KR20020096181A - Producing method of pteridine compounds using recombinant bacteria - Google Patents

Abstract

PURPOSE: A method for producing pteridine compounds using a recombinant Escherichia coli is provided, thereby mass producing the pteridine compounds easily in Escherichia coli. CONSTITUTION: The method for producing pteridine compounds using a recombinant Escherichia coli comprises the steps of: constructing a recombinant vector containing an enzyme gene producing the pteridine compound; transforming Escherichia coli with the recombinant vector to prepare transformed E. coli; and culturing the transformed E. coli, wherein the enzyme gene is selected from GTPCH (GTP cyclohydrolase I) gene, PTPS gene (6-pyruvoyltetrahydropterin synthase), SR gene (sepiapterin reductase), GHluT gene (BH4:UDP-glucose-alpha -glucosyltransferase), Chlorobium tepidum SR gene and pteridine glucotransferase; the pteridine compound is sepiapterin, BH4(6R)-5,6,7,8- tetrahydro-L -biopterin, BH4-alpha -glucoside, L-threo -BH4 and BH4-(alpha or beta)-saccharide; the recombinant vector is pET-GTPCH/PTPS, pET-BH4 or pET-BH4/BGluT; and the transformed E. coli is pET-GTPCH/PTS E. coli(KCTC 1020BP), pET-BH4 E. coli, or pET-BH4/BGluT E. coli.

Description

Production method of terridine compound by recombinant E. coli {PRODUCING METHOD OF PTERIDINE COMPOUNDS USING RECOMBINANT BACTERIA}

The present invention relates to a method for preparing a terridine compound by recombinant E. coli, and more particularly, GTPCH (GTP cyclohydrolase I), PTPS (6-pyruvoyltetrahydropterin synthase), SR (sepiapterin reductase) or BGluT (BH4: UDP-glucose- The present invention relates to a method of producing α-glucosyltransferase gene in E. coli to produce sepiaterin, bioopterin and bioterin-α-glycoside.

Pteridine compounds are cyclic compounds having 2-amino-4-oxo-pyrimidine as a basic structure and there are various kinds of derivatives. Terridine is active in vivo in the fully reduced form of tetrahydro and readily converts to dihydro or fully oxidized form when exposed to air (Nichol, CA, GK Smith, and DS Duch. 1985). Ann. Rev. Biochem. 54, 729-764). In addition, terridine is characterized by fluorescence in an oxidized state.

A, (hereinafter referred to as 'BH4' and full reduction of biopterin (6 R) -5,6,7,8-tetrahydro -L-biopterin) The most well-known of the terry Compound tetrahydro bio aminopterin. The function, biosynthesis and related enzymes and genes of BH4 are known (Thony B, Auerbach, G, Blau N (2000) Biochem. J. 347, 1-16). BH4 acts as a coenzyme of aromatic amino acid hydrosilases. The deficiency of the coenzyme decreases the activity of phenylalanine hydrosilase in the liver of newborns, increasing the concentration of phenylalanine in the blood, and as a result, atypical phenylketonuria (PKU), which leads to decreased development of the central nervous system. Disease may occur (Duch DS, Smith GK (1991) J. Nutr. Biochem. 2, 411-423). Because tyrosine hydrosilase and tryptophan hydrosilase act on the biosynthesis of neurotransmitters such as dopamine and serotonin in the brain, deficiency of BH4 can lead to various neurological diseases. Deficiency of BH4 also leads to dopa-responsive dystonia, an inherited neurological disorder (Ichinose H, Ohye T, Takahashi E, Seki N, Hori T, Segawa M, Nomura Y, Endo K, Tanaka H). , Tsuji S, Fujita K, Nagatsu T (1994) Nat. Genet. 8, 236-242). In addition, research on the BH4 concentration in the body fluids in patients with Parkinson's disease, Alzheimer's disease, depression, etc. is known to be lower than normal.

Another disease associated with BH4 deficiency is melanoma, which is deficient in melanocytes in epithelial cells (Schallreuter KU, Wood JM, Pittelkow MR, Gulich M, Lemke KR, Rodl W, Swanson NN, Hitzemann K, Ziegler I (1994)). Science 263, 1444-1446), BH4 also acts as a coenzyme of nitric oxide synthase (NOS), which plays an important role in the cardiovascular system (Kaufman S (1993) Annu. Rev. Nutr. 13, 261-286). ) Is believed to cause other diseases.

The in vivo biosynthesis of the terrydine compound starts from guanosine triphosphate (hereinafter referred to as 'GTP'). (FIG. 1) GTP is GTP cyclohydrolase I (hereinafter referred to as 'GTPCH'). Is converted to dihydroneopterin triphosphate (hereinafter referred to as "H2-NTP"), which in turn is called 6-pyruvoyltetrahydropterin synthase (hereinafter referred to as "PTPS"). ) To 6-pyruvoyltetrahydropterin (hereinafter referred to as 'PPH4'). PPH4 is converted to BH4 by the coordination of aldose reductase (AR, or PPH4 reductase) and sepiaterin reductase (hereinafter referred to as SR), or by the SR enzyme alone. When only AR is present without SR, only C2 'in the side chain of PPH4 is converted to reduced 6-lactoyltetrahydropterin (hereinafter referred to as' LPH4'), and LPH4 is easily dehydrogenated by dissolved oxygen. It is oxidized to (dihydro) sepiaterin. BH4 is also produced from sepiaterin by regeneration pathways. Sepiaterin can be reduced to 7,8-dihydrobiroterin by SR in vivo and then converted to BH4 by dihydrofolate reductase (hereinafter referred to as 'DHFR'). Therefore, when added in place of BH4 in animal tissue culture, it is known to exert the same effect as the administration of BH4.

In addition, BH4-glycosides (sugar compounds) are synthesized through a reaction in which sugars are bonded to BH4. BH4-sugar compounds have been found in some anaerobic photosynthetic bacteria and cyanobacteria (Forrest HS, Van Baalen C (1970) Ann. Rev. Microbiol. 24, 91-108; Cha KW, Pfleiderer W, Yim J (1995) Helv. Chim.Acta 78, 600-614; Cho SH, Na JU, Youn H, Hwang CS, Lee CH, Kang SO. (1998) Biochim. Biophys.Acta 1379, 53-60; Chung HJ, Kim Y, Kim YJ, Choi YK, Hwang YK, Park YS (2000) Biochim. Biophys. Acta 1524, 183-18). BH4: UDP-glucose-α-glucosyltransferase (BH4: UDP-glucose-α-glucosyltransferase, hereinafter referred to as 'BGluT'), which transfers UDP-glucose sugars to BH4, is a genus of Synechococcus sp.) purified from PCC 7942 and cloned (Choi YK (2001) Cyanobacteria Synechococcus sp. Cloning of the teridine glycotransferase gene in PCC 7942. MS thesis, Inje University; Choi YK, Hwang YK, Park YS (2001) FEBS Lett. In submission). The physiological activity of BH4-glycosides has not been revealed and it is not confirmed whether they have the same activity as BH4, but it is assumed that there are pharmacological advantages such as increased water solubility and stability when sugars are bound.

BH4 is used as a treatment for atypical PKU disease (Blau N, Barnes I, Dhondt JL (1996) J. Inherit. Metab. Dis. 19, 8-14), and several mental illnesses such as Parkinson's disease, autism, depression, and Alzheimer's disease. Potential as a therapeutic for vascular disorders resulting from diseases (Thony B, Auerbach, G, Blau N (2000) Biochem. J. 347, 1-16) and various diseases such as atherosclerosis, hypertension, hypercholesterolemia, diabetes Tiefenbacher CP (2001) Am. J. Physiol. Heart Circ. Physiol. 280, H2484-H2488), and BH4 is sold as a research reagent for these diseases.

Sepiaterin can also be converted into BH4 in vivo, and results from tissue culture or animal experiments related to vascular disorders have been reported to have the same effect as BH4 (Tiefenbacher CP (2001) Am. J. Physiol. Heart Circ.Physiol. 280, H2484-H2488). There are no reports that sepiaterin has been used for clinical purposes for vascular or malformed PKU disease. However, sepiaterin has the advantage of being more stable than BH4, so if it is possible to produce cheaper than BH4, it has the potential to replace BH4. BH4 and sepiaterin are produced by organic synthesis, and mass production using microorganisms has not been reported. BH4 biosynthesis does not occur in most bacteria, such as E. coli, except for some cyanobacteria including bacteria in which BH4 sugar compounds are present.

The terridine compound is also produced by organic synthesis as a compound used for medicines and reagents. However, it is not easy to purify only one useful form of the terridine compound present as stereoisomers by organic synthesis, and it is sold at a high price, and the sugar compound is not easy to prepare by organic synthesis.

Therefore, in order to solve the above problems biotechnologically, recombinant vectors into which genes involved in terridine biosynthesis were inserted were prepared, and E. coli transformants were also prepared.

An object of the present invention is to provide a method for producing a large amount of a terridine compound in E. coli.

It is another object of the present invention to provide a recombinant vector containing a physiological enzyme for producing a terridine compound.

It is another object of the present invention to provide a transformant capable of producing a terridine compound.

It is another object of the present invention to provide a recombinant terridine compound prepared by expression in E. coli.

1 is a biosynthetic pathway of BH4 and BH4-α-glucoside,

Figure 2 shows the manufacturing process of the pET-GTPCH / PTPS vector,

Figure 3 shows the manufacturing process of the pET-BH4 vector,

Figure 4 shows the manufacturing process of the pET-BH4 / BGluT vector,

5 is a solid culture photograph of BL21 (DE3) (A), pET-GTPCH / PTPS transformant (B), pET-BH4 transformant (C), and pET-BH4 / BGluT transformant (D) ego,

6 is a liquid culture photograph of BL21 (DE3) (A), pET-GTPCH / PTPS transformant (B), pET-BH4 transformant (C), and pET-BH4 / BGluT transformant (D) ego,

7 is a graph analyzed by HPLC of the liquid medium of the pET-GTPCH / PTPS transformant,

8 is an HPLC graph of the sepiaterin on the liquid medium of the pET-GTPCH / PTPS transformant.

9 is a graph analyzing the liquid medium of the pET-BH4 transformant by HPLC,

10 is a graph analyzed by HPLC of the liquid medium of the pET-BH4 / BGluT transformant,

Figure 11 shows the absorbance of the production of sepiaterin according to the growth of the pET-GTPCH / PTPS transformant.

In order to achieve the above object, the present invention provides a recombinant vector comprising a physiological enzyme gene for producing a terridine compound. Preferably pET-GTPCH / PTPS, pET-BH4, and pET-BH4 / BGluT are provided.

The present invention also provides a transformant transformed with the recombinant vector, pET-GTPCH / PTPS transformant (KCTC 1020BP), pET-BH4 transformant, and pET-BH4 / BGluT transformant.

The present invention also provides a terridine compound or recombinant enzyme produced by the transformant.

In another aspect, the present invention is characterized by producing a recombinant vector by inserting a bioenzyme gene for producing a terridine compound in the vector, and by producing a recombinant physiological compound by simultaneously expressing the bioenzyme in a transformant containing the recombinant vector Provided is a method for producing a teridine compound using E. coli.

Hereinafter, the present invention will be described in detail.

In the present invention, a recombinant vector was prepared to produce a terridine compound in E. coli. Recombinant vectors of the invention are pET-GTPCH / PTPS, pET-BH4, and pET-BH4 / BGluT.

The pET-GTPCH / PTPS includes a GTPCH enzyme gene for converting GTP to H2-NTP and a PTPS enzyme gene for converting H2-NTP to PPH4, and PTPS cDNA (SEQ ID NO: 1) and pET15b-GTPCH of pMal-hPTPS. PET-GTPCH / PTPS was prepared by inserting the promoter and GTPCH (SEQ ID NO: 2) into the pET-28a vector. The structure of pET-GTPCH / PTPS is such that GTPCH and PTPS are expressed in each promoter as shown in FIG.

The pET-BH4 includes a GTPCH enzyme gene for converting GTP to H2-NTP, a PTPS enzyme gene for converting H2-NTP to PPH4, and an SR gene for converting PPH4 to BH4, and the pET-GTPCH / PTPS vector pET-BH4 was prepared by further inserting the SR gene (SEQ ID NO: 3) of pET15b-mSR. The structure of pET-BH4 is such that GTPCH, PTPS and SR are expressed in each promoter as shown in FIG.

The pET-BH4 / BGluT is a GTPCH enzyme gene for converting GTP to H2-NTP, a PTPS enzyme gene for converting H2-NTP to PPH4, an SR gene for converting PPH4 to BH4, and BH4 to BH4-α-glucoside BGluT was included, and pET-BH4 / BGluT was prepared by inserting the BGluT gene (SEQ ID NO: 4) of pET28a-BGluT into pET-BH4. The structure of pET-BH4 / BGluT is such that GTPCH, PTPS, SR and BGluT are expressed in each promoter as shown in FIG.

The recombinant vector was transduced into E. coli to prepare a transformant. E. coli XL-1 blue, JM101, DH5F ', TG1, M5219, G1724, BL21 (DE3), B834 (DE3), BLR (DE3), HMS174 (DE3), AD494 (DE3), NovaBlue (DE3), Origami (DE3), OrigamiB (DE3), Rosetta (DE3) and Tuner (DE3) is preferably selected from the group consisting of, more preferably those containing (DE3), most preferably BL21 (DE3) .

In the present invention, pET-GTPCH / PTPS transformant (KCTC 1020BP), pET-BH4 transformant, and pET-BH4 / BGluT transformant were prepared.

The pET-GTPCH / PTPS transformant (KCTC 1020BP) produces GTPCH and PTPS. GTPCH converts GTP to H2-NTP and PTPS converts H2-NTP to PPH4. Therefore, the pET-GTPCH / PTPS transformant (KCTC 1020BP) preferably produces PPH4, but the pET-GTPCH / PTPS transformant produced sepiaterin. Since sepiaterin is a form in which PPH4 is modified by the enzymatic reaction of AR, it is estimated that LPH4 is produced by the mechanism in the pET-GTPCH / PTPS transformant, and the sepiaterin is produced as it is oxidized.

The pET-GTPCH / PTPS transformants are also useful strains that produce 32.66 mg of sepiaterin per liter of medium, as well as optimized expression systems for producing terridine derivatives. That is, by simultaneously expressing a number of enzymes involved in the generation of the terridine derivative in the pET-GTPCH / PTPS vector it is possible to produce the final terridine derivative by the mechanism of the expressed enzymes.

In addition, the pET-BH4 transformant of the present invention express GTPCH, PTPS and SR, respectively. Thus, the pET-GTPCH / PTPS transformant only produces sepiaterin while the pET-BH4 transformant can convert sepiaterin to BH4 using the expressed SR. As a result, the pET-BH4 transformant produced BH4 together with sepiaterin.

In addition, the pET-BH4 / BGluT transformant of the present invention express GTPCH, PTPS, SR and BGluT, respectively. Therefore, the pET-BH4 / BGluT transformant produced the final product BH4-α-glucoside by GTPCH, PTPS, SR and BGluT enzymatic action of E. coli GTP, and also produced the intermediate sepiaterin.

The present invention also provides a method for producing recombinant compounds in E. coli by co-expressing a plurality of physiological enzymes in the vector. Preferably, a recombinant vector is prepared by inserting a gene of physiological enzymes involved in generating a compound in vivo into a pET vector, selecting a transformant including the recombinant vector, and then expressing each of the enzymes in the transformant. And recombinant compounds are produced by the expressed enzymes. That is, the initial material of the recombinant compound is a biomaterial of E. coli, and the enzyme is used to express the gene inserted into the recombinant vector to produce the desired compound. Preferably terridine compound, BH4, sepiaterin, BH4-α-glucoside, Chlorobium tepidum SR gene, L-threo-BH4, and BH4 sugar compounds (BH4- (α or β) -sugars To produce one or more enzyme genes selected from the group consisting of GTPCH, PTPS, SR, and BGluT (BH4: UDP-glucose-α-glucosyltransferase) and other theridine glycotransferases by recombinant vectors. It is produced by transformants.

The present invention also provides a recombinant terridine compound and a recombinant physiological enzyme. The recombinant terridine compound is preferably BH4, sepiaterin and BH4-α-glucoside, and the recombinant physiases are GTPCH, PTPS, SR, and BGluT. The BH4, sepiaterin and BH4-α-glucoside can be used for medical use, health foods, and the like for the treatment of diseases.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

(1) vectors and strains

PGEM-T Easy vector (Promega) was used for the cloning of PCR products, and pET-28a (Novagen) was used as the overexpression vector. As a cloning strain for plasmid amplification, XL-1 Blue was used and BL21 (DE3) strain was used as a host.

XL-1 Blue and BL21 strains were cultured in LB medium, and the transformant of XL-1 Blue was added to ampicillin (Am) at a concentration of 50 µg / ml, and the transformant of BL21 was kanamycin ( kanamycin; Km) was incubated in medium added at a concentration of 50 μg / ml.

(2) genes and cDNA clones

Genes and cDNA clones of GTPCH, PTPS, SR, and BGluT enzymes have already been reported and cloned into E. coli overexpression vectors pET (Novagen) or pMal-c2 (New England Biolabs) to confirm the activity of the recombinant protein. In Table 1, the vector containing each enzyme gene is shown.

enzyme Overexpression vector clone origin references Remarks GTPCH pET15b-cGTPCH Synechocystis sp. PCC6803 (ORF slr0426) Lee SW, Lee HW, Chung HJ, Kim YA, Kim YJ, Chung JH, Park YS (1999) FEMS Microbiol. Lett. 176 (1), 169-176. PTPS pMal-hPTPS Human fibroblast cDNA Burgisser DM, Thony B, Redweik U, Hunziker P, Heizmann, Blau N (1994) Eur. J. Biochem. 219, 497-502. Dr. Nenad Blau, University of Zurich, Switzerland SR pET15b-mSR Mouse Liver cDNA Seong C, Kim YA, Chung HJ, Park D, Yim J, Baek K, Park YS, Han K, Yoon J (1998) Biochim. Biophys. Acta, 1443, 239-244; Kim, YA, Chung HJ, Kim YJ, Choi YK, Hwang YK, Lee SW, Park YS (2000) Mol. Cells 10, 405-410 BGluT pET28a-BGluT Synechococcus sp. PCC 7942 Choi YK (2001) Cyanobacteria Synechococcus sp. Cloning of the Terriin Glycotransferase Gene in PCC 7942. MS thesis, Inje University; Choi YK, Hwang YK, Park YS (2001) FEBS Lett. in submission. GenBank Accession No. AF331846

Example 1-1 . pET-GTPCH / PTPS Manufacturing

The vector producing the sepiaterin protein was prepared to include a GTPCH enzyme gene that converts GTP to H2-NTP and a PTPS enzyme gene that converts H2-NTP to PPH4.

2 shows the preparation of the pET-GTPCH / PTPS vector.

The PTPS cDNA of pMal-hPTPS was PCR by using a primer of SEQ ID NO: 5 and a primer of Reverse 6. PCR reactions were performed using 10 × reaction buffer (100 mM Tris-HCl, pH 9.0, 25 ° C., 500 mM KCl, 1.0% Triton X-100), 1.5 mM MgCl _2, 0.2 mM dNTP, 0.5 pmol primer each, pMal-hPTPS The final volume was 50 μl with DNA and 2.5 units of DNA polymerase. PCR conditions were pre-denaturation (pre-denaturation: 95 ° C, 4 min), denaturation (Denaturation: 95 ° C, 1 min), annealing (55-57 ° C, 1 min), amplification (Extension: 72 ° C, 1 min) 25-30 times), and finally, the PCR reaction was terminated by final extension at 72 ° C. for 10 minutes. The reaction was confirmed by electrophoresis on 1-2% agarose gel. The amplified PTPS gene was cloned into pGEM-T Easy vector (Promega) and then cut into NdeI / BamHI to clone into pET-28a vector to prepare pET28a-hPTPS.

pET15b-GTPCH was cut with BglII / HindIII to recover the cGTPCH fragment containing the promoter site and then inserted into the pET28a-PTPS plasmid cut with BamHI / HindIII to prepare pET-GTPCH / PTPS.

Example 1-2 . Preparation of pET-BH4

PET-BH4 was prepared by further inserting the SR gene of pET15b-mSR into the pET-GTPCH / PTPS vector. pET-BH4 manufacturing process is shown in FIG.

pET15b-mSR was cut with BglII / HindIII to recover mSR fragments containing the promoter site, and then inserted into pET-GTPCH / PTPS cut with BamHI / HindIII to prepare pET-BH4. The pET-BH4 was identified in XL-1 Blue, and transformed into BL21 (DE3) to obtain a pET-BH4 transformant.

Example 1-3 . Preparation of pET-BH4 / BGluT

PET-BH4 / BGluT was prepared by inserting the BGluT gene of pET28a-BGluT into the pET-BH4 vector. The manufacturing process is as shown in FIG.

pET28a-BGluT was cut into BglII / HindIII to recover the BGluT fragment including the promoter site, and then inserted into pET-BH4 cut with BamHI / HindIII to prepare pET-BH4 / BGluT.

Example 2

(1) pET-GTPCH / PTPS transformant preparation

PET-GTPCH / PTPS prepared in Example 1-1 was transformed into XL-1 Blue (Sambrook et al., 2001) and incubated at 37 ° C. for 16 hours, followed by IPB and X-Gal plated LB- White colonies were selected in Amp ⁺ (50 μg / ml) solid medium. Selected cells were purified by vector purification kit (QIAprep spin miniprep kit, QIAGEN) and then confirmed the nucleotide sequence, and transduced to the BL21 (DE3) strain to prepare a pET-GTPCH / PTPS transformant.

(2) pET-BH4 transformant preparation

PET-BH4 prepared in Example 1-2 was transformed into XL-1 Blue, incubated at 37 ° C. for 16 hours, and then IPB and X-Gal smeared LB-Amp ⁺ (50 μg / ml) solid White colonies were selected in the medium. Selected cells were purified by vector purification kit (QIAprep spin miniprep kit, QIAGEN) and then confirmed the nucleotide sequence, and transduced to the BL21 (DE3) strain to prepare a pET-BH4 transformant.

(3) pET-BH4 / BGluT transformant preparation

PET-BH4 / BGluT prepared in Example 1-3 was transformed into XL-1 Blue, cultured at 37 ° C. for 16 hours, and then LB-Amp ⁺ (50 μg / ml smeared with IPTG and X-Gal). ) White colonies were selected in solid medium. Selected cells were purified by the purification kit (QIAprep spin miniprep kit, QIAGEN) vector DNA and then confirmed the base sequence, and transduced to the BL21 (DE3) strain to prepare a pET-BH4 / BGluT transformant.

Experimental Example 1

pET-GTPCH / PTPS transformant, pET-BH4 transformant and pET-BH4 / BGluT transformant were cultured to confirm whether recombinant protein was produced.

pET-GTPCH / PTPS transformants, pET-BH4 transformants, and pET-BH4 / BGluT transformants were grown in LB-Km ⁺ solid medium, respectively, and colonies became yellow within 24 hours without addition of IPTG. Later on, the color of the medium was observed to turn yellow. This indicates that the yellow compound is released into the medium, and the same result was obtained in the liquid culture. On the other hand, no color was observed in the culture of the untransformed BL21 strain, and the pET-BH4 transformant and the pET-BH4 / BGluT transformant showed weak yellow color. Sepiaterin is a yellow compound, and it is assumed that the transformant colony is yellow because it produces sepiaterin.

5 shows solid cultured BL21 strain (a), pET-GTPCH / PTPS transformant (b), pET-BH4 transformant (c) and pET-BH4 / BGluT transformant (d). PET-GTPCH / PTPS transformant (b), pET-BH4 transformant (c) and pET-BH4 / BGluT transformant except for the strain are yellow.

6 shows liquid cultured BL21 strain (a), pET-GTPCH / PTPS transformant (b), pET-BH4 transformant (c) and pET-BH4 / BGluT transformant (d). PET-GTPCH / PTPS transformant (b), pET-BH4 transformant (c) and pET-BH4 / BGluT transformant except for the strain are yellow.

Experimental Example 2 Confirmation of the expression of the terridine compound of the transformant

(1) pET-GTPCH / PTPS transformants

The terridine compounds present in the transformant culture were analyzed using HPLC and spectrophotometer. The terridine compound is fluorescent by acidic iodine while the sepiaterin is not fluorescent by acidic iodine. In order to identify the terridine compound in the transformant culture, acid iodine was treated and HPLC was performed in fluorescence wavelength. Sepiaterin which does not fluoresce reacted with SR and acidified iodine was again treated with acid iodine. Analysis confirmed.

After 24 hours of inoculation of the pET-GTPCH / PTPS transformant into the liquid medium, the culture was centrifuged and the supernatant was mixed with an equal volume of acidic iodine solution (2% KI / 1% I ₂ in 1 N HCl) for 1 hour. It was left at room temperature in the dark for a while. After centrifugation, the vitamin C solution was added to the supernatant to a final concentration of 0.5%, followed by HPLC analysis. For HPLC analysis, a C18 column (Inertsil ODS-3, 5 μm, 150 × 2.3 mm, GL Sci., Japan) was used and 10 mM potassium phosphate (pH 6.0) solvent was flowed at a rate of 1.2 ml per minute. The eluted terridine compounds were measured at a wavelength of 350 nm / 450 nm (Ex / Em) with an HP Model 1046A fluorescence detector. The concentrations of sepiaterin and BH4 were determined from standard samples purchased (Schircks Lab., Switzerland), and bioterin-α-glucoside was purified from the laboratory (Chung et al., 2000).

The identification of sepiaterin present in the medium is also described in the method described in Kim (YA, Chung HJ, Kim YJ, Choi YK, Hwang YK, Lee SW, Park YS (2000) Mol. Cells 10, 405-410). Following reaction with recombinant SR of purified mice, they were oxidized with acidic iodine solution and analyzed as presence of biopterin using HPLC. Acidic iodine oxidizes the terrydine compound, causing the terrydine compound to fluoresce in the oxidized state. Quantitative analysis media was analyzed directly as absorbance at 420 nm or by HPLC. HPLC was used for the same column as above and 12% methanol solvent was flowed at a rate of 1.2 ml / min and analyzed at 420 nm.

FIG. 7 is a graph of HPLC of a culture of a pET-GTPCH / PTPS transformant, A is a HPLC graph of a standard terridine compound, B is a HPLC chart of a culture solution treated with an acidic iodine solution, and C is reacted with an SR enzyme. After is an HPLC graph of the culture treated with acidic iodine solution. A is neoopterin, b is pterin, c is biopterin, d is 6-hydroxymethylpterin, e is dictyopterin And the arrow points to the virotherin position.

As shown in FIG. 7, the medium of the pET-GTPCH / PTPS transformant was treated with acidic iodine solution and analyzed by HPLC. As a result, no peak of the other terridine compound was observed, but the SR enzyme and NADPH of the mouse were directly added to the medium. As a result of the reaction, the yellow color of the culture solution disappeared, and the peak corresponding to bioterin was high. This means the presence of sepiaterin in the culture fluid.

To reconfirm the culture was analyzed directly by HPLC without treatment with acidic iodine solution. FIG. 8 is an HPLC graph of the sepiaterin present in the liquid medium of the pET-GTPCH / PTPS transformant, A is a graph of the standard sepiaterin, and B is the liquid medium of the pET-GTPCH / PTPS transformant. This is a graph analyzed. As a result, many compounds having absorbance at 420 nm were detected, and the elution position was confirmed to be the same position as that of the standard sepiaterin, indicating that the pET-GTPCH / PTPS transformant produced recombinant sepiaterin.

(2) pET-BH4 transformants

Experiments for identifying the terridine compound were performed in the same manner for the pET-BH4 transformant.

9 is a graph of HPLC of pET-BH4 transformant culture, A is HPLC graph of standard terridine compound, B is HPLC chart of culture solution treated with acidic iodine solution, and C is acidic iodine after reaction with SR enzyme HPLC graph of the solution treated solution. A is neoopterin, b is pterin, c is biopterin, d is 6-hydroxymethylpterin, e is dictyopterin And the arrow points to the virotherin position. As a result, the pET-BH4 transformant culture showed a bioterin peak when the acidic iodine solution was treated (FIG. 9B). This means that the pET-BH4 transformant expressed all GTPCH, PTPS and SR to generate BH4. It is.

In addition, it was confirmed that the peak of the bioterin was increased by HPLC by reacting the pET-BH4 transformant culture with the SR enzyme (FIG. 9C). This indicates that most of the pET-GTPCH / PTPS transformants secrete sepiaterin. Therefore, the secreted sepiaterin reacts with the added SR to form bioterin. Therefore, it was confirmed that the yellow color of the pET-GTPCH / PTPS transformant medium was due to sepiaterin.

(3) pET-BH4 / BGluT transformants

BGluT is an enzyme that attaches glucose in α-coordination to the C2'-OH group of the 6th side chain of BH4 and examined whether BH4-α-glucose can be produced by expression of the BGluT gene. The experiment was performed in the same manner as the experiment for identifying the terridine compound.

FIG. 10 is a graph of HPLC of a culture medium of a pET-BH4 / BGluT transformant, A is an HPLC graph of a standard terridine compound, B is a culture HPLC chart of an acidic iodine solution, and C is a bioterin-α- An HPLC graph of a sample in which glucoside was mixed with a culture solution treated with an acidic iodine solution. D is an HPLC graph of a culture solution oxidized with an acidic iodine solution after reacting with an SR enzyme. A is neoopterin, b is pterin, c is biopterin, d is 6-hydroxymethylpterin, e is dictyopterin Arrow a indicates bioterin and arrow b indicates the location of bioterin-α-glucoside.

As shown in FIG. 10, after the culture of the pET-BH4 / BGluT transformant was treated with an acidic iodine solution, the result of analysis by HPLC showed little bioterin peak and a small amount of new compound at the elution site of about 18 minutes. (Arrow b) is shown. (FIG. 10B) The culture solution of the pET-BH4 / BGluT transformant was treated with acidic iodine solution, and shown in FIG. 10C when bioterin-α-glucoside was mixed. This proves that arrow b is bioterin-α-glucoside. In addition, when treated with SR in the culture broth bioterin eluted (Fig. 10D), pET-BH4 / BGluT transformant was also found to express sepiaterin like the pET-BH4 transformant. Thus, the yellow color of the pET-BH4 / BGluT transformant colonies is due to sepiaterin.

Example 3

Each transformant prepared in Example 2 was inoculated in LB-Km ⁺ liquid medium and shaken at 37 rpm to culture at 200 rpm to overexpress the recombinant protein and purify it. The expression method of the recombinant protein of the pET-GTPCH / PTPS transformant, the pET-BH4 transformant and the pET-BH4 / BGluT transformant is the same.

Transformants were LB-Km⁺(50 ㎍ / ml) O.D. by inoculating liquid medium₆₀₀After culturing until the value is between 0.6 and 1, the expression of recombinant protein was induced by adding IPTG to a final concentration of 50 uM. Overexpression of recombinant proteins was analyzed by SDS-PAGE. As a result, the molecular weight including His-tag was 27.5 kD for GTPCH, 18.6 kD for PTPS, 30 kD for SR, and 40.2 kD for BGluT. When the expression levels of recombinant proteins were analyzed by SDS-PAGE, the expression rate was good when IPTG was not added.

In addition, the amount of sepiaterin produced by the growth of the pET-GTPCH / PTPS transformant was analyzed by absorbance at 600 nm and 420 nm. As a control, the BL21 (DE3) strain and the pET-GTPCH / PTPS transformant were inoculated in LB-Km ⁺ liquid medium, respectively, and the absorbance at 600 nm and 420 nm was measured every 30-60 minutes while shaking at 37 ° C. . Also, when the pET-GTPCH / PTPS transformant was OD ₆₀₀ 0.6, the experimental group to which 50 uM IPTG was added was also tested. The results are shown graphically in FIG. Is a BL21 strain, ○ is a pET-GTPCH / PTPS transformant, ▼ is a pET-GTPCH / PTPS transformant + 50 uM IPTG, dotted line is absorbance measured at 600 nm, solid line is sepia at 420 nm The yellow absorbance of terrin was measured.

According to FIG. 11, the amount of sepiaterin produced by the growth of the pET-GTPCH / PTPS transformant was generated without addition of IPTG, reaching saturation within 24 hours, and showing absorbance values close to 2.0. (FIG. 11-solid line ○) (Fig. 11-solid line) The absorbance value at 23 hours was 1.84 when the absorbance value of the BL21 control was subtracted, and 1.84 was the absorbance of sepiaterin at 420 nm. The value corresponds to a concentration of 177 uM when the count value 10400 is applied. HPLC analysis showed a sepiaterin peak at a concentration corresponding to 77% level of 177 uM. Therefore, even if the corrected value was applied, the concentration of sepiaterin in the medium was calculated to be a high concentration corresponding to 137.7 uM. 137.7 uM was calculated with a yield of 32.66 mg per liter of pET-GTPCH / PTPS transformant medium when molecular weight 237.2 of sepiaterin was applied.

As mentioned above, the present invention establishes a method for producing a terridine compound using E. coli, so that a large amount of sepiaterin can be easily obtained from E. coli. In addition, a large amount of sepiaterin, BH4 and BH4-α-glucoside was prepared using the pET-GTPCH / PTPS transformant (KCTC 1020BP), the pET-BH4 transformant and the pET-BH4 / BGluT transformant of the present invention. Can be used for purposes.

Claims (8)

Recombinant vector comprising a physiase gene for producing a terridine compound. The recombinant vector of claim 1, wherein the recombinant vector is pET-GTPCH / PTPS, pET-BH4, or pET-BH4 / BGluT. A transformant transformed with the recombinant vector of claim 1. The transformant of claim 3, wherein the transformant is a pET-GTPCH / PTPS transformant (KCTC 1020BP), a pET-BH4 transformant, or a pET-BH4 / BGluT transformant. A terridine compound or recombinant enzyme produced by the transformant of claim 3. A terry using E. coli to produce a recombinant vector by inserting a physiological enzyme gene to generate a terridine compound in the vector, and to produce a recombinant physiological compound by simultaneously expressing the physiological enzyme in the transformant containing the recombinant vector Method for producing din compound. According to claim 6, wherein the physiological enzyme gene is GTPCH (GTP cyclohydrolase I) gene, PTPS gene (6-pyruvoyltetrahydropterin synthase), SR gene (sepiapterin reductase), BGluT gene (BH4: UDP-glucose-α-glucosyltransferase), chloro Method for producing a teridine compound using E. coli, characterized in that at least one selected from the group consisting of chlorotepium (Chlorobium tepidum) SR gene and the teridine glycotransferase gene. The method of claim 6, wherein the terry Compound sepia aminopterin (sepiapterin), BH4 ((6 R) -5,6,7,8-tetrahydro-L-biopterin), BH4-α- glucoside (BH4-α- glucoside), L-threo-BH4 and a method of producing a terridine compound using E. coli, characterized in that the sugar-binding BH4 (BH4- (α or β) -sugar).