WO2020004936A1 - Système d'inhibition d'expression génique cible multiplex basé sur un arns régulateur de synthèse et procédé pour sa production - Google Patents

Système d'inhibition d'expression génique cible multiplex basé sur un arns régulateur de synthèse et procédé pour sa production Download PDF

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WO2020004936A1
WO2020004936A1 PCT/KR2019/007729 KR2019007729W WO2020004936A1 WO 2020004936 A1 WO2020004936 A1 WO 2020004936A1 KR 2019007729 W KR2019007729 W KR 2019007729W WO 2020004936 A1 WO2020004936 A1 WO 2020004936A1
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target gene
srna
genes
expression
gene
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이상엽
유승민
양동수
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한국과학기술원
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a multiplex target gene expression suppression system and its preparation based on synthetic sRNA, and more particularly, to a combination and preparation of a vector comprising a variety of antibiotic resistance genes, origins of replication and synthetic regulatory sRNA, the vector It relates to an sRNA library consisting of a combination of and screening methods for producing useful substances using the same.
  • the technology overcomes many of the shortcomings and limitations of traditional genome-based engineering, including knockout techniques (long time, labor intensive, skilled skill requirements, low success rates, and the difficulty of concurrent large-volume experiments).
  • the time, efficiency and possibility of large-scale experiments could all be dramatically improved (Datsenko et al, Proc. Natl. Acad. Sci. USA (2000), 97 (12), 6640-6645).
  • the present inventors have made efforts to develop a variety of synthetic regulatory sRNA expression platforms.
  • the synthetically regulated sRNA expression vectors differ from antibiotic resistance genes and origins of replication. (golden gate assembly), ligation, homologous recombination, sequence and ligation-independent cloning (SLIC), seamless ligation cloning extract (SLiCE), circular polymerase extension cloning (CPEC), or gene synthesis methods
  • SLIC homologous recombination
  • SLiCE seamless ligation cloning extract
  • CPEC circular polymerase extension cloning
  • An object of the present invention is to provide a recombinant microorganism having a synthetic sRNA-based simultaneous multiple target gene expression suppression system and a recombinant microorganism having improved threonine, proline, indigo or violaserine production capacity using the recombinant microorganism having the system. will be.
  • the present invention is a first antibiotic resistance gene; First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; A recombinant microorganism into which a host multi-target gene expression suppression system is introduced, wherein the host vector is introduced, comprising: a q vector comprising a synthetic sRNA coding region that inhibits expression of the p target gene;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • the antibiotics are ampicillin, kanamycin, kanamycin, chloroamphenicol, apramycin, streptomycin, specrepomycin, spectinomycin, tetracycline and erythromycin.
  • the replication origin is selected from the group consisting of CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101, RSF1030 (RSF), and RK2,
  • the synthetic sRNA coding region comprises a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And a terminator
  • Simultaneous multiple target gene expression suppression systems provide recombinant microorganisms introduced into host cells.
  • the present invention also provides a method for improving a strain of useful substance producing strain comprising the following steps:
  • step (c) introducing the first vector library into a target strain to produce a useful material and identifying a candidate group of genes whose expression is suppressed when the production of the useful material is improved, and determining 2 to 500 expression inhibitory genes. step;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin and the first target gene and 2 to 500 expression suppression genes are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • q is an integer from 2 to 500.
  • the present invention also relates to a host cell having a threonine biosynthetic pathway
  • Antibiotic resistance genes First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; A recombinant microorganism into which a simultaneous multi-target gene expression suppression system of a prokaryote is introduced, comprising: a q vector comprising a synthetic sRNA coding region that inhibits p-target gene expression;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin, and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • the antibiotics are ampicillin, kanamycin, kanampycin, chloramphenicol, apramycin, streptomycin, spectiomycin, tetracycline, tetracycline and erythromycin.
  • the origin of replication is selected from the group consisting of CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101, RSF1030 (RSF), and RK2,
  • the synthetic sRNA coding region comprises a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And terminator,
  • the present invention also provides a host cell having a proline biosynthetic pathway
  • Antibiotic resistance genes First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; A recombinant microorganism into which a simultaneous multi-target gene expression suppression system of a prokaryote is introduced, comprising: a q vector comprising a synthetic sRNA coding region that inhibits p-target gene expression;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin, and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • the antibiotics are ampicillin, kanamycin, kanampycin, chloramphenicol, apramycin, streptomycin, spectiomycin, tetracycline, tetracycline and erythromycin.
  • the origin of replication is selected from the group consisting of CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101, RSF1030 (RSF), and RK2,
  • the synthetic sRNA coding region comprises a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And terminator,
  • the vector prepared by inserting a sequence complementary to the mRNA of the serC, murE, aspC, metB, fadR, fur or chpA gene in the region complementary to the mRNA of the target gene of the first to q vector Introduced, the expression of two or more genes selected from the group consisting of serC, murE, aspC, metB, fadR, fur and chpA is suppressed to provide a recombinant microorganism characterized by improved proline production capacity.
  • the present invention also provides a proline production method comprising culturing the recombinant microorganism through fed-batch fermentation in a medium containing a trace metal solution.
  • the present invention also provides a host cell having an indigo biosynthetic pathway, asnA, hisJ, yneH, to a region complementarily binding to the mRNA of the target gene of the first to q vectors of the simultaneous multiple target gene expression suppression system.
  • a vector prepared by inserting a sequence complementarily binding to mRNA of the napG, kdsA, ygfA, ftsl, aceF or ostB genes is introduced, and asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF and ostB It provides a recombinant microorganism with improved indigo production capacity, characterized in that the expression of two or more genes selected from the group consisting of is suppressed.
  • the invention also relates to a host cell having an indigo biosynthetic pathway
  • Antibiotic resistance genes First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; A recombinant microorganism into which a simultaneous multi-target gene expression suppression system of a prokaryote is introduced, comprising: a q vector comprising a synthetic sRNA coding region that inhibits p-target gene expression;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin, and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • the antibiotics are ampicillin, kanamycin, kanampycin, chloramphenicol, apramycin, streptomycin, spectiomycin, tetracycline, tetracycline and erythromycin.
  • the origin of replication is selected from the group consisting of CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101, RSF1030 (RSF), and RK2,
  • the synthetic sRNA coding region comprises a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And terminator,
  • the invention also relates to a first antibiotic resistance gene; First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; A recombinant microorganism into which a simultaneous multi-target gene expression suppression system of a prokaryote is introduced, comprising: a q vector comprising a synthetic sRNA coding region that inhibits p-target gene expression;
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin, and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • the antibiotics are ampicillin, kanamycin, kanampycin, chloramphenicol, apramycin, streptomycin, spectiomycin, tetracycline, tetracycline and erythromycin.
  • the replication origin is selected from the group consisting of CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101, RSF1030 (RSF), and RK2,
  • the synthetic sRNA coding region comprises a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And a terminator
  • the host cell has a violacein (violacein) biosynthesis pathway
  • a vector prepared by inserting a binding sequence has been introduced so that expression of two or more genes selected from the group consisting of ytfR, hybG, ampG, potG, caiF, minD, ilvM, yfbR, rfe, atpH, tiaE and murI
  • a vector prepared by inserting a binding sequence has been introduced so that expression of two or more genes selected from the group consisting of ytfR, hybG, ampG, potG, caiF, minD, ilvM, yfbR, rfe, atpH, tiaE and murI
  • a recombinant microorganism having improved violacein / deoxybiolacein production capacity, which is inhibited.
  • the present invention also provides a violacein / deoxybiolacein production method comprising culturing the recombinant microorganism through fed-batch fermentation in a medium containing a trace metal solution.
  • the present invention also provides a method for screening multiple inhibitor gene combinations comprising the following steps:
  • the first antibiotic resistance gene and the n antibiotic resistance gene, the first replication origin and the m replication origin and the first target gene and the p target gene are different from each other,
  • n is an integer from 2 to 50
  • m is an integer from 2 to 10
  • q is an integer from 2 to 500
  • p is an integer from 2 to 20000
  • r and s are an integer from 2 to 500.
  • Figure 2 compares the efficiency of DsRed2 fluorescent protein knockdown of each of the sRNA-regulated sRNA platforms for (A) pACYC184-DsRed2 (pLac), (B) pTac15K-DsRed2 (pTrc), (C) pTrc99A-DsRed2 (pTac) reporter plasmids.
  • A pACYC184-DsRed2 (pLac)
  • B pTac15K-DsRed2
  • pTrc pTrc99A-DsRed2
  • Figure 3 is a graph of the time-dependent growth curve measured after introduction of synthetically regulated sRNA expression platform plasmids (a) one by one, (b) two by two (c) three in the E. coli strain carrying a fluorescent protein reporter plasmid .
  • Reporter plasmids were expressed as pTac notation: pTac15K-dsRed2, pTrc notation: pTrc99A-dsRed2, pACYC: pACYC184-dsRed2.
  • Each graph is divided into three parts (blue, gray and red boxes), each representing Day 1, Day 2 (first pass) and Day 3 (second pass) from the front.
  • Colony PCR was performed after (a) 1st day, (b) 2nd day, and (c) 3rd day passage culture of the strains used in the experiment of FIG. 3 to confirm the presence of sRNA platform plasmids in the strain.
  • A represents pACYC184-DsRed2 (pLac)
  • R represents pTrc99A-DsRed2 (pTrc)
  • T represents pTac15K-DsRed2 (pTac) plasmid, 1: ColA-Sm, 2: pBBR1-Am, 3: pBBR1-Tc, 4: pBBR1-Sm, 5: CDF-Tc, 6: CDF-Sm.
  • the dashed combination means that the plasmids were transformed at the same time.
  • colony PCR was performed targeting the origin of replication on each plasmid, and the size of the PCR product was as follows: pBBR1, 1 kb; ColA, 0.6 kb; CDF, 0.3 kb.
  • 5 is a diagram showing the change in the threonine production according to the application of the synthetic regulatory sRNA library.
  • Figure 6 is a diagram showing the change in threonine production through the application of a double combination of synthetic regulatory sRNAs showing increased threonine production in FIG.
  • Figure 7 is a diagram showing the change in proline production according to the application of synthetic regulatory sRNA library.
  • FIG. 8 is a diagram showing a change in proline yield through the application of a double combination of synthetic regulatory sRNAs showing increased proline production in FIG. 7.
  • (d) shows the results when the strain NMH26 p15PP3533 pKKtrcSargF-trcSglnA was cultured under the fermentation conditions of (c) as a control experiment.
  • the red circle represents cell growth (OD600) and the blue triangle represents proline concentration (g / L).
  • FIG. 10 shows the result of fermenting four strains showing the highest production capacity in FIG. 7 (single knockdown application) under optimized fermentation conditions.
  • (a) is NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur strain
  • (b) is NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fadR strain
  • (c) is NMH26 p15PPgl33 pKKtFTScArc-pCrcTrcSargAsp trcSglnA pCDFTc-mazF strain.
  • the red circle shows cell growth and the blue triangle shows proline concentration.
  • FIG. 11 shows the results of fermentation under optimized fermentation conditions of four strains showing the highest productivity in FIG.
  • (a) is NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur pColASm-metB strain
  • (b) is NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur pColASm-murE strain pKtCDSrc p pt15
  • the red circle represents cell growth (OD600) and the blue triangle represents proline concentration (g / L).
  • Figure 12 shows the colony PCR results to confirm the retention status of the sRNA plasmids in the exponential phase (E) and stationary phase (S) state of all proline-producing strains subjected to fermentation.
  • strains 1 to 9 are the same as the strain numbers in Table 2.
  • the upper DNA band represented by the red triangle indicates the presence of ColA-Sm platform based sRNA
  • the lower DNA band indicates the presence of CDF-Tc platform based sRNA.
  • FIG. 13 is a diagram showing a method for transferring a synthetic regulatory sRNA library of Escherichia coli genome level to a novel sRNA expression platform in the present invention and applying it to strains.
  • Figure 14 shows the results of culturing in a test tube after introduction of the E. coli genome-level synthetic regulatory sRNA library based on the ColA-Sm platform, and screening the strains through color screening.
  • FIG. 15 is a result of culturing nine strains showing high yield in FIG. 14 in a flask containing 50 mL MR medium.
  • Figure 16 shows the results of culturing in a test tube after introduction of the E. coli genome-level synthetic regulatory sRNA library based on the ColA-Sm platform, and screening the strains through color screening.
  • FIG. 17 is a graph in which 12 strains showing a high yield in FIG. 16 are cultured in a flask containing 50 mL MR medium, and (a) is a graph measuring the yield of each strain. (b) is a photograph of each flask after incubation. Strains that produce high concentrations of violacein / deoxybiolacein are darker in color.
  • FIG. 18 is a result of fed-batch fermentation of strains showing high violacein / deoxybiolacein production ability in FIG. 17.
  • (a) is a strain in which the pColA-ytfR plasmid is inserted into the E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain, and
  • the pColA-minD plasmid is added to the E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain. Inserted strain.
  • the red circle represents cell growth
  • the blue triangle represents the violacein concentration
  • the purple triangle represents the deoxybiolacein concentration.
  • the red arrows in (a) and (b) indicate the time points induced by IPTG.
  • (c) shows the colony PCR results to confirm the retention status of the sRNA plasmids in the exponential phase (E) and stationary phase (S) state of all the violacein producing strains subjected to fermentation.
  • strain 1 control strain
  • strain 2 strain of (a)
  • strain 3 strain of (b).
  • the DNA band represented by the red triangle indicates the presence of the ColA-Sm platform based sRNA.
  • Figure 19 shows the results of the stability test of the synthetic regulatory sRNA expression plasmids in each strain, two E. coli W3110 strains carrying two reporter plasmids, pTac15K-DsRed2 ("Tac”) and pTrc99A-DsRed2 (“Trc”), respectively.
  • Tac pTac15K-DsRed2
  • Trc pTrc99A-DsRed2
  • sRNA small RNA
  • sRNA small RNA
  • ribosome binding site refers to the site where the ribosomes bind on the mRNA for transcription of the mRNA.
  • gene should be considered in the broadest sense and may encode a structural or regulatory protein.
  • the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA / RNA replication, transcription and / or translation.
  • the target gene to be suppressed expression may exist as an extrachromosomal component.
  • the present invention is intended to develop a synthetic regulatory sRNA-based expression control system that can be applied to various kinds of prokaryotes and at the same time can suppress the expression of a plurality of target genes.
  • sRNAs which are independently included and complementarily binds to the RBS of DsRed2 mRNA, is formed by Gibson assembly, golden gate assembly, ligation, homologous recombination, and SLIC (A combination of sequence and ligation-independent cloning (SLiCE), seamless ligation cloning extract (SLiCE), circular polymerase extension cloning (CPEC), or gene synthesis was used to prepare a total of nine types of synthetically regulated sRNA expression vectors for inhibiting DsRed2 expression (Fig.
  • the present invention provides in one aspect, a first antibiotic resistance gene; First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; And n antibiotic resistance genes; Mth replication origin; P-target vector expression suppression system of a prokaryotic organism comprising a q vector comprising a synthetic sRNA coding region that inhibits p-target gene expression, wherein the first antibiotic resistance gene, the n-antibiotic resistance gene, and the first copy
  • the origin, the m replication origin, the first target gene and the p target gene are different from each other, n is an integer of 2 to 50, m is an integer of 2 to 10, q is 2 to It is characterized in that the integer of 500, p is an integer of 2 to 20000 relates to a simultaneous multiple target gene expression suppression system.
  • p refers to the number of target genes
  • the target gene may include an integer range of 2 to 20000 since all genes may be used as target genes regardless of the type of vector.
  • the antibiotic may be used without limitation as long as it is an antibiotic for inducing prokaryotic death, preferably ampicillin, kanamycin, chloroamphenicol, apramycin ), Streptomycin, spectyomycin, tetracycline, erythromycin, neomycin, penicillin, actinomycin, actinomycin, and gavenicillin garbenicillin, gentamicin, blasticidin, mycophenolic acid, puromycin, zeocin, borrelidin, ionomycin, Daunorubicin, doxorubicin, ivermectin, evermectin, mitramycin, mitomycin, mitomycin, nalidixic acid, novobiocin , Nystatin ( nystatin, oxytetracycline, paclitaxel, polymyxin, rifampicin, salinomycin, tylosin, valinomycin, banomycin Comycin (van
  • the origin of replication can be used without limitation as long as the sequence can start replication of the plasmid introduced into the prokaryote, but preferably CloDF13 (CDF), pBBR1, ColA, ColE1, pUC, pBR322, SC101 , RSF1030 (RSF), and may be selected from the group consisting of RK2, but is not limited thereto.
  • CDF CloDF13
  • pBBR1, ColA, ColE1, pUC, pBR322, SC101 , RSF1030 (RSF) and may be selected from the group consisting of RK2, but is not limited thereto.
  • the prokaryote may be used as long as it is a prokaryote capable of expressing sRNA, without limitation, E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida (Candida), Erwinia, Enterobacter, Pasterella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas , Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zimomonas Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptobacterium Mises (Streptomyces), Bifidobacterium (Bifidobacterium), Cyanobacterium (cyanobacterium) and cyclobacterium (Cy
  • the synthetic sRNA coding region is a promoter; Hfq binding site derived from sRNA of any of MicC, SgrS and MicF; A region forming complementary binding with a target gene mRNA; And it may be characterized in that it comprises a terminator.
  • the promoter is available to all kinds of promoters capable of inducing the expression of sRNA, preferably selected from the group consisting of tac, trc, T7, BAD, ⁇ PR and Anderson synthetic promoter And, most preferably, it may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 7.
  • the Hfq binding site may be preferably derived from MicC, and most preferably may be represented by the nucleotide sequence of SEQ ID NO: 8.
  • terminators capable of terminating transcription of the sRNA are available, and preferably the T1 / TE terminator, and most preferably, those represented by the nucleotide sequence of SEQ ID NO. It can be characterized.
  • the region forming the complementary bond with the target gene mRNA is sufficient if the minimum length that can form a complementary bond according to the target gene, for example 20 to 50 base, preferably 19 It may be characterized by being at least 37 bases.
  • the region forming the complementary binding with the target gene mRNA forms a complementary binding in whole or in part with the protein coding sequence starting with the ribosome binding site or the start codon of the target gene mRNA It may be characterized by.
  • complementary binding refers to basepairing between nucleic acid sequences, wherein the sequence of some regions of the mRNA of the target gene and the region forming the complementary bond with the target gene mRNA is about 70-80%. Or more preferably about 80-90% or more, even more preferably about 95-99% or more complementary to each other.
  • vector refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
  • Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors.
  • Typical plasmid vectors that can be used for this purpose include (a) the origin of replication so that replication can be efficiently performed to include several to hundreds of plasmid vectors per host cell, and (b) host cells transformed with plasmid vectors will be selected. It has a structure that includes an antibiotic resistance gene that allows it and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no suitable restriction enzyme cleavage site is present, synthetic oligonucleotide adapters or linkers according to conventional methods can be used to facilitate ligation of the vector and foreign DNA. After ligation, the vector should be transformed into the appropriate host cell.
  • Transformation can be readily accomplished using calcium chloride methods or electroporation (Neumann, et al., EMBO J., 1: 841, 1982) and the like.
  • an expression vector known in the art may be used as the vector used for the expression of the sRNA according to the present invention.
  • nucleotide sequences are "operably linked” when placed in a functional relationship with other nucleic acid sequences.
  • This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s).
  • the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame.
  • enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
  • the target gene may be present in the prokaryote or may be introduced without limitation, preferably DsRed2, LuxR, AraC, KanR (kanamycin resistance gene), tyrR (tyrosine regulator), ppc (phosphoenolpyruvate) carboxylase), csrA (carbon storage regulator), pgi (glucose-6-phosphate isomerase), glt (citrate synthase), accA (acetyl-CoA carboxyltransferase, alpha-subunit), accB (biotinylated biotin-carboxyl carrier protein), accC ( acetyl-CoA carboxylase), accD (acetyl-CoA carboxyltransferase, beta-subunit), aceE (subunit of E1p component of pyruvate dehydrogenase complex), aceF (pyruvate dehydrogenase), ackA (propionate kina
  • CsiD is the product of a gene induced by carbon starvation), csiR (DNA-binding transcriptional repressor), cytR (transcription factor required for transport and utilization of ribonucleosides and deoxyribonucleosides), dcuA (The DcuA transporter is one of three transporters known to be responsible for the uptake of C4-dicarboxylates such as fumarate under anaerobic conditions), deoB (phosphopentomutase), deoC (deoxyribose-phosphate aldolase), deoR (The transcriptional repressor DeoR, for "Deoxyribose Regulator,” is involved in the negative expression of genes related to transport and catabolism of deoxyribonucleoside nucleotides, fabH (KASIII, -ketoacyl-ACP synthases), fadD (fatty acyl-CoA synthetase), fadR (Fa
  • phoQ / phoP Member of the two-component regulatory system phoQ / phoP involved in adaptation to low Mg2 + environments and the control of acid resistance genes
  • pnuC PnuC NMN transporter
  • ppsA phosphoenolpyruvate synthetase
  • pta Phosphate acetyltransferase
  • purA adenylosuccinate synthetase
  • purB adenylosuccinate lyase
  • purR PurRHypoxanthine DNA-binding transcriptional repressor.PurR dimer controls several genes involved in purine nucleotide biosynthesis and its own synthesis
  • puuE (4-aminobutyrate aminotransferase
  • rbsA ribose ABC transporter
  • rbsB ribose ABC transporter
  • rbsD ribose pyranase
  • zwf glucose 6-phosphate-1-dehydrogenase
  • tktA transketolase I
  • tktB transketolase II
  • pgi glucose-6-phosphate isomerase
  • fbp fructose-1,6-bisphosphatase
  • serC phosphohydroxythreonine aminotransferase / 3-phosphoserine aminotransferase
  • murE UP-N-acetylmuramoylalanyl-Dglutamate 2,6-diaminopimelate ligase
  • pps phosphoenolpyruvate synthetase
  • aceE subunit of E1p component of pyruvate dehydrogenasetransfer
  • pta purA
  • ackA propionate kinase / acetate kinase activity
  • pck phosphoenolpyruvate synthe
  • genes are involved in amino acid biosynthesis and catabolism, nutrient) transport, pili synthesis, and other cellular functions, including 1-carbon metabolism), gltA (citrate synthase), pdhR (PdhR, "pyruvate dehydrogenase complex regulator,” regulates genes involved in the pyruvate dehydrogenase complex, tyrR (tyrosine regulator), csrA (carbon storage regulator), lexA (LexA represses the transcription of several genes involved in the cellular response to DNA damage), aroF (3-deoxy-D-arabino-heptulosonate -7-phosphate synthase, tyrosine-repressible), aroA (5-enolpyruvylshikimate-3-phosphate synthetase), aroC (Chorismate synthase), pheA (chorismate mutase and prephenate dehydratase, P-protein), trp
  • the present invention also relates to a nucleic acid fragment comprising: a first nucleic acid fragment encoding a first antibiotic resistance gene; A second nucleic acid fragment encoding a first replication origin; And a third nucleic acid fragment encoding a synthetic sRNA coding region that inhibits the expression of a first target gene, the Gibson assembly, golden gate assembly, ligation, homologous recombination,
  • the present invention relates to a method for producing a simultaneous multiple target gene expression suppression system comprising the step of connecting by sequence and ligation-independent cloning (SLIC), seamless ligation cloning extract (SLiCE), circular polymerase extension cloning (CPEC), or gene synthesis.
  • SLIC sequence and ligation-independent cloning
  • SLiCE seamless ligation cloning extract
  • CPEC circular polymerase extension cloning
  • the present invention also relates to a recombinant microorganism in which the simultaneous multiple target gene expression suppression system is introduced.
  • transformation means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.
  • the present invention also provides a method of introducing a co-targeted gene expression inhibitory system into a prokaryote or expressing in a prokaryote; And it relates to a method for inhibiting simultaneous expression of multiple target genes comprising the step of inhibiting the mRNA expression of the target gene.
  • the present invention also relates to a composition for inhibiting simultaneous expression of a target gene comprising the system for inhibiting expression of a target expression gene.
  • the present invention in another aspect, (a) preparing any base sequence having 20 to 50 bases; (b) inserting the nucleotide sequence into a region complementarily binding to the target gene mRNA of the first vector of the simultaneous multiple target gene expression suppression system to prepare a first vector library comprising a first sRNA library; (c) introducing the first vector library into a target strain to produce a useful material and identifying a candidate group of genes whose expression is suppressed when the production of the useful material is improved, and determining 2 to 500 expression inhibitory genes.
  • q relates to a method for improving a useful substance producing strain, characterized in that the integer of 2 to 500.
  • the improvement method of the useful substance producing strain including the step can also be elicited, and the method of improving the useful substance producing strain can be elicited.
  • q, r and s may be an integer of 2 to 500.
  • the present invention by introducing a simultaneous multi-target gene expression suppression system according to the present invention to the modified prokaryote, which has been improved metabolically and introduced plasmid, to confirm whether the production of the target substance can be further improved. It was.
  • a strain that enhances the threonine production pathway in the conventional threonine production research improves the enzyme on the threonine production pathway with a feedback-resistant enzyme and prevents the activity from being inhibited by the product, and overexpresses the threonine transporter.
  • T28C-pBRThrABCR3, KH Lee et al., Mol. Syst. Biol. 2007, 3 (149) when processing the sRNA library produced in the present invention, found eight genes that further increase the threonine production (FIG. 5), through the combination of these, the most excellent combination of threonine production capacity was found to find a gene combination, and it was confirmed that 30% or more of threonine transpiration was possible (FIG. 6).
  • the present invention provides a host cell having a threonine biosynthetic pathway, which is complementarily bound to an mRNA of a target gene of the first to q vectors of the simultaneous multiple target gene expression suppression system.
  • a vector prepared by inserting a sequence complementarily binding to the mRNA of the tktA, aroF, pta, ilvH, ilvE, glnA, fur or chpA genes was introduced, and thus, tktA, aroF, pta, ilvH, ilvE, glnA fur and chpA It relates to a recombinant microorganism with improved threonine production capacity, characterized in that the expression of two or more genes selected from the group consisting of is suppressed.
  • the recombinant microorganism is lactose operon repressor (lacI), homoserine O-succinyltransferase (metA), lysA (Diaminopimelate decarboxylase), tdh (L-threonine dehydrogenase), iclR (AceBAK operon repressor) and tdcC (Threonine / ser).
  • lacI lactose operon repressor
  • metalA homoserine O-succinyltransferase
  • metalA homoserine O-succinyltransferase
  • lysA Diaminopimelate decarboxylase
  • tdh L-threonine dehydrogenase
  • iclR AceBAK operon repressor
  • tdcC Threonine / ser
  • the recombinant microorganism is a mutation in which the 1034 base sequence of thrA (Bifunctional aspartokinase / homoserine dehydrogenase 1) is changed from C to T, and the 1055 base sequence of lysine-lysine-sensitive aspartokinase 3 (lysC) is changed from C to T, ilvA ( thrABC operon, ppc (phosphoenolpyruvate carboxylase), characterized by the manipulation of one or more genes selected from the group consisting of mutations in which the 290th sequence of l-threonine dehydratase, biosynthetic; also known as threonine deaminase has been changed from C to T , characterized in that the promoter of one or more genes selected from the group consisting of acs (acetyl-CoA synthetase) is substituted with trc promoter, rhtA (threonine and
  • the host cell is E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteur Pastaella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Azotobacter Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus) Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium cyanoba cterium) and cyclobacterium (Cyclobacterium) may be characterized in that it is selected from the group consisting of.
  • E. coli strains (NMH26-p15PP3533, pKKtrcSargF-trcSglnA, M. Noh et al., Cell Systems (2017), 5, 1-9), which increased the production of proline by introducing a conventional synthetic regulatory sRNA
  • seven candidate gene groups were derived (FIG. 8), and a combination of these to discover gene combinations that further improve proline production is possible to further increase proline production of 30% or more. It was confirmed (Fig. 8).
  • the present invention is directed to a region of the host cell having a proline biosynthetic pathway, which complementarily binds to the mRNA of the target gene of the first to q vectors of the simultaneous multiple target gene expression suppression system.
  • a vector prepared by inserting a sequence that complementarily binds to the mRNA of the serC, murE, aspC, metB, fadR, fur, or chpA genes has been introduced, and is a group consisting of serC, murE, aspC, metB, fadR, fur, and chpA. It relates to a recombinant microorganism having improved proline production capacity, characterized in that the expression of two or more genes selected from is suppressed.
  • the recombinant microorganism is characterized in that one or more genes selected from the group consisting of lacI, speE, speG, argI, puuP, puuA, putA, putP, proP, speC, potE, and speF are further deleted. can do.
  • the recombinant microorganism may be characterized in that the promoter of one or more genes selected from the group consisting of argECBH operon, speF-potE, and argD is substituted with trc promoter.
  • the recombinant microorganism may be characterized in that the PP3533 gene is introduced or amplified.
  • the recombinant microorganism may be characterized in that the expression of the argF and glnA gene is further suppressed, the expression of the gene is characterized in that it is made through a synthetic regulatory sRNA in the present invention
  • the host cells are Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella , Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter (Acinetobacter), Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, B
  • the present invention also relates to a method for producing proline comprising culturing the recombinant microorganism having improved proline production capacity through fed-batch fermentation in a medium containing a trace metal solution.
  • the existing constructed sRNA library is transferred to the sRNA expression vector according to the present invention quickly, accurately and efficiently using Gibson assembly, and then indigo or via viola screening for indigo and violacein producing strains.
  • the production capacity of indigo or violacein significantly increased (Figs. 16 to 18).
  • the present invention provides a host cell having an indigo biosynthetic pathway, which is complementarily bound to an mRNA of a target gene of the first to q vectors of the simultaneous multiple target gene expression suppression system.
  • a vector prepared by inserting a sequence complementarily binding to mRNA of the asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF or ostB genes was introduced, and asnA, hisJ, yneH, napG, kdsA, ygfA, It relates to a recombinant microorganism having improved indigo production capacity, characterized in that the expression of two or more genes selected from the group consisting of ftsl, aceF and ostB is suppressed.
  • the recombinant microorganism is further deleted one or more genes selected from the group consisting of trpR, pykF and pykA, the promoter of tktA is substituted with the trc promoter, tnaA, fmo, aroGfbr, trpEfbr, and aroL
  • genes selected from the group consisting of may be characterized in that the introduced or amplified.
  • the fbr means no feedback resistance (feedback resistance).
  • the host cell is E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteur Pastaella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Azotobacter Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus) Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium cyanoba cterium) and cyclobacterium (Cyclobacterium) may be characterized in that it is selected from the group consisting of.
  • the present invention provides a host cell having a violasine biosynthetic pathway, ytfR in a region complementarily binding to mRNA of a target gene of the first to q-vectors of the simultaneous multiple target gene expression suppression system.
  • a vector prepared by inserting a sequence complementarily binding to mRNA of hybG, ampG, potG, caiF, minD, ilvM, yfbR, rfe, atpH, tiaE or murI gene was introduced, and ytfR, hybG, ampG, potG , caiF, minD, ilvM, yfbR, rfe, atpH, tiaE and murI expression of two or more genes selected from the group is suppressed in the recombinant microorganism with improved violacein / deoxybiolacein production capacity It is about.
  • the recombinant microorganism may be characterized in that one or more genes selected from the group consisting of vioA, vioB, vioC, vioD and vioE are introduced or amplified.
  • the host cell is E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteur Pastaella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Azotobacter Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus) Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium cyanoba cterium) and cyclobacterium (Cyclobacterium) may be characterized in that it is selected from the group consisting of.
  • the present invention also relates to a violacein / deoxybiolacein production method comprising culturing the recombinant microorganism through fed-batch fermentation in a medium containing a trace metal solution.
  • [SEQ ID NO 1] is the CDF replication origin
  • [SEQ ID NO 2] is the pBBR1 replication origin
  • [SEQ ID NO 3] is the ColA replication origin
  • [SEQ ID NO 4] is the Am antibiotic marker
  • [SEQ ID NO 5] is the Sm antibiotic
  • the marker, [SEQ ID NO: 6] refers to Tc antibiotic marker DNA sequences.
  • Synthetic regulatory sRNA used in the present invention utilized a micC scaffold (SEQ ID NO: 8) and T1 / TE terminator (SEQ ID NO: 9) based on the PR promoter (SEQ ID NO: 7).
  • the nine synthetic regulatory sRNA platform plasmids were constructed via a Gibson assembly consisting of three DNA fragments consisting of sRNA fragments, antibiotic marker (antibiotic resistance gene) fragments and origin of replication fragments (DGGibson et al., Nature Methods). (2009), 6 (5), 343-345.
  • sRNA fragments were obtained by PCR using [SEQ ID NO: 10 / SEQ ID NO: 11] as a template using the pWAS plasmid in the existing patent (KR 10-1575587 B1).
  • the Am fragment is via [SEQ ID NO: 12 / SEQ ID NO: 13]
  • the Sm fragment is via [SEQ ID NO: 14 / SEQ ID NO: 15]
  • the Tc fragment is via [SEQ ID NO: 16 / SEQ ID NO: 17]. Obtained.
  • the CDF fragments of the replication origin fragments are through [SEQ ID NO: 18 / SEQ ID NO: 19]
  • the pBBR1 fragments are through [SEQ ID NO: 20 / SEQ ID NO: 21]
  • the ColA fragments are [SEQ ID NO: 22 / SEQ ID NO: 23]. Obtained.
  • reporter plasmids containing the DsRed2 gene were first constructed.
  • pACYC184-DsRed2 utilized the same plasmid as produced in the literature (M. Noh et al., Cell Systems (2017), 5, 1-9).
  • pTac15K-DsRed2 (p15A replication origin, kanamycin -Km-antibiotic marker), pTrc99A-DsRed2 (ColE1 replication origin, ampicilin -Ap-antibiotic marker) Construction of the reporter plasmid was performed using pTac15K and pTrc99A as a template.
  • Example 1-1 Using various synthetic sRNA platform plasmids and DsRed2 fluorescent protein expression reporter plasmids constructed in Example 1-1, the knockdown test for DsRed2 fluorescent protein of synthetic sRNA and the performance of each platform were performed as follows.
  • the base strain was Escherichia coli W3110.
  • various synthetic regulatory sRNA platform plasmids were used as templates, using an inverse PCR reaction using [SEQ ID NO 28 / SEQ ID NO 29].
  • the template was removed with DpnI restriction enzyme (Engineics, Korea), and the anti-completed reaction was completed by reaction with T4 polynucleotide kinase (Engineics, Korea) and T4 ligase (Elpis Biotech, Korea).
  • DsRed2-sRNA plasmids were constructed.
  • DNA sequence identification on all cloning was carried out through Cosmogenetech (South Korea). Using nine anti-DsRed2-sRNA plasmids and three DsRed2-reporter plasmids constructed as specified above for three reporter plasmids frequently used in metabolic engineering, each with three different antibiotic markers To determine the knockdown performance of various synthetic regulatory sRNAs.
  • OD600 2 fluorescent protein
  • emission wavelength 603 nm
  • cutoff value was set at 590 nm.
  • the nine anti-DsRed2-sRNAs were transformed into the E. coli W3110 strain together with the pTac15K-DsRed2 reporter plasmid and cultured, and the fluorescence intensity was measured as described above.
  • the nine anti-DsRed2-sRNAs were transformed into the E. coli W3110 strain along with the pTrc15K-DsRed2 reporter plasmid and cultured, and the fluorescence intensity was measured as described above.
  • a total of six platforms (CDF-Sm, CDF-Tc, pBBR1-Am, pBBR1-Sm, pBBR1-Tc, ColA-Sm) were selected as platform candidates that can be used in actual metabolic engineering.
  • Selected platforms showed effective target gene expression inhibition in all of the DsRed2 expressing strains in various environments (FIG. 2).
  • ColA-Tc and ColA-Am platforms were less stable when transformed with reporter plasmids during cloning, CDF-Am platforms exhibited growth inhibition problems in liquid media and poor knockdown performance for pTrc99A-DsRed2 reporters. Excluded from platform candidates.
  • strains were prepared: Tac-pBBR1Am, Tac-pBBR1Tc, Tac-pBBR1Sm, Tac-ColASm, Tac-CDFTc, Tac-CDFSm, Trc-pBBR1Am, Trc-pBBR1Tc, Trc-pBBR1Sm, Trc-ColASc , Trc-CDFSm, ACYC-pBBR1Am, ACYC-pBBR1Tc, ACYC-pBBR1Sm, ACYC-ColASm, ACYC-CDFTc, ACYC-CDFSm.
  • the prepared strains and the respective control strains (indicated by Ctrl; strains in which only the reporter plasmid was introduced without introducing the sRNA platform) were inoculated into test tubes containing 5 mL of LB medium, respectively, at 37 ° C for 16 hours. , 200 RPM, and all strains were tested by inoculating three species. Subsequently, all the strains were passaged for 24 hours on a microplate in which 200 ⁇ L of LB medium was introduced into each well (day 1). At this time, OD600 of all strains was simultaneously agitated using Bioscreen C. Measurement was made every hour. After 24 hours of incubation, all strains were further passaged for 24 hours on the same microplate (day 2). At this time, strain growth was also measured using Bioscreen C. Day 3 passages were also performed in the same way.
  • Example 1.4 each time the 1st day, 2nd day, and 3rd day culture was terminated, a certain amount of each cell was obtained and colony PCR was performed to test whether the sRNA platforms were stably present in the strain.
  • the different origins of replication used the following six primers to produce PCR products of different lengths.
  • [SEQ ID NO: 30], [SEQ ID NO: 31] are specific to the CDF replication origin, and generate a PCR product of 0.34 kb.
  • [SEQ ID NO: 32] and [SEQ ID NO: 33] are specific to the pBBR1 replication origin. kb PCR products were generated, and [SEQ ID NO: 34] and [SEQ ID NO: 35] were specific to ColA replication origin and designed to generate 0.65 kb PCR products.
  • the experiment was carried out as follows. First, the strain was incubated in a 25 mL test tube containing 5 mL LB medium at 37 degrees Celsius for at least 12 hours, and about 1000 cells were passaged in freshly prepared LB medium and cultured at 37 degrees Celsius for about 20 generations. It was. Appropriate concentrations of antibiotics were used as needed. Subsequently, the cultures were incubated by diluting them on LB agar plates containing no antibiotics, and 50 colonies of each plate were randomly selected and incubated on LB agar plates containing respective antibiotics (Tc, Sm, or Am). It was.
  • the plasmid retention rate was determined using the colony viability of each strain on LB agar plates containing each antibiotic. The result is shown in FIG. 19. As can be seen in Figure 19, most of the strains (25 of 28 strains) showed a plasmid retention of 90% or more in more than 80 generations. The other three strains (strains with the following plasmid combinations: pColA-SmR-pBBR1-AmR, pCDF-SmR-pBBR1-TcR, and pCDF-TcR-pBBR1-SmR) also showed plasmid retention of at least 80%. .
  • the platform vectors for constructing the library were selected as ColA-Sm and CDF-Tc, and a synthetic regulatory sRNA was constructed to inhibit the expression of a total of 61 major genes on each of these platforms.
  • Target genes of the synthetic regulatory sRNA were screened for genes involved in 1) major metabolic pathways (major pathways derived from that pathway and the TCA circuit), 2) metabolite transporters, 3) cell death, and 4) metabolic circuit regulation (Table 1).
  • the same method as in the anti-DsRed2 sRNA was constructed in Example 1.3. In other words, the target binding sequence 24mer was inserted between promoter and micC by inverse PCR, followed by ligation by DpnI restriction enzyme and T4 PNK and T4 ligase, and then the correct clone was selected through sequencing.
  • the target genes corresponding to the 61 types of sRNAs thus constructed and the corresponding target binding sequences are disclosed in Table 1 below.
  • sRNA library prepared in Example 2 Using the sRNA library prepared in Example 2, a production strain that was not easily engineered using the existing pWAS based sRNA library was developed. It is difficult to apply the existing sRNA system, as described in the background. 1) The antibiotic resistance marker or the replication origin of the plasmid contained in the production strain is overlapped, or 2) The multiple knockdown gene targets are easily applied in various combinations. It means when you want to. The researchers solved these limitations using the new sRNA platform.
  • the threonine (L-threonine) producing strain is a strain showing a very high threonine production capacity of 82.4 g / L through fed-batch fermentation despite the 100% rational metabolic improved strain. It was anticipated that the production capacity would be improved by applying the randomized-regulated sRNA of the random-rational method to strains that already produce high concentrations of threonine by the 100% rational method.
  • Threonine production has been successful with high g / L concentrations and high yields of 0.393 g / g (threonine / glucose) (KH Lee et a., Mol. Syst. Biol. (2007), 3 ( 149)).
  • Flask culture was performed by applying 55 synthetic regulatory sRNA libraries applicable to threonine overproduction among the CDF-Tc based sRNA libraries constructed in Example 2 to the threonine producing strain.
  • Glycerol cell stock was inoculated into a test tube containing 5 mL LB for threonine flask culture, and passaged into flask after 16 hours of incubation.
  • Flask incubation was performed at 31 ° C., 250 rpm, for 48 hours in a baffle flask containing 30 mL of 50 g / L glucose added TPM1 medium, TPM1 medium containing the following components per liter: 2 g yeast extract, 4 g KH 2 PO 4, 14 g (NH 4 ) 2 SO 4 , 30 g CaCO 3 , 2 g MgSO 4 , 1 g betaine, 5 mL trace metal solution, 2 mM L-methionine, 2 mM L-lysine.
  • a single gene expression inhibition experiment was able to select eight gene targets to increase the threonine 20% or more out of a total of 55 gene targets. These eight knockdown targets are: tktA, aroF, pta, ilvH, ilvE, glnA, fur, chpA.
  • tktA aroF
  • pta ilvH
  • ilvE glnA
  • fur chpA
  • strains corresponding to a total of 28 double gene knockdown combinations were produced very easily, and flask culture results of the thus constructed strains are disclosed in FIG. 6.
  • the simultaneous double gene expression inhibition experiment was able to observe an increase of more than 30% compared to the existing parent strain in three of the total 28 strains.
  • about 37% of the increased threonine was obtained in the strains simultaneously down-glnA-tktA, and a maximum of 22.94 g / L of threonine was obtained.
  • the three gene knockdown combinations mentioned are: ilvE-tktA, glnA-tktA, chpA-pta.
  • the synthetically regulated sRNA platform was used to develop proline transcripts.
  • E. coli strains (NMH26-p15PP3533, pKKtrcSargF-trcSglnA strains) capable of producing large amounts of proline (L-proline), which are recently used in the animal feed, food additives, medicine and compound markets, have been developed using synthetic sRNA.
  • KR 10-1750855 B1 M. Noh et al., Cell Systems (2017), 5, 1-9.
  • This strain has two plasmids, one is a pTac15K based vector for the production of proline, and the other is a synthetic sRNA platform vector with ColE1 origin of replication and Ap antibiotics. Therefore, the researchers introduced a newly constructed synthetic regulatory sRNA platform to the above strains, but tried to increase the production capacity even though the strain was already expanded by the synthetic regulatory sRNA.
  • Previously reported strains (NMH26-p15PP3533, pKKtrcSargF-trcSglnA strains) produced 12.7 g / L of proline at 195 g / L, fed-batch fermentation on flasks (KR 10-1750855 B1, M. Noh et al. , Cell Systems (2017), 5, 1-9).
  • the flask culture was carried out by applying 60 sRNA libraries applicable to proline overproduction among CDF-Tc based sRNA libraries already constructed.
  • glycerol cell stock was preferentially inoculated into a test tube containing 5 mL LB, and cultured for 16 hours before passage to the flask.
  • the flask culture was carried out for 24 hours at 37 ° C., 200 rpm in a baffle flask containing 50 mL of R / 2 medium (pH 6.8) added with 10 g / L glucose, 3 g / L (NH 4 ) 2 SO 4 .
  • R / 2 medium contains the following components per liter: 6.75 g KH 2 PO 4 , 2 g (NH 4 ) 2 HPO 4 , 0.8 g MgSO 4 .7H 2 O, 0.85 g citric acid, and 5 mL trace metal solution.
  • the gene knockdown targets corresponding to these strains were as follows: serC, murE, aspC, metB, fadR, fur, and chpA. .
  • the fur-down strain showed an increased proline production capacity of about 38.4% compared to the existing strain, confirming that it can produce up to 2.72 g / L on the flask (FIG. 7).
  • the fed-batch fermentation was performed on strains with greatly improved proline production ability.
  • the fur drop down strain which produced the highest transpiration by suppressing single gene expression, was used.
  • the fed-batch fermentation was carried out using the previously reported proline strain fermentation conditions (KR 10-1750855 B1).
  • NMH26 p15PP3533 pKKtrcSargF-trcSglnA strain contains the corresponding antibiotic (Km, Ap, Tc) containing the pCDFTc-fur plasmid (sRNA vector for fur knockdown) Inoculated into a test tube containing 5 mL LB was incubated for 16 hours at 37 ° C. Subsequently, passage in two baffle flasks containing 50 mL of R / 2 medium (pH 6.8) with 10 g / L glucose and 3 g / L (NH 4 ) 2 SO 4 until OD600 reached 2 Cultivation was performed. 100 mL of this strain was inoculated into a fermentor containing 1.9 L of R / 2 (pH 6.8) medium supplemented with 10 g / L glucose and 3 g / L (NH 4 ) 2 SO 4 .
  • the pH was fixed at 6.8 through 28% (v / v) ammonia solution and the dissolved oxygen concentration (DO) was set to 40% of air saturation, at which point the speed of the stirrer and air to oxygen entering the fermentor DO was constantly adjusted by the ratio of.
  • DO dissolved oxygen concentration
  • the feeding solution in fed-batch fermentation was entered according to the pH-stat strategy.
  • the pH of the fermenter system rose above 6.83, a certain amount of feeding solution was set to be automatically introduced.
  • the feeding solution for fermentation contains the following components per liter: 650 g glucose, 85 g (NH 4 ) 2 SO 4 , 8 g / L MgSO 4 ⁇ 7H 2 O.
  • the fed-batch fermentation was carried out on the double knockdown strains, and the result was NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur pColASm-metB strain (55.3 g / L) of FIG. 11-a, and NMH26 p15PP3533 pKKtrcSargF- of FIG. 11-b.
  • the most proline-producing strain at this time was NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur pColASm-metB strain (strain with double knockdown of fur and metB), high production concentration of 55.3 g / L, 1.053 g / L / h High production capacity of, and yield of 0.200 g proline / g glucose. In terms of production concentration, this is a 63.6% increase in production compared to the previously reported proline production (33.8 g / L) (Noh et al., Cell Systems, 2017, KR 10-1750855 B1). The results for fed-batch fermentations for all mentioned strains are summarized in Table 2 below.
  • colony PCR was performed to confirm that sRNA remained stable in cells in the exponential and stationary phases during the fed-batch fermentation of the proline-producing strains. As shown in FIG. 12, all sRNA vectors were stably maintained. It was confirmed to be maintained.
  • FIG. 13 a method for easily transferring the genome-level sRNA expression platform to another platform was developed (FIG. 13).
  • a synthetic sRNA was transferred from a platform plasmid (pWAS) having a ColE1 origin of replication and an Ap antibiotic marker (pWAS) to a newly prepared ColA-Sm platform plasmid.
  • pWAS platform plasmid
  • pWAS Ap antibiotic marker
  • This strategy uses the Gibson assembly and PCR amplifies the ColA-Sm platform plasmid using the [SEQ ID NO: 36] and [SEQ ID NO: 37] primers.
  • all other DNA sequences except for the mRNA target binding sequence 24 bp are identical among the different sRNA vectors, so that the pWAS sRNA library is used as a template [SEQ ID NO 38] / [SEQ ID NO 39]
  • the primers were used to PCR amplify sRNA fragments containing 1,858 different mRNA target binding sequences. Thereafter, the sRNA library fragments and the ColA-Sm fragments were combined into a circular vector through a gibson assembly, and then transformed into the E. coli DH5 ⁇ strain.
  • the sRNA library constructed in Example 4 was introduced into indigo producing strains to select indigo producing strains with increased production capacity.
  • Indigo is a natural dye produced by Indigofera plant and is an aromatic amino acid compound mainly used for dyeing jeans. Originally produced through natural extraction, almost all demand is now met by chemical synthesis.
  • Previously reported indigo producing strains (IND5-pTrc-TF1, pTac-GEL strains) produced indigo at a concentration of 0.640 g / L in fed-batch fermentation and 0.108 g / L at the flask level (J. Du et. al., J Biotechnol., 2018, 267, 19-28).
  • Previously reported indigo-producing strains (IND5-pTrc-TF1, pTac-GEL strains) are not immediately applicable because they have the same vector as the origin of replication and the pWAS and antibiotic resistance markers.
  • the colony was blue color has the advantage that you can easily see the increase and decrease of production capacity. Therefore, a newly constructed sRNA library was introduced into an indigo producing strain to select an improved strain having improved indigo productivity through colony screening.
  • the (ColA-Sm) sRNA library of the platform constructed in Example 4 was transformed into an indigo producing strain, and about 20,000 or more colonies were obtained. Of these, 84 colonies of darker color were selected, and the selected strains were pre-cultivated for 16 hours at 37 ° C 200 rpm in 3 mL LB medium, followed by 3 mL of 10 g / L glucose.
  • MR medium was incubated at 250 RPM for 48 hours at 30 ° C through small scale cultivation. When the OD600 of the cells reached about 0.6-0.8, foreign gene expression was induced with 1 mM IPTG. At this time, MR medium was used as the MR medium of the composition as reported in the previous paper (J.
  • MR medium was Contains: 6.67 g of KH 2 PO 4, 4 g of (NH 4 ) 2 HPO 4 , 0.8 g of MgSO 4 7H 2 O, 0.8 g of citric acid, 5 mL of trace metal solution.
  • the culture results are as shown in Figure 14, through this initial screening was able to select up to 246% increased strain (81.9 mg / L compared to the existing strain production of 33.3 mg / L). At this time, the top 9 strains with the highest indigo production capacity were selected and incubated for 72 hours at 30 ° C and 200 rpm in 50 mL MR medium to which 10 g / L glucose was added. At about 0.6-0.8, foreign gene expression was induced with 1 mM IPTG.
  • Example 6 Violacein transpiration using platform transferred sRNA library and high speed screening
  • the E. coli genome level sRNA library transferred to the ColA-Sm platform prepared in Example 4 was applied to another secondary metabolite, violacein.
  • Violacein (violacein) and its isomer deoxyviolacein (deoxyviolacein) has many medical effects, such as anti-cancer effects, but the research has been lacking in power due to too low production. Therefore, we tried to solve the above problem through the excessive production of violacein and deoxybiolacein.
  • iGEM fragment containing vioABDE was used (Part BBa_K598019; MIT Registry of Standard Biological Parts).
  • the plasmid is based on pSB1C3 (chloramphenicol resistant antibiotic marker, pUC-based replication origin). Therefore, the following method was used to clone the remaining necessary vioC gene into pTac15K vector.
  • genomic DNA of Janthonobacterium lividum was extracted, and then amplified vioC DNA fragment using [SEQ ID NO 40] and [SEQ ID NO 41] as a primer. Then, vioC fragments were inserted through Gibson assembly through EcoRI and KpnI sites.
  • E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain was used as the base strain, and the strain was transformed into the ColA-Sm-based E. coli genome level sRNA library prepared in Example 4, followed by more than 20,000 colonies. Got. Afterwards, the strain selection process with improved violacein production was carried out in the same manner as in Example 5. More specifically, 32 of the darker purple colonies could be selected, and the selected strains were pre-cultivated for 16 hours at 37 ° C 200 rpm in 3 mL LB medium, followed by 3 mL of 10 g / L. MR medium supplemented with glucose was incubated at 250 RPM for 48 hours at 30 ° C through small scale cultivation.
  • the culture results are as shown in Figure 16, through this initial screening was able to select strains maximal 545% of viola sane, up to 784% deoxybiolasein. At this time, the top 12 strains with the highest violacein / deoxybiolacein production capacity were selected and incubated for 48 hours at 30 ° C and 200 rpm in 50 mL MR medium to which 10 g / L glucose was added. When the OD600 of the cells was about 0.6-0.8, foreign gene expression was induced with 1 mM IPTG.
  • the strain with the highest increase in violacein production was the ytfR knockdown strain, which showed increased violacein production capacity up to 0.656 g / L (based strain production of 0.116 g / L).
  • the strain showing the highest deoxybiolacein production capacity was a minD knockdown strain and showed a deoxybiolacein production capacity increased to 52.1 mg / L (based strain 21.7 mg / L production).
  • the fed-batch fermentation was carried out on strains with greatly improved violasane production ability.
  • the ytfR knockdown strain having the best violacein production capacity and the minD knockdown strain having the best dioxybiolacein production ability were each subjected to fed-batch fermentation.
  • PColA-ytfR plasmid sRNA vector for ytfR knockdown
  • coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain (sRNA vector for minD knockdown) was inoculated into a test tube containing 5 mL LB containing the corresponding antibiotics (Cm, Km, Spc) and incubated for 16 hours at 30 ° C. Subsequently, passages were performed in two baffle flasks containing 50 mL of MR medium (pH 7.0) added with 20 g / L glucose and 3 g / L (NH 4 ) 2 SO 4 until the OD600 reached 4. Was performed. 100 mL of this strain was inoculated into a fermentor containing 1.9 L of MR (pH 7.0) medium containing 20 g / L glucose and 3 g / L (NH 4 ) 2 SO 4 .
  • the pH was fixed at 7.0 through 28% (v / v) ammonia solution and the DO (dissolved oxygen concentration) was set to 40% of air saturation, at which time the speed of the stirrer and the air to oxygen entering the fermentor DO was constantly adjusted by the ratio of.
  • the feeding solution in fed-batch fermentation was entered according to the pH-stat strategy. When the pH of the fermenter system rose above 7.05, a certain amount of feeding solution was set to be automatically introduced. At this time, the feeding solution for fermentation contains the following components per liter: 650 g glucose, 85 g (NH 4 ) 2 SO 4 , 8 g / L MgSO 4 ⁇ 7H 2 O, 6 mL trace metal solution.
  • colony PCR was performed to confirm that sRNA remained stable in cells in the exponential phase and stationary phase during the fed-batch fermentation of the violacein-producing strains. As shown in FIG. 18-C, all sRNA vectors were disclosed. It was confirmed that they remained stable.
  • Simultaneous multiple target gene expression suppression system of the present invention unlike the existing gene deletion method is a synthetic regulatory sRNA-based gene expression control system that can effectively suppress the expression of a specific gene without modifying the sequence of the target gene, a variety of vectors It can be used to be compatible with other plasmids available in prokaryotes, including, and designed to enable simultaneous multi-introduction is useful for fast and efficient recombinant microbial production.
  • Recombinant microorganisms for the production of high efficiency proline or threonine developed through microbial metabolic flow regulation according to the present invention is useful as animal feed and industrial microorganisms.
  • E. coli genome-level synthetic regulatory sRNA library transfer technology with the high-speed screening technique, a dramatic increase in productivity was impossible.
  • Indigo or violacein highly efficient production strains according to the invention are also very useful as pharmaceutical and industrial microorganisms. Therefore, the present invention is useful because it can be used for the production of recombinant strains for the efficient production of a variety of industrial and medically useful metabolites and to establish an efficient production method.

Abstract

La présente invention concerne un système d'inhibition d'expression multiplex basé sur un ARNs régulateur de synthèse et un procédé pour sa production et, plus particulièrement, une combinaison de vecteurs comprenant divers gènes de résistance aux antibiotiques, une origine de réplication et un ARNs régulateur de synthèse ; un procédé pour sa production ; une bibliothèque d'ARNs constituée par ladite combinaison de vecteurs ; et un procédé de criblage d'une souche bactérienne produisant une substance utile l'utilisant. Le système d'inhibition d'expression génique cible multiplex selon la présente invention est un système de régulation d'expression génique basé sur un ARNs régulateur de synthèse qui, contrairement aux procédés d'inactivation de gènes existants, peut inhiber efficacement l'expression d'un gène spécifique sans modifier la séquence d'un gène cible ; qui comprend divers vecteurs et qui peut donc être utilisé de manière compatible avec d'autres plasmides utilisables dans les procaryotes ; et qui est conçu pour permettre une introduction multiplex et est ainsi avantageux dans la production rapide et efficace de micro-organismes recombinants. En outre, la présente invention concerne une technologie qui permet le transfert simple et rapide d'une bibliothèque d'ARNs régulateur de synthèse au niveau du génome à une nouvelle plateforme et ladite technologie est utile dans la sélection de micro-organismes recombinants produisant des substances utiles à grande capacité. Par conséquent, la présente invention est utile dans la production de souches bactériennes recombinantes capables d'augmenter considérablement l'efficacité de production de divers métabolites médicalement utiles.
PCT/KR2019/007729 2018-06-27 2019-06-26 Système d'inhibition d'expression génique cible multiplex basé sur un arns régulateur de synthèse et procédé pour sa production WO2020004936A1 (fr)

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