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WO2023065725A1 - Procédé d'édition multiplex du génome bactérien fondé sur la technique de recombinaison de l'adn double brin et son application - Google Patents

Procédé d'édition multiplex du génome bactérien fondé sur la technique de recombinaison de l'adn double brin et son application Download PDF

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WO2023065725A1
WO2023065725A1 PCT/CN2022/103604 CN2022103604W WO2023065725A1 WO 2023065725 A1 WO2023065725 A1 WO 2023065725A1 CN 2022103604 W CN2022103604 W CN 2022103604W WO 2023065725 A1 WO2023065725 A1 WO 2023065725A1
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double
delet
stranded dna
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卞小莹
王雪
郑文韬
涂强
张友明
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Shandong University
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Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a bacterial genome multiple editing method and its application.
  • the Cas nuclease of the CRISPR-Cas system uses a short guide RNA (gRNA) to recognize the target DNA region and create double strand breaks (double strand breaks, DSB) there.
  • DSB can be repaired by non-homologous end joining (NHEJ) to achieve gene knockout, and can also be repaired by homologous recombination (HR) to achieve gene replacement and insertion.
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • DSBs are harmful to most cells, and the introduction of too many DSBs at one time will often lead to cell death. Therefore, currently, the CRISPR-Cas system can be used to simultaneously edit no more than three sites.
  • Red/ET recombination technology was first used in Escherichia coli. It can use short homology arms ( ⁇ 50bp) to replicate in the genome replication stage. The fork can achieve genetic modification through homologous recombination without causing double-strand breaks in the genome.
  • Recombinant engineering is based on the expression of red ⁇ /red ⁇ derived from the Red operon of Escherichia coli lambda phage or the expression of recE/recT of Rac prophage.
  • ssDNA-based recombination engineering generally only requires the presence of Red ⁇ or RecT to complete precise genome editing, so ssDNA recombination engineering is widely used in multiple genome editing work, MAGE, TRMR, and DlvERGE are all automated through Red ⁇ -mediated ssDNA recombination With the assistance of the equipment, the technology of genome multiple editing is realized by using ssDNA that can initiate the synthesis of Okazaki fragments as a substrate.
  • Double-stranded DNA (double strand DNA, dsDNA) can be easily obtained by PCR reaction (up to 10kb), using dsDNA as a recombination substrate can greatly extend the insertion length, and the target fragment and selection marker can be put together to improve Screening efficiency of multi-region modified mutants.
  • dsDNA recombination engineering has been widely used in bacterial genome modification.
  • the phage recombinase Red ⁇ 7029 derived from S. brevitalea DSM7029 can effectively edit the genome of the strain itself and several other Burkholderiales strains.
  • the lambda Red-like recombinase-BAS derived from Pseudomonas aeruginosa phage Ab31 can complete the genome editing work of 4 species of Pseudomonas.
  • the characteristics of dsDNA recombination engineering make it a potential tool to mediate genome multi-region replacement, deletion and insertion, while ssDNA recombination engineering is inefficient in this regard.
  • no multiple genome editing method based on dsDNA recombination engineering has been established in E. coli so far.
  • the purpose of the present invention is to provide a dsDNA-recombineering assisted Multiplex Genome Editing method (dsDNA-recombineering assisted Multiplex Genome Editing, dReaMGE) applicable to various bacteria and its application in multiple genome editing of five kinds of bacteria.
  • dsDNA-recombineering assisted Multiplex Genome Editing dReaMGE
  • concentration of deoxyribonucleotide dNTP, deoxynucleoside
  • the efficiency of three homologous recombinases from different sources in mediating dsDNA recombination engineering in five host cells was improved.
  • the technical solution first adopted by the present invention is: a method for multiple editing of bacterial genomes based on double-stranded DNA recombination engineering, comprising:
  • the recovery time is 1-4 hours.
  • the GC content in the added dNTPs is close to or the same as the host genome GC content.
  • the host is E.coli GB2005, and the recovery time at 30°C is 1 hour.
  • the host is Schlegelella brevitalea DSM7029 or Paraburkholderia.megapolitana DSM 23488, which is recovered at 22°C for 4 hours.
  • the host is Pseudomonas.putida KT2440, which is recovered at 22°C for 1 hour.
  • the host is P. syringae DC3000, which is recovered at 22°C for 3 hours.
  • the host is E.coli GB2005, and the original promoter of its deoxynucleic acid reductase is replaced by the gentamicin promoter P genta , and the recovery time at 30° C. is 1 hour.
  • the dsDNA substrates targeting different target sequences carry the same resistance gene.
  • the dsDNA substrates targeting different target sequences have short homology arms capable of anchoring different target sites.
  • the present invention further provides the application of the method to mediate multiple genome editing in a bacterial host.
  • the method is used to replace multiple promoters of the gene cluster of the anti-tumor compound in the bacterial host to obtain various derivatives of the anti-tumor compound.
  • the method is used to modify the bacterial metabolic network to realize genome mining and target metabolite yield optimization.
  • the present invention has the advantages of low substrate preparation cost, unlimited length of inserted gene, non-dependence on double-strand DNA breaks, no need to construct multiple guide RNA expression vectors, no need to
  • the host bacterial restriction repair system has the advantages of inactivation, etc., and can effectively mediate E.
  • the dReaMGE method established by the present invention is a potential and convenient tool for detecting the relationship between genotype and phenotype, enhancing chemical production and strain optimization, and is an ideal supplement to current genome engineering technology.
  • Figure 1 The determinants of dsDNA recombineering mediating the simultaneous editing of E.coli genome dual regions; among them,
  • b dsDNA recombineering-mediated double-region replacement efficiency at different recovery temperatures (16°C, 20°C, 24°C, 28°C, 30°C, 32°C, 37°C) at 1 hour and 6 hours of recovery time;
  • Figure 2 is the establishment of dReaMGE in Escherichia coli GB2005;
  • Figure 3 is the application of the dReaMGE method in the genome streamlining of Paraburkholderia.megapolitana DSM 23488; wherein,
  • b Efficiency of dReaMGE-mediated simultaneous knockdown of 5 biosynthetic gene clusters (BGCs) in DSM23488 after recovery at 22°C for 12 h with or without the addition of 10 nM dNTPs: Single - only one gene cluster was knocked out Knockout mutants, double - mutants with two gene clusters completely knocked out, triple - mutants with three gene clusters completely knocked out, quadruple - mutants with four gene clusters completely knocked out, quintuple - a mutant strain with five gene clusters completely knocked out. The ordinate indicates the number of different mutant strains among 10 8 cells.
  • Figure 4 is the application of dReaMGE in KT2440; among them,
  • M DL5000 DNA ladder
  • detection primers p1-genta-5out, p2-KT2440-regionA-check-5, p3-KT2440-regionB-check-5, p4-KT2440- regionC-check-5;
  • Figure 5 shows the application of dReaMGE in DC3000; among them,
  • PCR detection results of three region replacements M: DL5000 DNA ladder, detection primers: p1-genta-5out, p2-DC3000-regionA-check-5, p3-DC3000-regionB-check-5, p4-DC3000- regionC-check-5;
  • Figure 6 is the application of the dReaMGE method of the present invention in the replacement of multiple promoters of the glidobactin biosynthesis gene cluster of DSM7029 and the combined knockout of multiple negative transcriptional regulators; wherein,
  • c A novel derivative obtained by double-promoter replacement of the glidobactin biosynthetic gene cluster: luminmicinF-luminmicinI;
  • d Schematic diagram of combined knockout of four lysine family negative transcriptional regulators in glidobactin biosynthesis gene cluster: lysR2-AAW51_4987; lysR18-AAW51_5436; lysR23-AAW51_2727 and lysR29-AAW51_4579;
  • Figure 7 is the application of dReaMG in the knockout of DSM7029 multiple transcriptional regulators
  • glbB, glbC and glbF Comparison of transcript levels of glb gene cluster core genes (glbB, glbC and glbF) in DSM 7029 wild type, 7029::Papra glbB and 7029 ⁇ 4LTTRs;
  • M DL5000 DNA ladder
  • detection primers p1-genta-5out, p4-7029-lysR2-check-5, p5-7029-lysR18-check-5, p6-7029-lysR23-check-5, p7-7029-lysR29 -check-5.
  • the experimental methods all adopt laboratory routine methods, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods.
  • Reagents in this example are mainly molecular biology experimental reagents, single-stranded nucleotides are synthesized by Shanghai Sangong Biotechnology Co., Ltd., restriction enzymes, DNA polymerases and DNA markers are from New England Biolabs (New England Biolabs) , antibiotics were purchased from Invitrogen, and Promega’s luciferase detection kit was purchased from Promega Bioreagents.
  • Escherichia coli involved in the present invention GB05-dir purchased from GeneBridges, Germany; Burkholderia E.coli GB2005, Pseudomonas.putida KT2440, Pseudomonas.syringae DC3000, Burkholderiales strain DSM 7029, Paraburkholderia.megapolitana DSM 23488 , were purchased from the German Culture Collection of Microorganisms (DSMZ).
  • Embodiment 1 A dsDNA-recombineering assisted Multiplex Genome Editing (dReaMGE) method applicable to various bacteria provided by the present invention, the specific implementation steps are as follows:
  • a dsDNA substrate with short homology arms (100 bp) capable of anchoring different target sites and the same resistance gene (for example: chloramphenicol resistance gene) was obtained by PCR.
  • substrates with short homology arms capable of anchoring different target sites were obtained by phosphorylating the 5' end of the complementary strand in the dsDNA substrate to the strand that primed the synthesis of the Okazaki fragment Modification, promotes the degradation of non-target strands by 5'-3' exonuclease Red ⁇ , accelerates the formation of target ssDNA substrates in host cells, and enables host cells to utilize dsDNA substrates more fully and rapidly.
  • the host cell with the recombinase plasmid in step (1) is cultivated to the logarithmic growth phase with a suitable medium, an inducer is added to induce the expression of the recombinase, and the recombinant plasmid is prepared according to the characteristics of different host cells.
  • an inducer is added to induce the expression of the recombinase
  • the recombinant plasmid is prepared according to the characteristics of different host cells.
  • dsDNA substrates targeting different target sequences are mixed and then introduced into competent cells by electroporation.
  • the recovery time and recovery temperature of different host cells are different: E.coli GB2005, 30°C, 1 hour; Schlegelella brevitalea DSM7029, 22°C, 4 hours; Pseudomonas.putida KT2440, 22°C, 1 hour; Paraburkholderia.megapolitana DSM 23488, 22 °C, 4 hours P.syringae DC3000, 22 °C, 3 hours);
  • the detection primers are designed to amplify the 0.5-1.5kb fragment at the junction between the exogenous gene and the genome.
  • Example 2 dsDNA-mediated simultaneous editing of dual regions of the E.coli genome
  • Primer sequence the lowercase part is used to mediate sufficient homology arms, and the uppercase part is the primer for amplifying the resistance gene.
  • Pgenta-nrdAB-DH10B-3 gcagtccttctgccgCccaatccagaacgcgatgGattttgtcgagattgatgcgctctgtgctaccgtcgcgctttgtcaccagcagattctgattcatATGTATATCTCCTTTAGGTG;
  • P genta -nrdAB-DH10B-5 tcgcttatatattgaccacaactgatacatcagattatgtgatgactcgtgcttagatcaatttttgcaatcattagcaaaaagattaataagccatctaAGGCACGAACCCAGTTGACA;
  • GB2005-regionA-delet-3 tgcacttcctgccggatatctacgtgccgtgcgaccagtgcaaaggtaacgctataaccgtgaaacgctggagattaagtacaaaggcaaaaccatccaTTACGCCCCGCCCTGCCACT;
  • GB2005-regionA-delet-5 gctgccagattcgcaaatctattatctggtggatgcgtcttatcagcaggcggtgaatttactgccggaagaaaaacgtaaattgctggtgcaactctgaCGTTGATCGGCACGTAAGAG;
  • GB2005-regionB-delet-3 taatgttggcaactgcgccaccatttggcatgtagcgtacttccgggtcctgacccagattaccaacgagaataaccttgttttacgcctctgctggccatTTACGCCCCGCCCTGCCACT;
  • GB2005-regionB-delet-5 ccaacgcgggcgaataacaaacgcaaatagtcgtggatttcggtgattgtcccaccgtagaacgcgggttatgagacgtcgatttctgctcaattgagaCGTTGATCGGCACGTAAGAG.
  • the above primers were used to amplify the dsDNA whose 5' end was modified by phosphorylation and carried homology arms targeting two regions and the chloramphenicol resistance gene respectively, and 2 ⁇ g of the dsDNA substrate mixture was electrotransformed into competent cells. After electrotransformation, use 1ml LB medium without antibiotics to recover at different temperatures (16°C, 20°C, 24°C, 28°C, 30°C, 32°C, 37°C) for different time (1-12 hours) , and then spread on LB solid medium plates containing 15 ⁇ g/mL chloramphenicol and 15 ⁇ g/mL kanamycin, cultured at 37°C for 12 hours, and 48 single clones were selected for each biological repetition for colony PCR detection.
  • the number of clones (954 correct clones per 108 cells) of the double-region mutation mutant (GB2005 ⁇ regionA::cm- ⁇ B::cm) obtained by the single-resistance gene strategy was determined by double
  • the number of clones (340 correct clones per 10 8 cells) of the double-region mutant (GB2005 ⁇ regionA::cm- ⁇ B::amp) obtained by the resistance gene mutation strategy was 3 times. This result demonstrates that multi-region editing using the same resistance gene is sufficient to select cell subpopulations available for recombination and can prevent recombinants from being killed by multiple antibiotics. This finding suggests that while the use of resistance genes is not a determinant of dsDNA recombineering-mediated multiplex genome editing, it is indeed an important influencing factor, both affecting efficiency and addressing the lack of selectable markers.
  • the recovery time is the determinant factor affecting dsDNA recombineering-mediated multiplex genome editing, and the realization of dsDNA recombineering-mediated double-region replacement in E. coli is at the expense of prolonged time, which hinders the widespread use of this technology in microbial cell construction.
  • the bottleneck of the application, so the next step of the present invention is to shorten the working time by speeding up the reorganization. Recombination occurs at the replication fork during DNA replication, and the speed of recombination is determined by the movement speed of the replication fork, which is limited by the dNTP level in the body.
  • Ribonucleotide reductase can catalyze nucleotide diphosphate (ribonucleotide diphosphate, NDP) to form deoxyribonucleotide diphosphate (deoxyribonucleoside diphosphate, dNDP), dNDP is deoxynucleotide triphosphate (deoxynucleoside triphosphate, rate-limiting condition for the formation of dNTPs.
  • Escherichia coli maintains subsaturated levels of dNTPs by maintaining subexpression levels of RNRs, resulting in a slower rate of DNA replication fork movement.
  • this embodiment replaces the original promoter encoding the RNR gene (nrdAB) with the constitutive strong promoter P genta of the gentamicin resistance gene, and accelerates the moving speed of the replication fork by overexpressing RNR, thereby accelerating recombination, thereby To shorten the time of dsDNA recombineering-mediated double-region replacement.
  • the results of this example prove that long enough recovery time is the decisive factor for dsDNA recombineering to mediate double-region replacement.
  • RNR overexpression and addition of dNTP can greatly shorten the recovery time and improve the efficiency of double-region replacement.
  • Simultaneous recovery temperature and the strategy of using resistance selection markers affect the efficiency of dsDNA recombineering-mediated double-region replacement to a certain extent.
  • Embodiment three Six regions are replaced simultaneously in Escherichia coli GB2005
  • This example tests the ability of dsDNA recombineering to mediate the simultaneous replacement of six regions of the genome (0.5kb) in wild-type E.coli GB2005 and GB2005-P genta -nrdAB under the condition of adding/not adding dNTPs, as shown in Figure 2a.
  • GB2005-regionC-delet-3 attgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccTTACGCCCCGCCCTGCCACT;
  • GB2005-regionC-delet-5 gccagctggcagttcaggccaatccgcgccggatgcggtgtatcgctcgccacttcaacatcaacggtaatcgccatttgaccactaccatcaatccggtCGTTGATCGGCACGTAAGAG;
  • GB2005-regionD-delet-3 cgcctaatacatctacactttctatttattgacaagtgatacgttgcaaaggagcaacacccccacagactcgatgactgcgcagtcatacagtgaaattTTACGCCCCGCCCTGCCACT;
  • GB2005-regionD-delet-5 taggaatttcggacgcgggttcaactcccgccagctccaccaaaattctccatcggtgattaccagagtcatccgatgaagtcctaagagcccgcacggcCGTTGATCGGCACGTAAGAG;
  • GB2005-regionE-delet-3 atcaatttatagctaaattaccgcctttcagccaatttgatcgagaacaatttatctctttttgatgcccatttccaagacttatacattgataaatatcTTACGCCCCGCCCTGCCACT;
  • GB2005-regionE-delet-5 atagcaatcaaaccgaagccacatatgcgcggccagattgttgacaaagggcgctttgttcatgccggatacggcatgaacgctttatcggtctacaaCGTTGATCGGCACGTAAGAG;
  • GB2005-regionF-delet-3 tttccatttctcaatgaatcaagggcgtattgcaatgacagatggcgacaaaaatagcgtcagaaggagattgcaaaaacatgcactaccattgagatTTACGCCCCGCCCTGCCACT;
  • GB2005-regionF-delet-5 tgagattaatgacgaagtggtcatatcacaatgataaaagtgacacaattcttataacaatttttcgtgcacatttcgttctggcgataataattaatcaCGTTGATCGGCACGTAAGAG.
  • the above primer sequences were used to amplify dsDNA whose 5' end was phosphorylated and carried homology arms targeting six regions and the chloramphenicol resistance gene respectively, and 6 ⁇ g of dsDNA substrate mixture was electrotransformed into competent cells.
  • the cells after electrotransformation were incubated with 1ml of antibiotic-free LB medium (with/without adding 10nM dNTPs with GC content of 50%) at 950rpm at 30°C for 1h, and then spread on 15 ⁇ g/mL chloramphenicol solid medium plate , and cultivated at 37°C for 12h. For each biological repeat, 48 single clones were selected for colony PCR detection. The results are shown in Figure 2b.
  • the dReaMGE technology was successfully established in Escherichia coli GB2005 through the optimization of key factors and the regulation of dNTP in vivo, and the inventors are encouraged to establish the dReaMGE technology in more bacteria through similar strategies.
  • Example 4 The dReaMGE method of the present invention realizes genome simplification of DSM23488
  • the dReaMGE method was successfully implemented in Burkholderia DSM 23488 using Red ⁇ -Red ⁇ 7029 recombinase with dNTP addition, recovery time and temperature adjustment, and genome reduction was achieved, as shown in Figure 3a.
  • 23488-delet-BGC1-5 gctgctgctcgtgatgcttgtctatccggtcggccagttgttgttgctgagcatgcacagaaggcacgaacccagttgaCCAACCGCGTGGCACAACAA;
  • 23488-delet-BGC1-3 gctgcgcacgcatcatctgtttgggcgggatggcgcgcgttgcgacacggtgattggcgtcggcttgaacgaattgttaCAACTTAAATGTGAAAGTGG;
  • 23488-delet-BGC3-3 cgtcacagccggcacgacgattacctgcgctaccggttccaacaccaatagcttttccatcggcttgaacgaattgttaCAACTTAAATGTGAAAGTGG;
  • 23488-delet-BGC5-5 gcaacgcggcgaatgacgggcccatcggacgcacgcggatctgcggatcgaggtgttgcgaaggcacgaacccagttgaCCAACCGCGTGGCACAACAA;
  • 23488-delet-BGC5-3 cgtgcgcggcctgccgcacttcgatcacacgctgcacgaaaattccgggcgtgacgatctcggcttgaacgaattgttaCAACTTAAATGTGAAAGTGG;
  • 23488-delet-BGC6-5 ggtcactgatagtgacgaagttctggccgtgattgaacgccgtggctgcagtcttgcccgaaggcacgaacccagttgaCCAACCGCGTGGCACAACAA;
  • 23488-delet-BGC6-3 cacccggcgccacgcctgtccagctacggaacgcgcgatagaacgacgggatatcgccgtcggcttgaacgaattgttaCAACTTAAATGTGAAAGTGG;
  • 23488-delet-BGC8-5 cagcgaggcgcctgcggcgacgcccgacaccgcacttccagcgttcggtggggctgtgaaggcacgaacccagttgaCCAACCGCGTGGCACAACAA;
  • 23488-delet-BGC8-3 cgccgctgcgctcggcgcaatcggttctccatcggtttccggcgcggtggctgtggcggtcggcttgaacgaattgttaCAACTTAAATGTGAAAGTGG.
  • the implementation steps of the dReaMGE method in DSM 23488 are as follows: use R6K-loxM-genta as a template, use the above primer sequence to amplify the 5' end modification and target five BGC regions ( BGC 1,3,6,8,9) homology arm of the gentamicin resistance gene, 5 ⁇ g of dsDNA substrate mixture was electrotransformed into competent cells.
  • Embodiment Five Three regions are replaced simultaneously in P.putida KT2440
  • KT2440-regionA-delet-3 ggtgccatcttctcgaatagatactgatttaaggagatgctggctaagcatggaggggtgtgaaggggtataaccctgctcgaaagcttcagtctctttttCAACTTAAATGTGAAAGTGG;
  • KT2440-regionA-delet-5 ccagctccgcgatatccaaccgaaggccgcataagagatctacgaccttagcgaggcaagcgtactgccgggccactcgaaagagggatcgcaaagccCCAACCGCGTGGCACAACAA;
  • KT2440-regionB-delet-3 gtcatccgtagcgaggctggatattgtgaagcccgcctttcccaaggatagatcaatttctttaggtatcgtcaaggagttcaaaaatgggggtgtagtgCAACTTAAATGTGAAAGTGG;
  • KT2440-regionB-delet-5 cccgcgccattctgagtagcacaaactttggcgagcatcagtccagcgtatacgatctagattaatttatggataaatcatcaatgagccacgctccagaCCAACCGCGTGGCACAACAA;
  • KT2440-regionC-delet-3 cacgatgtaggcgcctattcacctttccgcccttccggcgggttactttggtcgtggccaaagtaaccaaaaccgtcggctcccatcatccggccctacCAACTTAAATGTGAAAGTGG;
  • KT2440-regionC-delet-5 tcgacggatcgggtcatcaaagccgccaaaaccaaaagaccttgaaagggatggcccttacggcgggtcccttttgtcgtggcaaaaaggaaccaaaaCCAACCGCGTGGCACAACAA.
  • the dReaMGE method was improved in P. putida by prolonging the recovery time (Fig. 4a), optimizing the recovery temperature (Fig. KT2440 can also be applied.
  • the dsDNA substrate was amplified with the above primers.
  • KT2440 pBBR1-Rha-BAS-kan
  • transfer the overnight cultured bacterial solution to 1.3ml fresh LB medium add 10 ⁇ g/mL kanamycin (initial OD600 is 0.15), 30°C, 950rpm culture for 2h , adding L-rhamnose to induce the expression of the recombinase for 60 min.
  • Embodiment six Three regions are replaced simultaneously in P.syringae DC3000
  • DC3000-regionA-delelt-3 gaaaacaccgataatttagttaggagcaacattgttagtgagaatattaatagcttgctaactaatatctcgtaacaagatcttacggtcaacccgtaacCAACTTAAATGTGAAAGTGG;
  • DC3000-regionA-delelt-5 DC3000-regionA-delelt-5: gattctgcgaggtaaattttttcgtaaagcttgtactcttcgtcatctgacttcatgtgcgtacagttaccatccgaggtttgagctgtatcatagagtcCCAACCGCGTGGCACAACAA;
  • DC3000-regionB-delelt-3 gaaaacaccgataatttagttaggagcaacattgttagtgagaatattaatagcttgctaactaatatctcgtaacaagatcttacggtcaacccgtaacCAACTTAAATGTGAAAGTGG;
  • DC3000-regionB-delelt-5 DC3000-regionB-delelt-5: gattctgcgaggtaaattttttcgtaaagcttgtactcttcgtcatctgacttcatgtgcgtacagttaccatccgaggtttgagctgtatcatagagtcCCAACCGCGTGGCACAACAA;
  • DC3000-regionC-delelt-3 aggaacgtgggtaagtctgagggttttcatgcgctaatcctttatccttacggctcaatacttggcgagggttggtcggtactttgcgtgtgcaaggctgCAACTTAAATGTGAAAGTGG;
  • DC3000-regionC-delelt-5 attggcagctcaaagccgccagttttacccaggggggtcaattctaggttggcgttagggggtcatttttacagtggcggtgacaactacgtactgtcttCCAACCGCGTGGCACAACAA.
  • the dReaMGE technology has been improved in P. It can be applied in DC3000.
  • the steps of simultaneous editing of three genome regions in P. syringae DC3000 (pBBR1-Rha-BAS-kan) are similar to those of KT2440.
  • R6K-loxM-genta was amplified with the above primers.
  • Example 7 dReaMGE replaces both glbB and glbC promoters in DSM 7029
  • glbB-genta-GFP-P apra -5 tgctacagcttaacgagtccgtaaagatgtccaacaagtcgacaacacaagcagcaggcaagcgcccgcggtcttgaccgcgagcctcaacccacaacgtTTACTTGTACAGCTCGTCCA;
  • glbB-genta-GFP-P apra -3 cccatccgcgttgctgcaaggctgcgcgcacgtcgccgggttccgtttccggtgctgcggcagcccattcctgtcctgtcatttttgacatgctttcccgGACATTGCACTCCACCGCTG;
  • glbC-genta-P apra -5 ggctgtcagctgcagccccggcgcctcacacggcacgcaccgtgatcttgatcgacttctcgcccttgaaccctgcgtccagcgcgcaacccacctgaTTACTTGTACAGCTCGTCCA;
  • glbC-genta-P apra -3 gggctccagcgagacgacggtggtggcgccggtggtgtcgtggcgatcggggctggcggaggaggagatgtcagaacgttggctcacggtggtcgattccGACATTGCACTCCACCGCTG.
  • the glb gene cluster in DSM 7029 can produce a class of proteasome inhibitors glidobactin A and its derivatives (glidobactin BI and luminmycin AE) with good antitumor activity.
  • the glb gene cluster is composed of 8 genes (glbA-glbH), and glbC and glbF are assigned to code polypeptide modules to synthesize the core skeleton of glidobactin A.
  • the glbB code is responsible for cyclization of lysine 4, which is the rate-limiting step in glidobactin A synthesis138.
  • glbB and glbC belong to different transcription units, we increased the production of glidobactin A by replacing the promoters of glbB transcription unit and glbC-glbF transcription unit with exogenous strong promoters.
  • the above primers were used to amplify the genta-P apra components targeting different homology arms of the DSM 7029 glbB and glbC promoter regions, and 2 ⁇ g of dsDNA substrate was electroporated into Induced DSM 7029 (pBBR1-rha-Red ⁇ -Red ⁇ 7029-kan).
  • the electrotransformed cells were incubated with 1 ml of antibiotic-free CYMG medium at 950 rpm at 30°C for 4 hours, then spread on 15 ⁇ g/mL gentamicin solid medium plate, and incubated at 30°C for 48 hours. For each biological repeat, 24 single clones were selected for colony PCR detection. As shown in Figure 6a, we successfully replaced the promoters of the glbB transcription unit and the glbC-glbF transcription unit with the constitutive promoter (P apra ) using dReaMGE technology, and obtained three promoter replacement mutants.
  • Example 8 Application of dReaMGE in simultaneous knockout of four negatively regulated lysine family transcriptional regulators (lysR transcriptional regulator, LTTR) in the glidobactins synthetic gene cluster of DSM 7029
  • the present invention expects to increase the production of glidobactin A by knocking out the four negative regulatory LTTRs (lysR2-AAW51_4987, lysR18-AAW51_5436, lysR23-AAW51_2727 and lysR29-AAW51_4579) that have the strongest inhibitory effect on the glbB gene in the DSM 7029 genome.
  • 7029-delet-lysR1-5 ataaatttcgcgcatccgaaactgttggttttgaatcgatggacaagctgcgcagcatggaggttttcgtcgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR1-3 tgggcagggcctgccgatcgggcagatagacgaggtgcaccggccgcggtgccggcaggaagtcgtccagcaccaacTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR2-5 agcctgctaaggttgcttcattcccaactggcggtgttgaatatggaccggatcgagagcctgcaagtcttcgtCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR2-3 tcgggcaggtagagcaagtgtgccggccgctccggcggcggcgcgtaacgaggcaacaccctcaccagccggcgtAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR3-5 atggatcgtctgacttccatgcgggcgtttgcgaaagtgatcgatgaaggcggcttcgcggccgcggcacgggcCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR3-3 gcacgaacttgcgcgagggcatggcggcatagagcgtgaaggtgggggtgcgccact gcggcagctgcaccAGATCCTTTCTCCTCTTTAG;
  • 7029-delet-lysR4-5 aaagaaatagttcactgcgagagaatagtgcgatggatcgctataccgccctccagggtgtttcgccatgtCCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR4-3 tcgagaaaaacgcggatctttggagcgaggtgctggcgagacggatagacggcgtagagcgtggtttccacAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR5-5 acaaaggcggtcatcacggcgacgatcaggttcttcttggcgttcatggtgcttctccgttcacaagggtCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR5-3 tccatgaaggctcgggtgcgggccggcacgtacttgcggctgggcatcgccgcatagatcgtgtagctcatgaCAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR6-5 tggcggcatttgagcgccacactgttgccacagaggcgccaataatcgggcgatgaacgaccgattgaatggCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR6-3 ttgggggatgtgttcgaccagcgtgtcgatggccaaacgggtcttggccgacaggtaccggctgcgtggcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR7-5 ggccctcttgctcagacgctggacggtcggtaatgttgctcaacgagcaacaatacgggtgacacagaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR7-3 ctgctgcaagaagtcgacgaacgcagttgtcttcggcgccagccagtggcgggcgggatagacggcgtagatcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR8-5 tcgcagcgcttgaatagactcggcgccagaacaggagctgtgcacggtgagaacagatgactttctggaggtgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR8-3 gccagccgcatctcgctgtggttgacggtgtagcggccggcgacggccacctcgacctcgtcgtcgccgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR9-5 accgcaagatcggtgttgatttttactttttgacgcgtagtctcgcggcgtgctgggcaagctgaacgtgcaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR9-3 tcaggaaatcgatccacagccgcacccgcaacggcagatgcttgcgctgcgggaacacggcatagatgccAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR10-5 acccacaacccttcctccgatggatcgattccaggccatgcaggtgttcagcaaggtcgccgaacaccgcCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR10-3 tggcggtggtgcgcatagatcaccgacaccggcagcgcctgcggacgccagcgcggcagcacttcttcgagcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR11-5 agcattccaccgcgtggaatagcgacatggaccgtctcgacaccttgcgcgtgttttgtcgccgtggccgaaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR11-3 atggaccggtgtgggcggcggttcgtgctcggccagcacgatctgcaagcgccctgccaccacgtcagcggAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR12-5 tgtggcggaatgctgcggtgccgcaaacgatatgagttctcgcgtggcgaataaatactgcagatctggccGCTCAGTGGAACGAGGTTC;
  • 7029-delet-lysR12-3 atcggcttcggcgccgagatccactggcacagcaccggctgcaaggcgccgctgctcagatgttgctgcatCAGCCAATCGACTGGCGAG;
  • 7029-delet-lysR13-5 ttgttcaactcattgaacaacgaggccgaccgtcgtcgcctgcccagatggaccgcgtggctcccgtgctcgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR13-3 agtccggcagcaaggccgtcaggctgccgtcgcgcaagccatgcgcagcgctgaagtcgggcagtagcgcaAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR14-5 agacgggcaagtggataacattgttccgctactggaacaatcatgcagatcgatcccaacgaccttttgtCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR14-3 ccaggcggatcagcaacggcggggaattcgccaacgcccgcggcggcgccagcccggcccaacgctctccgTACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR16-5 ataatgaatgctgaaacgtctagtaggcaggctaatcaatctgagggcaaaccgcatggccaggaggaaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR16-3 aagcgcagcgcctcgaccacaagggaaaggcaggagaaggttgtcgccggctcgggtagtagtaggtggtAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR17-5 aagaggccgtgacaggtgcaagccagcctcgcagccctgctgctgcccacatcacacgcttcagatgtcgaaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR17-3 acgaggaagtgcacgaaggcctgcgtgctccccgggttgggcccgtggtctggcatggcggcatacaaggtCAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR18-5 agcgcatggccgtgtttgccgaagtggtcgaacacggctcgatgagcgccgcccgcacgctgggcaccaccccaACCGCGTGGCACAACAA;
  • 7029-delet-lysR18-3 accgcggcgatcgcgtggcgcaccttggccggctgggtgtcgcggcgcggcgtcacgacatacatcggcaaAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR19-5 tcgccgttcgtttgataatccgttcaaagctcacttgttaagtgccatgacggaacaaatcagcctggagcCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR19-3 acctcctcaaggaacgagatgaaagccttgacgttcgggcttccgcgtcggctctcgggccaaacagcagtAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR20-5 tcggcaagcaataatccggcaagcgactatttccttggaggaactgatgatcgaccggccggacctgttgctgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR20-3 tggcaaaccactgcagcaagtggtcttgcagcagccgcagcggcgcgctgtcggcctgtgcgcgctAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR21-5 aacaatcttcgaagggtcaggagcagagatggaccgtttcgatgcgcgggcttttgtccgtgtggtggaaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR21-3 acccagtcgatcaacacccgcagcttcgcgctgacatgccggttcgggaggtacgccacgtacagcggcatcggCAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR22-5 aagcaactcattcaaatgaaaattgaaaatacccaggagctgcgcctgatcgtggcctgtgcggctggcggcCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR22-3 tgggcagcaccgcatgcagcggaatcgcctcccctgccagccgggaaacaagccaggcggcccgcgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR23-5 aggaacgataagtcgttgcgattgataaacacagacgccaagaactggacgatgcgtcccagatttgCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR23-3 accgccgccggaacgggcacggtcccgccgtcggatgtcggcatgggaatcgacggcgcggccgctgcgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR24-5 tccgctcctcctatgcgcacgctgtcattcgtccctcgcgccttgcttccatgatcgaactgctgcgccatctaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR24-3 atgcaactcgctccacaagcccttgcggccgatgcgcttggccacgacgtagtcgtaatcgacatagttcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR25-5 gacgcacacgcgcccgacagagacaaccagactttgaggaagcgatcgactagcatcaccggatgggaaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR25-3 ggctccatcggctgcggcgtcgcgtcgcagccgctcactgttccaatagacatcgtcgccgctgcgtccgAACTTAAATGTGAAAGTGGG
  • 7029-delet-lysR26-5 tcgcgacagacgcacacgcgcccgacagagacaaccagactttgaggaagcgatcgactagcatcaccggCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR26-3 tgcggcgtcgcgtcgcagccgctcactgttccaatagacatcgtcgccgctgcgtccgtccagattggcgcgCAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR27-5 acaacaacccacaacaccctcaccgagcccctgctaggagcccccatgagccgtcaccacgccctcgccCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR27-3 ggggcgtcgagagcggcgttcggtcggtcgatcgatcaggccagcgccaggccggagggcgtgcccgcgtAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR28-5 aggcggcctgcgggccgtcccttaccgcagcgtcgcagatgaccttcacacagctccaggtgttcgccaccCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR28-3 tgaggctgcgatagacgatgccggggtgatcgtcgggcagggccagccgtgcggcgaccgagatggccagCACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR29-5 tgcgccctgcgccacggctgcggccatttgttcgatgtgcccataggacgagtagtagagtagaggacgaggatccCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR29-3 cgctcatccccgtttcggcccgtcgaaatgccacgtatttctgagacaggacactagcggctcCAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR30-5 cgccggcccgctctgcccttgggcccccggcgccatgccactccagctccgccagcttcaggtgttcgtcCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR30-3 accgtctggcctgacaccacctctcgcgccgtttgcagaaacacccgcagcgccggggcaggtcgccgctggccaacTTAAATGTGAAAGTGG;
  • 7029-delet-lysR31-5 gggagcactgtagaaaggctgaaggctcaggtacattcaaagatcgttagccatcgattaagcgcgcctgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR31-3 atagcgaaacgcttcggcggcgggcgacaacaccttggacgacggacgcacgacgttccaggtgcgcatcacAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR32-5 atgaattgaactcatgtgacgccccatgttcgaaacctgccgatgaccgcattgcgcaccttcgaggcatcgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR32-3 atgccgctcgcggcccggcacgcacagcgtggtagctcgccccggcacgctgatgtcggggcggcacAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR33-5 tggccctccacctcgctgtgcgagccgggtggaggcctgcagacgatgtgcgacgtgccccggagttgttgccCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR33-3 tgcggctggggatcgcggggcacgctcaactggggcaccacggtgatgcccatgccggaagccaccatgtgttAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR34-5 ttgtgtgaatcttcgacattcaccaatcgcataacttcgatgaacttccgcaccctcgacctcaacctgtCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR34-3 cgcttgggcgacctcggctgtcgcgctgggcgccggcggccaagatctgctcgcgcatccaacgctgtgcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR35-5 ataaatggcgataaatttcgcgcatccgaaactgttggttttgaatcgatggacaagctgcgcagcatggaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR35-3 gcggtgccggcaggaagtcgtccagcaccggcaccagtcgccccgccgccaggtcgtcggccaccagcacAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR36-5 tcgcccacctgctgccgccaccgcggccaatgccgctgctgccttcgactgggatgacctgcggtatgcgctggCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR36-3 acgagctcgggctcctgccgtgcgacgaagctgggcaacaggccgacaccgagcccggtgcgcagcatcgccaaaCTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR37-5 aatcccttcgatcgtcgcgcacagccattgcaccgcatggtcccggtcccgggtggcgtggtgtgcgatgCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR37-3 gccgcgggcactggaactgcaaagcgcgctgatcccggcgctcgagatgatccaggcggtggtgtcggatAGATCCTTTCTCCTTTTAG;
  • 7029-delet-lysR38-5 aaactttagggccgcgtggcaggccagtccaatgagaacggcgacccgcattgatgacgtaatcgcatgaatCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR38-3 atccagtcacagaactgcgccacctcgggccgctcgcgcgccacggccggcgccaccatccagtacaccgtgcgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR39-5 aaagtgcgccggctcgcacacgatacggaggtgacgctgtggacagcttgtactcgatgcgcgttttcgtCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR40-5 ctggtcgccgtcaccgaactcggcagcttttccgcggccgcccgggccttgcacctggcgcagccgacggCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR40-3 acggtgccggcacgctgggccaccgcgagccgccgcgtcaccgcgggccgcagcggcaccacctgtatctAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR41-5 ctgtgggggtgacgatggccgcactctatgattgcgaggacgcatccaaaataggcgccatcgcaaccgaCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR41-3 agcacccacacagcgaagtctgtgcgtcctcgagcggttccgaaatctgccgcacctcgcgccggcctgcggcaGAACTTAAATGTGAAAGTGG;
  • 7029-delet-lysR42-5 cgcaggcctgtcgggcgtcgctacactccgccctcgtggaaaccaagtggctcgaagacttcgtcagcctCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR42-3 acggacatgcttggccgcttcggggcgctcgcggtagacgcgcacgtccatcgtgacttcgaacgacccgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR43-5 atggccgcgcgagtcgatttcgacaacattgagcttcgcctcgtcagggtcttgcaacacattggtcaccgCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR43-3 actgggcgacataacgcgagcagaactggcgcccggtggtcagcaccagcaggctgtgcgccaccatctgAGATCCTTTCTCCTCTTTAG;
  • 7029-delet-lysR44-5 attgttccgctactggaacaatcatgcagatcgatcccaacgaccttttgtttgcccgggtcgccgagCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR44-3 cgggcatcaccgcccatgccgggatggccggcagacaccattgcggcagcacccgcaccagctggccgctgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR45-5 tcagaaaaacttagggccgtggatgccagaatcgccgccatgcaactgccgctcaacgccttgcgcgcctCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR45-3 gcttcgacttcagccacgtcagccaatagcgcccggtcgacacctcgatctcgaacggccgcaccagacgAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR46-5 atgagcggcgttttcgagttgcggcagttgcgccagttcgtcgcgctggcagaggaactgcacttcggccCAACCGCGTGGCACAACAAC;
  • 7029-delet-lysR46-3 cgggtgaccgactccggcacccaggccacgcccatgcccgccgacaccaggttgacgatggtctgcatctAGATCCTTTCTCCTCTTTAG;
  • 7029-delet-lysR47-5 gcttaccagtcatgcgcttgcccctgcaaacattgcatgcctttcgagccgcgcgactgcaaaacctCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR47-3 tcgctcagcccgagcgcaagtgtcggcgccccggcgcaactcgtccttgagccagacgcgcaaggccgcAACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR48-5 tacgctgcatatcgttttgatatagagcacgatggcgaagctggatgtcgactggctcgacgtgttcgtcgCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR48-3 tccaggttgaaccgctcatgccaatgctgcttgaccttgtacgacggcagcggcaccggcggctccagcaCACTTAAATGTGAAAGTGGG;
  • 7029-delet-lysR49-5 ttcacgaaggttgtcgacctgggcgccttgacgaaggccgccgaatcgctcgggagcgccgcgtcCCAACCGCGTGGCACAACAA;
  • 7029-delet-lysR49-3 atggtcggggccgccgaaggtggtcgtcaagaactgaacgaaggcgcgcgtactggcggggatgtaccgatagAACTTAAATGTGAAAGTGGG.
  • the above primers were used to amplify dsDNA substrates with gentamicin resistance genes targeting four glidobactin synthesis gene clusters and four negative regulatory LTTRs in DSM 7029, and 4 ⁇ g of dsDNA substrates
  • the mixture was electroporated into induced DSM 7029 (pBBR1-rha-Red ⁇ -Red ⁇ 7029-kan).
  • the electrotransformed cells were incubated with 1 ml of antibiotic-free CYMG medium at 950 rpm at 30°C for 4 hours, then spread on 15 ⁇ g/mL gentamicin solid medium plate, and incubated at 30°C for 48 hours.
  • transcript level analysis it can be seen that the transcript level of glbB gene in 7029 ⁇ 4 LTTRs::genta is significantly higher than that of DSM 7029 wild-type strain and 7029 P apra glbB, as shown in Figure 7a, which proves that the glbB suppressed state has been significantly relieved .
  • LC-MS analysis showed that the production of glidobactin A in 7029 ⁇ 4 LTTRs::genta was 4-fold higher than that of the wild-type strain, as shown in Figure 7f.
  • the results of this example prove that the dReaMGE method of the present invention has application potential in systematically modifying the bacterial metabolic network, realizing genome mining and optimizing the yield of target metabolites.

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Abstract

La présente invention concerne un procédé d'édition multiplex du génome bactérien fondé sur l'ingénierie de recombinaison de l'ADN double brin et son application. Le procédé comprend les étapes suivantes : (1) mélange de substrats d'ADNdb ciblant différentes séquences cibles, puis introduction desdits substrats dans des cellules compétentes hôtes portant un plasmide de recombinase ; (2) réanimation des cellules compétentes à une température inférieure à la température de croissance optimale ; ajout de dNTP ayant une concentration finale de 10 nM pendant le processus de réanimation ; et (3) criblage des mono-colonies possédant un marqueur à partir des cellules compétentes réanimées pour leur identification.
PCT/CN2022/103604 2021-10-20 2022-07-04 Procédé d'édition multiplex du génome bactérien fondé sur la technique de recombinaison de l'adn double brin et son application Ceased WO2023065725A1 (fr)

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