CN116478976A - yejG gene and mutant thereof and application of yejG gene and mutant thereof in preparation of amino acid - Google Patents

yejG gene and mutant thereof and application of yejG gene and mutant thereof in preparation of amino acid Download PDF

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Publication number
CN116478976A
CN116478976A CN202310297416.3A CN202310297416A CN116478976A CN 116478976 A CN116478976 A CN 116478976A CN 202310297416 A CN202310297416 A CN 202310297416A CN 116478976 A CN116478976 A CN 116478976A
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ycjg
protein
recombinant
amino acid
microorganism
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孟刚
赵春光
魏爱英
苏厚波
田斌
张晓琴
张英
王吉玮
马聪玲
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Ningxia Eppen Biotech Co ltd
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Ningxia Eppen Biotech Co ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/01—Racemaces and epimerases (5.1) acting on amino acids and derivatives (5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The invention discloses a yejG gene and a mutant thereof and application thereof in preparing amino acid. The present invention provides ycjG W96 Proteins (obtained by mutating amino acid residue 96 of ycjG protein from W to other amino acid residues) or comprising ycjG W96 Fusion proteins of the proteins, and related biological materials are also provided. ycjG W96 Use of a protein, fusion protein or said biological material: use in increasing amino acid production by a microorganism or cell; use in the production of amino acids; use in the construction of recombinant microorganisms or recombinant cells for the production of amino acids. The invention obtains various mutant forms of ycjG through point mutation W96 Proteins that function better than the ycjG protein. The invention obtains the ycjG with optimal function through saturation mutation W96L And (3) protein. The invention prepares engineering bacteria with various mutant proteins, and compared with starting bacteria, the engineering bacteria has obviously improved capability of producing amino acid. The invention has great application value for the industrial production of amino acid or upstream and downstream products thereof.

Description

yejG gene and mutant thereof and application of yejG gene and mutant thereof in preparation of amino acid
Technical Field
The invention belongs to the field of biotechnology, and relates to a yejG gene, a mutant thereof and application thereof in preparing amino acids (such as L-alanine, L-valine, L-arginine, L-threonine, L-tryptophan and the like).
Background
L-amino acids have been used in the animal feed, pharmaceutical and cosmetic industries. The microbial fermentation method for producing L-amino acid is the most widely used amino acid production method at present, and the fermentation production performance of amino acid production bacteria is a key factor influencing whether the fermentation method can realize large-scale industrial application. At present, few amino acid varieties are not produced by a fermentation method due to the lack of a production strain with excellent fermentation performance. For the amino acid production strain which has been produced by fermentation, the acid production level and the sugar acid conversion rate thereof have yet to be further improved in order to save production costs.
The excellent production strain is a guarantee for improving the output and quality of amino acid. With the development of recombinant DNA technology and the acquisition of related microbial genome information, genetic engineering breeding technology based on the principle of metabolic engineering is becoming the mainstream. The metabolic pathway and metabolic network of the microorganism are purposefully modified, the metabolic regulation mechanism of the strain is artificially changed, the metabolic flow in the microorganism is carried out according to the required direction, the amino acid is excessively accumulated, the yield of the amino acid is greatly improved, the cost is reduced, and the method has important significance for accelerating the process of L-amino acid industrialization.
The protein encoded by ycjG has the function of degrading peptidoglycan to supplement nitrogen source against nitrogen source starvation environment, and there is no research on the function of ycjG in L-amino acid production.
Disclosure of Invention
The invention aims to provide yejG gene and mutant thereof and application thereof in preparing amino acid.
The present invention provides ycjG W96 Proteins or comprise ycjG W96 Fusion proteins of proteins;
the ycjG W96 The protein is a protein, and is 96 th amino acid of ycjG proteinResidues are obtained by mutating W to other amino acid residues.
The ycjG protein is (a 1) or (a 2) or (a 3) as follows:
(a1) Comprising SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no;
(a2) A protein derived from a microorganism or a cell and having 80% or more identity to (a 1) and being related to amino acid production;
(a3) And (b) a protein derived from (a 1) and related to amino acid production, wherein the protein shown in (a 1) is obtained by substitution and/or deletion and/or addition of one or more amino acid residues.
"comprising SEQ ID NO:2, "in particular" the protein of the amino acid sequence shown in SEQ ID NO:2, and a protein represented by formula 2.
Specifically, the ycjG W96 The protein is (b 1) or (b 2) or (b 3) or (b 4) or (b 5) or (b 6) or (b 7) or (b 8) as follows:
(b1) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to L (ycjG W96L A protein);
(b2) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to P (ycjG W96P A protein);
(b3) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to R (ycjG W96R A protein);
(b4) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to S (ycjG W96S A protein);
(b5) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to F (ycjG W96F A protein);
(b6) Protein obtained by mutating the 96 th amino acid residue of the ycjG protein from W to T (ycjG W96T A protein);
(b7) A protein derived from a microorganism or a cell and having 80% or more identity to (b 1) or (b 2) or (b 3) or (b 4) or (b 5) or (b 6) and being related to amino acid production;
(b8) And (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein shown in (b 1) or (b 2) or (b 3) or (b 4) or (b 5) or (b 6) and is related to amino acid production.
The term "identity" as used in reference to amino acid sequences refers to sequence similarity to the native amino acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences. Any of the above 80% identity may specifically be 85% identity or more, 90% identity or more, 95% identity or more, 96% identity or more, 97% identity or more, 98% identity or more, 99% identity or more, or 99.5% identity or more.
The invention also provides related biological materials, which are (c 1) or (c 2) or (c 3) or (c 4) or (c 5) or (c 6) as follows:
(c1) Encoding the ycjG W96 A nucleic acid molecule of a protein;
(c2) A nucleic acid molecule encoding the fusion protein;
(c3) An expression cassette having the nucleic acid molecule of (c 1) or (c 2);
(c4) A recombinant vector having the nucleic acid molecule of (c 1) or (c 2);
(c5) A recombinant microorganism having the nucleic acid molecule of (c 1) or (c 2);
(c6) A recombinant cell having the nucleic acid molecule of (c 1) or (c 2).
Comprising ycjG W96 Fusion proteins of proteins other than ycjG W96 The protein has other amino acid residues.
The additional amino acid residues may constitute additional functional segments, such as protein tags, additional functional proteins, connecting peptides, and the like.
The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag, etc.
EncodingycjG W96 The nucleic acid molecule of the protein may specifically be a nucleic acid molecule obtained by subjecting a nucleic acid molecule encoding an ycjG protein to a codon mutation.
The nucleic acid molecule encoding the ycjG protein is (d 1) or (d 2) or (d 3) as follows:
(d1) A DNA molecule with a coding region shown as SEQ ID NO. 1;
(d2) A DNA molecule derived from a microorganism or cell and having more than 80% identity to (d 1) and encoding said protein;
(d3) A DNA molecule which hybridizes under stringent conditions to (d 1) and which encodes said protein.
The term "identity" as used in reference to nucleotide sequences refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences. Any of the above 80% identity may specifically be 85% identity or more, 90% identity or more, 95% identity or more, 96% identity or more, 97% identity or more, 98% identity or more, 99% identity or more, or 99.5% identity or more.
The stringent conditions may be hybridization and washing of the membrane in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.
Illustratively, the codon usage may be specifically any of the following: setting SEQ ID NO:1 from TGG to TTG or CCG or CGG or TCG or ACG or TTC.
The present invention also protects the ycjG W96 The application of the protein, the fusion protein or the biological material is as follows (I) or (II) or (III):
use of (i) to increase the amino acid production of a microorganism or cell;
(II) use in the production of amino acids;
(III) use in the construction of recombinant microorganisms or recombinant cells for the production of amino acids.
The invention also provides the use of specific substances, as follows (I) or (II) or (III):
use of (i) to increase the amino acid production of a microorganism or cell;
(II) use in the production of amino acids;
(III) use in the construction of recombinant microorganisms or recombinant cells for the production of amino acids;
the specific substance comprises the following (d 1), (d 2) or (d 3):
(d1) A substance for increasing expression of a nucleic acid molecule encoding a protein of interest;
(d2) A substance for increasing the abundance of a target protein;
(d3) A substance for improving the activity of a target protein.
The invention also provides a recombinant microorganism or recombinant cell obtained by overexpressing a nucleic acid molecule encoding a protein of interest in the starting microorganism or starting cell.
Expression regulatory sequences of the polynucleotides may be modified. Expression regulatory sequences control the expression of a polynucleotide to which they are operably linked and may include, for example, promoters, terminators, enhancers, silencers, and the like. The polynucleotide may have a change in the initiation codon. Polynucleotides may be incorporated into specific sites of the chromosome, thereby increasing copy number. Herein, a specific site may include, for example, a transposon site or an intergenic site. Alternatively, the polynucleotide may be incorporated into an expression vector, which is introduced into a host cell or into a starting bacterium, thereby increasing the copy number.
In one embodiment of the invention, the copy number is increased by incorporating a nucleic acid molecule encoding a protein of interest into a specific site of a chromosome of a microorganism or cell.
In one embodiment of the invention, the nucleic acid sequence is overexpressed by incorporating a nucleic acid molecule encoding the protein of interest with a promoter sequence into a specific site of the chromosome of the microorganism or cell.
In one embodiment of the invention, the nucleic acid molecule encoding the protein of interest is incorporated into an expression vector, which is introduced into a microorganism or cell, thereby increasing the copy number.
In one embodiment of the invention, a nucleic acid molecule encoding a protein of interest with a promoter sequence is incorporated into an expression vector, which is introduced into a microorganism or cell, whereby the nucleic acid sequence is overexpressed.
As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a polynucleotide sequence whereby the regulatory sequence controls transcription and/or translation of the polynucleotide sequence. The regulatory sequence may be a strong promoter capable of increasing the expression level of the polynucleotide. The regulatory sequence may be a promoter derived from a microorganism belonging to the genus Corynebacterium or may be a promoter derived from other microorganisms. For example, the promoter may be a trc promoter, a gap promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, an araBAD promoter or a cj7 promoter.
As used herein, the term "vector" refers to a polynucleotide construct containing regulatory sequences and gene sequences of a gene and configured to express a target gene in a suitable host cell. Alternatively, a vector may in turn refer to a polynucleotide construct containing sequences that can be used for homologous recombination, whereby due to the vector introduced into the host cell, the regulatory sequences of endogenous genes in the genome of the host cell can be altered, or the target gene that can be expressed can be inserted into a specific site in the genome of the host. In this regard, the vector used in the present invention may further comprise a selectable marker to determine the introduction of the vector into a host cell or the insertion of the vector into a chromosome of the host cell. The selectable marker may comprise a marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein. In an environment treated with such a selection agent, transformed cells may be selected because only cells expressing the selection marker may survive or exhibit a different phenotypic trait.
In some embodiments of the invention, the vector used is a pET28a (+) vector.
Illustratively, the recombinant microorganism may be each recombinant bacterium prepared in the examples.
The invention also protects Escherichia coli CGMCC26289, which is totally called Escherichia coli YP007-1 and is preserved in China general microbiological culture Collection center (CGMCC for short, address: north Xielu No. 1, 3 of the Beijing Korea, university of China institute of microorganisms) at the month of 12 and 26 of 2022, and the preservation registration number is CGMCC No.26289.
The invention also protects the application of the recombinant microorganism or the recombinant cell or the escherichia coli CGMCC26289 in the preparation of amino acid.
When the recombinant microorganism is used for preparing amino acid, the specific method comprises the following steps: fermenting the recombinant microorganism.
The person skilled in the art can carry out the fermentation culture by fermentation methods known in the art. Optimization and improvement of the fermentation process can also be carried out by routine experimentation. The fermentation of the microorganism may be performed in a suitable medium under fermentation conditions known in the art. The medium may comprise: carbon source, nitrogen source, trace elements, and combinations thereof. During the culture, the pH of the culture may be adjusted. In addition, the culture may include prevention of bubble generation, for example, by using an antifoaming agent. In addition, a gas may be injected into the culture during the cultivation. The gas may comprise any gas capable of maintaining aerobic conditions of the culture.
In the culture, the temperature of the culture may be 20-45 ℃ (e.g., 35 ℃ -37 ℃).
The method may further comprise the steps of: amino acids are obtained from the culture. The amino acid may be obtained from the culture by a variety of means including, but not limited to: the culture is treated with sulfuric acid or hydrochloric acid or the like, followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.
Illustratively, the recombinant microorganism is inoculated at 10% during fermentation culture.
Illustratively, the fermentation medium is an L-alanine fermentation medium (see examples for illustrative formulations).
Illustratively, the fermentation medium is an L-tryptophan fermentation medium (see examples for illustrative formulations).
Illustratively, the fermentation medium is an L-arginine fermentation medium (see examples for illustrative formulations).
Illustratively, the fermentation medium is an L-threonine fermentation medium (see examples for exemplary formulations).
Illustratively, the fermentation medium is L-valine fermentation medium (see examples for illustrative formulations).
For example, the culture conditions for fermentation culture are described in the examples.
The invention also provides a method for increasing the amino acid yield of a microorganism or cell, comprising the steps of: replacement of a nucleic acid molecule encoding an ycjG protein in the genome of a microorganism or cell with a nucleic acid molecule encoding ycjG W96 A nucleic acid molecule encoding a protein, or a nucleic acid molecule encoding an ycjG protein in the genome of a microorganism or cell is replaced with a nucleic acid molecule encoding a fusion protein.
The invention also provides a method for increasing the amino acid yield of a microorganism or cell, comprising the steps of: over-expressing a nucleic acid molecule encoding a protein of interest in a microorganism or cell, or increasing the abundance of the protein of interest in a microorganism or cell, or increasing the activity of the protein of interest in a microorganism or cell.
The invention also protects the application of the target protein in regulating and controlling the amino acid yield of microorganisms or cells.
The regulation is positive regulation, namely, the content of target protein is increased, and the amino acid yield of microorganisms or cells is increased.
Any one of the above proteins of interest is the ycjG W96 A protein, said fusion protein or said ycjG protein.
Any of the above amino acids may also be replaced with shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, etc.
The amino acids include, but are not limited to, any one of the following or a combination of any two or more of the following: glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, valine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, lysine, glutamic acid, ornithine, citrulline. Any of the above amino acids may be an L-amino acid.
Any of the above cells may be plant cells or animal cells.
Any of the above microorganisms may be bacteria, algae or fungi. In particular, the fungus may be a yeast.
Any of the above bacteria may be Escherichia coli, corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium lactofermentum, brevibacterium flavum (brevibacterium flavum), corynebacterium beijing (Corynebacterium pekinense), brevibacterium ammoniaphaga, corynebacterium crenatum or Pantoea (Pantoea), etc.
The Escherichia coli can be Escherichia coli CGMCC26289, escherichia coli CGMCC22721, escherichia coli CGMCC25402, escherichia coli CGMCC25404, escherichia coli CGMCC25403, escherichia coli MG1655 or Escherichia coli W3110.
The invention obtains various mutant forms of ycjG through point mutation W96 Proteins that function better than the ycjG protein. Furthermore, the invention obtains the ycjG with optimal function through saturation mutation W96L And (3) protein. Furthermore, the engineering bacteria with various mutant proteins are prepared, and compared with the starting bacteria, the capability of the engineering bacteria for producing amino acids is obviously improved. The invention has great application value for the industrial production of amino acid or upstream and downstream products thereof.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. pGRB plasmid (ampicillin resistance gene contained in this plasmid): adedge corporation, cat# 71539.pREDCas9 plasmid (containing the spectinomycin resistance gene): adedge corporation, cat# 71541.2-YT medium (pH 7.0): 16g/L peptone, 10g/L yeast powder, 5g/L sodium chloride and the balance of water.
The ycjG protein, which is known as L-alanine-D (L) -glutamate differential enzyme. The ycjG gene, i.e. the gene encoding the ycjG protein. The ycjG protein from Escherichia coli (Escherichia coli) is shown as SEQ ID NO:2 (called wild type ycjG protein), the ycjG gene is shown as SEQ ID NO:1 (referred to as wild-type ycjG gene). Through sequencing, the genome DNA of the escherichia coli MG1655 and the escherichia coli CGMCC26289 both have wild ycjG genes.
The Escherichia coli CGMCC26289, which is totally called Escherichia coli YP007-1, is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (address: beicheng Kogyo Mitsui No. 1, no. 3, china academy of sciences microbiological culture Collection center) at 12 months and 26 days of 2022, and has a preservation registration number of CGMCC No.26289. The escherichia coli CGMCC26289 is an L-alanine producing strain.
The Escherichia coli CGMCC22721, which is called Escherichia coli (Escherichia coli) YP045, is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (address: north Chen West road No. 1, 3, china academy of sciences microbiological study, gmbH) of Beijing, and has a preservation registration number of CGMCC No.22721. The escherichia coli CGMCC22721 is L-valine producing bacterium.
The Escherichia coli CGMCC25402, which is totally called Escherichia coli (Escherichia coli) YP004-8, is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (address: beicheng Kogyo North Xiyang No. 1, national institute of microorganisms, national academy of sciences of China) for 2022, and has a preservation registration number of CGMCC No.25402. Coli CGMCC25402 is L-arginine producing strain.
The Escherichia coli CGMCC25404, which is totally called Escherichia coli (Escherichia coli) YP0158, is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (address: north Chen West road No. 1, 3 of Beijing area, chachiensis university microbiological institute of China) in 2022, and has a preservation registration number of CGMCC No.25404. Coli CGMCC25404 is L-threonine producing bacterium.
The Escherichia coli CGMCC25403, which is called Escherichia coli YP006D, is preserved in China general microbiological culture Collection center (CGMCC) for 25 days in 2022, namely, the North Chen Xiyu No. 1, the North Xiyu No. 3 of the Beijing Korea, and the China academy of sciences microbiological study, and the preservation registration number is CGMCC No.25403. The escherichia coli CGMCC25403 is an L-tryptophan-producing strain.
Example 1, acquisition of muteins and genes encoding them
1. Construction of recombinant plasmid expressing wild-type ycjG Gene
1. The genome DNA of the escherichia coli MG1655 is used as a template, and a primer pair consisting of pET28-PF and pET28-PR is adopted for PCR amplification, and an amplification product is recovered. Sequencing, the amplified product is shown as SEQ ID NO: 3.
pET28-PF：5'-ACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCGATTATCTTTCCTGTTTAC-3'；
pET28-PR：5'-GGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTCTAAAGATGCAATTCGCCCGTCG-3'。
In the primer, the underlined part is the homology arm for recombination into the pET28a (+) vector.
SEQ ID NO:3, nucleotides 37 to 154 constitute the ycjG promoter and nucleotides 155 to 1120 constitute the wild-type ycjG gene.
2. The pET28a (+) vector was digested with restriction enzymes EcoRI and HindIII, and a large linear fragment of about 5.3kb was recovered.
3. And (3) carrying out homologous recombination on the amplification product recovered in the step (1) and the linear large fragment recovered in the step (2) to obtain a recombinant plasmid pET28 (a) -ycjG. And (5) sequencing and verifying the recombinant plasmid. Sequencing results showed that the recombinant plasmid pET28 (a) -ycjG only differed in the substitution of a small fragment between EcoR I and Hind III cleavage recognition sequences to SEQ ID NO:3, from positions 37 to 1120.
2. Preparation of plasmids expressing the respective mutants
The recombinant plasmid pET28 (a) -ycjG is used as a template, and a random mutagenesis kit (Agilent Technologies, USA) is adopted to introduce random point mutation into the ycjG gene, so that each mutant plasmid is obtained.
3. Preparation of recombinant bacteria and detection of L-amino acid production
The plasmids tested were: recombinant plasmid pET28 (a) -ycjG or individual mutant plasmids.
The test plasmids were each subjected to the following procedure:
1. the test plasmid was introduced into E.coli MG1655 to obtain a recombinant bacterium. Coli MG1655 was used as a starting control strain for the recombinant bacteria.
2. Inoculating the recombinant strain obtained in step 1 into 500mL triangular flask containing 30mL of rich culture medium, shake culturing at 37deg.C and 200rpm to OD 600nm ＝0.2。
3. After completion of step 2, IPTG was added and the mixture was cultured at 37℃and 200rpm with shaking at a concentration of 0.1mM in the system for 30 hours.
The rich medium consisted of additives and water, pH 7.0. The additives and the additive amounts in each liter of rich medium are as follows: glucose 30g, (NH) 4 ) 2 SO 4 2g、H 3 PO 4 0.5g、KCl 0.8g、MgSO 4 ·7H 2 O 0.8g、FeSO 4 ·7H 2 O 0.05g、MnSO 4 ·H 2 0.05g of O, 1.5g of FM902 yeast powder, 5g of corn steep liquor, 17g of molasses, 0.5g of betaine, 2g of citric acid, 20mg of vitamin H and 20mg of vitamin B 1 1.5mg, vitamin B 3 1.5mg, vitamin B 12 1.5mg。
4. After the completion of step 3, the concentration of each L-amino acid in the fermentation system was detected by High Performance Liquid Chromatography (HPLC).
The results show that the yield of the L-alanine of the partially recombinant bacterium is higher than that of the Escherichia coli MG1655.
The results of exemplary partially recombinant bacteria are shown in Table 1. Recombinant 0 represents the result of the above procedure for recombinant plasmid pET28 (a) -ycjG, and recombinant 1 to recombinant 5 represent the result of the above procedure for the different mutant plasmids.
TABLE 1 analytical results of high performance liquid chromatography for detecting L-amino acids
5. According to the result of the step 4, selecting the recombinant bacterium with the highest L-alanine yield, namely recombinant bacterium 2, extracting plasmids and sequencing. According to the sequencing result, 1 mutant protein and corresponding coding gene thereof are obtained. The mutein was designated ycjG W96L Protein, its coding gene is named ycjG W96L And (3) a gene.
6. According to ycjG W96L The mutation site of the protein designs another 5 mutation forms, and 5 kinds of mutant proteins and corresponding coding genes are obtained.
1 mutein of step 5 plus 5 muteins of step 6, totaling 6 muteins. Relative to SEQ ID NO:2, the 6 mutant proteins were mutated at the 96 th amino acid residue (no other amino acid residues were changed), and the specific mutated forms are shown in Table 2. Relative to SEQ ID NO:1, the coding genes of the 6 mutant proteins were mutated (no other nucleotide changes) at the position of the codon encoding the 96 th amino acid residue, and the specific mutated forms are shown in Table 2.
TABLE 2
EXAMPLE 2 construction of recombinant bacteria and detection of L-amino acid production
1. Construction of expression plasmids
6 recombinant plasmids were constructed separately for expression of the 6 muteins in Table 2.
The 6 recombinant plasmids differ only in the sequence set forth in SEQ ID NO:1, and a specific mutation form is shown in Table 2.
According to the principle corresponding to the mutant protein name, the 6 recombinant plasmids are named as recombinant plasmids pET28 (a) -W96L, recombinant plasmids pET28 (a) -W96P, recombinant plasmids pET28 (a) -W96R, recombinant plasmids pET28 (a) -W96S, recombinant plasmids pET28 (a) -W96T and recombinant plasmids pET28 (a) -W96F in sequence.
2. Preparation of recombinant bacteria and detection of L-amino acid production
The plasmids tested were: recombinant plasmid pET28 (a) -ycjG, recombinant plasmid pET28 (a) -W96L, recombinant plasmid pET28 (a) -W96P, recombinant plasmid pET28 (a) -W96R, recombinant plasmid pET28 (a) -W96S, recombinant plasmid pET28 (a) -W96T or recombinant plasmid pET28 (a) -W96F. A total of 7 test plasmids.
The test plasmids were each subjected to the following procedure:
1. the test plasmid was introduced into E.coli MG1655 to obtain a recombinant bacterium.
The recombinant strain obtained by the recombinant plasmid pET28 (a) -ycjG is named MG1655-pET28 (a) -ycjG. According to the principle corresponding to the mutant protein names, 6 recombinant bacteria are named recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria MG1655-pET28 (a) -W96P, recombinant bacteria MG1655-pET28 (a) -W96R, recombinant bacteria MG1655-pET28 (a) -W96S, recombinant bacteria MG1655-pET28 (a) -W96T and recombinant bacteria MG1655-pET28 (a) -W96F in sequence.
2. Inoculating the recombinant strain obtained in step 1 into 500mL triangular flask containing 30mL of rich culture medium, shake culturing at 37deg.C and 200rpm to OD 600nm ＝0.2。
3. After completion of step 2, IPTG was added and the mixture was cultured at 37℃and 200rpm with shaking at a concentration of 0.1mM in the system for 30 hours.
4. After the completion of step 3, the concentration of each L-amino acid in the fermentation system was detected by High Performance Liquid Chromatography (HPLC).
The results are shown in Table 3. Recombinant MG1655-pET28 (a) -W96L producing L-alanine is higher than recombinant MG1655
pET28 (a) -W96P, MG1655-pET28 (a) -W96R, MG1655-pET28 (a) -W96S, MG1655-pET28 (a) -W6T and MG1655-pET28 (a) -W96F are stronger.
TABLE 3 analytical results of high performance liquid chromatography for detecting L-amino acids
EXAMPLE 3 preparation of recombinant YPAA-ycjG-001
1. Construction of sgRNA
1. The sgRNA-1F and the sgRNA-1R were annealed to obtain double-stranded DNA molecules.
sgRNA-1F：
5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTtgaccgagggtgacgtcagaGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'；
sgRNA-1R：
5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACtctgacgtcaccctcggtcaACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'。
Both sgRNA-1F and sgRNA-1R are single stranded DNA molecules, underlined are homology arms for recombination into pGRB plasmid.
2. pGRB plasmid was taken, digested with restriction enzyme SpeI, dephosphorylated and linearized.
3. And (3) taking the double-stranded DNA molecule obtained in the step (1) and the linearization plasmid obtained in the step (2), and recombining by using a Gibson Assembly kit (New England Co.) to obtain a recombinant plasmid pGRB-sgRNA-1.
2. Preparation of recombinant bacteria
1. And (3) introducing pREDCas9 plasmid into escherichia coli CGMCC26289 competent cells to obtain recombinant escherichia coli containing pREDCas9 plasmid.
2. Competent cells of the recombinant E.coli obtained in step 1, recombinant plasmid pGRB-sgRNA-1 and DNA fragment Up-ycjG W96L Down co-incubation and then plating onto 2-YT solid medium plates containing 100mg/L spectinomycin and 100mg/L ampicillin, and incubation at 32 ℃. And (5) picking single bacterial colonies, and carrying out PCR identification to obtain the target recombinant bacteria.
DNA fragment Up-ycjG W96L Down is a double stranded DNA molecule as shown in SEQ ID NO: 4.
And (3) PCR identification: PCR amplification was performed using a primer pair consisting of P5 and P6 (target sequence: 420 bp), followed by denaturation at 95℃for 10min and ice bath for 5min, followed by SSCP (Single-Strand Conformation Polymorphis) electrophoresis. During electrophoresis, the DNA fragment Up-ycjG was used W96L Down is used as a positive control, an amplification product obtained by PCR amplification of escherichia coli MG1655 by using a primer pair consisting of P5 and P6 is used as a negative control, water is used as a blank control, and the electrophoresis positions are different due to different fragment structures. The test bacteria satisfying the following conditions are target recombinant bacteria: the electrophoresis position of the PCR amplification product is inconsistent with the negative control and consistent with the positive control, and the strain is the strain with successful allelic replacement, namely the target recombinant strain.
P5：5'-AACTGGAAGAAGAGGGTATT-3'；
P6：5'-GCGTCGCATCGGGCACAGCT-3'。
3. The target recombinant bacteria obtained in the step 2 are inoculated to a 2-YT solid culture medium plate containing 100mg/L spectinomycin and 0.2g/100ml arabinose and cultured at 37 ℃. Then, the strain is transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate containing 100mg/L of ampicillin respectively, cultured at 37 ℃, and the strain growing in the spectinomycin culture medium and not growing in the ampicillin culture medium is selected, namely the target recombinant strain with the plasmid pGRB-sgRNA-1 removed.
4. Inoculating the target recombinant bacteria obtained in the step 3 to a 2-YT solid culture medium plate, and culturing at 42 ℃. Then, the strain is respectively transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate not containing spectinomycin, cultured at 37 ℃, and the strain which does not grow on the spectinomycin culture medium and grows on the spectinomycin-free culture medium is selected, namely the target recombinant strain for removing pREDCas9 plasmid.
5. And (3) taking the target recombinant strain obtained in the step (4), carrying out PCR amplification by adopting a primer pair consisting of P5 and P6, and then recovering and sequencing an amplification product to obtain the recombinant strain YPAa-ycjG-001. Sequencing results show that compared with the escherichia coli CGMCC26289, the recombinant bacterium YPHA-ycjG-001 is only different in that a codon mutation occurs in the ycjG gene in the genome DNA, namely, the codon encoding the 96 th amino acid residue of the ycjG protein is mutated from TGG to TTG.
EXAMPLE 4 preparation of recombinant YPAA-ycjG-002 and recombinant YPAA-ycjG-003
1. Construction of sgRNA
1. Annealing of sgRNA-2F and sgRNA-2R gives a double stranded DNA molecule.
sgRNA-2F：
5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTggcaactatgtaaactatagGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'；
sgRNA-2R：
5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACctatagtttacatagttgccACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'。
Both sgRNA-2F and sgRNA-2R are single stranded DNA molecules, underlined are homology arms for recombination into pGRB plasmid.
2. pGRB plasmid was taken, digested with restriction enzyme SpeI, dephosphorylated and linearized.
3. And (3) taking the double-stranded DNA molecule obtained in the step (1) and the linearization plasmid obtained in the step (2), and recombining by using a Gibson Assembly kit (New England Co.) to obtain a recombinant plasmid pGRB-sgRNA-2.
2. Preparation of recombinant YPAA-ycjG-002
1. And (3) introducing pREDCas9 plasmid into escherichia coli CGMCC26289 competent cells to obtain recombinant escherichia coli containing pREDCas9 plasmid.
2. Competent cells of the recombinant E.coli obtained in step 1, recombinant plasmid pGRB-sgRNA-2 and DNA fragment Up yaiT -ycjG-Down yaiT Co-incubation was followed by plating onto 2-YT solid medium plates containing 100mg/L spectinomycin and 100mg/L ampicillin and incubation at 32 ℃. And (5) picking single bacterial colonies, and carrying out PCR identification to obtain the target recombinant bacteria.
DNA fragment Up yaiT -ycjG-Down yaiT Is a double-stranded DNA molecule, as shown in SEQ ID NO: shown at 5. SEQ ID NO: in 5, nucleotides 1 to 570 constitute the upstream homology arm (570 bp), nucleotides 571 to 688 constitute the ycjG promoter (118 bp), nucleotides 689 to 1654 constitute the ycjG gene (966 bp), and nucleotides 1655 to 2239 constitute the downstream homology arm (585 bp).
And (3) PCR identification: and (3) carrying out PCR amplification by adopting a primer pair consisting of P15 and P16, wherein a characteristic band is 1346bp (shown as SEQ ID NO: 7), and if the amplification product has the characteristic band, the PCR identification is positive, namely the target recombinant bacterium.
P15：5'-CAACGAAACCTACACTACCG-3'；
P16：5'-TTCACTTTCAGTAATTTCGCG-3'。
3. The target recombinant bacteria obtained in the step 2 are inoculated to a 2-YT solid culture medium plate containing 100mg/L spectinomycin and 0.2g/100ml arabinose and cultured at 37 ℃. Then, the strain is transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate containing 100mg/L of ampicillin respectively, cultured at 37 ℃, and the strain growing in the spectinomycin culture medium and not growing in the ampicillin culture medium is selected, namely the target recombinant strain with the plasmid pGRB-sgRNA-2 removed.
4. Inoculating the target recombinant bacteria obtained in the step 3 onto a 2-YT solid culture medium plate, and culturing at 42 ℃. Then, the strain is respectively transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate not containing spectinomycin, cultured at 37 ℃, and the strain which does not grow on the spectinomycin culture medium and grows on the spectinomycin-free culture medium is selected, namely the target recombinant strain for removing pREDCas9 plasmid.
5. And (3) taking the target recombinant strain obtained in the step (4), carrying out PCR amplification by adopting a primer pair consisting of P17 and P18, and then recovering and sequencing an amplification product to obtain the recombinant strain YPAa-ycjG-002. Sequencing results show that compared with the escherichia coli CGMCC26289, the recombinant bacterium YPIa-ycjG-002 only has the difference that a wild-type ycjG gene is used for replacing yaiT part of the coding region. The recombinant strain YPAA-ycjG-002 has two copies of the wild-type ycjG gene in its genomic DNA.
P17：5'-TGGGGAAAGTGATGCCTCGG-3'；
P18：5'-CGACCTGTAGTATCCCATTC-3'。
3. Preparation of recombinant YPAA-ycjG-003
Basically, the same procedure as in step two, only the difference procedure is described as follows:
2. competent cells of the recombinant E.coli obtained in step 1, recombinant plasmid pGRB-sgRNA-2 and DNA fragment Up yaiT -ycjG W96L -Down yaiT Co-incubation was followed by plating onto 2-YT solid medium plates containing 100mg/L spectinomycin and 100mg/L ampicillin and incubation at 32 ℃. And (5) picking single bacterial colonies, and carrying out PCR identification to obtain the target recombinant bacteria.
DNA fragment Up yaiT -ycjG W96L -Down yaiT Is a double-stranded DNA molecule, as shown in SEQ ID NO: shown at 6. SEQ ID NO:6, nucleotide 1-570 constitutes the upstream homology arm (570 bp), nucleotide 571-688 constitutes the ycjG promoter (118 bp), nucleotide 689-1654 constitutes the ycjG W96L Gene (966 bp) with nucleotides 1655-2239 constituting the downstream homology arm (585 bp).
Recombinant YPAA-ycjG-003 was obtained. Sequencing results show that compared with the escherichia coli CGMCC26289, the recombinant bacterium YPIa-ycjG-003 only has the difference of ycjG W96L The yaiT part coding region is replaced by the gene. In the genomic DNA of recombinant bacterium YPAA-ycjG-003, there are 1 copy of wild-type ycjG gene and 1 copy of ycjG W96L And (3) a gene.
EXAMPLE 5 construction of recombinant YPAA-ycjG-004
1. Construction of sgRNA
1. The sgRNA-3F and the sgRNA-3R were annealed to give double-stranded DNA molecules.
sgRNA-3F：
5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTtttgcgcttaatccgcagacGTTTTAGAGCT AGAAATAGCAAG TTAAAATAAGG-3'；
sgRNA-3R：
5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACgtctgcggattaagcgcaaaACTAGTATTAT ACCTAGGACTGA GCTAGCTGTCA-3'。
Both sgRNA-3F and sgRNA-3R are single stranded DNA molecules, underlined are homology arms for recombination into pGRB plasmid.
2. pGRB plasmid was taken, digested with restriction enzyme SpeI, dephosphorylated and linearized.
3. And (3) taking the double-stranded DNA molecule obtained in the step (1) and the linearized plasmid obtained in the step (2), and recombining by using a Gibson Assembly kit (New England Co.) to obtain a recombinant plasmid pGRB-sgRNA-3.
2. Preparation of recombinant YPAA-ycjG-004
1. And (3) introducing pREDCas9 plasmid into escherichia coli CGMCC26289 competent cells to obtain recombinant escherichia coli containing pREDCas9 plasmid.
2. The competent cells of the recombinant E.coli obtained in step 1, the recombinant plasmid pGRB-sgRNA-3 and the DNA fragment DeltaycjG-Up-Dwon were incubated together, and then plated onto a 2-YT solid medium plate containing 100mg/L spectinomycin and 100mg/L ampicillin, and cultured at 32 ℃. And (5) picking single bacterial colonies, and carrying out PCR identification to obtain the target recombinant bacteria.
The DNA fragment delta ycjG-Up-Dwon is a double-stranded DNA molecule, and is shown in SEQ ID NO: shown at 8.
And (3) PCR identification: and (3) carrying out PCR amplification by adopting a primer pair consisting of P19 and P22, wherein the characteristic band is 1610bp, and if the amplification product has the characteristic band, the PCR identification is positive, namely the target recombinant bacterium.
P19：5'-TCGGTATTAACCCAGCTTTATC-3'；
P22：5'-GCAAAAAAGTGATACAAGATC-3'。
3. The target recombinant bacteria obtained in the step 2 are inoculated to a 2-YT solid culture medium plate containing 100mg/L spectinomycin and 0.2g/100ml arabinose and cultured at 37 ℃. Then, the strain is transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate containing 100mg/L of ampicillin respectively, cultured at 37 ℃, and the strain growing in the spectinomycin culture medium and not growing in the ampicillin culture medium is selected, namely the target recombinant strain with the plasmid pGRB-sgRNA-3 removed.
4. Inoculating the target recombinant bacteria obtained in the step 3 onto a 2-YT solid culture medium plate, and culturing at 42 ℃. Then, the strain is respectively transferred to a 2-YT solid culture medium plate containing 100mg/L of spectinomycin or a 2-YT solid culture medium plate not containing spectinomycin, cultured at 37 ℃, and the strain which does not grow on the spectinomycin culture medium and grows on the spectinomycin-free culture medium is selected, namely the target recombinant strain for removing pREDCas9 plasmid.
5. And (3) taking the target recombinant strain obtained in the step (4), carrying out PCR amplification by adopting a primer pair consisting of P19 and P22, and then recovering and sequencing an amplification product to obtain the recombinant strain YPAa-ycjG-004. Sequencing results show that compared with the escherichia coli CGMCC26289, the recombinant bacterium YPIa-ycjG-004 only has the difference that the sequence of SEQ ID NO:1, and a wild-type ycjG gene shown in seq id no.
EXAMPLE 6 construction of recombinant YPAA-ycjG-005 and recombinant YPAA-ycjG-006
The recombinant plasmid pET28 (a) -ycjG prepared in example 1 was introduced into E.coli CGMCC26289 to obtain recombinant E.coli containing the recombinant plasmid pET28 (a) -ycjG, which was named recombinant strain YPAA-ycjG-005.
The recombinant plasmid pET28 (a) -W96L prepared in example 2 is introduced into escherichia coli CGMCC26289 to obtain recombinant escherichia coli containing the recombinant plasmid pET28 (a) -W96L, which is named as recombinant bacterium YPAA-ycjG-006.
Example 7L-alanine fermentation experiment
The test bacteria are respectively the following starting bacteria or recombinant bacteria: escherichia coli MG1655, recombinant strain MG1655-pET28 (a) -ycjG, recombinant strain MG1655-pET28 (a) -W96L, recombinant strain MG1655-pET28 (a) -W96P, recombinant strain MG1655-pET28 (a) -W96R, recombinant strain MG1655-pET28 (a) -W96S, escherichia coli CGMCC26289, recombinant strain YPIa-ycjG-001, recombinant strain YPIa-ycjG-002, recombinant strain YPIa-ycjG-003, recombinant strain YPIa-ycjG-004, recombinant strain YPIa-ycjG-005 or recombinant strain YPIa-ycjG-006.
Among the above recombinant bacteria, partial recombinationThe bacteria are recombinant bacteria which over-express target genes by utilizing recombinant plasmids. These recombinant plasmids are obtained by inserting the target gene by taking the pET28a (+) vector as a starting vector. These recombinant bacteria are recombinant bacteria MG1655-pET28 (a) -ycjG, recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria MG1655-pET28 (a) -W96P, recombinant bacteria MG1655-pET28 (a) -W96R, recombinant bacteria MG1655-pET28 (a) -W96S, recombinant bacteria YPAA-ycjG-005, recombinant bacteria YPAA-ycjG-006. The recombinant bacteria need IPTG induction in the fermentation process, namely when the system OD 600nm IPTG was added at=0.2 and the concentration in the system was set to 0.1mM.
Among the above recombinant bacteria, a part of the recombinant bacteria is integrated with the target gene in the genomic DNA, and thus, no additional IPTG induction is required. These recombinant bacteria are recombinant bacteria YPAA-ycjG-001, recombinant bacteria YPAA-ycjG-002, recombinant bacteria YPAA-ycjG-003 and recombinant bacteria YPAA-ycjG-004.
Coli MG1655, E.coli CGMCC26289 without additional IPTG induction.
The seed solution of the test strain was inoculated at an inoculum size of 10% into a 5L fermenter (Shanghai Bai Biotechnology Co., ltd.) of the type BLBIO-5GC-4-H containing L-alanine fermentation medium for fermentation.
The OD600nm value of the seed solution of the test bacteria is 10, and the seed solution is obtained by inoculating the test bacteria into a 2-YT culture medium and culturing.
The L-alanine fermentation medium consisted of additives and water, pH 7.0. The additives and the addition amounts of the additives in each liter of the L-alanine fermentation medium are as follows: glucose 13g/L, (NH) 4 ) 2 SO 4 1g/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 0.01g/L，MnSO 4 ·H 2 O0.01 g/L, FM902 yeast powder 1.5g/L, corn steep liquor 5g/L and molasses 17g/L.
During the fermentation, the pH of the system was maintained at 7.0 by passing ammonia gas.
Correction of DO 100%: the temperature is 37 ℃, the air quantity is 5L/min, the rotating speed is 800rpm, the tank pressure is 0mpa, and the calibration is carried out after 5 min.
Fermentation temperature: 37 ℃.
Fermentation time: and 30h.
Initial conditions: the tank pressure is 0Mpa, the air quantity is 0.5L/min, and the rotating speed is 400rpm.
And (3) whole-process control:
1. when the dissolved oxygen is less than 30%, the rotation speed is sequentially increased by 500rpm, 600rpm, the air quantity is increased by 1L/min, 700rpm and 800rpm;
2. fermenting for 8h, extracting and pressing for 0.01Mpa; the tank extracting pressure is 0.02 Mpa-0.03 Mpa-0.04 Mpa-0.05 Mpa for 12 h.
Residual sugar control: 0.1-0.5% before F12 h; after F12h, controlling the residual sugar by 0.1-0.3% in combination with DO requirement;
feeding: 25% ammonia water, 55% concentrated sugar and 10% dichlord.
The control process takes dissolved oxygen of 20-30% as the standard of air quantity reduction.
After the fermentation, the L-alanine content was measured by High Performance Liquid Chromatography (HPLC). Three replicates were set and the results averaged. The L-alanine concentration in the fermentation system at the end of the fermentation is shown in Table 4. For both the high-yield L-alanine strain and the model strain MG1655, the 96 th tryptophan of the amino acid sequence of the ycjG gene is replaced by leucine, which is helpful for improving the yield of L-alanine; for high L-alanine producing strains, wild-type ycjG genes and mutant ycjG W96L The overexpression of (C) contributes to an increase in L-alanine production, whereas the knock-out of the ycjG gene does not contribute to an increase in its production.
TABLE 4L-alanine production by strains
Example 8 preparation and use of engineering bacteria for valine production
1. Preparation of engineering bacteria
The procedure of example 3, step two, was followed using E.coli CGMCC22721 instead of E.coli CGMCC26289 to obtain recombinant YPEL-ycjG-001. Sequencing results show that compared with the escherichia coli CGMCC22721, the recombinant bacterium YPLA-ycjG-001 is only different in that a codon mutation is generated in the ycjG gene in the genome DNA, namely, the codon encoding the 96 th amino acid residue of the ycjG protein is mutated from TGG to TTG.
The procedure of example 4, step two, was followed using E.coli CGMCC22721 instead of E.coli CGMCC26289 to obtain recombinant strain YPEL-ycjG-002. Sequencing results show that compared with the escherichia coli CGMCC22721, the recombinant bacterium YPLA-ycjG-002 only has the difference that the wild-type ycjG gene replaces the yaiT part coding region. The recombinant strain YPVal-ycjG-002 has two copies of the wild-type ycjG gene in its genomic DNA.
The procedure of step three of example 4 was followed using E.coli CGMCC22721 instead of E.coli CGMCC26289 to obtain recombinant strain YPEL-ycjG-003. Sequencing results showed that the recombinant strain YPLA-ycjG-003 was different from E.coli CGMCC22721 only in the use of ycjG W96L The yaiT part coding region is replaced by the gene. In the genomic DNA of recombinant bacterium YVal-ycjG-003, there are 1 copy of the wild-type ycjG gene and 1 copy of ycjG W96L And (3) a gene.
The procedure of example 5, step two, was followed using E.coli CGMCC22721 instead of E.coli CGMCC26289 to obtain recombinant YPEL-ycjG-004. Sequencing results show that compared with the escherichia coli CGMCC22721, the recombinant bacterium YPLA-ycjG-004 only has the difference that SEQ ID NO is deleted: 1, and a wild-type ycjG gene shown in seq id no.
The recombinant plasmid pET28 (a) -ycjG prepared in example 1 was introduced into E.coli CGMCC22721 to obtain a recombinant E.coli containing the recombinant plasmid pET28 (a) -ycjG, which was designated as recombinant strain YPVal-ycjG-005.
The recombinant plasmid pET28 (a) -W96L prepared in example 2 was introduced into E.coli CGMCC22721 to obtain recombinant E.coli containing the recombinant plasmid pET28 (a) -W96L, which was designated as recombinant strain YPVal-ycjG-006.
2. Production of valine
The test bacteria are respectively the following starting bacteria or recombinant bacteria: escherichia coli MG1655, recombinant strain MG1655-pET28 (a) -ycjG, recombinant strain MG1655-pET28 (a) -W96L, escherichia coli CGMCC22721, recombinant strain YPVal-ycjG-001, recombinant strain YPVal-ycjG-002, recombinant strain YPVal-ycjG-003, recombinant strain YPVal-ycjG-004, recombinant strain YPVal-ycjG-005 or recombinant strain YPVal-ycjG-006.
Some recombinant bacteria need IPTG induction in the fermentation process, namely when the system OD 600nm IPTG was added at=0.2 and brought to a concentration of 0.1mM in the system; these recombinant bacteria refer to recombinant bacteria MG1655-pET28 (a) -ycjG, recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria YPVal-ycjG-005 and recombinant bacteria YPVal-ycjG-006. The remaining test bacteria do not require additional IPTG induction during fermentation.
The seed solution of the test strain was inoculated at an inoculum size of 10% into a 5L fermenter (Shanghai Bai Biotechnology Co., ltd.) of the type BLBIO-5GC-4-H containing L-valine fermentation medium for fermentation.
The OD600nm value of the seed solution of the test bacteria is 10, and the seed solution is obtained by inoculating the test bacteria into a 2-YT culture medium and culturing.
The L-valine fermentation medium consisted of additives and water, and had a pH of 7.0. The additives and the addition amounts of the L-valine in each liter of the fermentation medium are as follows: yeast extract 4g/L, corn steep liquor dry powder 2g/L, peptone 4g/L, methionine 2g/L, KH 2 PO 4 ·3H 2 O7g/L，MgSO 4 ·7H 2 O 2g/L，CoCl 2 20mg/L，(NH 4 ) 2 SO 4 3g/L, 2g/L of citric acid and 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, defoamer 0.3mL/L, (NH) 4 ) 2 SO 4 3g/L。
Fermentation culture conditions (two-stage, aerobic-limited oxygen fermentation): firstly, aerobic fermentation is carried out, and the air quantity rotation speed and the sugar supplementing rate are adjusted in the early stage to control the dissolved oxygen to be about 25%; waiting for OD 600 When the value is 50-60, the rotating speed is reduced to 400rpm, the air quantity is reduced to 2L/min, and the aerobic fermentation is converted into oxygen-limited fermentation. The dissolved oxygen electrode calibration method comprises the following steps: zero is marked in saturated sodium sulfite solution, and hundred points are marked in air.
After the fermentation, the L-valine content was measured by High Performance Liquid Chromatography (HPLC). Three replicates were set and the results averaged. The L-valine concentration in the fermentation system at the end of the fermentation is shown in Table 5. Whether for high-yield L-valine bacteriaThe strain is also a model strain MG1655, and the 96 th tryptophan of the amino acid sequence of the ycjG gene is replaced by leucine, so that the improvement of the L-valine yield is facilitated; for high L-valine-producing strains, wild-type ycjG gene and mutant ycjG W96L The overexpression of (C) contributes to an increase in L-valine production, whereas the knock-out of the ycjG gene does not contribute to an increase in its production.
TABLE 5L-valine production of strains
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Example 9 preparation and use of engineering bacteria for arginine production
1. Preparation of engineering bacteria
The procedure of example 3, step two, was followed using E.coli CGMCC25402 instead of E.coli CGMCC26289 to obtain recombinant YPAG-ycjG-001. Sequencing results show that compared with the escherichia coli CGMCC25402, the recombinant bacterium YPAG-ycjG-001 is only different in that a mutation of a codon occurs in the ycjG gene in the genome DNA, namely, the codon encoding the 96 th amino acid residue of ycjG protein is mutated from TGG to TTG.
The procedure of example 4, step two, was followed using E.coli CGMCC25402 instead of E.coli CGMCC26289 to obtain recombinant YPAG-ycjG-002. Sequencing results show that compared with the escherichia coli CGMCC25402, the recombinant bacterium YPAG-ycjG-002 only has the difference that a wild-type ycjG gene replaces a yaiT part coding region. The recombinant strain YPAG-ycjG-002 has two copies of the wild-type ycjG gene in its genomic DNA.
The procedure of step three of example 4 was followed using E.coli CGMCC25402 instead of E.coli CGMCC26289 to obtain recombinant YPAG-ycjG-003. Sequencing results show that compared with the escherichia coli CGMCC25402, the recombinant bacterium YPAG-ycjG-003 only has the difference of ycjG W96L Gene replacement yaiT partial codeA zone. In the genomic DNA of recombinant bacterium YPAG-ycjG-003, there are 1 copy of wild-type ycjG gene and 1 copy of ycjG W96L And (3) a gene.
The procedure of example 5, step two, was followed using E.coli CGMCC25402 instead of E.coli CGMCC26289 to obtain recombinant YPAG-ycjG-004. Sequencing results show that compared with the escherichia coli CGMCC25402, the recombinant bacterium YPAG-ycjG-004 only has the difference that the sequence of SEQ ID NO is deleted: 1, and a wild-type ycjG gene shown in seq id no.
The recombinant plasmid pET28 (a) -ycjG prepared in example 1 was introduced into E.coli CGMCC25402 to obtain recombinant E.coli containing the recombinant plasmid pET28 (a) -ycjG, which was named recombinant strain YPAG-ycjG-005.
The recombinant plasmid pET28 (a) -W96L prepared in example 2 is introduced into escherichia coli CGMCC25402 to obtain recombinant escherichia coli containing the recombinant plasmid pET28 (a) -W96L, which is named as recombinant bacterium YPAG-ycjG-006.
2. Arginine production
The test bacteria are respectively the following starting bacteria or recombinant bacteria: escherichia coli MG1655, recombinant strain MG1655-pET28 (a) -ycjG, recombinant strain MG1655-pET28 (a) -W96L, escherichia coli CGMCC25402, recombinant strain YPAG-ycjG-001, recombinant strain YPAG-ycjG-002, recombinant strain YPAG-ycjG-003, recombinant strain YPAG-ycjG-004, recombinant strain YPAG-ycjG-005 or recombinant strain YPAG-ycjG-006.
Some recombinant bacteria need IPTG induction in the fermentation process, namely when the system OD 600nm IPTG was added at=0.2 and brought to a concentration of 0.1mM in the system; these recombinant bacteria are recombinant bacteria MG1655-pET28 (a) -ycjG, recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria YPAG-ycjG-005 and recombinant bacteria YPAG-ycjG-006. The remaining test bacteria do not require additional IPTG induction during fermentation.
The seed solution of the test strain was inoculated at an inoculum size of 10% into a 5L fermenter (Shanghai Bai Biotechnology Co., ltd.) of the type BLBIO-5GC-4-H containing L-arginine fermentation medium for fermentation.
The OD600nm value of the seed solution of the test bacteria is 10, and the seed solution is obtained by inoculating the test bacteria into a 2-YT culture medium and culturing.
The L-arginine fermentation medium consisted of additives and water, pH 7.2. The additives and the addition amounts of the additives in each liter of the L-arginine fermentation medium are as follows: glucose 8g/L, FM902 yeast powder 3g/L, K 2 HPO 4 ·3H 2 O 6g/L，MgSO 4 ·7H 2 O 1g/L，FeSO 4 ·7H 2 O0.05 g/L, betaine 0.5g/L, VB 12 0.005g/L, defoamer 0.3mL/L, ammonium sulfate 3g/L.
Fermentation culture conditions:
correction of DO 100%: the temperature is 35 ℃; ph7.2; the rotation speed is 100rpm; the air quantity is 6L/min; tank pressure is 0.00Mpa;
initial conditions: the temperature is 35 ℃, the pH is 7.2, the tank pressure is 0.01mpa, the air quantity is 1.5L/min, and the rotating speed is 350rpm;
and (3) whole-process control: controlling DO at 20-30%; when the dissolved oxygen is less than or equal to 25 percent, the rotating speed is increased every time
300rpm→400rpm→2.0L/min→500rpm→0.02Mpa→600rpm→3.0L/min→0.03Mpa→700rpm→3.5L/min→0.04Mpa→800rpm→900rpm→4.0L/min→0.05Mpa→1000rpm；
Residual sugar control: controlling the residual sugar to be 0.05-0.1% in the whole process;
feeding: 25% ammonia water, 80% concentrated sugar and 10% dichlord;
fermentation period: about 50 hours, the control process takes 20-30% of dissolved oxygen as the standard of air volume reduction.
After the fermentation is completed, the L-arginine content is detected by High Performance Liquid Chromatography (HPLC). Three replicates were set and the results averaged. The L-arginine concentration in the fermentation system at the end of the fermentation is shown in Table 6. For both the high-yield L-arginine strain and the model strain MG1655, the 96 th tryptophan of the amino acid sequence of the ycjG gene is replaced by leucine, which is helpful for improving the yield of L-arginine; for high L-arginine producing strains, wild-type ycjG gene and mutant ycjG W96L The overexpression of (C) contributes to an increase in L-arginine production, whereas the knock-out of the ycjG gene does not contribute to an increase in its production.
TABLE 6L-arginine production by strains
Example 10 preparation and use of engineering bacteria for threonine production
1. Preparation of engineering bacteria
The procedure of example 3, step two, was followed using E.coli CGMCC25404 instead of E.coli CGMCC26289 to obtain recombinant YPTR-ycjG-001. Sequencing results show that compared with the escherichia coli CGMCC25404, the recombinant bacterium YPTR-ycjG-001 is different only in that a codon mutation occurs in the ycjG gene in the genomic DNA, namely, the codon encoding the 96 th amino acid residue of ycjG protein is mutated from TGG to TTG.
The procedure of example 4, step two, was followed using E.coli CGMCC25404 instead of E.coli CGMCC26289 to obtain recombinant YPTR-ycjG-002. Sequencing results show that compared with the escherichia coli CGMCC25404, the recombinant bacterium YPTR-ycjG-002 only has the difference that the wild-type ycjG gene replaces the yaiT part coding region. The recombinant strain YPTR-ycjG-002 has two copies of the wild-type ycjG gene in its genomic DNA.
The procedure of step three of example 4 was followed using E.coli CGMCC25404 instead of E.coli CGMCC26289 to obtain recombinant YPTR-ycjG-003. Sequencing results showed that the recombinant strain YPTR-ycjG-003 was different from the E.coli CGMCC25404 only in the use of ycjG W96L The yaiT part coding region is replaced by the gene. In the genomic DNA of recombinant bacterium YPTR-ycjG-003, there are 1 copy of the wild-type ycjG gene and 1 copy of ycjG W96L And (3) a gene.
The procedure of example 5, step two, was followed using E.coli CGMCC25404 instead of E.coli CGMCC26289 to obtain recombinant YPTR-ycjG-004. Sequencing results show that compared with the escherichia coli CGMCC25404, the recombinant bacterium YPTR-ycjG-004 only has the difference that the sequence of SEQ ID NO:1, and a wild-type ycjG gene shown in seq id no.
The recombinant plasmid pET28 (a) -ycjG prepared in example 1 was introduced into E.coli CGMCC25404 to obtain recombinant E.coli containing the recombinant plasmid pET28 (a) -ycjG, which was named recombinant strain YPTR-ycjG-005.
The recombinant plasmid pET28 (a) -W96L prepared in example 2 is introduced into escherichia coli CGMCC25404 to obtain recombinant escherichia coli containing the recombinant plasmid pET28 (a) -W96L, which is named as recombinant strain YPTR-ycjG-006.
2. Threonine production
The test bacteria are respectively the following starting bacteria or recombinant bacteria: escherichia coli MG1655, recombinant strain MG1655-pET28 (a) -ycjG, recombinant strain MG1655-pET28 (a) -W96L, escherichia coli CGMCC25404, recombinant strain YPTR-ycjG-001, recombinant strain YPTR-ycjG-002, recombinant strain YPTR-ycjG-003, recombinant strain YPTR-ycjG-004, recombinant strain YPTR-ycjG-005 or recombinant strain YPTR-ycjG-006.
Some recombinant bacteria need IPTG induction in the fermentation process, namely when the system OD 600nm IPTG was added at=0.2 and brought to a concentration of 0.1mM in the system; these recombinant bacteria refer to recombinant bacteria MG1655-pET28 (a) -ycjG, recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria YPTR-ycjG-005 and recombinant bacteria YPTR-ycjG-006. The remaining test bacteria do not require additional IPTG induction during fermentation.
The seed solution of the test strain was inoculated at an inoculum size of 10% into a 5L fermenter (Shanghai Bai Biotechnology Co., ltd.) of the type BLBIO-5GC-4-H containing L-threonine fermentation medium for fermentation.
The OD600nm value of the seed solution of the test bacteria is 10, and the seed solution is obtained by inoculating the test bacteria into a 2-YT culture medium and culturing.
The L-threonine fermentation medium consists of additives and water, and has a pH of 7.0. The additives and the addition amounts of the additives in each liter of the L-threonine fermentation medium are as follows: glucose 13g/L, (NH 4) 2 SO 4 1g/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 0.01g/L，MnSO 4 ·H 2 O0.01 g/L, FM902 yeast powder 1.5g/L, corn steep liquor 5g/L and molasses 17g/L.
Fermentation culture conditions:
correction of DO 100%: the temperature is 37 ℃, the air quantity is 5L/min, the rotating speed is 800rpm, the tank pressure is 0mpa, and the calibration is carried out after 5 min;
initial conditions: pH7.0, culture temperature 37 ℃, tank pressure 0Mpa, air volume 0.5L/min, and rotation speed 400rpm;
And (3) whole-process control: 1. when the dissolved oxygen is less than 30%, the rotation speed is sequentially increased by 500rpm, 600rpm, the air quantity is increased by 1L/min, 700rpm and 800rpm; 2. fermenting for 8h, extracting and pressing for 0.01Mpa; extracting the tank for 12 hours, wherein the tank pressure is 0.02 Mpa-0.03 Mpa-0.04 Mpa-0.05 Mpa;
residual sugar control: 0.1-0.5% before F12 h; after F12h, controlling the residual sugar by 0.1-0.3% in combination with DO requirement;
feeding: 25% ammonia water, 55% concentrated sugar and 10% dichlord;
fermentation period: about 30 hours, the control process takes 20-30% of dissolved oxygen as the standard of air volume reduction.
After the fermentation is completed, the L-threonine content is detected by High Performance Liquid Chromatography (HPLC). Three replicates were set and the results averaged. The L-threonine concentration in the fermentation system at the end of the fermentation is shown in Table 7. For both the high-L threonine-producing strain and the model strain MG1655, the tryptophan at position 96 of the amino acid sequence of the ycjG gene is replaced by leucine, which contributes to the improvement of L-threonine production; for high L-threonine-producing strains, wild-type ycjG genes and mutant ycjG W96L The overexpression of (A) contributes to an increase in the production of L-threonine, whereas the knock-out of the ycjG gene does not contribute to an increase in its production.
L-threonine production by strains of Table 7
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Example 11 preparation and use of engineering bacteria for tryptophan production
1. Preparation of engineering bacteria
The procedure of example 3, step two, was followed using E.coli CGMCC25403 instead of E.coli CGMCC26289 to obtain recombinant YPTRP-ycjG-001. Sequencing results show that compared with the escherichia coli CGMCC25403, the recombinant bacterium YPTRP-ycjG-001 is different only in that a codon mutation occurs in the ycjG gene in the genome DNA, namely, the codon encoding the 96 th amino acid residue of the ycjG protein is mutated from TGG to TTG.
The procedure of example 4, step two, was followed using E.coli CGMCC25403 instead of E.coli CGMCC26289 to obtain recombinant strain YPTRP-ycjG-002. Sequencing results show that compared with the escherichia coli CGMCC25403, the recombinant bacterium YPTRP-ycjG-002 only has the difference that a wild-type ycjG gene is used for replacing yaiT part of the coding region. The recombinant strain YPTRP-ycjG-002 has two copies of the wild-type ycjG gene in its genomic DNA.
The procedure of step three of example 4 was followed using E.coli CGMCC25403 instead of E.coli CGMCC26289 to obtain recombinant YPTRP-ycjG-003. Sequencing results show that compared with the escherichia coli CGMCC25403, the recombinant bacterium YPTRP-ycjG-003 only has the difference of ycjG W96L The yaiT part coding region is replaced by the gene. In the genomic DNA of recombinant strain YTrp-ycjG-003, there are 1 copy of wild-type ycjG gene and 1 copy of ycjG W96L And (3) a gene.
The procedure of example 5, step two, was followed using E.coli CGMCC25403 instead of E.coli CGMCC26289 to obtain recombinant YPTRP-ycjG-004. Sequencing results show that compared with the escherichia coli CGMCC25403, the recombinant bacterium YPTRP-ycjG-004 only has the difference that SEQ ID NO is deleted: 1, and a wild-type ycjG gene shown in seq id no.
The recombinant plasmid pET28 (a) -ycjG prepared in example 1 was introduced into E.coli CGMCC25403 to obtain recombinant E.coli containing the recombinant plasmid pET28 (a) -ycjG, which was designated as recombinant strain YPTRP-ycjG-005.
The recombinant plasmid pET28 (a) -W96L prepared in example 2 is introduced into escherichia coli CGMCC25403 to obtain recombinant escherichia coli containing the recombinant plasmid pET28 (a) -W96L, which is named as recombinant strain YPTRP-ycjG-006.
2. Production of tryptophan
The test bacteria are respectively the following starting bacteria or recombinant bacteria: escherichia coli MG1655, recombinant strain MG1655-pET28 (a) -ycjG, recombinant strain MG1655-pET28 (a) -W96L, escherichia coli CGMCC25403, recombinant strain YPTRP-ycjG-001, recombinant strain YPTRP-ycjG-002, recombinant strain YPTRP-ycjG-003, recombinant strain YPTRP-ycjG-004, recombinant strain YPTRP-ycjG-005 or recombinant strain YPTRP-ycjG-006.
Some recombinant bacteria need IPTG induction in the fermentation process, namely when the system OD 600nm IPTG was added at=0.2 and brought to a concentration of 0.1mM in the system; these recombinant bacteria refer to recombinant bacteria MG1655-pET28 (a) -ycjG, recombinant bacteria MG1655-pET28 (a) -W96L, recombinant bacteria YPTRP-ycjG-005 and recombinant bacteria YPTRP-ycjG-006. The remaining test bacteria do not require additional IPTG induction during fermentation.
The seed solution of the test strain was inoculated at an inoculum size of 10% into a 5L fermenter (Shanghai Bai Biotechnology Co., ltd.) of the type BLBIO-5GC-4-H containing an L-tryptophan fermentation medium for fermentation.
The OD600nm value of the seed solution of the test bacteria is 10, and the seed solution is obtained by inoculating the test bacteria into a 2-YT culture medium and culturing.
The L-tryptophan fermentation medium consisted of additives and water, and had a pH of 7.0. The additives and the addition amounts of the additives in each liter of the L-tryptophan fermentation medium are as follows: glucose 7g/L, FM902 yeast powder 1g/L, (NH 4) 2 SO 4 1.2g/L, citric acid 1.2g/L, mgSO 4 ·7H 2 O 1.5g/L,K 2 HPO 4 ·3H 2 O5.5 g/L, defoamer 0.2mL/L.
Culture conditions:
correction of DO 100%: the temperature is 35 ℃, the rotating speed is 800rpm, the air quantity is 5L/min, and the tank pressure is 0.00Mpa;
initial conditions: the temperature is 35 ℃, the pH is 7.0, the air quantity is 1.0L/min, and the rotating speed is 350rpm;
and (3) whole-process control: when dissolved oxygen is less than or equal to 20 percent before the bottom sugar is consumed, sequentially extracting 400rpm to 450rpm; the bottom sugar is consumed, and the dissolved oxygen is controlled to be 15-30% by supplementing sugar; pH7.0 before F24h, 6.7 after F24 h;
Residual sugar control: 0.1-0.5% before F12 h; after F12h, controlling the residual sugar by 0.1-0.3% in combination with DO requirement;
feeding: 25% ammonia water, 55% concentrated sugar and 10% dichlord;
fermentation period: about 34 hours, the control process takes 15-30% of dissolved oxygen as the standard of air volume reduction.
Detecting L-tryptamine by High Performance Liquid Chromatography (HPLC) after fermentationAcid content. Three replicates were set and the results averaged. The L-tryptophan concentration in the fermentation system at the end of the fermentation is shown in Table 8. For both the high-yield L-tryptophan strain and the model strain MG1655, the 96 th tryptophan of the amino acid sequence of the ycjG gene is replaced by leucine, which is helpful for improving the yield of L-tryptophan; for high L-tryptophan-producing strains, wild-type ycjG gene and mutant ycjG W96L The overexpression of (C) contributes to an increase in L-tryptophan production, whereas the knock-out of the ycjG gene does not contribute to an increase in its production.
TABLE 8L-tryptophan production by strains
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1.ycjG W96 Proteins or comprise ycjG W96 Fusion proteins of proteins;
the ycjG W96 The protein is a protein, and is obtained by mutating the 96 th amino acid residue of ycjG protein from W to other amino acid residues;
the ycjG protein is (a 1) or (a 2) or (a 3) as follows:
(a1) Comprising SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no;
(a2) A protein derived from a microorganism or a cell and having 80% or more identity to (a 1) and being related to amino acid production;
(a3) And (b) a protein derived from (a 1) and related to amino acid production, wherein the protein shown in (a 1) is obtained by substitution and/or deletion and/or addition of one or more amino acid residues.
2. A biomaterial which is (c 1) or (c 2) or (c 3) or (c 4) or (c 5) or (c 6) as follows:
(c1) Encoding the ycjG of claim 1 W96 A nucleic acid molecule of a protein;
(c2) A nucleic acid molecule encoding the fusion protein of claim 1;
(c3) An expression cassette having the nucleic acid molecule of (c 1) or (c 2);
(c4) A recombinant vector having the nucleic acid molecule of (c 1) or (c 2);
(c5) A recombinant microorganism having the nucleic acid molecule of (c 1) or (c 2);
(c6) A recombinant cell having the nucleic acid molecule of (c 1) or (c 2).
3. The ycjG of claim 1 W96 The use of a protein, a fusion protein according to claim 1 or a biomaterial according to claim 2 as follows (i) or (ii) or (iii):
Use of (i) to increase the amino acid production of a microorganism or cell;
(II) use in the production of amino acids;
(III) use in the construction of recombinant microorganisms or recombinant cells for the production of amino acids.
4. The application of the specific substances is as follows (I) or (II) or (III):
use of (i) to increase the amino acid production of a microorganism or cell;
(II) use in the production of amino acids;
(III) use in the construction of recombinant microorganisms or recombinant cells for the production of amino acids;
the specific substance comprises the following (d 1), (d 2) or (d 3):
(d1) A substance for increasing expression of a nucleic acid molecule encoding a protein of interest;
(d2) A substance for increasing the abundance of a target protein;
(d3) A substance for increasing the activity of a target protein;
the protein of interest being the ycjG of claim 1 W96 A protein, a fusion protein according to claim 1 or an ycjG protein according to claim 1.
5. A recombinant microorganism or recombinant cell obtained by overexpressing a nucleic acid molecule encoding a protein of interest in the microorganism or cell; the protein of interest being the ycjG of claim 1 W96 A protein, a fusion protein according to claim 1 or an ycjG protein according to claim 1.
6. Escherichia coli (Escherichia coli) YP007-1 having a accession number of CGMCC No. 26089.
7. Use of the recombinant microorganism of claim 5 or the recombinant cell of claim 5 or escherichia coli CGMCC26289 in the preparation of amino acids; the escherichia coli CGMCC26289 is escherichia coli YP007-1 as set forth in claim 6.
8. A method for increasing amino acid production by a microorganism or cell comprising the steps of: replacement of a nucleic acid molecule encoding an ycjG protein in the genome of a microorganism or cell with a nucleic acid molecule encoding ycjG W96 A nucleic acid molecule encoding a protein, or alternatively, a nucleic acid molecule encoding an ycjG protein in the genome of a microorganism or cell is replaced with a nucleic acid molecule encoding a fusion protein; the ycjG protein is the ycjG protein of claim 1; the ycjG W96 The protein being the ycjG of claim 1 W96 A protein; the fusion protein is the fusion protein of claim 1.
9. A method for increasing amino acid production by a microorganism or cell comprising the steps of: overexpression of a nucleic acid molecule encoding a protein of interest in a microorganism or cell, or, alternatively, enhancement of the middle order of a microorganism or cellOr, increasing the activity of a protein of interest in a microorganism or cell; the protein of interest being the ycjG of claim 1 W96 A protein, a fusion protein according to claim 1 or an ycjG protein according to claim 1.
10. Application of target protein in regulating amino acid yield of microorganism or cell; the protein of interest being the ycjG of claim 1 W96 A protein, a fusion protein according to claim 1 or an ycjG protein according to claim 1.
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