CN116555150B - Recombinant Escherichia coli for fermentative production of L-valine - Google Patents

Recombinant Escherichia coli for fermentative production of L-valine Download PDF

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CN116555150B
CN116555150B CN202310765899.5A CN202310765899A CN116555150B CN 116555150 B CN116555150 B CN 116555150B CN 202310765899 A CN202310765899 A CN 202310765899A CN 116555150 B CN116555150 B CN 116555150B
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amino acid
primer
valine
escherichia coli
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CN116555150A (en
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苏厚波
孟刚
魏爱英
赵春光
毕国东
张英
张晓琴
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Beijing Zhongke Yipin Biotechnology Co ltd
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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
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

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Abstract

The invention discloses recombinant escherichia coli for producing L-valine by fermentation. Recombinant E.coli is an engineering bacterium obtained by "knocking out adhE gene", "knocking out adhE gene and knocking out ilvE (E) gene while inserting ilvE (B) gene from Bacillus subtilis", "knocking out adhE gene and thiE gene and knocking out ilvE (E) gene while inserting ilvE (B) gene from Bacillus subtilis" or "knocking out adhE gene, thiE gene and mdh gene and knocking out ilvE (E) gene while inserting ilvE (B) gene from Bacillus subtilis" in E.coli producing valine. The fermentation recombinant escherichia coli can improve the yield of the L-valine. The invention has important application value.

Description

Recombinant escherichia coli for fermentative production of L-valine
Technical Field
The invention belongs to the technical field of biology, and particularly relates to recombinant escherichia coli for producing L-valine by fermentation.
Background
L-valine has important application in the fields of food, medicine, health care products, feeds and the like. At present, a plurality of enterprises in China perform L-valine production by fermentation, and a plurality of laboratories are also developing research on L-valine fermentation processes. Researchers find that the process of secreting intracellular L-valine to the outside of cells through cell membranes requires the participation of specific transport proteins, but in the process, the activity of the specific transport proteins cannot meet the requirement of timely secreting the intracellular L-valine to the outside of cells through cell membranes, so that the intracellular L-valine is accumulated; meanwhile, the acetohydroxy acid synthase is contained in the bacterial cells, and has a catalytic effect on the biosynthesis of L-valine, so that the acid production can be promoted, but when the L-valine synthesized in the bacterial cells cannot be timely discharged and accumulated, the acetohydroxy acid synthase is strongly inhibited by the L-valine, the catalytic effect is reduced, the L-valine cannot be further catalyzed and synthesized, the continuous synthesis of the L-valine is limited, and finally the yield is low. In addition, it was found that Escherichia coli is very sensitive to L-valine, and when L-valine in L-valine-producing bacteria cannot be timely discharged to accumulate, growth of Escherichia coli is severely inhibited, and synthesis of L-valine in bacteria cells is inhibited, resulting in low yield.
Disclosure of Invention
The main object of the present invention is how to increase the yield of L-amino acids, which is not limited to the described subject matter, and other objects not mentioned herein will be apparent to those skilled in the art from the following description.
The invention firstly protects an engineering bacterium which can be a microorganism for inhibiting or down-regulating the expression quantity and/or activity of alcohol dehydrogenase in vivo;
the microorganism can produce valine.
The alcohol dehydrogenase is B1), B2) or B3):
b1 Amino acid sequence shown in SEQ ID No: 11;
b2 A protein which has 90% or more identity with the protein shown in B1) and has the same function as the protein shown in B1) and is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in B1);
b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).
In the engineering bacteria, the inhibition or the downregulation of the expression quantity and/or the activity of the alcohol dehydrogenase is realized by knocking out or knocking down the encoding gene (namely adhE gene) of the alcohol dehydrogenase in the microorganism.
The inhibition or downregulation of the expression level and/or activity of alcohol dehydrogenase is achieved by introducing into the microorganism the pGRB-adhE sgRNA plasmid mentioned in the examples and the sequence of SEQ ID No:3 and the ΔadhE-Up-Down fragment shown in FIG. 3.
The engineering bacteria also inhibit or down regulate the expression quantity and/or activity of the microbial branched chain amino acid aminotransferase in vivo and contain or express the bacillus subtilis branched chain amino acid aminotransferase.
The microbial source branched chain amino acid transaminase is C1), C2) or C3):
c1 Amino acid sequence shown in SEQ ID No: 12;
c2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in the C1), has more than 90% of identity with the protein shown in the B1) and has the same function;
c3 Fusion proteins with the same function obtained by ligating a tag at the N-terminal and/or C-terminal of C1) or C2).
The branched chain amino acid transaminase derived from bacillus subtilis is D1), D2) or D3):
d1 Amino acid sequence shown in SEQ ID No: 13;
d2 A protein which has 90% or more identity with the protein shown in B1) and has the same function, and is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in D1);
d3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of D1) or D2).
In the engineering bacteria, the inhibition or down-regulation of the expression level and/or activity of the branched-chain amino acid transaminase from the microorganism is achieved by knocking out or knocking down the encoding gene of the branched-chain amino acid transaminase (i.e., ilvE (E) gene) in the microorganism.
In the above engineering bacterium, the expression or expression of the branched chain amino acid aminotransferase derived from Bacillus subtilis is achieved by knocking in or introducing a gene encoding the branched chain amino acid aminotransferase derived from Bacillus subtilis (i.e., ilvE (B) gene) into the microorganism.
In the engineering bacterium, the knocking-in or introducing of the coding gene of the branched-chain amino acid transaminase derived from bacillus subtilis into the microorganism is realized by introducing an expression cassette into the microorganism; the expression cassette comprises a promoter and a coding gene of branched chain amino acid aminotransferase derived from bacillus subtilis. The promoter may be an inducible promoter. The inducible promoter may specifically be the ptrc promoter.
The inhibition or downregulation of the expression level and/or activity of a branched-chain amino acid transaminase of microbial origin and the inclusion or expression of a branched-chain amino acid transaminase of Bacillus subtilis origin can be achieved by introducing into the microorganism the pGRB-ilvE sgRNA plasmids mentioned in the examples and the sequence of SEQ ID No:6, the ptrc-ilvE (B) -Up-Down fragment is implemented.
The expression level and/or activity of the thiamine phosphate synthase can be inhibited or down-regulated in the body of the engineering bacteria.
The phosphorothioate synthase is E1), E2) or E3):
e1 Amino acid sequence shown in SEQ ID No: 14;
e2 A protein which has 90% or more identity with the protein shown in B1) and has the same function, and is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in E1);
e3 Fusion proteins with the same function obtained by ligating a tag at the N-terminal and/or C-terminal of E1) or E2).
In the engineering bacteria, the inhibition or the down-regulation of the expression quantity and/or the activity of the thiamine phosphate synthase is realized by knocking out or knocking down a coding gene (namely, thiE gene) of the thiamine phosphate synthase in the microorganism.
The inhibition or downregulation of the expression level and/or activity of the phosphorothioate-thioic acid synthase can be achieved by introducing into the microorganism the pGRB-thiE sgRNA plasmids mentioned in the examples and SEQ ID No: 8. DELTA.thiE-Up-Down fragment.
The expression level and/or activity of malate dehydrogenase is also inhibited or down-regulated in the body of the engineering bacteria.
The malate dehydrogenase is F1), F2) or F3):
f1 Amino acid sequence shown in SEQ ID No:15, a protein represented by formula (i);
F2 A protein which is obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence of the protein shown in the F1), has more than 90% of identity with the protein shown in the B1) and has the same function;
f3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of F1) or F2).
In the engineering bacteria, the inhibition or the downregulation of the expression quantity and/or the activity of the malate dehydrogenase is realized by knocking out or knocking down a coding gene (namely mdh gene) of the malate dehydrogenase in the microorganism.
The inhibition or downregulation of the expression level and/or activity of malate dehydrogenase can be achieved by introducing into the microorganism the pGRB-mdh sgRNA plasmids mentioned in the examples and SEQ ID No:10, and the Δmdh-Up-Down fragment shown in FIG. 10.
The present invention also provides a method for producing an engineering bacterium for producing valine, comprising the steps of (a 1): reducing the expression level and/or activity of any one of the above-mentioned alcohol dehydrogenases in a microorganism;
the microorganism can produce valine.
In the method, the reduction of the expression amount and/or activity of the alcohol dehydrogenase in the microorganism is achieved by knocking out or knocking down the gene encoding the alcohol dehydrogenase in the microorganism (i.e., adhE gene).
The reduction of the expression level and/or activity of alcohol dehydrogenase in microorganisms is achieved by introducing into the microorganism the pGRB-adhE sgRNA plasmid mentioned in the examples and the sequence of SEQ ID No:3 and the ΔadhE-Up-Down fragment shown in FIG. 3.
The method further comprises the step (a 2): after completion of step (a 1), the expression level and/or activity of the branched chain amino acid transaminase derived from the microorganism is reduced and the branched chain amino acid transaminase derived from Bacillus subtilis is expressed in the microorganism.
The microbial source branched chain amino acid transaminase is C1), C2) or C3):
c1 Amino acid sequence shown in SEQ ID No: 12;
c2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in the C1), has more than 90% of identity with the protein shown in the B1) and has the same function;
c3 Fusion proteins with the same function obtained by ligating a tag at the N-terminal and/or C-terminal of C1) or C2).
The branched chain amino acid transaminase derived from bacillus subtilis is D1), D2) or D3):
d1 Amino acid sequence shown in SEQ ID No: 13;
d2 A protein which has 90% or more identity with the protein shown in B1) and has the same function, and is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence of the protein shown in D1);
D3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of D1) or D2).
In the method, the reduction of the expression level and/or activity of the branched-chain amino acid transaminase in the microorganism is achieved by knocking out or knocking down a gene encoding the branched-chain amino acid transaminase in the microorganism, i.e., ilvE (E) gene. The expression of the branched-chain amino acid aminotransferase derived from Bacillus subtilis in the microorganism is achieved by knocking in or introducing a gene encoding the branched-chain amino acid aminotransferase derived from Bacillus subtilis, i.e., ilvE (B) gene, into the microorganism. The knocking-in or introducing of the encoding gene of the branched-chain amino acid transaminase derived from bacillus subtilis into the microorganism is realized by introducing an expression cassette into the microorganism; the expression cassette comprises a promoter and a coding gene of branched chain amino acid aminotransferase derived from bacillus subtilis. The promoter may be an inducible promoter. The inducible promoter may specifically be the ptrc promoter.
The reduction of the expression level and/or activity of the branched-chain amino acid aminotransferase from the microorganism and the expression of the branched-chain amino acid aminotransferase from Bacillus subtilis in the microorganism can be achieved by introducing into the microorganism the pGRB-ilvE sgRNA plasmid and the SEQ ID No:6, the ptrc-ilvE (B) -Up-Down fragment is implemented.
The method further comprises the step (a 3): after step (a 2) is completed, the expression level and/or activity of any one of the above-mentioned thiamine phosphate synthases in the microorganism is reduced.
In the method, the reduction of the expression level and/or activity of the thiamine phosphate synthase in the microorganism is achieved by knocking out or knocking down the gene encoding the thiamine phosphate synthase in the microorganism (i.e., the thiE gene).
Said reducing the expression level and/or activity of said phosphorothioate thiamine enzyme of any of the above-mentioned microorganisms can be achieved by introducing into the microorganism the pGRB-thiE sgRNA plasmid and SEQ ID No: 8. DELTA.thiE-Up-Down fragment.
The method further comprises step (a 4): after step (a 3) is completed, the expression level and/or activity of any one of the malate dehydrogenases in the microorganism is reduced.
In the method, the reduction of the expression level and/or activity of malate dehydrogenase in the microorganism is achieved by knocking out or knocking down the gene encoding malate dehydrogenase in the microorganism (i.e., mdh gene).
The reduction of the expression level and/or activity of malate dehydrogenase in the microorganism can be achieved by introducing into the microorganism the pGRB-mdh sgRNA plasmid mentioned in the examples and the sequence of SEQ ID No:10, and the Δmdh-Up-Down fragment shown in FIG. 10.
The invention also protects the engineering bacteria described above or the application of the engineering bacteria prepared by any one of the methods described above, and the engineering bacteria can be A1), A2) or A3):
a1 Production of L-amino acids;
a2 Preparing a product for producing an L-amino acid;
a3 Preparing food, feed or medicine containing L-amino acid.
The invention also provides a method for producing or preparing L-amino acid, comprising the following steps: fermenting and culturing the engineering bacteria or the engineering bacteria prepared by the method, and collecting fermentation products to obtain L-amino acid.
The culture may be performed according to a conventional method in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, fed-batch culture, etc., and various culture conditions such as temperature, time, pH of the medium, etc., may be appropriately adjusted according to the actual situation.
Herein, the L-amino acid may include L-valine, L-isoleucine, L-threonine, L-tryptophan, L-arginine, L-lysine, L-glutamic acid, L-glycine, L-alanine, L-leucine, L-methionine, L-proline, L-serine, L-tyrosine, L-cysteine, L-phenylalanine, L-asparagine, L-glutamine, L-aspartic acid and/or L-histidine.
Any of the above L-amino acids may be specifically L-valine.
Herein, identity refers to identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria are derived from the genus EscherichiaEscherichia sp.) Genus ErwiniaErwinia sp.) Genus AgrobacteriumAgrobacterium sp.) The genus FlavobacteriumFlavobacterium sp.) Genus AlcaligenesAlcaligenes sp.) Genus PseudomonasPseudomonas sp.) Bacillus genusBacillus sp.) Genus BrevibacteriumBrevibacterium sp.) Genus CorynebacteriumCorynebacterium sp.) The genus Aerobacter Aerobacter sp.) The enterobacter genusEnterobacteria sp.) Micrococcus genusMicrococcus sp.) Serratia genusSerratia sp.) Salmonella genusSalmonella sp.) Streptomyces genusStreptomyces sp.) Provedsia species @Providencia sp.) And the like, but is not limited thereto.
Further, the bacteria may be Escherichia coli @, or a mixture thereofEscherichia coli) Corynebacterium glutamicumCorynebacterium glutamicum) Brevibacterium lactofermentumBrevibacterium lactofermentum) Brevibacterium flavum (Brevibacterium flavum) and preparation method thereofBrevibacterium flavum) Beijing corynebacteriumCorynebacterium pekinense) Brevibacterium ammoniagenesBrevibacterium ammoniagenes，Brevibacterium ammoniagenes and corynebacterium crenatumCorynebacterium crenatum) Or Pantoea (L.) DielsPantoea) But is not limited thereto.
In one or more embodiments of the present application, the microorganism is Escherichia coliEscherichia coli). Specifically, the Escherichia coli may be Escherichia coli @, or a mixture thereofEscherichia coli) YP045 CGMCC No.22721 or E.coli W3110.
The inventors of the present application have found through a large number of experiments that, in Escherichia coli (e.g., escherichia coli @Escherichia coli) YP045 CGMCC No.22721 or E.coli W3110) by "knockoutThe engineering bacteria obtained by inserting ptrc-initiated ilvE (B) gene from bacillus subtilis into the adhE gene, the adhE gene and the ilvE (E) gene, inserting ptrc-initiated ilvE (B) gene from bacillus subtilis into the adhE gene and the thiE gene and inserting ptrc-initiated ilvE (E) gene from bacillus subtilis into the ilvE gene, or inserting ptrc-initiated ilvE (B) gene from bacillus subtilis into the adhE gene, thiE gene and mdh gene and inserting ptrc-initiated ilvE (E) gene into the ilvE (B) gene from bacillus subtilis can remarkably improve the yield of L-amino acid (such as L-valine). The application has important application value.
Preservation description:
strain name: escherichia coli
Latin name:Escherichia coli
strain number: YP045
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2021, 06, 15
Accession numbers of the preservation center: CGMCC No.22721
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.
Escherichia coli @Escherichia coli) YP045 is useful for the production of valine which has been deposited in China at 2021, month 06 and 15The general microbiological culture collection center (CGMCC for short, the address is 1 # 3 of North West Lu of the Chaoyang area of Beijing city), and the collection number is 22721. Escherichia coli @ Escherichia coli) YP045 is called Escherichia coliEscherichia coli) YP045 CGMCC No.22721, hereinafter referred to as valine-producing bacterium CGMCC 22721.
Example 1 engineering bacterium transformation starting from valine-producing bacterium CGMCC 22721
Through a large number of experiments, the inventor of the invention starts from valine-producing bacteria CGMCC 22721 and carries out transformation to obtain engineering bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03 and YPVal-adhE04. Genotypes of engineering bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03 and YPVal-adhE04 are shown in Table 1.
1. Acquisition of engineering bacterium YPVal-adhE01
Coli according to NCBI publicationEscherichia coli) The W3110 genome sequence is used for knocking out adhE genes in a valine-producing bacterium CGMCC 22721 genome by using CRISPR/Cas9 gene editing technology.
The adhE Gene codes for alcohol dehydrogenase, the Gene ID of which is 945837, and the amino acid sequence of which is shown in SEQ ID No: 11.
The method comprises the following specific steps:
1. construction of pGRB-adhE sgRNA plasmid
Coli according to NCBI publicationEscherichia coli) W3110 genomic sequence, using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer/design of the sgRNA target sequence for knocking out adhE gene (SEQ ID No: 2), linearized pGRB vector homology arm sequences were added at the 5 'and 3' ends of the target sequence for construction of the sgRNA plasmid.
(1) Primer sgRNA-2F was synthesized by the company Invitrogen:
5’-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTaagaaccacaacccagagtcaggGTTTTAGA GCTAGAAATAGCAAGTTAAAATAAGG-3' (underlined as pGRB vector homology arm sequence), primer sgRNA-2R:
5’-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcctgactctgggttgtggttcttACTAGTAT TATACCTAGGACTGAGCTAGCTGTCA-3' (underlined as pGRB vector homology arm sequence), primer sgRNA-PF:
5'-GTCTCATGAGCGGATACATATTTG-3' and primer sgRNA-PR:
5’-ATGAGAAAGCGCCACGCT-3’。
(2) Annealing the primer sgRNA-2F and the primer sgRNA-2R (reaction procedure: denaturation at 95 ℃ C. For 5min, annealing at 50 ℃ C. For 1 min), then recovering the target fragment by using a DNA purification kit, measuring the DNA concentration, and diluting the concentration to 100 ng/. Mu.L to obtain an annealed product. The annealed product contains a nucleotide sequence shown as SEQ ID No:2, and a sgRNA-2 fragment shown in FIG. 2.
(3) By restriction enzymesSpeThe pGRB vector was digested and the approximately 2700bp DNA fragment was recovered using DNA recovery kit (QIAGEN Gel Extraction Kit).
The cleavage system was 50. Mu.L, and the cleavage system was composed of 5. Mu.L of 10 XBuffer (restriction enzymeSpeI self-contained), 2.5. Mu.L restriction enzymeSpeI. 3000-5000ng pGRB vector and ddH 2 O composition.
And (3) enzyme cutting: 3h at 37 ℃.
(4) And (3) carrying out dephosphorylation reaction (aiming at preventing self-connection of pGRB vectors) on the DNA fragment recovered in the step (3), and recovering the DNA fragment by using a DNA recovery kit to obtain the linearized pGRB vector.
The dephosphorylation system was 50. Mu.L, and the DNA fragment recovered from step (3) was 5. Mu.L of 10 XBuffer (CIAP self-contained), 1000-2000ng, 2.5. Mu.L of CIAP and ddH 2 O composition.
Dephosphorylation procedure: and 1h at 37 ℃.
(5) And (3) recombining the linearized pGRB vector obtained in the step (4) and the annealed product obtained in the step (2) by using a Gibson Assembly kit, and then converting the recombinant product into E.coli DH5 alpha competent cells to obtain the pGRB-adhE sgRNA plasmid.
The recombination system was 5. Mu.L, consisting of 2. Mu.L of linearized pGRB vector, 0.5. Mu.L of annealed product and 2.5. Mu.L of assembled enzyme (Gibson Assembly kit self-contained).
Recombination procedure: assembling at 50deg.C for 30min.
2. Acquisition of ΔadhE-Up-Down fragment
(1) Coli according to NCBI publicationEscherichia coli) The W3110 genomic sequence, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by the company Invitrogen.
The primer pair for amplifying the upstream homology arm consists of primer P5:5'-GTGCCAGTCATCCTTCAGGT-3' and primer P6: 5'-CGTTCCGACCACTAACCCGACTTGGGTATTCCGAAATCTATCC-3'.
The primer pair for amplifying the downstream homology arm consists of primer P7:
5'-GGATAGATTTCGGAATACCCAAGTCGGGTTAGTGGTCGGAACG-3' and primer P8:
5'-AAGCGATGCTGAAAGGTGTC-3'.
(2) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P5 and a primer P6 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering the upstream homology arm with the size of 765 bp.
(3) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P7 and a primer P8 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering the downstream homology arm with the size of 643 bp.
(4) Mixing the upstream homology arm recovered in the step (2) and the downstream homology arm recovered in the step (3) and using the mixture as a template, and performing overlap PCR by using a primer pair consisting of a primer P5 and a primer P8 to obtain SEQ ID No: 3. DELTA.adhE-Up-Down fragment shown in FIG. 3.
3. Acquisition of CGMCC 22721-Cas9 Strain
(1) Plasmid pREDCas9 (product catalog number 71541; containing spectinomycin resistance gene) was transformed into competent cells of valine-producing bacterium CGMCC 22721, and then spread on 2-YT agar plates containing 100mg/L spectinomycin, and cultured at 32℃to obtain single colonies resistant to spectinomycin.
The preparation method of the 2-YT agar plate is as follows: 16g of tryptone, 10g of yeast extract powder, 5g of sodium chloride and 16g of agar powder are dissolved in a proper amount of water, the volume is fixed to 1L by water, the pH value is regulated to 7.0 by sodium hydroxide, and the mixture is sterilized for 20 minutes at 121 ℃.
(2) And (3) respectively taking the single colonies obtained in the step (1) as templates, and adopting a primer pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' and primer pRedCas9-PR: and 5'-CCTTGGTGATCTCGCCTTTC-3', performing PCR amplification to obtain a PCR amplification product. If the PCR amplified product obtained by a single colony contains a nucleotide sequence shown as SEQ ID No:1, the colony contains plasmid pREDCas9. The strain of the colony is named CGMCC 22721-Cas9 strain.
4. Acquisition of engineering bacterium YPVal-adhE01
(1) Culturing CGMCC 22721-Cas9 strain; when CGMCC 22721-Cas9 strain grows to OD 600nm Adding IPTG at 0.1 and making the concentration of IPTG in the system to be 0.1mM, and continuing to culture to induce lambda-Red mediated homologous recombination; when CGMCC 22721-Cas9 strain grows to OD 600nm At 0.6, the thalli are collected and CGMCC 22721-Cas9 competent cells are prepared.
(2) The pGRB-adhE sgRNA plasmid obtained in the step 1 and the delta adhE-Up-Down fragment obtained in the step 2 are transformed into CGMCC 22721-Cas9 competent cells, coated on a 2-YT agar plate containing 100mg/L spectinomycin and 100mg/L ampicillin, and cultured at 32 ℃ to obtain single colonies.
(3) And (3) respectively taking the single colony obtained in the step (2) as a template, and adopting a primer pair consisting of a primer P5 and a primer P8 to carry out PCR amplification to obtain a PCR amplification product. If a single colony obtained PCR amplification product contains a DNA fragment of 1365bp in size, the colony is initially identified as a positive transformant.
(4) Inoculating the positive transformant obtained in the step (3) to a 2-YT agar plate containing 100mg/L spectinomycin and 0.2% (m/v) arabinose, and culturing at 32 ℃ (for eliminating pGRB-adhE sgRNA plasmid); colonies that grew on the 2-YT agar plates containing 100mg/L spectinomycin but did not grow on the 2-YT agar plates containing 100mg/L ampicillin were then selected, and these colonies were transferred to the 2-YT agar plates and cultured at 42℃for elimination of plasmid pREDCas 9; finally, colonies were selected which did not grow on 2-YT agar plates containing 100mg/L spectinomycin, but which grew on 2-YT agar plates without resistance.
(5) And (3) taking the single colony obtained in the step (4) as a template, and adopting a primer pair consisting of a primer P5 and a primer P8 to carry out PCR amplification to obtain a PCR amplification product. If a single colony resulted in a PCR amplification product containing a DNA fragment of 1365bp in size, the colony was identified as a positive transformant. The positive transformant is obtained by knocking out the adhE gene of the valine-producing bacterium CGMCC 22721 genome, and is named as engineering bacterium YPVal-adhE01.
2. Obtaining engineering bacterium YPVal-adhE02
Coli according to NCBI publicationEscherichia coli) Genomic sequence of W3110 and bacillus subtilis Bacillus subtilis subsp.subtilis str.168The ilvE (E) gene of the engineering bacteria YPVal-adhE01 genome obtained in the first step is knocked out by using CRISPR/Cas9 gene editing technology, and simultaneously the ilvE gene of bacillus subtilis (namely, ptrc-ilvE (B) sequence) started by a ptrc promoter is knocked in, so that engineering bacteria YPVal-adhE02, which is hereinafter called YPVal-adhE02, is obtained. The nucleotide sequence of the ptrc-ilvE (B) sequence is shown as SEQ ID No:4, SEQ ID No:4, from the 5' -end, the nucleotide sequence of the ptrc promoter is located at 1096-1169 and the bacillus subtilis ilvE (B) gene is located at 1-1095.
The ilvE (E) Gene of engineering bacterium YPVal-adhE01 codes branched chain amino acid aminotransferase, the Gene ID is 948278, and the amino acid sequence is shown in SEQ ID No: shown at 12.
The bacillus subtilis ilvE (B) Gene codes branched chain amino acid aminotransferase, the Gene ID is 938420, and the amino acid sequence is shown in SEQ ID No: shown at 13.
The method comprises the following specific steps:
1. construction of pGRB-ilvE sgRNA plasmid
(1) Primer sgRNA-3F was synthesized by the company Invitrogen:
5’-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTgaaagcagcgataatcacgtcggGTTTTAGA GCTAGAAATAGCAAGTTAAAATAAGG-3' (underlined as pGRB vector homology arm sequence) and primer sgRNA-3R:
5’-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACccgacgtgattatcgctgctttcACTAGTAT TATACCTAGGACTGAGCTAGCTGTCA-3' (underlined as pGRB vector homology arm sequence).
(2) Annealing the primer sgRNA-3F and the primer sgRNA-3R (reaction procedure: denaturation at 95 ℃ C. For 5min, annealing at 50 ℃ C. For 1 min), then recovering the target fragment by using a DNA purification kit, measuring the DNA concentration, and diluting the concentration to 100 ng/. Mu.L to obtain an annealed product. The annealed product contains a nucleotide sequence shown as SEQ ID No:5, and a sgRNA-3 fragment shown in FIG. 5.
(3) By restriction enzymesSpeI (product of Takara Co., ltd., catalog No. 1631) and pGRB vector (product of addgene Co., catalog No. 71539) were digested, and a DNA fragment of about 2700bp was recovered by using a DNA recovery kit (QIAGEN Gel Extraction Kit).
The cleavage system was 50. Mu.L, and the cleavage system was composed of 5. Mu.L of 10 XBuffer (restriction enzymeSpeI self-contained), 2.5. Mu.L restriction enzymeSpeI. 3000-5000ng pGRB vector and ddH 2 O composition.
And (3) enzyme cutting: 3h at 37 ℃.
(4) And (3) carrying out dephosphorylation reaction (aiming at preventing self-connection of pGRB vectors) on the DNA fragment recovered in the step (3), and recovering the DNA fragment by using a DNA recovery kit to obtain the linearized pGRB vector.
The dephosphorylation system was 50. Mu.L, and the DNA fragment recovered in step (3) was 5. Mu.L of 10 XBuffer (CIAP self-contained), 1000-2000ng, 2.5. Mu.L of CIAP (product of Takara Co., ltd., catalog number 2250A) and ddH 2 O composition.
Dephosphorylation procedure: and 1h at 37 ℃.
(5) And (3) recombining the linearized pGRB vector obtained in the step (4) and the annealed product obtained in the step (2) by using a Gibson Assembly kit (New England Co.), and then transforming the recombinant product into escherichia coli DH5 alpha competent cells (TAKARA) to obtain the pGRB-ilvE sgRNA plasmid.
The recombination system was 5. Mu.L, consisting of 2. Mu.L of linearized pGRB vector, 0.5. Mu.L of annealed product and 2.5. Mu.L of assembled enzyme (Gibson Assembly kit self-contained).
Recombination procedure: assembling at 50deg.C for 30min.
2. Acquisition of the ptrc-ilvE (B) -Up-Down fragment
(1) Coli according to NCBI publicationEscherichia coli) The W3110 genomic sequence, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by the company Invitrogen. Bacillus subtilis according to NCBI publicationBacillus subtilis subsp.subtilis str.168Genomic sequences, primer pairs for amplifying ptrc-ilvE (B) were designed and synthesized by the company Invitrogen.
The primer pair for amplifying the upstream homology arm consists of primer P9: 5'-CAGGCAGTTCATTGAGTTAGCG-3' and primer P10: 5'-CACAGTGTATTAAGCAGACGTTAAATACAAAAAATGGGACGGCAC-3'.
The primer pair for amplifying the downstream homology arm consists of primer P13:
5’-GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTTTATATTCCTTTTGCGCTC-3' (underlined as part of the sequence of the ptrc promoter) and primer P14:
5'-ACGGTTAGGGATGGTTCGAC-3'.
The primer pair for amplifying ptrc-ilvE (B) consists of primer P11:
5'-GTGCCGTCCCATTTTTTGTATTTAACGTCTGCTTAATACACTGTG-3' and primer P12:
5’-GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAACTTTTTAAATATATGGAG-3' (underlined as part of the sequence of the ptrc promoter).
(2) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P9 and a primer P10 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering an upstream homology arm with the size of 709 bp.
(3) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P13 and a primer P14 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering a downstream homology arm with the size of 611 bp.
(4) By bacillus subtilisBacillus subtilis subsp.subtilis str.168The genome DNA of (2) is used as a template, a primer pair consisting of a primer P11 and a primer P12 is adopted to carry out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is adopted to recover ilvE (B) fragments with the size of 1168 bp.
(5) Mixing the upstream homology arm recovered in the step (2), the downstream homology arm recovered in the step (3) and the ilvE (B) fragment recovered in the step (4) and using the mixture as a template, and performing overlap PCR by using a primer pair consisting of a primer P9 and a primer P14 to obtain SEQ ID No:6, a ptrc-ilvE (B) -Up-Down fragment.
3. Obtaining of YPVal-adhE01-Cas9 Strain
(1) Plasmid pREDCas9 is transformed into engineering bacteria YPVal-adhE01 competent cells, and then the engineering bacteria are coated on a 2-YT agar plate containing 100mg/L spectinomycin, and cultured at 32 ℃ to obtain single colony resisting the spectinomycin.
(2) And (3) respectively taking the single colony obtained in the step (1) as a template, and adopting a primer pair consisting of a primer pRedCas9-PF and a primer pRedCas9-PR to carry out PCR amplification to obtain a PCR amplification product. If the PCR amplified product obtained by a single colony contains a nucleotide sequence shown as SEQ ID No:1, the colony contains plasmid pREDCas9. The strain of this colony was designated as YPEL-adhE 01-Cas9 strain.
4. Obtaining engineering bacterium YPVal-adhE02
(1) Culturing a YPVal-adhE01-Cas9 strain; when YPEL-adhE 01-Cas9 strain grows to OD 600nm Adding IPTG at 0.1 and making the concentration of IPTG in the system to be 0.1mM, and continuing to culture to induce lambda-Red mediated homologous recombination; when YPEL-adhE 01-Cas9 strain grows to OD 600nm At 0.6, the cells were collected and YPEL-adhE 01-Cas9 strain competent cells were prepared.
(2) pGRB-ilvE sgRNA plasmid obtained in step 1 and ptrc-ilvE (B) -Up-Down fragment obtained in step 2 were transformed into YVal-adhE 01-Cas9 strain competent cells, plated on 2-YT agar plates containing 100mg/L spectinomycin and 100mg/L ampicillin, and cultured at 32℃to obtain single colonies.
(3) And (3) respectively taking the single colony obtained in the step (2) as a template, and adopting a primer pair consisting of a primer P11 and a primer P12 to carry out PCR amplification to obtain a PCR amplification product. If a single colony obtained PCR amplification product contains a 1168bp DNA fragment, the colony is initially identified as a positive transformant.
(4) Inoculating the positive transformant obtained in the step (3) to a 2-YT agar plate containing 100mg/L spectinomycin and 0.2% (m/v) arabinose, and culturing at 32 ℃ (for eliminating pGRB-ilvE sgRNA plasmid); colonies that grew on the 2-YT agar plates containing 100mg/L spectinomycin but did not grow on the 2-YT agar plates containing 100mg/L ampicillin were then selected, and these colonies were transferred to the 2-YT agar plates and cultured at 42℃for elimination of plasmid pREDCas 9; finally, colonies were selected which did not grow on 2-YT agar plates containing 100mg/L spectinomycin, but which grew on 2-YT agar plates without resistance.
(5) And (3) taking the single colony obtained in the step (4) as a template, and adopting a primer pair consisting of a primer P11 and a primer P12 to carry out PCR amplification to obtain a PCR amplification product. If a single colony resulted in a PCR amplification product containing a DNA fragment of 1168bp, the colony was identified as a positive transformant. The positive transformant is obtained by knocking out ilvE (E) genes of engineering bacterium YPVal-adhE01 genome and knocking in bacillus subtilis ilvE (B) genes (namely ptrc-ilvE (B) sequences) started by ptrc promoters, and is named as engineering bacterium YPVal-adhE02.
3. Acquisition of engineering bacterium YPVal-adhE03
Engineering bacterium YPLA-adhE 02 is taken as starting bacterium, and the escherichia coli is published according to NCBIEscherichia coli) The W3110 genome sequence is used for knocking out the thiE gene in the engineering bacterium YPVal-adhE02 genome by using CRISPR/Cas9 gene editing technology.
the thiE Gene codes for thiamine phosphate synthase, the Gene ID is 948491, and the amino acid sequence is shown in SEQ ID No: 14.
The method comprises the following specific steps:
1. construction of pGRB-thiE sgRNA plasmid
Coli according to NCBI publicationEscherichia coli) W3110 genomic sequence, using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer/design of the sgRNA target sequence for knockout of the thiE gene (SEQ ID No: 7), linearized pGRB vector homology arm sequences were added at the 5 'and 3' ends of the target sequence for construction of the sgRNA plasmid.
(1) Primer sgRNA-4F was synthesized by the company Invitrogen:
5’-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTcgcccctcttatatcgcgctgggGTTTTAGA GCTAGAAATAGCAAGTTAAAATAAGG-3' (underlined as pGRB vector homology arm sequence) and primer sgRNA-4R:
5’-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcccagcgcgatataagaggggcgACTAGTAT TATACCTAGGACTGAGCTAGCTGTCA-3' (underlined as pGRB vector homology arm sequence).
(2) Annealing the primer sgRNA-4F and the primer sgRNA-4R (reaction procedure: denaturation at 95 ℃ C. For 5min, annealing at 50 ℃ C. For 1 min), then recovering the target fragment by using a DNA purification kit, measuring the DNA concentration, and diluting the concentration to 100 ng/. Mu.L to obtain an annealed product. The annealed product contains a nucleotide sequence shown as SEQ ID No:7, a fragment of sgRNA-4.
(3) By restriction enzymesSpeThe pGRB vector was digested and the approximately 2700bp DNA fragment was recovered using DNA recovery kit (QIAGEN Gel Extraction Kit).
The cleavage system was 50. Mu.L, and the cleavage system was composed of 5. Mu.L of 10 XBuffer (restriction enzymeSpeI self-contained), 2.5. Mu.L restriction enzymeSpeI. 3000-5000ng pGRB vector and ddH 2 O composition.
And (3) enzyme cutting: 3h at 37 ℃.
(4) And (3) carrying out dephosphorylation reaction (aiming at preventing self-connection of pGRB vectors) on the DNA fragment recovered in the step (3), and recovering the DNA fragment by using a DNA recovery kit to obtain the linearized pGRB vector.
The dephosphorylation system was 50. Mu.L, and the DNA fragment recovered from step (3) was 5. Mu.L of 10 XBuffer (CIAP self-contained), 1000-2000ng, 2.5. Mu. L CIAP and ddH 2 O composition.
Dephosphorylation procedure: and 1h at 37 ℃.
(5) And (3) recombining the linearized pGRB vector obtained in the step (4) and the annealed product obtained in the step (2) by using a Gibson Assembly kit, and then converting the recombinant product into E.coli DH5 alpha competent cells to obtain the pGRB-thiE sgRNA plasmid.
The recombination system was 5. Mu.L, consisting of 2. Mu.L of linearized pGRB vector, 0.5. Mu.L of annealed product and 2.5. Mu.L of assembled enzyme (Gibson Assembly kit self-contained).
Recombination procedure: assembling at 50deg.C for 30min.
2. Acquisition of the DeltathiE-Up-Down fragment
(1) Coli according to NCBI publicationEscherichia coli) The W3110 genomic sequence, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by the company Invitrogen.
The primer pair for amplifying the upstream homology arm consists of primer P15:5'-TTCTATTCAGGACGCCAACG-3' and primer P16: 5'-GCTATAACGCATAAAGTCACGGCACGCTTCCTCCTTACGCAGG-3'.
The primer pair for amplifying the downstream homology arm consists of primer P17:
5'-CCTGCGTAAGGAGGAAGCGTGCCGTGACTTTATGCGTTATAGC-3' and primer P18:
5'-GCCTGCAAAGTGCCCATAACCC-3'.
(2) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P15 and a primer P16 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering an upstream homology arm with the size of 721 bp.
(3) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P17 and a primer P18 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering the downstream homology arm with the size of 618 bp.
(4) Mixing the upstream homology arm recovered in the step (2) and the downstream homology arm recovered in the step (3) and using the mixture as a template, and performing overlap PCR by using a primer pair consisting of a primer P15 and a primer P18 to obtain SEQ ID No: 8. DELTA.thiE-Up-Down fragment shown in FIG.
3. Obtaining of YPVal-adhE02-Cas9 Strain
(1) Plasmid pREDCas9 is transformed into engineering bacteria YPVal-adhE02 competent cells, and then the engineering bacteria are coated on a 2-YT agar plate containing 100mg/L spectinomycin, and cultured at 32 ℃ to obtain single colony resisting the spectinomycin.
(2) And (3) respectively taking the single colony obtained in the step (1) as a template, and adopting a primer pair consisting of a primer pRedCas9-PF and a primer pRedCas9-PR to carry out PCR amplification to obtain a PCR amplification product. If the PCR amplified product obtained by a single colony contains a nucleotide sequence shown as SEQ ID No:1, the colony contains plasmid pREDCas9. The strain of this colony was designated as YPEL-adhE 02-Cas9 strain.
4. Acquisition of engineering bacterium YPVal-adhE03
(1) Culturing a YPEL-adhE 02-Cas9 strain; when YPEL-adhE 02-Cas9 strain grows to OD 600nm Adding IPTG at 0.1 and making the concentration of IPTG in the system to be 0.1mM, and continuing to culture to induce lambda-Red mediated homologous recombination; when YPEL-adhE 02-Cas9 strain grows to OD 600nm At 0.6, the cells were collected and YPEL-adhE 02-Cas9 strain competent cells were prepared.
(2) pGRB-thiE sgRNA plasmid obtained in step 1 and ΔthiE-Up-Down fragment obtained in step 2 were transformed into YVal-adhE 02-Cas9 competent cells, plated on 2-YT agar plates containing 100mg/L spectinomycin and 100mg/L ampicillin, and cultured at 32℃to obtain single colonies.
(3) And (3) respectively taking the single colony obtained in the step (2) as a template, and adopting a primer pair consisting of a primer P15 and a primer P18 to carry out PCR amplification to obtain a PCR amplification product. If a single colony obtained PCR amplification product contains a DNA fragment of 1296bp, the colony is initially identified as a positive transformant.
(4) Inoculating the positive transformant obtained in the step (3) to a 2-YT agar plate containing 100mg/L spectinomycin and 0.2% (m/v) arabinose, and culturing at 32 ℃ (for eliminating pGRB-thiE sgRNA plasmid); colonies that grew on the 2-YT agar plates containing 100mg/L spectinomycin but did not grow on the 2-YT agar plates containing 100mg/L ampicillin were then selected, and these colonies were transferred to the 2-YT agar plates and cultured at 42℃for elimination of plasmid pREDCas 9; finally, colonies were selected which did not grow on 2-YT agar plates containing 100mg/L spectinomycin, but which grew on 2-YT agar plates without resistance.
(5) And (3) taking the single colony obtained in the step (4) as a template, and adopting a primer pair consisting of a primer P15 and a primer P18 to carry out PCR amplification to obtain a PCR amplification product. If the PCR amplified product obtained from a single colony contains a DNA fragment of 1296bp, the colony is identified as a positive transformant with deletion of the thiE gene on the engineering bacterium YPLA-adhE 02 genome. The positive transformant was designated as engineering bacterium YPLA-adhE 03.
4. Acquisition of engineering bacterium YPLA-adhE 04
Engineering bacteria YPLA-adhE 03 is taken as a starting strain, and the strain is published according to NCBIEscherichia coli) The mdh gene in the engineering bacterium YPVal-adhE03 genome is knocked out by using a W3110 genome sequence and using CRISPR/Cas9 gene editing technology.
The mdh Gene codes for malate dehydrogenase, the Gene ID of which is 947854, and the amino acid sequence of which is shown in SEQ ID No: 15.
The method comprises the following specific steps:
1. construction of pGRB-mdh sgRNA plasmid
Coli according to NCBI publicationEscherichia coli) W3110 genomic sequence, using CRISPR RGEN Tools (http:// www.rgenome.net/cas-designer/design of the sgRNA target sequence for knockout of mdh gene (SEQ ID No: 9), linearized pGRB vector homology arm sequences were added at the 5 'and 3' ends of the target sequence for construction of the sgRNA plasmid.
(1) Primer sgRNA-5F was synthesized by the company Invitrogen:
5’-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTgcctttcagttccgcaacaaaggGTTTTAGA GCTAGAAATAGCAAGTTAAAATAAGG-3' (underlined as pGRB vector homology arm sequence) and primer sgRNA-5R:
5’-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcctttgttgcggaactgaaaggcACTAGTAT TATACCTAGGACTGAGCTAGCTGTCA-3' (underlined as pGRB vector homology arm sequence).
(2) Annealing the primer sgRNA-5F and the primer sgRNA-5R (reaction procedure: denaturation at 95 ℃ C. For 5min, annealing at 50 ℃ C. For 1 min), then recovering the target fragment by using a DNA purification kit, measuring the DNA concentration, and diluting the concentration to 100 ng/. Mu.L to obtain an annealed product. The annealed product contains a nucleotide sequence shown as SEQ ID No:9, and a sgRNA-5 fragment as shown in FIG. 9.
(3) By restriction enzymesSpeThe pGRB vector was digested and the approximately 2700bp DNA fragment was recovered using DNA recovery kit (QIAGEN Gel Extraction Kit).
The cleavage system was 50. Mu.L, and the cleavage system was composed of 5. Mu.L of 10 XBuffer (restriction enzymeSpeI self-contained), 2.5. Mu.L restriction enzymeSpeI. 3000-5000ng pGRB vector and ddH 2 O composition.
And (3) enzyme cutting: 3h at 37 ℃.
(4) And (3) carrying out dephosphorylation reaction (aiming at preventing self-connection of pGRB vectors) on the DNA fragment recovered in the step (3), and recovering the DNA fragment by using a DNA recovery kit to obtain the linearized pGRB vector.
The dephosphorylation system was 50. Mu.L, and the DNA fragment recovered from step (3) was 5. Mu.L of 10 XBuffer (CIAP self-contained), 1000-2000ng, 2.5. Mu.L of CIAP and ddH 2 O composition.
Dephosphorylation procedure: and 1h at 37 ℃.
(5) And (3) recombining the linearized pGRB vector obtained in the step (4) and the annealed product obtained in the step (2) by using a Gibson Assembly kit, and then converting the recombinant product into E.coli DH5 alpha competent cells to obtain the pGRB-mdh sgRNA plasmid.
The recombination system was 5. Mu.L, consisting of 2. Mu.L of linearized pGRB vector, 0.5. Mu.L of annealed product and 2.5. Mu.L of assembled enzyme (Gibson Assembly kit self-contained).
Recombination procedure: assembling at 50deg.C for 30min.
2. Acquisition of the Δmdh-Up-Down fragment
(1) Coli according to NCBI publicationEscherichia coli) W3110 genome sequence, set upPrimer pairs for amplifying the upstream homology arm and primer pairs for amplifying the downstream homology arm were calculated and synthesized by the company invitrogen.
The primer pair for amplifying the upstream homology arm consists of primer P19:5'-AACTTCCTCCAAACCGATGC-3' and primer P20: 5'-CAATATAATAAGGAGTTTAGGTTGATTAGCGGATAATAAAAAACC-3'.
The primer pair for amplifying the downstream homology arm consists of primer P21:
5'-GGTTTTTTATTATCCGCTAATCAACCTAAACTCCTTATTATATTG-3' and primer P22:
5'-TCTTCAATGGACTGGAGGTG-3'.
(2) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P19 and a primer P20 is used for carrying out PCR amplification by using high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering an upstream homology arm with the size of 590 bp.
(3) The genome DNA of the escherichia coli W3110 is used as a template, a primer pair consisting of a primer P21 and a primer P22 is used for carrying out PCR amplification by using a high-fidelity amplifying enzyme KAPA HiFi HotStart, and a DNA recovery kit is used for recovering the downstream homology arm with the size of 708 bp.
(4) Mixing the upstream homology arm recovered in the step (2) and the downstream homology arm recovered in the step (3) and using the mixture as a template, and performing overlap PCR by using a primer pair consisting of a primer P19 and a primer P22 to obtain SEQ ID No: 10. DELTA.mdh-Up-Down fragment shown in FIG.
3. Acquisition of YPVal-adhE03-Cas9 Strain
(1) Plasmid pREDCas9 is transformed into engineering bacteria YPVal-adhE03 competent cells, and then the engineering bacteria are coated on a 2-YT agar plate containing 100mg/L spectinomycin, and the engineering bacteria are cultured at 32 ℃ to obtain single colony resisting the spectinomycin.
(2) And (3) respectively taking the single colony obtained in the step (1) as a template, and adopting a primer pair consisting of a primer pRedCas9-PF and a primer pRedCas9-PR to carry out PCR amplification to obtain a PCR amplification product. If the PCR amplified product obtained by a single colony contains a nucleotide sequence shown as SEQ ID No:1, the colony contains plasmid pREDCas9. The strain of this colony was designated as YPEL-adhE 03-Cas9 strain.
4. Acquisition of engineering bacterium YPLA-adhE 04
(1) Culturing a YPEL-adhE 03-Cas9 strain; when YPEL-adhE 03-Cas9 strain grows to OD 600nm Adding IPTG at 0.1 and making the concentration of IPTG in the system to be 0.1mM, and continuing to culture to induce lambda-Red mediated homologous recombination; when YPEL-adhE 03-Cas9 strain grows to OD 600nm At 0.6, the cells were collected and YPEL-adhE 03-Cas9 strain competent cells were prepared.
(2) pGRB-mdh sgRNA plasmid obtained in step 1 and Δmdh-Up-Down fragment obtained in step 2 were transformed into YPVal-adhE03-Cas9 strain competent cells, plated on 2-YT agar plates containing 100mg/L spectinomycin and 100mg/L ampicillin, and cultured at 32℃to obtain single colonies.
(3) And (3) respectively taking the single colony obtained in the step (2) as a template, and adopting a primer pair consisting of a primer P19 and a primer P22 to carry out PCR amplification to obtain a PCR amplification product. If a single colony obtained PCR amplification product contains a 1253bp DNA fragment, the colony is initially identified as a positive transformant.
(4) Inoculating the positive transformant obtained in the step (3) to a 2-YT agar plate containing 100mg/L spectinomycin and 0.2% (m/v) arabinose, and culturing at 32 ℃ (for eliminating pGRB-thiE sgRNA plasmid); colonies that grew on the 2-YT agar plates containing 100mg/L spectinomycin but did not grow on the 2-YT agar plates containing 100mg/L ampicillin were then selected, and these colonies were transferred to the 2-YT agar plates and cultured at 42℃for elimination of plasmid pREDCas 9; finally, colonies were selected which did not grow on 2-YT agar plates containing 100mg/L spectinomycin, but which grew on 2-YT agar plates without resistance.
(5) And (3) taking the single colony obtained in the step (4) as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer P19 and a primer P22 to obtain a PCR amplification product. If a single colony obtained PCR amplification product contains a 1253bp DNA fragment, the colony is identified as a positive transformant with deletion of the thiE gene on the engineering bacterium YPLA-adhE 03 genome. The positive transformant was designated as engineering bacterium YPLA-adhE 04.
EXAMPLE 2 engineering bacteria transformation with E.coli W3110
The inventor of the present invention performed a great deal of experiments, and modified by starting with E.coli W3110 to obtain engineering bacteria YPVal-adhE05, YPVal-adhE06, YPVal-adhE07 and YPVal-adhE08. Genotypes of engineering bacteria YPVal-adhE05, YPVal-adhE06, YPVal-adhE07 and YPVal-adhE08 are shown in Table 2.
1. Obtaining engineering bacterium YPVal-adhE05
1. Construction of pGRB-adhE sgRNA plasmid
Step 1 was the same as in example 1.
2. Acquisition of ΔadhE-Up-Down fragment
Step one 2 of example 1.
3. Acquisition of W3110-Cas9 Strain
According to the procedure of step 3 of example 1, the competent cells of valine-producing strain CGMCC 22721 were replaced with competent cells of E.coli W3110, and the other procedures were unchanged, thus obtaining strain W3110-Cas 9.
4. Obtaining engineering bacterium YPVal-adhE05
According to the step 4 in the step one of example 1, the CGMCC 22721-Cas9 strain is replaced by a W3110-Cas9 strain, and other steps are unchanged, so as to obtain the engineering bacterium YPVal-adhE05.
2. Acquisition of engineering bacterium YPLA-adhE 06
1. Construction of pGRB-ilvE sgRNA plasmid
Step two, 1, of example 1.
2. Acquisition of the ptrc-ilvE (B) -Up-Down fragment
Step 2 of example 1.
3. Obtaining of YPVal-adhE05-Cas9 Strain
According to the procedure of step 3 of example 1, the engineering bacterium YPVal-adhE01 competent cells were replaced with the engineering bacterium YPVal-adhE05 competent cells, and the other procedures were unchanged, to obtain YPVal-adhE05-Cas9 strain.
4. Acquisition of engineering bacterium YPLA-adhE 06
According to the procedure of step 4 of example 1, the engineering bacterium YPVal-adhE06 was obtained by replacing the YPVal-adhE01-Cas9 strain with the YPVal-adhE05-Cas9 strain in the same manner.
3. Acquisition of engineering bacterium YPLA-adhE 07
1. Construction of pGRB-thiE sgRNA plasmid
As in step three of example 1, 1.
2. Acquisition of the DeltathiE-Up-Down fragment
As in step three 2 of example 1.
3. Acquisition of YPVal-adhE06-Cas9 Strain
According to the step 3 in the step three of example 1, the engineering bacterium YPVal-adhE02 competent cells are replaced by the engineering bacterium YPVal-adhE06 competent cells, and other steps are unchanged, so as to obtain the YPVal-adhE06-Cas9 strain.
4. Acquisition of engineering bacterium YPLA-adhE 07
According to the procedure of step three 4 of example 1, the engineering bacterium YPVal-adhE07 was obtained by replacing the YPVal-adhE02-Cas9 strain with the YPVal-adhE06-Cas9 strain, all other steps being unchanged.
4. Acquisition of engineering bacterium YPLA-adhE 08
1. Construction of pGRB-mdh sgRNA plasmid
1 in step four of example 1.
2. Acquisition of the Δmdh-Up-Down fragment
As in step four 2 of example 1.
3. Obtaining of YPVal-adhE07-Cas9 Strain
According to the procedure of step 3 of example 1, the engineering bacterium YPVal-adhE03 competent cells were replaced with engineering bacterium YPVal-adhE07 competent cells, and the other procedures were unchanged, to obtain YPVal-adhE07-Cas9 strain.
4. Acquisition of engineering bacterium YPLA-adhE 08
According to the procedure of step 4 of example 1, the engineering bacterium YPVal-adhE08 is obtained by replacing the YPVal-adhE03-Cas9 strain with the YPVal-adhE07-Cas9 strain, and the other steps are unchanged.
Example 3 fermentation production of L-valine Using engineering bacteria modified from example 1 and example 2
1. Engineering bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, YPVal-adhE04, YPVal-adhE05, YPVal-adhE06, YPVal-adhE07 and YPVal-adhE08 obtained by modification of example 1 and example 2, and a starting bacterium valine-producing bacterium CGMCC 22721 and Escherichia coli W3110 were fermented in a fermenter (model BLBIO-5GC-4-H, shanghai Bailan Biotechnology Co., ltd.) to obtain a fermentation broth.
Each strain was fermented in triplicate.
The composition of the fermentation medium used in the fermentation is shown in Table 3.
The control process of the fermentation is shown in Table 4.
2. And (3) respectively detecting the L-valine yield in the fermentation liquid by adopting high performance liquid chromatography.
The results of three fermentations of the engineering bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03 and YPVal-adhE04 obtained from the valine-producing bacterium CGMCC 22721 are shown in Table 5 (P value <0.01 indicates that the difference is very significant). The results show that compared with valine-producing bacterium CGMCC 22721, the YPVal-adhE01, YPVal-adhE02, YPVal-adhE03 and YPVal-adhE04 can obviously improve the yield of L-valine. Namely, the engineering bacteria obtained by "knocking out adhE gene", "knocking out adhE gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis", "knocking out adhE gene and thiE gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis" or "knocking out adhE gene, thiE gene and mdh gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis" in valine-producing bacteria CGMCC 22721 can all improve the yield of L-valine.
The results of three fermentations of engineering bacteria YPVal-adhE05, YPVal-adhE06, YPVal-adhE07 and YPVal-adhE08 obtained from E.coli W3110 are shown in Table 6 (P value <0.01 indicates that the difference is extremely remarkable). The results showed that YPVal-adhE05, YPVal-adhE06, YPVal-adhE07 and YPVal-adhE08 each significantly improved the yield of L-valine as compared with E.coli W3110. That is, the engineering bacteria obtained by "knocking out adhE gene", "knocking out adhE gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis", "knocking out adhE gene and thiE gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis" or "knocking out adhE gene, thiE gene and mdh gene and knocking out ilvE gene (E) while inserting ptrc-initiated ilvE (B) from Bacillus subtilis" in E.coli W3110 can increase the yield of L-valine.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (6)

1. The method for preparing the engineering bacteria for producing valine comprises the following steps:
(a1) Knocking out or knocking down a coding gene of alcohol dehydrogenase in escherichia coli;
(a2) Knocking out or knocking down coding genes of branched chain amino acid aminotransferase in escherichia coli, and knocking in or introducing coding genes of branched chain amino acid aminotransferase from bacillus subtilis into escherichia coli;
the escherichia coli produces valine;
the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID No: 11. shown;
the amino acid sequence of the branching chain amino acid transaminase in the escherichia coli is shown as SEQ ID No: shown at 12;
The amino acid sequence of the branched chain amino acid aminotransferase derived from the bacillus subtilis is shown as SEQ ID No: 13. as shown.
2. The method according to claim 1, characterized in that: the method further comprises the step (a 3): knocking out or knocking down coding genes of thiamine phosphate synthase in escherichia coli;
the amino acid sequence of the thiamine phosphate synthase is shown as SEQ ID No: 14.
3. The method according to claim 2, characterized in that: the method further comprises step (a 4): knocking out or knocking down coding genes of malate dehydrogenase in escherichia coli;
the amino acid sequence of the malate dehydrogenase is shown in SEQ ID No: 15.
4. An engineered bacterium produced by the method of any one of claims 1 to 3.
5. The use of the engineering bacterium of claim 4, which is A1), A2) or A3):
a1 Producing valine;
a2 Preparing a product for producing valine;
a3 Preparing food, feed or medicine containing valine.
6. A method for producing valine, comprising the steps of: the engineering bacterium according to claim 4 is cultured by fermentation, and the fermentation product is collected to obtain valine.
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