KR101190879B1 - Recombinant microorganism containning indole oxygenase derived from Corynebacterium and its using method of producing indirubin - Google Patents

Abstract

The present invention relates to a process for producing indirubin (indirubin) using a recombinant microorganism containing a novel indole oxygenase gene derived from Corynebacterium glutamicum , and indole oxygenase. To prepare a recombinant vector expressing tryptophanase from Escherichia coli at the same time, using this to develop a transformed recombinant Corynebacterium glutamicum KS405 (Accession No .: KCTC-11795BP) And a method for producing an indigoid compound, in particular, indirubin by culturing in a medium containing tryptophan.

Description

Recombinant microorganism containning indole oxygenase derived from Corynebacterium and its using method of producing indirubin}

The present invention relates to a method for producing indirubin using a recombinant microorganism containing a novel indole oxygenase gene derived from Corynebacterium glutamicum , and more specifically, Recombinant Corynebacterium glutamicum KS405 (Accession No .: KCTC-) was prepared using a recombinant vector expressing indole oxygenase and tryptophanase from Escherichia coli . 11795BP), and incubated in a medium containing tryptophan to produce indigoid compounds, in particular indirubin.

Indirubin is an indigoide compound that exhibits a red color that is very similar in chemical structure to indigo. In general, a small amount of by-product is produced in the process of producing indigo, a blue dye using polygonum tinctorium and isatis tinctoria. The traditional herbal medicine, Danggui Longhui Wan, consists of 11 kinds of medicines that have been used to treat chronic leukemia. Among them, indirubin has been found to be an effective drug, and recently, it is a chronic cell cycle inhibitor. It is known to have a very high medical value such as leukemia medication and neurodegenerative diseases such as Alzheimer's disease (Bri. J. Haemato., 130: 681-690, 2005 Nature Cell Biol., 1: 60-67, 1999). .

Indirubin can be prepared as a by-product in the process of producing indigo by chemical synthesis, plant extraction, microbiological biological method. In the case of the chemical synthesis method, a small amount of indirubin is synthesized in the process of synthesizing the indigo starting from o-nitrobenzaldehyde (http://en.wikipedia.org/wiki/Baeyer-Drewson_indigo_synthesis Bioorg.Med. Chem., 14: 237246, 2006). In the case of plant extraction, indigo is produced by extracting an indyl glycoside called indicant, which is a precursor of blue pigment, which is freed from fermentation and liberated indoxyl by fermentation. After the room is converted to isatin, a small amount of indirubin is produced by combining the room with isatin. However, in the case of the chemical synthesis method, there is a problem of environmental pollution due to toxic substances used in the reaction process or by-products, and the natural pigment extracted from plants has problems in terms of mass production, product standardization and economics. Therefore, as a strategy for overcoming these shortcomings, many well-known techniques for producing indigo from indole have been reported by introducing various oxygenases derived from microorganisms and animal origin into microorganisms in a biological method (J. Indus). Microbiol.Biotech., 28: 127-133, 2002; Korean Patent No. 10-0494764; Appl. Microbiol.Biotechnol. 63, 1997; Science.222: 167-169, 1983). Representative enzyme types involved in this reaction include a multicomponent system consisting of oxygenase, oxidoreductase, electron transfer protein, and the like (J. Bacteriol. 184: 344-349, 2002; Appl. Environ.Microbiol., 71: 5476-5483, 2005; Lett. Appl.Microbiol., 31: 5-9, 2000; Appl. Microbiol. Biotechnol., 66: 422-429, 2005), FAD-dependent monooxygena FAD-dependent monooxygenase (Biochem. Biophy. Res. Comm., 306: 930-936, 2003), having no homology with the above-mentioned enzymes and consisting of a single component. Environ. Microbiol., 71: 7768-7777, 2005; Kor. J. Microbiol. Biotechnol., 37: 197203, 2009; Appl. Microbiol. Biotechnol., 79: 417-422, 2008). Most oxygenases produce indigo as a main component from indole and a small amount of indirubin as a byproduct. Among them, the highest production concentration of indirubin is 35 mg / L level (Republic of Korea Patent Registration 10-0494764). On the other hand, some enzymes have been reported to form a relatively high indolebin from indole, the level is 65mg / L (Korea Patent 10-0590053), a technique that can produce indierubin with high purity is known yet none. In addition, most of the E. coli is used as a production microorganism, E. coli has a disadvantage of being unsuitable for use as a pharmaceutical production microorganism because the lipid A (lipid A) component of the extracellular membrane acts as an endotoxin. That is, indirubin has been found to be a high value-added bioactive substance with high value for various applications, but until now, it can be obtained as a by-product by indigo by chemical synthesis method, plant extraction method, and microorganism, especially, 65mg by biological method. Techniques for producing indirubin at levels below / L are known.

Therefore, the problem to be solved in the future is the development of technology capable of producing indirubin in high concentration and high purity while minimizing the concentration of indigo by using GRAS (generally regarded as safe) microorganism, which is traditionally regarded as safe for production. .

The present invention has been made in order to solve the above problems, the present inventors have been studied for the mass production of indirubin using microorganisms for many years, a novel indole oxygenase gene derived from Corynebacterium glutamicum Cloning and overexpressing in the GRAS microorganism Corynebacterium glutamicum together with E. coli-derived tryptophanase to incubate in a medium containing L-tryptophan to produce indirubin at high purity and high concentration The present invention has been completed by developing a technique capable of.

The present invention according to a preferred embodiment of the present invention to achieve the above object is an indole oxygenase gene of SEQ ID NO: 1 derived from the genus Corynebacterium and tryptopanase of SEQ ID NO: 4 derived from E. coli Provided is a method for producing indirubin using a recombinant microorganism containing a corynebacterium-derived indole oxygenase gene, characterized by generating indirubin by using a recombinant corynebacterium transformed with a vector expressing a gene. .

In addition, the transformed Corynebacterium, the indole oxygenase gene and tryptopanase gene is recombined with the "tac promoter" of SEQ ID NO: 3 and the "ilvC promoter" of SEQ ID NO: 5, respectively, the two genes at the same time Corynebacterium glutamicum ATCC13032 ( Coynebacterium glutamicum ATCC13032) transformed by the overexpressed recombinant vector is characterized in that the recombinant microorganisms (Accession Number: KCTC-11795BP).

In addition, the recombinant microorganism is characterized by producing indirubin in a medium containing tryptophan.

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 As described above, according to the present invention, a recombinant vector expressing a novel indole oxygenase gene derived from Corynebacterium glutamicum and a tryptopanase gene derived from E. coli and a recombinant corynebacterium containing the same It is possible to produce a large amount of high value-added red indirubin compound with high value for medical use by biological method.

1 is a view showing the manufacturing process of the plasmid pPIO1 of the present invention.
2 is a view showing the manufacturing process of the plasmid pCOX2 of the present invention.
Figure 3 is a view showing the manufacturing process of the plasmid pTn3 of the present invention.
4 is a view showing the manufacturing process of the plasmid pTO2C of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an indole oxygenase gene and protein having a DNA sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 isolated from Corynebacterium glutamicum ATCC13032, and having a promoter activity in Corynebacterium Provided is an indole oxygenase overexpressing recombinant vector produced by recombination with the "tac promoter" of SEQ ID NO: 3.

The present inventors have analyzed the Corynebacterium glutamicum ATCC13032 genome information. As a result, the function of the protein is not experimentally verified and only the function is estimated to be iron transport flavoprotein or a flavin-containing monojade. There were two genes named flavin-containing monooxygenase 3 (GenBank accession number CAF19847, CAF20910). Among them, the CAF19847 gene was amplified through a polymerase chain reaction (hereinafter, referred to as PCR), and introduced into an E. coli expression vector pKK223-3 containing a "tac promoter" to prepare plasmid pPIO1. As a result, the color of colonies and liquid culture liquid of recombinant E. coli cells containing pPIO1 changed to dark blue jeans. In order to express this gene in Escherichia coli and Corynebacterium glutamicum, E. coli / Corynebacterium shuttle vector pCES208 (J. Microbiol. Biotechnol., 18: 639-647, 2008) SEQ ID NO: 6 And primers described in SEQ ID NO: 7 were used to amplify by PCR using plasmid pPIO1 as a template, and then introduced into pCES208 to prepare plasmid pCOX1. E. coli W3110 containing pCOX1 was incubated for 2 days in a LB (Lolea cloning: a laboratory manual, 3rd ed. 2001) containing tryptophan. As a result of analysis by chromatography, high performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS), the resulting red compound is indirubin and the blue compound is indigo. It was confirmed that it is an indoleoxygenase that produces a colored indigoid compound (hereinafter, the gene is referred to as iog 1). The DNA base sequence of the gene consists of a total of 1,413 base pairs described in SEQ ID NO: 1, and consists of a total of 470 amino acids specified in SEQ ID NO: 2.

Gene sequences of the invention can be readily modified by one or more bases by substitutions, deletions, insertions or combinations thereof. Thus, DNAs or proteins having high homology with SEQ ID NO: 1 and SEQ ID NO: 2, such as DNAs and proteins having high homology of 70% or more, preferably 80% or more, are also within the scope of the present invention. It should be interpreted as being included.

The present invention also provides a tryptopanase gene having a DNA base sequence of SEQ ID NO: 4 isolated from E. coli. In addition, the present invention is a recombinant trippypanase gene derived from E. coli, which has the activity to make indole by dissolving tryptophan in a tripine (proteter) having a promoter (promoter) of SEQ ID NO: 5 trips produced by recombinant Provides topanase overexpressed recombinant vector.

Since Corynebacterium glutamicum lacks tryptopanases that break down tryptophan into indole, it is derived from Corynebacterium using the primers described in SEQ ID NOS: 10-15. The ilv C promoter fragment, the tnaA gene fragment from Escherichia coli and the rrnB transcriptional terminator fragment in pKK223-3 were amplified by PCR, respectively. Two overlap PCRs were performed using the amplified DNA fragments to obtain DNA fragments to which the ilv C promoter- tnaA gene-transcription terminator was linked. This fragment after double-cut with restriction enzymes Sal I, Kpn I by introducing the recombinant plasmid was prepared pCES208 pTn3.

In another aspect, the present invention provides a recombinant vector and transformed Corynebacterium glutamicum transformed with the indole oxygenase and tryptopanase gene simultaneously overexpressed. Furthermore, the present invention converts tryptophan to indole by tryptophanase activity while culturing the recombinant Corynebacterium glutamicum in a tryptophan-containing medium, and indole oxygenase activity is induced by indole oxygenase activity. Provided is a method of converting to chitin and combining indoxin and isatin to produce indirubin.

That is, the plasmid pTO2C in which iog1 and tnaA are simultaneously expressed in the pCES vector was prepared using the iog1 expression vector and tnaA expression vector described above. After incubation for 3 days in LB medium containing 10 g / L tryptophan using recombinant Corynebacterium KS405 transformed with plasmid pTO2C (Accession No .: KCTC-11795BP), 509 mg / L indigo was 45 mg / L. Confirmed to be produced. This completed the present invention by confirming that the highest concentration of high purity indirubin production technology compared to the indirubin production technology reported so far.

The host cell used in the present invention is preferably bacterial cells belonging to the genus Corynebacterium or E. coli, but is not limited to these examples.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Cloning of iog1 gene encoding indole oxygenase from Corynebacterium glutamicum.

Corynebacterium glutamicum ATCC13032 cells cultured in LB agar medium were put in 100μl of sterile tertiary distilled water, left in boiling water for about 2 minutes, suspended well, centrifuged for 1 minute, and the supernatant was taken to take genomic DNA. Used as an extract. PCR was performed using Pfu-X DNA polymerase (solgent, Inc.), 5 μl of template DNA, and 20 pmol of each primer were added. PCR conditions were repeated 30 times for 2 minutes denaturation (deaturation) at 95 ℃, denatured for 20 seconds at 95 ℃, annealing for 40 seconds at 50 ℃, (extension) for 1 minute 30 seconds at 72 ℃. Amplified DNA fragments were electrophoresed on 0.8% agarose gel to recover only about 1.4 kb (kilo base) fragments, digested with restriction enzymes Eco RI and Hind III, and then digested with Eco RI / Hind III. Recombinant plasmid pPIO1 was prepared by inserting into the cut plasmid pKK223-3 (Pharmacia Co., Ltd.) (FIG. 1).

PPIO1 was further purified from the transformant obtained by introducing the prepared plasmid into E. coli TOP10, which is an E. coli host, and then analyzed the nucleotide sequence. It was confirmed that it consists of a total of 470 amino acids.

primer  1, 2 nucleotide sequence

Primer 1 (SEQ ID NO: 6) 5'-CCCGGAATTC ATGGAGATGGTTATGAAGAA-3 '

Primer 2 (SEQ ID NO: 7) 5'-CCCCAAGCTT TTAGGCTTTATCGCGGACTT-3 '

Example 2 Preparation of Recombinant Plasmid for Indole Oxygenase Expression in Corynebacterium

In order to overexpress Iog1 in Corynebacterium, a recombinant plasmid inserted into Escherichia coli / Corynebacterium sher. PCR was carried out by adding 20 pmol of 100ng pPIO1 and primers 3 and 4 as template DNA. PCR conditions were repeated 30 times for 2 minutes denaturation at 95 ℃, 20 seconds at 95 ℃, 40 seconds at 55 ℃ decompression, extended for 2 minutes at 72 ℃. The amplified DNA fragments were electrophoresed on agarose gel to recover only 1.98 kb fragments, digested with restriction enzymes Xba I and Bam HI, and then inserted into plasmid pCES208 digested with Xba I / Bam HI to insert recombinant plasmid pCOX2. It was produced (Fig. 2). Purified pCOX1 from the transformant obtained by introducing the prepared plasmid into E. coli TOP10, which is an E. coli host, and analyzed the base sequence to confirm that there was no sequence modification. E. coli W3110 transformed with plasmid pCOX1 was finally named recombinant E. coli WCX12.

primer  3 and 4 base sequences

Primer 3 (SEQ ID NO: 8) 5'-CTAGTCTAGATCAAGGCGCACTCCCGTTCT-3 '

Primer 4 (SEQ ID NO: 9) 5'-CCGCGGATCCGCAAAAAGGCCATCCGTCAG-3 '

Example 3 Indigooid Compound Analysis

In order to analyze the indigoid compound produced by Iog1 derived from Corynebacterium, it was analyzed using HPLC and GC-MS.

Recombinant Escherichia coli WCX12 was inoculated into 500 ml LB medium (2,000 ml Erlenmeyer flask) to which 50 µg / ml of kanamycin was added and 5 g / L of tryptophan were inoculated and shaken at 33 ° C. When the OD (optical density) of the cell was 0.6, 0.1 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to incubate for 48 hours while inducing protein expression to obtain indigoid.

100 μl of the culture was taken and mixed with 900 μl DMF (dimethylformamide), and further diluted if necessary. The diluted solution was centrifuged at 13,000 rpm for 1 minute to obtain a supernatant. The supernatant was injected into HPLC Capcell Pak C-18 column (5μm, 4.6x250mm) and separated using 70% methanol as mobile phase and flowing at a flow rate of 1.0ml / min.Indirubin was measured at 540nm and 600nm at indigo. Analyzed. The results were compared with the standard synthetic indirubin and indigo, and it was confirmed that indigo showed the maximum absorbance values at the positions of 9 to 11 minutes and indirubin at the positions of 17 to 19 minutes for both the standard synthetic product and the sample obtained in the powder invention.

The indigodiate fermentation broth was centrifuged at 7,000 rpm for 20 minutes to recover the cells and indigoid precipitates, and then suspended in 40 ml of distilled water and centrifuged again. The washed precipitate was suspended in 40 ml of distilled water, and the cells and the precipitate were crushed finely with an ultrasonic wave and extracted three times with ethyl acetate. After drying it, it was dissolved in a small amount of chloroform and separated from blue pigment and red pigment by flowing a 1: 1 mixed solvent of n-hexane and ethyl acetate in a silica gel column (3.6x60 cm, 235x440 mesh). It was. The separated pigments were dissolved in chloroform and purified again in silica gel column, respectively, to prepare red pigment and blue pigment solution, which were finally dissolved in chloroform. This was analyzed using an Agilent 5973N GC-MS (USA) equipped with a VF-1 ms capillary column. The red pigment had the same retention time (18.4 minutes) as the standard synthetic indirubin and had a molecular weight of 262 Da. The blue pigment has the same retention time (17.94 minutes) as the standard synthetic indigo and its molecular weight was confirmed as 262 Da, finally confirming that the red and blue pigments produced from tryptophan in the present invention were indirubin and indigo, respectively. In addition, the iog1 gene derived from Corynebacterium glutamicum cloned and expressed in the present invention proved to be a gene encoding indole oxykenase .

Example 4 Cloning of the tnaA Gene Encoding Tryptopanase from E. Coli

TnaA from E. coli to break down tryptophan into indole in Corynebacterium glutamicum Gene, ilvC works well in Corynebacterium The transcription freezing factor rrnB present in the promoter of the gene, pKK223-3, was amplified by PCR, respectively, and then cloned into pCES208 by connecting through duplicate PCR.

The E. coli chromosome used for PCR is placed in 100 μl of E. coli W3110 cells cultured in LB agar medium in sterile tertiary distilled water and left in boiling water for about 2 minutes. The Corynebacterium chromosome was used as a DNA extract, and the Coryne genomic DNA extract obtained in [Example 1] was used.

In order to amplify the ilv C promoter, PCR was performed by adding 5 μl of coryneglutacomb chromosome extract and 20 pmol of primers 5 and 6 as template DNA. PCR conditions were repeated 30 times to denature for 2 minutes at 95 ° C, 20 seconds at 95 ° C, 40 seconds at 55 ° C, 30 seconds at 72 ° C, and electrophoresed amplified DNA fragments on an agarose gel. Fragment 1 of kb size was purified.

tnaA In order to amplify the gene, PCR was performed by adding 20 pmol of E. coli chromosome extract 5, and primers 7, 8 as template DNA. PCR conditions were performed by electrophoresis on the amplified DNA fragments on agarose gel for 30 min denaturation at 95 ° C., denaturation at 95 ° C. for 20 sec, annealing at 43 ° C. for 40 sec, and extension at 72 ° C. for 1 min 30 sec. Fragment 2 of about 1.4 kb in size was purified.

In order to amplify the transcription terminator rrnB, PCR was performed by adding 20 pmol of plasmid pKK223-3 100ng and primers 9 and 10 as template DNA. PCR conditions were repeated 30 times to denature for 2 minutes at 95 ° C, 20 seconds at 95 ° C, 40 seconds at 55 ° C, 30 seconds at 72 ° C, and electrophoresed the amplified DNA fragments on agarose gel. 0.4 kb fragment 3 was purified.

Duplicate PCR was performed over two steps to connect each amplified fragment. As a template DNA, PCR was performed by adding 100 ng of 0.3 kb fragment 1 and about 1.4 kb fragment 2, respectively, and 20 pmol of primers 5 and 8, respectively. PCR conditions were amplified by repeating the procedure of denaturation at 95 ° C. for 2 minutes, denaturation at 95 ° C. for 20 seconds, annealing at 55 ° C. for 40 seconds, stretching at 72 ° C. for 2 minutes, and then purified fragment 4 having a size of about 1.7 kb. . Then, duplicate PCR was performed by adding 100 ng of fragment 4 and fragment 0.4 of about 0.4 kb and 20 pmol of primers 5 and 10, respectively. PCR conditions were repeated 30 times for 2 minutes denaturation at 95 ℃, 20 seconds denatured at 95 ℃, 40 seconds unwinded at 55 ℃, elongation 2 minutes 10 seconds at 72 ℃. The amplified DNA fragments after the electrophoresis and recovered the fragment of about 2.1kb in size from agarose gel with restriction enzymes Sal I and Kpn I cut to the next, and then inserted into the plasmid pCES208 cut with Sal I / Kpn I recombinant plasmid pTn3 Was prepared (FIG. 3). Purified pTna3 from the transformant obtained by introducing the prepared plasmid into E. coli TOP10, an E. coli host, and analyzed the sequencing to confirm that there was no sequence modification.

primer  5 ~ 10 base sequences

Primer 5 (SEQ ID NO: 10) 5'-CCGCGGATCCCCAGGCAAGCTCCGCGCACT-3 '

Primer 6 (SEQ ID NO: 11) 5'-GGGAGAAAATCTCGCCTTTC-3 '

Primer 7 (SEQ ID NO: 12) 5'-GAAAGGCGAGATTTTCTCCCATGGAAAACTTTAAACATCT-3 '

Primer 8 (SEQ ID NO: 13) 5'-TCTCTCATCCGCCAAAACAGTTAAACTTCTTTAAGTTTTG -3 '

Primer 9 (SEQ ID NO: 14) 5'-CTGTTTTGGCGGATGAGAGA-3 '

Primer 10 (SEQ ID NO: 15) 5'-CGGGGTACCGCAAAAAGGCCATCCGTCAG-3 '

[Example 5]: Preparation of iog1 and tnaA gene co-expression plasmid

Recombinant plasmid pCOX1 was digested with restriction enzymes Xba I and Bam HI, followed by electrophoresis on agarose gel to recover only about 2 kb fragments, and digested with restriction enzymes Xba I and Bam HI, followed by Xba I / Bam. Recombinant plasmid pTO2C was constructed by inserting into recombinant plasmid pTn3 digested with HI. The prepared plasmid was introduced into E. coli TOP10, an E. coli host, to prepare a transformant obtained (FIG. 4). Finally, recombinant Corynebacterium glutamicum KS405 transformed with pTO2C was developed.

Example 6 Indigoid Production in Recombinant Escherichia Coli

Recombinant E. coli WCX12 prepared in Example 2 was added to the antibiotic kanamycin 50μg / ml, tryptophan was shaken incubation for 30 hours at 33 ℃, 30ml LB medium added 0.5 ~ 10g / L. Indigoid was produced by inducing protein expression by adding IPTG when the OD of the cells was 0.6 in the middle of the culture. 100 μl of culture was taken and mixed with 900 μl DMF, further diluted if necessary. The supernatant obtained by centrifuging the diluted solution at 13,000 rpm and 1 minute was taken and quantitatively analyzed indirubin and indigo by HPLC. As a result, it was confirmed that 319 mg / L of indirubin and 529 mg / L of indigo accumulated in the LB medium to which tryptophan was added 2.5 g / L.

Indigoid production using E. coli WCX12 in LBP medium supplemented with tryptophan Added to LB medium
Tryptophan Concentration (g / L) Generated indirubin concentration (mg / L) Indigo concentration produced (mg / L) 0.5 29 206 One 155 381 2.5 319 529 5 279 392 7.5 281 280 10 191 169

Example 7 Indigoid Production in Recombinant Corynebacterium glutamicum

Using the recombinant Corynebacterium glutamicum KS405 developed in Example 4, 50 μg / ml of kanamycin was added, and tryptophan was shaken at 33 ° C. for 72 hours in a 30 ml LB medium added with 0.5-10 g / L. 100 μl of the culture was taken and mixed with 900 μl DMF, further diluted if necessary. The supernatant obtained by centrifuging the diluted solution at 13,000 rpm and 1 minute was taken and quantitatively analyzed indirubin and indigo by HPLC. As a result, it was confirmed that 509 mg / L of indirubin and 45 mg / L of indigo accumulated in LB medium to which 10 g / L of tryptophan was added. Indigo was also produced in small amounts as a by-product and was found to produce mostly indirubin.

Indigoid production using Corynebacterium glutamicum KS405 in LB medium supplemented with tryptophan Added to LB medium
Tryptophan Concentration (g / L) Generated indirubin concentration (mg / L) Indigo concentration produced (mg / L) 0.5 30 One One 58 5 2.5 76 12 5 160 22 7.5 311 36 10 509 45

Korea Biotechnology Research Institute KCTC11795 20101022

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Claims (10)

Indyrubin using recombinant Corynebacterium transformed with a vector expressing the indole oxygenase gene of SEQ ID NO: 1 from the genus Corynebacterium and the tryptopanase gene of SEQ ID NO: 4 from E. coli Method for producing indirubin using a recombinant microorganism containing the corynebacterium-derived indole oxygenase gene, characterized in that to produce a.
delete delete delete delete delete The method of claim 1,
In the transformed recombinant Corynebacterium, the indole oxygenase gene and tryptopanase gene are recombined with the "tac promoter" of SEQ ID NO: 3 and the "ilvC promoter" of SEQ ID NO: 5, respectively, to overexpress both genes simultaneously. Corynebacterium -derived indole oxygenase gene, characterized in that it is a recombinant microorganism (Accession Number; KCTC-11795BP) based on Corynebacterium glutamicum ATCC13032 transformed by a recombinant vector Indirubin production method using a recombinant microorganism containing.
The method of claim 7, wherein
The recombinant microorganism is a method for producing indirubin using a recombinant microorganism containing the corynebacterium-derived indole oxygenase gene, characterized in that to produce indirubin in a medium containing tryptophan.
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