (purchased from NEB company), a DH5 alpha competent cell is transformed by the connection product, the cell is coated on a 2-YT agar plate containing kanamycin (50 mg/L) and cultured for 12h at 37 ℃, and PCR identification is carried out on primers T7:5'-GCTAGTTATTGCTCAGCGG-3' and T7T:5'-TAATACGACTCACTATAGGGGGAAT-3' for the single clone grown by culture, and PCR can amplify a fragment of 1348bp (the sequence is SEQ ID No. 4) and is a positive transformant pET28a-yedZ containing a yedZ promoter and a coding region sequence.

To obtain mutants encoding the yedZ gene, plasmids of the yedZ mutant gene were prepared using a random mutagenesis kit (Agilent Technologies, USA). The constructed plasmid pET28a-yedZ is taken as a template, and a primer yedZ-F/yedZ-R is used for PCR amplification to obtain a yedZ gene coding region containing random point mutation and a promoter region fragment (the sequence is shown as SEQ ID No.3, but random point mutation exists in the yedZ coding region).

PCR amplification System: 5 × HiFi with Mg ²⁺ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) each 1.6. Mu.L, KAPA HiFi HotStart (1U/. Mu.L) 0.5. Mu.L, and complementary ddH ₂ O to a total volume of 50. Mu.L.

PCR amplification procedure: pre-denaturation at 95 ℃ for 5min, (denaturation at 98 ℃ for 20s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30 cycles), and overextension at 72 ℃ for 5min.

The recovered DNA fragment was ligated with the expression vector pET28a (purchased from TaKaRa, containing kanamycin resistance) recovered by EcoRI/HindIII digestion at 50 ℃ for 30min using NEBuilder enzyme (purchased from NEB), the ligation product was transformed into DH 5. Alpha. And spread on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃ for 12h. The cultured single clone is identified by PCR (polymerase chain reaction) through T7 (5'-GCTAGTTATTGCTCAGCGG-3')/T7T (5'-TAATACGACTCACTATAGGGGGAAT-3'), and the PCR can amplify a pET28a-yedZ-MT positive transformant containing the random mutation of the yedZ gene, wherein the fragment has the size of 1348bp (the sequence is shown as SEQ ID No.4, but the random point mutation exists in the yedZ coding region).

PCR amplification System: 2.5. Mu.L of 2 XPremixrTaq, 1. Mu.L of each primer (10 pM), and a complementary ddH ₂ O to a total volume of 25. Mu.L.

PCR amplification procedure: pre-denaturation at 94 ℃ for 15min, and denaturation at 94 ℃ for 30s; annealing at 56 ℃ for 30s; extension at 72 ℃ for 90s (30 cycles) and over-extension at 72 ℃ for 10min.

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

yedZ-F:5'-GACTGGTGGACAGCAAATGGGTCGCGGATCCTGTTTATG

GCAAGGCGTTAC-3' (underlined nucleotide sequence is pET28 (a) homologous arm sequence),

yedZ-R:5'-CAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCCCTG

ATGCGATCTGTGTATG-3' (underlined nucleotide sequence is pET28 (a) homology arm sequence).

2. Construction of Strain containing mutant yedZ Gene plasmid

In order to identify the productivity of the mutant vector pET28a-yedZ-MT constructed in the first step in L-tyrosine, specifically, different yedZ random mutant plasmids constructed in the first step are transformed into Escherichia coli W3110 strain (the same transformation and identification are carried out in the same step), positive transformants are continuously passaged three times on 2-YT agar plates containing kanamycin (50 mg/L), the obtained product is inoculated into 500mL triangular flasks containing 30mL of rich medium and subjected to shake flask fermentation at 37 ℃ for 24 hours, and the fermentation culture bacteria grow to OD ₆₀₀ And 0.1mM IPTG was added to induce yedZ protein overexpression at the final concentration of 0.1 mM. After completion of the fermentation culture, the concentration of L-amino acid was measured by High Performance Liquid Chromatography (HPLC), and as shown in Table 1, a strain having an L-amino acid-producing ability superior to that of the W3110 control was selected as W3110-yedZ mutant strain 1-5.

Enriching a culture medium: the solvent is water, the solute and the concentration thereof are 30g/L (NH 4) of glucose ₂ SO ₄ 2g/L，H ₃ PO ₄ 0.5g/L，KCl0.8g/L，MgSO ₄ ·7H ₂ O0.8g/L，FeSO ₄ ·7H ₂ O0.05g/L，MnSO ₄ ·H ₂ O0.05g/L, FM902 yeast powder 1.5g/L, corn steep liquor 5g/L, molasses 17g/L, betaine 0.5g/L, citric acid 2g/L, VH20mg/L, VB ₁ 1.5mg/L，VB ₃ 1.5mg/LVB ₁₂ 1.5g/L, pH7.0 adjusted with sodium hydroxide.

TABLE 1 results of L-amino acid analysis by high performance liquid chromatography of W3110-yedZ mutant strain

As shown in Table 1, the E.coli W3110-yedZ mutant strain of the present disclosure has an ability to produce a part of L-amino acids, wherein the W3110-yedZ mutant strain 3 is more superior in the ability to produce L-tyrosine, indicating that the yedZ mutant strain 3 has an activity of synthesizing L-tyrosine.

Extracting plasmid from W3110-yedZ mutant 3, sequencing yedZ gene, and determining that 539-th cytosine (C) in nucleotide sequence of coding region of yedZ gene is mutated into thymine (T) (shown as SEQ ID No. 5), 180-th alanine (A) in corresponding amino acid sequence is mutated into valine (V) (shown as SEQ ID No. 6), and the mutant protein is designated as yedZ ^A180V 。

3. Construction of yedZ Gene mutant vector

The W3110-yedZ mutant 3 was obtained by random mutagenesis using wild-type E.coli W3110. In order to obtain more yedZ mutants to increase L-tyrosine productivity, mutants having the same amino acid positions as those of the above yedZ mutants were constructed. Specifically, the plasmid pET28a-yedZ sequenced in the second step ^A180V As a template, 5 mutants in which the 180 th amino acid in yedZ was substituted with a different amino acid were constructed. The substituted amino acids are all hydrophobic amino acids, and the mutant substituted amino acids and the primer names used in the respective mutants are shown in table 2.

TABLE 2 amino acids substituted by yedZ mutants and the primer names used in the respective mutants

W-PR-1:5'-aagcagtacagccagcccccagtagatgagcggctgcggtg-3'，

W-PR-2:5'-caccgcagccgctcatctactgggggctggctgtactgctt-3'，

F-PR-1:5'-aagcagtacagccagcccaaagtagatgagcggctgcggtg-3'，

F-PR-2:5'-caccgcagccgctcatctactttgggctggctgtactgctt-3'，

L-PR-1:5'-aagcagtacagccagcccaaggtagatgagcggctgcggtg-3'，

L-PR-2:5'-caccgcagccgctcatctaccttgggctggctgtactgctt-3'，

I-PR-1:5'-aagcagtacagccagcccaatgtagatgagcggctgcggtg-3'，

I-PR-2:5'-caccgcagccgctcatctacattgggctggctgtactgctt-3'，

M-PR-1:5'-aagcagtacagccagccctacgtagatgagcggctgcggtg-3'，

M-PR-2:5'-caccgcagccgctcatctacatggggctggctgtactgctt-3'，

taking a wild Escherichia coli W3110 genome as a template, and carrying out PCR amplification by using primers yedZ-F/W-PR-1 and KAPA HiFi HotStart in a table 2 to obtain an Up DNA fragment 895bp with yedZ mutant bases; PCR amplification was performed with primers W-PR-2/yedZ-R and KAPA HiFi HotStart to obtain a 201bp Down DNA fragment with yedZ mutant bases. After the PCR reaction was completed, the recovered DNA fragments were respectively recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit, and the recovered DNA fragments were ligated with expression vector pET28 (a) digested and recovered with EcoRI/HindIII by NEBuilder enzyme (available from NEB) at 50 ℃ for 30min, and the ligation products were transformed into DH 5. Alpha. And spread on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃. The single clone grown by the culture is identified by a primer T7/T7T PCR, and a positive transformant pET28 (a) -yedZ of pET28 (a) which contains 1348bp fragments and is obtained by mutating 180-bit alanine of yedZ gene into tryptophan is amplified by a Taq PCR ^A180W The other four strains were constructed in the same manner. Wherein alanine at position 180 is substituted with the amino acids in Table 2The 5 yedZ mutant vectors of (a) are named as the names listed in table 2.

PCR amplification procedure: pre-denaturation at 95 ℃ for 5min, (denaturation at 98 ℃ for 20s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30 cycles), and over-extension at 72 ℃ for 5min.

PCR amplification System: 2 × Premix r Taq12.5 μ L, primers (10 pM) each 1 μ L, complement ddH ₂ The total volume of O was 25. Mu.L.

PCR amplification procedure: pre-denaturation at 94 ℃ for 5min, and denaturation at 94 ℃ for 30s; annealing at 56 ℃ for 30s; extension at 72 ℃ for 90s (30 cycles) and over-extension at 72 ℃ for 10min.

4. Construction of yedZ mutant strains

In order to identify the L-tyrosine productivity of the mutant vectors constructed in step three, specifically, 5 plasmids constructed in step three were transformed into E.coli W3110 strain, respectively (transformation and identification same as in step one), and after three serial passages of positive transformants on 2-YT agar plates containing kanamycin (50 mg/L), respectively, the transformants were inoculated into 500mL Erlenmeyer flasks containing 30mL of rich medium and subjected to shake flask fermentation at 37 ℃ for 24h.

After the fermentation culture was completed, the concentration of L-tyrosine was analyzed by High Performance Liquid Chromatography (HPLC), and as shown in Table 3, the mutant strain W3110-pET28 (a) -yedZ ^A180V The ratio of L-tyrosine-producing ability W3110-pET28 (a) -yedZ ^A180W 、W3110-pET28(a)-yedZ ^A180F 、W3110-pET28(a)-yedZ ^A180L 、W3110-pET28(a)-yedZ ^A180I And W3110-pET28 (a) -yedZ ^A180M More preferably.

TABLE 3 high Performance liquid chromatography of W3110-yedZ mutant for L-tyrosine detection

Example 2 construction of an engineered Strain comprising a mutant yedZ Gene

According to an Escherichia coli (Escherichia coli) W3110 genome sequence published by NCBI, point mutation is carried out on yedZ gene of an L-tyrosine high-yield strain CGMCC No.25231 by using CRISPR/Cas9 gene editing technology (after sequencing, wild type yedZ gene is reserved on chromosome of the L-tyrosine production strain), so that the L-tyrosine production capacity of the strain is further improved.

Introducing a point mutation into a coding region (SEQ ID No. 1) of the yedZ gene, wherein the point mutation is used for mutating 539 th cytosine (C) in a nucleotide sequence of the coding region of the yedZ gene into thymine (T) (the sequence is shown as SEQ ID No.5 and is named as mutant yedZ ^A180V A gene).

Wherein, the DNA molecule coding protein amino acid sequence shown in SEQ ID No.1 is SEQ ID No.2 (the protein is named as wild type yedZ protein). The amino acid sequence of the DNA molecule coding protein shown in SEQ ID No.5 is SEQ ID No.6 (the name of the mutant protein is mutant yedZ) ^A180V Protein), said mutant protein yedZ ^A180V Valine (V) at position 180 in the amino acid sequence (SEQ ID No. 6) is mutated from alanine (A).

1. Construction of sgRNA

The sgRNA target sequence was designed using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer /) according to the genomic sequence of E.coli (Escherichia coli) W3110 published by NCBI, and after selecting an appropriate sgRNA target sequence, the terminal sequences of linearized pGRB cloning vectors were added to the 5 'and 3' ends of the target sequence to form a complete sgRNA plasmid by recombination.

The sgRNA fragment is amplified without a template, and only a PCR annealing process is needed, and the system and the procedure are as follows. And (3) PCR reaction system: sgRNA-1F 10. Mu.L, sgRNA-1R 10. Mu.L; PCR reaction procedure: denaturation at 95 ℃ for 5min, and annealing at 50 ℃ for 1min. After completion of annealing, the objective fragment was recovered using a DNA purification kit, the DNA concentration thereof was determined, and the concentration was diluted to 100 ng/. Mu.L.

pGRB plasmid (Addgene, cat # 71539) was extracted and digested with Spe I and dephosphorylated to prevent self-ligation of pGRB plasmid. Enzyme digestion system: 10xBuffer 5. Mu.L, speI 2.5. Mu.L, pGRB plasmid DNA 3000-5000ng, complement ddH ₂ O to 50. Mu.L. Carrying out enzyme digestion at 37 ℃ for 3h, carrying out agarose gel electrophoresis, cutting gel, recovering, carrying out dephosphorizing reaction, and carrying out dephosphorizing reaction: 10xBuffer 5. Mu.L, linearized pGRB plasmid DNA 1000-2000ng, CIAP 2.5. Mu.L, complemented H ₂ O to 50. Mu.L. After 1h of treatment at 37 ℃ the linear pGRB plasmid was recovered using a DNA purification kit. Recombination of sgRNA and pGRB plasmids was then carried out using a Gibson Assembly kit (New England Co.). A recombination system: NEB assembly enzyme 2.5 μ L, linearized cloning vector 2 μ L, sgRNA 0.5 μ L. And (3) after assembling at 50 ℃ for 30min, transforming the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The correctly sequenced plasmid was stored and named pGRB-sgRNA-1.

The primers used in this experiment were designed as follows (synthesized by shanghai invitrogen), the underlined bases were pGRB cloning vector homologous arm sequences, the lower case bases were sgRNA sequences:

sgRNA-1F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTcgcagccgctcatctacgctGT TTTAGAGC TAGAAATAGCAAGTTAAAATAAGG-3'

sgRNA-1R:

5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACagcgtagatgagcggctgcgACTAGTATTAT ACCTAGGACTGAGCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-ACTGGCACTGCTGTGCGCCG-3'

2. mutant Gene yedZ ^A180V Amplification of DNA

Using W3110 genome DNA as template, and primer P1/P2, P3/P4 and KAPA HiFi HotStart polymerase to perform PCR amplification to obtain two yedZ with mutant bases of 563bp and 310bp respectively ^A180V DNA fragment (yedZ) ^A180V Up and yedZ ^A180V -Down). After the PCR reaction is finished, the column type DNA gel recovery kit is adopted to recover the fragment yedZ ^A180V Up and yedZ ^A180V Down was recovered by agarose gel electrophoresis, respectively. The recovered DNA fragment was subjected to overlap PCR using primers P1/P4 to obtain integrated homologous arm DNA fragment Up-yedZ containing point mutation ^A180V -Down(SEQ ID No.7)742bp。

The primers were designed as follows (synthesized by Shanghai Invitrogen corporation) with the base in lower case red bold as the mutation position:

P1:5'-ATCAATCACGGTGGACTGG-3'，

P2:

P3:

P4:5'-GCAACAACTCTTAGGAAACGAG-3'，

3. preparation and transformation of competence

pREDCas9 plasmid (containing spectinomycin resistance gene, purchased from addrene, cat No. 371541) is extracted, and is respectively transformed into L-tyrosine producing bacteria CGMCC No.25231 and Escherichia coli W3110 competent cells, the competent cells are spread on a 2-YT agar plate containing spectinomycin (100 mg/L) for culture at 32 ℃, single colonies of anti-spectinomycin (100 mg/L) are selected and subjected to PCR identification by using a primer pREDCas9-PF/pREDCas9-PR, and 943bp (SEQ ID No. 8) YPTyr-Cas9 and W3110-Cas9 transformants containing the pREDCas9 plasmid are obtained.

L-tyrosine YPTyr-Cas9 and W3110-Cas9 competent cells were prepared. When the bacterial body grows to OD ₆₀₀ =0.1 IPTG was added to a final concentration of 0.1mM to induce λ -Red mediated homologous recombination. When OD is reached ₆₀₀ When the expression is not less than 0.4, collecting thalli to prepare competent cells, and respectively transforming pGRB-sgRNA-1 plasmid and point mutation recombinant DNA fragment Up-yedZ ^A180V Down, spread on 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L), and cultured at 32 ℃ for 12h. 10 transformants were picked up and inoculated with spectinomycin (100 mg/L) and final concentrationTo 0.2% arabinose in 2-YT medium to eliminate plasmid pGRB-sgRNA-1; colonies that grew on spectinomycin (100 mg/L) but not ampicillin (100 mg/L) were picked and transferred to 2-YT medium for culture at 42 ℃ to eliminate the pREDCas9 plasmid. Respectively selecting 10 colonies which do not grow on spectinomycin (100 mg/L) but grow on 2-YT without resistance, carrying out PCR amplification and sequencing identification through primers P1/P4, comparing the sequencing result with a yedZ gene sequence of a wild type W3110 genome, and mutating a No. 539 cytosine (C) of a nucleotide sequence of a coding region of the yedZ gene into thymine (T) (the sequence is shown as SEQ ID No. 5) to be a gene mutant type yedZ ^A180V And (4) positive transformants. Will contain mutant gene yedZ ^A180V The L-tyrosine-producing strain CGMCC No.25231 and Escherichia coli W3110 are respectively named YPTyr-yedZ-01 and W3110-yedZ-01;

pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3'

pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3'

PCR amplification System: 2 XPremix r Taq 12.5. Mu.L, primers (10 pM) each 1. Mu.L, complement ddH ₂ Total volume of O was 25. Mu.L.

PCR amplification procedure: pre-denaturation at 94 ℃ for 10min and denaturation at 94 ℃ for 30s; annealing at 56 ℃ for 30s; extension at 72 ℃ for 90s (30 cycles) and over-extension at 72 ℃ for 10min.

Example 3 construction of overexpression of the yedZ Gene or yedZ on the genome ^A180V Engineered strains of genes

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, a CRISPR/Cas9 gene editing technology is utilized to integrate wild type yedZ gene or mutant type yedZ gene into coding regions of L-tyrosine producing bacteria CGMCC No.25231 and yaiT gene of Escherichia coli W3110 respectively ^A180V The gene, and the yedZ gene and mutant yedZ gene have been studied further ^A180V Influence of the genes on the amount of L-tyrosine synthesis.

1. Construction of sgRNA

The sgRNA target sequences were designed according to the genomic sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer /), and after selecting the appropriate sgRNA target sequences, linearized pGRB cloning vector end sequences were added at the 5 'and 3' ends of the target sequences to form complete sgRNA plasmids by recombination.

The sgRNA fragment is amplified without a template, and only a PCR annealing process is needed, and the system and the procedure are as follows. And (3) PCR reaction system: sgRNA-2F 10. Mu.L, sgRNA-2R 10. Mu.L; PCR reaction procedure: denaturation at 95 ℃ for 5min, and annealing at 50 ℃ for 1min. After completion of annealing, the objective fragment was recovered using a DNA purification kit, the DNA concentration thereof was measured, and the concentration was diluted to 100 ng/. Mu.L.

pGRB plasmid was extracted and digested with Spe I and dephosphorylated to prevent self-ligation of pGRB plasmid. Enzyme digestion system: 10xBuffer 5. Mu.L, speI 2.5. Mu.L, pGRB plasmid DNA 3000-5000ng, complement ddH ₂ O to 50. Mu.L. Carrying out enzyme digestion at 37 ℃ for 3h, carrying out agarose gel electrophoresis, cutting gel, recovering, carrying out dephosphorizing reaction, and carrying out dephosphorizing reaction: 10xBuffer 5. Mu.L, pGRB plasmid DNA 1000-2000ng, CIAP 2.5. Mu.L, complement ddH ₂ O to 50. Mu.L. After 1h of treatment at 37 ℃ the linear pGRB plasmid was recovered using a DNA purification kit. Recombination of sgRNA and pGRB plasmids was then carried out using a Gibson Assembly kit (New England Co.). A recombination system: NEB assembly enzyme 2.5 μ L, linearized cloning vector 2 μ L, sgRNA 0.5 μ L. And (3) after assembling at 50 ℃ for 30min, transforming the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The correctly sequenced plasmid was named pGRB-sgRNA-2.

The primers used in this experiment were designed as follows (synthesized by shanghai invitrogen), the underlined bases were the pGRB cloning vector homology arm sequences, the bases in lower case highlighted font were the sgRNA sequences:

sgRNA-2F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTggcaactatgtaaactatagGT TTTAGAGC TAGAAATAGCAAGTTAAAATAAGG-3'

sgRNA-2R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACctatagtttacatagttgccAC TAGTATTA TACCTAGGACTGAGCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-ACTGGCACTGCTGTGCGCCG-3'

2. PCR amplification of genomic over-expressed DNA sequences

Three pairs of amplified upstream and downstream homology arm sequences and yedZ or yedZ are designed and synthesized according to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI ^A180V Primers of a gene coding region and a promoter region are respectively introduced into a coding region of L-tyrosine production bacteria CGMCC No.25231 and an escherichia coli W3110 yaiT by a CRISPR/Cas9 gene editing mode ^A180V A gene.

P7:5'-AAGAGAATGGAAGAGAGGCC-3'，

P8:5'-GTAACGCCTTGCCATAAACACCCAATCAAGTGCTGTAACG-3'，

P9:5'-CGTTACAGCACTTGATTGGGTGTTTATGGCAAGGCGTTAC-3'，

P10:5'-CGGTAGTGTAGGTTTCGTTGCCTGATGCGATCTGTGTATG-3'，

P11:5'-CATACACAGATCGCATCAGGCAACGAAACCTACACTACCG-3'，

P12:5'-CGACCTGTAG TATCCCATTC-3'。

using W3110 genome DNA as template, respectively using primers P7/P8 and P11/P12 and KAPA HiFi HotStart to perform PCR amplification, and obtaining 590bp (SEQ ID No. 91-590) of the upper homologous arm and 605bp (SEQ ID No. 91606-2210) of the lower homologous arm; using W3110 genome DNA as template, using primer P9/P10 and KAPA HiFi HotStart to PCR amplify yedZ promoter and coding region fragment 1095bp (SEQ ID No. 9551-1645); as plasmid pET28a-yedZ ^A180V As a template, yedZ was PCR amplified with primers P9/P10 and KAPA HiFi HotStart ^A180V The promoter and coding region fragment 1095bp (SEQ ID No. 10551-1645). After the PCR reaction is finished, the agarose gel electrophoresis recovery is respectively carried out by adopting a column type DNA gel recovery kit. The recovered DNA was subjected to PCR with primers P7 and P12 overlap to obtain recombinant DNA fragments Up-yedZ-Down (SEQ ID No. 9) and Up-yedZ, respectively, which were overexpressed in the genome ^A180V -Down(SEQ ID No.10)2210bp。

PCR amplification System: 5 × HiFi with Mg ²⁺ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) each 1.6. Mu.L, KAPA HiFi HotStart(1U/. Mu.L) 0.5. Mu.L, supplement ddH ₂ O to a total volume of 50. Mu.L.

3. Preparation and transformation of competence

L-tyrosine YPTyr-Cas9 and W3110-Cas9 competent cells were prepared. When the bacterial body grows to OD ₆₀₀ =0.1 IPTG was added to a final concentration of 0.1mM to induce λ -Red mediated homologous recombination. When OD is reached ₆₀₀ When the expression vector is not less than 0.4, collecting thalli to prepare competent cells, and respectively transforming pGRB-sgRNA-2 plasmid and genome over-expression DNA fragment Up-yedZ-Down or Up-yedZ ^A180V Down, spread on 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L), and cultured at 32 ℃ for 12h. After the single colony generated by culture is passaged, PCR identification is carried out through a primer P7/P12, and a fragment with the size of 2210bp (a sequence without point mutation is shown as SEQ ID No.9, and a sequence with point mutation is shown as SEQ ID No. 10) is amplified by PCR and is taken as a positive transformant.

Inoculating positive transformants in a 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% to eliminate plasmid pGRB-sgRNA-2, selecting colonies that grow on spectinomycin (100 mg/L) but do not grow on ampicillin (100 mg/L), transferring the colonies to a 2-YT medium at 42 ℃ to eliminate pREDCas9 plasmid, selecting colonies that do not grow on spectinomycin (100 mg/L) but grow on nonresistant 2-YT, performing PCR identification again by using primer P7/P12, amplifying by size 2210bp as positive seeds, sending the positive seeds to sequence, and designating strains with correct sequencing results as Tyr-yedZ-02 (containing no mutation point), YPTyr-yedZ-03 (containing mutation point), W3110-yedZ-02 (containing no mutation point) and W3110-yedZ-03 (containing mutation point);

the recombinant bacteria YPTyr-yedZ-02 and W3110-yedZ-02 contain double copies of yedZ gene shown in SEQ ID No. 1; specifically, the recombinant bacteria YPTyr-yedZ-02 and W3110-yedZ-02 are obtained by replacing yaiT partial coding regions on the genome of the L-tyrosine producing bacteria CGMCC No.25231 and the wild type Escherichia coli W3110 with yedZ genes and promoters thereof and keeping other nucleotides in the genome unchanged. The recombinant bacterium containing double copies of the yedZ gene can obviously and stably improve the expression quantity of the yedZ gene.

Recombinant bacteria YPTyr-yedZ-03 and W3110-yedZ-03 contain double copies of yedZ shown in SEQ ID No.5 ^A180V A gene; specifically, recombinant bacteria YPTyr-yedZ-03 and W3110-yedZ-03 are obtained by replacing yaiT partial coding region on genome of L-tyrosine producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 with yedZ ^A180V Gene and its promoter, and recombinant bacterium obtained by keeping other nucleotides in its genome unchanged. Containing double copies yedZ ^A180V The recombinant strain of the gene can obviously and stably improve the expression quantity of the yedZ gene.

PCR amplification System: 2 × Premix r Taq12.5 μ L, primers (10 pM) each 1 μ L, complement ddH ₂ Total volume of O was 25. Mu.L.

Example 4 construction of an engineered Strain with deletion of yedZ Gene on genome

According to a genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, a CRISPR/Cas9 gene editing technology is utilized to knock out yedZ genes in L-tyrosine-producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 genes (complete yedZ genes are reserved on chromosomes of strains through sequencing confirmation), so that the influence of the E.coli yedZ genes on the synthesis of L-tyrosine is further studied.

1. Construction of sgRNA

The sgRNA target sequence was designed using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer /) according to the E.coli (Escherichia coli) W3110 genomic sequence published by NCBI, and after selecting the appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5 'and 3' ends of the target sequence to form a complete sgRNA plasmid by recombination.

The sgRNA fragment is amplified without a template, and only a PCR annealing process is needed, and the system and the program are as follows: and (3) PCR reaction system: sgRNA-3F 10. Mu.L, sgRNA-3R 10. Mu.L; PCR reaction procedure: denaturation at 95 ℃ for 5min, and annealing at 50 ℃ for 1min. After completion of annealing, the objective fragment was recovered using a DNA purification kit, the DNA concentration thereof was determined, and the concentration was diluted to 100 ng/. Mu.L.

pGRB plasmid was extracted and treated with Spe I digestion and dephosphorylation to prevent self-ligation of pGRB plasmid. Enzyme digestion system: 10xBuffer 5. Mu.L, speI 2.5. Mu.L, pGRB plasmid DNA 3000-5000ng, complement ddH ₂ O to 50. Mu.L. Carrying out enzyme digestion at 37 ℃ for 3h, carrying out agarose gel electrophoresis, cutting gel, recovering, carrying out dephosphorylation reaction, and carrying out dephosphorylation system: 10xBuffer 5. Mu.L, linearized pGRB plasmid DNA 1000-2000ng, CIAP 2.5. Mu.L, complemented H ₂ O to 50. Mu.L. After 1h of treatment at 37 ℃ the linear pGRB plasmid was recovered using a DNA purification kit. Recombination of sgRNA and pGRB plasmids was then carried out using a Gibson Assembly kit (New England Co.). A recombination system: NEB assembly enzyme 2.5 μ L, linearized cloning vector 2 μ L, sgRNA 0.5 μ L. And (3) after assembling at 50 ℃ for 30min, transforming the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The correctly sequenced plasmid was named pGRB-sgRNA-3.

sgRNA-3F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTcggtggactgggtgccgatcGT TTTAGAGCTAGA AATAGCAAGTTAAAATAAGG-3'

sgRNA-3R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACgatcggcacccagtccaccgAC TAGTATTATACC TAGGACTGAGCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-GCGTCAGGTGCATAAACAGA-3'

2. PCR amplification of genomic deleted recombinant DNA fragments

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, two pairs of primers for amplifying upstream and downstream homologous arm sequences are designed and synthesized, and the yedZ gene in the genes of L-tyrosine producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 is knocked out in a CRISPR/Cas9 gene editing mode.

P13:5'-ATCCGCTCACACTGATGAC-3'，

P14:5'-GTGAACCTGCTTGCGTAACAGAAACGGCAACAATCCG-3'，

P15:5'-CGGATTGTTGCCGTTTCTGTTACGCAAGCAGGTTCAC-3'，

P16:5'-CCTCAATGCCAATCATCTC-3'。

performing PCR amplification by using W3110 genome DNA as a template and using primers P13/P14, P15/P16 and KAPA HiFi HotStart to obtain fragments of an upper homology arm and a lower homology arm with the sizes of 433bp and 627bp respectively; after the PCR reaction is finished, the 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 using primers P13/P16 to obtain a recombinant DNA fragment Δ yedZ-Up-Dwon (SEQ ID No. 11) with a size of 1060bp, in which yedZ was deleted from the genome.

3. Preparation and transformation of competence

L-tyrosine YPTyr-Cas9 and W3110-Cas9 competent cells were prepared. When the bacterial body grows to OD ₆₀₀ =0.1 IPTG was added to a final concentration of 0.1mM to induce λ -Red mediated homologous recombination. When OD is measured ₆₀₀ When the expression density is not more than 0.4, competent cells were collected, and the pGRB-sgRNA-3 plasmid and the recombinant DNA fragment Δ yedZ-Up-Dwon with yedZ deleted from the genome were transformed, respectively, and plated on a 2-YT agar plate containing spectinomycin (100 mg/L) and ampicillin (100 mg/L) and cultured at 32 ℃ for 12 hours. After the single colony generated by culture is passed, PCR identification is carried out through a primer P13/P16, a positive transformant with a 1060bp (SEQ ID No. 11) fragment is amplified by PCR, and a raw bacterium with a 1588bp fragment is amplified.

Inoculating the positive transformant to a 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% to eliminate plasmid pGRB-sgRNA-3, selecting colonies which grow on spectinomycin (100 mg/L) but do not grow on ampicillin (100 mg/L), transferring the colonies to a 2-YT medium at 42 ℃ to eliminate pREDCas9 plasmid, selecting colonies which do not grow on spectinomycin (100 mg/L) but grow on 2-YT without antibiotics, performing PCR identification again by using a primer P13/P16, amplifying a 1060bp fragment in size to be a positive seed, sending the positive seed for sequencing, and respectively naming the strains with correct sequencing results as YPTyr-yedZ-04 and W3110-yedZ-04;

the recombinant bacteria YPTyr-yedZ-04 and W3110-yedZ-04 contain deleted yedZ genes; specifically, the recombinant bacteria YPTyr-yedZ-04 and W3110-yedZ-04 are recombinant bacteria obtained by knocking out part of coding regions of yedZ genes on the genome of L-tyrosine producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 and keeping other nucleotides in the genome unchanged.

Example 5 construction of plasmids overexpressing yedZ or yedZ ^A180V Of (2) an engineered strain

The wild-type yedZ gene or mutant yedZ gene was introduced into E.coli expression vector pET28a (purchased from TaKaRa, containing kanamycin resistance) according to the genomic sequence of E.coli (Escherichia coli) W3110 published by NCBI ^A180V The gene coding region and the promoter region are introduced into the L-tyrosine production bacterium CGMCC No.25231 and the wild type Escherichia coli W3110, so as to further research the multi-copy yedZ gene or mutant yedZ ^A180V Influence of the Gene on the production of L-tyrosine.

L-tyrosine CGMCC No.25231 and wild type W3110 competent cells were prepared and plasmids pET28a-yedZ and pET28a-yedZ constructed in example 1 were transformed, respectively ^A180V Spread on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃. The single colony generated by culture is identified by PCR through primer T7T/T7, and the PCR is used for amplifying a sequence containing 1348bp (without point mutation, such as SE)Q ID No.4 shows that the 284 th site of the sequence containing the point mutation is A, the rest fragments shown as SEQ ID No. 4) are positive transformants, and the original strain is not amplified.

PCR amplification System: 2 XPremix r Taq 12.5. Mu.L, primers (10 pM) each 1. Mu.L, complement ddH ₂ The total volume of O was 25. Mu.L.

Wild type yedZ gene and mutant yedZ gene overexpressed by L-tyrosine CGMCC No.25231 and wild type Escherichia coli W3110 plasmid ^A180V The genes were named YPTyr-yedZ-05 (without mutation), YPTyr-yedZ-06 (with mutation), W3110-yedZ-05 (without mutation) and W3110-yedZ-06 (with mutation), respectively.

The recombinant bacteria YPTyr-yedZ-05 and W3110-yedZ-05 contain pET28a-yedZ over-expressed yedZ gene shown in SEQ ID No.1, and the genome sequence of the recombinant bacteria is kept unchanged to obtain the recombinant bacteria. The recombinant bacterium overexpressed by pET28a-yedZ can obviously and stably improve the expression quantity of yedZ gene.

The recombinant bacteria YPTyr-yedZ-06 and W3110-yedZ-06 contain pET28a-yedZ ^A180V Overexpressed yedZ as shown in SEQ ID No.5 ^A180V Gene, and recombinant bacteria obtained by keeping the genome sequence unchanged. pET28a-yedZ ^A180V The over-expressed recombinant bacteria can obviously and stably improve the expression quantity of the yedZ gene.

Example 6 fermentation experiment of L-tyrosine

The strain constructed in the above example, the tyrosine-producing strain CGMCC No.25231 and the wild type Escherichia coli W3110 were inoculated into 5L fermentors (BaiBIO-5 GC-4-H, tokyo Biotech Co., ltd.) of BLBIO type 5GC-4-H, respectively, and fermentation experiments were carried out with L-tyrosine fermentation medium (see Table 4) and fermentation protocol (see Table 5) in triplicate for each strain. Wherein YPTyr-yedZ-05, YPTyr-yedZ-06, W3110-yedZ-05 and W3110-yedZ-06 are strains containing pET28a overexpression, IPTG induction is required in the fermentation process, and IPTG with the final concentration of 0.1mM is added when the fermentation culture bacteria grow to OD600=0.1 to induce yedZ protein overexpression. After the fermentation was completed, the L-tyrosine content was measured by High Performance Liquid Chromatography (HPLC), and the results were averaged over three replicates as shown in Table 6.

TABLE 4L tyrosine fermentation Medium

Reagent	Concentration (g/L)
		Glucose	30
Yeast powder	8
		Ammonium sulfate	6
Potassium dihydrogen phosphate	6
		Magnesium sulfate heptahydrate	3
Glutamic acid	2
		Citric acid monohydrate	4
Methionine	0.5
		Phenylalanine (PHE)	0.8
Manganese sulfate monohydrate	20mg/L
		Ferrous sulfate heptahydrate	40mg/L
VH	2mg/L
		VB1、3、5、12	Each 0.3mg/L
MnSO4·H2O	3mg/L
		ZnSO4	4mg/L
Gamma-aminobutyric acid	1g/L

TABLE 5L-tyrosine fermentation protocol

TABLE 6L-tyrosine production and significance analysis of the yedZ engineered strain

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As shown by the fermentation results, the strain CGMCC No.25231 with high yield of L-tyrosine is adopted, and the strain CGMCC No. 5363 is also usedIs a model strain W3110, and the alanine at the 180 th site of the amino acid sequence of yedZ gene is replaced by valine, which is favorable for improving the yield of L-tyrosine; for the strain with high L-tyrosine yield, wild type yedZ gene and mutant yedZ ^A180V The overexpression of (a) contributes to the increase of the L-tyrosine production, while the knockout of yedZ gene reduces the L-tyrosine production.

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. 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.

Claims

1. A protein, wherein the protein is any one of:

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has the identity of more than 80 percent with the protein shown in A1) and can regulate and control the yield of the microorganism L-tyrosine;

2. The protein of claim 1, wherein said protein is a yedZ protein or wherein the alanine residue at position 180 of the amino acid sequence of said yedZ protein is replaced with any of;

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

b1 Nucleic acid molecules encoding the protein of claim 1 or 2;

b2 An expression cassette containing the nucleic acid molecule of B1);

b5 A nucleic acid molecule that inhibits or reduces or down regulates expression of a gene encoding the protein;

b6 Expressing a gene encoding the RNA molecule of B5);

b7 An expression cassette containing the gene according to B6);

4. The biomaterial of claim 3, wherein the nucleic acid molecule of B1) is any one of the following:

z1) the coding sequence is a DNA molecule shown in SEQ ID No. 1;

z2) the coding sequence is a DNA molecule shown in SEQ ID No. 5;

z4) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with a thymine deoxyribonucleotide residue and the 539 st cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z6) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue and the 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue;

z7) the coding sequence is a DNA molecule obtained by replacing the 538 st guanine deoxyribonucleotide residue of SEQ ID No.1 with an adenine deoxyribonucleotide residue, 539 th cytosine deoxyribonucleotide residue with a thymine deoxyribonucleotide residue and 540 th thymine deoxyribonucleotide residue with a guanine deoxyribonucleotide residue;

z8) the nucleotide sequence is a DNA molecule shown in SEQ ID No. 1;

z9) the nucleotide sequence is a DNA molecule shown in SEQ ID No. 5;

5. The use of any one of the following materials in regulating the L-tyrosine yield of a microorganism, or in preparing a microorganism for regulating the L-tyrosine yield, or in breeding a microorganism:

c1 A protein according to claim 1 or 2;

6. The use according to claim 5, wherein the substance regulating the expression of the gene encoding the protein is any one of:

d1 Nucleic acid molecules encoding the protein of claim 1 or 2;

d5 A nucleic acid molecule that inhibits or reduces or downregulates expression of a gene encoding a protein according to claim 1 or sequence 2;

d6 Expressing a gene encoding the RNA molecule of D5);

d7 An expression cassette containing the gene according to D6);

7. A method for controlling the production of L-tyrosine by a microorganism, which comprises controlling the production of L-tyrosine by a microorganism by controlling the expression of a gene encoding the protein of claim 1 or 2 or the activity or content of the protein in the microorganism of interest.

8. A method for constructing a recombinant microorganism, which comprises controlling the expression of a gene encoding the protein of claim 1 or 2 or the activity or content of the protein in a microorganism of interest to obtain a recombinant microorganism having a modified amino acid production, wherein the L-tyrosine production of the recombinant microorganism is higher or lower than that of the microorganism of interest.

9. The method according to claim 7 or 8, wherein the regulation of the expression of the gene encoding the protein according to claim 1 in the microorganism of interest is carried out by any one of the following methods:

e1 Introducing an expression cassette of a gene encoding the protein of claim 1 or 2 into the microorganism of interest;

10. The method as claimed in claim 6 or 7, wherein the regulation of the activity or content of the protein of claim 1 or 2 in the microorganism of interest is carried out by mutating yedZ gene in the genome of the microorganism of interest by a mutation from a codon for alanine at position 180 of the amino acid sequence encoded by said yedZ gene to a valine codon; the yedZ gene encodes any one of the following proteins:

m1) the amino acid sequence is the protein of sequence 2;

m2) protein obtained by substituting and/or deleting and/or adding amino acid residues to the protein of M1), has the identity of more than 80 percent with the protein shown by M1), and has the function of regulating and controlling the yield of microbial amino acids.

11. A method for producing an L-amino acid, which comprises producing an L-amino acid using the protein of claim 1 or 2, the biological material of claim 3 or 4, or the recombinant microorganism of claim 3 or 4.

12. The biomaterial of claim 3 or 4, the use of claim 5 or 6 or the method of any one of claims 6 to 11, wherein the microorganism is any one of:

c1 Bacteria kingdom);

c2 Enterobacteriaceae;

c3 Escherichia coli;

c4 Coli).