KR101844452B1 - Enhanced nar Promoter and Method for Producing Biochemical Using the Same - Google Patents

Abstract

The present invention relates to improvement of a nar promoter which is operated depending on the concentration of dissolved oxygen. More specifically, the present invention relates to a nar promoter in which strength of the promoter is improved by randomly mutating the -10 and -35 conserved sequences on the nar promoter, and to an increase in biochemical production using the same. According to the present invention, the nar promoter whose promoter strength is improved by randomly mutating the -10 and -35 conserved sequences excellently expresses metabolic pathway protein involved in the production of target substances, thereby being effectively useful for producing industrially useful biochemicals through the metabolic pathway activated by the protein.

Description

[0001] The present invention relates to a modified Nalg promoter and a method for producing the Nalg promoter,

The present invention relates to an improved Nal promoter which is activated according to the concentration of dissolved oxygen. More specifically, the present invention relates to a Nal promoter which improves the promoter strength by causing random mutations in the -10 and -35 conserved sequences of the Nal promoter, It is about increase.

Carry a promoter (nar promoter) is narGHIJ present in Escherichia coli (E. coli) It is a transcription-inducible promoter that regulates operons and is dependent on dissolved oxygen concentration under anaerobic conditions. Previously, promoters of pMW618 engineered to induce transcription in micro-aerobic conditions by site-directed mutagenesis in -10 and -35 conserved sequences have been used to detect beta-galactosidase, GFP (D-lactic acid) and ( R, R ) -2,3-butanediol (2,3-butanediol, 2,3-BDO) through gene expression as well as various proteins And research to produce biochemicals. However, existing mutant promoters are limited in the strength of the promoter.

These narGHIJ The sequence of the promoter region is known (Margaret S. Walker et al., J. Bacteriol 174 (4); 1119-1123, 1992). With respect to the Nal promoter, Korean Patent Laid-Open Publication No. 97-61912 discloses "a method for producing a protein by an oxygen-dependent inducible promoter ", Korean Patent Publication No. 99-38719 discloses" A method for producing a protein from a dependent inducible promoter ". Also, the nar promoter or nar operon described in the above literatures means narG promoter, narGHIJ operon or narG operon among various nar operons, and Korean Patent No. 10 -0985746 discloses an expression vector containing the narK promoter or the mutated narK promoter and a method for mass production of a target protein using the same.

On the other hand, biochemicals such as 2,3-butanediol can be applied as an octane booster by mixing with gasoline (Celinska et al., Biotechnol. Adv ., 27: 715, 2009). However, 2,3-butanediol production through the microbial fermentation process is still difficult to apply directly to commercial processes due to low productivity and yield. Therefore, research on the development of microorganisms related to biochemical production such as 2,3-butanediol is needed.

Thus, the present inventors have made intensive efforts to develop a strong-strength Nal promoter applicable to a biochemical production-related biosynthetic metabolic circuit gene such as 2,3-butanediol. As a result, it has been found that the Nal promoter- ( R, R ) -2,3-BDO production was checked by selecting one of the strongest promoter, mid-intensity promoter, and weakest promoter in a mutagenic library obtained by randomly mutating 15 base pairs of the region Thus completing the present invention.

It is an object of the present invention to provide a synthetic nar-promoter containing a polynucleotide represented by any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 4.

It is another object of the present invention to provide a vector containing the synthetic Nal promoter and a recombinant microorganism into which the vector is introduced.

Yet another object of the present invention is to provide a method for producing a recombinant microorganism, which comprises culturing the recombinant microorganism to express a metabolic pathway protein involved in production of a target substance; And recovering the target substance produced through the metabolic pathway activated by the expressed protein.

In order to achieve the above object, the present invention provides a synthetic Nalpromoter comprising a polynucleotide represented by any one of SEQ ID NOS: 1 to 4.

The present invention also provides a vector containing the synthetic nar-promoter and a gene encoding a metabolic pathway protein involved in the production of the target substance.

The present invention also provides a recombinant microorganism into which said vector is introduced.

The present invention also provides a method for producing a recombinant microorganism, comprising the steps of: culturing the recombinant microorganism to express a metabolic pathway protein involved in production of a target substance; And recovering the target substance produced through the metabolic pathway activated by the expressed protein.

The Nal promoter improved in promoter strength by the random mutation in the -10 and -35 conservative sequences according to the present invention is excellent in the expression of the metabolic pathway protein involved in the production of the target substance, Can be effectively used for the production of useful biochemicals.

Figure 1 is a schematic diagram for constructing a synthetic Nal promoter library.
FIG. 2 shows FACS analysis for screening of the synthetic Nal promoter, wherein (A) is a strong promoter and (B) is a sorting result for weak and intermediate promoter selection. 1: negative control (pSTVM2), 2: positive control (pSTVN-gfpmhis), 3: original library, 4: first round sorted cells, 5: 2nd round sorted cells, 6: 3rd round sorted cells.
Fig. 3 shows the fluorescence intensity of the synthetic Nalpromoter. Red arrow: negative control (pSTVM2); Blue arrow: positive control (pSTVN-gfpmhis); Green bar graph: strong promoter; Yellow bar graph: intermediate promoter; Red bar graph: weak promoter
FIG. 4 is a graph comparing the intensity of the synthetic Nal promoter with the relationship between the amount of mRNA expression (X axis), the amount of protein expression (Y axis), and the fluorescence intensity of GFP (Z axis) (greenish: strong promoter, yellow: intermediate promoter, red: weak promoter, white: constitutive promoter, black: normal nar promoter).
FIG. 5 shows the nucleotide sequence of each promoter. The yellow region corresponds to the randomly mutated spacer, and the light blue region represents the region having the same sequence as the normal Nal promoter.
Fig. 6 shows the plasmids (A) pUCNrS, (B) pUCNrI and (C) pUCNrW containing the respective promoters.
7 shows the plasmid (A) NrL and (B) NrSL containing the ldhD gene.
Fig. 8 compares the production of D-lactic acid according to each promoter at 4, 8 and 20 hours.
Figure 9 compares the production of D-lactic acid by fed-batch culture using (A) NrL and (B) NrSL (red arrow: the portion that changed the dissolved oxygen; gray circles: dissolved oxygen; white circles: Growth curve: black square: ( R, R ) -2,3-BDO yield; white triangle: residual glucose concentration).
FIG. 10 shows plasmids (A) Si, (B) Ii, (C) Wi, (D) NrSa, (E) NrIa and (F) NrWa for producing acetone.
11 compares the yield of acetone according to the combination of promoters.
FIG. 12 shows plasmids (A) SiWa, (B) IiIa, (C) SiWaSb, (D) NrSb, (E) NrIb and (F) NrWb for producing 2,3-BDO.
Figure 13 compares the ( R, R ) -2, 3-BDO yields according to the promoter combination.
Figure 14 compares the production of ( R, R ) -2,3-BDO by fed-batch culture using (A) NiNaNb and (B) SiWaSb (red arrow: Circle: Dissolved Oxygen; White circles: Cell growth curve; Black square: ( R, R ) -2,3-BDO yield; white triangle: residual glucose concentration).

Unless otherwise defined, 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.

In the present invention, in order to improve the strength of the existing pMW618 Nal promoter, mutations were generated in 15 base pairs of the spacer region existing in the -10 and -35 conservative sequences, which are known to cause mutation in order to regulate the promoter intensity. The intensity of the promoter was determined by fluorescence intensity using GFP as a marker protein. The library constructed on the plasmid was transformed into E. coli and transformed into a strain library, which was then screened by FACS. ( R, R ) -2,3-BDO production-related biosynthetic metabolic pathway gene by selecting one of the strongest promoter, mid-intensity promoter and weakest promoter in the obtained mutant library, Respectively.

Accordingly, the present invention relates to a synthetic Nalpromoter containing a polynucleotide represented by any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 4 in one aspect.

In the present invention, the polynucleotide may be characterized by being located between -35 and -10 conservative sequences, and may be characterized by being 15 base pairs. In addition, the polynucleotide between the existing pMW618 Nal promoter-35 and -10 conserved sequence can be represented by SEQ ID NO: 62 (Table 1).

In another aspect, the present invention relates to a vector containing a synthetic Nal promoter containing a polynucleotide represented by any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 4.

In the present invention, a gene encoding a metabolic pathway protein involved in the production of a target substance is preferably included downstream of the synthetic nar promoter sequence, but is not limited thereto.

In the present invention, the target material may be D-lactic acid, acetoin, ( R, R ) -2,3-butanediol, 2,3-BDO, 1,3-propanediol, 1,4-butanediol, succinic acid, isobutanol, 1-butanol / n-butanol ), Fatty acid, hydrogen, 1-propanol, and acetone, and more preferably selected from the group consisting of D-lactic acid, acetone or ( R, R ) 2,3-butanediol, but is not limited thereto.

In the present invention, the target substance is preferably a hormone, an immune-related protein, or an enzyme that can be used as a therapeutic protein, and the hormone may be parathyroid hormone, insulin, erythropoietin , The immuno-related protein may be interferon alpha, interferon beta, interferon gamma, interleukin-2 (IL-2), granulocyte colony-stimulating factor (human granulocyte colony-stimulatory factor) But are not limited to, asparaginase, alkaline phosphatase, levan fructotransferase, or streptokinase. The term " inhibitor "

In the present invention, the gene coding for the metabolic pathway protein involved in D-lactic acid production is ldhD (lactate dehydrogenase), and the gene encoding the metabolic pathway protein involved in the production of ( R, R ) -2,3-butanediol is ilvBN (acetohydroxyacid synthase, acetohydroxan-producing enzyme), aldB (acetolactate decarboxylase) or BDH1 (butanediol dehydrogenase), and the gene coding for the metabolic pathway protein involved in the production of 1,3-propanediol includes dhaB (glycerol dehydratase), yqhD (aldehyde dehydrogenase, aldehyde Dehydrogenase) or gdrAB (glycerol dehydratase reactivation factors), and the gene coding for the metabolic pathway protein involved in 1,4-butanediol production is cat1 (succinate CoA transferase; succinic acid CoA transferase), sucD (CoA-dependent succinic semialdehyde dehydrogenase; CoA- dependent succinic semi-aldehyde dehydrogenase), 4 hbd (NAD-dependent 4-hydroxybutyrate dehydrogenase; NAD -dependent 4-hydroxybutyrate dehydrogenase), cat2 (4 -hydroxybutyryl CoA: acetyl-CoA transferase, 4-hydroxybutyryl CoA: acetyl-CoA transferase), bld (butyraldehyde dehydrogenase (butyraldehyde dehydrogenase), bdh (butanol dehydrogenase), adhE2 (bifunctional aldehyde / alcohol dehydrogenase), or sucA (alpha-keto glutarate, ), And the gene coding for the metabolic pathway protein involved in the production of succinic acid is pyc (pyruvate carboxylase), fdh (formate dehydrogenase), gapA (glyceraldehyde phosphate dehydrogenase), gltA (citrate synthase), or sucE (succinate exporter). A gene coding for a metabolic pathway protein involved in isobutanol production may be selected from the group consisting of alsS (acetolactate synthase; acetolactate synthase), ilvC (2,3-dihydroxy isovalerate oxidoreductase; 2, Dihydroxyisovalerate oxidoreductase), ilvD (2,3-dihydroxy isovalerate dehydratase (2,3-dihydroxyisovalerate dehydratase), kivD (alpha-ketoisovalerate decarboxylase) or an adhE (alcohol dehydrogenase), and the gene coding for the metabolic pathway protein involved in 1-butanol production is atoB (acetyl-CoA acetyltransferase; acetyl -CoA acetyltransferase), thl (acetoacetyl-CoA thiolase ; acetoacetyl -CoA up tee claim), hbd (3-hydroxybutyryl- CoA dehydrogenase; 3- hydroxy-butyryl -CoA dehydrogenase ), crt (crotonase; crotonase), bcd (butyryl-CoA dehydrogenase, butyryl-CoA dehydrogenase) The gene coding for the metabolic pathway protein involved in the production of fatty acids may be characterized by being an electron transfer flavoprotein (ETF) or an aldehyde / alcohol dehydrogenase ( adhE ) -COA carboxylase, malonyl-CoA (ACP transacylase), fabBFH (beta-ketoacyl-ACP synthases, beta-ketoacyl-ACP synthase ), fabG (ketoacyl reductase), fabAZ (beta-hydroxyacyl-acyl carrier protein dehydratases), fabI (enoyl-ACP reductase, enoyl-ACP reductase ) Or tesA (thioesterase; thioesterase). The gene encoding the metabolic pathway protein involved in hydrogen production may be hya (hydrogenase), and 1-propanol Genes that encode metabolic pathway proteins involved in production include cimA (citramalate synthase), kivD (alpha-ketoisovalerate decarboxylase), or yqhD / adhE (alcohol dehydrogenase; (Acetyl CoA-acetyltransferase or thiorase), ctfAB (acetyl-CoA-acetyltransferase or thiorase), and the like. acetoacetyl-CoA: acetate / butyrate: CoA-transferase; acetoacetyl-CoA: acetate / butyrate: CoA-transferase), adc (acetoacetate decarboxylase; Acetoacetate dicarboxylase), teIIsrf (thioesterase II; thioesterase II), or ybgC (thioesterase; thioesterase), but the present invention is not limited thereto.

The term "vector" of the present invention means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. As the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Although there is no appropriate truncated enzyme cleavage site, the synthetic oligonucleotide adapters according to conventional methods can easily ligate the vector and foreign DNA using a linker.

After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook, et al., Supra. Alternatively, electroporation (Neumann, et al., EMBO J. , 1: 841, 1982) can also be used to transform these cells.

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification control regulatory sequence that functions within the selected expression host. Preferably, the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector should further comprise a useful expression marker in the eukaryotic expression host.

The host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term transformation refers to the introduction of DNA into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

In another aspect, the present invention relates to a recombinant microorganism into which said vector has been introduced.

The vector should be transformed into an appropriate host cell so that the desired protein can be expressed. In the present invention, the preferred host cell is a prokaryotic cell. Suitable prokaryotic host cells include E. coli DH5α, E. coli JM101, E. coli TOP10, E. col i K12, E.coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B , E. coli BL21, and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and species and genera of other prokaryotes and the like can also be used. In addition to the above-mentioned E. coli, Lactobacillus bacteria (lactobacillus) into Agrobacterium (Agrobacterium) genus Pseudomonas (Pseudomonas), A separation tank emptying (Rhizobium), An and the rest Nella (Shewanella) in, also Pseudomonas (Rhodopseudomonas) sp May be used as a host cell. Preferably, Agrobacterium tumefaciens, Pseudomonas fluorescens, Rhizobium meliloti, Shewanella putrefaciens, Pseudomonas aeruginosa, Pseudomonas stutzeri, Rhodopseudomonas palustris, Lactobacillus casei can be used.

The host cell used in the preferred embodiment of the present invention is Escherichia coli. Escherichia coli is one of the most physiologically, biochemically and genetically studied bacteria, and it is a useful microorganism for producing recombinant proteins and useful metabolites. Production of recombinant proteins using E. coli has many advantages such as high production efficiency, shortened time and cost, and thus occupies an important position in the development of biotechnology. In particular, since DNA double helix structure has been revealed by Watson and Crick, microbial DNA manipulation techniques have been used to produce a variety of useful materials. This DNA manipulation technique has made possible the wide industrial use of microorganisms, and accordingly, a technique for more efficiently producing a variety of useful substances has been developed. Biotechnology technology, which has been started from simple protein production, .

The wild type (WT) Nal promoter can be applied to Agrobacterium tumefaciens, Pseudomonas fluorescens, Rhizobium meliloti, etc. The Nal promoter regulated by FNR protein can be applied to hewanella putrefaciens , Pseudomonas aeruginosa , Pseudomonas stutzeri, Rhodopseudomonas palustris, Lactobacillus casei .

It will be apparent to those skilled in the art that the gene used in the present invention may be present in the host cell either independently in the plasmid or inserted into the chromosome of the host.

The term " micro-aerobic condition " of the present invention means that the dissolved oxygen amount (DO level) in the culture medium is in the range of 1-5%.

In one embodiment of the present invention, the DO level was maintained at 1-2% for induction during the batch and fed-batch processes using the bioreactor. The standard of induction was OD600 = 1.0, and before that, aerobic conditions of DO level 80% or more were maintained.

In another aspect of the present invention, there is provided a method for producing a recombinant microorganism, comprising: culturing the recombinant microorganism to express a metabolic pathway protein involved in production of a target substance; And recovering the target substance produced through the metabolic pathway activated by the expressed protein.

In the present invention, the target material may be at least one selected from the group consisting of D-lactic acid, acetone, ( R, R ) -2,3-butanediol, 1,3-propanediol, 1,4-butanediol, succinic acid, isobutanol, The present invention also relates to a pharmaceutical composition for preventing or treating inflammation caused by fatty acid, hydrogen, 1-propanol, acetone, parathyroid hormone, insulin, erythropoietin, interferon alpha, interferon beta, interferon gamma, interleukin- 2, granulocyte macrophage colony stimulating factor, Lane paracase, and streptokinase, but the present invention is not limited thereto.

The recombinant microorganism of the present invention can be cultured as needed through a culturing method conventionally used in the art, and the culture medium to be used and the culture period can be arbitrarily selected by a person skilled in the art if necessary.

Preferably, the present invention induces the production of the target protein by culturing the transformed E. coli in LB (Luria Bertani) medium.

The target substance produced by culturing in the present invention may be obtained from a culture obtained by culturing the host cell into which the vector has been introduced. The culture may contain both the host cell itself and the culture medium, and the host cell May include any material that occurs during the course of culturing.

[ Example ]

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

Example  1: Synthesis Narr  Construction of a promoter library

Sequence with Dalgarno sequence (Shine-Dalgano sequence, SD sequence, AGGAGG) and a spacer sequence (ATTACAAA) with overhangs (overhang) - XmaI recognition sequence, Shine the GFP for use as a marker protein for confirming the strength of the promoter (NotI-gfpm-R) of SEQ ID NO: 66 having the forward primer (XmaI-gfpm-F) of SEQ ID NO: 65 and the NotI recognition sequence as an overhang.

SEQ ID NO: 65 (XmaI-gfpm-F):

5'-TCCC CCCGGG AGGAGGATTACAAAATGAGTAAAGGAGAAGAACTTTT-3 '

SEQ ID NO: 66 (NotI-gfpm-R):

5'-TAAGAAT GCGGCCGC TTAGTGGTGGTGGTGGTGGTGTTTGTAGAGCTCATCGATGC-3 '

The amplified gene fragment was Xma I and to carry a promoter DNA sequence of pMW618 treated with Not I, treated with the same enzymes: was inserted into the vector containing the pUCN (positive control promoter, positive control nar promoter SEQ ID NO: 62). Xma I (XmaI-SNPL-gfpm-F) and Sph I (SNF-gfpm-F) of SEQ ID NO: 67, which have a nal promoter made by randomly sequencing the spacer sequence of 15 bp existing between the -10 and -35 conserved sequences of the promoter GFP was amplified by PCR using as template DNA the pUCN-gfpm plasmid obtained with the reverse primer (SphI-gfpm-R) of SEQ ID NO: 68 having the recognition sequence as an overhang.

SEQ ID NO: 67 (XmaI-SNPL-gfpm-F):

5'-TCCC CCCGGG CTCTTGATCGTTATCAAATCCCANNNNNNNNNNNNNNNGTATAATGCCCTT AAAAGGAGGATTACAAAATGAGTAAAGGAGAAG-3 '

SEQ ID NO: 68 (SphI-gfpm-R)

5'-ACAT GCATGC TTAGTGGTGGTGGTGGTGGTGTTTGTAGAGCTCATCGATGC-3 '

The amplified library gene fragments were inserted into the Xma I and Sph I was treated pSTV29 Xma I vector of pSTVM2 improved by removing the lac promoter (TAKARA, Japan) and Sph I seat. The obtained pSTVM2-SNPL-gfpm library plasmid was transformed into E. coli TOP10 to construct a strain library (Fig. 1).

Example  2: Synthesis Narr  Screening of Promoter Libraries

Escherichia coli having the pSTVM2-SNPL-gfpm library plasmid prepared in Example 1 was inoculated into 40 ml of LB medium (10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of yeast extract) supplemented with 50 ug / ml of chloramphenicol, Sodium chloride 5 g / L) in a 100 ml flask containing LBC medium for 12 hours at 30 DEG C and 100 rpm. The cultured strain libraries were centrifuged for 10 minutes at 4 ° C and 7000 rpm on a centrifuge to collect only the cells. After washing twice with PBS buffer solution (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM sodium phosphate diphosphate (Na ₂ HPO ₄ ), 2 mM potassium phosphate monobasic (KH ₂ PO ₄ )), The suspension was made. They were sorted using FL1 channel of flow cytometry (FACS), and the sorted cells were plated on LBC agar plates. The colonies were scraped and resuspended in a 100 ml flask containing 40 ml of LBC medium at 30 ° C, 100 rpm. This procedure was repeated three more times, and colonies grown by harvesting the obtained cells were randomly selected and cultured in 96 deep well plates to measure the fluorescence intensity thereof (FIG. 2).

Example  3: Synthesis Narr  Strength analysis of promoter

A spectrofluorometer was used to measure the fluorescence intensities of over 300 colonies randomly selected from the library of Example 2. The colonies cultured on a 96 well deep well plate were collected by centrifugation at 4 ° C and 4000 rpm, and a cell suspension was prepared with PBS buffer to measure a part of the colonies. The excitation wavelength was set to 470 nm, and the emission wavelength was set to 510 nm. Fluorescence was measured.

The RBS (ribosomal binding site) or GFP sequence was removed and the 68 clones isolated were divided into three groups. The strong promoter is a cell with an RFU / OD600 value of over 5000, and the intermediate and weak promoters are cells with a value of 1000-2000, 1000 or less, respectively, for RFU / OD600. Here, S3-2-64 (strong), W2U-30 (intermediate), and W2L-29 (weak) were selected as representatives of each group (FIG. 3).

Example  4: Synthesis Narr  Characterization of promoter

4-1: Analysis at the enterprise level

Each of the clones selected in Example 3 was collected from the cells that reached the log phase in LBC medium, and the whole RNA was extracted and subjected to real-time RT-PCR (qRT-PCR). Hybrid-R RNA purification kit from GeneAll Inc. was used for total RNA, and SensiFAST ^™ SYBR No-ROX One-step kit for qRT-PCR. The PCR was carried out using 5.0 μl of 2X SensiFAST ^™ SYBR No-ROX One-step mix, 0.1 μl of reverse transcriptase, 0.2 μl of RNas inhinitor, forward and reverse primers of 0.4 mM each gene, 2.0 μl of isolated total RNA , And 1.9 ul diethylpyrocarbonate (DEPC). The qRT-PCR conditions were 40 × cycle at 45 ° C. for 10 minutes, 95 ° C. for 2 minutes, (95 ° C. for 5 seconds, 60 ° C. for 10 seconds, and 72 ° C. for 5 seconds). The obtained qRT-PCR information was calculated as ΔΔCt. cysG is a reference gene and the gene expressed by the existing nar promoter is used as a calibrator.

4-2: Confirmation of protein expression

SDS-PAGE and western blotting were performed to confirm the expression level of GFP, a marker protein expressed by the control of a synthetic nar promoter. The cultured cells were collected and dissolved in 20 mM Tris-HCl (pH 8.0) to make a cell suspension, and the cells were disrupted by sonication. The cell lysate was centrifuged and supernatant was taken and loaded on 15% SDS-PAGE. (Horseradish peroxidase-conjugated anti-mouse IgG) conjugated with a monoclonal anti-polyhistidine antibody recognizing his-tag and horseradish peroxidase. . GAPDH was used as the reference protein.

4-3: Synthesis Narr  Sequence analysis of promoter

(SEQ ID NO: 1), W2U-30 (SEQ ID NO: 23) and W2L-29 (SEQ ID NO: 64) selected in Example 3 and the nucleotide sequence of pMW618 The Nal promoter (SEQ ID NO: 62: positive / WT) was analyzed (Figure 5 and Table 1). Followed by four strongest promoters with strongest centroids (SEQ ID NOS: 1 to 4).

SEQ ID NO: order Synthetic promoter Average intensity (RFU / OD600) Standard Deviation One ATACTGTGTTAGAGA S3-2-64 5834 575 2 ATTCGACAGATGTAA S3-2-72 5593 370 3 CAACTCATATCGAAC S3-2-24 4616 36 4 ATATAGAACGAAATG S3-2-91 4578 590 5 GACTAAACCTACCTA S3-2-12 4559 255 6 AAATCGCACTGCGCC S3-2-93 4539 325 7 CTCACAAGATACCTG S3-2-27 4414 99 8 GGACCGATCGGAGCG S3-2-15 3513 371 9 TTAGGGACACTTTAG S3-1-09 3380 420 10 TTACGTGGTGTGAGA S3-2-62 3210 15 11 ATGGAAGACCAAAGG S3-2-40 3089 245 12 AAACAAGCTAATAGG W1-03 2794 185 13 CAAAGAGTAAACTAT S3-1-25 2758 128 14 ATATGGTACGAAGGG W2U-07 2680 198 15 ATAGGTAAATTCCTG S3-1-06 2562 353 16 GCAAGATTTTTTAAA S3-1-08 2418 463 17 CGCATGATGAATGGT S3-1-18 2359 144 18 CAAGTGGTTCAAGAT W2U-01 2307 429 19 ATTTACATTCCAACG N-07 2002 352 20 CATGTGGATTGGAAT W2U-06 1981 43 21 GAGAGAGTCAAAAAT N-11 1863 69 22 GAGACGCGAAGAAAC W2U-28 1862 265 23 TCGGGTTACTAGATT W2U-30 1837 85 24 GCGTTACCGCTCAGA W2U-16 1757 214 25 TCAAGGGGAAATTGA W1-08 1751 226 26 TCCGGTGAATATAGA W2U-14 1741 280 27 CATACAATGCAGAAA W1-58 1738 193 28 ACCTGTCAGCAAATG N-13 1713 176 29 GGCCTAAGCAGAACG W2U-05 1619 213 30 GAATCACAATACGGA N-16 1586 281 31 ACGCCTAAGGCGTTC N-04 1509 187 32 ATGCAAAGAACTTAT N-09 1502 112 33 CGACAAGCCATATAC W2U-22 1492 166 34 ACCGAATGTAACGAG W2U-02 1476 18 35 ATAACGCACAGACAA W1-44 1438 151 36 CAAGAGGCAGCGCAG W1-73 1421 219 37 AGCATAGCAACAGCT W2U-12 1404 284 38 GAAAATAGGCAGTAA W1-18 1311 105 39 GGAAAATTCACAAAT N-03 1288 156 40 TAGCGTACTGGTCCA W2U-13 1257 92 41 CGAGATGAAGCCTAA W2U-24 1257 146 42 ATACAGAGCCGGAAC W2U-15 1226 207 43 AATAAATACCAAAAA W1-21 1225 39 44 GTGAGAAGCAACCAA W2U-21 1142 113 45 TATCATGTGAGAATA N-15 1087 271 46 CAAAACGACGGACCT W1-13 1067 34 47 CAGGAGTGCAAGCCA W2U-29 1053 94 48 TAAAACACCATGATC W1-89 1017 104 49 GCTGGAAATAGCCCG N-01 983 266 50 TCTAGTAGAGACAAC W1-19 963 69 51 ACGCCCGGAGCGGTC W2U-11 927 46 52 TTTGGCATCGCCCGA W1-04 864 74 53 AAGACAGGTAAAAAT N-14 852 48 54 GACCCACCATCGAAA W2U-04 786 59 55 GCCCAATAATCGTCC W2U-27 765 41 56 AATAAGGTGCACATG W2U-08 686 16 57 TGGGTCTCCAGGAGG W2U-25 677 56 58 CAAGAAAGTACAGGA W1-48 676 19 59 GGTCACGAAACGATC W1-06 595 94 60 GAGTAGAAAAACAAT N-10 591 212 61 ATGCATCGATAGTCT W1-49 555 102 62 CGCTGTTTCAGAGCG Positive 294 8 63 ACATGAGACTGTTAG N-06 218 56 64 TATTGTAACATCTCC W2L-29 161 2

Example  5: Synthesis Narr  Production of recombinant vector containing promoter

5-1: Synthesis Narr  The promoter-containing vector

To construct a plasmid containing the three kinds of promoters (S3-2-64, W2U-30, W2L-29) selected in Example 4, the nal promoter of the pUCNr plasmid was replaced with each promoter. (SEQ ID NO: 69: PnarS-R, SEQ ID NO: 70: PnarI-R, SEQ ID NO: 71: PnarW-R) containing a replaced portion of each promoter and a forward promoter attached to the -10 portion of the promoter (SEQ ID NO: 72: -10Pnar-F), the PCR was carried out using the pUCNr plasmid as a template.

SEQ ID NO: 69 (PnarS-R):

5'-TCTCTAACACAGTATTGGGATTTGATAACGATCAAG-3 '

SEQ ID NO: 70 (PnarI-R):

5'-AATCTAGTAACCCGATGGGATTTGATAACGATCAAG-3 '

SEQ ID NO: 71 (PnarW-R):

5'-GGAGATGTTACAATATGGGATTTGATAACGATCAAG-3 '

SEQ ID NO: 72 (-10 Pnar-F):

5'-GTATAATGCCCTTAAATCTAGA-3 '

Each of these plasmids was named pUCNrS (including the S3-2-64 strong nar promoter), pUCNrI (including the W2U-30 intermediate nar promoter), and pUCNrW (including the W2L-29 weak nar promoter) (Fig. 6).

5-2: Vector for the production of lactic acid type D

In order to produce type D lactic acid, in order to clone ldhD , a gene encoding lactate dehydrogenase derived from Ryukonosost Citrium , gDNA of Leuconostoc citreum is used as a template and Xba I (LdhD-citreum-F-XbaI) of SEQ ID NO: 73 having an overlapping sequence of the recognition sequence and the SD sequence and the spacer sequence, and Not I The ldhD gene was amplified using the reverse primer (ldhD-citreum-R-Notl) of SEQ ID NO: 74 with the recognition sequence as an overhang.

SEQ ID NO: 73 (ldhD-citreum-F-XbaI):

5'-CTAG TCTAGA AGGAGGATTACAAAATGAAGATTTTTGCTTATGGT-3 '

SEQ ID NO: 74 (ldhD-citreum-R-Notl):

5'-TTCCCTT GCGGCCGC TTAATACTTTACAGCAATACTT-3 '

The plasmids obtained by inserting the ldhD amplified at positions Xba I and Not I of pUCNrS, pUCNrI and pUCNrW prepared in Example 5-1 were named NrL, NrSL, NrIL and NrWL (FIG. 7).

5-3: ( R, R ) Type 2,3- BDO  Vector for production

IlvBN encoding an acetohydrolic acid- producing enzyme derived from E. coli to produce (R, R) type 2,3-BDO, acetolactate decarboxylase derived from Lactococcus lactis AldB encoding yeast , bacillus yeast ( Saccharomyces The gene was amplified using BDH1 encoding butanediol dehydrogenase derived from S. cerevisiae as a template of gDNA of each strain. ilvBN the Xba I recognition sequence and the SD sequence, having a spacer sequence as an overhang sequence number 75 of the forward primer (ilvB-EC-F-XbaI ) and a Not I recognition having the sequence as an overhang sequence number 76 reverse primers (ilvN-EC of -R-NotI), aldB is the Xba I recognition sequence and the SD sequence, the forward primer of SEQ ID NO: 77 having a spacer sequence with overhangs (aldB-LL-F-XbaI ) SEQ ID NO: 78 having a sequence with overhangs that the Not I reverse primer (aldB-LL-R-NotI ), bDH1 is Xma I recognition sequence and the SD sequence, the overhang of SEQ ID NO: 79 of the forward primer sequence that the (bdh1-SC-F-XmaI ) and Not I having the spacer sequence with overhangs (Bdh1-SC-R-NotI) of SEQ ID NO: 80 was used as the primer.

SEQ ID NO: 75 (ilvB-EC-F-XbaI):

5'-CTAG TCTAGA AGGAGGATTACAAAATGGCAAGTTCGGGCA-3 '

SEQ ID NO: 76 (ilvN-EC-R-Notl):

5'-TTCCCTT GCGGCCGC TTACTGAAAAAACACCGCGAT-3 '

SEQ ID NO: 77 (aldB-LL-F-XbaI):

5'-CTAG TCTAGA AGGAGGATTACAAAATGACAGAAATCACACAACTT-3 '

SEQ ID NO: 78 (aldB-LL-R-NotI):

5'-TTCCCTT GCGGCCGC TCATTCAGCTACATCGATATC-3 '

SEQ ID NO: 79 (bdh1-SC-F-XmaI):

5'-TCCC CCCGGG AGGAGGATTACAAAATGAGAGCTTTGGCATATTTC-3 '

SEQ ID NO: 80 (bdh1-SC-R-NotI):

5'-TTCCCTT GCGGCCGC TTACTTCATTTCACCGTGATT-3 '

The gene amplified by PCR was inserted into the restriction enzyme cleaved site in pUCNrS, pUCNrI, and pUCNrW prepared in Example 5-1.

In order to sub-clone ilvBN into pSTVM2 among the genes used in the 2,3-BDO biosynthetic metabolic pathway, the plasmid contains the forward primer (pSTVM2-pUC-sub-USER-3-F) PUCNrI-ilvBN, pUCNrI-ilvBN and pUCNrW-ilvBN were used as templates for amplification of each promoter, ilvBN and terminator together, using the reverse primer (pSTVM2-pUC-sub-USER-1-R) The gene was amplified using the forward primer (pUC-sub-USER-1-F) of SEQ ID NO: 83 and the reverse primer (pUC-sub-USER-3-R)

SEQ ID NO: 81 (pSTVM2-pUC-sub-USER-3-F):

5'-AGACAGUCATAAGTGCGG-3 '

SEQ ID NO: 82 (pSTVM2-pUC-sub-USER-1-R):

5'-ATGCAACUCGTAGGACAG-3 '

SEQ ID NO: 83 (pUC-sub-USER-1-F):

5'-AGTTGCAUCCCGACTGGAAAGCG-3 '

SEQ ID NO: 84 (pUC-sub-USER-3-R):

5'-ACTGTCUATGCGGTGTGAAATACCG-3 '

The amplified fragments were ligated using USER enzyme to obtain a plasmid. The subcloned plasmids were designated Si, Ii, and Wi, respectively (FIGS. 10A, 10B, and 10C).

In order to secure the SiWa plasmid, pUCNrS-ilvBN was used as a template and S3-2-64 (SEQ ID NO: 1: strong nar promoter) and ilvBN (PUC-sub-USER-1-F) of Sequence No. 83 and a reverse primer (pUC-sub-USER-2-R) of SEQ ID NO: 85 were used for amplification of the pUCNrW- W2L-29 (SEQ ID NO: 64: weak nar promoter) and aldB (PUC-sub-USER-2-F) of SEQ ID NO: 86 and a reverse primer (pUC-sub-USER-3-R) of SEQ ID NO: 84 to amplify the gene and the terminator together Respectively.

SEQ ID NO: 85 (pUC-sub-USER-2-R):

5'-ACATGGAUATGCGGTGTGAAATACC-3 '

SEQ ID NO: 86 (pUC-sub-USER-2-F):

5'-ATCCATGUCCCGACTGGAAAGCG-3 '

Similarly, ilvBN and aldB amplified, and pSTVM2, ilvBN and aldB PCR fragments amplified were ligation using USER enzyme to obtain final SiWa plasmid (FIG. 12A).

The SWS plasmid was constructed by amplifying the promoter, the gene, and the terminator portion at once using the pUCNrS-ilvBN, pUCNrW-aldB and pUCNrS-bdh1 plasmids as templates. (pUC-sub-USER-1-F) of SEQ ID NO: 83, reverse primer (pUC-sub-USER-2-R) of SEQ ID NO: 85, aldB is a forward primer of SEQ ID NO: 86 (PUC-sub-USER-5-F) of SEQ ID NO: 88 and pUC-sub-USER-5-F of SEQ ID NO: PCR was carried out using the reverse primer (pUC-sub-USER-3-R). The PCR products of pSTVM2, ilvBN, aldB, and BDH1 were ligated using USER enzyme and the final SWS plasmid was obtained.

SEQ ID NO: 87 (pUC-sub-USER-5-R):

5'-ATCGCATAUATGCGGTGTGAAATACCG-3 '

SEQ ID NO: 88 (pUC-sub-USER-5-F):

5'-ATATGCGAUCCCGACTGGAAAGCGACATGGAUATGCGGTGTGAAATACC-3 '

Example  6: Production of D-lactic acid using synthetic promoter

Escherichia coli W023 into which the recombinant plasmid prepared in Example 5 was introduced was transformed into 3 ml of medium containing 100 ug / ml of ampicillin or 50 ug / ml of chloramphenicol. After incubation at 37 ° C and 250 rpm for 12 hours, 100 ml of a 100 ml flask containing 100 μg / ml of ampicillin or 100 μg / ml of ampicillin and 40 ml of LB medium containing 50 μg / ml of chloramphenicol had an initial OD ₆₀₀ value of 0.05 to inoculation to produce the D-type lactic acid to proceed with the cultivation by Miho group condition OD ₆₀₀ value when while cultured in aerobic conditions until reaching a 1.0 OD ₆₀₀ value reaches 1.0 switched to 100 rpm at 30 ℃, 250 rpm did. In the case of the production of lactic acid D, calcium carbonate (CaCO ₃ ) was added to the medium prior to incubation to maintain the pH of the medium.

As a result, the concentrations of D-lactic acid produced by NrL, NrSL, NrIL and NrWL were 18.56 ± 0.59, 18.66 ± 0.42, 18.51 ± 0.38, and 18.27 ± 0.07 g / L, respectively (Fig. 8).

NrSL produced higher yields than other promoters and fed-batch fermentation was performed to control the dissolved oxygen (DO) in the medium. 1 L culture medium was cultured in a 3 L capacity fermenter. The initial dissolved oxygen level was kept at 80% at 30 ° C and 500 rpm. When the OD ₆₀₀ value reached 10.0, the dissolved oxygen level was maintained at 1-2%. The culture medium used for fed-batch culture was MR medium containing 13.5 g / L potassium diphosphate (KH ₂ PO ₄ ), 4.0 g / L ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), 1.7 g / L citric acid), was added at 1.2 g / L magnesium sulfate _{(MgSO4 · 7H 2 O),} 100X Trace metal solution (TMS) is added to 20 g / L glucose (glucose). 100X TMS was prepared by adding 10 g / L FeSO ₄ · 7H ₂ O, 2.0 g / L calcium chloride (CaCl ₂ · 2H ₂ O), 2.25 g / L zinc sulfide (ZnSO ₄ · 7H ₂ O) (MnSO ₄ .5H ₂ O), 1.0 g / L copper sulfide (CuSO ₄ .5H ₂ O), 0.1 g / L ammonium molybdate ((NH ₄ ) ₆ Mo ₇ O ₂₄ .7H ₂ O) g / L sodium tetraborate (Na ₂ B ₄ O ₇ · 10H ₂ O), and 10 ml of TMS was added per 1 L of culture medium. 5 N ammonia water was used to maintain the pH of the medium at 7.0. When the concentration of glucose 10 g / L or less in order to maintain the carbon source and nitrogen source, a certain amount 800 g / L glucose concentrate and 50 g / L yeast extract, 15 g / L tryptone, 15 g / L magnesium sulfate (MgSO ₄ · 7H ₂ O) and 5 g / L potassium phosphate (KH ₂ PO ₄ ) mixed concentrate were added to the culture solution.

In order to observe the growth of the cells, a bacterial mass spectrophotometer was used, and in some cultures, some fermentation broth was taken and the absorbance was measured at a wavelength of 600 nm. In addition, HPLC was used for the analysis of various organic acids including glucose consumption, D-lactic acid, and ( R, R ) -2,3-BDO, and RID was used as a detector. The fermentation liquid obtained by each hour was centrifuged to separate the cells and the culture medium, and the culture medium was secured. The obtained culture medium was analyzed through filtration and filtration.

As a result, NrL, which was grown as a positive control, produced 79.00 g / L of D-lactic acid at 23 hours after cultivation. The yield and productivity of the NrL were 0.67 g / g-glucose and 3.47 g / L / h . In the case of NrSL, the yield, productivity and productivity were measured as 105.56 g / L, 0.71 g / g-glucose and 4.59 g / L / h, respectively, when cultured for 23 hours (FIG.

Example  7: Synthesis of 2,3- BDO's  production

( R, R ) -2,3-BDO biosynthetic circuit, ilvBN, aldB, and bdh1 were synthesized under the control of a synthetic promoter and cloned. The intermediate of 2,3-BDO biosynthesis circuit IlvBN was subcloned into the pSTVM2 plasmid in order to find a combination that produced a large amount of acetone. Thus, the plasmids subcloned in Example 5 were named Si, Ii, and Wi, respectively (Fig. 10). They were expressed with NrSa, NrIa, and NrWa to compare the production of acetoin. Acetone was converted to some ( R, R ) -2,3-BDO by the reaction of alcohol dehydrogenases of Escherichia coli.

As a result, in the positive control group Ni + NrNa, 6.66 g / L of acetone and 1.30 g / L of ( R, R ) -2, 3-BDO. ( R, R ) -2,3-BDO and Ii + NrIa showed the highest amount of acetone and acetoin, respectively. (Fig. 11).

Next, the production of 2,3-BDO was compared in combination with NrSb, NrIb, and NrWb by making two combinations each as one plasmid. The production of ( R, R ) -2,3-BDO showed the highest yield of 8.17 ± 0.85 g / L for the NiNa + NrNb combination used as the positive control, while the highest for the SiWa + NrSb combination was 9.67 ± 0.18 g / (Fig. 12).

Finally, the above combination was made into a single plasmid to obtain SiWaSb, followed by the same fed-batch culture as in Example 6. The fed-batch culture was initially performed in MR medium containing 20 g / L glucose. When the glucose concentration dropped below 10 g / L, glucose and medium were added.

As a result, the productivity, yield and productivity of ( R, R ) -2,3-BDO obtained through 48-hour cultivation were measured as 87.95 g / L, 0.35 g / g-glucose and 1.87 g / L / h, respectively (Fig. 14).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION &Lt; 120 > Enhanced Narn Promoter and Method for Producing Biochemical Using          the Same &Lt; 130 > P16-B311 <160> 88 <170> KoPatentin 3.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-64 <400> 1 atactgtgtt agaga 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-72 <400> 2 attcgacaga tgtaa 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-24 <400> 3 caactcatat cgaac 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-91 <400> 4 atatagaacg aaatg 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-12 <400> 5 gactaaacct accta 15 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-93 <400> 6 aaatcgcact gcgcc 15 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-27 <400> 7 ctcacaagat acctg 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-15 <400> 8 ggaccgatcg gagcg 15 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-1-09 <400> 9 ttagggacac tttag 15 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-62 <400> 10 ttacgtggtg tgaga 15 <210> 11 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-2-40 <400> 11 atggaagacc aaagg 15 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-03 <400> 12 aaacaagcta atagg 15 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-1-25 <400> 13 caaagagtaa actat 15 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-07 <400> 14 atatggtacg aaggg 15 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-1-06 <400> 15 ataggtaaat tcctg 15 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-1-08 <400> 16 gcaagatttt ttaaa 15 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S3-1-18 <400> 17 cgcatgatga atggt 15 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-01 <400> 18 caagtggttc aagat 15 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-07 <400> 19 atttacattc caacg 15 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-06 <400> 20 catgtggatt ggaat 15 <210> 21 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-11 <400> 21 gagagagtca aaaat 15 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-28 <400> 22 gagacgcgaa gaaac 15 <210> 23 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-30 <400> 23 tcgggttact agatt 15 <210> 24 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-16 <400> 24 gcgttaccgc tcaga 15 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-08 <400> 25 tcaaggggaa attga 15 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-14 <400> 26 tccggtgaat ataga 15 <210> 27 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-58 <400> 27 catacaatgc agaaa 15 <210> 28 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-13 <400> 28 acctgtcagc aaatg 15 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-05 <400> 29 ggcctaagca gaacg 15 <210> 30 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-16 <400> 30 gaatcacaat acgga 15 <210> 31 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-04 <400> 31 acgcctaagg cgttc 15 <210> 32 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-09 <400> 32 atgcaaagaa cttat 15 <210> 33 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-22 <400> 33 cgacaagcca tatac 15 <210> 34 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-02 <400> 34 accgaatgta acgag 15 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-44 <400> 35 ataacgcaca gacaa 15 <210> 36 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-73 <400> 36 caagaggcag cgcag 15 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-12 <400> 37 agcatagcaa cagct 15 <210> 38 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-18 <400> 38 gaaaataggc agtaa 15 <210> 39 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-03 <400> 39 ggaaaattca caaat 15 <210> 40 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-13 <400> 40 tagcgtactg gtcca 15 <210> 41 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-24 <400> 41 cgagatgaag cctaa 15 <210> 42 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-15 <400> 42 atacagagcc ggaac 15 <210> 43 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-21 <400> 43 aataaatacc aaaaa 15 <210> 44 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-21 <400> 44 gtgagaagca accaa 15 <210> 45 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-15 <400> 45 tatcatgtga gaata 15 <210> 46 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-13 <400> 46 caaaacgacg gacct 15 <210> 47 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-29 <400> 47 caggagtgca agcca 15 <210> 48 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-89 <400> 48 taaaacacca tgatc 15 <210> 49 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-01 <400> 49 gctggaaata gcccg 15 <210> 50 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-19 <400> 50 tctagtagag acaac 15 <210> 51 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-11 <400> 51 acgcccggag cggtc 15 <210> 52 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-04 <400> 52 tttggcatcg cccga 15 <210> 53 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-14 <400> 53 aagacaggta aaaat 15 <210> 54 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-04 <400> 54 gacccaccat cgaaa 15 <210> 55 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-27 <400> 55 gcccaataat cgtcc 15 <210> 56 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-08 <400> 56 aataaggtgc acatg 15 <210> 57 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2U-25 <400> 57 tgggtctcca ggagg 15 <210> 58 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-48 <400> 58 caagaaagta cagga 15 <210> 59 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-06 <400> 59 ggtcacgaaa cgatc 15 <210> 60 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-10 <400> 60 gagtagaaaa acaat 15 <210> 61 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W1-49 <400> 61 atgcatcgat agtct 15 <210> 62 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Positive <400> 62 cgctgtttca gagcg 15 <210> 63 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> N-06 <400> 63 acatgagact gttag 15 <210> 64 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> W2L-29 <400> 64 tattgtaaca tctcc 15 <210> 65 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> XmaI-gfpm-F <400> 65 tccccccggg aggaggatta caaaatgagt aaaggagaag aactttt 47 <210> 66 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> NotI-gfpm-R <400> 66 taagaatgcg gccgcttagt ggtggtggtg gtggtgtttg tagagctcat cgatgc 56 <210> 67 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> XmaI-SNPL-gfpm-F <400> 67 tccccccggg ctcttgatcg ttatcaaatc ccannnnnnn nnnnnnnngt ataatgccct 60 taaaaggagg attacaaaat gagtaaagga gaag 94 <210> 68 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> SphI-gfpm-R <400> 68 acatgcatgc ttagtggtgg tggtggtggt gtttgtagag ctcatcgatg c 51 <210> 69 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PnarS-R <400> 69 tctctaacac agtattggga tttgataacg atcaag 36 <210> 70 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PnarI-R <400> 70 aatctagtaa cccgatggga tttgataacg atcaag 36 <210> 71 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PnarW-R <400> 71 ggagatgtta caatatggga tttgataacg atcaag 36 <210> 72 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> -10 Pnar-F <400> 72 gtataatgcc cttaaatcta ga 22 <210> 73 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ldhD-citreum-F-XbaI <400> 73 ctagtctaga aggaggatta caaaatgaag atttttgctt atggt 45 <210> 74 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> ldhD-citreum-R-NotI <400> 74 ttcccttgcg gccgcttaat actttacagc aatactt 37 <210> 75 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ilvB-EC-F-XbaI <400> 75 ctagtctaga aggaggatta caaaatggca agttcgggca 40 <210> 76 <211> 36 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > ilvN-EC-R-NotI <400> 76 ttcccttgcg gccgcttact gaaaaaacac cgcgat 36 <210> 77 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > aldB-LL-F-XbaI <400> 77 ctagtctaga aggaggatta caaaatgaca gaaatcacac aactt 45 <210> 78 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> aldB-LL-R-NotI <400> 78 ttcccttgcg gccgctcatt cagctacatc gatatc 36 <210> 79 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> bdh1-SC-F-XmaI <400> 79 tccccccggg aggaggatta caaaatgaga gctttggcat atttc 45 <210> 80 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> bdh1-SC-R-NotI <400> 80 ttcccttgcg gccgcttact tcatttcacc gtgatt 36 <210> 81 <211> 18 <212> DNA <213> Artificial Sequence <220> PSTVM2-pUC-sub-USER-3-F <400> 81 agacagucat aagtgcgg 18 <210> 82 <211> 18 <212> DNA <213> Artificial Sequence <220> PSTVM2-pUC-sub-USER-1-R <400> 82 atgcaacucg taggacag 18 <210> 83 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-1-F <400> 83 agttgcaucc cgactggaaa gcg 23 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-3-R <400> 84 actgtcuatg cggtgtgaaa taccg 25 <210> 85 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-2-R <400> 85 acatggauat gcggtgtgaa atacc 25 <210> 86 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-2-F <400> 86 atccatgucc cgactggaaa gcg 23 <210> 87 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-5-R <400> 87 atcgcataua tgcggtgtga aataccg 27 <210> 88 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> pUC-sub-USER-5-F <400> 88 atatgcgauc ccgactggaa agcgacatgg auatgcggtg tgaaatacc 49

Claims (10)

A synthetic nar promoter comprising a 15 base pair polynucleotide represented by any one of SEQ ID NOS: 1 to 4 at a position between -35 and -10 conserved sequences.
delete A vector containing the synthetic Nal promoter of claim 1.
4. The vector according to claim 3, further comprising a gene encoding a metabolic pathway protein involved in the production of the target substance downstream of the synthetic nar promoter sequence.
The method of claim 4, wherein the target material is selected from the group consisting of D-lactic acid, acetone, ( R, R ) -2,3-butanediol, 1,3-propanediol, 1,4-butanediol, succinic acid, isobutanol, , Interferon alpha, interferon beta, interferon gamma, interleukin-2, granulocyte macrophage colony stimulating factor, asparaginase, alkaline phosphatase, levan fructose, Wherein the vector is selected from the group consisting of a tocopherolase, a tocopherolase and a streptokinase.
The method of claim 4, wherein the gene encoding a metabolic pathway protein involved in the target substance production is ldhD (lactate dehydrogenase; lactate dehydrogenase), ilvBN (acetohydroxyacid synthase; acetonitrile hydroxide Roxanne enzyme), aldB (acetolactate decarboxylase), BDH1 (butanediol dehydrogenase), dhaB (glycerol dehydratase), yqhD (aldehyde dehydrogenase), gdrAB (glycerol dehydratase reactivation factors), cat1 (succinate CoA transferase; acid CoA transferase), sucD (CoA-dependent succinic semialdehyde dehydrogenase; CoA- dependent succinic semi-aldehyde dehydrogenase), 4 hbd (NAD-dependent 4-hydroxybutyrate dehydrogenase; NAD -dependent 4-hydroxybutyrate dehydrogenase), cat2 ( 4-hydroxybutyryl CoA: acetyl-CoA transferase; 4- hydroxy-butyryl CoA: acetyl -CoA transferase), bld (butyraldehyde dehydrogenase; butyraldehyde dehydrogenase), bdh (butanol dehydrogenase; butanol dehydrogenase), adhE2 (bifunctional aldehyde / alcohol dehydrogenase, two functional aldehyde / alcohol dehydrogenase), sucA (alpha-keto glutarate), pyc (pyruvate carboxylase), fdh (formate dehydrogenase), gapA (glyceraldehyde phosphate dehydrogenase), gltA (citrate synthase), sucE (succinate exporter) Succinate exosphere ), alsS (acetolactate synthase), ilvC (2,3-dihydroxy isovalerate oxidoreductase, 2,3-dihydroxyisovalerate oxidoreductase), ilvD (2,3-dihydroxy isovalerate dehydratase; 2,3-dihydroxyisovalerate dehydratase), kivD (alpha-ketoisovalerate decarboxylase), adhE (alcohol dehydrogenase), atoB (acetyl-CoA acetyltransferase, acetyl-CoA acetyltransferase), thl (acetoacetyl-CoA thiolase; acetoacetyl -CoA up tee claim), hbd (3-hydroxybutyryl- CoA dehydrogenase; 3- hydroxy-butyryl -CoA dehydrogenase), crt (crotonase; chroman appear claim), bcd (butyryl-CoA dehydrogenase ; butyryl -CoA Dehydrogenase), etf (electron transfer flavoprotein; electron transfer flavin protein), adhE (aldehyde / alcohol dehydrogenase ; aldehyde / alcohol dehydrogenase), accABCD (acetyl-CoA carboxylase ; acetyl -CoA acid decarboxylase), fabD (malonyl-CoA: ACP transacylase , Malonyl-CoA: ACP transacylase), fabBFH (beta-ketoacyl-ACP synthase, beta-ketoacyl-ACP synthase), fabG (ketoacyl reductase) carrier protein dehydratases (beta-hydroxyacyl-acyl carrier protein dehydratase), fabI (enoyl-ACP reductase), tesA (thioesterase), hya (hydrogenase) (cimA (citramalate synthase), kivD (alpha-ketoisovalerate decarboxylase), yqhD / adhE (alcohol dehydrogenase), thlA / atoB (Acetyl coenzyme A acetyltransferase or Thiolase; acetyl CoA-acetyltrans Acetoacetyldicarboxylase), adc (acetoacetate dicarboxylase), ctfAB (acetoacetyl-CoA: acetate / butyrate: CoA-transferase; acetoacetyl- CoA: acetate / a thioesterase II (thioesterase II), and a ybgC (thioesterase; thioesterase).
A recombinant microorganism into which a vector of any one of claims 3 to 6 has been introduced.
The method of claim 7, wherein the Escherichia coli (E. coli), Lactobacillus bacteria (lactobacillus), An Agrobacterium (Agrobacterium) genus Pseudomonas (Pseudomonas), A separation tank emptying (Rhizobium), An and the rest Nella (Shewanella) and also in Wherein the recombinant microorganism is selected from the group consisting of strains belonging to the genus Rhodopseudomonas .
A method for producing a target material comprising the steps of:
(a) culturing a recombinant microorganism into which a vector of any one of (4) to (6) is introduced to express a metabolic pathway protein involved in production of a target substance; And
(b) recovering the target substance produced through the metabolic pathway activated by the expressed protein.
The method of claim 9, wherein the target material is selected from the group consisting of D-lactic acid, acetone, ( R, R ) -2,3-butanediol, 1,3-propanediol, 1,4-butanediol, succinic acid, isobutanol, , Interferon alpha, interferon beta, interferon gamma, interleukin-2, granulocyte macrophage colony stimulating factor, asparaginase, alkaline phosphatase, levan fructose, &Lt; / RTI &gt; tolus lanceolase and streptokinase.

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