WO2019231149A1 - Kit pour la sélection d'une cellule hôte transformée par un gène cible à l'aide d'un système crispri et son utilisation - Google Patents

Kit pour la sélection d'une cellule hôte transformée par un gène cible à l'aide d'un système crispri et son utilisation Download PDF

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WO2019231149A1
WO2019231149A1 PCT/KR2019/005960 KR2019005960W WO2019231149A1 WO 2019231149 A1 WO2019231149 A1 WO 2019231149A1 KR 2019005960 W KR2019005960 W KR 2019005960W WO 2019231149 A1 WO2019231149 A1 WO 2019231149A1
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host cell
gene
plasmid
sgrna
kit
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이대희
이승구
김성근
우의전
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한국생명공학연구원
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    • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)

Definitions

  • the present invention was made with the support of the Korean government according to project number KGM2121733, "Biopart / Circuit and Synthetic Biology Technology Development Project” of the Ministry of Science, ICT and Future Planning.
  • Plasmids refer to DNA molecules other than chromosomes that can replicate and propagate independently in bacterial cells. They are not essential for the survival of bacteria and can also be transferred to bacterial cells of other species. Genetic recombination technology is used to separate the plasmid in the bacteria by using such a characteristic, cut it with restriction enzymes, insert the target gene to be expressed, and put it back into the culture.
  • the plasmid is a structure that includes a replication origin at which DNA replication begins, a multiple cloning site (MCS), which is a site where a restriction enzyme recognition sequence is collected, and an antibiotic resistance gene consist of.
  • Antibiotic resistance genes express antibiotic degrading enzymes and are used for the selection and proliferation of plasmids themselves or cells containing plasmids by culturing bacteria in culture conditions containing antibiotics.
  • the use of antibiotics to screen plasmids has the problem that antibiotic resistance genes migrate to the microbial community of the surrounding environment through horizontal gene transfer (HGT).
  • HAT horizontal gene transfer
  • the production cost of the final product is increased due to the high price of antibiotics and the increase in costs associated with the removal of antibiotics.
  • Another object of the present invention is to provide a method for producing a host cell transformed with the gene of interest using the kit and a mass production method of the desired product.
  • kits comprising: (a) a first nucleic acid fragment encoding a Cas (CRISPR associated protein) protein operably linked with an inducible promoter expressed by an inducer; At least one second nucleic acid fragment encoding a single guide RNA (sgRNA) that inhibits expression of a gene essential for host cell survival; And a first vector comprising a third nucleic acid fragment encoding a selection protein for confirming the transformation of the first vector; And (b) one or more gene fragments of interest encoding one or more proteins to be expressed in the host cell; And at least one second vector comprising a fourth nucleic acid fragment encoding an sgRNA targeting the second nucleic acid fragment, a kit for selecting a host cell transformed with the gene of interest.
  • sgRNA single guide RNA
  • the host cell may be a microorganism that exhibits trophic composition.
  • the nutritional component may be one that requires D-glutamate.
  • the inducer may be selected from the group consisting of rhamnose, arabinose and lactose.
  • the selection protein of (a) may be a D-amino acid transferase or antibiotic degradation protein.
  • the antibiotic may be selected from the group consisting of kanamycin, ampicillin, tetracycline and chloramphenicol.
  • the sgRNA of the first vector and the sgRNA of the second vector may be expressed by a constitutive promoter.
  • the expression inducing substance may be selected from the group consisting of rhamnose, arabinose and lactose.
  • the antibiotic may be selected from the group consisting of kanamycin, ampicillin, tetracycline and penicillin.
  • the expression inducing substance may be selected from the group consisting of rhamnose, arabinose and lactose.
  • FIG. 5 shows the structure of pSLiP (A2) and pgRNA (rA2) plasmids.
  • FIG. 9 shows the structures of pSLiP (A1 + A2), pA-RFP (rA1) and pT-GFP (rA2) plasmids.
  • FIG. 17 shows a schematic of a CRISPRi-based double inverter system (a: using aphA gene, b: using gapA gene) including a gene family encoding enzymes involved in the mevalonate pathway as the gene of interest.
  • FIG. 20 shows the correlation of (-)- ⁇ -bisabobolol with cell growth, production and fluorescence levels of (-)- ⁇ -bissabolol after co-transformation of pTM-BBS (rG2) plasmid into E. coli It is a graph.
  • FIG. 21 shows a schematic of the structure of a pSLiP (G2) -DAAT plasmid and a CRISPRi-based antibiotic-free single inverter system using the same.
  • Figure 22 shows the extent of growth in LB medium of Escherichia coli transformed with pSLiP (G2) -DAAT plasmid depending on the presence of L-rhamose.
  • 25 and 26 show that pSLiP (G2) -DAAT and pSNAG-MrBBS-IspA (rG2) plasmids were transformed into Escherichia coli, followed by the production, fluorescence level and (-)- ⁇ -bisabobolol according to the selection method. It is a graph showing the correlation of) - ⁇ -bisabolol.
  • At least one second nucleic acid fragment encoding an sgRNA that inhibits expression of a gene essential for host cell survival
  • the Cas protein may be used Cas9 (Type II CRISPRi), Cse1, Cse2, Cas7, Cas5, Cas6e (Type I CRISPRi) or Cpf1 (CXXC finger protein 1; Type V CRISPRi).
  • Cas9 Type II CRISPRi
  • Cse1, Cse2, Cas7, Cas5, Cas6e Type I CRISPRi
  • Cpf1 CXXC finger protein 1; Type V CRISPRi
  • dCas9 or dCpf1 may be used in which any mutation has been introduced into the nucleic acid fragment to remove endonuclease activity required for gene shearing.
  • the inducible promoter means a promoter that operates when the inducer is present, the inducer may be selected from the group consisting of rhamnose, arabinose and lactose.
  • the inducer may be selected from the group consisting of rhamnose, arabinose and lactose.
  • rhamnose when rhamnose is present in the culture medium, dCas9 is expressed, and dCas9 and sgRNA complex exhibit CRISPRi activity, thereby inhibiting expression of genes essential for host cell survival.
  • the gene essential for the survival of the host cell of (a) may be a replication origin from which replication of the plasmid is initiated, and includes ColE1, pBR322, p15A, RK2, pBBR1, R6K and RSF1010. It may be selected from the group consisting of.
  • the gene essential for the survival of the host cell of (a) may be a gene involved in glycolysis.
  • Glycolysis is a metabolic process that oxidizes glucose into pyruvate and obtains energy in the form of ATP (adenosine triphophate) and NADH (oxidized form of Nicotinamide Adenine Dinucleotide (NAD)).
  • the gene involved in the glycolysis is hexokinase (hexokinase), phosphoglucose isomerase (phosphoglucose isomerase), phosphofructo kinase-1 (phosphofructo kinase-1), aldolase ( aldolase), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase ( gapA ), phosphoglycerate kinase, phosphoglyceromutase It can be selected from the group consisting of phosphoglyceromutase, enolase and pyruvate kinase, for example using the gapA gene (SEQ ID NO: 58) encoding glyceraldehyde-3-phosphate dehydrogenase. have.
  • selected protein refers to a protein for use in confirming whether the first vector is transformed and maintaining the first vector in the host cell after the transformation. Since the growth of the host cell depends on the expression of the selection protein, it is possible to determine whether the first vector is present and maintained in the host cell.
  • one or more sgRNAs of the first vector may be included.
  • sgRNA (G1) SEQ ID NO: 51
  • G2 SEQ ID NO: 52 sequences that inhibit expression of the gapA gene can be used as sgRNAs of the first vector.
  • the second vector may be present in one or more, each second vector is sgRNA (rG1) (SEQ ID NO: 55) or sgRNA that inhibits the expression of sgRNA (G1) or sgRNA (G2) (rG2) (SEQ ID NO: 56) sequence.
  • the inventors of the present invention provide a CRISPRi-based double inverter system that mimics the correlation between antibiotics and antibiotic-degrading enzymes in order to solve the problem of a method for screening host cells transformed with plasmids with antibiotics. Devised.
  • host cells are transformed with a first vector and a second vector, and then cultured in the presence of an inducer rhamnose.
  • dCas9 is expressed, and a complex of dCas9 and sgRNA inhibits the growth of host cells by inhibiting the expression of genes essential for the survival of the host cell.
  • the second vector is present, the expression of the sgRNA is blocked by the sgRNA expressed in the second vector, so that the growth of the host cell is restored. Therefore, the host cell transformed with the plasmids can be selected without antibiotics.
  • both the first vector and the second vector are required for normal growth of the host cell, both vectors can be maintained in the host cell.
  • the expression inducing substance may be selected from the group consisting of rhamnose, arabinose and lactose
  • the antibiotic may be selected from the group consisting of kanamycin, ampicillin, tetracycline and penicillin.
  • a host cell transformed with a plurality of plasmids including one or more target genes can be selected with high efficiency without antibiotics.
  • the term "object product” means a product produced in the host cell by the expression of the target gene, and may be a product produced by the protein encoded by the target gene or a protein encoded by the target gene.
  • the target product may be ( ⁇ )- ⁇ -bisabobolol produced by the mevalonate metabolic pathway gene, which is the target gene.
  • the expression inducing substance may be selected from the group consisting of rhamnose, arabinose and lactose
  • the antibiotic may be selected from the group consisting of kanamycin, ampicillin, tetracycline and penicillin.
  • the step of recovering the desired product may use a conventional method known in the art.
  • a kit for selecting a host cell transformed with the target gene of the present invention By using a kit for selecting a host cell transformed with the target gene of the present invention, a plurality of plasmids containing one or more target genes can be stably maintained in the host cell, thereby increasing production of the target product.
  • Another object of the present invention is to provide a third object of the present invention.
  • kits for selecting a host cell transformed with a gene of interest comprising one or more second vectors comprising a fourth nucleic acid fragment encoding an sgRNA targeting the second nucleic acid fragment.
  • the method for introducing the nucleic acid construct into the chromosome may use a conventional method known in the art.
  • the kit host cells transformed with a plurality of plasmids containing one or more target genes can be efficiently selected without antibiotics.
  • (-)- ⁇ - bisabolol production includes terrific broth medium containing glycerol; 12 g / L enzymatic casein digest, 24 g / L yeast extract, 9.4 g / LK 2 HPO 4 , 2.2 g / L KH 2 PO 4 and 3.5% (w / v) glycerol; Hereinafter described as TBG].
  • Recovery medium after bacterial transformation is SOC medium [Super Optimal broth with Catabolite repression medium; 20 g / L tryptone, 5 g / L yeast extract and 0.5 g / L sodium chloride, 2.4 g / L magnesium sulfate, 186 mg / L potassium chloride, 4 g / L glucose].
  • L-Rhamnose and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ampicillin, chloramphenicol and kanamycin were used at concentrations of 100 ⁇ g / ml, 34 ⁇ g / ml, and 25 ⁇ g / ml, respectively, unless otherwise noted.
  • the high fidelity KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan) was used for the polymerase chain reaction (PCR). All restriction enzymes and modification enzymes used in the experiment were purchased from New England BioLabs (Ipswich, Mass., USA).
  • the CRISPRi plasmids pSECRi and pSLiP plasmids used in the present invention are expressed by the L-rhamnose-induced dCas9 expression cassette and the sgRNA cassette expressed by the constitutive promoter BioBrick BBa_J23119 promoter.
  • the CRISPRi plasmid is derived from pSEVA221 and thus contains a kanamycin resistant aphA gene with a low-copy RK2 origin (Silva-Rocha, R., et al., Nucleic Acids Res, 2013, 41). , D666-675).
  • the entire sequence of the pSECRi plasmid was amplified by PCR using CRI-F and CRI-R primers to alter the spacer sequence within the sgRNA sequence.
  • PAM-F and PAM-R primers were used instead of CRI-F and CRI-R primers when introducing PAM (protospacer adjacent motif) sequences into the BBa_J23119 promoter sequence.
  • PCR products amplified to the correct size were extracted and purified with Wizard ® SV Gel and PCR Clean-Up System (Promega, Madison, Wis., USA).
  • the purified PCR product was treated with DpnI and then ligated with T4 DNA ligase and T4 polynucleotide kinase to prepare pSLiP plasmids with altered PAM sequences (pSLiP (A1) and pSLiP (A2) plasmids).
  • a BBa_J23119 promoter comprising a PAM sequence could be constructed without impairing CRISPRi function, and the plasmid with the BBa_J23119 promoter comprising a PAM sequence was named pSLiP ( p lasmid containing an s gRNA l and i ng p ad). .
  • the pSLiP plasmid has the following characteristics: i) can express sgRNA complexing with dCas9 (eg sgRNA (A2)) to inhibit host cell growth, and ii) within the promoter of growth-inhibiting sgRNA The presence of the PAM sequence allows other sgRNAs (eg, sgRNA (rA2)) to bind and to restore cell growth by blocking growth-inhibiting sgRNA expression.
  • dCas9 eg sgRNA (A2)
  • Plasmids containing a plurality of sgRNAs were produced as follows.
  • the pSLiP (A1) plasmid was treated with XmaI and NcoI to purify fragments containing the sgRNA (A1) cassette.
  • the pSLiP (A2) plasmid was treated with AgeI and NcoI to purify fragments containing the sgRNA (A2) cassette.
  • the purified sgRNA (A1) cassette fragment and the sgRNA (A2) cassette fragment were ligated to prepare a pSLiP (sgRNA1 + sgRNA2) plasmid.
  • sgRNA (G1) or sgRNA (G2) that inhibits the expression of the gapA gene (SEQ ID NO: 58) encoding glyceraldehyde 3-phosphate dehydrogenase (GapA) in the same manner as described above Introduced pSECRi (G1), pSECRi (G2), pSLiP (G1) and pSLiP (G2) and pSLiP (G1 + G2) plasmids.
  • the spacer sequence of the pgRNA-bacteria plasmid was modified to construct a sgRNA (rA2) expression plasmid that inhibits the expression of sgRNA (A2) present in the pSLiP (A2) plasmid.
  • rA2 expression plasmid that inhibits the expression of sgRNA (A2) present in the pSLiP (A2) plasmid.
  • the entire region of the pgRNA-bacteria plasmid was amplified with pg-F (SEQ ID NO: 29) and pg-R primer (SEQ ID NO: 30) to purify amplification products of the correct size. Purified amplification products were ligated with T4 DNA ligase and T4 polynucleotide kinase to prepare pgRNA (rA2) plasmids.
  • a pSLiP plasmid expressing D-amino acid aminotransferase (DAAT: SEQ ID NO: 59) was prepared as follows.
  • the BBa_J23100 promoter was inserted before the DAAT gene of the pGESS-DAAT plasmid.
  • amplification products were purified after amplifying the entire region of the pGESS-DAAT plasmid with pDAAT-F (SEQ ID NO: 25) and pDAAT-R (SEQ ID NO: 26) primers containing the BBa_J23100 promoter sequence.
  • Purified amplification products were ligated with T4 DNA ligase / T4 polynucleotide kinase to obtain the pGESS-J23100-DAAT plasmid.
  • the J23114-DAAT cassette comprising the BBa_J23114 promoter sequence was amplified with DAAT-F (SEQ ID NO: 27) and DAAT-R (SEQ ID NO: 28) primers.
  • the amplified product and the PSLI-treated pSLiP (G2) plasmid were linked using the Gibson Assembly Method (NEB, Ipswich, MA, USA).
  • pT-GFP plasmid was prepared as follows.
  • the backbone region of the pT-BBS plasmid was amplified with pTG-VF (SEQ ID NO: 31) and pTG-VR (SEQ ID NO: 32) primers, and the superfolder GFP region of the pK7 sfGFP plasmid was converted to pTG-IF (SEQ ID NO: 23). Number 33) and pTG-IR (SEQ ID NO: 34) primers. Thereafter, the two amplification products were linked by Gibson Assembly Method.
  • a CRISPRi plasmid capable of inhibiting sgRNA expression on the pSLiP plasmid was constructed using the pSECRi plasmid and rCRI-F and rCRI-R primers.
  • the sgRNA cassette region of the CRISPRi plasmid was then amplified with pTsg-F (SEQ ID NO: 39) and pTsg-R (SEQ ID NO: 40) primers, and the amplification product was linked to the XTay treated pT-GFP plasmid with Gibson Assembly Method.
  • the pA-RFP plasmid into which sgRNA was introduced was constructed as follows.
  • the sgRNA cassette region of the CRISPRi plasmid is amplified with pAsg-IF (SEQ ID NO: 43) and pAsg-IR (SEQ ID NO: 44) primers, and the backbone sites of the pA-RFP plasmid are pAsg-VF (SEQ ID NO: 41) and pAsg-VR (SEQ ID NO: 42) Amplified with a primer.
  • the amplification products were then linked by Gibson Assembly Method.
  • the gfp gene and sgRNA were introduced into the pTM-BBS or pSNA-MrBBS-IspA plasmid in the following manner.
  • the gfp gene and sgRNA cassette were amplified from pT-GFP (rG2) plasmid with pBGsg-F (SEQ ID NO: 47) and pBGsg-R (SEQ ID NO: 48) primers, and the amplification products were purified. Purified amplification products were linked to pTM-BBS or pSNA-MrBBS-IspA plasmids treated with SbfI using the Gibson Assembly Method.
  • sgRNA name Target Spacer sequence (5 ' ⁇ 3') PAM sequence (5 ' ⁇ 3') SEQ ID NO: sgRNA (A1) aphA agctcgtttcacgctgaata TGG 49 sgRNA (A2) aphA tgttgctgttcagacgcgga CGG 50 sgRNA (G1) gapA ggaaacaatgcgaccgata CGG 51 sgRNA (G2) gapA ccatgtaatcagcgtctaac AGG 52 sgRNA (rA1) sgRNA (A1) tcagcgtgaaacgagctact AGG 53 sgRNA (rA2) sgRNA (A2) gcgtctgaacagcaacaact AGG 54 sgRNA (rG1) sgRNA (G1) cggtcgcattgttttccact A
  • E. coli DH5 ⁇ was transformed with the pSLiP (A2) plasmid, and then the transformed strain with the plasmid was selected on an LB plate containing kanamycin (25 ⁇ g / ml). Selected single colonies were inoculated in LB medium containing kanamycin (25 ⁇ g / mL) and L-rhamose (1 mM) and incubated overnight at 30 ° C. and 200 rpm. Thereafter, 0.1% of the cultured cells were inoculated in LB medium containing various concentrations of kanamycin and L-rhamose (1 mM) and incubated at 30 ° C. and 200 rpm for 11 hours. Absorbance (Optical density, OD) of LB medium was measured by plate leader Victor V (Perkin-Elmer).
  • E. coli DH5 ⁇ was transformed with pSLiP (A2) and pgRNA (rA2) plasmids, and transformed strains with the plasmid were selected in LB plates containing kanamycin (25 ⁇ g / ml) and ampicillin (100 ⁇ g / ml). Selected single colonies were inoculated in LB medium containing kanamycin (25 ⁇ g / ml), ampicillin (100 ⁇ g / ml) and L-rhamose (1 mM) and incubated overnight at 30 ° C. and 200 rpm.
  • Escherichia coli DH5 ⁇ was transformed with a pSLiP (G2) plasmid, and the transformed strain with the plasmid was selected on an LB plate containing kanamycin (25 ⁇ g / ml). Selected single colonies were inoculated in LB medium or EZ medium containing kanamycin (25 ⁇ g / ml) and L-rhamose (4 mM) and incubated overnight at 30 ° C. and 200 rpm. Thereafter, 0.1% of the cultured cells were inoculated in LB medium or EZ medium containing kanamycin (25 ⁇ g / ml) and L-rhamose (4 mM) and incubated at 30 ° C. and 200 rpm for 11 hours. The absorbance of LB medium was measured by plate reader Victor V.
  • the degree of cell growth with time was confirmed as follows.
  • E. coli WM335 When E. coli WM335 was used, E. coli DH5 ⁇ strain with pSLiP (G2) -DAAT and pT-GFP (rG2) plasmids and E. coli WM335 strain with pSLiP (G2) -DAAT and pTrc99A plasmids were mixed at a ratio of 1:10. Thereafter, 40 ⁇ l of the mixed solution was inoculated into 5 ml of LB medium containing L-rhamose (4 mM) and incubated overnight at 30 ° C. and 200 rpm. The procedure was repeated twice and the fluorescence level of single cells was checked with a flow cytometer (BD FACSCalibur) during the culture.
  • BD FACSCalibur flow cytometer
  • E. coli DH5 ⁇ with pSLiP plasmid was transformed with pT-GFP (rA2) and pA-RFP (rA1) plasmids and incubated for 1 hour with the addition of ml of SOC medium. Thereafter, 40 ⁇ l of the transformed cell solution was inoculated in 5 ml of LB medium (selective medium) containing kanamycin (200 ⁇ g / ml) and L-rhamnose (4 mM) and incubated overnight at 30 ° C and 200 rpm. . After repeating the above procedure twice, the culture was plated on a plate containing kanamycin (200 ⁇ g / ml) and L-rhamose (4 mM) and cultured.
  • LB medium selective medium
  • kanamycin 200 ⁇ g / ml
  • L-rhamnose 4 mM
  • kanamycin 25 ⁇ g / ml
  • ampicillin 100 ⁇ g / ml
  • L-Rhamnose 4 mM
  • n-dodecane 1/5 of the culture volume. Inoculation was incubated for 48 hours at 37 °C and 200 rpm conditions.
  • a medium to which kanamycin was added at a concentration of 200 ⁇ g / ml was used.
  • the WM335 strain When using the E. coli WM335 strain, the WM335 strain was co-transformed with pSNAG-MrBBS-IspA (rG2) and pSLiP (G2) -DAAT plasmids. Transformants with these two plasmids were selected on LB plates containing kanamycin (25 ⁇ g / ml) and ampicillin (100 ⁇ g / ml) (4 mM L-rhamose was also added for CRISPRi-based selection). . Six colonies were inoculated in different selective LB media and incubated overnight at 30 ° C.
  • n-dodecane overlaid on E. coli culture was recovered by centrifugation (3 min at 14,500 ⁇ g), and the amount of (-)-bisabolol contained in n-dodecane was quantified.
  • a gas chromatography system equipped with a flame ionization detector (FID) and an HP-5 column (30 m ⁇ 0.320 mm ⁇ 0.25 ⁇ m) were used, and the flow rate of the solvent was maintained at 1 ml / min. .
  • the temperature change of the detector oven was set as follows: the starting temperature was maintained at 60 ° C. for 2 minutes, raised to 200 ° C. at a rate of 5 ° C./min, held at 200 ° C.
  • pSECRi (A1) or pSECRi (A2) plasmid was transformed into DH5 ⁇ , and the growth of E. coli was measured.
  • pSEVA221 control
  • pSLiP A2-AGG
  • pSLiP A2-TGG
  • kanamycin when used at a concentration of 500 ⁇ g / ml, the growth of Escherichia coli is significantly reduced by high concentrations of antibiotics. Therefore, kanamycin was used at a concentration of 200 ⁇ g / ml for the experiment of CRISPRi-based inverter system using aphA gene.
  • pSLiP (A2) and pT-GFP (rA2) plasmids were transformed together with E. coli. Subsequently, the transfected cells were passaged three times in LB medium containing 200 ⁇ g / ml kanamycin and 1 mM L-rhamose, and the green fluorescence was measured at the single cell level to detect the cells having the GFP expression plasmid during the culturing process. The distribution was confirmed.
  • PSLiP (A1 + A2), pT-GFP (rA2), and pA-RFP (rA1) plasmids were co-transformed into E. coli DH5 ⁇ , and passaged cultures were followed by cell fluorescence microscopy.
  • Adding L-Ranose to the medium to operate a CRISPRi-based double inverter system inhibits the expression of both sgRNAs targeting the aphA gene in the pSLiP plasmid, thereby inhibiting pA-RFP (rA1) and pT-GFP (rA2). Only cells with both plasmids can grow.
  • kanamycin In a CRISPRi-based double inverter system using aphA gene, high concentration (200 ⁇ g / ml) of kanamycin should be used to effectively select cells transformed with sgRNA-containing plasmids. In order to alleviate the kanamycin dependency, essential genes of the host cell were selected as targets for CRISPRi instead of the aphA gene.
  • Glyceraldehyde-3-phosphate dehydrogenase is a glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1.3-bisphophoglycerate) It is catalyzed by and is an essential enzyme of glycolysis. Knockout microorganisms lacking the gapA gene cannot grow in glucose minimal media unless supplemented with other carbon sources.
  • pSLiP (G1) and pSLiP (G2) plasmids targeting the gapA gene were transformed into DH5 ⁇ , respectively, and cultured in EZ or LB complex medium containing kanamycin (25 ⁇ g / ml), and then sgRNA (G1) or sgRNA. It was confirmed whether cell growth was inhibited by (G2). This concentration of kanamycin was used to maintain the pSLiP plasmid.
  • G1 or G2 expression was grown at 11% or 5% level, respectively, compared to the control group, and in LB medium, at 18% or 10% level.
  • antibiotic-based plasmid selection systems operate by enzymes that degrade antibiotics
  • antibiotics present in the medium are degraded by antibiotic degradation enzymes produced from cells having plasmids, resulting in overgrowth of cells without plasmids.
  • This phenomenon is particularly problematic when the protein produced in the plasmid inhibits the growth of the cell, or when the produced protein produces a toxic metabolite, which inhibits the growth of the cell with the plasmid.
  • the CRISPRi-based double inverter system disclosed in the present invention has an advantage of solving the above problem because it does not depend on a diffusible enzyme.
  • a CRISPRi-based double interverter selection system was applied to a strain having a plasmid for producing (-)- ⁇ -bisabobolol (pTM-BBS) including a mevalonate metabolic pathway.
  • the strain is known to be able to produce only small amounts of (-)- ⁇ -bisabobolol due to instability of the pTM-BBS plasmid (Kim, S.K., et al., Metab Eng, 2016b, 38, 228-240).
  • an sgRNA (rA2) sequence or sgRNA (rG2) sequence was introduced into the pTM-BBS plasmid, respectively.
  • pTM-BBS (rA2) and pSLiP (A2) plasmids or pTM-BBS (rG2) and pSLiP (G2) plasmids were co-transformed into E. coli, followed by antibiotics from seed culture to main culture. Plasmids were maintained using either a single inverter system based or a combination of antibiotics and a CRISPRi-based double inverter system (double screening). Since (-)- ⁇ -bisabolol production and cell growth was confirmed.
  • E. coli was transformed with the pSLiP (G2) plasmid by inserting a GFP sequence into the pTM-BBS (rG2) plasmid. After the cell growth, (-)- ⁇ -bisabolol production was confirmed.
  • the cells selected by the double selection produced significantly higher amounts of (-)- ⁇ -bisabobolol and increased cell growth as compared to the control cells selected with antibiotics alone.
  • (-)- ⁇ -bisabolol production was positively correlated with the percentage of fluorescent cells.
  • the D-glutamate auxotrophic property of E. coli WM335 strain was used.
  • the WM335 strain can survive only when D-glutamate is externally supplied or expresses a gene capable of synthesizing D-glutamate from intracellular resources (Liu, L., et al., Biosci Biotechnol Biochem, ( 1998, 62, 193-5).
  • DAAT D-amino acid aminotransferase
  • the cells having the pT-GFP (rG2) plasmid were sufficiently selected by only two passages when L-rhamose was added.
  • the pSLiP (G2) -DAAT plasmid was used to produce (-)- ⁇ -bisabolol without adding antibiotics to the medium.
  • the gfp gene and sgRNA (rG2) cassette were inserted into the pSNA-MrBBS-IspA plasmid containing the ispA (geranyl diphosphate synthase) gene (Han, GH, et al. , Microb Cell Fact, 2016, 15, 185).
  • Cells with both pSLiP (G2) -DAAT and pSNAG-MrBBS-IspA (rG2) plasmids were then selected by three methods (antibiotic-based, CRISPRi-based, antibiotic-based and CRISPRi-based double selection), respectively. By comparison.

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Abstract

La présente invention concerne un kit pour la sélection d'une cellule hôte transformée par un gène cible et un procédé pour la construction d'une cellule hôte transformée utilisant celui-ci. Lorsqu'ils sont utilisés, le kit et le procédé selon un mode de réalisation de la présente invention permettent de sélectionner des cellules transformées par de multiples plasmides portant au moins un gène cible avec une efficacité élevée à l'aide d'un système CRISPRi en l'absence d'un antibiotique et permettent de maintenir de manière stable les multiples plasmides portant au moins un gène cible au sein des cellules, ce qui permet d'augmenter la production d'un produit cible exprimé par le gène cible.
PCT/KR2019/005960 2018-05-30 2019-05-17 Kit pour la sélection d'une cellule hôte transformée par un gène cible à l'aide d'un système crispri et son utilisation WO2019231149A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574467A (zh) * 2022-01-21 2022-06-03 华南农业大学 一种基因表达调控系统及其应用
CN114574467B (zh) * 2022-01-21 2023-05-23 华南农业大学 一种基因表达调控系统及其应用

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