KR102006904B1 - Microorganism including genetic modification that increase productivity of deoxyviolacein and method for producing deoxyviolacein using the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism having increased deoxybioracene production capacity and a method for producing deoxybioracene using the same. When using a recombinant microorganism having increased deoxybioracene production capacity according to one aspect, the growth rate of the microorganisms is not delayed, thereby reducing the production stage of the deoxybioracene. It can produce casein.

Description

Microorganism including genetic modification that increase productivity of deoxyviolacein and method for producing deoxyviolacein using the same}

The present invention relates to a recombinant microorganism having increased deoxybioracene production capacity and a method for producing deoxybioracene using the same.

From the use of antibiotics in humans, the problem of superbacteria that are resistant to antibiotics has emerged, and the amount and amount of superbacteria increases with increasing use of antibiotics. In the case of antibiotic resistant strains, effective bactericidal and bacteriostatic effects cannot be achieved using conventional antibiotics, and as a result, infection in hospitals is greatly increased, thereby increasing the demand for substances capable of antibiotic action against antibiotic resistant bacteria.

Deoxyviolacein, on the other hand, is a co-metabolite of bioracein and has antitumor activity. It is known to have a biological activity against. As deoxybioracene effectively removes MRSA, which currently accounts for most of hospital infections, the demand for deoxybioracene is increasing. However, deoxybioracene is produced endogenously from wild-type bacteria, but is produced in very low concentrations, so it cannot be separated and purified and mass produced.

Therefore, in order to mass-produce deoxybioracene capable of effectively removing MRSA, studies on recombinant microorganisms having increased deoxybioracene production capacity and methods for producing deoxybioracene from such microorganisms are needed.

One aspect is to provide a recombinant microorganism comprising genetic modifications such that the activity of the vioABCE gene cluster is increased relative to the parent cell.

Another aspect provides a method of culturing the microorganism to produce deoxybioracene.

Another aspect provides a method for producing microorganisms with increased deoxybioracene production capacity.

As used herein, the term "increase in activity", or "increased activity" may refer to a detectable increase in the activity of a cell, polynucleotide, protein, or enzyme. An "increase in activity", or "increased activity" means a cell, polynucleotide, protein, or enzyme (eg, native or "wild type" that does not have a given genetic modification. -type) cells, polynucleotides, proteins, or enzymes), modified (eg, genetically engineered) cells, polynucleotides that are higher than the level of the comparison cell, polynucleotide, protein, or enzyme of the same type , Protein, or enzyme activity. "Cell activity" may refer to the activity of a particular polynucleotide, protein or enzyme in a cell. For example, the activity of the modified or engineered cell, polynucleotide, protein, or enzyme may be the same type of unengineered cell, polynucleotide, protein, or enzyme, for example, wild-type cell, polynucleotide, protein, Or at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, or at least about 100% of the activity of the enzyme. It may be increased. The activity of a particular polynucleotide, protein or enzyme in the cell is at least about 5%, at least about 10%, at least about 15%, at least about 20 than the activity of the same polynucleotide, protein or enzyme in the parent cell, for example, an unengineered cell. Or at least about 30%, at least about 50%, at least about 60%, at least about 70%, or at least about 100%. Cells with increased activity of polynucleotides, proteins or enzymes can be identified using any method known in the art. The cells with increased activity may have genetic modifications that increase the activity of one or more polynucleotides, enzymes or polypeptides as compared to cells without genetic modifications.

As used herein, the term “genetic modification” refers to a modification that introduces a polynucleotide encoding a polypeptide (eg, increases the copy number of a gene), substitution, addition of one or more nucleotides to the genetic material of the parent cell. , Insertion, or deletion, or chemical alterations to the genetic material of the parent cell. Such genetic modifications include those that are heterologous, homologous, or for coding regions and functional fragments thereof for heterologous and homologous polypeptides to the referenced species. do. In addition, the genetic modification includes modification of non-coding regulatory regions that alter the expression of a gene or operon. Non-coding regions include 5'-non coding sequences and / or 3'-non coding sequences.

The term "copy number increase" may be by introduction or amplification of a gene, including the case where the genetic manipulation of a gene that does not exist in an unengineered cell. Introduction of the gene may be via a vehicle such as a vector. The introduction may be a transient introduction in which the gene is not integrated into the genome or inserted into the genome. The introduction may be made, for example, by introducing a vector into which the polynucleotide encoding the desired polypeptide is inserted, and then the vector is replicated in the cell or the polynucleotide is integrated into the genome.

Polynucleotides that are introduced externally or whose copy number is increased can be endogenous or exogenous. The endogenous gene refers to a gene existing on the genetic material contained in the microorganism. An exogenous gene means that the gene is introduced into a host cell such as to be integrated into the host cell genome, and the gene to be introduced may be homologous or heterologous to the host cell to be introduced.

"Heterologous" may mean a foreign that is not native.

The term “gene” refers to a nucleic acid fragment capable of producing an expression product, eg, an mRNA or a protein, by one or more of transcription and translation, and refers to a 5'-non-coding sequence outside the coding region or coding region (5'- and a regulatory sequence such as a non coding sequence and a 3'-non coding sequence.

"Cell", "strain", or "microorganism" are interchangeable and can include E. coli, yeast, bacteria, fungi, and the like.

In contrast, the terms "decrease in activity" or "decreased activity" as used herein are lower polynucleotides, enzymes than those measured in parental cells (eg, genetically unengineered cells). Or a cell having the activity of the polypeptide. In addition, "decrease in activity" or "decreased activity" refers to an isolated poly that has a lower activity than the original or wild-type polynucleotide, enzyme or polypeptide. Nucleotide, enzyme or polypeptide. Reduced or reduced activity includes no activity. For example, an enzyme conversion activity from a substrate to a product for a modified (eg genetically engineered) cell, or for an enzyme, is a cell or enzyme that does not have the modification, for example, a parent cell or a "wild-type. About 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 75% At least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. Reduced activity of enzymes or cells can be identified using any method known in the art. The decrease in activity is inherently genetically engineered, even when the enzyme is expressed or inactive, even when the enzyme is expressed or when the activity of the enzyme is reduced or reduced compared to cells having unmodified genes, eg, parental or wild type cells. This includes cases where the amount of expression is reduced compared to genes that are not. The cells with reduced activity may be those with genetic modifications that reduce the activity of one or more polynucleotides, enzymes or polypeptides as compared to cells without genetic modifications.

The term "parent cell" refers to an original cell, eg, a genetically unengineered cell of the same type relative to an engineered E. coli cell. For a particular genetic modification, the "parent cell" is a cell that does not have the specific genetic modification, but may be the same for other situations. Thus, the parental cells may be cells used as starting materials to produce genetically engineered E. coli cells with increased activity of a given polynucleotide, protein.

The term "parent cell" or "parent strain" may be used for the subject genetic modification. Since the parent cell is the same as the subject cell except for the genetic modification, it may be a reference cell for the genetic modification. The "genetic modification" refers to an artificial alteration of the composition or structure of the genetic material of the cell. The parent cell may be a cell that does not have a genetic modification of interest, for example, an genetic modification that causes increased activity. The parent cell may be a parental Escherichia cell.

The term “wild-type” polypeptide or polynucleotide may be a polypeptide or polynucleotide that does not have a particular genetic modification, and the genetic modification may be such that one can obtain a genetically engineered polypeptide or polynucleotide. have.

The term "disruption" refers to genetic modifications that cause the expression of a referenced gene to be reduced. The disruption may be a genetic modification that results in no expression of the gene mentioned (hereinafter referred to as "inactivation" of the gene) or a genetic modification that causes expression of the gene but is expressed at a reduced level (hereinafter referred to as a gene). Is referred to as "attenuation". The inactivation includes not only the functional product of the gene is expressed but also the expressed but not the functional product of the gene. The attenuation involves a decrease in the amount of expression of the functional product of the gene. That is, the attenuation includes a decrease in the expression level of the functional product even though the net expression level of the gene is increased. The functional product of the gene herein refers to having the biochemical or physiological function (eg, enzyme activity) of the product (eg, enzyme) of the gene in the parent cell or wild-type cell. Thus, the disruption involves functional disruption of the gene. Such genetic modifications include modifications that introduce a polynucleotide encoding a polypeptide, substitution, addition, insertion, or deletion of one or more nucleotides into a genetic material of a parental cell, or chemical variation of a genetic material of a parental cell. Such genetic modifications include those that are heterologous, homologous, or for coding regions and functional fragments thereof for heterologous and homologous polypeptides to the referenced species. do. In addition, the genetic modification includes modification of non-coding regulatory regions that alter the expression of a gene or operon. The non-coding region comprises a 5'-non coding sequence and / or a 3'-non coding sequence.

The disruption of the gene can be accomplished by genetic manipulation such as homologous recombination, directed mutagenesis, or molecular evolution. If the cell comprises a plurality of identical genes, or two or more paralogs of genes, one or more genes may be destroyed. For example, the genetic modification may be performed by transforming a cell containing a vector containing a partial sequence of a gene into the cell and culturing the cell to cause the sequence to be destroyed by homologous recombination of the sequence with the endogenous gene of the cell, Cells in which homologous recombination has occurred can be made by selecting using a selection marker.

 As used herein, the term "gene" refers to a nucleic acid fragment that expresses a particular protein, and refers to a 5'-non coding sequence and / or a 3'-non coding sequence (3'-non coding). It may or may not include a regulatory sequence of the sequence.

"Sequence identity" of a polypeptide or polynucleotide as used in the present invention means the same degree of amino acid residues or bases between the sequences after aligning the two sequences as closely as possible in a specific comparison region. Sequence identity is a value measured by optimally aligning two sequences in a specific comparison region, and a portion of the sequence in the comparison region may be added or deleted in comparison with a reference sequence. The percent sequence identity is, for example, comparing two optimally aligned sequences throughout the comparison region, determining the number of positions where identical amino acids or nucleic acids appear in both sequences to obtain the number of matched positions. By dividing the number of matched positions by the total number of positions in the comparison range (ie, range size), and multiplying the result by 100 to obtain a percentage of sequence identity. The percent sequence identity can be determined using known sequence comparison programs, and examples include BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlign ™ (DNASTAR Inc), and the like.

Different levels of sequence identity can be used to identify polypeptides or polynucleotides having the same or similar functions or activities of different species. For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% Sequence identity including at least 98%, at least 99%, at least 100%, and the like.

As used herein, the term “exogenous” refers to the introduction of a referenced molecule or referenced activity into a host cell. Molecules can be introduced, for example, by introduction of encoding nucleic acid into a host genetic material, such as by insertion into a host chromosome, or as a non-chromosomal genetic material, such as a plasmid. With respect to the expression of a coding nucleic acid, the term “exogenous” refers to the introduction of the coding nucleic acid in an expressible form into an individual. With respect to biosynthetic activity, the term “exogenous” refers to the activity introduced into host parental cells. The source may be, for example, a homologous or heterologous coding nucleic acid which expresses the mentioned activity after introduction into the host parental cell. Therefore, the term "endogenous" refers to the mentioned molecule or activity present in the host cell. Similarly, with respect to the expression of a coding nucleic acid, the term "endogenous" refers to the expression of a coding nucleic acid contained within an individual. The term "heterologous" refers to a molecule or activity from a source other than the species mentioned and the term "homologous" refers to a molecule or activity from a host parental cell. Thus, exogenous expression of a coding nucleic acid can utilize either or both heterologous or homologous coding nucleic acids.

Also, as used herein, the terms "genetic engineering" or "genetically engineered" refer to the act of introducing one or more genetic modifications to a cell or to a cell made thereby. Indicates.

As used herein, the term deoxyviolacein is a co-metabolizer of Biolacein, and the IUPAC name is (3E) -3- [5- (1H-indol-3-yl) -2. -oxo-1H-pyrrol-3-ylidene] -1H-indol-2-one and has a structure as follows.

As used herein, the term gene cluster may refer to a group of genes in which genes related to each other in a specific metabolism are closely connected in the genome.

One aspect provides recombinant microorganisms comprising genetic modifications that allow for increased activity of the vioABCE gene cluster compared to the parental cell.

The vioABCE gene cluster may be a gene cluster involved in deoxybioracene production. The vioABCE gene cluster may refer to the vioA, vioB, vioC, and vioE genes. Increasing activity of the vioABCE gene cluster may include an increase in vioABCE gene cluster expression.

The recombinant microorganism may include a genetic modification to increase the activity of the vioABCE gene cluster, or may include an exogenous vioABCE gene.

The recombinant microorganism may be a genetically engineered cell in which the activity of the vioABCE gene cluster is increased compared to the parent cell. The genetically engineered cell can produce deoxybioracene. The cell may comprise a gene encoding a vioABCE gene cluster.

The recombinant microorganism may have an increase in the number of copies of the vioABCE gene. The recombinant microorganism may include an exogenous gene encoding the vioABCE gene. That is, the copy number increase may be due to the introduction of the vioABCE gene from outside the cell into the inside. The vioABCE gene cluster is at least about 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polynucleotide sequence of SEQ ID NO: 2. It may be a polynucleotide having base sequence identity.

The exogenous gene may be appropriately regulated by an exogenous promoter operably linked to the gene. The promoter will be described later.

In addition, the recombinant microorganism may have a modification of the expression control sequence of the vioABCE gene cluster. The expression control sequence of the gene may be a promoter or terminator for the gene expression. The expression control sequence may be a sequence encoding a motif that can affect gene expression. The motif can be, for example, a secondary structure-stabilizing motif, an RNA destabilizing motif, a splice-activating motif, a polyadenylation motif, an adenine-rich sequence, or an endonuclease recognition site.

The promoter may be an exogenous promoter operably linked with the vioABCE gene. The promoter may be a constitutive promoter. The promoter may be at least about 50%, 60%, 70%, 80%, 90%, 95% or 95% homologous to the native promoter with respect to the host cell gene. In addition, the promoter may be at least about 50%, 60%, 70%, 80%, 90%, 95% or 95% homologous to a promoter for a native gene to the host cell.

The terminator may be at least about 50%, 60%, 70%, 80%, 90%, 95% or 95% homologous to the native terminator with respect to the host cell gene. In addition, the promoter may be at least about 50%, 60%, 70%, 80%, 90%, 95%, or 95% homology with a terminator for a native gene to the host cell.

The microorganism may belong to the genus Escherichia .

The microorganism is Escherichia It can be coli). The E. coli may comprise E. coli W3110, GT 1002, MG1655, or BL21 species.

The vioABCE gene cluster may include those derived from bacteria, E. coli, fungi, mammals or reptiles. The gene cluster is Escherichia , Chromobacterium , such as Escherichia coli. Genus, or Janthinobacterium It may be the vioABCE gene from the genus but is not limited now.

The vioABCE gene cluster may be included in a vector. The vector may include an origin of replication, a promoter, a polynucleotide encoding the vioABCE gene, and a terminator. The promoter is the same as described above. The terminator is the same as described above. The vector may have a sequence of SEQ ID NO: 1. Specifically, the vector may include plasmid BBa_J72214-BBa_J72090 (Addgene Plasmid # 40782). The plasmid BBa_J72214-BBa_J72090 may be prepared by introducing the vioABCE gene into the BBa_J72214 vector. In addition, the plasmid BBa_J72214-BBa_J72090 may have the structure of FIG. 7. When a polynucleotide encoding the vioABCE gene functions to produce an active polynucleotide in a cell, the polynucleotide is considered to be "functional" in the cell.

The recombinant microorganism may include a single vioABCE gene cluster, or a plurality of vioABCE gene clusters of 2 to 10 copies. The recombinant microorganism may include, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 copies of the vioABCE gene cluster. When the recombinant microorganism comprises a plurality of vioABCE gene clusters, each polynucleotide may be the same or a combination of two or more different vioABCE genes. Multiple copies of the exogenous vioABCE gene may be included in the same locus or multiple loci within the genome of the host cell, and the promoter or terminator of each copy may be the same or different.

The microorganism may be one having an increased deoxybioracene production capacity compared to the parent cell.

As used herein, the term "recombinant microorganism having increased deoxybioracene production capacity" means a microorganism or a microorganism to which deoxybioracene production capacity is imparted to a wild type microorganism that does not naturally have deoxybioracene production capacity. It refers to a microorganism having increased production capacity of deoxybioracene to wild-type microorganisms having oxybioracene production ability.

The term "increase in productivity" in the present invention may refer to a detectable increase in deoxybioracene production. Increased deoxybioracene production can be confirmed using any method known in the art.

The recombinant microorganism can shorten the time required for the step of deoxyaviorase production because the growth rate is not delayed compared to the parent cell by genetic modification. Thus, it can be used to produce deoxybioracene with high efficiency and high yield for a short time.

Another aspect provides a method of producing deoxybioracene comprising culturing the recombinant microorganism described above to produce deoxybioracene.

The "combined microorganism with increased deoxybioracene production capacity" is as described above.

The culturing can be carried out in a medium containing a carbon source, for example glucose. The medium used for culturing the microorganism may be any conventional medium suitable for the growth of host cells, such as minimal or complex medium containing appropriate supplements. Suitable media are available from commercial vendors or can be prepared according to known recipes.

The medium may be a medium that can satisfy the requirements of specific microorganisms depending on the product selected for the culture. The medium may be a medium selected from the group consisting of carbon sources, nitrogen sources, salts, trace elements, and combinations thereof. The medium may be, for example, MR medium, LB medium, HS medium or a combination thereof.

Culture conditions mean conditions for culturing microorganisms. Such culture conditions may be, for example, carbon sources, nitrogen sources or oxygen conditions used by microorganisms. Carbon sources available to microorganisms may include monosaccharides, disaccharides or polysaccharides. The carbon source may be a magnetizable sugar and may include glucose, fructose, mannose, or galactose. The nitrogen source may be an organic nitrogen compound, or an inorganic nitrogen compound. The nitrogen source may be an amino acid, an amide, an amine, a nitrate, or an ammonium salt. Oxygen conditions for culturing microorganisms include aerobic conditions of normal oxygen partial pressure, low oxygen conditions containing 0.1% to 10% oxygen in the atmosphere, or anaerobic conditions without oxygen. Metabolic pathways can be modified to suit the carbon and nitrogen sources actually available to the microorganism.

The culture conditions may be appropriately adjusted to suit the production of the product of choice, for example, deoxybioracene. The culture may be made in aerobic or anaerobic conditions for cell proliferation. The culture may be a static culture without stirring.

The method includes the step of separating deoxybioracene from the culture.

The separation can be carried out, for example, by extraction by adding ethanol to the precipitated deoxybioracene.

The separating may include a purification process. Purification can be carried out by selecting a purification process commonly used in the art.

Another aspect provides a method of making a microorganism having increased deoxybioracene production capacity, comprising introducing a gene encoding a vioABCE gene cluster into a microorganism.

The step of introducing the gene may be to introduce a plasmid containing the gene into the microorganism. In the method, the genetic modification may comprise amplifying the gene, manipulating the regulatory sequence of the gene, or manipulating the sequence of the gene itself. The gene amplification may be due to an increase in the number of copies of the gene. The manipulation can be insertion, substitution, conversion or addition of nucleotides.

According to a recombinant microorganism according to one aspect, it can be used to produce deoxybioracene with high efficiency.

According to the method for producing deoxybioracene according to another aspect, it is possible to efficiently produce deoxybioracene.

According to the method for producing a microorganism having increased production capacity of deoxybioracene according to another aspect, it is possible to efficiently produce a microorganism having an increased production capacity of deoxybioracene.

By using a recombinant microorganism with increased bioracein production capacity according to one aspect, the growth rate of the microorganisms is not delayed, thereby shortening the production step of the second metabolite, deoxybioracene, and thus, high efficiency and high yield for a short time. Deoxybioracene can be produced.

1 is a photograph of microorganisms produced in a culture medium in which the E. coli w3110 Dvio strain of one embodiment is precipitated and deoxybioracene.
FIG. 2 is a graph showing the total amount of deoxybioracene produced per 1 L (g / L) obtained by culturing the E. coli w3110 Dvio strain of one embodiment for 24 hours.
3 is a graph of growth curve (OD600) and production curve of deoxybioracene (deoxybioracene (g / L)) over time of the E. coli w3110 Dvio strain of one embodiment.
Figure 4 is a graph showing the growth of four recombinant E. coli strains (MG1655, W3110, BW25113, BL21) (No BH: BH plasmid not inserted, BH1: BH1 plasmid inserted, BH2: BH2 plasmid) .
5 is a graph showing the production amount of deoxybioracein in recombinant E. coli strain inserted with four deoxybioracein producing plasmids (No BH: BH plasmid not inserted, BH1: BH1 plasmid inserted, BH2 : Insert the BH2 plasmid).
Figure 6 is a graph showing the growth of the GPT Dvio strain over time (OD600) and deoxybioracene production (mg / L) in the GPT Dvio strain.
7 is a structural diagram of a deoxybioracene production plasmid.

It will be described in more detail through the following examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

Example  One. vioABCE  Production of Escherichia Coli with Gene Cluster

In this example, it was confirmed whether deoxybioracene production ability was increased in the recombinant microorganism prepared by introducing the vioABCE gene cluster into E. coli W3110, a wild type E. coli strain. The specific process is as follows.

After washing the E. coli W3110 strain (ATCC 17325) in a 10% solution of glycerol to remove salts, plasmid BBa_J72214-BBa_J72090 (Addgene, Plasmid # 40782) (SEQ ID NO: 1) was inserted into the strain using 1800V electroporation. It was. After insertion, strains in which the vioABCE gene cluster was inserted were selected by screening performed by culturing in a medium containing chloramphenicol.

The strain with increased production capacity of deoxybioracene prepared as described above was named E. coli w3110 Dvio.

Example  2. vioABCE  Of the E. coli gene cluster Deoxybioracene  Check production

In order to analyze the deoxybioracene productivity of E. coli introduced vioABCE gene cluster prepared in Example 1 was cultured using a 3L fermenter. The specific process is as follows.

2.1 Preparation of microorganisms

E. coli w3110 Dvio, a microorganism to be inoculated by streaking, was prepared by incubating in an LB plate. One colony of the cultured strain was harvested and cultured. In a 250 ml flask, 100 ml of LB culture solution was added, and pre-culture was prepared by incubating at 250 RPM for 24 hours at 37 ° C.

2.2 Preparation of Media

A culture medium of 0.9L was prepared using peptone 35g / L, yeast extract 20g / L, sodium chloride 5g / L, and tryptone 1g / L. The culture was titrated to 7.2 via sodium hydroxide.

After the liquid prepared as described above was added to the incubator, chloramphenicol was added at a concentration of 100 mg / L, and 0.5 mL of antifoam 204 was added to prepare a final culture solution.

2.3 Cultivation of Microorganisms

100 ml of LB preculture prepared in 2.1 was added to the culture prepared in 2.2 above and cultured for 24 hours at 37 ° C. and 500 RPM at 1.5vvm by air supply. The pH was adjusted to within pH 7.2 ± 0.4 through sodium hydroxide and hydrochloric acid.

2.4 Growth and Growth of Microorganisms Deoxybioracene  Production concentration check

Cultures of E. coli w3110 Dvio were collected and measured with OD600 to determine the growth of Escherichia coli. Specifically, the growth of Escherichia coli was measured according to the value of OD based on OD600. When the OD600 absorbance is 1, there is about 8 × 10 ⁸ CFU / ml, which has an arithmetically increasing value.

In addition, 1 mL of the culture solution was centrifuged and deoxybioracene was extracted using the same volume of ethanol, and the concentration was measured. Specifically, the pellet formed through the centrifuged culture was washed with ethanol. After washing 2-3 times, the color of the pellets turned white, and when the production was large, the number of washings was increased to extract deoxybioracene. The washed deoxybioracene extracted ethanol solution was then measured for absorbance at OD575. The concentration was confirmed through an absorption wavelength of 51.056 L / (g * cm).

1 is a photograph of microorganisms produced in the incubator and precipitated deoxybioracene.

FIG. 2 is a graph showing the total amount of deoxybioracene produced per 1 L for 24 hours (calculated by measuring 12 times in total, N = 12).

3 is a graph of the growth curve of E. coli w3110 Dvio and the production curve of deoxybioracene.

As shown in Figure 1, it was confirmed that the recombinant microorganism produced in the present invention produced an excessive amount of deoxybioracein to show purple and precipitated.

As shown in FIG. 2, the recombinant microorganism prepared in one example produced 1.12 g / L of deoxybioracene for 24 hours.

As shown in FIG. 3, the recombinant microorganism prepared in one example produced deoxybioracene without delay in growth rate.

Therefore, E. coli w3110 Dvio of the present invention was confirmed that the deoxybioracene production ability is significantly improved compared to wild type E. coli. In addition, E. coli w3110 Dvio can produce does not delay the growth speed of the secondary metabolite deoxy bio with high efficiency and high yield for a short period of time it is possible to shorten the production of the La-deoxy Bio La is.

Comparative Example  One. E. coli  By species Deoxybioracene Production capacity  compare

The production capacity of deoxybioracene in four different E. coli was compared. The concrete comparison process is as follows.

As described in Example 1, plasmid BBa_J72214-BBa_J72090 was inserted into wild-type E. coli strains MG1655, W3110, BW25113, and BL21 to prepare recombinant E. coli with the vioABCE gene cluster. At the same time, to determine whether additional oxygen utilization via bacterial hemoglobin is related to deoxybioracene production, bacterial hemoglobin plasmid BH1 (pUC8: 16 vgb, SC Liu et al. 1994), and BH2 (pTrc_VHb_His, Byung Hoon Jo et al . 2014) were introduced to produce recombinant E. coli. Each of the recombinant Escherichia coli was cultured for 24 hours or 48 hours to measure the growth of microorganisms and the production of deoxybioracene, and the growth of the microorganisms is shown in FIG. 4 and the production of deoxybioracene is shown in FIG. 5.

As shown in Figure 4, the growth of the microorganisms did not significantly change by the additional plasmid insertion, it was confirmed that the insertion of the vioABCE gene cluster did not significantly affect the growth of E. coli.

In addition, as shown in FIG. 5, since the degree of production of deoxybioracein did not change significantly by additional plasmid insertion, it was confirmed that additional oxygen utilization through bacterial hemoglobin was not related to deoxybioracene production. It was.

In addition, as a result of comparing the growth of microorganisms and the production of deoxybioracein, it was confirmed that the production capacity of deoxybioracene compared to the growth degree was the best in the W3110 strain of the four E. coli species.

Comparative Example  2. From Tryptophan-producing E. coli strains Deoxybioracene Production capacity  compare

Tryptophan produced E. coli strains were confirmed the deoxybioracene production capacity. The specific confirmation process is as follows.

Recombinant Escherichia coli was prepared by inserting plasmid BBa_J72214_BBa_J72090 as described in Example 1 to GPT 1002, an E. coli strain that produces tryptophan prepared by modifying some genes in E. coli w3110 strain, and named it GPT Dvio. Growth of the GPT Dvio strain (OD600) and the deoxybioracein production (deoxybioracein (mg / L)) in the GPT Dvio strain was measured and shown in Figure 6.

As shown in FIG. 6, after introducing the vioABCE gene cluster into the E. coli strain overproducing tryptophan, deoxybioracene production decreased compared to before introduction (FIG. 2, about 1.1 g / L) (about 0.55 g / L). Was confirmed.

<110> ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Microorganism including genetic modification that increase          productivity of deoxyviolacein and method for producing          deoxyviolacein using the same <130> PN119243 <150> KR 17/086614 <151> 2017-07-07 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 12793 <212> DNA <213> Artificial Sequence <220> <223> plasmid BBa_J72214-BBa_J72090 <400> 1 gaattcatga gatctttaag gaggtaaaaa aaatgaaaca ttcttccgat atctgcattg 60 ttggtgctgg tatttctggt ttgacgtgcg caagccatct gctggacagc ccggcatgcc 120 gtggtctgag cctgcgtatc tttgacatgc agcaagaagc cggtggccgt atccgcagca 180 aaatgctgga tggtaaggca agcattgaac tgggcgcagg tcgctactcc cctcagttgc 240 acccgcattt ccaaagcgca atgcagcact atagccaaaa gagcgaagtc tatccgttca 300 cccagttgaa gttcaaatct cacgtgcagc aaaagctgaa gcgcgccatg aatgaactgt 360 ccccgcgtct gaaagagcat ggtaaagaga gctttttgca gtttgtcagc cgttatcaag 420 gtcacgatag cgcggttggt atgatccgct ctatgggtta cgacgcactg ttcctgccgg 480 atatcagcgc agaaatggcc tacgacattg tgggtaagca cccggagatc cagagcgtga 540 cggacaacga cgcgaaccaa tggtttgcag cggaaacggg ctttgctggt ctgattcagg 600 gcatcaaggc taaggttaag gcggcaggtg cgcgttttag cctgggttat cgtctgctga 660 gcgtccgtac cgacggtgac ggctacctgc tgcaactggc aggtgacgac ggctggaaac 720 tggagcaccg tacccgccat ctgattctgg cgattccgcc gagcgcgatg gcgggtttga 780 atgttgattt tccagaagcc tggtccggtg cgcgctatgg cagcctgccg ctgtttaagg 840 gctttctgac gtacggtgag ccgtggtggt tggactacaa actggacgat caggtgctga 900 ttgttgacaa cccgctgcgc aaaatctatt tcaaaggcga taagtacctg ttcttctata 960 ccgatagcga gatggcgaat tactggcgcg gttgtgtcgc ggagggcgag gacggttacc 1020 tggagcaaat tcgcacccat ttggctagcg cactgggtat cgtccgtgaa cgtatcccgc 1080 aaccgctggc acacgttcac aagtattggg cgcacggcgt tgagttttgc cgtgattctg 1140 atattgacca cccgagcgca ctgtctcatc gcgacagcgg tatcatcgcg tgctccgatg 1200 cgtacacgga gcattgtggt tggatggagg gcggtctgct gagcgcccgt gaggcaagcc 1260 gtctgctgtt gcagcgtatc gccgcgtgat taaggaggta aaaaaaatga gcattctgga 1320 tttcccgcgt atccacttcc gtggctgggc ccgtgtcaat gcgccgaccg cgaaccgcga 1380 tccgcacggc cacatcgata tggccagcaa taccgtggcg atggcgggtg agccgttcga 1440 cctggcacgc catcctacgg agttccaccg tcacctgcgc tccctgggtc cgcgcttcgg 1500 cttggatggt cgtgctgacc cggaaggccc gttcagcctg gccgagggct acaacgctgc 1560 cggtaacaac cacttttcgt gggagagcgc aaccgttagc cacgtgcaat gggatggcgg 1620 tgaggcggat cgtggtgacg gtctggtcgg tgctcgtttg gcactgtggg gtcactacaa 1680 tgattatctg cgtaccacct tcaatcgtgc tcgttgggtc gacagcgacc cgacgcgccg 1740 tgacgctgca caaatctatg cgggccaatt caccattagc ccggctggtg ccggtccggg 1800 tacgccgtgg ctgtttacgg cagacattga tgatagccat ggtgcacgtt ggacgcgtgg 1860 cggccacatt gcagagcgtg gcggccactt cttggatgaa gagtttggtc tggcacgcct 1920 gtttcagttc tctgtgccga aagatcaccc acattttctg tttcacccgg gtccgtttga 1980 ttccgaggcc tggcgtcgtc tgcaattggc tctggaggat gacgacgttc tgggtctgac 2040 cgtgcaatat gcgttgttca atatgagcac cccgcctcag ccgaacagcc cggtttttca 2100 cgatatggtc ggtgttgtcg gtctgtggcg tcgtggtgaa ctggcgagct acccggctgg 2160 tcgtctgctg cgtccgcgtc aaccgggtct gggtgacctg accctgcgcg tcaacggtgg 2220 tcgcgttgcg ctgaatttgg cgtgtgccat tccgttcagc actcgtgccg cgcagccaag 2280 cgcaccggac cgcctgaccc cggacctggg tgccaaactg ccgctgggcg atctgctgct 2340 gcgtgatgag gacggcgcac tgttggcacg tgtgccgcag gctctgtacc aagactattg 2400 gacgaatcac ggtattgtgg acctgccgct gctgcgcgaa ccgcgtggta gcttgaccct 2460 gagcagcgaa ctggcggagt ggcgtgagca agactgggtc acccaaagcg acgcgtctaa 2520 cctgtacctg gaggcaccgg atcgccgtca cggtcgcttt ttccctgaga gcatcgcgct 2580 gcgcagctac tttcgcggtg aagcgcgtgc gcgtccggat atcccgcatc gtatcgaggg 2640 catgggcctg gtcggcgtcg aatctcgtca ggatggcgac gctgcggaat ggcgtctgac 2700 gggtctgcgt ccgggtccgg cacgcattgt tctggacgat ggtgccgagg cgatccctct 2760 gcgtgttctg cctgacgatt gggcgctgga tgacgcgacc gtcgaagaag tggattacgc 2820 ctttttgtac cgccacgtta tggcgtatta cgagctggtg tatccattca tgagcgacaa 2880 ggtgttttcc ctggctgatc gttgcaaatg tgaaacgtac gcacgtctga tgtggcagat 2940 gtgtgatccg cagaaccgca acaagtccta ttacatgccg agcacccgcg aactgtcggc 3000 accgaaagct cgtttgttct tgaagtatct ggcccacgtg gaaggccagg cacgcctgca 3060 agcacctccg ccagcgggtc cggcacgcat tgaatctaaa gcccagttgg cggcagagct 3120 gcgtaaagcc gtcgacctgg agctgtctgt gatgctgcaa tacctgtacg cggcgtatag 3180 cattccgaac tatgcacagg gccaacaacg tgttcgtgac ggtgcgtgga ccgccgagca 3240 gctgcaactg gcgtgcggta gcggtgaccg tcgccgtgat ggcggtattc gtgcagcact 3300 gctggaaatt gctcatgaag aaatgattca ttacctggtc gttaacaacc tgctgatggc 3360 cctgggcgag ccgttctacg cgggtgtccc gctgatgggc gaagcggcac gtcaggcgtt 3420 tggcctggac accgagttcg ctctggaacc gtttagcgaa agcacgctgg cacgttttgt 3480 tcgtctggaa tggccgcact ttatcccagc accgggcaaa tccatcgcgg actgctatgc 3540 cgccattcgt caggcgtttt tggatctgcc ggacttgttt ggtggcgagg caggtaagcg 3600 tggcggtgaa caccacctgt tcctgaatga gctgaccaac cgtgcgcatc cgggttatca 3660 actggaagtt ttcgatcgcg actcggcgct gtttggtatt gcatttgtga ccgatcaggg 3720 cgaaggtggc gctctggaca gcccgcacta cgaacatagc cattttcaac gtctgcgtga 3780 aatgagcgcg cgtatcatgg ctcaaagcgc accgttcgaa ccggcgctgc cggcgttgcg 3840 taatccggtt ctggatgaga gcccgggttg ccaacgtgtc gcagacggtc gtgcgcgtgc 3900 gctgatggca ttgtaccaag gcgtttatga gctgatgttt gcgatgatgg cgcagcactt 3960 cgccgtgaaa ccgctgggta gcttgcgtcg cagccgcctg atgaacgcag caatcgatct 4020 gatgaccggt ctgttgcgtc cgctgagctg cgcgctgatg aacctgccaa gcggcatcgc 4080 cggtcgcacg gccggtccgc cgctgccggg tccggttgac acccgtagct atgacgacta 4140 cgcgctgggc tgtcgcatgc tggcacgccg ttgcgagcgt ctgctggagc aggcgagcat 4200 gctggaaccg ggttggctgc cggatgcgca gatggagctg ctggatttct atcgtcgcca 4260 aatgctggac ttggcgtgcg gcaaactgag ccgcgaggcc taatgatcct taaggaggta 4320 aaaaaaatga aacgtgcgat tatcgttggt ggcggcctgg cgggtggcct gaccgcgatc 4380 tacctggcga agcgtggcta cgaagtgcac gtcgtggaga agcgtggtga tcctctgcgc 4440 gatctgagct cttacgtgga cgttgttagc agccgtgcga tcggcgtgag catgaccgtt 4500 cgtggtatca agagcgtttt ggctgcgggc attccgcgtg cagagctgga tgcgtgtggc 4560 gaaccgatcg tggcaatggc tttctccgtg ggtggtcagt atcgcatgcg cgaactgaag 4620 ccgttggagg atttccgtcc gctgagcttg aaccgtgcgg cgtttcaaaa gctgctgaac 4680 aaatacgcga acctggcagg cgttcgttac tactttgagc ataagtgcct ggatgttgac 4740 ctggatggta agagcgtgtt gattcagggc aaagatggtc agccgcagcg tctgcaaggt 4800 gacatgatta tcggtgcgga tggcgcccac agcgccgtcc gtcaggcgat gcagagcggc 4860 ctgcgtcgtt tcgagttcca gcaaacgttc ttccgccatg gctacaaaac cctggttttg 4920 ccggacgcgc aagcactggg ttaccgtaaa gacacgctgt actttttcgg catggattcc 4980 ggtggcctgt tcgcgggtcg tgcggctacg atcccagatg gtagcgtcag catcgccgtt 5040 tgcctgccgt actcgggtag cccttccctg acgaccaccg acgaaccgac gatgcgtgcg 5100 ttcttcgatc gttacttcgg tggcctgccg cgtgacgcgc gtgacgaaat gctgcgtcag 5160 tttctggcga agccgagcaa cgacctgatt aacgtgcgct ctagcacctt tcactataag 5220 ggtaatgtgc tgttgctggg tgatgctgcg catgcgactg cgccgttcct gggtcagggt 5280 atgaacatgg cgctggagga cgcccgcacg tttgtcgagc tgctggaccg ccaccagggc 5340 gaccaagaca aagcctttcc ggagttcacg gagctgcgca aagtccaggc agacgcaatg 5400 caagacatgg ctcgcgccaa ctatgacgtt ttgagctgct cgaacccgat ctttttcatg 5460 cgtgcgcgtt acacgcgtta catgcattcc aagtttccgg gcctgtatcc gccggatatg 5520 gccgagaaac tgtactttac gagcgagccg tacgatcgtc tgcaacaaat ccagcgtaaa 5580 cagaatgttt ggtacaagat tggtcgcgtg aattgatgat ctttaaggag gtaaaaaaaa 5640 tggagaaccg tgagccacca ctgttgccag cccgttggag cagcgcctat gtctcttatt 5700 ggagcccgat gctgccggat gaccagctga ccagcggcta ttgctggttc gactatgaac 5760 gtgacatctg tcgtattgac ggcctgttca atccgtggag cgagcgtgat actggttatc 5820 gcctgtggat gtcggaggtt ggtaatgcgg ccagcggccg tacctggaaa caaaaagtcg 5880 cctatggtcg tgagcgtacc gccctgggtg aacagctgtg tgagcgtccg ctggatgatg 5940 agactggccc ttttgccgaa ttgttcctgc cacgcgatgt cctgcgccgt ctgggtgccc 6000 gtcacattgg ccgtcgcgtg gttctgggtc gcgaagcgga cggttggcgt taccagcgcc 6060 caggtaaagg tccgagcacc ctgtacctgg atgcggcgag cggcactcca ctgcgcatgg 6120 tcaccggcga tgaagcgtcg cgtgcaagcc tgcgtgattt tccgaatgtg agcgaggcgg 6180 agatcccgga cgcggttttc gcggccaagc gctaaggatc cgaggcttgg attctcacca 6240 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc 6300 attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg 6360 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 6420 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 6480 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 6540 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 6600 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 6660 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 6720 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 6780 catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 6840 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 6900 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 6960 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc 7020 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg 7080 aaagttggaa cctcttacgt gcccgatcaa agatccgcac cgccggacat cagcgctagc 7140 ggagtgtata ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt 7200 ggcaggagaa aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc 7260 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct 7320 tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga 7380 gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct 7440 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7500 ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc 7560 tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc 7620 tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt 7680 aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact 7740 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag 7800 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc 7860 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat 7920 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta 7980 aggcctcccc tgattctgtg gataaccggg atctgtaagg atcaaccact ttgtacaaga 8040 aagctgggtc gaattgagat ccgaacggtt tattacgtac atcaggtaaa actgaccgat 8100 aagccgcttt cttttgggta tagtgtcgtg gacagtcatt catctttctg cccctccaaa 8160 agtaaaaacc cgccgaagcg ggtttttacg taaaacaggt gaaactgacc gataagccgc 8220 tttcttttgg gtatagtgtc gtggacagtc attcatcttt ctgcccctcc aaaagcaaaa 8280 acccgccgaa gcgggttttt acgtaaacca ggtgaaactg accgataagc cgctttcttt 8340 tgggtatagc gtcgtggaca gtcattcatc tttctgcccc tccaaaagca aaaacccgcc 8400 gaagcgggtt tttacgtaaa tcaggtgaaa ctgaccgata agccgggttc tgtcgtggac 8460 agtcattcat ctaggccagc aatcgctcag atccttaatc atcatccccg tacagtttgc 8520 gggtaacttc ttcgtcttcc tcctcgtctt cgtccagatc ctgttcaagc catgcttcgt 8580 ttgatatgcc ttccgcatcg acatcaccat aaccacctgt atcgacgata tcggcaattt 8640 cttctctacg atgctcaatc cacaatgcgg catcagcgcg gcggctggcg gcccgttctc 8700 gcgcgatttt atccagatcc tcaagtgatg gagcgccaga tgctgtttgg ttttcctcat 8760 catcggtatt ggtcttagga gcacgcagat cggctttgat ttgctccagc atcaggggcg 8820 gcacttttcc tccatgcgcc tcgatgaact cagccgcttc cagcactgac cagttatttt 8880 cacgctttcg ttcgtatgcc agcttaacaa tgccagcttg ccccatagat aaagcgtgct 8940 tttccgcctc ccggctttct tttcgatagt tattccggat gctgtaaatg gtgttgatca 9000 ggctgcttat ctgcgcggaa cagctgttta gcatgctcgc gatacggtat tcaggcggag 9060 taccttcatc atcgtctttt tgctgatcgc gcatttcctg caccaggcga atacacgtat 9120 ccctggcgtt ctccagcata aggagatgag aaagagactt ttccagaaga gtggtttcca 9180 gaacatcggc cccggaccga cgcaacatag cgcgcgcggc cttccgcgct tcaacgttat 9240 ctatcaggta atcgccaact tcgaactcaa agcgttcacc atcatcatcc agggtgtcgc 9300 gttccaggcg atcacgtaag gtccggtggg cgcgggtgat cacgtcatga tcatctgaac 9360 gatcatttat gcgcttattt tggcgcttcg cattctcgac tgcggcactg acaacggcat 9420 taactctttg tttttccgct atttcagccg caatgtgatc acctgcatgt tgatcattag 9480 agtgatcaat gatcatgctt tttagtggct tcctgactgg cttatttggc ttgcggctgt 9540 ccgtagtcct ggtgtcttct ttgaaggcac ggagataacg acgtgcggta ttagggttaa 9600 gattaaactc ggcggcatac tgtgcgatgg tgtaaccacc atctcgcgcc aggcgagcaa 9660 aattcttctt gtgatcgtcc caggtcactt atgcttcctt tcgagatccc aacaatattc 9720 ctactcaatg aacaaatgac tactcgtaga atcggttaac acaccagatt cagatcctta 9780 ccctctgaat cctgccggta taccccattg ttcgttatct ttatttttgg ctaaaaccgc 9840 attaagagct tcgtttaccg tcatgcaatg cggtaggtta tcgaagtttg atatcccgcc 9900 aatatcaggc gaacgcttgt ttttcaggta agcatatttc cgcgcagccg cctctacttt 9960 ctgcttgaac tcatgttttt gagtgcgttt tttggataac cgcagattgt cagcctttgc 10020 ttttgcctta gcgatccatg aagtcaattt tttgaggctg gttgttccgg caccgccgga 10080 aactgatctt tttgtttttt taacttgtga cttcttattc tttattgcca cgtcatcctg 10140 acagggggag ggggtatcat tttgacatgg gggtgtggat aaaaaattaa ataaagccaa 10200 tgtcttagcg agaacagctt taaccttggt tgccgctgac gaaatcttta atttgctttc 10260 tatcagcgca tttttggctt gttgtgcgaa ggccaaaaag gatggtgtaa accggtacag 10320 gttagcgcga cgttcacggt gatcgccgat aacaatctct acagacagaa tacctttgtt 10380 tacagcttca cggaatgcac gaacgacggt tgattggcta taaccagttt ctgccgcgat 10440 caggcggtga ggcttgtgaa tgaagtattc actggttgtt gccgcgagat ttgcacattg 10500 cgacaggata tgcccggcgc tacgggatag accggagtgt gttacaaagc aggccaattc 10560 atagccagaa aaagtaaaat cgctttaaga tccccgagtt ctcttaaacc aaggtttagg 10620 attgaaatga tgacgccgga aacttcttat aaagcgtgga aacagccaca tcatagatga 10680 ttgcaacctg cttacggggg atgcccttct ccagcaatcg ccgcatttgc tgccatgttt 10740 cttcttggta tttaggccga cgcccaccta tacgaccttc tgcgcgagct gcatcaagtc 10800 cagcgcgtgt acgttcaacg ataagctcac gttccatttc tgccagcgcc cccattacgt 10860 gaaagaaaaa gcgccccatt ggtgtactgg tgtcgatgga gtcagtgaga ctccggaagt 10920 taatgcctct gtcacgcagc tcttccacca gcacaactaa gtgacgcatg ctgcgcccaa 10980 gacggtctaa cttccatacg accagggtat cacctctgga aagttacgga gtaccttttt 11040 taacccaggg cgctcagcct ttttgccgct cgccttgtcc tcaaaaatta gctcacatcc 11100 tgcgctttca agagcgtttc gttgtaaagc agtgttttgt tcatttgttg atacgcgtac 11160 atagcctatt agagatccta taaacgcaga aaggcccacc cgaaggtgag ccagtgtgac 11220 tctagtagag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact 11280 gagcctttcg ttttatttga tgcctggaga tccttattgt gagtctggct ggttagtggc 11340 catgagatat tcgattgtgt cagtgagatc atccaggtcg tcttgggtga tgcggtactc 11400 ctgattggat atctttgagt agtgttcagc aatggcgcgg gcagcgtcgg tttcggcggg 11460 gtctacagat aaagcgttag agcaatgtct aacgtcgtcg atggttggtg gaatgaaagc 11520 cataattatg cctcactgta agatccgggt atggagaaac agtagagagt tgcgataaaa 11580 agcgtcaggt aagatccgct aatcttatgg ataaaaatgc tatggcatag caaagtgtga 11640 cgccgtgcaa ataatcaatg tggacttttc tgccgtgatt atagacactt ttgttacgcg 11700 tttttgtcat ggctttggtc ccgctttgtt acagaatgct tttaataagc ggggttaccg 11760 gtttggttag cgagaagagc cagtaaaaga cgcagtgacg gcaatgtctg atgcaatatg 11820 gacaattggt ttcttctctg aatggcggga gtatgaaaag tatggctgaa gcgcaaaatg 11880 atcccctgct gccgggatac tcgtttaatg cccatctggt ggcgggttta acgccgattg 11940 aggccaacgg ttatctcgat ttttttatcg accgaccgct gggaatgaaa ggttatattc 12000 tcaatctcac cattcgcggt cagggggtgg tgaaaaatca gggacgagaa tttgtttgcc 12060 gaccgggtga tattttgctg ttcccgccag gagagattca tcactacggt cgtcatccgg 12120 aggctcgcga atggtatcac cagtgggttt actttcgtcc gcgcgcctac tggcatgaat 12180 ggcttaactg gccgtcaata tttgccaata cggggttctt tcgcccggat gaagcgcacc 12240 agccgcattt cagcgacctg tttgggcaaa tcattaacgc cgggcaaggg gaagggcgct 12300 attcggagct gctggcgata aatctgcttg agcaattgtt actgcggcgc atggaagcga 12360 ttaacgagtc gctccatcca ccgatggata atcgggtacg cgaggcttgt cagtacatca 12420 gcgatcacct ggcagacagc aattttgata tcgccagcgt cgcacagcat gtttgcttgt 12480 cgccgtcgcg tctgtcacat cttttccgcc agcagttagg gattagcgtc ttaagctggc 12540 gcgaggacca acgtatcagc caggcgaagc tgcttttgag caccacccgg atgcctatcg 12600 ccaccgtcgg tcgcaatgtt ggttttgacg atcaactcta tttctcgcgg gtatttaaaa 12660 aatgcaccgg ggccagcccg agcgagttcc gtgccggttg tgaagaaaaa gtgaatgatg 12720 tagccgtcaa gttgtcataa ttggtaacga atcagacaat tgacggctcg atggagtagc 12780 atagagatct cat 12793 <210> 2 <211> 6743 <212> DNA <213> Unknown <220> <223> vioABCE <400> 2 gaattcatga gatctttaag gaggtaaaaa aaatgaaaca ttcttccgat atctgcattg 60 ttggtgctgg tatttctggt ttgacgtgcg caagccatct gctggacagc ccggcatgcc 120 gtggtctgag cctgcgtatc tttgacatgc agcaagaagc cggtggccgt atccgcagca 180 aaatgctgga tggtaaggca agcattgaac tgggcgcagg tcgctactcc cctcagttgc 240 acccgcattt ccaaagcgca atgcagcact atagccaaaa gagcgaagtc tatccgttca 300 cccagttgaa gttcaaatct cacgtgcagc aaaagctgaa gcgcgccatg aatgaactgt 360 ccccgcgtct gaaagagcat ggtaaagaga gctttttgca gtttgtcagc cgttatcaag 420 gtcacgatag cgcggttggt atgatccgct ctatgggtta cgacgcactg ttcctgccgg 480 atatcagcgc agaaatggcc tacgacattg tgggtaagca cccggagatc cagagcgtga 540 cggacaacga cgcgaaccaa atgaaacatt cttccgatat ctgcattgtt ggtgctggta 600 tttctggttt gacgtgcgca agccatctgc tggacagccc ggcatgccgt ggtctgagcc 660 tgcgtatctt tgacatgcag caagaagccg gtggccgtat ccgcagcaaa atgctggatg 720 gtaaggcaag cattgaactg ggcgcaggtc gctactcccc tcagttgcac ccgcatttcc 780 aaagcgcaat gcagcactat agccaaaaga gcgaagtcta tccgttcacc cagttgaagt 840 tcaaatctca cgtgcagcaa aagctgaagc gcgccatgaa tgaactgtcc ccgcgtctga 900 aagagcatgg taaagagagc tttttgcagt ttgtcagccg ttatcaaggt cacgatagcg 960 cggttggtat gatccgctct atgggttacg acgcactgtt cctgccggat atcagcgcag 1020 aaatggccta cgacattgtg ggtaagcacc cggagatcca gagcgtgacg gacaacgacg 1080 cgaaccaatg gtttgcagcg gaaacgggct ttgctggtct gattcagggc atcaaggcta 1140 aggttaaggc ggcaggtgcg cgttttagcc tgggttatcg tctgctgagc gtccgtaccg 1200 acggtgacgg ctacctgctg caactggcag gtgacgacgg ctggaaactg gagcaccgta 1260 cccgccatct gattctggcg attccgccga gcgcgatggc gggtttgaat gttgattttc 1320 cagaagcctg gtccggtgcg cgctatggca gcctgccgct gtttaagggc tttctgacgt 1380 acggtgagcc gtggtggttg gactacaaac tggacgatca ggtgctgatt gttgacaacc 1440 cgctgcgcaa aatctatttc aaaggcgata agtacctgtt cttctatacc gatagcgaga 1500 tggcgaatta ctggcgcggt tgtgtcgcgg agggcgagga cggttacctg gagcaaattc 1560 gcacccattt ggctagcgca ctgggtatcg tccgtgaacg tatcccgcaa ccgctggcac 1620 acgttcacaa gtattgggcg cacggcgttg agttttgccg tgattctgat attgaccacc 1680 cgagcgcact gtctcatcgc gacagcggta tcatcgcgtg ctccgatgcg tacacggagc 1740 attgtggttg gatggagggc ggtctgctga gcgcccgtga ggcaagccgt ctgctgttgc 1800 agcgtatcgc cgcgtgatta aggaggtaaa aaaaatgagc attctggatt tcccgcgtat 1860 ccacttccgt ggctgggccc gtgtcaatgc gccgaccgcg aaccgcgatc cgcacggcca 1920 catcgatatg gccagcaata ccgtggcgat ggcgggtgag ccgttcgacc tggcacgcca 1980 tcctacggag ttccaccgtc acctgcgctc cctgggtccg cgcttcggct tggatggtcg 2040 tgctgacccg gaaggcccgt tcagcctggc cgagggctac aacgctgccg gtaacaacca 2100 cttttcgtgg gagagcgcaa ccgttagcca cgtgcaatgg gatggcggtg aggcggatcg 2160 tggtgacggt ctggtcggtg ctcgtttggc actgtggggt cactacaatg attatctgcg 2220 taccaccttc aatcgtgctc gttgggtcga cagcgacccg acgcgccgtg acgctgcaca 2280 aatctatgcg ggccaattca ccattagccc ggctggtgcc ggtccgggta cgccgtggct 2340 gtttacggca gacattgatg atagccatgg tgcacgttgg acgcgtggcg gccacattgc 2400 agagcgtggc ggccacttct tggatgaaga gtttggtctg gcacgcctgt ttcagttctc 2460 tgtgccgaaa gatcacccac attttctgtt tcacccgggt ccgtttgatt ccgaggcctg 2520 gcgtcgtctg caattggctc tggaggatga cgacgttctg ggtctgaccg tgcaatatgc 2580 gttgttcaat atgagcaccc cgcctcagcc gaacagcccg gtttttcacg atatggtcgg 2640 tgttgtcggt ctgtggcgtc gtggtgaact ggcgagctac ccggctggtc gtctgctgcg 2700 tccgcgtcaa ccgggtctgg gtgacctgac cctgcgcgtc aacggtggtc gcgttgcgct 2760 gaatttggcg tgtgccattc cgttcagcac tcgtgccgcg cagccaagcg caccggaccg 2820 cctgaccccg gacctgggtg ccaaactgcc gctgggcgat ctgctgctgc gtgatgagga 2880 cggcgcactg ttggcacgtg tgccgcaggc tctgtaccaa gactattgga cgaatcacgg 2940 tattgtggac ctgccgctgc tgcgcgaacc gcgtggtagc ttgaccctga gcagcgaact 3000 ggcggagtgg cgtgagcaag actgggtcac ccaaagcgac gcgtctaacc tgtacctgga 3060 ggcaccggat cgccgtcacg gtcgcttttt ccctgagagc atcgcgctgc gcagctactt 3120 tcgcggtgaa gcgcgtgcgc gtccggatat cccgcatcgt atcgagggca tgggcctggt 3180 cggcgtcgaa tctcgtcagg atggcgacgc tgcggaatgg cgtctgacgg gtctgcgtcc 3240 gggtccggca cgcattgttc tggacgatgg tgccgaggcg atccctctgc gtgttctgcc 3300 tgacgattgg gcgctggatg acgcgaccgt cgaagaagtg gattacgcct ttttgtaccg 3360 ccacgttatg gcgtattacg agctggtgta tccattcatg agcgacaagg tgttttccct 3420 ggctgatcgt tgcaaatgtg aaacgtacgc acgtctgatg tggcagatgt gtgatccgca 3480 gaaccgcaac aagtcctatt acatgccgag cacccgcgaa ctgtcggcac cgaaagctcg 3540 tttgttcttg aagtatctgg cccacgtgga aggccaggca cgcctgcaag cacctccgcc 3600 agcgggtccg gcacgcattg aatctaaagc ccagttggcg gcagagctgc gtaaagccgt 3660 cgacctggag ctgtctgtga tgctgcaata cctgtacgcg gcgtatagca ttccgaacta 3720 tgcacagggc caacaacgtg ttcgtgacgg tgcgtggacc gccgagcagc tgcaactggc 3780 gtgcggtagc ggtgaccgtc gccgtgatgg cggtattcgt gcagcactgc tggaaattgc 3840 tcatgaagaa atgattcatt acctggtcgt taacaacctg ctgatggccc tgggcgagcc 3900 gttctacgcg ggtgtcccgc tgatgggcga agcggcacgt caggcgtttg gcctggacac 3960 cgagttcgct ctggaaccgt ttagcgaaag cacgctggca cgttttgttc gtctggaatg 4020 gccgcacttt atcccagcac cgggcaaatc catcgcggac tgctatgccg ccattcgtca 4080 ggcgtttttg gatctgccgg acttgtttgg tggcgaggca ggtaagcgtg gcggtgaaca 4140 ccacctgttc ctgaatgagc tgaccaaccg tgcgcatccg ggttatcaac tggaagtttt 4200 cgatcgcgac tcggcgctgt ttggtattgc atttgtgacc gatcagggcg aaggtggcgc 4260 tctggacagc ccgcactacg aacatagcca ttttcaacgt ctgcgtgaaa tgagcgcgcg 4320 tatcatggct caaagcgcac cgttcgaacc ggcgctgccg gcgttgcgta atccggttct 4380 ggatgagagc ccgggttgcc aacgtgtcgc agacggtcgt gcgcgtgcgc tgatggcatt 4440 gtaccaaggc gtttatgagc tgatgtttgc gatgatggcg cagcacttcg ccgtgaaacc 4500 gctgggtagc ttgcgtcgca gccgcctgat gaacgcagca atcgatctga tgaccggtct 4560 gttgcgtccg ctgagctgcg cgctgatgaa cctgccaagc ggcatcgccg gtcgcacggc 4620 cggtccgccg ctgccgggtc cggttgacac ccgtagctat gacgactacg cgctgggctg 4680 tcgcatgctg gcacgccgtt gcgagcgtct gctggagcag gcgagcatgc tggaaccggg 4740 ttggctgccg gatgcgcaga tggagctgct ggatttctat cgtcgccaaa tgctggactt 4800 ggcgtgcggc aaactgagcc gcgaggccta atgatcctta aggaggtaaa aaaaatgaaa 4860 cgtgcgatta tcgttggtgg cggcctggcg ggtggcctga ccgcgatcta cctggcgaag 4920 cgtggctacg aagtgcacgt cgtggagaag cgtggtgatc ctctgcgcga tctgagctct 4980 tacgtggacg ttgttagcag ccgtgcgatc ggcgtgagca tgaccgttcg tggtatcaag 5040 agcgttttgg ctgcgggcat tccgcgtgca gagctggatg cgtgtggcga accgatcgtg 5100 gcaatggctt tctccgtggg tggtcagtat cgcatgcgcg aactgaagcc gttggaggat 5160 ttccgtccgc tgagcttgaa ccgtgcggcg tttcaaaagc tgctgaacaa atacgcgaac 5220 ctggcaggcg ttcgttacta ctttgagcat aagtgcctgg atgttgacct ggatggtaag 5280 agcgtgttga ttcagggcaa agatggtcag ccgcagcgtc tgcaaggtga catgattatc 5340 ggtgcggatg gcgcccacag cgccgtccgt caggcgatgc agagcggcct gcgtcgtttc 5400 gagttccagc aaacgttctt ccgccatggc tacaaaaccc tggttttgcc ggacgcgcaa 5460 gcactgggtt accgtaaaga cacgctgtac tttttcggca tggattccgg tggcctgttc 5520 gcgggtcgtg cggctacgat cccagatggt agcgtcagca tcgccgtttg cctgccgtac 5580 tcgggtagcc cttccctgac gaccaccgac gaaccgacga tgcgtgcgtt cttcgatcgt 5640 tacttcggtg gcctgccgcg tgacgcgcgt gacgaaatgc tgcgtcagtt tctggcgaag 5700 ccgagcaacg acctgattaa cgtgcgctct agcacctttc actataaggg taatgtgctg 5760 ttgctgggtg atgctgcgca tgcgactgcg ccgttcctgg gtcagggtat gaacatggcg 5820 ctggaggacg cccgcacgtt tgtcgagctg ctggaccgcc accagggcga ccaagacaaa 5880 gcctttccgg agttcacgga gctgcgcaaa gtccaggcag acgcaatgca agacatggct 5940 cgcgccaact atgacgtttt gagctgctcg aacccgatct ttttcatgcg tgcgcgttac 6000 acgcgttaca tgcattccaa gtttccgggc ctgtatccgc cggatatggc cgagaaactg 6060 tactttacga gcgagccgta cgatcgtctg caacaaatcc agcgtaaaca gaatgtttgg 6120 tacaagattg gtcgcgtgaa ttgatgatct ttaaggaggt aaaaaaaatg gagaaccgtg 6180 agccaccact gttgccagcc cgttggagca gcgcctatgt ctcttattgg agcccgatgc 6240 tgccggatga ccagctgacc agcggctatt gctggttcga ctatgaacgt gacatctgtc 6300 gtattgacgg cctgttcaat ccgtggagcg agcgtgatac tggttatcgc ctgtggatgt 6360 cggaggttgg taatgcggcc agcggccgta cctggaaaca aaaagtcgcc tatggtcgtg 6420 agcgtaccgc cctgggtgaa cagctgtgtg agcgtccgct ggatgatgag actggccctt 6480 ttgccgaatt gttcctgcca cgcgatgtcc tgcgccgtct gggtgcccgt cacattggcc 6540 gtcgcgtggt tctgggtcgc gaagcggacg gttggcgtta ccagcgccca ggtaaaggtc 6600 cgagcaccct gtacctggat gcggcgagcg gcactccact gcgcatggtc accggcgatg 6660 aagcgtcgcg tgcaagcctg cgtgattttc cgaatgtgag cgaggcggag atcccggacg 6720 cggttttcgc ggccaagcgc taa 6743

Claims (11)

A recombinant microorganism comprising a genetic modification to increase the activity of the vioABCE gene cluster compared to the parent cell, wherein the vioABCE gene cluster has a polynucleotide sequence of SEQ ID NO. The microorganism of claim 1, wherein the genetic modification is an increase in the number of copies of the vioABCE gene cluster, a modification of an expression control sequence of the vioABCE gene cluster, or a combination thereof. The microorganism of claim 2, wherein the copy number increase is by introduction of the vioABCE gene cluster from outside to inside of the cell. delete The microorganism of claim 1, wherein the microorganism belongs to the genus Escherichia . The method of claim 5, wherein the microorganism is Escherichia coli ) . The microorganism of claim 1, wherein the microorganism has an increased deoxyviolacein production capacity as compared to the parent cell. A method of producing deoxybioracene comprising culturing the microorganism of claim 1 to produce deoxyviolacein. The method of claim 8, further comprising recovering deoxybioracene from the culture. The method of claim 8, wherein the microorganism is Escherichia coli ) . A method for producing a microorganism having increased deoxybioracene production ability, comprising introducing a gene encoding a vioABCE gene cluster into a microorganism, wherein the vioABCE gene cluster has a polynucleotide sequence of SEQ ID NO: 2. 9.

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