WO2020004936A1

WO2020004936A1 - Multiplex target gene expression inhibition system based on synthesis regulator srna and method of producing same

Info

Publication number: WO2020004936A1
Application number: PCT/KR2019/007729
Authority: WO
Inventors: 이상엽; 유승민; 양동수
Original assignee: 한국과학기술원
Priority date: 2018-06-27
Filing date: 2019-06-26
Publication date: 2020-01-02
Also published as: KR102037958B1

Abstract

The present invention relates to a multiplex expression inhibition system based on a synthesis regulator sRNA and a method of producing same, and more specifically, to a combination of vectors comprising a variety of antibiotic-resistance genes, an origins of replication, and a synthesis regulator sRNA; a method of producing same; an sRNA library consisting of said combination of vectors; and a method of screening for a bacterial strain producing an useful substance by using same. The multiplex target gene expression inhibition system according to the present invention is a gene expression regulation system based on a synthesis regulator sRNA that, unlike existing gene knock-out methods, can effectively inhibit the expression of a specific gene without altering the sequence of a target gene; comprises various vectors and thus can be used compatibly with other plasmids usable in prokaryotes; and is designed to enable multiplex introduction, and thus is advantageous in a fast and efficient production of recombinant microorganisms. Furthermore, the present invention provides a technology that enables simple and fast transferring a genome-level synthesis regulation sRNA library to a new platform, and said technology is useful in selection of recombinant microorganisms producing useful substances in high capacity. Accordingly, the present invention is useful in the production of recombinant bacterial strains capable of dramatically increasing the production efficiency of a variety of medically useful metabolites.

Description

Synthesis-regulated SRNA-based simultaneous multiple target gene expression suppression system and its preparation

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.

Due to global environmental problems, limited resource depletion and demand for environmentally friendly energy sources, there is a growing interest in building cell plants based on reproducible organisms. These cell factories can manufacture a metabolic network within cells to optimize their production of metabolites (bio-energy, green chemicals, new drug formulations, etc.), which require a variety of molecular biology techniques.

In order for the organism to produce the desired metabolites with high efficiency, optimization of metabolic flow is essential, which is possible through optimization of expression of necessary enzymes. If the reaction is to occur quickly and in large quantities, the amount of enzyme must be large, and the less reactive enzyme must be expressed in large amounts to improve the reaction rate, which is essential for cell survival, but causes metabolic reactions that inhibit the production of the desired product. The expression of enzymes needs to be optimized at low levels.

Recently, synthetic regulatory sRNA systems have been developed and applied to various kinds of microorganisms, so that the genes in the metabolic circuits can be easily knocked down, enabling efficient gene expression control (KR 10-1575587 B1, US 9388417, EP 13735942.8, CN 201380012767.X, KR 10-1690780 B1, KR 10-1750855 B1, US 15317939, CN 201480081132.X, D. Na et al., Nat.Biotechnol. (2013), 31 (2), 170-174 , SM Yoo et al., Nat. Protoc. (2013), 8 (9), 1694-707, C. Cho et al., Biotechnol. Bioeng. (2017), 11 (2), 374-383, M. Noh et al., Cell Systems (2017), 5, 1-9).

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). In addition, it was possible to downgrade survival essential genes, which were impossible or difficult in the existing gene deletion engineering, and thus, it was possible to find a knockdown target gene that can unexpectedly increase the productivity of the target compound through systemic application. .

Various expression platforms are necessary to utilize the synthetic regulatory sRNA system having these advantages in the development of more various strains. If the origin or antibiotic marker in the synthetic regulatory sRNA expression platform is the same as the origin or antibiotic marker of the plasmid already present in the metabolically improved strain, it is difficult to apply the sRNA library system directly. Therefore, under the existing synthetic regulatory sRNA platform, it was difficult to immediately apply to various strains, and new cloning had to be carried out depending on the situation. In addition, in order to confirm the simultaneous knockdown effect of several genes in one cell, it is preferable to apply a corresponding synthetic regulatory sRNA library at once, but since both the origin of replication and the antibiotic marker in this library are the same, Additional work is needed to clone several synthetic regulatory sRNAs. This also required a new cloning operation depending on the situation, there was a difficult time consuming in some cases.

These limitations include: 1) developing a variety of synthetic regulatory sRNA expression platforms that are compatible and compatible with most commonly used plasmids; and 2) transferring E. coli genome-level synthetic regulatory sRNA libraries to various synthetic regulatory sRNA expression platforms easily and conveniently. If the technology is developed, it will be overcome. If these technologies are developed, it will be possible to control gene expression in a wider variety of environments. Furthermore, it will be possible to develop microbial cell plants that can produce useful chemicals more easily and conveniently in a short time. Will be able to maximize. In the present invention, various replication origin and antibiotic markers were selected and combined to construct synthetic regulatory sRNA expression plasmids that are compatible for use with existing vectors.

Accordingly, the present inventors have made efforts to develop a variety of synthetic regulatory sRNA expression platforms. As a result, 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 When prepared by the present invention, not only efficient expression inhibition of multiple target genes is possible, but also does not affect the growth of the target microorganism, and completed the present invention by confirming that the stability is high in the target microorganism.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

발명의 요약Summary of the Invention

Disclosure of the Invention 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.

In order to achieve the above object, 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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin in, salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine, ,

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:

(A) preparing any base sequence having a size of 20 ~ 50bp;

(b) the nucleotide sequence of the first antibiotic resistance gene; First replication origin; Preparing a first vector library comprising a first sRNA library by inserting into a region complementarily binding to a target gene mRNA of a first vector comprising a synthetic sRNA coding region that inhibits expression of the first target gene;

(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;

(d) the 2 to 500 expression inhibitory genes determined as the n antibiotic resistance genes; Mth replication origin; Inserting each of the q-vectors including the synthetic sRNA coding region that inhibits the expression of the 2 to 500 genes to inhibit expression; And

(e) preparing a recombinant strain into which q vectors containing the expression inhibitory gene are introduced,

here,

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,

The antibiotics are ampicillin, kanamycin, kanampycin, chloramphenicol, apramycin, streptomycin, spectiomycin, tetracycline, tetracycline and erythromycin. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin n), salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine; ,

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,

A sequence prepared by inserting a sequence complementarily binding to mRNA of a tktA, aroF, pta, ilvH, ilvE, glnA, fur, or chpA gene into a region complementarily binding to mRNAs of the target genes of the first to q vectors Since the vector is introduced, expression of two or more genes selected from the group consisting of tktA, aroF, pta, ilvH, ilvE, glnA fur and chpA is suppressed, thereby providing a recombinant microorganism characterized by improved threonine production capacity. .

The present invention also provides a host cell having a proline biosynthetic pathway,

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,

Inserting a sequence complementary to the mRNA of the asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF or ostB gene in the region complementary to the mRNA of the target gene of the first to q vector Since the prepared vector is introduced, expression of two or more genes selected from the group consisting of asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF, and ostB is suppressed, thereby improving indigo production capacity. Provide a recombinant microorganism.

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;

In recombinant microorganisms in which a simultaneous multiple target gene expression suppression system is introduced into a host cell,

The host cell has a violacein (violacein) biosynthesis pathway,

Complementary to the mRNA of the target gene of the first to q vector complementary to the mRNA of ytfR, hybG, ampG, potG, caiF, minD, ilvM, yfbR, rfe, atpH, tiaE or murI gene 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 Provided is 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:

(A) preparing any base sequence having a size of 20 ~ 50bp;

(b) the nucleotide sequence of the first antibiotic resistance gene; First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; To n-n antibiotic resistance genes; Mth replication origin; A q vector comprising a synthetic sRNA coding region that inhibits p target gene expression; a first to r vector library comprising first to r sRNA libraries inserted into a region complementarily binding to a target gene mRNA Preparing a; And

(c) introducing the first to r vector library into the target strain to produce a useful material, and when the production of the useful material is improved, to identify a group of gene candidates with suppressed expression, s expression suppression gene combinations are identified Determining;

Here, 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.

1 shows each replication origin and antibiotic markers used in combination with various synthetic regulatory sRNA expression platforms.

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. One graph.

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.

4 is a result confirming the stability (stability) of the synthetic regulatory sRNA fragment platform plasmid. 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. Where 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. In addition, 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.

9 are graphs obtained through the fed-batch fermentation conditions optimization process of proline production strains. Using the NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur strain (a) under fermentation conditions in the existing patents and articles (10 g / L glucose, 3 g / L (NH ₄ ) ₂ SO ₄ addition 2 LR / 2 medium) , (b) added 1.6 g / L yeast extract to the existing medium, (c) added 3 g / L yeast extract to the existing medium and added 6 mL trace metal solution to the existing feeding solution. Expression fermentation was carried out. (d) shows the results when the strain NMH26 p15PP3533 pKKtrcSargF-trcSglnA was cultured under the fermentation conditions of (c) as a control experiment. In each graph, 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. In each graph, 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 , (d) NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-chpA pColASm-fadR strain. In each graph, 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. In this case, strains 1 to 9 are the same as the strain numbers in Table 2. At this time, the upper DNA band represented by the red triangle indicates the presence of ColA-Sm platform based sRNA, and 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 (b) the pColA-minD plasmid is added to the E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain. Inserted strain. In each of the graphs, the red circle represents cell growth, the blue triangle represents the violacein concentration, and 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. At this time, strain 1: control strain, strain 2: strain of (a), strain 3: strain of (b). At this time, 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. These are graphs measuring the growth curve by transforming the corresponding sRNA plasmids of (A)-(F) single, (G)-(M) double, and (N) triple combinations. At this time, each sRNA does not contain the target attachment sequence. Each experiment was done twice to show the average.

발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, “sRNA (small RNA)” is short RNA, usually no more than 200 nucleotides in length, that is not translated into protein and effectively inhibits translation of a particular mRNA through complementary binding.

As used herein, the term "ribosome binding site" refers to the site where the ribosomes bind on the mRNA for transcription of the mRNA.

As used herein, "gene" should be considered in the broadest sense and may encode a structural or regulatory protein. At this time, the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA / RNA replication, transcription and / or translation. In the present invention, the target gene to be suppressed expression may exist as an extrachromosomal component.

In the present invention, it 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.

That is, in one embodiment of the present invention, three antibiotic markers (apramycin, streptomycin (or specidomycin) and tetracycline) that can coexist with each other and three origins of replication (CDF, pBBR1 and ColA) that can coexist with each other Each of the 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. 1). When cultured by introducing the reporter plasmid into Escherichia coli, the growth and expression of Escherichia coli may be achieved even if three kinds of synthetic regulatory sRNA expression vectors are introduced. It was confirmed that there is no problem in the stability of the emitter (2 to 4).

Accordingly, 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.

In the present invention, p refers to the number of target genes, and 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.

In the present invention, 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 (vancomycin), vinblastine (vinblastine), and vincristine (vincristine) may be characterized in that it is selected from the group consisting of, but is not limited thereto.

In the present invention, 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.

In the present invention, 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 (Cyclobacterium) may be characterized in that it is selected from the group consisting of, but is not limited thereto.

In the present invention, 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.

In the present invention, 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, λPR and Anderson synthetic promoter And, most preferably, it may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 7.

In the present invention, 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.

In the present invention, all kinds of 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.

In the present invention, 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.

In the present invention, 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.

As used herein, “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.

As used herein, "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. As the vector used for the expression of the sRNA according to the present invention, an expression vector known in the art may be used.

In 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). For example, 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; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "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. However, 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.

In the present invention, 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 kinase / acetate kinase activity), adiY (AdiY is a positive DNA-binding transcriptional regulator that controls the arginine) decarboxylase (adi) system, argB (acetylglutamate kinase), argC (N-acetylglutamylphosphate reductase), argG (argininosuccinate synthase), argH (argininosuccinate lyase), asnC (transcriptiona) regulator that activates the expression of asnA, a gene involved in the synthesis of asparagine), aspA (aspartate ammonia-lyase), crp (CRP transcriptional dual regulator), csiD (predicted protein. 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 (FadR Fatty acid degradation Regulon, is a multifunctional dual regulator that exerts negative control over the fatty acid degradative regulon [Simons80, Simons80a] and acetate metabolism), fbp (fructose-1,6-bisphosphatase), fnr (FNR is the primary transcriptional regulator that me diates the transition from aerobic to anaerobic growth), fruR (FruR is a dual transcriptional regulator that plays a pleiotropic role to modulate the direction of carbon flow through the different metabolic pathways of energy metabolism, but independently of the CRP regulator), ftsL (essential cell division protein FtsL), ftsQ (essential cell division protein FtsQ), ftsW (essential cell division protein FtsW), ftsZ (essential cell division protein FtsZ), fur (Fur-Fe + 2 DNA-binding transcriptional dual regulator), gabD ( succinate semialdehyde dehydrogenase, NADP + -dependent), gabP (APC transporter), gabT (4-aminobutyrate aminotransferase), gadA (glutamate decarboxylase A subunit), gadB (glutamate decarboxylase B subunit), gadC (GABA APC transporter), glcC (GntR family transcriptional regulator, glc operon transcriptional activator, glpK (glycerol kinase), glpR (sn-Glycerol-3-phosphate repressor), glpX (fructose 1,6-bisphosphatase II), gltA (citrate synthase), hflD (lysogenization regula tor), ihfA (IHF, Integration host factor, is a global regulatory protein), ihfB (IHF, Integration host factor, is a global regulatory protein), ilvB (acetohydroxybutanoate synthase / acetolactate synthase), ilvC (acetohydroxy acid isomeroreductase), ilvD (dihydroxy acid dehydratase), ilvG_1 (acetolactate synthase II, large subunit, N-ter fragment (pseudogene)), ilvG_2 (acetolactate synthase II, large subunit, C-ter fragment (pseudogene)), ilvH (acetolactate synthase / acetohydroxybutanoate synthase) , ilvL (ilvGEDA operon leader peptide), ilvM (acetohydroxybutanoate synthase / acetolactate synthase), ilvN (acetohydroxybutanoate synthase / acetolactate synthase), ilvX (Predicted small protein), lexA (LexA represses the transcription of several genes involved in the cellular response to DNA damage), lpxC (UDP-3-O-acyl-N-acetylglucosamine deacetylase), marA (MarA participates in controlling several genes involved in resistance to antibiotics, oxidative stress, organic solvents and heavy metals.) , metJ (MetJ transcriptional repressor), modE (ModE is the principal regulator that controls the transcription of operons involved in the transport of molybdenum and synthesis of molybdoenzymes and molybdate-related functions), nadB (L-aspartate oxidase), narL (nitrate / nitrite response regulator), pck (phosphoenolpyruvate carboxykinase), PdhR (PdhR, "pyruvate dehydrogenase complex regulator," regulates genes involved in the pyruvate dehydrogenase complex), phoP (PhoP-Phosphorylated DNA-binding transcriptional dual regulator. 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), rbsK (ribokinase), rbsR (The transcription factor RbsR, for "Ribose Repressor," is negatively autoregulated and controls the transcription of the operon involved in ribose catabolism and transport), rcsB (RcsB -BglJ DNAbinding transcriptional activator.RcsB protein for "Regulator capsule synthesis B," is a response regulator that belongs to the multicomponent RcsF / RcsC / RcsD / RcsA-RcsB phosph orelay system and is involved in the regulation of the synthesis of colanic acid capsule, cell division, periplasmic proteins, motility, and a small RNA), rutR (RutR regulates genes directly or indirectly involved in the complex pathway of pyrimidine metabolism), serA ( alpha-ketoglutarate reductase / D-3-phosphoglycerate dehydrogenase), serC (phosphohydroxythreonine aminotransferase / 3-phosphoserine aminotransferase), soxS (dual transcriptional activator and participates in the removal of superoxide and nitric oxide), sroD (SroD small RNA), zwf ( glucose 6-phosphate-1-dehydrogenase), asnA (asparagine synthetase A), asnB (asparagine synthetase B), carA (carbamoyl phosphate synthetase), carB (carbamoyl phosphate synthetase), ddlB (D-alanine-D-alanine ligase B) , deoA (thymidine phosphorylase / uracil phosphorylase), deoD (purine nucleoside phosphorylase deoD-type), dpiA (dual transcriptional regulator involved in anaerobic citrate catabolism), fis (Fis, "factor for inversion stimulation" , is a small DNA-binding and bending protein whose main role appears to be the organization and maintenance of nucleoid structure), gadE (GadE controls the transcription of genes involved in glutamate dependent system), gadW (GadW controls the transcription of genes involved in glutamate dependent system), gadX (GadX controls the transcription of genes involved in glutamate dependent system), glpF (GlpF glycerol MIP channel), ilvY (IlvY DNA-binding transcriptional dual regulator), ivbL (The ilvB operon leader peptide (IvbL)) , lhgO (L-2-hydroxyglutarate oxidase), lpd (Lipoamide dehydrogenase), lrp (Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These genes are involved in amino acid biosynthesis and catabolism, nutrient) transport, pili synthesis, and other cellular functions, including 1-carbon metabolism), metB (O-succinylhomoserine lyase / Osuccinylhomoserine (thiol) -lyase), metL (aspartate kinase / homoserine dehydrogenase), mraY (phospho-Nacetylmuramoyl-pentapeptide transferase), mraZ (Unknown function), murE (UDP-N-acetylmuramoylalanyl-Dglutamate 2,6-diaminopimelate ligase), murF (D-alanyl-D-alanine-adding enzyme) , murG (Nacetylglucosaminyl transferase), nac (Nacregulates, without a coeffector, genes involved in nitrogen metabolism under nitrogen-limiting conditions), nadA (quinolinate synthase), nsrR (NsrR, the "nitritesensitive repressor" regulates genes involved in cell protection against nitric oxide (NO)), panC (pantothenate synthetase), panD (Aspartate 1-decarboxylase), pgl (6-phosphogluconolactonase), pyrB (aspartate carbamoyltransferase, PyrB subunit), pyrC (dihydroorotase), pyrL (aspartate carbamoyltransferase, PyrI subunit), rob (Rob is a transcriptional dual regulator. Its N-terminal domain shares 49% identity with MarA and SoxS. These proteins activate a common set of about 50 target genes, the marA / soxS / rob regulon, involved in antibiotic resistance, superoxide resistance, and tolerance to organic solvents and heavy metals.), Rpe (ribulose phosphate 3-epimerase), talA ( transaldolase A), thrA (aspartate kinase / homoserine dehydrogenase), thrB (homoserine kinase), thrC (threonine synthase), thrL (thr operon leader peptide), tthrA (transketolase I), tktB (transketolase II), torR (two-component system, OmpR family, torCAD operon response regulator TorR), 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), trpD (fused glutamine amidotransferase (component II) of anthranilate synthase / anthranilate phosphoribosyl transferase), dapA (4-hydroxy-tetrahydrodipicolinate synthase), ilvE (Branched-chain-amino -acid aminotransferase), lysA (Diaminopi melate decarboxylase), gltA (cistrate synthase), fumA (Fumarate hydratase class I, aerobic), glnA (glutamine synthetase), mdh (malate dehydrogenase), gdhA (NADP-specific glutamate dehydrogenase), argA (amino acid N-acetyltransferase and inactive acetylglutamate kinase, metA (Homoserine O-succinyltransferase), tdh (L-threonine dehydrogenase), livJ (branched-chain amino acid ABC transporter periplasmic binding protein), rhtC (Threonine efflux protein), yohJ (UPF0299 membrane protein), chpA ( antitoxin of the ChpA-ChpR toxin-antitoxin system), aspC (aspartate aminotransferase), pfkA (ATP-dependent 6-phosphofructokinase isozyme 1), parE (DNA topoisomerase 4 subunit B), ytfR (predicted sugar transporter subunit), ilvA (L -threonine dehydratase, biosynthetic; also known as threonine deaminase, ilvG (Acetolactate synthase), minD (inhibitor of FtsZ ring polymerization; chromosome-membrane tethering protein; membrane ATPase of the MinCDEE system), hisJ (histidine transport system substrate-binding protein), yneH (Glutaminase) , ferpedoxin-type protein NapG (napG), 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA), ygfA (5-formyltetrahydrofolate cyclo-ligase), cell division protein FtsI (penicillin-binding protein 3) ftsI, trehalose 6-phosphate phosphatase, hybG (hydrogenase expression / formation protein HypC), ampG (MFS transporter, PAT family, beta-lactamase induction signal transducer AmpG), potG (spermidine / putrescine transport system permease protein), caiF (transcriptional activator CaiF) , yfbR (5'-deoxynucleotidase YfbR), rfe (UDP-GlcNAc: undecaprenyl-phosphate / decaprenyl-phosphate GlcNAc-1-phosphate transferase), atpH (F-type H + -transporting ATPase subunit delta), tiaE (glyoxylate / hydroxypyruvate 2-ketogluconate reductase) and murI (glutamate rac) emase) can be selected from the group consisting of

More preferably 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 (UDP-N-acetylmuramoylalanyl-Dglutamate 2,6-diaminopimelate ligase), pps (phosphoenolpyruvate synthetase), aceE (subunit of E1p component of pyruvate dehydrogenasetransfer) pta, pta purA (adenylosuccinate synthetase), ackA (propionate kinase / acetate kinase activity), pck (phosphoenolpyruvate carboxykinase), ppc (phosphoenolpyruvate carboxylase), accA (acetyl-CoA carboxyltransferase, alpha-subunit), fadD (fatthetylase-CoA synfabase) (KASIII, -ketoacyl-ACP synthases), aspA (aspartate ammonia-lyase), carB (carbamoyl phosphate synthetase), ilvH (acetolactate synthase / acetohydroxybutanoate synthase), ilvM (acetohydroxybutanoate synthase / acetolactate synthase), ilvM te synthase / acetolactate synthase), ilvC (acetohydroxy acid isomeroreductase), asnA (asparagine synthetase A), asnB (asparagine synthetase B), argH (argininosuccinate lyase), deoA (thymidine phosphorylase / uracil phosphorylase (partase deserine) ), metB (O-succinylhomoserine lyase / Osuccinylhomoserine (thiol) -lyase), metA, tdh, ilvL (ilvGEDA operon leader peptide), crp (CRP transcriptional dual regulator), fadR (FadR Fatty acid degradation Regulon, is a multifunctional dual regulator that exerts negative control over the fatty acid degradative regulon [Simons80, Simons80a] and acetate metabolism), fur (Fur-Fe + 2 DNA-binding transcriptional dual regulator), lrp (Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These 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), trpD (fused glutamine amidotransferase (component II ) of anthranilate synthase / anthranilate phosphoribosyl transferase), dapA (4-hydroxy-tetrahydrodipicolinate synthase), ilvE (Branched-chain-amino-acid aminotransferase), lysA (Diaminopimelate decarboxylase), gltA (cistrate synthase), fumA (F umarate hydratase class I, aerobic), glnA (glutamine synthetase), mdh (malate dehydrogenase), gdhA (NADP-specific glutamate dehydrogenase), argA (amino acid N-acetyltransferase and inactive acetylglutamate kinase), metA (Homoserine O-succinyltransase) tdh (L-threonine dehydrogenase), livJ (branched-chain amino acid ABC transporter periplasmic binding protein), rhtC (Threonine efflux protein), yohJ (UPF0299 membrane protein), chpA (antitoxin of the ChpA-ChpR toxin-antitoxin system), aspate (aspartate aminotransferase), pfkA (ATP-dependent 6-phosphofructokinase isozyme 1), parE (DNA topoisomerase 4 subunit B), ytfR (predicted sugar transporter subunit), ilvA (L-threonine dehydratase, biosynthetic; also known as threonine deaminase, ilvG (Acetolactate synthase), minD (inhibitor of FtsZ ring polymerization; chromosome-membrane tethering protein; membrane ATPase of the MinCDEE system), hisJ (histidine transport system substrate-binding protein), yneH (Glutaminase) , ferpedoxin-type protein NapG (napG), 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA), ygfA (5-formyltetrahydrofolate cyclo-ligase), cell division protein FtsI (penicillin-binding protein 3) ftsI, trehalose 6-phosphate phosphatase, hybG (hydrogenase expression / formation protein HypC), ampG (MFS transporter, PAT family, beta-lactamase induction signal transducer AmpG), potG (spermidine / putrescine transport system permease protein), caiF (transcriptional activator CaiF) , yfbR (5'-deoxynucleotidase YfbR), rfe (UDP-GlcNAc: undecaprenyl-phosphate / decaprenyl-phosphate GlcNAc-1-phosphate transferase), atpH (F-type H + -transporting ATPase subunit delta), tiaE (glyoxylate / hydroxypyruvate 2-ketogluconate reductase) and murI (glutamate rac) emase) may be selected from the group consisting of, but is not limited thereto.

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.

The present invention also relates to a recombinant microorganism in which the simultaneous multiple target gene expression suppression system is introduced.

As used herein, the term “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.

In another aspect, the present invention, (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. step; (d) inserting the determined 2 to 500 expression inhibitory genes into the q vector of the simultaneous multiple target gene expression suppression system, respectively; And (e) preparing a recombinant strain into which q vectors including the expression inhibitory gene are introduced; Here, q relates to a method for improving a useful substance producing strain, characterized in that the integer of 2 to 500.

In addition, those skilled in the art, through the method of the present invention, the steps of (a) preparing any base sequence having 20 to 50 bases; (b) inserting an arbitrary base sequence into a region complementarily binding to the target gene mRNA of the first vector to the q-th vector of the simultaneous multiple target droplet expression suppression system to include a first to r sRNA library Preparing a first to r-th vector library; (c) When the first to r-vector library is introduced into a target strain to produce a useful material to improve the production of the useful material, identifying a candidate group of genes whose expression is suppressed to determine s expression suppression gene combinations 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.

Here, q, r and s may be an integer of 2 to 500.

This means that multiple libraries can be screened at the same time, so that the most effective gene combinations can be directly derived in one screening without complicated steps, thereby dramatically improving the existing genetic circuit improvement method.

On the other hand, in 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.

That is, in another embodiment of the present invention, 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. (TH28C-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).

Accordingly, in another aspect, 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.

In the present invention, 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 One or more genes selected from the group consisting of transporters may be further deleted.

In addition, 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 homoserine efflux system), rhtB (homoserine, homoserine lactone and S-methyl one selected from the group consisting of -methionine efflux pump, rhtC (threonine efflux pump), thrAC1034T, thrB (homoserine kinase), and thrC (L-threonine synthase) On the gene it can be characterized in that the plasmid-based overexpression.

In the present invention, 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.

In another embodiment of the present invention, 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 By processing the sRNA library according to the present invention, 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).

Therefore, in another aspect, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, the recombinant microorganism may be characterized in that the PP3533 gene is introduced or amplified.

In the present invention, 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, Bifidobacterium It may be characterized in that it is selected from the group consisting of (cyanobacterium) and cyclobacterium (Cyclobacterium).

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.

In another embodiment of the present invention, 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. By discovering a gene combination that can improve the violacein production capacity, it was confirmed that the production capacity of indigo or violacein significantly increased (Figs. 16 to 18).

Accordingly, in another aspect, 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.

In the present invention, 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 One or more genes selected from the group consisting of may be characterized in that the introduced or amplified. The fbr means no feedback resistance (feedback resistance).

In another aspect, 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.

In the present invention, 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 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.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Performance Verification of a Simultaneous Multiple Target Gene Expression Suppression System

1.1. Production of various synthetic regulatory sRNA platforms

In the present invention, the following experiments were conducted to construct various synthetic regulatory sRNA platforms. Designed to be used simultaneously with several plasmids for the production of useful compounds, more specifically three antibiotic markers that can coexist with each other (denoted Apramycin, Am; streptomycin or spectinomycin, Sm; tetracycline, Tc) In total, nine vectors (CDF-Am, CDF-Sm, CDF-Tc, pBBR1-Am, pBBR1-Sm, pBBR1-Tc, using three coexisting replication origins (CDF, pBBR1, ColA)) were used. ColA-Am, ColA-Sm, ColA-Tc) were constructed (FIG. 1). At this time, [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, and [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). Among the antibiotic marker fragments, 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], and 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], and the ColA fragments are [SEQ ID NO: 22 / SEQ ID NO: 23]. Obtained.

1.2. Reporter plasmid construction for various synthetic regulatory sRNA efficiency tests

In order to confirm gene expression inhibition of various platform sRNAs, reporter plasmids containing the DsRed2 gene were first constructed. Among the reporter plasmids, 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. 24 / SEQ ID NO 25] to prepare a plasmid fragment for gibson assembly by PCR, and then obtain the DsRed2 gene by PCR reaction via [SEQ ID NO 26 / SEQ ID NO 27], and then the two fragments PTac15K-DsRed2, pTrc99A-DsRed2 reporter plasmids were constructed by assembling through a Gibson assembly.

1.3. Knockdown efficiency test and performance investigation of DsRed2 fluorescent protein of various synthetic regulatory sRNAs

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. First, in constructing synthetic regulatory sRNAs to down-express the expression of DsRed2 fluorescent protein, various synthetic regulatory sRNA platform plasmids were used as templates, using an inverse PCR reaction using [SEQ ID NO 28 / SEQ ID NO 29]. Among the DNA generated by PCR, 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.

Constructed 9 anti-DsRed2-sRNAs-inserted into CDF-Am, CDF-Sm, CDF-Tc, pBBR1-Am, pBBR1-Sm, pBBR1-Tc, ColA-Am, ColA-Sm, ColA-Tc platform plasmids, respectively Was transformed into the E. coli W3110 strain with the pACYC184-DsRed2 reporter plasmid, incubated at 37 ° C. at 200 RPM for 24 hours inoculated into a test tube containing 5 mL of LB (Luria-Bertani) medium. After OD600 was calibrated (OD600 = 2) to 96 well standard opaque microplates, fluorescence intensity was measured through a microplate reader. The excitation wavelength of DsRed2 fluorescent protein was set to 562 nm, emission wavelength was 603 nm, and cutoff value was set at 590 nm.

In addition, 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.

In addition, 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.

As a result, 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.

In the present invention, three platforms of ColA-Sm, CDF-Tc, and pBBR1-Am were selected in order to simultaneously knock down three genes in a strain and used in a later metabolic example. One of these platforms was selected for primary library production. At this time, the primary screening platform is generally designated by the present inventors for convenience of use, and it will be obvious that the usage of the present invention is not limited.

1.4. Effects of Various Combination Synthetic Regulatory sRNA Platforms on Growth of Host Escherichia Coli

1) single sRNA platform

When the synthetic regulatory sRNA platform plasmids prepared in Examples 1-3 were introduced into E. coli strains in various combinations, the effect on the growth of host cells was examined. First, the strain growth test was performed after the introduction of a single sRNA platform, and each sRNA platform was introduced into an E. coli strain, in which one species of reporter plasmid was already introduced to test in an environment as close as possible to the actual environment used for metabolic engineering. Therefore, each of the six E. coli W3110 strains carrying the pTac15K-dsRed2, pTrc99A-dsRed2, and pACYC184-dsRed2 reporter plasmids mentioned in the above examples was introduced into each of the six sRNA platforms selected in Example 1.3. Species of 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 μL 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.

As a result, it was confirmed that the introduction of a single sRNA platform does not affect the growth of host E. coli, as disclosed in the top three graphs (a) of FIG.

2) dual sRNA platform

We also wanted to analyze the growth curve when E. coli strains had two sRNA platforms simultaneously. Therefore, as mentioned above, 21 strains transformed with the sRNA platform in the E. coli W3110 strain into which three reporter plasmids were introduced were prepared: Tac-ColASm-pBBR1Am and Tac-ColASm-. pBBR1Tc, Tac-ColASm-CDFTc, Tac-CDFSm-pBBR1Am, Tac-CDFSm-pBBR1Tc, Tac-CDFTc-pBBR1Am, Tac-CDFTc-pBBR1Sm, Trc-ColASm-pBBR1Am, Trc-ColASc-TcB-ColASc Trc-CDFSm-pBBR1Am, Trc-CDFSm-pBBR1Tc, Trc-CDFTc-pBBR1Am, Trc-CDFTc-pBBR1Sm, ACYC-ColASm-pBBR1Am, ACYC-ColASm-pBBR1Tc, ACYCFTCmA-ColAm-ColAm CDFSm-pBBR1Tc, ACYC-CDFTc-pBBR1Am, ACYC-CDFTc-pBBR1Sm.

Growth tests on the produced strains were performed in the same manner as the test on the single sRNA platform.

As a result, it was confirmed that the introduction of two sRNA platforms did not significantly affect the growth of the host E. coli strain, as disclosed in the middle three graphs (b) of FIG. 3.

3) Triple sRNA Platform

Finally, we wanted to investigate the effect of simultaneous introduction of three sRNA platforms on the growth of host E. coli strains. Therefore, three strains transformed with the sRNA platform in the E. coli W3110 strain into which three reporter plasmids were introduced as mentioned above were prepared: Tac-ColASm-pBBR1Am-CDFTc, Trc- ColASm-pBBR1Am-CDFTc, ACYC-ColASm-pBBR1Am-CDFTc.

As a result, as shown in the bottom three graphs (c) of FIG. 3, the introduction of three sRNA platforms did not significantly affect the growth of the host E. coli strain.

1.5. Stability in Escherichia coli Hosts of Various Combinations of Synthetic Regulatory sRNA Platforms

In proceeding with 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. At this time, in order to distinguish each platform more easily, 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.

As a result of confirming the PCR product by electrophoresis, as shown in FIG. 4, all of the sRNAs were retained until the 3rd day of passage, but on the 3rd day, sRNA was lost in several strains. However, most strains were confirmed to have stable sRNA platforms.

Although the stability of the synthetic regulatory sRNA platforms of the present invention in the host cell was tested as described above, the simultaneous application of several synthetic regulatory sRNA expression vectors proved to be more stable or at least similarly stable compared to utilizing one vector. In order to perform the following experiment.

As shown by way of example in this patent specification, up to three synthetic regulatory sRNA platform vectors (except for vectors that are already included in host cells) were transformed simultaneously. Two strains of Escherichia coli W3110, each containing two reporter plasmids, pTac15K-dsRed2 and pTrc99A-dsRed2, were transformed into a single, double, and triple combinations to synthesize 28 strains. The thus prepared strains were passaged for> 80 generations and the passage and plasmid retention rates were measured about once every 20 generations.

In order to measure the plasmid retention ratio, 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%. .

This proved that the co-synthesized regulatory sRNA expression plasmids developed in the present invention showed very high stability. In this example, cells were cultured up to about 80 generations, which is much larger than about 15, which is usually the number of generations grown in industry (assuming fed-batch cultures start at 0.05 cells OD600 and end at 1,000). Bioprocesses also show that the corresponding sRNA expression plasmids can be stable.

Example 2 sRNA Library Construction Using ColA-Sm and CDF-Tc Platforms for Genes on Main Metabolic Circuits

In order to confirm the applicability of the synthetically regulated sRNA expression platform selected in Examples 1-3 to the practically useful substance producing strain, genes on major metabolic circuits in Escherichia coli were selected and a synthetic regulatory sRNA library was constructed to suppress the expression of these genes. .

First, 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). One). In constructing the sRNA library, 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.

Example 3. Production of useful materials using the constructed sRNA library Improvement of microbial plant production capacity

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.

3.1. Enrichment of L-threonine Using Various Synthetic Regulatory sRNA Platforms

1) single sRNA platform

Among the previously developed strains, 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.

Existing threonine production studies strengthened the threonine production pathway, improved it with feedback-resistant enzymes to prevent enzymes in the threonine production pathway from being inhibited by the product, and through fed-batch fermentation through strategies such as threonine transporter overexpression. 82.4 Threonine production (TH28C-pBRThrABCR3 strain) 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.

As a result of the flask culture, about 16.68 g / L of threonine was obtained by performing flask culture on the previously reported production strain (TH28C-pBRThrABCR3 strain) as disclosed in FIG. 5, while applying 55 synthetic regulatory sRNA libraries. In this case, it was confirmed that threonine production could increase to 21.68 g / L.

Given that the most prolific knockdown gene target for a threonine was aroF and pheA also produced significant transpiration, it was thought that the metabolic flow to threonine could be significantly increased by inhibiting metabolic flow to aromatic products (Fig. 5). In addition, remarkable threonine transpiration was observed when ilvH on the metabolic pathway that produces isoleucine from threonine and pta involved in acetic acid accumulation were suppressed.

2) multiple sRNA platform

Although significant transpiration could be observed even through the inhibition of single gene expression, the team used two compatible synthetic sRNA plasmids to maximize the development of the synthetic sRNA platform. Was to be observed.

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. Using our synthetic sRNA platform, we can easily test for simultaneous double-knockdown by combining two of the eight knockdown gene targets mentioned above, thereby selecting strains that produce higher yields of threonine. Expected to be able.

Existing synthetically regulated sRNA platform has a disadvantage in that it takes a long time and it is difficult to produce several strains at once because of multiple recombination of single sRNA in a single vector for simultaneous multiple knockdown, but using this platform, simple transformation Through this, the advantage of being able to produce several strains at once without genetic recombination is highlighted.

Therefore, 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. Interestingly, 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. In particular, 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.

As described in the above examples, multiple knockdown tests on already metabolically modified strains could be performed easily, quickly and effectively by utilizing the synthetic regulatory sRNA libraries previously built on CDF-Tc and ColA-Sm platforms. . In addition, it is remarkable that the industry has increased the production capacity by 37% by applying the newly constructed sRNA to the strain that already produces rational high concentration of threonine comparable to the industrial strain.

3.2. Proliferation of Proline (L-proline) Using Various Synthetic Regulatory sRNA Platforms

1) single sRNA platform

In the present invention, 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. There are reports (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. For flask culture, 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.

As a result of the flask culture, as shown in FIG. 7, a total of seven strains increased 20% or more, and the gene knockdown targets corresponding to these strains were as follows: serC, murE, aspC, metB, fadR, fur, and chpA. . In particular, 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).

2) multiple sRNA platform

To further increase proline by utilizing a synthetic sRNA platform, we observed proline transpiration through simultaneous double knockdown using two compatible synthetic regulatory sRNA plasmids. Simultaneous double knockdown was tested by combining two of the seven gene targets showing proline 20% increase in yield, and flask culture results for these strains are shown in FIG. 8.

As a result, it was confirmed that the strain in which fur-metB was simultaneously knocked down showed proline transpiration of 40.8% (2.90 g / L) in flask culture (FIG. 8).

Taken together, 5 out of a total of 21 strains were observed to be increased more than 30% compared to the existing parent strain. The five gene knockdown combinations mentioned are: fur-murE, fur-metB, fur-fadR, chpA-fadR, fur-chpA. This indicates that a total of three sRNA plasmids, including the sRNA plasmids originally contained in the parent strain, were introduced, and thus a high efficiency production strain can be produced quickly and easily through a simultaneous multiple sRNA knockdown system.

3.3. Development of a fed-batch fermentation process for the production of expanded proline (L-proline) strains using synthetic sRNA platform

1) single sRNA platform

The fed-batch fermentation was performed on strains with greatly improved proline production ability. In order to optimize the fermentation process, the fur drop down strain, which produced the highest transpiration by suppressing single gene expression, was used. First, the fed-batch fermentation was carried out using the previously reported proline strain fermentation conditions (KR 10-1750855 B1).

NMH26 p15PP3533 pKKtrcSargF-trcSglnA strain (proline-producing strain in existing patent KR 10-1750855 B1) 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 .

During fermentation, 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. In addition, the feeding solution in fed-batch fermentation was entered according to the pH-stat strategy. When the pH of the fermenter system rose above 6.83, 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. As a result, Fig. 9-a As disclosed in the strain fermented to the fermenter almost did not grow and stopped growing. This time, 1.6 g / L yeast extract was added to the flask and the fermentation medium, respectively. Although the growth of the strain was somewhat recovered, it was confirmed that the level is still relatively low (Fig. 9-b). In addition, 3 g / L yeast extract was added to the fermentation medium, and 6 mL trace metal solution was added to the feeding solution because the intracellular metal ion-related metabolic circuit could not work properly when the fur fell down.

As a result, as shown in Fig. 9-c it was possible to observe a significantly improved cell growth, the concentration of proline was also greatly increased to 51.6 g / L. This is a 52.7% increase in production compared to the previously reported proline production (33.8 g / L) (Noh et al., Cell Systems, 2017, KR 10-1750855 B1).

In this optimized fed-batch fermentation conditions, it was confirmed that even when the previously reported strain was cultured, proline similar to the previously reported concentration was produced (FIG. 9-d).

The researchers cultured the high-proline production strains produced in Example 3.2 under optimized fed-batch fermentation conditions and measured proline production. The fed-batch fermentation was performed on four single knockdown strains, and the result was NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-fur strain (51.6 g / L) of FIG. 10-a, and NMH26 p15PP3533 pKKtrcSargF-trcSglnA of FIG. 10-b. pCDFTc-fadR strain (33.8 g / L), NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-aspC strain (41.4 g / L) of FIGS. Same as L).

2) dual sRNA platform

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. trcSglnA pCDFTc-fur pColASm-murE strain (41.3 g / L), NMH26 p15PP3533 pKKtrcSargF-trcSglnA pCDFTc-chpA pColASm-fur strain (47.1 g / L) in FIGS. -chpA pColASm-fadR strain (38.6 g / L).

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.

In addition, 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.

As described in the above examples, multiple knockdown tests for already metabolically modified strains could be easily, quickly and effectively carried out using the synthetic regulatory sRNA libraries previously built on CDF-Tc and ColA-Sm platforms. The strains thus constructed showed very good performance in fed-batch fermentation, showing great potential for development into industrial strains. Moreover, strains containing a total of four synthetic regulatory sRNAs, up to two synthetic regulatory sRNAs introduced through existing metabolic strategies and two newly introduced synthetic regulatory sRNAs, showed very good growth and very high proline production in fed-batch cultures. It is very noteworthy in the art, it can be seen as an example showing the utility as an industrial strain in the future.

Example 4 Platform Transfer of Genome-Level sRNA Libraries and Fast Screening Techniques Using the Same

In order to apply the synthetic regulatory sRNA to various environments and various production strains, a method for easily transferring the genome-level sRNA expression platform to another platform was developed (FIG. 13). In this example, 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. We have established an E. coli genome-level synthetic regulatory sRNA library (for 1,858 target genes) (D. Yang et al., Proc Natl Acad Sci USA (2018), 115 (40): 9835). -9844).

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. As compared to each individual in the sRNA library, 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 DH5α strain.

In order to accommodate all 1,858 sRNAs on the new platform, it was repeated until about 25,000 colonies (approximately 13.5 times library size) were obtained. After mixing all the colonies, the maxiprep based on the new ColA-Sm platform was used. Successfully obtained sRNA library (FIG. 13). The time required to transfer the genome-level synthetic regulatory sRNA library to the new platform is two days, which is significantly longer than the weeks or months required to build a new genome-level synthetic regulatory sRNA library with limited manpower. It is noteworthy in that it saves manpower and resources.

Example 5 Indigo Expansion Using Platform-Transferd sRNA Libraries and Fast Screening

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. In addition, 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.

First, 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. Du et al., J Biotechnol., 2018, 267, 19-28), 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.

As a result, it was confirmed that the indigo yield increased to 134.84 mg / L in the asnA knockdown strain as disclosed in FIG. 15.

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.

6.1. Production of violacein using platform-shifted sRNA libraries and high-speed screening

Five genes of vioABCDE are required for the production of viola sane. First of all, 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. First, 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.

As a result, as shown in FIG. 17, 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). In addition, 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).

6.2. Development of fed-batch fermentation process of violacein-producing strains cultivated using synthetic sRNA platform

The fed-batch fermentation was carried out on strains with greatly improved violasane production ability. At this time, in the flask culture in Example 6.1, 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) inserted into E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain and pColA-minD plasmid into E. 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 .

During fermentation, 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. In addition, 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.

As a result of fed-batch fermentation of the strain in which the pColA-ytfR plasmid was inserted into the E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain, 1.80 g / L of violasane and 0.55 g / L of diol as shown in FIG. Oxyviolacein was produced. In addition, as a result of fed-batch fermentation of the strain in which the pColA-minD plasmid was inserted into the E. coli BL21 pSB1C3-vioABDE pTac15K-Jli-vioC strain, as described in Fig. 18-b, 0.78 g / L of 1.10 g / L of violacein Of deoxybiolacein was produced.

In addition, 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.

As described in the above examples, it was possible to transfer genome level synthetic regulatory sRNA libraries to new platforms in a very short time, which proved very effective in the production of indigo and violacein. Moreover, in the case of violasane producing strains, the screening method was proved to be very effective through fed-batch fermentation of strains containing the screened synthetic regulatory sRNA, and experimented for several months to several years through existing metabolic strain development methods. It is remarkable in the art that the violacein / deoxybiolacein production concentrations obtained by performing were obtained in a matter of days.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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. In addition, by incorporating the 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.

Electronic file attached.

Claims

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 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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin in, salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine, ,

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

A recombinant microorganism in which a simultaneous multiple target gene expression suppression system is introduced into a host cell.
Method of improving strains of useful material production comprising the following steps:

(A) preparing any base sequence having a size of 20 ~ 50bp;

(b) the nucleotide sequence of the first antibiotic resistance gene; First replication origin; Preparing a first vector library comprising a first sRNA library by inserting into a region complementarily binding to a target gene mRNA of a first vector comprising a synthetic sRNA coding region that inhibits expression of the first target gene;

(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;

(d) the 2 to 500 expression inhibitory genes determined as the n antibiotic resistance genes; Mth replication origin; Inserting each of the q-vectors including the synthetic sRNA coding region that inhibits the expression of the 2 to 500 genes to inhibit expression; And

(e) preparing a recombinant strain into which q vectors containing the expression inhibitory gene are introduced,

here,

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 method of claim 1, wherein the prokaryote is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Parfait. Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter , Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium (cyanobacte Recombinant microorganisms having a simultaneous multi-target gene expression suppression system introduced into the host cell, characterized in that selected from the group consisting of rium) and Cyclobacterium.
The recombinant microorganism of claim 1, wherein the promoter is selected from the group consisting of tac, trc, T7, BAD, λPR, and Anderson synthetic promoters.
5. The recombinant microorganism of claim 4, wherein the promoter is expressed by a nucleotide sequence of SEQ ID NO. 7.
The recombinant microorganism according to claim 1, wherein the Hfq binding site is represented by a nucleotide sequence of SEQ ID NO: 8.
The recombinant microorganism of claim 1, wherein the terminator is represented by a nucleotide sequence of SEQ ID NO: 9. A multi-target gene expression suppression system is introduced into a host cell.
The method of claim 1, wherein the target gene is 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 kinase / acetate kinase activity), adiY (AdiY is a positive DNA-binding transcriptional regulator that controls the arginine) decarboxylase (adi) system) , argB (acetylglutamate kinase), argC (N-acetylglutamylphosphate reductase), argG (argininosuccinate synthase), argH (argininosuccinate lyase), asnC (transcriptional regulator that activates the expression of asnA, a gene involved in the synthesis of asparagine), aspA ( aspartate ammonia-lyase), crp (CRP transcriptional dual regulator), and csiD (predicted protein. 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 (FadR Fatty acid degradation Regulon, is a multifunctional dual regulator that exerts negative control over the fatty acid degradative regulon [Simons80, Simons80a] and acetate metabolism), fbp (fructose-1,6-bisphosphatase), fnr (FNR is the primary transcriptional regulator that me diates the transition from aerobic to anaerobic growth), fruR (FruR is a dual transcriptional regulator that plays a pleiotropic role to modulate the direction of carbon flow through the different metabolic pathways of energy metabolism, but independently of the CRP regulator), ftsL (essential cell division protein FtsL), ftsQ (essential cell division protein FtsQ), ftsW (essential cell division protein FtsW), ftsZ (essential cell division protein FtsZ), fur (Fur-Fe + 2 DNA-binding transcriptional dual regulator), gabD ( succinate semialdehyde dehydrogenase, NADP + -dependent), gabP (APC transporter), gabT (4-aminobutyrate aminotransferase), gadA (glutamate decarboxylase A subunit), gadB (glutamate decarboxylase B subunit), gadC (GABA APC transporter), glcC (GntR family transcriptional regulator, glc operon transcriptional activator, glpK (glycerol kinase), glpR (sn-Glycerol-3-phosphate repressor), glpX (fructose 1,6-bisphosphatase II), gltA (citrate synthase), hflD (lysogenization regula tor), ihfA (IHF, Integration host factor, is a global regulatory protein), ihfB (IHF, Integration host factor, is a global regulatory protein), ilvB (acetohydroxybutanoate synthase / acetolactate synthase), ilvC (acetohydroxy acid isomeroreductase), ilvD (dihydroxy acid dehydratase), ilvG_1 (acetolactate synthase II, large subunit, N-ter fragment (pseudogene)), ilvG_2 (acetolactate synthase II, large subunit, C-ter fragment (pseudogene)), ilvH (acetolactate synthase / acetohydroxybutanoate synthase) , ilvL (ilvGEDA operon leader peptide), ilvM (acetohydroxybutanoate synthase / acetolactate synthase), ilvN (acetohydroxybutanoate synthase / acetolactate synthase), ilvX (Predicted small protein), lexA (LexA represses the transcription of several genes involved in the cellular response to DNA damage), lpxC (UDP-3-O-acyl-N-acetylglucosamine deacetylase), marA (MarA participates in controlling several genes involved in resistance to antibiotics, oxidative stress, organic solvents and heavy metals.) , metJ (MetJ transcriptional repressor), modE (ModE is the principal regulator that controls the transcription of operons involved in the transport of molybdenum and synthesis of molybdoenzymes and molybdate-related functions), nadB (L-aspartate oxidase), narL (nitrate / nitrite response regulator), pck (phosphoenolpyruvate carboxykinase), PdhR (PdhR, "pyruvate dehydrogenase complex regulator," regulates genes involved in the pyruvate dehydrogenase complex), phoP (PhoP-Phosphorylated DNA-binding transcriptional dual regulator. 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 (PurR Hypoxanthine 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), rbsK (ribokinase), rbsR (The transcription factor RbsR, for "Ribose Repressor," is negatively autoregulated and controls the transcription of the operon involved in ribose catabolism and transport), rcsB ( RcsB-BglJ DNAbinding transcriptional activator.RcsB protein for "Regulator capsule synthesis B," is a response regulator that belongs to the multicomponent RcsF / RcsC / RcsD / RcsA-RcsB phosp horelay system and is involved in the regulation of the synthesis of colanic acid capsule, cell division, periplasmic proteins, motility, and a small RNA), rutR (RutR regulates genes directly or indirectly involved in the complex pathway of pyrimidine metabolism), serA ( alpha-ketoglutarate reductase / D-3-phosphoglycerate dehydrogenase), serC (phosphohydroxythreonine aminotransferase / 3-phosphoserine aminotransferase), soxS (dual transcriptional activator and participates in the removal of superoxide and nitric oxide), sroD (SroD small RNA), zwf ( glucose 6-phosphate-1-dehydrogenase), asnA (asparagine synthetase A), asnB (asparagine synthetase B), carA (carbamoyl phosphate synthetase), carB (carbamoyl phosphate synthetase), ddlB (D-alanine-D-alanine ligase B) , deoA (thymidine phosphorylase / uracil phosphorylase), deoD (purine nucleoside phosphorylase deoD-type), dpiA (dual transcriptional regulator involved in anaerobic citrate catabolism), fis (Fis, "factor for inversion stimulation ", is a small DNA-binding and bending protein whose main role appears to be the organization and maintenance of nucleoid structure), gadE (GadE controls the transcription of genes involved in glutamate dependent system), gadW (GadW controls the transcription of genes involved in glutamate dependent system), gadX (GadX controls the transcription of genes involved in glutamate dependent system), glpF (GlpF glycerol MIP channel), ilvY (IlvY DNA-binding transcriptional dual regulator), ivbL (The ilvB operon leader peptide (IvbL) ), lhgO (L-2-hydroxyglutarate oxidase), lpd (Lipoamide dehydrogenase), lrp (Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These genes are involved in amino acid biosynthesis and catabolism, nutrient) transport, pili synthesis, and other cellular functions, including 1-carbon metabolism), metB (O-succinylhomoserine lyase / Osuccinylhomoserine (thiol) -lyase), metL (aspartate kinase / homoserine dehydrogenase), mraY (phospho-Nacetylmuramoyl-pentapeptide transferase), mraZ (Unknown function), murE (UDP-N-acetylmuramoylalanyl-Dglutamate 2,6-diaminopimelate ligase), murF (D-alanyl-D-alanine-adding enzyme) , murG (Nacetylglucosaminyl transferase), nac (Nacregulates, without a coeffector, genes involved in nitrogen metabolism under nitrogen-limiting conditions), nadA (quinolinate synthase), nsrR (NsrR, the "nitritesensitive repressor" regulates genes involved in cell protection against nitric oxide (NO)), panC (pantothenate synthetase), panD (Aspartate 1-decarboxylase), pgl (6-phosphogluconolactonase), pyrB (aspartate carbamoyltransferase, PyrB subunit), pyrC (dihydroorotase), pyrL (aspartate carbamoyltransferase, PyrI subunit), rob (Rob is a transcriptional dual regulator. Its N-terminal domain shares 49% identity with MarA and SoxS. These proteins activate a common set of about 50 target genes, the marA / soxS / rob regulon, involved in antibiotic resistance, superoxide resistance, and tolerance to organic solvents and heavy metals.), Rpe (ribulose phosphate 3-epimerase), talA ( transaldolase A), thrA (aspartate kinase / homoserine dehydrogenase), thrB (homoserine kinase), thrC (threonine synthase), thrL (thr operon leader peptide), tthrA (transketolase I), tktB (transketolase II), torR (two-component system, OmpR family, torCAD operon response regulator TorR), 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), trpD (fused glutamine amidotransferase (component II) of anthranilate synthase / anthranilate phosphoribosyl transferase), dapA (4-hydroxy-tetrahydrodipicolinate synthase), ilvE (Branched-chain-amino -acid aminotransferase), lysA (Diaminopi melate decarboxylase), gltA (cistrate synthase), fumA (Fumarate hydratase class I, aerobic), glnA (glutamine synthetase), mdh (malate dehydrogenase), gdhA (NADP-specific glutamate dehydrogenase), argA (amino acid N-acetyltransferase and inactive acetylglutamate kinase, metA (Homoserine O-succinyltransferase), tdh (L-threonine dehydrogenase), livJ (branched-chain amino acid ABC transporter periplasmic binding protein), rhtC (Threonine efflux protein), yohJ (UPF0299 membrane protein), chpA ( antitoxin of the ChpA-ChpR toxin-antitoxin system), aspC (aspartate aminotransferase), pfkA (ATP-dependent 6-phosphofructokinase isozyme 1), parE (DNA topoisomerase 4 subunit B), ytfR (predicted sugar transporter subunit), ilvA (L -threonine dehydratase, biosynthetic; also known as threonine deaminase, ilvG (Acetolactate synthase), minD (inhibitor of FtsZ ring polymerization; chromosome-membrane tethering protein; membrane ATPase of the MinCDEE system), hisJ (histidine transport system substrate-binding protein), yneH (Glutaminase) , ferpedoxin-type protein NapG (napG), 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA), ygfA (5-formyltetrahydrofolate cyclo-ligase), cell division protein FtsI (penicillin-binding protein 3) ftsI, trehalose 6-phosphate phosphatase, hybG (hydrogenase expression / formation protein HypC), ampG (MFS transporter, PAT family, beta-lactamase induction signal transducer AmpG), potG (spermidine / putrescine transport system permease protein), caiF (transcriptional activator CaiF) , yfbR (5'-deoxynucleotidase YfbR), rfe (UDP-GlcNAc: undecaprenyl-phosphate / decaprenyl-phosphate GlcNAc-1-phosphate transferase), atpH (F-type H + -transporting ATPase subunit delta), tiaE (glyoxylate / hydroxypyruvate 2-ketogluconate reductase) and murI (glutamate rac) E. coli is a recombinant microorganism which is introduced into the host cell is a simultaneous multiple target gene expression suppression system, characterized in that selected from the group consisting of.
The method of claim 8, wherein the target gene is 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 (UDP-N-acetylmuramoylalanyl-Dglutamate 2,6-diaminopimelate ligase), pps (phosphoenolpyruvate synthetase), aceE (subunit of E1p component of pyruvate) pta (Phosphate acetyltransferase), purA (adenylosuccinate synthetase), ackA (propionate kinase / acetate kinase activity), pck (phosphoenolpyruvate carboxykinase), ppc (phosphoenolpyruvate carboxylase), accA (acetyl-CoA carboxyltransunitty -CoA synthetase), fabH (KASIII, -ketoacyl-ACP synthases), aspA (aspartate ammonia-lyase), carB (carbamoyl phosphate synthetase), ilvH (acetolactate synthase / acetohydroxybutanoate synthase), ilvM (acetohydroxyaceanotolacase synthase synthase N (acetohydroxybutanoate synthase / acetolactate synthase), ilvC (acetohydroxy acid isomeroreductase), asnA (asparagine synthetase A), asnB (asparagine synthetase B), argH (argininosuccinate lyase), deoA (thymidine phosphorylase / part, uracil kinase homoserine dehydrogenase), metB (O-succinylhomoserine lyase / Osuccinylhomoserine (thiol) -lyase), metA, tdh, ilvL (ilvGEDA operon leader peptide), crp (CRP transcriptional dual regulator), fadR (FadR Fatty acid degradation Regulon, is a multifunctional dual regulator that exerts negative control over the fatty acid degradative regulon [Simons80, Simons80a] and acetate metabolism), fur (Fur-Fe + 2 DNA-binding transcriptional dual regulator), lrp (Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These 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), trpD (fused glutamine amidotransferase (component II ) of anthranilate synthase / anthranilate phosphoribosyl transferase), dapA (4-hydroxy-tetrahydrodipicolinate synthase), ilvE (Branched-chain-amino-acid aminotransferase), lysA (Diaminopimelate decarboxylase), gltA (cistrate synthase), fumA (F umarate hydratase class I, aerobic), glnA (glutamine synthetase), mdh (malate dehydrogenase), gdhA (NADP-specific glutamate dehydrogenase), argA (amino acid N-acetyltransferase and inactive acetylglutamate kinase), metA (Homoserine O-succinyltransase) tdh (L-threonine dehydrogenase), livJ (branched-chain amino acid ABC transporter periplasmic binding protein), rhtC (Threonine efflux protein), yohJ (UPF0299 membrane protein), chpA (antitoxin of the ChpA-ChpR toxin-antitoxin system), aspate (aspartate aminotransferase), pfkA (ATP-dependent 6-phosphofructokinase isozyme 1), parE (DNA topoisomerase 4 subunit B), ytfR (predicted sugar transporter subunit), ilvA (L-threonine dehydratase, biosynthetic; also known as threonine deaminase, ilvG (Acetolactate synthase), minD (inhibitor of FtsZ ring polymerization; chromosome-membrane tethering protein; membrane ATPase of the MinCDEE system), hisJ (histidine transport system substrate-binding protein), yneH (Glutaminase) , ferpedoxin-type protein NapG (napG), 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA), ygfA (5-formyltetrahydrofolate cyclo-ligase), cell division protein FtsI (penicillin-binding protein 3) ftsI, trehalose 6-phosphate phosphatase, hybG (hydrogenase expression / formation protein HypC), ampG (MFS transporter, PAT family, beta-lactamase induction signal transducer AmpG), potG (spermidine / putrescine transport system permease protein), caiF (transcriptional activator CaiF) , yfbR (5'-deoxynucleotidase YfbR), rfe (UDP-GlcNAc: undecaprenyl-phosphate / decaprenyl-phosphate GlcNAc-1-phosphate transferase), atpH (F-type H + -transporting ATPase subunit delta), tiaE (glyoxylate / hydroxypyruvate 2-ketogluconate reductase) and murI (glutamate rac) E. coli is a recombinant microorganism in which a simultaneous multiple target gene expression suppression system is introduced into a host cell.
Culturing a recombinant microorganism having the simultaneous multiple target gene expression suppression system of any one of claims 1 and 3 to 9 introduced into a host cell; And inhibiting mRNA expression of the first and p target genes.
In 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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin n), salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine; ,

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,

A sequence prepared by inserting a sequence complementarily binding to mRNA of a tktA, aroF, pta, ilvH, ilvE, glnA, fur, or chpA gene into a region complementarily binding to mRNAs of the target genes of the first to q vectors A recombinant microorganism, wherein a vector is introduced and the expression of two or more genes selected from the group consisting of tktA, aroF, pta, ilvH, ilvE, glnA fur, and chpA is suppressed to improve threonine production capacity.
The method of claim 11, wherein the lacI (Lactose-inducible lac operon transcriptional repressor), metA (Homoserine O-succinyltransferase / O-acetyltransferase), lysA (Diaminopimelate decarboxylase), tdh (L-threonine dehydrogenase), iclR (AceBAK operon repressor) Recombinant microorganisms, characterized in that one or more genes selected from the group consisting of tdcC (Threonine / serine transporter) is further deleted.
The mutation of claim 10, wherein the 1034 base sequence of thrA (Bifunctional aspartokinase / homoserine dehydrogenase 1) is changed from C to T, the 1055 base sequence of lysine-sensitive aspartokinase 3 (lysC) is changed from C to T, ilvA (l-threonine dehydratase, biosynthetic; also known as threonine deaminase), characterized in that one or more genes selected from the group consisting of mutations in which the 290th sequence of the nucleotide sequence is changed from C to T has been engineered, thrABC operon, ppc ), a promoter of one or more genes selected from the group consisting of acs (acetyl-CoA synthetase) is substituted with trc promoter, rhtA (threonine and homoserine efflux system), rhtB (homoserine, homoserine lactone and S-methyl One or more genes selected from the group consisting of -methionine efflux pump, rhtC (threonine efflux pump), thrAC1034T, thrB (homoserine kinase), and thrC (L-threonine synthase) Recombinant microorganisms, characterized in that plasmid-based overexpression.
The host cell of claim 11, wherein the host cell is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, or Farobacter. Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter , Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium (cyanobact erium) and cyclobacterium (Cyclobacterium) is selected from the group consisting of recombinant microorganisms.
In host cells with 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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin n), salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine; ,

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 A recombinant microorganism which is introduced, wherein the expression of two or more genes selected from the group consisting of serC, murE, aspC, metB, fadR, fur, and chpA is suppressed to improve proline production capacity.
The recombinant microorganism according to claim 15, wherein at least one gene selected from the group consisting of lacI, speE, speG, argI, puuP, puuA, putA, putP, proP, speC, potE, and speF is further deleted. .
The recombinant microorganism according to claim 15, wherein the PP3533 gene is introduced or amplified.
The recombinant microorganism according to claim 15, wherein expression of the argF and glnA genes is further inhibited.
16. The recombinant microorganism of claim 15, wherein the promoter of at least one gene selected from the group consisting of argECBH operon, speF-potE, and argD is substituted with trc promoter.
A proline production method comprising culturing the recombinant microorganism of claim 15 through a fed-batch fermentation in a medium containing a trace metal solution.
The host cell of claim 15, wherein the host cell is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, or Farobacter. Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter , Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium (cyanobact erium) and cyclobacterium (Cyclobacterium) is selected from the group consisting of recombinant microorganisms.
In 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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin n), salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine; ,

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,

Inserting a sequence complementary to the mRNA of the asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF or ostB gene in the region complementary to the mRNA of the target gene of the first to q vector Since the prepared vector is introduced, expression of two or more genes selected from the group consisting of asnA, hisJ, yneH, napG, kdsA, ygfA, ftsl, aceF, and ostB is suppressed, thereby improving indigo production capacity. Recombinant microorganisms.
The group of claim 22, wherein at least one gene selected from the group consisting of trpR, pykF and pykA is further deleted, the promoter of tktA is substituted with the trc promoter, and the group consisting of tnaA, fmo, aroGfbr, trpEfbr, and aroL Recombinant microorganisms, characterized in that one or more genes selected from the introduced or amplified.
The host cell of claim 22, wherein the host cell is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, or Farobacter. Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter , Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium (cyanobact erium) and cyclobacterium (Cyclobacterium) is selected from the group consisting of recombinant microorganisms.
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. Erythromycin, neomycin, penicillin, axinomycin, actinomycin, garbenicillin, gentamicin, blasticidin, mycophenolic acid ), Puromycin, zeocin, borelidin, borrelidin, ionomycin, daunorubicin, doxorubicin, doxorubicin, ivermectin, avermectin Mithramycin, mitomycin, mitomycin, nalidixic acid, novobiocin, nystatin, oxytetracycline, paclitaxel, polymycin (polymyxin), rifampicin n), salinomycin, tylosin, valinomycin, vancomycin, vinblastine, and vincristine; ,

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

In recombinant microorganisms in which a simultaneous multiple target gene expression suppression system is introduced into a host cell,

The host cell has a violacein (violacein) biosynthesis pathway,

Complementary to the mRNA of the target gene of the first to q vector complementary to the mRNA of ytfR, hybG, ampG, potG, caiF, minD, ilvM, yfbR, rfe, atpH, tiaE or murI gene 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 recombinant microorganism according to claim 25, wherein at least one gene selected from the group consisting of vioA, vioB, vioC, vioD, and vioE is introduced or amplified.
The host cell of claim 25, wherein the host cell is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Parfait. Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter , Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus (Lactococcus), Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Bifidobacterium (cyanobact erium) and cyclobacterium (Cyclobacterium) is selected from the group consisting of recombinant microorganisms.
A method for producing a violacein / deoxybiolacein comprising culturing the recombinant microorganism of claim 25 through a fed-batch fermentation in a medium containing a trace metal solution.
Screening method for multiple inhibitory gene combinations comprising the following steps:

(A) preparing any base sequence having a size of 20 ~ 50bp;

(b) the nucleotide sequence of the first antibiotic resistance gene; First replication origin; A first vector comprising a synthetic sRNA coding region that inhibits expression of a first target gene; To n-n antibiotic resistance genes; Mth replication origin; A q vector comprising a synthetic sRNA coding region that inhibits p target gene expression; a first to r vector library comprising first to r sRNA libraries inserted into a region complementarily binding to a target gene mRNA Preparing a; And

(c) introducing the first to r vector library into the target strain to produce a useful material, and when the production of the useful material is improved, to identify a group of gene candidates with suppressed expression, s expression suppression gene combinations are identified Determining;

Here, 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.