CN115947804A

CN115947804A - L-tyrosine yield-related protein yccE, and biological material and application thereof

Info

Publication number: CN115947804A
Application number: CN202211590632.9A
Authority: CN
Inventors: 孟刚; 赵春光; 魏爱英; 米杰; 贾慧萍; 田斌; 毕国东
Original assignee: Ningxia Eppen Biotech Co ltd
Current assignee: Ningxia Eppen Biotech Co ltd
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-04-11

Abstract

The invention discloses an L-tyrosine yield-related protein yccE, and 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 A yccE protein or replacing the arginine residue 415 of the amino acid sequence of the yccE protein with either: a phenylalanine residue, a leucine residue, isoleucine, a methionine residue, a valine residue, a serine residue, a proline residue, a threonine residue, an alanine 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 cysteine residue, a tryptophan residue, a serine residue, or a glycine residue; the yccE protein is a protein having the amino acid sequence of sequence 2; a2 A protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has 80% or more of identity with the protein shown in A1), and has the function of regulating and controlling the yield of the L-tyrosine of the microorganism; a3 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2). Substances that regulate the expression of the yccE protein gene can be used to regulate the L-tyrosine production of microorganisms or the breeding of L-tyrosine producing strains.

Description

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

Technical Field

The application belongs to the field of biotechnology. In particular to a protein yccE related to the yield of L-tyrosine, 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 yccE and a biological material and application thereof, and solves the technical problem of how to regulate or improve 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:

a1 A yccE protein or a yccE mutein obtained by replacing the arginine residue at position 415 of the amino acid sequence of said yccE protein with any one of the following amino acid residues:

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, an alanine 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 yccE protein is a protein with an amino acid sequence of sequence 2;

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

a3 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2).

The above protein is yccE protein or the 415 th arginine residue of the amino acid sequence of the yccE protein is replaced by any one of the following;

the amino acid residue is cysteine residue, glycine residue, serine residue, threonine residue, tyrosine residue or alanine residue; the yccE protein is a protein having the amino acid sequence of SEQ ID No. 2.

The amino acid sequence of the yccE mutein may be one or more of sequences 6, 8, 10, 12, 14 and 16, supra. As described above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of SEQ ID NO.2 to a cysteine residue is SEQ ID NO. 6. As described above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of SEQ ID NO.2 to a glycine residue was SEQ ID NO. 8. As described above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of SEQ ID NO.2 to a serine residue was 10. As described above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of SEQ ID NO.2 to a threonine residue is 12. In the above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of the sequence 2 to a tyrosine residue is 14. In the above, the amino acid sequence of the protein obtained by mutating the 415 th arginine residue of SEQ ID No.2 to an alanine residue was 16.

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 the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the 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, the sequence 2 (SEQ ID No. 2) is composed of 419 amino acid residues.

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

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

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 shown in SEQ ID No. 7;

z4) the coding sequence is a DNA molecule shown in SEQ ID No. 9;

z5) the coding sequence is a DNA molecule shown in SEQ ID No. 11;

z6) the coding sequence is a DNA molecule shown in SEQ ID No. 13;

z7) the coding sequence is a DNA molecule shown in SEQ ID No. 15;

z8) the nucleotide sequence is a DNA molecule shown as 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 shown in SEQ ID No. 7;

z11) the nucleotide sequence is a DNA molecule shown as SEQ ID No. 9;

z12) the nucleotide sequence is a DNA molecule shown as SEQ ID No. 11;

z13) the nucleotide sequence is a DNA molecule shown as SEQ ID No. 13;

z14) nucleotide sequence is the DNA molecule shown in SEQ ID No. 15.

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

the use of any one of the following materials in the regulation of the L-tyrosine production of microorganisms, or in the preparation of a microorganism for the regulation of the L-tyrosine production of microorganisms, or in the breeding of microorganisms:

c1 Protein) which is the protein described above:

c2 Substances which regulate the expression of genes encoding said proteins;

c3 Substances which regulate the activity or content of the protein.

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

d1 Nucleic acid molecules encoding the protein of C1);

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).

B1 Or D5), the nucleotide sequence of the invention coding for the protein yccE can easily be mutated by a person skilled in the art using known methods, for example directed evolution or point mutation. Those nucleotides which have been artificially modified to have 80% or more than 80% identity with the nucleotide sequence of protein yccE isolated according to 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 protein yccE and have the function of protein yccE.

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 the advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost, and the Lambda ratio are set to 11,1, and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of the amino acid sequence, and then the value (%) of identity can be obtained.

In the above biological material, the nucleic acid molecule of B1) or D1) may be a gene encoding the protein.

As above, the sequence 6 shows the protein with the 415 th arginine mutated into the cysteine in the sequence 2. The coding gene is shown as a sequence 5.

As above, the sequence 8 shows the protein with the 415 th arginine mutated into the glycine shown in the sequence 2. The coding gene is shown as a sequence 7.

As above, the sequence 10 shows the protein with the 415 th arginine mutated into serine in the sequence 2. The coding gene is shown as a sequence 9.

In the above, the protein having the sequence in which the 415 th arginine of the sequence 2 is mutated into threonine is shown as the sequence 12. The coding gene is shown as a sequence 11.

As above, the sequence 14 shows the protein obtained by mutating the 415 th arginine of the sequence 2 into the tyrosine. The coding gene is shown as sequence 13.

As above, the sequence 16 shows the protein with the 415 th arginine mutated into alanine in the sequence 2. The coding gene is shown as sequence 15.

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 B2) or D2) 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 according to B5) 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.

B5 In the case of the nucleic acid molecules described, the nucleotide sequence of the invention which codes for the protein yccE can easily be mutated by a person skilled in the art using known methods, for example directed evolution or point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence of the isolated protein yccE of the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention, as long as they encode the protein yccE and have the function of the protein ycE.

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

In order to solve the above problems, the present application provides a method for regulating the production of amino acids from microorganisms.

The method for regulating the microbial amino acid production comprises regulating the microbial amino acid production by regulating the expression of the coding gene of the protein or the activity or content of the protein in a target microorganism.

In order to solve the above problems, the present application 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 the coding gene of the protein or the activity or the content of the protein in a target microorganism to obtain the recombinant microorganism with the changed amino acid yield, wherein the amino acid 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 downregulating or attenuating or reducing microbial amino acid production.

As described 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 Introducing a gene encoding the above-mentioned protein into the target microorganism;

e2 Introducing a gene encoding any one of the following proteins into the target microorganism:

e3 Knock-out or down-regulation or attenuation or reduction of the expression of the gene encoding E2) 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 yaiT coding region. E1 can also be achieved by the expression of the coding gene recombinant plasmid into the microorganisms of interest, the recombinant plasmid can be as extrachromosomal genetic factor. The nucleotide sequence of the encoding gene of the recombinant plasmid can be at least one of sequence 1, sequence 5, sequence 7, sequence 9, sequence 11, sequence 13 and sequence 15.

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

In the method, the regulation and control of the activity or the content of the protein in the target microorganism are realized by mutating the yccE gene in the genome of the target microorganism, wherein the mutation is realized by changing the codon of 415 th arginine of the amino acid sequence coded by the yccE gene into a cysteine codon; the yccE gene encodes any one of the following proteins:

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

m2) protein which is 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 the yield of the L-tyrosine.

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 coding the protein of which the amino acid sequence is the sequence 6 can be obtained by mutating cytosine (C) at the 1243 th position of the sequence into thymine (T) and keeping other nucleotides of the sequence 1 unchanged.

In order to solve the above problems, the present application 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 biological material or the above-mentioned recombinant microorganism.

In the protein biomaterial, the use thereof, and the method, the microorganism may be any of:

c1 Bacteria kingdom);

c2 Enterobacteriaceae;

c3 Escherichia coli;

c4 Coli).

The Escherichia coli can be W3110 and 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: the above-mentioned biological material is introduced into a biological cell capable of synthesizing the objective amino acid to obtain a recombinant biological cell, and the recombinant biological cell is cultured to obtain the objective 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 application, the Escherichia coli is preferred, any type of Escherichia coli can be used, and the selected strains in the examples are only representative strains, so that the general practicability of the Escherichia coli is realized.

Advantageous effects

Plasmid expression vectors pET28 (a) -yccER415C, pET28 (a) -yccER415G, pET28 (a) -yccER415S, pET28 (a) -yccER415T, pET28 (a) -yccE ^R415Y And pET28 (a) -yccE ^R415A The recombinant strain is introduced into W3110, and the result shows that the L-tyrosine yield of the recombinant strain is remarkably improved after the plasmid is introduced.

Further, the yccE gene in the L-tyrosine producing strain CGMCC No.25231 is replaced by yccE by gene knockout technology ^R415CL The obtained recombinant strain was named YPThr-ycE 001. The results showed that the yccE gene was replaced with yccE ^R415C The L-tyrosine yield of the related high-yield strain of the gene is obviously improved.

Further, yccE gene and yccE were constructed ^R415C Genomic overexpression vector of Gene, yccE Gene and yccE ^R415C The gene is inserted into the yaiT gene region of the strain CGMCC No.25231 to obtain yccE genome over-expression strain (YPTyr-002) and yccE ^R415C The genome overexpression strain (YPTyr-003) results show that: to be provided withOverexpression of yccE Gene and yccE by means of integration by staining ^R415C The gene can obviously improve the L-tyrosine yield of the strain.

Further, an yccE gene knockout vector is constructed and is introduced into a strain of CGMCC No.25231 to obtain a strain (YPTyr-yccE-004) with the yccE gene deleted. The results show that: the L-tyrosine production of related high-producing strains with yccE gene knockout is remarkably reduced.

Further, yccE gene and yccE were constructed ^R415C Plasmid overexpression vectors for genes, yccE gene and yccE ^R415C The gene is integrated into pET28 (a) and is introduced into strain CGMCC No.25231, and the result of obtaining yccE plasmid over-expression strain (YPTyr-yccE-005) and ycER 415C genome over-expression strain (YPTyr-yccE-006) shows that: overexpression of yccE Gene and yccE by means of plasmid ^R415C The gene can obviously improve the L-tyrosine yield of the strain.

The result shows that the 415 th arginine of the amino acid sequence of the yccE gene is replaced by cysteine, glycine residue, serine residue, threonine residue, tyrosine residue or alanine residue, which is beneficial to the improvement of the L-tyrosine yield no matter the high-yield L-tyrosine strain or the model strain W3110, and especially the L-tyrosine yield is obviously improved after the substitution by cysteine; wild-type yccE gene and mutant yccE for high L-amino acid tyrosine-producing strains ^R415C Mutant yccE ^R415G Mutant yccE ^R415S Mutant yccE ^R415T Mutant yccE ^R415Y And mutant yccE ^R415A The overexpression of (A) all contributed to the improvement of L-tyrosine production, while the knockout of yccE gene was not favorable for the improvement of L-tyrosine production.

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: 7/2022/month 04;

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 specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. 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 W3110 was purchased from CGMCC (China general microbiological culture Collection center) with the number of CGMCC1.7052.

Example 1 construction of yccE mutant W3110 Strain of unknown protein

1. Construction of mutant plasmids of unknown protein yccE

For ease of investigation, the wild-type yccE gene (sequence as SEQ ID No. 1) was first cloned into expression vector pET28 (a). The wild-type yccE gene fragment is obtained by PCR amplification with primers pET28-PF/yccE-PF and pET 28-PR/ycE-PR by using a genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI as a template. After recovery, the recovered expression vector pET28 (a) (purchased from TaKaRa, containing kanamycin resistance) was ligated with NEBuilder enzyme (purchased from NEB) at 50 ℃ for 30min, and the ligation product was applied to 2-YT agar plate containing kanamycin (50 mg/L) and cultured at 37 ℃ to obtain pET28 (a) transformant containing yccE gene, i.e., pET28 (a) -yccE (sequence shown in SEQ ID No. 3). The single clone grown by culture is identified by PCR with primers T7-F/T7-R and R Taq, and PCR is used for amplifying pET28 (a) positive transformant pET28 (a) -ycE containing ycE gene and containing 1616bp (sequence is shown as SEQ ID No. 4) fragment.

To obtain mutants encoding the yce gene, yce mutant gene plasmids were prepared using a random mutagenesis kit (Agilent Technologies, USA). Plasmid pET28 (a) -yccE is taken as a template, and primers pET28-PF/pET28-PR are respectively used for PCR amplification to obtain a 1257bp pET28 (a) positive transformant pET28 (a) -ycE-MT (the sequence is shown as SEQ ID No.3, but random point mutation exists in a ycC E coding region) containing a ycC gene of the ycC gene fragment containing random point mutation.

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 expression vector pET28 (a) (purchased from TaKaRa, containing kanamycin resistance) recovered by the Nco I/BamH I digestion at 50 ℃ for 30min using NEBuilder enzyme (purchased from NEB), and the ligation product was transformed into DH 5. Alpha. And plated on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃. The single clone grown by culture is identified by PCR with primers T7-F/T7-R and R Taq, and PCR is used for amplifying a pET28 (a) positive transformant containing yccE random mutation, wherein the pET28 (a) positive transformant contains a fragment with the size of 1616bp (the sequence is shown as SEQ ID No.4, but random point mutation exists in the yccE coding region).

PCR amplification System: 2 × Premix r Taq 12.5 μ L, primer(10 pM) 1. Mu.L each, supplemented with ddH ₂ O to a total volume of 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.

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

pET28-PF:5'-GCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAAC

TTTAAGAAGGAGATATACCATGGGGatgacacgaactattattgtga-3' (the underlined nucleotide sequence is the pET28 (a) homology arm sequence),

pET28-PR:5'-GGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGG

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

T7-F:5'-GCTAGTTATTGCTCAGCGG-3'

T7-R:5'-TAATACGACTCACTATAGGGGGAAT-3'

2. Construction of Strain containing mutant yccE Gene plasmid

In order to identify the L-tyrosine production performance of the mutant vector constructed in the first step, specifically, the yccE random mutant plasmid constructed in the first step is transformed into Escherichia coli W3110 strain (the same transformation and identification steps are carried out as in the first step), after the positive transformant is continuously passaged three times on a 2-YT agar plate containing kanamycin (50 mg/L), the plate is inoculated into a 500mL triangular flask containing 30mL of rich medium and is subjected to shake flask fermentation at 37 ℃ for 24 hours, and the fermentation culture bacterium grows to OD ₆₀₀ And when the concentration is 0.1, IPTG is added to the concentration of 0.1mM to induce YCCE protein overexpression.

After completion of the fermentation culture, the concentration of L-amino acid was analyzed by High Performance Liquid Chromatography (HPLC) as shown in Table 1. The strain having an L-amino acid-producing ability superior to that of the W3110 control was selected as W3110-yccE 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，KCl 0.8g/L，MgSO ₄ ·7H ₂ O 0.8g/L，FeSO ₄ ·7H ₂ O 0.05g/L，MnSO ₄ ·H ₂ 0.05g/L of O, 1.5g/L of FM902 yeast powder, 5g/L of corn steep liquor, 17g/L of molasses, 0.5g/L of betaine, 2g/L of citric acid, 20mg/L of VH and VB ₁ 1.5mg/L，VB ₃ 1.5mg/LVB ₁₂ 1.5g/L, pH7.0 adjusted with sodium hydroxide.

TABLE 1 high Performance liquid chromatography L-amino acid analysis results of W3110-yccE mutants

As shown in Table 1, the E.coli W3110-yccE mutant strain of the present disclosure has an ability to produce partial L-amino acids, wherein the W3110-yccE mutant strain 2 is more superior in its ability to produce L-tyrosine. Indicating that yccE mutant 2 has activity in synthesizing L-tyrosine.

The result of sequencing yccE gene by extracting plasmid from W3110-yccE mutant strain 2 confirms that the 1243 rd cytosine (C) of yccE mutant nucleotide sequence is mutated into thymine (T), the 415 th arginine (R) of mutant protein yccE amino acid sequence is mutated into cysteine (C), the plasmid is pET28 (a) -R415C (the sequence is shown as SEQ ID No.3, the 1243 th arginine is mutated into C, wherein, the DNA sequence shown as SEQ ID No.1 is wild type yccE gene, the coding protein amino acid sequence is SEQ ID No.2 (the protein name is wild type yccE protein), the DNA sequence shown as SEQ ID No.5 is mutant yccE ^R415C Gene, the mutant yccE ^R415C The 1243 rd cytosine (C) in the gene sequence (SEQ ID No. 5) is mutated into thymine (T), and the amino acid sequence of the coding protein is SEQ ID No.6 (the name of the mutant protein is mutant yccE) ^R415C Protein), the mutant protein yccE e ^R415C The cysteine (C) at position 415 in the amino acid sequence (SEQ ID No. 6) was mutated from arginine (R).

3. Construction of yccE Gene mutant plasmid

The W3110-yccE mutant 2 was obtained by random mutagenesis using wild-type E.coli W3110. In order to obtain more yccE mutants to increase their L-tyrosine productivity, mutants were constructed in which the amino acid at the yccE mutation position was replaced with a different amino acid. Specifically, 5 mutants in which amino acid 415 of yccE was substituted with a different amino acid were constructed using the plasmid sequenced in step two as a template. 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 2yccE mutant substituted amino acids and primer names used in the respective mutants

G-PR-1:5'-Catttaaatagtcgagttgcgcatt-3'，

G-PR-2:5'-ttaaattgcataacCatttaaatag-3'，

S-PR-1:5'-Tatttaaatagtcgagttgcgcatt-3'，

S-PR-2:5'-ttaaattgcataacTatttaaatag-3'，

T-PR-1:5'-GTatttaaatagtcgagttgcgcat-3'，

T-PR-2:5'-ttaaattgcataaGTatttaaatag-3'，

Y-PR-1:5'-TAatttaaatagtcgagttgcgcat-3'，

Y-PR-2:5'-ttaaattgcataaTAatttaaatag-3'，

A-PR-1:5'-GCatttaaatagtcgagttgcgcat-3'，

A-PR-2:5'-ttaaattgcataaGCatttaaatag-3'，

wild Escherichia coli W3110 genome is used as template, primer and KAPA HiFi HotStart in Table 2 are respectively used for PCR amplification to obtain an Up DNA fragment 1243bp with yccE mutation base, and the amplified 1243bp fragment is used as template to amplify 1257bp fragment again by using primer. After the PCR reaction was completed, the column type DNA gel recovery kit was used to recover the 1257bpDNA fragment obtained by the second amplification by agarose gel electrophoresis. The recovered DNA fragment was ligated with the expression vector pET28 (a) recovered by EcoR I/Hind III digestion at 50 ℃ for 30min using NEBuilder enzyme (purchased from NEB Co., ltd.), and the ligation product was transformed into DH 5. Alpha. And plated on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃. The single clone grown out by culture is identified by PCR with a primer T7-F/T7-R, a positive transformant which is yccE mutant pET28 (a) and contains a 1616bp fragment is amplified by PCR with rTaq, and other four strains are constructed in the same way. The 5 ycc e mutant vectors in which the threonine at position 415 was substituted with each amino acid in table 2 were named as listed in table 2.

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

4. Construction of yccE mutant strains

In order to identify the L-amino acid productivity of the mutant vector constructed in step three, specifically, the plasmid constructed in step three is transformed into Escherichia coli W3110 strain (transformation and identification are performed in the same step one), positive transformants are respectively subcultured on 2-YT agar plates of kanamycin (50 mg/L) for three times in succession, inoculated into 500mL triangular flasks filled with 30mL of rich medium and subjected to shake flask fermentation at 37 ℃ for 24h, and the fermentation culture bacteria grow to OD ₆₀₀ And when the concentration is 0.1, IPTG is added to the concentration of 0.1mM to induce YCCE protein overexpression.

After completion of the fermentation culture, the concentration of L-amino acid was analyzed by High Performance Liquid Chromatography (HPLC), as shown in Table 3. Mutant W3110-pET28 (a) -yccE ^R415C 、W3110-pET28(a)-yccE ^R415G 、W3110-pET28(a)-yccE ^R415S 、W3110-pET28(a)-yccE ^R415T 、W W3110-pET28(a)-yccE ^R415Y And W3110-pET28 (a) -ycE ^R415A Can improve cheese qualityProduction of amino acid, wherein W3110-pET28 (a) -yccE ^R415C Increased yield, intended to be yccE ^R415C Mutants were further validated on engineered strain genomes.

TABLE 3 high Performance liquid chromatography of W3110-yccE mutants for L-tyrosine detection

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

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, point mutation is carried out on ycE genes (sequence confirmation shows that wild type yedZ genes are reserved on chromosomes of L-tyrosine production strains) of L-tyrosine high-yield strains CGMCC No.25231 and wild type W3110 by using a CRISPR/Cas9 gene editing technology, so that the influence of the ycE genes and mutant types on the L-tyrosine yield is further studied.

Introducing point mutation into a coding region (SEQ ID No. 1) of the yccE gene, wherein the point mutation is to mutate the 1243 rd cytosine (C) in the nucleotide sequence (SEQ ID No. 1) of the yccE gene into thymine (T) to obtain a DNA molecule (mutant yccE gene, named mutant yccE) shown in SEQ ID No.5 ^R415C A gene).

Wherein, the DNA molecule coding protein amino acid sequence shown in SEQ ID No.1 is SEQ ID No.2 (the protein name is wild type yccE 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 yccE) ^R415C Protein), the mutant protein yccE e ^R415C Arginine (R) 415 to cysteine (C) in the amino acid sequence (SEQ ID No. 6) was mutated.

1. Construction of sgRNA

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, the target sequence of sgRNA is designed by using CRISPR RGEN Tools (http:// www.rgeno.net/cas-designer /), after selecting the appropriate target sequence of sgRNA, the terminal sequence of linearized pGRB cloning vector (Addge company, cat # 71539) is added at the 5 'and 3' extreme ends of the target sequence, so as to form the complete sgRNA plasmid through 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 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, 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 for 30min at 50 ℃, converting the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The constructed plasmid was named pGRB-sgRNA-1.

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

sgRNA-1F:

5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTcacgtcggataggattaaagGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'

sgRNA-1R:

5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACctttaatcctatccgacgtgACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-ACTGGCACTGCTGTGCGCCG-3'

2. gene mutant yccE ^R415C Amplification of DNA

Using W3110 genome DNA as template, and primers P1/P2, P3/P4 and KAPA HiFi HotStart to perform PCR amplification to obtain two ycE with mutation base sizes of 1503bp and 331bp respectively ^R415C DNA fragment (yccE) ^R415C Up and ycE ^R415C Down). After the PCR reaction is finished, the column type DNA gel recovery kit is adopted to respectively carry out agarose gel electrophoresis and recover yccE ^R415C Up and yccE ^R415C And Down. The recovered DNA is subjected to point mutation integration homologous arm DNA fragment Up-yccE by using primer P1/P4 overlap PCR ^R415C -Down(SEQ ID No.17)1809bp。

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.

The primers were designed as follows (synthesized by Shanghai Invitrogen corporation), and the base in lower case and bold font is the mutation position:

P1:5'-AGCGTTATCTCGCGTAAATCAACAC-3'，

P2:5'-aATTTAAATAGTCGAGTTGCGCATT-3'，

P3:5'-AATGCGCAACTCGACTATTTAAATt-3'，

P4:5'-TCCTGGCACCTCTTTTGTTATCAAT-3'，

3. preparation and transformation of competence

pREDCAS9 plasmid (containing spectinomycin resistance gene, purchased from addrene, cat No. 371541) was extracted, transformed into L-tyrosine-producing bacteria CGMCC No.25231 and E.coli W3110 competent cells, plated on a 2-YT agar plate containing spectinomycin (100 mg/L) and cultured at 32 ℃, and a single colony of anti-spectinomycin (100 mg/L) was selected and identified by rTaq PCR using a primer pReddAS 9-PF/pReddAS 9-PR, to obtain 943bp (SEQ ID No. 18) transformants of L-tyrosine-producing bacteria YPTyr-Cas9 and W3110-Cas9 containing pREDCAS9 plasmid.

Preparing L-tyrosine bacteria YPTyr-Cas9 and W3110-Cas9 competent cells, and culturing until the bacteria grow 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 cell number is not less than 0.4, collecting the thallus to prepare competent cells, and transforming pGRB-sgRNA-1 plasmid and the point mutation recombinant DNA fragment Up-yccE ^R415C Down, spread on 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L), and incubated at 32 ℃ for 12h. 10 transformants were picked up and inoculated into 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% for culture to eliminate the plasmid pGRB-sgRNA-1, respectively; 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. 10 colonies which did not grow on spectinomycin (100 mg/L) but did not grow on 2-YT without antibody were selected, the transformants were identified by PCR using primers P5/P6 and rTaq, and the resulting 1856bp (SEQ ID No. 19) PCR product was subjected to SSCP (Single-Strand formation polynucleotides) electrophoresis (with Up-yce E) after denaturation at 95 ℃ for 10min and ice-bath for 5min ^R415C The Down amplified PCR fragment is used as a positive control, the W3110 amplified PCR fragment is used as a negative control, and water is used as a blank control). Sequencing and identifying the amplified sequence, comparing the sequencing result with the W3110 genome sequence, and determining that the yccE gene has the 1243 rd base C mutated into the base T and is the gene mutant yccE ^R415C Positive transformants. Gene-containing mutant yccE ^R415C The L-tyrosine-producing bacteria CGMCC No.25231 and Escherichia coli W3110 are respectively named YPTyr-yccE-001 and W3110-yccE-001.

P5:5'-tggcgtaataatcctttaattccat-3'，

P6:5'-cggcgattagcgttttgttcat-3'，

pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3'

pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3'

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

Preparation of SSCP electrophoretic PAGE and electrophoresis conditions: 8mL of 40% acrylamide, 4mL of glycerol, 2mL of 10 XTBE, 40. Mu.L of TEMED, 10% APS 600. Mu.L of ddH ₂ O26 mL; the electrophoresis chamber was placed in ice and electrophoresed at 120V in 1 XTBE buffer for 10h.

Example 3 construction of genomic overexpression of yccE and yccE ^R415C 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 yccE gene and mutant type yccE gene in the yaiT gene coding regions of L-tyrosine producer CGMCC No.25231 and Escherichia coli W3110 (through sequencing, the wild type yaiT and yccE genes are reserved on the chromosomes of the amino acid producer strains) ^R415C The gene, which can be used to further study yccE gene and mutant yccE ^R415C Influence of the genes on the amount of L-tyrosine synthesis.

1. Construction of sgRNA

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, a target sequence of sgRNA is designed by using CRISPR RGEN Tools (http:// www.rgeno.net/cas-designer /), and after selecting a proper target sequence of sgRNA, end sequences of a linearized pGRB cloning vector are added to the 5 'and 3' ends of the target sequence so as to form a complete sgRNA plasmid through 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 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 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 for 30min at 50 ℃, converting the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The constructed plasmid was named pGRB-sgRNA-2.

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

sgRNA-2F:

5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTggcaactatgtaaactatagGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'

sgRNA-2R:

5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACctatagtttacatagttgccACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-ACTGGCACTGCTGTGCGCCG-3'

2. PCR amplification of genomic over-expressed DNA sequences

Based on the genome sequence of Escherichia coli W3110 published by NCBI, four pairs of amplification upstream and downstream homology arm sequences and yccE or yccE are designed and synthesized ^R415C Primers of gene coding region and promoter region are respectively edited in coding regions of L-tyrosine producing bacteria CGMCC No.25231 and escherichia coli W3110 yaiT in a mode of CRISPR/Cas9 geneIntroduction of yccE Gene or yccE ^R415C A gene.

P7:5'-AAGAGAATGGAAGAGAGGCC-3'，

P8:5'-GTGTTGATTTACGCGAGATAACGCTCCCAATCAAGTGCTGTAACG-3'，

P9:5'-CGTTACAGCACTTGATTGGGAGCGTTATCTCGCGTAAATCAACAC-3'，

P10:5'-TAATTCCATGTATATTACTACCCATATATAGCGTCTATAAAATTTAATAA-3'，

P11:5'-TTATTAAATTTTATAGACGCTATATATGGGTAGTAATATACATGGAATTA-3'，

P12:5'-TGGTTTTTAGCATATGCCTTTGCCATTAAATTGCATAACGATTTAAATAG-3'，

P13:5'-CTATTTAAATCGTTATGCAATTTAATGGCAAAGGCATATGCTAAAAACCA-3'，

P14:5'-CGACCTGTAGTATCCCATTC-3',

P15:5'-GTTATCCCAAGTATGAGATTTCCTGGCACCTCTTTTGTTATCAAT-3'。

P16:5'-CAATGTTCAGCGAAGAACCGTTAG-3',

P17:5'-GCTTGCTTTAGATACCGACACGTC-3'

P18:5'-TGGTTTTTAGCATATGCCTTTGCCATTAAATTGCATAACAATTTAAATAG-3',

P19:5'-ATTGATAACAAAAGAGGTGCCAGGAAATCTCATACTTGGGATAAC-3',

P20:5'-CTATTTAAATTGTTATGCAATTTAATGGCAAAGGCATATGCTAAAAACCA-3',

using W3110 genome DNA as template, and primer P7/P8 and P19/P14 and KAPA HiFi HotStart PCR amplification to obtain 595bp (SEQ ID No. 201-595) of upper homology arm and 590bp (SEQ ID No. 202355-2944) of lower homology arm fragment; amplifying a yccE promoter fragment 280bp (SEQ ID No. 20551-855) by using W3110 genome DNA as a template and using a primer P9/P10 and a KAPA HiFi HotStart PCR; PCR amplifying yccE (SEQ ID No. 20806-2112) and ycER 415C gene (SEQ ID No. 21806-2112) 1307bp by using W3110 genome DNA and plasmid pET28 (a) -ycE R415C as templates and primers P11/P12, P11/P18 and KAPA HiFi HotStart respectively; amplification of yccE terminator fragment 337bp (SEQ ID N) by PCR with primers P13/P15 and KAPA HiFi HotStart using W3110 genomic DNA as templateo.202062-2399 bits); PCR amplification of yccE with the genomic DNA of W3110 as template and primers P20/P15 and KAPA HiFi HotStart ^R415C A terminator fragment of 337bp (SEQ ID No. 202062-2399). 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 is subjected to PCR by using primers P7 and P14 overlap to respectively obtain DNA recombinant fragments Up-yccE-Down (SEQ ID No. 20) and Up-ycE with genome over-expression ^R415C -Down(SEQ ID No.21)2944bp。

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 cell number is not less than 0.4, collecting the thallus to prepare competent cells, and respectively transforming pGRB-sgRNA-2 plasmid and genome over-expression DNA fragments Up-yccE-Down and Up-ycE ^R415C Down, plated on 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L) and cultured at 32 ℃. The single colony produced by the culture is identified by a primer P17/P16 through a Taq PCR, a fragment with the size of 1256bp (the sequence without point mutation is shown as SEQ ID No.22, the 275 th site of the sequence with the point mutation is C, and the rest is shown as SEQ ID No. 22) is amplified by the PCR to be a positive transformant, and the fragment which can not be amplified is the original bacterium.

The positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% to eliminate plasmid pGRB-sgRNA-2, colonies growing on spectinomycin (100 mg/L) but not growing on ampicillin (100 mg/L) were selected, these colonies were transferred to 2-YT medium at 42 ℃ to eliminate pREDCas9 plasmid, colonies not growing on spectinomycin (100 mg/L) but growing on nonresistant 2-YT were selected, PCR-amplified by using rTaq PCR using primers P17/P16 to identify fragments containing 1256bp in size (the sequence not containing point mutation is shown in SEQ ID No.22, the fragment containing point mutation sequence is C at position 275, and the rest is SEQ ID No. 22) as positive transformants and the fragment not amplified is the original bacterium.

Wild yccE gene and mutant yccE gene overexpressed by L-tyrosine producing strain CGMCC No.25231 genome ^R415C The genes were designated YPTyr-yccE-002 (without mutation points) and YPTyr-yccE-003 (with mutation points), respectively, while the wild-type yccE gene and the mutant yccE gene were overexpressed on the genome of the W3110 strain ^R415C The genes were designated W3110-yccE-002 (without mutation points) and W3110-yccE-003 (with mutation points), respectively.

The recombinant bacteria YPTyr-yccE-002 and W3110-yccE-002 contain double copies of yccE gene shown in SEQ ID No. 1; specifically, the recombinant bacteria YPTyr-yccE-002 and W3110-yccE-002 are recombinant bacteria obtained by replacing yaiT partial coding regions on the genomes of L-tyrosine producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 with ycE genes and promoters thereof and keeping other nucleotides in the genomes unchanged. The recombinant bacteria containing double copies of yccE gene can obviously and stably improve the expression quantity of the yccE gene.

Recombinant bacteria YPTyr-yccE-003 and W3110-yccE-003 contain mutated yccE shown in SEQ ID No.3 ^R415C A gene; specifically, recombinant bacteria YPTyr-yccE-003 and W3110-yccE-003 are obtained by replacing yaiT partial coding regions on genomes of L-tyrosine-producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 with mutant ycE ^R415C Gene and its promoter, and recombinant bacterium obtained by keeping other nucleotides in its genome unchanged. Containing two copies of yccE ^R415C The recombinant strain of the gene can obviously and stably improve the expression quantity of the yccE gene.

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.

Example 4 construction of engineered strains with deletions of yccE Gene on the genome

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

1. Construction of sgRNA

According to the genome sequence of Escherichia coli (Escherichia coli) W3110 published by NCBI, a target sequence of sgRNA is designed by using CRISPR RGEN Tools (http:// www.rgeno.net/cas-designer /), and after selecting a proper target sequence of sgRNA, a linearized pGRB cloning vector homologous arm sequence is added to the 5 'and 3' extreme ends of the target sequence so as to form a complete sgRNA plasmid through recombination.

The sgRNA fragment is amplified without a template and only by carrying out a PCR annealing process, and the system and the procedure 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 measured, 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 dephosphorizing reaction, and carrying out dephosphorizing reaction: 10xBuffer 5. Mu.L, linearized 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.). And (3) 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 for 30min at 50 ℃, converting the product into DH5 alpha competent cells, extracting plasmids, and sequencing and identifying by using a sequencing primer sgRNA-PF/sgRNA-PR. The constructed plasmid was named pGRB-sgRNA-3.

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-3F:

5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTttaaaatatgctcctgtagaGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'

sgRNA-3R:

5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACtctacaggagcatattttaaACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'’

sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3'

sgRNA-PR:5'-GCGTCAGGTGCATAAACAGA-3'

2. PCR amplification of recombinant DNA fragments deleted from the genome

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 an yccE gene in L-tyrosine producing bacteria and wild Escherichia coli W3110 genes is knocked out in a CRISPR/Cas9 gene editing mode.

P21:5'-aacacttcccaggcttcagc-3'，

P22:5'-gagcaaaaagttcaggagggaggtatgtgttttcaaaaagcgtgattgaa-3'，

P23:5'-ttcaatcacgctttttgaaaacacatacctccctcctgaactttttgctc-3'，

P24:5'-gctcatcatgagcacttgctgtag-3'。

P25:5'-cttcggcgttaaaactctgaccg-3'

P26:5'-cctttggtggtgagttgcc-3'

taking W3110 genome DNA as a template, and performing PCR amplification by using primers P21/P22, P23/P24 and KAPA HiFi HotStart to obtain fragments of an upper homologous arm and a lower homologous arm with the sizes of 575bp and 524bp 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 PCR with primer P21/P24 overlap to obtain a recombinant DNA fragment Δ yccE-Up-Dwon (SEQ ID No. 23111-1159) lacking yccE on 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 less than 0.4, competent cells were prepared from collected cells, and the pGRB-sgRNA-3 plasmid and the recombinant DNA fragment Δ yccE-Up-Down of genomic deletion ycE were transformed, respectively, and plated on a 2-YT agar plate containing spectinomycin (100 mg/L) and ampicillin (100 mg/L) for culture at 32 ℃. PCR identification is carried out on the single colony generated by culture by using a primer P26/P27 and a primer rTaq, a positive transformant containing a 1269bp (SEQ ID No. 23) fragment is amplified by PCR, and a raw bacterium containing a 2310bp fragment is amplified.

Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg/L) and arabinose to a final concentration of 0.2% to eliminate plasmid pGRB-sgRNA-3, colonies growing on spectinomycin (100 mg/L) but not on ampicillin (100 mg/L) were selected, these colonies were transferred to 2-YT medium at 42 ℃ to eliminate pREDCas9 plasmid, colonies not growing on spectinomycin (100 mg/L) but growing on non-resistant 2-YT were selected, and PCR-amplified to positive transformants having a size of 1269bp (SEQ ID No. 23) again by using rTaq PCR identification using primers P26/P27.

Positive transformants with deletion of yccE gene on the genomes of L-tyrosine-producing bacteria CGMCC No.25231 and W3110 strains are named YPTyr-yccE-004 and W3110-yccE-004 respectively.

Recombinant bacteria YPTyr-yccE-004 and W3110-yccE-004 contain deleted yccE genes; specifically, the recombinant bacteria YPTyr-yccE-004 and W3110-yccE-004 are recombinant bacteria obtained by knocking out partial coding regions of yccE genes on genomes of L-tyrosine producing bacteria CGMCC No.25231 and wild type Escherichia coli W3110 and keeping other nucleotides in the genomes unchanged.

Example 5 overexpression of yccE or yccE on a construction plasmid ^R415C Engineered strains of genes

Wild-type yccE gene and mutant yccE gene were introduced into an E.coli expression vector pET28 (a) (purchased from TaKaRa, containing kanamycin resistance) according to the genomic sequence of E.coli (Escherichia coli) W3110 published by NCBI ^R415C The gene coding region and the promoter region are introduced into L-tyrosine production bacteria CGMCC No.25231 and wild type Escherichia coli W3110 (the wild type yccE gene is reserved on chromosomes of the amino acid production strains confirmed by sequencing), so that the multi-copy yccE gene and the mutant yccE gene can be further studied ^R415C Influence of the Gene on the production of L-tyrosine.

Preparing L-tyrosine CGMCC No.25231 and wild type W3110 competent cells. When OD is reached ₆₀₀ When =0.6, competent cells were prepared by collecting the cells and transforming the plasmids pET28 (a) -yccE and pET28 (a) -yccE constructed in example 1, respectively ^R415C Spread on 2-YT agar plates containing kanamycin (50 mg/L) and cultured at 37 ℃. The single colony generated by the culture is identified by a primer T7-F/T7-R and rTaq PCR, a positive transformant containing a fragment with the size of 1616bp (a sequence without point mutation is shown as SEQ ID No.4, the 1420 th position of the sequence containing the point mutation is T, and the rest is shown as SEQ ID No. 4) is amplified by the PCR, and the original bacterium without the amplified fragment is obtained.

The L-tyrosine production bacteria CGMCCNo.25231 and wild-type E.coli W3110 plasmids overexpressed wild-type yccE gene and mutant yccE ^R415C The genes were named YPTyr-yccE-005 (without mutation point), YPTyr-yccE-006 (with mutation point), W3110-yccE-005 (without mutation point) and W3110-yccE-006 (with mutation point), respectively.

The recombinant bacteria YPTyr-yccE-005 and W3110-yccE-005 contain an yccE gene which is expressed by pET28 (a) -yccE and is shown in SEQ ID No.1, and the genome sequence of the recombinant bacteria is kept unchanged to obtain the recombinant bacteria. The recombinant bacteria of pET28 (a) -yccE overexpression can obviously and stably improve the expression quantity of the yccE gene.

The recombinant bacteria YPTyr-yccE-006 and W3110-yccE-006 contain pET28 (a) -yccE ^R415C Overexpressed mutant yccE represented by SEQ ID No.5 ^R415C Gene, and recombinant bacteria obtained by keeping the genome sequence unchanged. pET28 (a) -yccE ^R415C The recombinant strain with over-expression can obviously and stably improve yccE ^R415C The expression level of the gene.

Example 6 fermentation experiment

1. L-tyrosine fermentation experiments

The strain constructed in the above example, the tyrosine-producing strain CGMCC No.25231 and the wild type Escherichia coli W3110 were inoculated into a 5L fermentor (Lobarland Biotech Co., ltd.) of BLBIO-5GC-4-H type, respectively, and fermentation experiments were carried out using L-tyrosine fermentation medium and culture conditions, and each strain was repeated three times. YPTyr-yccE-005, W3110-yccE-005, YPTyr-yccE-006 and W3110-ycE-006 are strains containing pET28 (a) over-expression, IPTG induction is needed 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 the yccE protein over-expression. 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 4.

L-tyrosine fermentation medium: the solvent is water, the solute and the concentration thereof are 4g/L of yeast extract powder, 2g/L of corn steep liquor dry powder, 4g/L of peptone, 2g/L of methionine and KH ₂ PO ₄ ·3H ₂ O 7g/L，MgSO ₄ ·7H ₂ O 2g/L，CoCl ₂ 20mg/L，

(NH ₄ ) ₂ SO ₄ 3g/L, citric acid 2g/L, feSO ₄ ·7H ₂ O 50mg/L，MnSO ₄ ·7H ₂ O 30mg/L，VH 20mg/L，VB ₁ 1.5mg/L，VB ₃ 1.5mg/L VB ₁₂ 1.5g/L, antifoam 0.3mL/L, (NH 4) ₂ SO ₄ 3g/L and pH value of 7.0.

L-tyrosine fermentation culture conditions: the calibration method of the dissolved oxygen electrode comprises the following steps: calibrating a zero point in a saturated sodium sulfite solution, and calibrating a hundred points in air;

the L-tyrosine fermentation comprises two-stage aerobic-oxygen-limited fermentation, the cells are firstly cultured under the aerobic fermentation, the air quantity rotating speed and the sugar supplement speed are adjusted in the early stage to control the dissolved oxygen to be about 25 percent, and the OD is waited ₆₀₀ When the value is 50-60, the rotating speed is reduced to 400rpm, and the air volume is reduced to 2L/min; and converting the aerobic fermentation into oxygen-limited fermentation.

TABLE 4L-tyrosine production and significance analysis of yccE engineered strains

As shown by the above fermentation results, the substitution of the 415 th arginine in the amino acid sequence of yccE gene by aspartic acid contributes to the improvement of L-tyrosine yield both for the high-yielding L-tyrosine strain and the model strain W3110; wild type yccE gene and mutant yccE for high L-tyrosine-producing strains ^R415C The overexpression of (a) all contributes to the improvement of the L-tyrosine yield, while the knockout of the yccE gene is not beneficial to the improvement of the L-tyrosine yield.

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:

2. The protein of claim 1, wherein said protein is an yccE protein or wherein the arginine residue at position 415 of the amino acid sequence of said yccE protein is replaced with any one of;

3. A biomaterial, characterized in that it 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);

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

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;

z3) the coding sequence is a DNA molecule shown in SEQ ID No. 7;

z4) the coding sequence is a DNA molecule shown in SEQ ID No. 9;

z5) the coding sequence is a DNA molecule shown in SEQ ID No. 11;

z6) the coding sequence is a DNA molecule shown in SEQ ID No. 13;

z7) the coding sequence is a DNA molecule shown in SEQ ID No. 15;

z8) the nucleotide sequence is a DNA molecule shown as 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 shown as SEQ ID No. 7;

z11) the nucleotide sequence is a DNA molecule shown in SEQ ID No. 9;

z12) the nucleotide sequence is a DNA molecule shown as SEQ ID No. 11;

z13) the nucleotide sequence is a DNA molecule shown as SEQ ID No. 13;

z14) nucleotide sequence is a DNA molecule shown in SEQ ID No. 15.

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 Protein according to claim 1 or 2, which is a protein according to claim 1 or 2:

c2 Substances which regulate the expression of genes encoding said proteins;

c3 Substances that regulate the activity or content of the protein.

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 C1);

d5 A nucleic acid molecule for inhibiting or reducing or down-regulating 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 an amino acid in a microorganism, which comprises controlling the production of an amino acid in 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 amino acid 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 a gene encoding the protein according to claim 1 or 2 into the microorganism of interest;

e3 Knock-out or down-regulate or attenuate or reduce the expression of the gene encoding E2) in the microorganism of interest.

10. The method according to claim 7 or 8, wherein the regulation of the activity or content of the protein according to claim 1 or 2 in the microorganism of interest is achieved by mutating the yccE gene in the genome of said microorganism of interest by changing the codon for arginine at position 415 of the amino acid sequence encoded by said yccE gene to a cysteine codon; the yccE 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 on 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 L-tyrosine.

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 protein according to claim 1 or 2, the biological material according to claim 3 or 4, the use according to claim 5 or 6, the method according to any one of claims 6 to 11, wherein the microorganism is any one of the following:

c1 Bacteria kingdom);

c2 Enterobacteriaceae;

c3 Escherichia coli;

c4 Coli).