METHOD FOR SCREENING TARGET GENE USING CRISPRI SYSTEM AND USES THEREOF

Abstract

The present invention relates to a method for screening a target gene using a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system and uses thereof. According to the present disclosure, it is expected that the method may be used in wide fields as a tool for genome-scale gene function profiling and studying organisms poorly developed in genetic technology, including pathogens or industrially useful strains or cell lines, and as a platform for electronic regulatory network engineering of genes.

Claims

1. A method for constructing a shortened sgRNA random library comprising the following steps: (a) synthesizing shortened crRNA that complementarily binds to target DNA, wherein the shortened crRNA comprises a shortened target recognition sequence (TRS) consisting of the length selected from the group consisting of 7 to 19 nucleotides from a 5′-terminal; and (b) generating a shortened sgRNA random library including the shortened crRNA by repeating the step of synthesizing the shortened crRNA one or more times.

2. The method for constructing the shortened sgRNA random library of claim 1, wherein the shortened TRS consists of 9 nucleotides from the 5′-terminal.

3. The method for constructing the shortened sgRNA random library of claim 1, wherein the complementary binding comprises complete complementary binding or one or more mismatch bindings to the target DNA.

4. The method for constructing the shortened sgRNA random library of claim 3, wherein the target DNA comprises a nucleotide of a complementary sequence to the TRS or sgRNA and a protospacer-adjacent motif (PAM).

5. The method for constructing the shortened sgRNA random library of claim 1, wherein the shortened sgRNA comprises crRNA including a shortened TRS and trRNA.

6. A method for screening a target gene based on a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system comprising the steps of: (a) introducing a shortened sgRNA random library prepared according to the method of claim 1 into plurality of cells in which a nuclease-deactivated Cas (dCas) protein is overexpressed, thereby generating a plurality of test transformants; (b) selecting a subject exhibiting a modified phenotype among the transformants, compared to a control group; and (c) confirming a sequence of a shortened TRS of the shortened sgRNA introduced into the selected subject, thereby screening a target gene exhibiting high activity.

7. The method for screening the target gene based on the CRISPRi system of claim 6, further comprising: (d) additionally screening a target gene based on a duplicated gene in a shortened sgRNA random library comprising shortened TRSs having the same length or a duplicated gene between shortened sgRNA random libraries comprising shortened TRSs having the various lengths, among the target genes screened in the step (c).

8. The method for screening the target gene based on the CRISPRi system of claim 6, wherein the dCas protein is a dead CRISPR/Cas enzyme selected from the group consisting of dead Cas9, dead Cas12a, dead Cas12b, and dead Cas12c.

9. The method for screening the target gene based on the CRISPRi system of claim 8, wherein the dCas protein is dCas9 comprising mutations in HNH and RuvC domains.

10. The method for screening the target gene based on the CRISPRi system of claim 9, wherein the dCas9 protein is derived from a bacterial species selected from the group consisting of Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua and Streptococcus mutans.

11. The method for screening the target gene based on the CRISPRi system of claim 10, wherein the dCas9 protein comprises at least one mutation selected from the group consisting of D10A, H840A and N863A in Streptococcus pyogenes Cas9.

12. The method for screening the target gene based on the CRISPRi system of claim 6, wherein the target gene is involved in xylose catabolism.

13. A method for mass-producing a target product of interest comprising the following steps: (a) deleting two or more target genes obtained according to the method for screening the target gene of claim 6 in a host cell to produce a target product of interest; and (b) incubating the host cell to obtain a mass-produced target product of interest.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG. 1A shows a CRISPRi system that represses the expression of xylA and xylB genes involved in D-xylose metabolism by binding to a xylA promoter of a dCas9-NG/sgRNA complex prepared from a dcas9-NG gene induced by L-arabinose and sgRNA targeting the xylA promoter. FIG. 1B shows sgRNA with a 20-nucleotide-long TRS targeting a −10 region of the xylA promoter. FIGS. 1C and 1D show results of phenotypic changes related to a D-xylose metabolism of a cell and the repression of a xylA gene expression according to various lengths of a TRS in sgRNA, respectively.

[0073] FIG. 2A shows a sgRNA random library constructed using a primer having a random sequence phosphorylated at a 5′-terminal, and FIG. 2B shows a screening method using the sgRNA random library. FIG. 2C shows a result of screening target genes associated with a D-xylose metabolism using a sgRNA random library. FIGS. 2D and 2E show results of transcriptional repression of xylA and xylB genes according to sgRNA binding sites (promoter and open reading frame).

[0074] FIG. 3A shows introduction of a violacein biosynthetic gene into an E. coli genome using a homologous recombination method. FIG. 3B shows a purple color change of recombinant E. coli colonies by violacein biosynthesis. FIG. 3C shows the screening of colonies with increased production of violacein through phenotypic changes of transformed colonies with the sgRNA random library. FIG. 3D shows results of screening target genes through redundancy analysis of many candidate genes.

[0075] FIGS. 4A and 4B show results of comparing phenotypes and violacein production amounts of strains deleted with 17 target genes screened through CRISPRi screening and a wild-type strain.

[0076] FIG. 5 shows target genes that enhance violacein production found through CRISPRi screening in an intracellular metabolic pathway.

DETAILED DESCRIPTION

[0077] In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0078] Hereinafter, Examples are to describe the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these Examples in accordance with the gist of the present disclosure.

Example 1. Confirmation of Length of Target Recognition Sequence (TRS) in sgRNA Required for CRISPR Interference

[0079] The present inventors used an E. coli HK1160 strain, in which the gene expression of a dCas9-NG protein was inducible by the addition of L-arabinose. The dCas9-NG protein was a protein having 5′-NG as a PAM sequence. The E. coli was streaked on an LB solid medium, and then grown colonies were inoculated into 50 ml of a LB liquid medium and incubated at 37° C. until OD.sub.600 nm became 0.4, centrifuged at 3500 rpm for 20 minutes and then washed twice with 40 ml of 10% glycerol to prepare electro-competent cells. Various lengths of sgRNAs recognizing a −10 region of PxylA, a promoter of a xylA gene, as a target sequence were prepared to confirm the length of the target recognition sequence of the sgRNA required for CRISPRi.

[0080] Various sgRNA expression plasmids with different lengths of TRS (5′-shortened; N.sub.7, N.sub.8, N.sub.9, N.sub.10, N.sub.11, N.sub.14, N.sub.17, and N.sub.20) having the promoter of the xylA gene as the target sequence were constructed as follows: Using a primer pair designed according to a length of the target sequence using plasmid pHK459 as a template, two pieces of DNA with overlapping sequences at both ends were amplified by PCR, and then the two pieces of DNA were connected using Gibson assembly Master Mix to prepare various sgRNA plasmids with different lengths of TRS (FIG. 1B). After the sgRNA expression plasmids with target recognition sequences of various lengths were inserted into HK1160 cells by electroporation, the cells were smeared on a MacConkey selective medium supplemented with 0.3% of D-xylose and L-arabinose, respectively, and then incubated at 37° C.

[0081] When sgRNA plasmids carrying the original length of the TRS (N.sub.20) and 5′-end-shortened TRS (N.sub.9, N.sub.10, N.sub.11, N.sub.14, and N.sub.17) were transformed, white colonies were observed, indicating the repression of D-xylose metabolic enzyme genes (FIG. 1C). HK1160 cells harboring either sgRNA-deleted or sgRNA plasmids carrying a relatively shorter TRS (N.sub.7 and N.sub.8) formed red colonies.

[0082] The repression of the xylA promoter by the dCas9-NG/sgRNA complex was verified by quantitative RT-qPCR (FIG. 1D). The relative abundance of xylA transcripts in cells transformed with various sgRNA plasmids was compared to that in cells transformed with the sgRNA-deleted plasmid and determined. The degree of xylA transcriptional repression did not change from N.sub.20 to N.sub.11 of the TRS in sgRNAs, but gradually decreased as the length of TRS was shortened from N.sub.11 to N.sub.9. The xylA repression was not observed by CRISPRi in N.sub.8 and N.sub.7. These results demonstrated that the minimum length of the TRS in sgRNAs required for transcriptional repression and phenotype change was confirmed to be 9 nt.

[0083] Primer sequences used in Example above were listed in Table 1.

TABLE-US-00001 TABLE 1 SEQ ID NO: Primer name Primer sequence (5′->3′)  1 Pxyl-20NG-F AAAGGGAGTGCCCAATATTAGTTTTAGA GCTAGAAATAGCAAG  2 Pxyl-20NG-R TAATATTGGGCACTCCCTTTACTAGTAT TATACCTAGGACTG  3 Pxyl-17NG-F AGTGGGAGTGCCCAATATTAGTTTTAGA GCTAGAAATAGCAAG  4 Pxyl-17NG-R TAATATTGGGCACTCCCACTAGTATTAT ACCTAGGACTG  5 Pxyl-14NG-F ACTAGTAGTGCCCAATATTA GTTTTAGAGCTAGAAATAGCAAG  6 Pxyl-14NG-R TAATATTGGGCACT ACTAGTATTATACCTAGGACTG  7 Pxyl-11NG-F AATACTAGTGCCCAATATTA GTTTTAGAGCTAGAAATAGCAAG  8 Pxyl-11NG-R TAATATTGGGC ACTAGTATTATACCTAGGACTG  9 Pxyl-10NG-F TAATACTAGTCCCAATATTAGTTTTAGA GCTAGAAATAGCAAG 10 Pxyl-10NG-R TAATATTGGGACTAGTATTATACCTAGG ACTG 11 Pxyl-9NG-F ATAATACTAGTCCAATATTAGTTTTAGA GCTAGAAATAGCAAG 12 Pxyl-9NG-R TAATATTGGACTAGTATTATACCTAGGA CTG 13 Pxyl-8NG-F TATAATACTAGTCAATATTAGTTTTAGA GCTAGAAATAGCAAG 14 Pxyl-8NG-R TAATATTGACTAGTATTATACCTAGGAC TG 15 Pxyl-7NG-F GTATAATACTAGTAATATTAGTTTTAGA GCTAGAAATAGCAAG 16 Pxyl-7NG-R TAATATTACTAGTATTATACCTAGGACT G 17 Sm-ATGout GATACTGGGCCGGCAGGCGCTCCATTGC CC 18 Sm-TAAout GCAATGGAGCGCCTGCCGGCCCAGTATC AG 19 pBR322ori_F GGGAAACGCCTGGTATCTTTATAGTC 20 N20_F NNNNNNNNNNNNNNNNNNNNGTTTTAG AGCTAGAAATAGCA 21 N12_F NNNNNNNNNNNNGTTTTAGAGCTAGAA ATAGCA 22 N11_F NNNNNNNNNNNGTTTTAGAGCTAGAAA TAGCA 23 N10_F NNNNNNNNNNGTTTTAGAGCTAGAAAT AGCA 24 N9_F NNNNNNNNNGTTTTAGAGCTAGAAATA GCA 25 N8_F NNNNNNNN GTTTTAGAGCTAGAAATAGCA 26 9NG_R ACTAGTATTATACCTAGGACTG 27 xylA_F CGTGGCGATGCGCAACTGGGCTGGGAC 28 xylA-vioAF- AAGGAACGATCGATATGACGAATTATTC OF TGACATTTGCATAG 29 xylA-vioAF- CAGAATAATTCGTCATATCGATCGTTC OR CTTAAAAAAATGCC 30 vioAF-KmR-OF CGCCATGGCTTACGACATTCCGGGGATC CGTCGACCTGCAG 31 vioAF-KmR- GGATCCCCGGAATGTCGTAAGCCATGGC OR GGCCGTTACGATC 32 vioAB_F CAGTGTTTCGCGAGCGCGAACAAGAGAA 33 vioB-CmR-OF GGAGACGCAATCCATCGGGATCCGTATA CCGTGTAGGCTGG 34 vioB-CmR-OR GTATACGGATCCCGATGGATTGCGTCTC CCGGCCCTCGCCC 35 vioBC_500up CCGTGTCGGAACAGCATCCAACCCATGC 36 vioBC-KmR- TGGAAGGGTAAATTAAATTCCGGGGATC OF CGTCGACCTGCAG 37 vioBC-KmR- GGATCCCCGGAATTTAATTTACCCTTC OR CAAGTTTGTACCAA 38 VioDE_F CCAACTACGAAACGCTGAGCAACCCGAA 39 vioDE-CmR-OF CATTTCATCCCGCTAGGGGATCCGTATA CCGTGTAGGCTGG 40 vioDE-CmR- GTATACGGATCCCCTAGCGGGATGAAAT OR GGCGCTTCTTTCC 41 KmR-xy1B-OF GCAGCTCCAGCCTACAACGTTATCCCCT GCCTGACCGGGTG 42 KmR-xylB-OR GGCAGGGGATAACGTTGTAGGCTGGAGC TGCTTCGAAGTTC 43 xylB_200dn CCAGATAAACCAGCGCCCCGACAACA 44 CmR-xylB_R ACTGAGATATATAGATGTGAATTATCCC CCACCCGGTCAGGCAGGGGATAACGTGG AATTCGTATACCGGGGATCGGTCGACGT 45 xylB_500dn GTTGCTCATGCCGAGCGAAACAAACG 46 BW25113_pyk CAGAGATAACTTGAAGCGGGTCAAAG A-F 47 BW25113_pykF CAGCGTATAATGCGCGCCAATTGACTC -F 48 BW25113_ptsG AATAAAGGGCGCTTAGATGCCCTGTA 49 BW25113_sdaA CTGGCGCTGCAAATTGGTGTGAAACC -F 50 BW25113_tnaA GCTTCGCTTCATTGTTACCACTCCTG 51 BW25113_ GACGATGACAAACCTCGCCTCGGGGA tyrR-F 52 BW25113_ GAGCAGATCGAAAAGCAATTACACAAA cra-F 53 BW25113_ppsR GTTAAACGCGTCGGCGGTTGTGGCGA -F 54 BW25113_sad- GGATAACGACGGTTGAATTCCGCCAG F 55 BW25113_sdaC CTCATCAACTCATTTCATTTGTTATA -F 56 BW25113_livG- GAAATACGATCCCTCCGATCGTGTCA F 57 BW25113_ubil- GGTGTCATCCACTGGAACGGCGCGAA F 58 BW25113_purL CAGCTGGCTGATATTCTGCCGCACGG -F 59 BW25113_met CGGCAATCGCAGACCTGGCGAAACTC N-F 60 galT_half_F GCAAATAGCTTCCTGCCTAACGAAGC 61 BW25113_rbsA GATCACATTTCCGTAACGTCACGATG -F 62 BW25113_gltD- GCAAGCATTATCGGCAACACCTGCCT F 63 KmR-ATGout ACCTGCGTGCAATCCATCTTGTTCAA TCAT

Example 2. Method for Constructing sgRNA Libraries with Shortened and Random TRS

[0084] The present inventors have constructed sgRNA expression plasmids having random sequences N.sub.8, N.sub.9, N.sub.10, N.sub.11, N.sub.12, and N.sub.20 as target recognition sequences using site-directed mutagenesis. DNA fragments having random regions of different lengths depending on the length of TRS were amplified by PCR using a plasmid pHK459 as a template and a pair of primers phosphorylated at the 5′-terminal. The two amplified fragments were treated with a DpnI restriction enzyme to remove the template and purified. The two purified fragments were ligated using T4 DNA ligase, transformed into a DH5a competent cell, smeared on an LB solid medium, and incubated at 37° C. To confirm the library quality, 10 colonies were randomly selected from random sgRNA libraries (TRS=N.sub.8, N.sub.9, N.sub.10, N.sub.11, N.sub.12, and N.sub.20), and TRS sequences of sgRNAs were confirmed by Sanger sequencing. It was confirmed that all 10 sgRNAs were different TRSs. All transformants formed on the solid medium were harvested and the plasmids were purified to obtain a random sgRNA library.

Example 3. Target Gene Screening Using Shortened sgRNA Random Library

[0085] The shortened sgRNA random library of Example 2 was inserted into the HK1160 strain overexpressed with dCas9-NG by electroporation, and then smeared on a MacConkey solid medium supplemented with D-xylose and L-arabinose, and incubated at 37° C. Transformants of about 10.sup.4 CFU/ligated DNA (μg) were obtained from all the sgRNA random libraries. In the transformed HK1160 cells, 1, 2, 8, and 11 white colonies were observed in the N.sub.12, N.sub.11, N.sub.10, and N.sub.9 sgRNA libraries, respectively (FIG. 2C). White colonies were not observed in transformed HK1160 cells in the N.sub.8 or N.sub.20 sgRNA library. For N.sub.8, it seems that the expression of the gene may not be repressed because it may not be tightly attached to the target. When the length of the target recognition sequence in sgRNA was 20 nt, the size of random sgRNA with all possible nucleotide sequences was 4.sup.20 (to 10.sup.12). Accordingly, it was unlikely that 20-nt-long TRS sequences in 10.sup.4 transformed cells will exactly match the genome sequence of the microorganism.

[0086] 22 different sgRNA plasmids were obtained from 1, 2, 8, and 11 white colonies. Since the target recognition of dCas9-NG was determined by the TRS and the PAM sequence, the TRS and the 5′-NG PAM sequence were confirmed in a D-xylose operon in the E. coli genome to identify target nucleotide sequences. As a result, it was confirmed that all 22 types of sgRNA plasmids targeted a regulatory region and a structural gene of genes involved in D-xylose metabolism. Among the 22 types of TRSs, 5 TRSs in the regulatory region and 17 TRSs in the structural gene were identified. Even when the sgRNA was bound to the structural gene using the RT-qPCR, whether the expression of the D-xylose operon gene could be effectively repressed was tested (FIG. 2D). When the sgRNA was bound to the xylA promoter (TRS (−10)), the expression of xylA and xylB was reduced by 2.1- and 1.3-fold, respectively, by CRISPRi. When the sgRNA was bound to the end (1118 to 1126) of the xylA gene, the expression of xylA was not repressed but instead increased by 0.5-fold, and the expression of xylB was decreased by 1.3-fold. When the dCas9-NG/sgRNA was bound to the xylA promoter, the transcripts of xylA and xylB were not formed, so that the repression degree of the gene expression was similarly shown. As a result, it was confirmed that it is possible to find target sequences in the genome of cells with changed phenotypes using a shortened random TRS library.

Example 4. Construction of Recombinant Strain in which Expression of Genes for Violacein Biosynthesis was Induced by D-Xylose

[0087] The present inventors have prepared an E. coli strain (E. coli MG1655 xylB::vio ABCDE-CmR) in which a vioABCDE operon was inserted into the genome through lambda-red recombination. The E. coli MG1655 strain was streaked on an LB solid medium, and then grown colonies were inoculated into 50 ml of a LB liquid medium and incubated at 37° C. until OD.sub.600 nm became 0.4, centrifuged at 3500 rpm for 20 minutes and then washed twice with 40 ml of 10% glycerol to prepare electro-competent cells.

[0088] The vioABCDE operon was divided and inserted into four DNA fragments vioAF, vioABF, vioBC, and vioDE. Some of the genes for violacein biosynthesis were amplified by PCR from genomic DNA of a Massilia sp. NR4-1 strain. A violacein biosynthetic gene part-antibiotic resistance gene cassette was amplified using overlap PCR so as to have a homologous sequence for recombination with a kanamycin or chloramphenicol gene. The amplified PCR product was purified and then inserted sequentially into E. coli MG1655 in which a lambda-red recombinant enzyme of a pKD46 plasmid was overexpressed by L-arabinose and located at the back of a promoter in which the gene expression was induced by D-xylose to prepare an SH148 strain. The SH148 strain was streaked on an LB solid medium and an LB solid medium added with D-xylose, and incubated at 30° C. for 24 hours. In the LB solid medium added with D-xylose, the SH148 strain formed purple colonies (FIG. 3B).

[0089] P1 transduction was used to construct a violacein-producing E. coli strain (E. coli MG1655 xylB::vioABCDE-CmR, araBAD::P.sub.BAD-dcas9-NG-KmR) in which the dcas9-NG gene was inserted into the genome. Single colonies formed by streaking the SH148 strain of Example 4 on the LB solid medium were inoculated into 30 ml of an LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2, incubated at 37° C. until OD.sub.600 nm became 0.4, and added with P1 vir 300 μl and further incubated for 3 hours until the cells were lysed. Then, the cells were added with 600 μl of chloroform, vortexed, and centrifuged at 3500 rpm for 20 minutes. The cells were completely lysed with chloroform once more to prepare a SH148 P1 lysate.

[0090] Single colonies grown by streaking the HK1160 strain having the dcas9-NG gene on the LB solid medium were inoculated into 10 ml of an LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2 and incubated at 37° C. 1 ml of the culture solution was transferred to a 1.5 ml tube, centrifuged at 12,000 rpm for 5 minutes, and a supernatant was removed. A cell pellet was resuspended in 100 μl of the LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2, and then added with 10 μl or 100 μl of the SH148 P1 lysate and reacted at 37° C. for 20 minutes. Thereafter, the cell pellet was added with 100 μl of 1 M sodium citrate, and smeared on an LB solid medium added with chloramphenicol and incubated at 37° C. The grown colonies were subjected to PCR to confirm whether the vioABCDE-CmR cassette was inserted at a position of the xylB gene, thereby preparing a SH150 strain.

Example 5. Screening of Target Genes with Increased Violacein Production Using Shortened Random TRS Library

[0091] The SH150 strain of Example 4 was streaked on the LB solid medium and then the grown single colonies were inoculated in 50 ml of an LB liquid medium and incubated at 37° C. until OD.sub.600 nm became 0.4, and added with L-arabinose at a concentration of 1 mM and further incubated for 3 hours to overexpress a dCas9-NG protein. The SH150 strain was centrifuged at 3500 rpm for 20 minutes, washed twice with 40 ml of 10% glycerol to prepare electrocompetent cells.

[0092] Shortened random sgRNA libraries N.sub.8, N.sub.9, N.sub.10, N.sub.11, N.sub.12, and N.sub.20 was inserted into the SH150 strain overexpressed with dCas9-NG by electroporation, and then smeared on the LB solid medium added with 0.3% of D-xylose and L-arabinose and then incubated at 25° C. Among 10.sup.4 colonies formed on the solid medium, colonies with increased violacein production were screened. Colony colors were extracted from colony images and converted to RGB values. Compared to other colonies, colonies with dark purple color were screened. The sgRNA plasmids were purified from the dark purple colonies and the TRSs of 68 sgRNAs were identified by Sanger sequencing. A total of 63 types (N.sub.9=46, N.sub.10=9, N.sub.11=5 and N.sub.12=3) of sgRNA target genes were screened, excluding 5 overlapping TRSs (FIG. 3D).

[0093] Since sgRNAs including shortened TRSs may bind to several different sites in the E. coli genome, it is difficult to identify which genes cause phenotypic changes among target candidate genes found by CRISPR screening. Accordingly, the present inventors have used a method of analyzing the redundancy of target candidate genes to select target genes. Target genes were screened according to the following criteria: 1) overlapping genes that are repeatedly targeted in the library with same TRS length, and 2) overlapping genes that were repeatedly found in libraries with different TRS length. For the N.sub.9 sgRNA library, 460 different sites were identified in the E. coli genome by querying 46 TRSs. In particular, 6 genes ptsG, pykA, pykF, sdaA, tnaA, and tyrR were identified five times or more. However, no repeated target genes were found in the N.sub.10, N.sub.11, and N.sub.12 libraries.

[0094] Next, overlapping target genes were selected among different sgRNA libraries N.sub.9, N.sub.10, N.sub.11, and N.sub.12. Although secA and ligA were found as target genes four and two times, the genes were excluded from the process of screening the target genes for further gene knockout because the genes were known as essential genes in cell growth. Finally, 17 target genes were selected based on the criteria, and each target gene was not biased in the genome (FIG. 3D). In 5 genes cra, ptsG, pykA, pykF, and tnaA, target sequences were identified only in the promoters. In 7 genes galK, gltD, livG, metN, purL, rbsA, and ubil, target sequences were identified only in the middle of the gene. In 5 genes ppsR, sad, sdaA, sdaC, and tyrR, target sequences were identified in both the promoter and the structural region.

Example 6. Construction of Strain with Single-Deleted Target Gene

[0095] It was tested whether violacein production was enhanced by repressing the expression of the target gene. The present inventors have used P1 transduction to construct 17 deletion strains in which the target gene was single-deleted. Single colonies formed by streaking kanamycin-resistant strains for 17 target genes in the Keio collection on an LB solid medium were inoculated into 30 ml of an LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2, incubated at 37° C. until OD.sub.600 nm became 0.4, and added with P1 vir 300 μl and further incubated for 3 hours until the cells were lysed. Then, the cells were added with 600 μl of chloroform, vortexed, and centrifuged at 3500 rpm for 20 minutes. The cells were completely lysed with chloroform once more to prepare a P1 lysate of a kanamycin-resistant strain from the Keio collection.

[0096] Single colonies grown by streaking the SH148 strain of Example 4 on the LB solid medium were inoculated into 10 ml of an LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2 and incubated at 37° C. 1 ml of the culture solution was transferred to a 1.5 ml tube, centrifuged at 12,000 rpm for 5 minutes, and a supernatant was removed. A cell pellet was resuspended in 100 μl of the LB liquid medium added with 10 mM MgSO.sub.4 and 5 mM CaCl.sub.2, and then 10 μl or 100 μl of the P1 lysate of the 17 kanamycin-resistant strains was added to each SH148 cell and reacted at 37° C. for 20 minutes. Thereafter, the cell pellet was added with 100 μl of 1 M sodium citrate, and smeared on an LB solid medium added with kanamycin and incubated at 37° C. The grown colonies were subjected to PCR to confirm whether the target gene was deleted in the SH148 strain.

Example 7. Enhancement of Violacein Production by Target Gene Deletion

[0097] A wild-type strain, SH148, and 17 strains with a single-gene deletion were streaked on a D-xylose-added LB solid medium and incubated at 25° C. Compared to the wild-type strain, all of the 17 deletion strains formed dark purple colonies (FIG. 4A). The wild-type strain and the 17 deletion strains were streaked on the LB solid medium and then the formed single colonies were inoculated into 10 ml of an LB liquid medium and incubated at 37° C. for 12 hours. The culture medium was inoculated into the LB liquid medium added with D-xylose, and then incubated at 25° C. for 48 hours.

[0098] For violacein extraction, cells were harvested by centrifugation (4° C., 12,000 rpm, 10 minutes) and the supernatant was discarded. The collected cell pellet was added with methanol (100%, the same volume as the culture medium) and vortexed to be released, and then reacted at room temperature for 20 minutes. Methanol containing violacein was extracted by centrifugation (4° C., 12,000 rpm, 10 minutes). The concentration of violacein in the supernatant was measured by high performance liquid chromatography (HPLC) using a C18 column. As a mobile phase, a mixture of methanol, acetonitrile, and distilled water (1:1:2, v/v) (adjusted to pH 3.6 with acetic acid) was used. Isocratic elution was performed at 30° C. at a flow rate of 0.5 ml/min, and violacein was detected using UV light at 575 nm.

[0099] The wild type SH148 strain produced 24.2 μg/ml of violacein, and all the 17 deletion strains showed higher violacein concentrations than the SH148 strain (FIG. 4B). In particular, a single deletion of livG produced 82.6 μg/ml of violacein, 3.4-fold increased compared to the wild-type cell. Among the 17 target genes, 7 genes tyR, pykF, cra, ptsG, pykA, sdaA and tnaA are known to increase the intracellular concentration of tryptophan, a precursor of violacein. Therefore, through CRISPR screening, it was possible to find genes known in tryptophan metabolism engineering in the related art as well as new target genes (FIG. 5), and by deleting the selected target gene, it was possible to prepare strains with enhanced violacein production.

[0100] As described above, specific parts of the present disclosure have been described in detail, and it will be apparent to those skilled in the art that these specific techniques are merely preferred exemplary embodiments, and the scope of the present disclosure is not limited thereto. Therefore, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.

[0101] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.