Engineering bacterium for producing l-valine, construction method, and use

Abstract

An engineering bacterium for producing L-valine, a construction method, and a use. After an yjiT gene in Escherichia coli is knocked out and brnF and brnE are expressed, an yjiV gene is knocked out and ilvE and ilvD are expressed, a trpR gene is knocked out and ilvH mutant protein is expressed, lacI and lacZ genes are knocked out and DNA polymerase is expressed, and / or after an ycgH gene is knocked out and ilvC is expressed, an obtained engineering bacterium can increase the yield of L-valine. The engineering bacterium can be used for producing L-valine and has good application prospects.

Description

L-valine-producing engineered bacteria and their construction method and application

[0001] Cross-reference to related applications: This application claims priority to five Chinese patent applications filed on July 4, 2023 (application numbers 2023108092706, 2023108092710, 2023108092744, 2023108092778, and 2023108092814, respectively). The entire contents of these five patent applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to L-valine-producing engineering bacteria, a construction method and an application thereof in the field of biotechnology. Background Art

[0003] In microorganisms, Valine (a kind of branched-chain amino acid) is from pyruvic acid, via acetolactate, dihydroxyisovalerate and ketoisovalerate biosynthesis.These intermediate metabolites are produced by the reaction of acetohydroxy acid synthase, acetohydroxy acid reductoisomerase, dihydroxy acid dehydratase and transaminase B catalysis. However, these enzymes also participate in the biosynthesis of the Valine starting from ketobutyric acid and pyruvic acid, and Valine also starts from ketoisovalerate (a kind of intermediate metabolite), via 2-isopropylmalic acid, 3-isopropylmalic acid and ketoisocaproic acid biosynthesis. Therefore, because branched-chain amino acid (i.e. Valine, Valine and Valine) use identical enzyme for its biosynthetic process, so the branched-chain amino acid of known industry by fermentation production a type has difficulty. In addition, one problem that exists is that industrial batch production is subject to the restriction of feedback inhibition caused by Valine (it is end product) or its derivatives.

[0004] Escherichia coli has a well-defined genetic background and is an attractive industrial production platform for amino acid production. However, compared to Corynebacterium glutamicum, there are fewer reports on Escherichia coli strains producing L-valine, which may be due to the more complex regulatory mechanism of L-valine biosynthesis in Escherichia coli. Acetohydroxyacid synthase (AHAS) is the rate-limiting enzyme in L-valine biosynthesis. Escherichia coli has three AHAS isoenzymes, encoded by ilvBN, ilvGM, and ilvIH, with different properties and regulatory mechanisms. Park et al. reported that through systematic metabolic engineering of Escherichia coli W3110 and Escherichia coli W, the final L-valine yield reached 60.7 g / L and the sugar-acid conversion rate was 0.22 g / g. In addition to mutagenesis breeding and conventional metabolic engineering modifications, cofactor balance is also considered to be a key bottleneck for increasing L-valine production. Because intracellular cofactors affect metabolic networks, signal transduction, and material transport, they further affect the physiological functions of microbial cells. In the process of microbial fermentation to produce chemicals, the potency and yield of the chemicals are often limited by cofactor imbalance, which is mainly caused by the unbalanced expression of cofactor-dependent enzymes in the synthesis pathway. Savrasova, Stoynova et al. constructed an L-valine engineered bacterium using Escherichia coli MG1655 as the starting strain by replacing the natural NADPH-dependent transaminase with a heterologous NADH-dependent leucine dehydrogenase. Under microaerobic conditions, the sugar-acid conversion rate of this strain (0.23g / g) is only 35.4% of the maximum theoretical yield of 0.65g / g. Developing high-throughput screening methods for biosensors, introducing exogenous coenzyme regeneration pathways to balance intracellular cofactors, and constructing efficient industrial chassis production strains are key scientific issues that need to be addressed.

[0005] Invention Disclosure

[0006] The technical problem to be solved by the present invention is how to increase the yield of L-valine.

[0007] To solve the above technical problems, the present invention first provides an engineered bacterium for producing valine, wherein the engineered bacterium is a recombinant bacterium obtained by transforming a recipient bacterium; the transformation includes A1), A2), A3), A4) or A5);

[0008] A1) includes A11), A12) and A13):

[0009] A11) knocking out the yjiT gene of the recipient bacteria, inhibiting the expression of the yjiT gene or inhibiting the activity of the protein encoded by the yjiT gene;

[0010] A12) increasing the content of the protein encoded by the brnF gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnF gene;

[0011] A13) increasing the content of the protein encoded by the brnE gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnE gene;

[0012] A2) includes A21), A22) and A23):

[0013] A21) knocking out the yjiV gene of the recipient bacteria, inhibiting the expression of the yjiV gene or inhibiting the activity of the protein encoded by the yjiV gene;

[0014] A22) increasing the content of the protein encoded by the ilvE gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvE gene;

[0015] A23) increasing the content of the protein encoded by the ilvD gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvD gene;

[0016] A3) includes A31) and A32):

[0017] A31) knocking out the trpR gene of the recipient bacteria, inhibiting the expression of the trpR gene or inhibiting the activity of the protein encoded by the trpR gene;

[0018] A32) increasing ilvH in the recipient bacteria G14D、S17F Gene encoding protein content or enhance the ilvH G14D、S17F Activity of the gene encoding protein; the ilvH G14D、S17F The gene was obtained by replacing the 14th glycine codon in the ilvH gene with an aspartic acid codon and the 17th serine codon with a phenylalanine codon;

[0019] A4) includes A41), A42) and A43):

[0020] A41) knocking out the lacI gene of the recipient bacterium, thereby inhibiting the expression of the lacI gene or the activity of the protein encoded by the lacI gene;

[0021] A42) knocking out the lacZ gene of the recipient bacteria, thereby inhibiting the expression of the lacZ gene or the activity of the protein encoded by the lacZ gene;

[0022] A43) increasing the content of DNA polymerase in the recipient bacterium or enhancing the activity of the DNA polymerase;

[0023] A5) includes A51) and A52):

[0024] A51) knocking out the ycgH gene of the recipient bacterium, inhibiting the expression of the ycgH gene or inhibiting the activity of the protein encoded by the ycgH gene;

[0025] A52) increasing the content of the protein encoded by the ilvC gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvC gene;

[0026] The recipient bacteria is Escherichia coli.

[0027] Specifically, the engineered bacteria for producing valine have characteristics B1), B2), B3), B4) or B5);

[0028] B1) includes B11), B12) and B13):

[0029] B11) The yjiT gene is not expressed or weakly expressed, or the activity of the protein encoded by the yjiT gene is weakened or lost;

[0030] B12) the content of the protein encoded by the brnF gene increases or the activity of the protein encoded by the brnF gene increases;

[0031] B13) the content of the protein encoded by the brnE gene increases or the activity of the protein encoded by the brnE gene increases;

[0032] B2) includes B21), B22) and B23):

[0033] B21) The yjiV gene is not expressed or weakly expressed, or the activity of the protein encoded by the yjiV gene is weakened or lost;

[0034] B22) the content of the protein encoded by the ilvE gene is increased or the activity of the protein encoded by the ilvE gene is enhanced;

[0035] B23) the content of the protein encoded by the ilvD gene is increased or the activity of the protein encoded by the ilvD gene is enhanced;

[0036] B3) includes B31) and B32):

[0037] B31) The trpR gene is not expressed or weakly expressed, or the activity of the protein encoded by the trpR gene is weakened or lost;

[0038] B32)ilvH G14D、S17F Increased levels of the gene encoding the protein or the ilvH G14D、S17F The activity of the gene encoding protein is enhanced; the ilvH G14D、S17FThe gene was obtained by replacing the 14th glycine codon in the ilvH gene with an aspartic acid codon and the 17th serine codon with a phenylalanine codon;

[0039] B4) includes B41), B42) and B43):

[0040] B41) the lacI gene is not expressed or weakly expressed, or the activity of the protein encoded by the lacI gene is weakened or lost;

[0041] B42) the lacZ gene is not expressed or weakly expressed, or the activity of the protein encoded by the lacZ gene is weakened or lost;

[0042] B43) increasing the content of DNA polymerase or enhancing the activity of the DNA polymerase;

[0043] B5) includes B51) and B52):

[0044] B51) The ycgH gene is not expressed or weakly expressed, or the activity of the protein encoded by the ycgH gene is weakened or lost;

[0045] B52) the content of the protein encoded by the ilvC gene is increased or the activity of the protein encoded by the ilvC gene is enhanced;

[0046] The engineered bacteria is Escherichia coli.

[0047] Among them, the recipient bacteria contain yjiT gene (GeneID: 945056, updated on April 14, 2023), yjiV gene (GeneID: 2847669, updated on April 14, 2023), trpR gene (GeneID: 948917, updated on April 14, 2023), lacI gene (GeneID: 945007, updated on April 14, 2023), lacZ gene (GeneID: 945006, updated on April 14, 2023) and ycgH gene (GeneID: 2847703, updated on April 14, 2023).

[0048] In the above-mentioned engineered bacteria, the brnF gene and the brnE gene may be derived from Corynebacterium glutamicum.

[0049] Furthermore, the brnF gene can encode the protein shown in SEQ ID No. 2 in the sequence table;

[0050] The brnE gene can encode the protein shown in SEQ ID No. 3 in the sequence list.

[0051] Furthermore, the brnF gene may be the DNA molecule shown in positions 832-1587 of SEQ ID No. 1 in the sequence listing;

[0052] The brnE gene can be the DNA molecule shown in positions 1584-1910 of SEQ ID No. 1 in the sequence listing.

[0053] In the above-mentioned engineered bacteria, the ilvE gene may be derived from Bacillus subtilis. The ilvD gene may be derived from Escherichia coli.

[0054] Furthermore, the ilvE gene can encode the protein shown in SEQ ID No. 6 in the sequence listing. The ilvD gene can encode the protein shown in SEQ ID No. 5 in the sequence listing.

[0055] Furthermore, the ilvE gene may be the DNA molecule shown in positions 808 to 1902 of SEQ ID No. 4 in the sequence listing. The ilvD gene may be the DNA molecule shown in positions 1977 to 3827 of SEQ ID No. 4 in the sequence listing.

[0056] In the above-mentioned engineered bacteria, the ilvH gene may be derived from Escherichia coli.

[0057] Furthermore, the ilvH G14D、S17F The gene can encode the protein shown in SEQ ID No. 8 in the sequence listing.

[0058] Furthermore, the ilvH G14D、S17F The gene may be the DNA molecule shown in positions 835-1326 of SEQ ID No. 7 in the sequence listing.

[0059] In the above-mentioned engineered bacteria, the DNA polymerase may be derived from Escherichia coli.

[0060] Furthermore, the DNA polymerase may be the protein shown in SEQ ID No. 10 in the sequence listing.

[0061] Furthermore, the gene encoding the DNA polymerase may be the DNA molecule shown in positions 701-3352 of SEQ ID No. 9 in the sequence listing.

[0062] In the above-mentioned engineered bacteria, the ilvC gene may be derived from Escherichia coli.

[0063] Furthermore, the ilvC gene can encode the protein shown in SEQ ID No. 12 in the sequence listing.

[0064] Furthermore, the ilvC gene may be the DNA molecule shown in positions 794-2269 of SEQ ID No. 11 in the sequence listing.

[0065] The brnF gene, the brnE gene, the ilvE gene, the ilvD gene, the ilvH gene G14D、S17F The expression of the gene, the DNA polymerase encoding gene and the ilvC gene is initiated by a promoter capable of initiating the expression of the corresponding genes in the engineered bacteria, including but not limited to a strong promoter and a constitutive promoter.

[0066] In one embodiment of the present invention, the expression of the brnF gene and the brnE gene is driven by the Ptrc promoter, and the Ptrc promoter is the DNA molecule shown at positions 758-831 of SEQ ID No. 1.

[0067] In one embodiment of the present invention, the expression of the ilvE gene is driven by the Ptrc promoter, and the Ptrc promoter is the DNA molecule shown at positions 1903-1976 of SEQ ID No.4.

[0068] In another embodiment of the present invention, the expression of the ilvD gene is driven by the PilvD promoter, and the PilvD promoter is the DNA molecule shown at positions 3828-3893 of SEQ ID No.4.

[0069] In another embodiment of the present invention, the ilvH G14D、S17F The expression of the gene is driven by the Ptrc promoter, which is the DNA molecule shown at positions 761-834 of SEQ ID No.7.

[0070] In another embodiment of the present invention, the expression of the DNA polymerase encoding gene is driven by the PxylF promoter, and the PxylF promoter is the DNA molecule shown at positions 3353-3617 of SEQ ID No.9.

[0071] In another embodiment of the present invention, the expression of the ilvC gene is driven by the Ptrc promoter, and the Ptrc promoter is the DNA molecule shown at positions 2270-2343 of SEQ ID No.11.

[0072] Specifically, A1) can be achieved by using the CRISPR / Cas9 system to perform gene editing using an sgRNA targeting the yjiT gene and the DNA fragment shown in SEQ ID No. 1 as a donor.

[0073] A2) Gene editing can be achieved through the CRISPR / Cas9 system using an sgRNA targeting the yjiV gene and the DNA fragment shown in SEQ ID No. 4 as a donor.

[0074] A3) Gene editing can be achieved through the CRISPR / Cas9 system using an sgRNA targeting the trpR gene and the DNA fragment shown in SEQ ID No. 7 as a donor.

[0075] A4) Gene editing can be achieved by using the CRISPR / Cas9 system, using sgRNA targeting the lacI and lacZ genes and the DNA fragment shown in SEQ ID No. 9 as a donor.

[0076] A5) Gene editing can be achieved through the CRISPR / Cas9 system using an sgRNA targeting the ycgH gene and the DNA fragment shown in SEQ ID No. 11 as a donor.

[0077] The recipient bacteria of the present invention include but are not limited to Escherichia coli. Any bacteria containing yjiT gene, yjiV gene, trpR gene, lacI and lacZ gene, ycgH gene and capable of synthesizing L-valine can utilize the method of the present invention to prepare recombinant bacteria and produce L-lysine. The bacteria can be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniaphagous, Corynebacterium blunt-toothed, Pantoea, Pantoea ananatis, Bacillus brevis, Brevibacterium lactis or Brevibacterium flavum. The yeast can be Saccharomyces cerevisiae or Pichia pastoris.

[0078] In one embodiment of the present invention, the Escherichia coli is Escherichia coli YP045 or Escherichia coli W3110.

[0079] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiT and W3110-yjiT. The recombinant bacteria CGMCC22721-yjiT and W3110-yjiT are respectively obtained by knocking out part of the coding region of the yjiT gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while keeping the other nucleotides in their genomes unchanged.

[0080] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiV and W3110-yjiV. The recombinant bacteria CGMCC22721-yjiV and W3110-yjiV are respectively obtained by knocking out the yjiV gene coding region on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110), while keeping the other nucleotides in their genomes unchanged.

[0081] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-trpR and W3110-trpR, and the recombinant bacteria CGMCC22721-trpR and W3110-trpR are respectively L-valine producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The trpR gene coding region is knocked out, and the mutant ilvH driven by the Ptrc promoter is inserted. G14D、S17F Gene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0082] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-lacIZ and W3110-lacIZ. The recombinant bacteria CGMCC22721-lacIZ and W3110-lacIZ are respectively obtained by knocking out part of the coding region of the lacI-lacZ gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21), while keeping the other nucleotides in their genomes unchanged.

[0083] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-ycgH and W3110-ycgH. The recombinant bacteria CGMCC22721-ycgH and W3110-ycgH are respectively obtained by knocking out part of the coding region of the ycgH gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter, while keeping the other nucleotides in the genome unchanged.

[0084] In the above-mentioned engineered bacteria, the transformation may also include any two, any three, any four or five of the above-mentioned five types A1), A2), A3), A4) and A5).

[0085] Specifically, the transformation may be: the above-mentioned A1) and A2), A1) and A3), A1) and A4), A1) and A5), A2) and A3), A2) and A4), A2) and A5), A3) and A4), A3) and A5), A4) and A5), A1), A2) and A3), A1), A2) and A4), A1), A2) and A5), A1), A3) and A4), A1), A3) and A5), A1) , A4) and A5), A2), A3) and A4), A2), A3) and A5), A2), A4) and A5), A3), A4) and A5), A1), A2), A3) and A4), A1), A2), A3) and A5), A1), A2), A4) and A5), A1), A3), A4) and A5), A2), A3), A4) and A5).

[0086] Specifically, the engineered bacteria for producing valine also has any two, any three, any four or five of the above characteristics B1), B2), B3), B4) and B5).

[0087] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV, and the recombinant bacteria CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV are respectively L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiT gene coding region is partially deleted and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; at the same time, the yjiV gene coding region is partially deleted and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli) driven by the PilvD promoter are inserted. coli)W3110), and the recombinant bacteria were obtained by keeping other nucleotides in their genome unchanged.

[0088] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiV-trpR and W3110-yjiV-trpR, and the recombinant bacteria CGMCC22721-yjiV-trpR and W3110-yjiV-trpR are respectively L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiV gene coding region is deleted, and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter are inserted; at the same time, the trpR gene coding region is deleted, and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17F Gene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0089] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ, and the recombinant bacteria CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ are respectively L-valine producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The trpR gene coding region is knocked out and the mutant ilvH driven by the Ptrc promoter is inserted. G14D、S17Fgene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0090] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH. The recombinant bacteria CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH are respectively obtained by knocking out part of the coding region of the lacI-lacZ gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); at the same time, the coding region of the ycgH gene is knocked out, and the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110) is inserted, while keeping the other nucleotides in the genome unchanged.

[0091] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR, and the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR are respectively L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiT gene coding region is partially deleted and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; the yjiV gene coding region is partially deleted and the ilvE gene (from Bacillus subtilis subsp. subtilis) driven by the Ptrc promoter is inserted. str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); at the same time, the coding region of the trpR gene was deleted and the mutant ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F Gene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0092] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ, and the recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ are respectively L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiV gene coding region is deleted, and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter are inserted; the trpR gene coding region is deleted, and the mutant ilvH driven by the Ptrc promoter is inserted. G14D、S17F gene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0093] In another embodiment of the present invention, the recombinant bacteria obtained are the recombinant bacteria CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH, respectively, in which the coding region of the trpR gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 is deleted, and the mutant ilvH driven by the Ptrc promoter is inserted. G14D、S17F The invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0094] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ, and the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ are respectively L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiT gene coding region is partially deleted and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; the yjiV gene coding region is partially deleted and the ilvE gene (from Bacillus subtilis subsp. subtilis) driven by the Ptrc promoter is inserted. str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); the coding region of the trpR gene was deleted, and the mutant ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F gene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0095] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH, and the recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH are L-valine producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiV gene coding region is deleted and the ilvE gene (from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli) driven by the PilvD promoter are inserted. coli)W3110); the coding region of the trpR gene was partially deleted and the mutant ilvH driven by the Ptrc promoter was inserted. G14D、S17FThe invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0096] In another embodiment of the present invention, the obtained recombinant bacteria are recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH, and the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH are L-valine producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genomes. The yjiT gene coding region is deleted and the brnF-brnE gene (from Corynebacterium glutamicum) driven by the Ptrc promoter is inserted. glutamicum) ATCC13032); the yjiV gene coding region was partially deleted, and the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110) were inserted; the trpR gene coding region was partially deleted, and the mutant ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F The invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0097] The present invention also provides a method for preparing an engineered bacterium for producing valine, the method comprising: performing the above-mentioned transformation A1), A2), A3), A4) or A5) on a recipient bacterium to obtain the target engineered bacterium; the recipient bacterium is Escherichia coli.

[0098] The above method may further comprise subjecting the recipient bacteria to any two, any three, any three, any four or five of the above five modifications A1), A2), A3), A4) and A5).

[0099] The present invention also provides a method for preparing L-valine, which comprises: culturing the engineered bacteria to obtain L-valine.

[0100] In the above method, the engineered bacteria may be cultured using a culture medium capable of growing the engineered bacteria;

[0101] And / or, the recombinant engineered bacteria are cultured under conditions that enable the engineered bacteria to grow.

[0102] The present invention also provides a product for producing L-valine, which is any one of P1), P2), P3), P4), and P5), or a product consisting of any two, three, four, or five of these five:

[0103] P1) is a substance that separately or simultaneously achieves A11), A12) and A13):

[0104] A11) knocking out the yjiT gene of the recipient bacteria or inhibiting the expression of the yjiT gene or inhibiting the activity of the protein encoded by the yjiT gene;

[0105] A12) increasing the content of the protein encoded by the brnF gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnF gene;

[0106] A13) increasing the content of the protein encoded by the brnE gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnE gene;

[0107] P2) is a substance that separately or simultaneously realizes A21), A22) and A23):

[0108] A21) knocking out the yjiV gene of the recipient bacteria or inhibiting the expression of the yjiV gene or inhibiting the activity of the protein encoded by the yjiV gene;

[0109] A22) increasing the content of the protein encoded by the ilvE gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvE gene;

[0110] A23) increasing the content of the protein encoded by the ilvD gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvD gene;

[0111] P3) is a substance that separately or simultaneously implements A31) and A32):

[0112] A31) knocking out the trpR gene of the recipient bacteria or inhibiting the expression of the trpR gene or inhibiting the activity of the protein encoded by the trpR gene;

[0113] A32) Increase ilvH in recipient bacteria G14D、S17F Gene encoding protein content or enhance the ilvH G14D、 S17F the activity of the protein encoded by the gene;

[0114] P4) is a substance that separately or simultaneously achieves A41), A42) and A43):

[0115] A41) knocking out the lacI gene of the recipient bacteria or inhibiting the expression of the lacI gene or inhibiting the activity of the protein encoded by the lacI gene;

[0116] A42) knocking out the lacZ gene of the recipient bacteria or inhibiting the expression of the lacZ gene or inhibiting the activity of the protein encoded by the lacZ gene;

[0117] A43) increasing the content of DNA polymerase in the recipient bacterium or enhancing the activity of the DNA polymerase;

[0118] P5) is a substance that separately or simultaneously realizes A51) and A52):

[0119] A51) knocking out the ycgH gene of the recipient bacteria or inhibiting the expression of the ycgH gene or inhibiting the activity of the protein encoded by the ycgH gene;

[0120] A52) increasing the content of the protein encoded by the ilvC gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvC gene.

[0121] The use of the engineered bacteria or the product in producing L-valine, or in preparing a product for producing L-valine, or in preparing a food, feed or medicine containing L-valine also falls within the scope of protection of the present invention.

[0122] The engineered bacteria of the present invention, the preparation method thereof, and the related products can be used to produce a variety of products, including but not limited to the valine in the examples. The products produced can also be glutamic acid, threonine, tryptophan, arginine, lysine, glycine, alanine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutaric acid, citric acid, ornithine, citrulline, etc.

[0123] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0124] Preservation Instructions

[0125] Classification name: Escherichia coli.

[0126] Strain number: YP045.

[0127] Name of depository unit: General Microbiology Center of China Culture Collection Administration of Microorganisms.

[0128] Abbreviation of the depository unit: CGMCC.

[0129] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0130] Date of preservation: June 15, 2021.

[0131] The registration number of the CGMCC Collection Center is: CGMCC No.22721. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1. Fermentation medium formula (the rest is water).

[0133] Figure 2. Fermentation control process.

[0134] Figure 3. L-valine production and significance analysis. Modes for Carrying Out the Invention

[0135] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The quantitative tests in the following examples were all repeated three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0136] In the following examples, the L-valine-producing strain CGMCC22721 is Escherichia coli YP045, which was deposited with the General Microbiology Center of the China Culture Collection Administration on June 15, 2021, with the deposit number CGMCC No. 22721. The depositor of Escherichia coli YP045, Ningxia Yipin Biotechnology Co., Ltd., has authorized Heilongjiang Yipin Biotechnology Co., Ltd. to use this strain.

[0137] Example 1: Construction of an engineered strain in which the yjiT gene is deleted from the genome and the brnF-brnE gene is overexpressed.

[0138] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the yjiT gene in the L-valine-producing strain CGMCC22721 and the wild-type E. coli W3110 genome (sequencing confirmed that these strains retain the intact yjiT gene on their chromosomes (GeneID: 945056, updated on April 14, 2023)). At the same time, the brnF-brnE genes (branched chain amino acid exporter, branched-chain amino acid exporter) driven by the Ptrc promoter from Corynebacterium glutamicum ATCC13032 were inserted to further study the effects of these genes on L-valine synthesis.

[0139] 1. Construction of sgRNA

[0140] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0141] Amplification of the sgRNA fragment requires no template, requiring only PCR annealing. The system and procedure are as follows: PCR reaction system: 10 μL sgRNA-1F, 10 μL sgRNA-1R; PCR reaction procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the sgRNA target DNA fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the DNA was diluted to 100 ng / μL.

[0142] The pGRB plasmid was digested with Spe I and then dephosphorylated. The sgRNA and dephosphorylated linearized plasmid were recombined using the Gibson Assembly Kit (New England Biolabs). The recombination system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of dephosphorylated linearized plasmid, and 0.5 μL of the sgRNA target DNA fragment. After assembly at 50°C for 30 minutes, the product was transformed into DH5α competent cells, and the plasmid was extracted and sequenced using the sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was named pGRB-sgRNA-1.

[0143] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). The underlined nucleotides are the homology arm sequences of the pGRB cloning vector, and the bold nucleotides are the sgRNA sequences:

[0144] sgRNA-1F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTGTACGCGTTGCCAATTCTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 14),

[0145] sgRNA-1R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACATAGAATTGGCAACGCGTACACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 15),

[0146] sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 16),

[0147] sgRNA-PR: 5'-GCGTCAGGTGCATAAACAGA-3' (SEQ ID No. 17).

[0148] 2. PCR amplification of homologous recombination fragments

[0149] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the yjiT gene were designed and synthesized. At the same time, primers for inserting the Ptrc-brnF-brnE gene sequence were used. The yjiT gene in the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 gene was knocked out by CRISPR / Cas9 gene editing, and the brnF-brnE gene driven by the Ptrc promoter (from Corynebacterium glutamicum ATCC13032) was inserted.

[0150] The primers are as follows (synthesized by Shanghai Invitrogen):

[0151] P1:5'-GAGTGATGAGCGGTTGAAG-3' (SEQ ID No. 18),

[0152] P2:5'-CTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCC-3' (SEQ ID No. 19),

[0153] P3:5'-CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCGTGCAAAAAACGCAAGAG-3' (SEQ ID No. 20),

[0154] P4:5'-CTTCACAGGTAGTGCTTTTAGTTAGAAAAGATTCACCAGTCCAAC-3' (SEQ ID No. 21),

[0155] P5:5'-GTGAATCTTTTCTAACTAAAAGCACTACCTGTGAAGG-3' (SEQ ID No. 22),

[0156] P6: 5'-CTGCGGCAATAATCAACG-3' (SEQ ID No. 23).

[0157] Using Escherichia coli W3110 genomic DNA as a template, PCR amplification was performed using primers P1 / P2, P5 / P6, and KAPA HiFi HotStart (Shanghai Huayasichuang Biotechnology Co., Ltd., KK2601). The upper homology arm fragment was 801 bp, and the lower homology arm fragment was 684 bp. Genomic DNA from Corynebacterium glutamicum ATCC13032 was used as a template, and PCR amplification using primers P3 / P4 and KAPA HiFi HotStart yielded a 1153 bp Ptrc-brnF-brnE gene fragment. After the PCR reaction, the DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR with primers P1 / P6 yielded the homologous recombinant DNA fragment ΔyjiT-Ptrc-brnF-brnE (SEQ ID No. 1), which is 2579 bp in size.

[0158] In SEQ ID No. 1, positions 758-831 represent the Ptrc promoter, and positions 832-1910 represent the brnF-brnE gene, which encodes the brnF protein shown in SEQ ID No. 2 (the coding sequence is positions 832-1587 of SEQ ID No. 1) and the brnE protein shown in SEQ ID No. 3 (the coding sequence is positions 1584-1910 of SEQ ID No. 1).

[0159] 3. Preparation and transformation of competent cells

[0160] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into L-valine-producing bacteria CGMCC22721 and Escherichia coli W3110 competent cells, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. The 943 bp transformants obtained were CGMCC22721-Cas9 and W3110-Cas9 transformants containing the pREDCas9 plasmid.

[0161] The primers are as follows (synthesized by Shanghai Invitrogen):

[0162] pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No. 24);

[0163] pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 25).

[0164] Prepare CGMCC22721-Cas9 and W3110-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were harvested to prepare competent cells and transformed with the pGRB-sgRNA-1 plasmid and the homologous recombinant DNA fragment ΔyjiT-Ptrc-brnF-brnE, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and incubated at 32°C for 12 hours. Individual colonies were subcultured and identified by PCR using primers P7 / P8. Positive transformants were identified by PCR amplification of a 1720 bp fragment.

[0165] The primers are as follows (synthesized by Shanghai Invitrogen):

[0166] P7:5'-GCTGTATTCCTTATGTGGACC-3' (SEQ ID No. 26),

[0167] P8: 5'-GCAGGAATCCAAAGTCAGC-3' (SEQ ID No. 27).

[0168] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-1. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified by PCR again using primers P1 / P6. Those that could amplify a fragment of 2579 bp (SEQ ID No. 1) were positive. The positive transformants were sent for sequencing, and the strains with correct sequencing results were named CGMCC22721-yjiT and W3110-yjiT, respectively.

[0169] The recombinant bacteria CGMCC22721-yjiT and W3110-yjiT both lack the yjiT gene and overexpress the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter; specifically, the recombinant bacteria CGMCC22721-yjiT and W3110-yjiT are obtained by knocking out part of the coding region of the yjiT gene on the genomes of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while keeping other nucleotides in their genomes unchanged.

[0170] Example 2: Construction of an engineered strain in which the yjiV gene is deleted from the genome and the ilvE-ilvD genes are overexpressed.

[0171] Based on the Escherichia coli W3110 genome sequence published by NCBI, the yjiV gene in the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110 was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that these strains retained the complete yjiV gene on their chromosomes (GeneID: 2847669, updated on 2023-04-14). At the same time, the ilvE gene (branched-chain amino-acid aminotransferase, branched-chain amino acid transaminase, from Bacillus subtilis subsp. subtilis str. 168) driven by the Ptrc promoter and the ilvD gene (dihydroxyacid dehydratase, dihydroxyacid dehydratase, from Escherichia coli W3110) driven by the PilvD promoter were inserted to further study the effects of these genes on L-valine synthesis.

[0172] 1. Construction of sgRNA

[0173] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0174] Amplification of the sgRNA fragment requires no template, requiring only PCR annealing. The system and procedure are as follows: PCR reaction system: 10 μL sgRNA-2F, 10 μL sgRNA-2R; PCR reaction procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the sgRNA target DNA fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng / μL.

[0175] The pGRB plasmid was digested with Spe I and then dephosphorylated. The sgRNA and dephosphorylated linearized plasmid were recombined using the Gibson Assembly Kit (New England Biolabs). The recombinant system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of dephosphorylated linearized plasmid, and 0.5 μL of the sgRNA target DNA fragment. After assembly at 50°C for 30 minutes, the product was transformed into DH5α competent cells, and the plasmid was extracted and sequenced using the sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was designated pGRB-sgRNA-2.

[0176] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). The underlined nucleotides are the homology arm sequences of the pGRB cloning vector, and the bold nucleotides are the sgRNA sequences:

[0177] sgRNA-2F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTCTATTGATATTATCAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 28),

[0178] sgRNA-2R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTGTATTGATAATATCAATAGACTAGTATTATACCTAGGACTGAGCTAGCCTTCA-3' (SEQ ID No. 29).

[0179] 2. PCR amplification of homologous recombination fragments

[0180] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the yjiV gene were designed and synthesized. At the same time, primers for inserting the Ptrc-ilvE gene and the PilvD-ilvD gene were inserted. The yjiV gene in the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genes was knocked out by CRISPR / Cas9 gene editing. At the same time, the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp. subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110) were inserted.

[0181] The primers are as follows (synthesized by Shanghai Invitrogen):

[0182] P9:5'-TGAATGGACTGCTATGCG-3' (SEQ ID No. 30),

[0183] P10:5'-CACAGTGTATTAAGCCAGACGTTAACAACGCAGTACTTCCTGCTG-3' (SEQ ID No. 31),

[0184] P11:5'-GAAGTACTGCGTTGTTAACGTCTGCTTAATACACTGTG-3' (SEQ ID No. 32),

[0185] P12:5'-GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAACTTTTTAAATATATGGAG-3' (SEQ ID No. 33),

[0186] P13:5'-CTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTAACCCCCCAGTTTCGATTTAT-3' (SEQ ID No. 34),

[0187] P14:5'-CCACCAGCACATCCGTTGAAATACAAAAAATGGGAC-3' (SEQ ID No. 35),

[0188] P15:5'-TCCCATTTTTTGTATTTCAACGGATGTGCTGGTGG-3' (SEQ ID No. 36),

[0189] P16: 5'-TGAAGACACGCTGGCTAAC-3' (SEQ ID No. 37).

[0190] Using Escherichia coli W3110 genomic DNA as a template, PCR amplification using primers P9 / P10, P13 / P14, P15 / P16, and KAPA HiFi HotStart yielded an 831-bp upper homology arm, a 1979-bp PilvD-ilvD gene, and an 856-bp lower homology arm. Using Bacillus subtilis subsp. subtilis str.168 genomic DNA as a template, PCR amplification using primers P11 / P12 and KAPA HiFi HotStart yielded an 1161-bp Ptrc-ilvE gene. After PCR, the recovered DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR using primers P9 / P16 yielded the homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD (SEQ ID No. 4), which is 4732 bp in size.

[0191] In SEQ ID No. 4, positions 3828-3893 represent the PilvD promoter, positions 1977-3827 represent the ilvD gene, encoding the ilvD protein shown in SEQ ID No. 5; positions 1903-1976 represent the Ptrc promoter, positions 808-1902 represent the ilvE gene, encoding the ilvE protein shown in SEQ ID No. 6.

[0192] 3. Preparation and transformation of competent cells

[0193] Competent cells were prepared by using CGMCC22721-Cas9 and W3110-Cas9 from Example 1. When the cells grew to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600=0.4, the cells were harvested to prepare competent cells and transformed with the pGRB-sgRNA-2 plasmid and the homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and incubated at 32°C for 12 hours. Individual colonies were subcultured and identified by PCR using primers P17 / P18. Positive transformants were identified by PCR amplification of a 1543 bp fragment.

[0194] The primers are as follows (synthesized by Shanghai Invitrogen):

[0195] P17:5'-AGCGAGTTTCCAATACCG-3' (SEQ ID No. 38),

[0196] P18: 5'-ATTTCTCCTGCTTTCGGC-3' (SEQ ID No. 39).

[0197] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-2. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P9 / P16. Those that could amplify a fragment of 4732 bp (SEQ ID No. 4) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiV and W3110-yjiV, respectively.

[0198] The recombinant bacteria CGMCC22721-yjiV and W3110-yjiV both deleted the yjiV gene and overexpressed the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter. Specifically, the recombinant bacteria CGMCC22721-yjiV and W3110-yjiV were respectively obtained by knocking out the yjiV gene coding region on the genomes of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter. coli)W3110), and the recombinant bacteria were obtained by keeping other nucleotides in their genome unchanged.

[0199] Example 3: Construction of an engineered strain in which the trpR gene is deleted and the ilvH gene is overexpressed in the genome.

[0200] Based on the Escherichia coli W3110 genome sequence published by NCBI, the trpR gene in the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110 gene was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that these strains retained the complete trpR gene (DNA-binding transcriptional repressor; GeneID: 948917, updated on April 14, 2023) on their chromosomes), and the ilvH gene driven by the Ptrc promoter was inserted. G14D、 S17F genes (acetolactate synthase, acetolactate synthase), in order to further study the impact of these genes on the synthesis of L-valine.

[0201] 1. Construction of sgRNA

[0202] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0203] Amplification of the sgRNA fragment requires no template, requiring only PCR annealing. The system and procedure are as follows: PCR reaction system: 10 μL sgRNA-3F, 10 μL sgRNA-3R; PCR reaction procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the sgRNA target DNA fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng / μL.

[0204] The pGRB plasmid was digested with Spe I and then dephosphorylated. The sgRNA and dephosphorylated linearized plasmid were recombined using the Gibson Assembly Kit (New England Biolabs). The recombination system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of dephosphorylated linearized plasmid, and 0.5 μL of the sgRNA target DNA fragment. After assembly at 50°C for 30 minutes, the product was transformed into DH5α competent cells, and the plasmid was extracted and sequenced using the sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was designated pGRB-sgRNA-3.

[0205] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). The underlined nucleotides are the homology arm sequences of the pGRB cloning vector, and the bold nucleotides are the sgRNA sequences:

[0206] sgRNA-3F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTCGTGAGTTAAAAAATGAACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 40),

[0207] sgRNA-3R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACAGTTCATTTTTTAACTCACGACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 41).

[0208] 2. PCR amplification of homologous recombination fragments

[0209] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the trpR gene and inserting the Ptrc-ilvH G14D、S17F The primers of gene sequence were used to knock out the trpR gene in L-valine producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 by CRISPR / Cas9 gene editing, and the ilvH gene driven by Ptrc promoter was inserted at the same time. G14D、S17F Gene.

[0210] The primers are as follows (synthesized by Shanghai Invitrogen):

[0211] P19:5'-CCAATCTGGTGAAGAGCAAG-3' (SEQ ID No. 42),

[0212] P20: 5'-CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGCTGAATAGGGTGATTGTTG-3' (SEQ ID No. 43),

[0213] P21:5'-CAATTTCACACAGGAAACAGACCATGCGCCGGATATTATCAGTCTTACTCGAAAATGAATCAGACGCGTTATTCCGC-3' (SEQ ID No. 44),

[0214] P22:5'-GTCTTATCATGCCTACCAAATCAACGCATTATTTTATCG-3' (SEQ ID No. 45),

[0215] P23:5'-CGATAAAATAATGCGTTGATTTGGTAGGCATGATAAGAC-3' (SEQ ID No. 56),

[0216] P24: 5'-GTGCGTCCTAAATCGCTAC-3' (SEQ ID No. 47).

[0217] Using Escherichia coli W3110 genomic DNA as a template, PCR amplification was performed with primers P19 / P20, P21 / P22, P23 / P24 and KAPA HiFi HotStart to obtain the upper homology arm of 831 bp and the Ptrc-ilvH of 535 bp. G14D、S17F The gene and the 801bp lower homology arm fragment were isolated. After the PCR reaction, the recovered DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR with primers P19 / P24 was performed to obtain the homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH (SEQ ID No. 7), which was 2108bp in size.

[0218] In SEQ ID No. 7, positions 761-834 represent the Ptrc promoter, and positions 835-1326 represent the ilvH G14D、S17F Gene encoding ilvH shown in SEQ ID No.8 G14D、S17F protein.

[0219] 3. Preparation and transformation of competent cells

[0220] Competent cells were prepared by using CGMCC22721-Cas9 and W3110-Cas9 from Example 1. When the cells grew to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were harvested to prepare competent cells and transformed with the pGRB-sgRNA-3 plasmid and the homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were subcultured and identified by PCR using primers P25 / P26. Positive transformants were identified by PCR amplification of a 1437 bp fragment.

[0221] The primers are as follows (synthesized by Shanghai Invitrogen):

[0222] P25:5'-CATCGGCGAAGAGTATGAG-3' (SEQ ID No. 48),

[0223] P26: 5'-AAAGTCCGACCACACCAGAG-3' (SEQ ID No. 49).

[0224] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-3. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified by PCR again using primers P19 / P24. Those that could amplify a fragment of 2108 bp (SEQ ID No. 7) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-trpR and W3110-trpR, respectively.

[0225] The recombinant strains CGMCC22721-trpR and W3110-trpR both lacked the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17F Gene; Specifically, the recombinant bacteria CGMCC22721-trpR and W3110-trpR are respectively L-valine production bacteria CGMCC22721 and wild-type Escherichia coli W3110 genome trpR gene partial coding region is knocked out, and the Ptrc promoter-driven mutant ilvH is inserted G14D、S17F Gene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0226] Example 4: Construction of an engineered strain with lacI-lacZ gene deletion and DNA polymerase gene overexpression in the genome.

[0227] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the lacI-lacZ genes in the L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110 (sequencing confirmed that these strains retain intact lacI-lacZ genes (DNA-binding transcriptional repressor, DNA-binding transcriptional repressor; beta-D-galactosidase, β-D-galactosidase; lacI GeneID: 945007, updated on 2023-04-14; lacZ GeneID: 945006, updated on 2023-04-14). Simultaneously, a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted to further investigate the effects of these genes on L-valine synthesis.

[0228] 1. Construction of sgRNA

[0229] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0230] Amplification of the sgRNA fragment requires no template, requiring only PCR annealing. The system and procedure are as follows: PCR reaction system: 10 μL sgRNA-4F, 10 μL sgRNA-4R; PCR reaction procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the sgRNA target DNA fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng / μL.

[0231] The pGRB plasmid was digested with Spe I and then dephosphorylated. The sgRNA and dephosphorylated linearized plasmid were recombined using the Gibson Assembly Kit (New England Biolabs). The recombinant system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of dephosphorylated linearized plasmid, and 0.5 μL of the sgRNA target DNA fragment. After assembly at 50°C for 30 minutes, the product was transformed into DH5α competent cells, and the plasmid was extracted and sequenced using the sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was designated pGRB-sgRNA-4.

[0232] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). The underlined nucleotides are the homology arm sequences of the pGRB cloning vector, and the bold nucleotides are the sgRNA sequences:

[0233] sgRNA-4F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTCACTGCCCGCTTTCCAGTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 50),

[0234] sgRNA-4R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGACTGGAAAGCGGGCAGTGAACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 51).

[0235] 2. PCR amplification of homologous recombination fragments

[0236] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the lacI-lacZ gene sequence and primers for inserting the PxylF-DNA polymerase gene sequence were designed and synthesized. The lacI-lacZ genes in the L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110 genes were knocked out using CRISPR / Cas9 gene editing, and the DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted.

[0237] The primers are as follows (synthesized by Shanghai Invitrogen):

[0238] P27:5'-CGGTAATAATCCACAGCAGG-3' (SEQ ID No. 52),

[0239] P28:5'-GACTTCGCGTTCGCGTAACAGGTAGCAGAGCGGGTA-3' (SEQ ID No. 53),

[0240] P29:5'-TACCCGCTCTGCTACCTGTTACGCGAACGCGAAGTC-3' (SEQ ID No. 54),

[0241] P30:5'-CTAACTACAGAAGGCCCTACACCATGAACACGATTAACATCGC-3' (SEQ ID No. 55),

[0242] P31:5'-GCGATGTTAATCGTGTTCATGGTGTAGGGCCTTCTGTAGTTAG-3' (SEQ ID No. 56),

[0243] P32:5'-CATTAAGTTCTGTCTCTCGGCGGAGATAATTCACAAGTGTGCG-3' (SEQ ID No. 57),

[0244] P33:5'-CGCACACTTGTGAATTATCTCCGCCGAGACAGAACTTAATG-3' (SEQ ID No. 58),

[0245] P34: 5'-GGTTACGGACAGAACTACCG-3' (SEQ ID No. 59).

[0246] Using E. coli W3110 genomic DNA as a template, PCR amplification with primers P27 / P28, P31 / P32, P33 / P34, and KAPA HiFi HotStart yielded a 718-bp upper homology arm, a 305-bp PxylF promoter, and a 928-bp lower homology arm. Using E. coli BL21 genomic DNA as a template, PCR amplification with primers P29 / P30 and KAPA HiFi HotStart yielded a 2693-bp DNA polymerase gene. After PCR, the DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR with primers P27 / P34 yielded the homologous recombinant DNA fragment ΔlacIZ-PxylF-DNA polymerase (SEQ ID No. 9), which is 4524 bp long.

[0247] In SEQ ID No. 9, positions 3353-3617 represent the PxylF promoter, and positions 701-3352 represent the DNA polymerase gene, encoding the DNA polymerase shown in SEQ ID No. 10.

[0248] 3. Preparation and transformation of competent cells

[0249] Competent cells were prepared by using CGMCC22721-Cas9 and W3110-Cas9 from Example 1. When the cells grew to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 When the concentration of dapoxetine (dapoxetine) was 0.4, competent cells were harvested and transformed with the pGRB-sgRNA-4 plasmid and the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase, respectively. The cells were then plated onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin and incubated at 32°C for 12 hours. Individual colonies were subcultured and identified by PCR using primers P35 / P36. Positive transformants were identified by PCR amplification of a 1655 bp fragment.

[0250] The primers are as follows (synthesized by Shanghai Invitrogen):

[0251] P35:5'-TTCGCCCATTGTCGTTAC-3' (SEQ ID No. 60),

[0252] P36: 5'-AGTTCCGCTTACAGCCTACC-3' (SEQ ID No. 61).

[0253] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-4. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified again by PCR using primers P27 / P34. Those that could amplify a fragment of 4524 bp (SEQ ID No. 9) were positive. The positive transformants were sent for sequencing, and the strains with correct sequencing results were named CGMCC22721-lacIZ and W3110-lacIZ, respectively.

[0254] The recombinant bacteria CGMCC22721-lacIZ and W3110-lacIZ both lack the lacI-lacZ genes and simultaneously overexpress a DNA polymerase gene driven by a PxylF promoter (from Escherichia coli BL21); specifically, the recombinant bacteria CGMCC22721-lacIZ and W3110-lacIZ are obtained by knocking out part of the coding region of the lacI-lacZ genes on the genomes of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, respectively, and inserting a DNA polymerase gene driven by a PxylF promoter (from Escherichia coli BL21), while keeping other nucleotides in their genomes unchanged.

[0255] Example 5: Construction of an engineered strain in which the ycgH gene is deleted from the genome and the ilvC gene is overexpressed.

[0256] Based on the Escherichia coli W3110 genome sequence published by NCBI, the ycgH gene in the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110 was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that these strains retained the complete ycgH gene on their chromosomes (GeneID: 2847703, updated on 2023-04-14)). At the same time, the ilvC gene (ketol-acid reductoisomerase, from Escherichia coli W3110) driven by the Ptrc promoter was inserted to further study the effects of these genes on L-valine synthesis.

[0257] 1. Construction of sgRNA

[0258] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector homology arm sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0259] Amplification of the sgRNA fragment requires no template, requiring only PCR annealing. The system and procedure are as follows: PCR reaction system: 10 μL sgRNA-5F, 10 μL sgRNA-5R; PCR reaction procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the sgRNA target DNA fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng / μL.

[0260] The pGRB plasmid was digested with Spe I and then dephosphorylated. The sgRNA and dephosphorylated linearized plasmid were recombined using the Gibson Assembly Kit (New England Biolabs). The recombination system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of dephosphorylated linearized plasmid, and 0.5 μL of the sgRNA target DNA fragment. After assembly at 50°C for 30 minutes, the product was transformed into DH5α competent cells, the plasmid was extracted, and sequenced using the sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was designated pGRB-sgRNA-5.

[0261] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). The underlined nucleotides are the homology arm sequences of the pGRB cloning vector, and the bold nucleotides are the sgRNA sequences:

[0262] sgRNA-5F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTCCCGTCCTGCGCCCCGAAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 62),

[0263] sgRNA-5R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGCTTCGGGGGCGCAGGACGGGACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 63).

[0264] 2. PCR amplification of homologous recombination fragments

[0265] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the ycgH gene and primers for inserting the Ptrc-ilvC gene were designed and synthesized. The ycgH gene in the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 gene was knocked out by CRISPR / Cas9 gene editing, and the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter was inserted.

[0266] The primers are as follows (synthesized by Shanghai Invitrogen):

[0267] P37:5'-AAATGGAGGGATAACAGCC-3' (SEQ ID No. 64),

[0268] P38:5'-GCTGTTGCGGGTTAACGCTCCATTTAGAATAGAGTCGGACGAAAT-3' (SEQ ID No. 65),

[0269] P39:5'-CTAAATGGAGCGTTAACCCGCAACAGCAATAC-3' (SEQ ID No. 66),

[0270] P40:5'-GCCATTTACGCCAAGTTTTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAG-3' (SEQ ID No. 67),

[0271] P41:5'-CAGTGTATTGAAGTAGTTAGCCATGGTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATAC-3' (SEQ ID No. 68),

[0272] P42: 5'-TTGCCTTCGGGCTTATCTC-3' (SEQ ID No. 69).

[0273] Using Escherichia coli W3110 genomic DNA as a template, PCR amplification was performed using primers P37 / P38, P39 / P40, and P41 / P42 with KAPA HiFi HotStart. The resulting fragments were an 808-bp upper homology arm, a 1580-bp Ptrc-ilvC gene, and a 944-bp lower homology arm. Following PCR, the recovered DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR with primers P37 / P42 yielded the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC (SEQ ID No. 11), which is 3189 bp in size.

[0274] In SEQ ID No. 11, positions 2270 to 2343 represent the Ptrc promoter, and positions 794 to 2269 represent the ilvC gene, encoding the ilvC protein shown in SEQ ID No. 12.

[0275] 4. Preparation and transformation of competent cells

[0276] Competent cells were prepared by using CGMCC22721-Cas9 and W3110-Cas9 from Example 1. When the cells grew to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 When the p-value was 0.4, the cells were harvested to prepare competent cells. Competent cells were transformed with the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. Individual colonies were subcultured and identified by PCR using primers P43 / P44. Positive transformants were identified by PCR amplification of a 1669 bp fragment.

[0277] The primers are as follows (synthesized by Shanghai Invitrogen):

[0278] P43:5'-AAGACCTCTGGAAGCGTATC-3' (SEQ ID No.70),

[0279] P44: 5'-CGTTGCGGAAGTGAAATC-3' (SEQ ID No. 71).

[0280] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-5. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P37 / P42. Those that could amplify a fragment of 3189 bp (SEQ ID No. 11) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-ycgH and W3110-ycgH, respectively.

[0281] The recombinant bacteria CGMCC22721-ycgH and W3110-ycgH both lack the ycgH gene and overexpress the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter; specifically, the recombinant bacteria CGMCC22721-ycgH and W3110-ycgH are obtained by knocking out part of the coding region of the ycgH gene on the genomes of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, respectively, and inserting the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter, while keeping the other nucleotides in their genomes unchanged.

[0282] Example 6: Construction of engineered strains CGMCC22721-yjiT and W3110-yjiT in which the yjiV gene was deleted and the ilvE-ilvD genes were overexpressed.

[0283] Based on the Escherichia coli W3110 genome sequence published by NCBI, the yjiV gene in the L-valine-producing strain CGMCC22721-yjiT and the wild-type Escherichia coli W3110-yjiT was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that the yjiV gene was retained intact on the chromosomes of these strains). At the same time, the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp. subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110) were inserted to further study the effects of these genes on L-valine synthesis.

[0284] 1. PCR amplification of homologous recombination fragments

[0285] Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the yjiV gene were designed and synthesized. At the same time, primers for inserting the Ptrc-ilvE gene and the PilvD-ilvD gene were inserted. The yjiV gene in the genomes of the L-valine-producing strain CGMCC22721-yjiT and the wild-type Escherichia coli W3110-yjiT was knocked out by CRISPR / Cas9 gene editing, and the ilvE gene (from Bacillus subtilis) driven by the Ptrc promoter and the ilvD gene driven by the PilvD promoter were simultaneously inserted.

[0286] The primers are as follows (synthesized by Shanghai Invitrogen):

[0287] P45: 5'-AGTTCGCCCTTTTGCTCTCTC-3' (SEQ ID No. 72).

[0288] PCR amplification was performed using Escherichia coli W3110-yjiT genomic DNA as a template with primers P45 / P10, P13 / P14, and P15 / P16, along with KAPA HiFi HotStart. An 812-bp upper homology arm, a 1979-bp PilvD-ilvD gene, and an 856-bp lower homology arm were obtained. A 1161-bp Ptrc-ilvE gene was obtained using genomic DNA from Bacillus subtilis subsp. subtilis str.168 as a template with primers P11 / P12 and KAPA HiFi HotStart. After PCR, the DNA was recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. The recovered DNA was subjected to overlap PCR using primers P45 / P16 to obtain a homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD-2 (SEQ ID No. 13) with a size of 4713 bp.

[0289] In SEQ ID No. 13, positions 3809-3874 represent the PilvD promoter, positions 1958-3808 represent the ilvD gene, encoding the ilvD protein shown in SEQ ID No. 5; positions 1884-1957 represent the Ptrc promoter, positions 789-1883 represent the ilvE gene, encoding the ilvE protein shown in SEQ ID No. 6.

[0290] 2. Preparation and transformation of competent cells

[0291] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiT and W3110-yjiT competent cells of Example 1, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp CGMCC22721-yjiT-Cas9 and W3110-yjiT-Cas9 transformants containing the pREDCas9 plasmid.

[0292] Prepare CGMCC22721-yjiT-Cas9 and W3110-yjiT-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600=0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-2 plasmid and the homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD-2 from Example 2 were transformed, respectively. The cells were spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies produced by the culture were passaged and identified by PCR using primers P46 / P18. Positive transformants were identified by PCR amplification of a 1561 bp fragment.

[0293] The primers are as follows (synthesized by Shanghai Invitrogen):

[0294] P46: 5'-AGCGTTTCACTCCTACTGGG-3' (SEQ ID No. 73).

[0295] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-2. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified by PCR again using primers P45 / P16. Those that could amplify a fragment of 4713 bp (SEQ ID No. 13) were positive. The positive transformants were sent for sequencing, and the strains with correct sequencing results were named CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV, respectively.

[0296] The recombinant strains CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV both lacked the yjiT gene and overexpressed the brnF-brnE genes driven by the Ptrc promoter (from Corynebacterium glutamicum ATCC13032); the recombinant strains CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV both lacked the yjiT gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp. subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli). coli)W3110); specifically, the recombinant bacteria CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV are respectively obtained by knocking out part of the coding region of the yjiT gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter; at the same time, part of the coding region of the yjiV gene is knocked out, and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli (Escherichia coli) W3110) driven by the PilvD promoter are inserted, and the other nucleotides in the genome are kept unchanged to obtain the recombinant bacteria.

[0297] Example 7: Construction of engineered strains in which the trpR gene is deleted and the ilvH gene is overexpressed in CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV.

[0298] Based on the Escherichia coli W3110 genome sequence published by NCBI, the trpR gene in the L-valine-producing strain CGMCC22721-yjiT-yjiV and the wild-type Escherichia coli W3110-yjiT-yjiV gene was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that these strains retained the complete trpR gene on their chromosomes), and the ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F genes, in order to further study the effects of these genes on the synthesis of L-valine.

[0299] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV competent cells of Example 6, respectively. The cells were spread on a 2-YT agar plate containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiT-yjiV-Cas9 and W3110-yjiT-yjiV-Cas9 transformants containing the pREDCas9 plasmid.

[0300] Prepare CGMCC22721-yjiT-yjiV-Cas9 and W3110-yjiT-yjiV-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-3 plasmid and the homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH from Example 3 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were subcultured and identified by PCR using primers P25 / P26. Positive transformants were identified by PCR amplification of a 1437 bp fragment.

[0301] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-3. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P19 / P24. Those that could amplify a fragment of 2108 bp (SEQ ID No. 7) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR, respectively.

[0302] The recombinant strains CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR both deleted the yjiT gene and overexpressed the brnF-brnE genes driven by the Ptrc promoter (from Corynebacterium glutamicum ATCC13032); deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); deleted the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17F Gene. Specifically, the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR are respectively the L-valine producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genomes on which the partial coding region of the yjiT gene is knocked out, and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; the partial coding region of the yjiV gene is knocked out, and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter are inserted; at the same time, the partial coding region of the trpR gene is knocked out, and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17F Gene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0303] Example 8: Construction of engineered strains in which the lacI-lacZ genes are deleted and the DNA polymerase gene is overexpressed in CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR.

[0304] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the lacI-lacZ genes in the L-valine-producing strain CGMCC22721-yjiT-yjiV-trpR and the wild-type Escherichia coli W3110-yjiT-yjiV-trpR genes (sequencing confirmed that these strains retain the intact lacI-lacZ genes on their chromosomes). At the same time, a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted to further study the effects of these genes on L-valine synthesis.

[0305] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR competent cells of Example 7, respectively. The cells were spread on a 2-YT agar plate containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiT-yjiV-trpR-Cas9 and W3110-yjiT-yjiV-trpR-Cas9 transformants containing the pREDCas9 plasmid.

[0306] Prepare CGMCC22721-yjiT-yjiV-trpR-Cas9 and W3110-yjiT-yjiV-trpR-Cas9 competent cells. 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were harvested to prepare competent cells. These cells were transformed with the pGRB-sgRNA-4 plasmid and the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase described in Example 4, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Individual colonies were subcultured and identified by PCR using primers P35 / P36. Positive transformants were identified by PCR amplification of a 1655 bp fragment.

[0307] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-4. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P27 / P34. Those that could amplify a fragment of 4524 bp (SEQ ID No. 9) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ, respectively.

[0308] The recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ both deleted the yjiT gene and overexpressed the brnF-brnE genes driven by the Ptrc promoter (from Corynebacterium glutamicum ATCC13032); deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); deleted the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17FGenes: lacI-lacZ genes were deleted, and the DNA polymerase gene driven by the PxylF promoter was overexpressed (from Escherichia coli BL21). Specifically, the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ are respectively the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genomes on which the partial coding region of the yjiT gene is knocked out, and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; the partial coding region of the yjiV gene is knocked out, and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter are inserted; the partial coding region of the trpR gene is knocked out, and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17F gene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0309] Example 9: Construction of engineered strains CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ in which the ycgH gene was deleted and the ilvC gene was overexpressed.

[0310] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the ycgH gene in the L-valine-producing strain CGMCC22721-yjiT-yjiV-trpR-lacIZ and the wild-type Escherichia coli W3110-yjiT-yjiV-trpR-lacIZ (sequencing confirmed that these strains retained the intact ycgH gene on their chromosomes). At the same time, the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter was inserted to further study the effects of these genes on L-valine synthesis.

[0311] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ competent cells of Example 8, respectively. The cells were spread on a 2-YT agar plate containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiT-yjiV-trpR-lacIZ-Cas9 and W3110-yjiT-yjiV-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0312] Prepare CGMCC22721-yjiT-yjiV-trpR-lacIZ-Cas9 and W3110-yjiT-yjiV-trpR-lacIZ-Cas9 competent cells. 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were passaged and identified by PCR using primers P43 / P44. Positive transformants were identified by PCR amplification of a 1669 bp fragment.

[0313] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-5. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P37 / P42. Those that could amplify a fragment of 3189 bp (SEQ ID No. 11) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH, respectively.

[0314] The recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH both deleted the yjiT gene and overexpressed the brnF-brnE genes driven by the Ptrc promoter (from Corynebacterium glutamicum ATCC13032); deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); deleted the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17F Genes; lacI-lacZ genes were deleted, and the DNA polymerase gene driven by the PxylF promoter was overexpressed (from Escherichia coli BL21); ycgH gene was deleted, and the ilvC gene driven by the Ptrc promoter was overexpressed (from Escherichia coli W3110). Specifically, the recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH are respectively the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genomes on which the partial coding region of the yjiT gene is knocked out and the brnF-brnE gene (from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter is inserted; the partial coding region of the yjiV gene is knocked out and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli) driven by the PilvD promoter are inserted. coli)W3110); the coding region of the trpR gene was partially deleted and the mutant ilvH driven by the Ptrc promoter was inserted. G14D、S17FThe invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0315] Example 10: Construction of engineered strains in which the trpR gene is deleted and the ilvH gene is overexpressed in CGMCC22721-yjiV and W3110-yjiV.

[0316] Based on the Escherichia coli W3110 genome sequence published by NCBI, the trpR gene in the L-valine-producing strain CGMCC22721-yjiV and the wild-type Escherichia coli W3110-yjiV gene was knocked out using CRISPR / Cas9 gene editing technology (sequencing confirmed that these strains retained the complete trpR gene on their chromosomes), and the ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F genes, in order to further study the effects of these genes on the synthesis of L-valine.

[0317] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiV and W3110-yjiV competent cells of Example 2, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiV-Cas9 and W3110-yjiV-Cas9 transformants containing the pREDCas9 plasmid.

[0318] Prepare CGMCC22721-yjiV-Cas9 and W3110-yjiV-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600=0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-3 plasmid and the homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH from Example 3 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were subcultured and identified by PCR using primers P25 / P26. Positive transformants were identified by PCR amplification of a 1437 bp fragment.

[0319] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-3. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P19 / P24. Those that could amplify a fragment of 2108 bp (SEQ ID No. 7) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiV-trpR and W3110-yjiV-trpR, respectively.

[0320] The recombinant strains CGMCC22721-yjiV-trpR and W3110-yjiV-trpR both deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); the trpR gene was deleted and the ilvH gene driven by the Ptrc promoter was overexpressed. G14D、S17F Specifically, the recombinant bacteria CGMCC22721-yjiV-trpR and W3110-yjiV-trpR are respectively the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genomes of which the yjiV gene coding region is deleted and the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter are inserted; at the same time, the trpR gene coding region is deleted and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17FGene, and recombinant bacteria obtained by keeping other nucleotides in its genome unchanged.

[0321] Example 11: Construction of engineered strains in which the lacI-lacZ genes were deleted and the DNA polymerase gene was overexpressed in CGMCC22721-yjiV-trpR and W3110-yjiV-trpR.

[0322] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the lacI-lacZ genes in the L-valine-producing strain CGMCC22721-yjiV-trpR and the wild-type Escherichia coli W3110-yjiV-trpR gene (sequencing confirmed that these strains retain the complete lacI-lacZ genes on their chromosomes). At the same time, a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted to further study the effects of these genes on L-valine synthesis.

[0323] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiV-trpR and W3110-yjiV-trpR competent cells of Example 10, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiV-trpR-Cas9 and W3110-yjiV-trpR-Cas9 transformants containing the pREDCas9 plasmid.

[0324] Prepare CGMCC22721-yjiV-trpR-Cas9 and W3110-yjiV-trpR-Cas9 competent cells. 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were harvested to prepare competent cells. These cells were transformed with the pGRB-sgRNA-4 plasmid and the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase described in Example 4, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Individual colonies were subcultured and identified by PCR using primers P35 / P36. Positive transformants were identified by PCR amplification of a 1655 bp fragment.

[0325] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-4. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P27 / P34. Those that could amplify a fragment of 4524 bp (SEQ ID No. 9) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ, respectively.

[0326] The recombinant strains CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ both deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); the trpR gene was deleted and the ilvH gene driven by the Ptrc promoter was overexpressed. G14D、S17F Gene; lacI-lacZ genes are deleted, and the DNA polymerase gene driven by the PxylF promoter is overexpressed (from Escherichia coli BL21). Specifically, the recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ are respectively the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110 genomes. The yjiV gene coding region is deleted, and the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110) are inserted; the trpR gene coding region is deleted, and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17Fgene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0327] Example 12: Construction of engineered strains CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ in which the ycgH gene is deleted and the ilvC gene is overexpressed.

[0328] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the ycgH gene in the L-valine-producing strain CGMCC22721-yjiV-trpR-lacIZ and the wild-type Escherichia coli W3110-yjiV-trpR-lacIZ (sequencing confirmed that these strains retained the intact ycgH gene on their chromosomes). At the same time, the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter was inserted to further study the effects of these genes on L-valine synthesis.

[0329] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-yjiV-trpR-lacIZ and wild-type Escherichia coli W3110-yjiV-trpR-lacIZ competent cells of Example 11, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-yjiV-trpR-lacIZ-Cas9 and W3110-yjiV-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0330] Prepare CGMCC22721-yjiV-trpR-lacIZ-Cas9 and W3110-yjiV-trpR-lacIZ-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600=0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were passaged and identified by PCR using primers P43 / P44. Positive transformants were identified by PCR amplification of a 1669 bp fragment.

[0331] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-5. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P37 / P42. Those that could amplify a fragment of 3189 bp (SEQ ID No. 11) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH, respectively.

[0332] The recombinant strains CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH both deleted the yjiV gene and overexpressed the ilvE gene driven by the Ptrc promoter (from Bacillus subtilis subsp.subtilis str.168) and the ilvD gene driven by the PilvD promoter (from Escherichia coli W3110); the trpR gene was deleted and the ilvH gene driven by the Ptrc promoter was overexpressed. G14D、S17FGenes; lacI-lacZ genes were deleted, and the DNA polymerase gene driven by the PxylF promoter was overexpressed (from Escherichia coli BL21); ycgH gene was deleted, and the ilvC gene driven by the Ptrc promoter was overexpressed (from Escherichia coli W3110). Specifically, the recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH respectively deleted the coding region of the yjiV gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserted the ilvE gene (from Bacillus subtilis subsp.subtilis str.168) driven by the Ptrc promoter and the ilvD gene (from Escherichia coli W3110) driven by the PilvD promoter; the coding region of the trpR gene was deleted and the mutant ilvH gene driven by the Ptrc promoter was inserted. G14D、S17F The invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0333] Example 13: Construction of engineered strains in which the lacI-lacZ genes were deleted and the DNA polymerase gene was overexpressed in CGMCC22721-trpR and W3110-trpR.

[0334] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the lacI-lacZ genes in the L-valine-producing strain CGMCC22721-trpR and the wild-type Escherichia coli W3110-trpR gene (sequencing confirmed that these strains retain the complete lacI-lacZ genes on their chromosomes). At the same time, a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted to further study the effects of these genes on L-valine synthesis.

[0335] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-trpR and W3110-trpR competent cells of Example 3, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain CGMCC22721-trpR-Cas9 and W3110-trpR-Cas9 transformants containing the pREDCas9 plasmid (943 bp).

[0336] Prepare CGMCC22721-trpR-Cas9 and W3110-trpR-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were harvested to prepare competent cells. These cells were transformed with the pGRB-sgRNA-4 plasmid and the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase described in Example 4, respectively. The cells were then plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Individual colonies were subcultured and identified by PCR using primers P35 / P36. Positive transformants were identified by PCR amplification of a 1655 bp fragment.

[0337] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-4. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified again by PCR using primers P27 / P34. Those that could amplify a fragment of 4524 bp (SEQ ID No. 9) were positive. The positive transformants were sent for sequencing, and the strains with correct sequencing results were named CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ, respectively.

[0338] The recombinant strains CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ both lacked the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17FGenes; lacI-lacZ genes are deleted, and the DNA polymerase gene driven by the PxylF promoter is overexpressed (from Escherichia coli BL21). Specifically, the recombinant bacteria CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ are respectively knocked out of the coding region of the trpR gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and the mutant ilvH driven by the Ptrc promoter is inserted. G14D、S17F gene; at the same time, the coding region of the lacI-lacZ gene was knocked out, and a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21) was inserted, while keeping other nucleotides in its genome unchanged to obtain a recombinant bacterium.

[0339] Example 14: Construction of engineered strains CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ in which the ycgH gene was deleted and the ilvC gene was overexpressed.

[0340] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the ycgH gene in the L-valine-producing strain CGMCC22721-trpR-lacIZ and the wild-type Escherichia coli W3110-trpR-lacIZ gene (sequencing confirmed that these strains retained the intact ycgH gene on their chromosomes). At the same time, the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter was inserted to further study the effects of these genes on L-valine synthesis.

[0341] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-trpR-lacIZ and wild-type Escherichia coli W3110-trpR-lacIZ competent cells of Example 13, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp of CGMCC22721-trpR-lacIZ-Cas9 and W3110-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0342] Prepare CGMCC22721-trpR-lacIZ-Cas9 and W3110-trpR-lacIZ-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were passaged and identified by PCR using primers P43 / P44. Positive transformants were identified by PCR amplification of a 1669 bp fragment.

[0343] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-5. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P37 / P42. Those that could amplify a fragment of 3189 bp (SEQ ID No. 11) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH, respectively.

[0344] The recombinant strains CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH both lacked the trpR gene and overexpressed the ilvH gene driven by the Ptrc promoter. G14D、S17FGenes; lacI-lacZ genes are deleted, and the DNA polymerase gene driven by the PxylF promoter is overexpressed (from Escherichia coli BL21); the ycgH gene is deleted, and the ilvC gene driven by the Ptrc promoter is overexpressed (from Escherichia coli W3110). Specifically, the recombinant bacteria CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH are respectively knocked out of the coding region of the trpR gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and the mutant ilvH gene driven by the Ptrc promoter is inserted. G14D、S17F The invention relates to a recombinant bacterium obtained by knocking out part of the coding region of the lacI-lacZ gene and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); and simultaneously knocking out part of the coding region of the ycgH gene and inserting the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110), while keeping other nucleotides in the genome unchanged.

[0345] Example 15: Construction of engineered strains CGMCC22721-lacIZ and W3110-lacIZ in which the ycgH gene is deleted and the ilvC gene is overexpressed.

[0346] Based on the Escherichia coli W3110 genome sequence published by NCBI, CRISPR / Cas9 gene editing technology was used to knock out the ycgH gene in the L-valine-producing strain CGMCC22721-lacIZ and the wild-type Escherichia coli W3110-lacIZ (sequencing confirmed that these strains retained the intact ycgH gene on their chromosomes). At the same time, the ilvC gene (from Escherichia coli W3110) driven by the Ptrc promoter was inserted to further study the effects of these genes on L-valine synthesis.

[0347] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into the CGMCC22721-lacIZ and wild-type Escherichia coli W3110-lacIZ competent cells of Example 4, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32 ° C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR to obtain 943 bp CGMCC22721-lacIZ-Cas9 and W3110-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0348] Prepare CGMCC22721-lacIZ-Cas9 and W3110-lacIZ-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. 600 =0.4, the cells were collected to prepare competent cells, and the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5 were transformed, respectively. The cells were plated onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 h. Single colonies were passaged and identified by PCR using primers P43 / P44. Positive transformants were identified by PCR amplification of a 1669 bp fragment.

[0349] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and a final concentration of 0.2% arabinose to eliminate the plasmid pGRB-sgRNA-5. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and identified by PCR again using primers P37 / P42. Those that could amplify a fragment of 3189 bp (SEQ ID No. 11) were positive, and the positive transformants were sent for sequencing. The strains with correct sequencing results were named CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH, respectively.

[0350] The recombinant bacteria CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH both lacked the lacI-lacZ genes and overexpressed the DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); they also lacked the ycgH gene and overexpressed the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110). Specifically, the recombinant bacteria CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH are respectively obtained by knocking out part of the coding region of the lacI-lacZ gene on the genome of the L-valine-producing bacteria CGMCC22721 and the wild-type Escherichia coli W3110, and inserting a DNA polymerase gene driven by the PxylF promoter (from Escherichia coli BL21); at the same time, part of the coding region of the ycgH gene is knocked out, and the ilvC gene driven by the Ptrc promoter (from Escherichia coli W3110) is inserted, while keeping other nucleotides in their genomes unchanged.

[0351] Example 16, L-valine fermentation experiment.

[0352] Fermentation experiments were conducted using the strains constructed in Examples 1-15 above, the valine-producing strain CGMCC22721, and wild-type Escherichia coli W3110 in a BLBIO-5GC-4-H fermentor (Shanghai Bailun Biotechnology Co., Ltd.) using the culture medium shown in Figure 1 and the control process shown in Figure 2. Each strain was repeated three times, and the results are shown in Figure 3. As shown in the above fermentation results, the modifications described in Examples 1-15 contributed to increased L-valine production in both the L-valine-producing strain CGMCC22721 and the model strain wild-type E. coli W3110.

[0353] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art. Industrial Applicability

[0354] Experiments have shown that the engineered bacteria modified by the present invention can increase the yield of L-valine, can be used to produce L-valine, and have good application prospects. Sequence Listing Free Content

[0355] SEQ ID No.1: Homologous recombination DNA fragment ΔyjiT-Ptrc-brnF-brnE sequence (2579 bp), artificial sequence

[0356] SEQ ID No. 2: brnF protein (from Corynebacterium glutamicum ATCC13032)

[0357] SEQ ID No. 3: brnE protein (from Corynebacterium glutamicum ATCC13032)

[0358] SEQ ID No.4: Homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD sequence (4732 bp), artificial sequence

[0359] SEQ ID No. 5: ilvD protein (from Escherichia coli W3110)

[0360] SEQ ID No. 6: ilvE protein (from Bacillus subtilis subsp. subtilis str. 168)

[0361] SEQ ID No.7: Homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH (2108 bp), artificial sequence

[0362] SEQ ID No.8:ilvH G14D、S17F Gene-encoded amino acid sequence (163aa), artificial sequence

[0363] SEQ ID No.9: Homologous recombinant DNA fragment ΔlacIZ-PxylF-DNA polymerase sequence (4524 bp), artificial sequence

[0364] SEQ ID No. 10: Amino acid sequence of DNA polymerase (from Escherichia coli BL21)

[0365] SEQ ID No.11: Homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC sequence (3189 bp), artificial sequence

[0366] SEQ ID No. 12: Amino acid sequence of ilvC protein (from Escherichia coli W3110)

[0367] SEQ ID No.13: Homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD-2 sequence (4713 bp), artificial sequence

Claims

1. An engineered bacterium for producing valine, which contains the characteristics of B1), B2), B3), B4) or B5); B1) includes B11), B12) and B13): B11) The yjiT gene is not expressed or weakly expressed, or the activity of the protein encoded by the yjiT gene is weakened or lost; B12) the content of the protein encoded by the brnF gene increases or the activity of the protein encoded by the brnF gene increases; B13) the content of the protein encoded by the brnE gene increases or the activity of the protein encoded by the brnE gene increases; B2) includes B21), B22) and B23): B21) The yjiV gene is not expressed or weakly expressed, or the activity of the protein encoded by the yjiV gene is weakened or lost; B22) the content of the protein encoded by the ilvE gene increases or the activity of the protein encoded by the ilvE gene increases; B23) the content of the protein encoded by the ilvD gene increases or the activity of the protein encoded by the ilvD gene increases; B3) includes B31) and B32): B31) the trpR gene is not expressed or weakly expressed, or the activity of the protein encoded by the trpR gene is weakened or lost; B32)ilvH G14D、S17F Increased levels of the gene encoding the protein or the ilvH G14D、S17F The activity of the gene-encoded protein is enhanced; the ilvH G14D、S17F The gene was obtained by replacing the 14th glycine codon in the ilvH gene with an aspartate codon and the 17th serine codon with a phenylalanine codon; B4) includes B41), B42) and B43): B41) the lacI gene is not expressed or weakly expressed, or the activity of the protein encoded by the lacI gene is weakened or lost; B42) the lacZ gene is not expressed or weakly expressed, or the activity of the protein encoded by the lacZ gene is weakened or lost; B43) the content of DNA polymerase increases or the activity of the DNA polymerase increases; B5) includes B51) and B52): B51) The ycgH gene is not expressed or weakly expressed, or the activity of the protein encoded by the ycgH gene is weakened or lost; B52) the content of the protein encoded by the ilvC gene increases or the activity of the protein encoded by the ilvC gene increases; The engineered bacteria is Escherichia coli.

2. The engineered bacteria for producing valine are recombinant bacteria obtained by transforming the recipient bacteria; the transformation includes A1), A2), A3), A4) or A5); A1) includes A11), A12) and A13): A11) knocking out the yjiT gene of the recipient bacteria, inhibiting the expression of the yjiT gene or inhibiting the activity of the protein encoded by the yjiT gene; A12) increasing the content of the protein encoded by the brnF gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnF gene; A13) increasing the content of the protein encoded by the brnE gene in the recipient bacteria or enhancing the activity of the protein encoded by the brnE gene; A2) includes A21), A22) and A23): A21) knocking out the yjiV gene of the recipient bacteria, inhibiting the expression of the yjiV gene or inhibiting the activity of the protein encoded by the yjiV gene; A22) increasing the content of the protein encoded by the ilvE gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvE gene; A23) increasing the content of the protein encoded by the ilvD gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvD gene; A3) includes A31) and A32): A31) knocking out the trpR gene of the recipient bacteria, inhibiting the expression of the trpR gene or inhibiting the activity of the protein encoded by the trpR gene; A32) increasing ilvH in the recipient bacteria G14D、S17F Gene encoding protein content or enhance the ilvH G14D、S17F The activity of the gene encoding the protein; the ilvH G14D、S17F The gene was obtained by replacing the 14th glycine codon in the ilvH gene with an aspartate codon and the 17th serine codon with a phenylalanine codon; A4) includes A41), A42) and A43): A41) knocking out the lacI gene of the recipient bacteria, inhibiting the expression of the lacI gene or inhibiting the activity of the protein encoded by the lacI gene; A42) knocking out the lacZ gene of the recipient bacterium, inhibiting the expression of the lacZ gene or inhibiting the activity of the protein encoded by the lacZ gene; A43) increasing the content of DNA polymerase in the recipient bacteria or enhancing the activity of the DNA polymerase; A5) includes A51) and A52): A51) knocking out the ycgH gene of the recipient bacteria, inhibiting the expression of the ycgH gene or inhibiting the activity of the protein encoded by the ycgH gene; A52) increasing the content of the protein encoded by the ilvC gene in the recipient bacterium or enhancing the activity of the protein encoded by the ilvC gene; The recipient bacteria is Escherichia coli.

3. The engineered bacteria according to claim 1 or 2, characterized in that: The brnF gene and the brnE gene are derived from Corynebacterium glutamicum.

4. The engineered bacteria according to claim 3, characterized in that: The brnF gene encodes the protein shown in SEQ ID No. 2 in the sequence table; The brnE gene encodes the protein shown in SEQ ID No. 3 in the sequence table.

5. The engineered bacteria according to claim 4, characterized in that: The brnF gene is a DNA molecule shown in positions 832-1587 of SEQ ID No. 1 in the sequence list; The brnE gene is a DNA molecule shown in positions 1584-1910 of SEQ ID No. 1 in the sequence table.

6. The engineered bacteria according to any one of claims 1 to 5, characterized in that: The i lvE gene is derived from Bacillus subtilis; The ilvD gene is derived from Escherichia coli.

7. The engineered bacteria according to claim 6, characterized in that: The ilvE gene encodes the protein shown in SEQ ID No.6 in the sequence table; The ilvD gene encodes the protein shown in SEQ ID No. 5 in the sequence table.

8. The engineered bacteria according to claim 7, characterized in that: The ilvE gene is a DNA molecule shown in positions 808-1902 of SEQ ID No. 4 in the sequence list; The ilvD gene is a DNA molecule shown in positions 1977-3827 of SEQ ID No. 4 in the sequence list.

9. The engineered bacteria according to any one of claims 1 to 8, characterized in that: The i lvH gene is derived from Escherichia coli.

10. The engineered bacteria according to claim 9, characterized in that: The ilvH G14D、S17F The gene encodes the protein shown in SEQ ID No.8 in the sequence table.

11. The engineered bacteria according to claim 10, characterized in that: The ilvH G14D、S17F The gene is a DNA molecule shown in positions 835-1326 of SEQ ID No. 7 in the sequence list.

12. The engineered bacteria according to any one of claims 1 to 11, characterized in that: The DNA polymerase is derived from Escherichia coli.

13. The engineered bacteria according to claim 12, characterized in that: The DNA polymerase is a protein shown in SEQ ID No. 10 in the sequence table.

14. The engineered bacteria according to claim 13, characterized in that: The coding gene of the DNA polymerase is the DNA molecule shown in positions 701-3352 of SEQ ID No. 9 in the sequence list.

15. The engineered bacteria according to any one of claims 1 to 14, characterized in that: The ilvC gene is derived from Escherichia coli.

16. The engineered bacteria according to claim 15, characterized in that: The ilvC gene encodes the protein shown in SEQ ID No. 12 in the sequence table.

17. The engineered bacteria according to claim 16, characterized in that: The ilvC gene is a DNA molecule shown in positions 794-2269 of SEQ ID No. 11 in the sequence list.

18. The engineered bacteria according to any one of claims 1, 3-17, characterized in that: The engineered bacteria also contain any two, any three, any four or five of the characteristics of B1), B2), B3), B4) and B5).

19. The engineered bacteria according to any one of claims 2 to 17, characterized in that: The transformation also includes any two, any three, any four or five of A1), A2), A3), A4) and A5).

20. A method for preparing an engineered bacterium for producing valine, comprising: The recipient bacteria are transformed according to any one of A1), A2), A3), A4) or A5) in claims 2-17 to obtain the target engineered bacteria, wherein the recipient bacteria is Escherichia coli.

21. The method according to claim 20, characterized in that: The method also includes subjecting the recipient bacteria to any two, three, four or five of the five modifications A1), A2), A3), A4) and A5) described in any one of claims 2-17.

22. A method for preparing L-valine, comprising: Cultivate the engineered bacteria according to any one of claims 1 to 19 to obtain L-valine.

23. Use of the engineered bacteria according to any one of claims 1 to 19 in producing L-valine, or in preparing a product for producing L-valine, or in preparing a food, feed or medicine containing L-valine.

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