KR20130081394A - A novel cytochrome p450 hydroxylase and process for preparing hydroxylated cyclosporin a employing the same - Google Patents

Abstract

The present invention is a novel cytochrome P450 hydroxylase (CYP) derived from Severkia Benihana, polynucleotide encoding the CYP, expression vector comprising the polynucleotide, transformation expression into which the expression vector is introduced Sieve, a method for producing the CYP using the transformant, an expression vector comprising respective polynucleotides encoding the CYP and an electron transfer enzyme, and each polynucleotide encoding the CYP and an electron transfer enzyme A method of producing 4- (γ-hydroxy) -CyA using a transformant having an expression vector introduced therein and a transformant having an expression vector containing the polynucleotide encoding the CYP and the electron transfer enzyme. It is about. By using the production method of the hydroxylated cyclosporin A of the present invention, since it can produce 4- (γ-hydroxy) -CyA with higher efficiency than the conventional method, it will be widely used in the development and production of various hair regrowth.

Description

A novel Cytochrome P450 hydroxylase and process for preparing hydroxylated Cyclosporin A employing the same

The present invention relates to a novel cytochrome P450 hydroxylase and a method for producing hydroxylated cyclosporin A using the same. More specifically, the present invention relates to a novel cytochrome P450 hydroxylase (Cytochrome P450 hydroxylase, CYP) derived from Severia benihana. ), A polynucleotide encoding the CYP, an expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, a method of producing the CYP using the transformant, the CYP and an electron transfer enzyme An expression vector comprising each polynucleotide encoding, a transformant into which an expression vector comprising each polynucleotide encoding the CYP and electron transfer enzyme is introduced, and each polynucleotide encoding the CYP and electron transfer enzyme 4- (γ-hydroxy) -Cy using a transformant having an expression vector including A method for producing A.

The human hair is about 100,000 to 150,000 pieces, and each hair has a different cycle and grows and drops out through the anagen, the catagen, and the telogen. This cycle is repeated over three to six years, with the result that on average 50 to 100 hairs fall out normally. In general, alopecia refers to an increase in the number of hairs that fall abnormally due to shortening of the hair growth rate in the growth phase and more hair in the degenerative or resting phase.

The causes of alopecia have been discussed, such as poor blood circulation, excessive male hormone action, excessive sebum secretion, peroxide, bacteria, hypodermic function, genetic factors, aging, stress. However, to date, no clear cause of hair loss is known, and in recent years, a growing number of people are suffering from hair loss due to changes in diet and stress caused by social conditions. It is increasing.

The most widely used formulations for the treatment or prevention of such alopecia to date are minoxidil-containing formulations, and are currently one of two hair repellent components approved by the US FDA. Minoxidil was a drug for hypertension that was developed for the purpose of lowering blood pressure, but with the side effect of use, hair growth phenomenon has now become a more famous drug for hair growth. Although the mechanism of minoxidil's hair growth is not known, it is thought to promote hair growth by nourishing hair roots by increasing blood flow through vasodilation effect.

In addition, Merck's recently launched propecia, finasteride, inhibits the conversion of androgen testosterone to the more potent form of dihydrotestosterone. In December 1997, a 1 mg tablet was approved by the FDA for the treatment of androgenetic alopecia and is currently on the market. It has been shown to have a significant clinical effect, but some male side effects are reported (J. Am. Acad. Dermatol). , 1998; 39; 578-589). However, this drug, like minoxidil, is not very effective in clinical research, and researches for more excellent hair repellents are being actively conducted due to concerns about side effects.

Cyclosporins, on the other hand, have various physiological effects such as renal toxicity, hepatotoxicity, hypertension, periodontal tissue enlargement, and hair growth effect as a representative immunosuppressive agent, and inhibiting viruses, fungi and protozoa (Advances in Pharmacol., 1996 35; 114-246 and Drug Safety, 1994; 10; 310-317). Cyclosporin A (CyA) is an oligopeptide with a ring of 11 amino acids and is a powerful immunosuppressant to treat rejection of organ transplant patients. It has various physiological effects such as hypertrophy and hair growth effect. CyA is the most abundant of the 24 structurally similar metabolites (cyclosporine A to Z) produced when incomplete fungi, Topopodium inflatum, are cultured in aerobic liquid culture. CyA and its analogs are biosynthesized by non-ribosomal mechanisms in which 11 amino acids are activated sequentially, N-methylated and formed peptide bonds by a multifunctional single enzyme called Cyclosporin Synthetase. do.

Among the various side effects of CyA, the possibility of development as a hair regrowth agent using the overgrowth side effects of the hair has been conducted by various studies. Among them, extensive researches on animal hair growth test, human alopecia areata, human androgenetic alopecia, and chemotherapy for hair loss control were performed, and the results of mouse backing experiments were about 100 times better than commercial minoxidil. Is showing.

CyA is converted to various derivatives through bioconversion by various bacteria and fungi. Derivatives having a hydroxyl group at position 4 by the rare actinomycetes Sebekia benihana (4- (γ-hydroxy) -CyA, 4- (γ-hydroxy) -4-N-demethyl-CyA , Di-4,6- (γ-hydroxy) -CyA, etc.) (Korean Patent Laid-Open Publication No. 2004-88593), among which 4- (γ-hydroxy) -CyA is an immune that CyA has It is known that the inhibitory activity is reduced and the hair growth effect is maintained as it is (Korean Patent No. 316604). This reaction is known to be carried out by the cytochrome P450 hydroxylase (CYP) and electron transferase expressed in the strain, the CYP is in all species of nature, such as plants, bacteria, animals, archaea, etc. Hemoprotein (hemoprotein) containing heme is expressed, the iron ion contained in the heme when combined with carbon monoxide (CO) exhibits a characteristic absorption at 450 nm, catalyzing various oxidation reactions in vivo Known as To date, five studies (CYP501, CYP502, CYP503, CYP504, and CYP506) that are expressed in Severia Benihana are known. It is frozen to be converted to (γ-hydroxy) -CyA, but the conversion yield is low, there is a problem that it is difficult to use industrially.

Under these circumstances, the present inventors have made diligent efforts to develop a method for improving the hydroxylation efficiency during the bioconversion of CyA by microorganisms. As a result, the present inventors have discovered a novel enzyme protein that mediates the hydroxylation of CyA and electron transfer the enzyme protein. When using a transformant transformed to be expressed with the enzyme, it was confirmed that the hydroxylation efficiency of CyA can be improved, the present invention was completed.

One object of the present invention is to provide a novel cytochrome P450 hydroxylase (CYP) that mediates the hydroxylation of CyA.

Another object of the present invention is to provide a polynucleotide encoding the CYP.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

Still another object of the present invention is to provide a method for producing the CYP using the transformant.

Still another object of the present invention is to provide an expression vector comprising each polynucleotide encoding the CYP and the electron transfer enzyme.

Still another object of the present invention is to provide a transformant into which an expression vector containing each polynucleotide encoding the CYP and the electron transfer enzyme is introduced.

Still another object of the present invention is to provide a method for producing 4- (γ-hydroxy) -CyA using a transformant into which an expression vector containing each polynucleotide encoding the CYP and an electron transfer enzyme is introduced. will be.

As one embodiment for achieving the object of the present invention, the present invention provides a novel cytochrome P450 hydroxylase (CYP) that mediates the hydroxylation of cyclosporin A (CyA).

The inventors of the present invention can hydroxylate CyA from Sebekia benihana , a type of actinomycetes capable of hydroxylating CyA to produce derivatives having reduced immunosuppressive activity (4- (γ-hydroxy) -CyA). We attempted to identify novel enzyme proteins. To this end, the genomic DNA of the strain was analyzed to screen for genes encoding novel CYP other than the five known species. As a result, genes expected to encode a total of 16 new CYPs (CYP001 to CYP016) were discovered, and among them, the genes encoding CYPs involved in the production of 4- (γ-hydroxy) -CyA were screened. As a result, it was confirmed that the expression level of 4- (γ-hydroxy) -CyA was reduced in the S. benihana strains in which CYP008 was deleted, compared to that of the wild type strain, and thus, the CYP008 exhibited an activity capable of hydroxylating CyA. Example 1).

As used herein, the term "cyclosporin A (Cyclosporin A, CyA)" is a cyclic peptide composed of one amino acid, which may exist in various forms depending on the structure of the amino acid, and means a compound having the structure of Formula 1 below. do. CyA is used as an immunosuppressive agent for inhibiting tissue transplant rejection following organ transplantation, and has an activity of promoting hair growth as a side effect. For the purposes of the present invention, CyA is used as a precursor for producing 4- (γ-hydroxy) -CyA, but is not particularly limited thereto.

The term "cytochrome P450 hydroxylase (CYP)" of the present invention is expressed in all species of nature, catalyzes various oxidation reactions, and refers to a kind of hemoprotein including heme, When iron ions contained in the heme are combined with carbon monoxide (CO), they exhibit a characteristic absorption of 450 nm and use various kinds of compounds including polyketides, fatty acids, vitamins, and steroid-based antibiotics as substrates. It is known. For the purposes of the present invention, the CYP is used to carry out a reaction for hydroxylating CyA, and is not particularly limited thereto. Preferably, the CYP may be CYP derived from actinomycetes, and more preferably, CYP501 derived from Severkia bennihana. , CYP502, CYP503, CYP504, CYP506, or any of the newly selected CYP001 to CYP016 in the present invention, most preferably a novel CYP008 derived from Severkia Benihana. In particular, the novel CYP008 is not particularly limited, but may be a protein having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, more preferably 80%, more preferably 90% or more homology thereof. Can be.

As used herein, the term "homology" refers to a similar degree of a nucleotide sequence or an amino acid sequence of a gene encoding a protein. When homology is sufficiently high, expression products of the gene may have the same or similar activity.

As another embodiment for achieving the above object, the present invention is a polynucleotide encoding the CYP008, an expression vector comprising the polynucleotide, a transformant capable of expressing CYP008 by introducing the expression vector and the transformation It provides a method for producing CYP008 using a sieve.

The polynucleotide encoding the novel CYP provided by the present invention is not particularly limited, but may be a polynucleotide derived from a Severkibenihana strain, preferably the polynucleotide sequence of SEQ ID NO: 2 or 70% thereof. As described above, the polynucleotide having a base sequence having more than 80% homology, more preferably at least 80%, more preferably at least 90% may be, but is not particularly limited thereto.

As used herein, the term “expression vector” refers to a DNA preparation containing a nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a gene of interest in a suitable host. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation, and other signals DNAs encoding peptides, enhancer sequences, untranslated regions on 5 'and 3' sides of the gene of interest, selection marker regions, or replicable units, etc. may be appropriately included. The expression vector can be prepared and purified using standard recombinant DNA techniques. The expression vector is not particularly limited as long as it expresses a desired gene in various host cells of prokaryotic and eukaryotic cells and produces a desired protein. However, the expression vector has a promoter that exhibits strong activity, A vector capable of producing a large amount of exogenous protein in a form similar to that of the wild type. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, [lambda] EMBL3, [lambda] EMBL4, [lambda] FIXII, [lambda] DASHII, [lambda] ZAPII, [lambda] gt10, [lambda] gt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector. , pBluescriptII system, pGEM system, pTZ system, pCL system and pET system can be used. The vector which can be used is not specifically limited, A well-known expression vector can be used. Preferably, a pDZ vector or the like can be used. For the purpose of the present invention, the expression vector may be used to express CYP008, which mediates the hydroxylation of CyA, including polynucleotides encoding CYP008, but is not particularly limited thereto.

As used herein, the term "transformer" refers to a cell that has been transfected so that a vector having a gene encoding one or more target proteins is introduced into the host cell to express the target protein, and all eukaryotic cells, prokaryotic cells, and the like. Cells, but are not particularly limited to bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells.

Method for producing a CYP008 provided by the present invention comprises the steps of (iii) culturing the transformant to obtain a culture; And (ii) recovering CYP008 from the culture.

As another embodiment for achieving the object of the present invention, the present invention is an expression vector comprising each polynucleotide encoding the CYP and electron transfer enzyme, the transformant is introduced with the expression vector and the transformant To provide a method for producing 4- (γ-hydroxy) -CyA.

In order to determine whether CYP008, a novel CYP derived from Severkia Benihana, can mediate the hydroxylation of CyA, we used a gene encoding two hydroxylases (CTY506 and CYP008) and three sets of electron transfer enzymes. An expression vector (pYJ1621, pYJ1623, pYJ1625) comprising a combination of coding genes was prepared (Example 2), and each of the prepared expression vectors was introduced into Severia benihana and transformants (YJ405, YJ407, YJ409). (Example 3), and confirmed whether the gene encoding the desired hydroxyl enzyme and the gene encoding the electron transfer enzyme to these transformants was normally introduced (Example 4, 5, 6), these As a result of culturing each transformant in a liquid medium or a solid medium, it was confirmed that 4- (γ-hydroxy) -CyA can be produced at a content of 1.1 to 1.8 times higher than that of wild-type Severkia Benihana. It was (Examples 7,8).

As used herein, the term "electron transfer enzyme" refers to an enzyme that provides electrons or hydrogen ions, etc., which are used as a driving force through NAD, NADP, etc., which are cofactors, to enzymes involved in the oxidation or reduction reaction in vivo. . Typically, putidaredoxin and putidaredoxin reductase, ferredoxin and perredoxin reductase, flavodoxin and flavodoxin reductase And two proteins are formed in pairs. For the purposes of the present invention, the electron transfer enzyme is used to supply hydrogen ions to CYP to provide a driving force for hydroxylating CyA by CYP.

The expression vector comprising each of the polynucleotides encoding the CYP and the electron transfer enzyme is constructed to include the polynucleotide encoding the CYP and the polynucleotide encoding the electron transfer enzyme, May be constructed such that polynucleotides encoding one or more CYPs are continuously connected, and more preferably, a polynucleotide encoding a variety of polynucleotides derived from Sebecia vanilan, which is used as a host cell It is preferable to use an expression vector constructed such that a polynucleotide encoding CYP506 and a polynucleotide encoding CYP008 are continuously connected to each other, and most preferably, a polynucleotide encoding CYP008 and a polynucleotide encoding CYP008 are continuously connected. In addition, the polynucleotide encoding the electron transfer enzyme is composed of two proteins constituting the enzyme (putidaredoxin and putidaredoxin reductase, perredoxin and perredoxin reductase, flavodoxin and flavodoxin reductase, etc.) The polynucleotide encoding the may be configured to be connected continuously, the electron transfer enzyme may include one or two or more, but is not particularly limited thereto.

The transformant into which the expression vector containing each polynucleotide encoding the CYP and the electron transfer enzyme is introduced is not particularly limited thereto, and the expression vector is introduced into a host cell capable of hydroxylating at least CyA. Host cells capable of becoming mutated cells to increase the production of (γ-hydroxy) -CyA, and which can hydroxylate the CyA are not particularly limited thereto, but preferably microorganisms of actinomycetes capable of hydroxylating CyA. It may be, and more preferably may be a Severkier microorganism capable of hydroxylating CyA, and most preferably it may be a Severkia benihana capable of hydroxylating CyA.

The method for producing 4- (γ-hydroxy) -CyA provided by the present invention comprises the steps of: (i) preparing an expression vector comprising each polynucleotide encoding a cytochrome P450 hydroxylase (CYP) and an electron transfer enzyme; ; (Ii) preparing a transformant by introducing the prepared expression vector into a host cell capable of hydroxylating CyA; (Iii) culturing the transformant to obtain a culture; And (iii) recovering 4- (γ-hydroxy) -CyA from the culture.

At this time, the CYP is not particularly limited thereto, but may preferably be a CYP derived from the genus Severkia, more preferably may be a CYP derived from Severkia benihana, and most preferably, Severkia benihana. Derived from CYP501, CYP502, CYP503, CYP504, CYP506, CYP008 and combinations thereof.

In addition, the electron transfer enzyme is not particularly limited, but putidaredoxin (putidaredoxin) and putidaredoxin reductase derived from P. putida , ferredoxin ( S. benihana ) and S. coelicolor A3 ( 2) can be derived from ferredoxin reductase (ferredoxin reductase), flavodoxin (flavodoxin) and flavodoxin reductase (flavodoxin reductas) derived from E. coli .

Preferably, a combination comprising CYP506 and CYP008 as CYP and containing putidaredoxin and putidaredoxin reductase as electron transfer enzyme, and CYP506 and CYP008 as CYP and peredoxin and peredoxin reductase as electron transfer enzyme Combinations including CYP506 and CYP008 as CYP, and combinations including flavodoxin and flavodoxin reductase as electron transfer enzymes, but are not particularly limited thereto.

In addition, the host cell is not particularly limited thereto, but may be a microorganism of actinomycetes capable of hydroxylating CyA, and more preferably, may be a microorganism of the genus Sebekia, which is capable of hydroxylating CyA, most preferably. It can be Severkia Benihana which can hydroxylate CyA.

The culturing of the transformant is not particularly limited thereto, but may be performed by liquid culture, solid culture, or the like according to the type of medium, and may be batch culture method, continuous culture method, or fed-batch type according to the medium component supply method. It is preferably carried out by a culturing method, and the culturing conditions are not particularly limited thereto, but using a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid) at an appropriate pH (pH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8), can be introduced to the culture to maintain aerobic conditions by introducing an oxygen or oxygen-containing gas mixture, the culture temperature is 20 to 45 ℃, Preferably 25 to 40 ℃ can be maintained, it is preferable to incubate for about 10 to 160 hours. Ornithine produced by the culture may be secreted into the medium or remain in the cells.

In addition, the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid), etc. may be used individually or in combination ; Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination; As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.

By using the production method of the hydroxylated cyclosporin A of the present invention, since it can produce 4- (γ-hydroxy) -CyA with higher efficiency than the conventional method, it will be widely used in the development and production of various hair regrowth.

1 is a schematic diagram showing the configuration of an expression vector for producing 4- (γ-hydroxy) -CyA.
Figure 2 is a schematic diagram showing a transformation method through conjugation of S. benihana .
Figure 3a is a schematic diagram showing the genes of interest (CYP506, CYP008, camA and camB ) contained in the expression vector pYJ1621,
Figure 3b is an electrophoresis picture showing the result of PCR amplification of genomic DNA of YJ405.
Figure 3c is an electrophoresis picture showing the results of analyzing the genomic DNA of YJ405 by southern blot hybridization method.
4A is a schematic diagram showing the genes of interest (CYP506, CYP008, FD506, and SCO0681) included in the expression vector pYJ1623.
Figure 4b is an electrophoresis picture showing the result of PCR amplification of genomic DNA of YJ407.
4C is an electrophoresis image showing the result of analysis of the genomic DNA of YJ407 by Southern blot hybridization.
Figure 5a is a schematic diagram showing the genes of interest (CYP506, CYP008, fldA and fpr ) contained in the expression vector pYJ1625.
Figure 5b is an electrophoresis picture showing the result of PCR amplification of genomic DNA of YJ409.
Figure 5c is an electrophoresis picture showing the results of analyzing the genomic DNA of YJ409 by southern blot hybridization method.
Figure 6 is the result of analyzing the 4- (γ-hydroxy) -CyA produced in the strain of the control by HPLC-ESI-MS.
7 shows the results of analyzing the hydroxylation rate of 4- (γ-hydroxy) -CyA produced by the liquid culture method.
8 is a result of analyzing the hydroxylation rate of 4- (γ-hydroxy) -CyA produced by the solid culture method.

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

Example  1: mediating the hydroxylation of cyclosporin A New CYP  Selection of genes

In order to improve the hydroxylation rate of cyclosporin A, 16 new CYP genes were selected in addition to the five known CYP genes through genome analysis of S. benihana (KTTC 9610) (Table 1).

CYP gene selected from S. benihana No. CYP name Description One
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21 CYP501
CYP502
CYP503
CYP504
CYP506
CYP001
CYP002
CYP003
CYP004
CYP005
CYP006
CYP007
CYP008
CYP009
CYP010
CYP011
CYP012
CYP013
CYP014
CYP015
CYP016 Accession no. DI130758.1
Accession no. DI131915.1
Accession no. DI126285.1
Accession no. DI129591.1
Accession no. DI139785.1

As shown in Table 1, 16 to 21 CYP genes were selected, and in order to select a gene encoding a protein expressing an activity capable of mediating the hydroxylation of cyclosporin A from the selected genes, Mutant strains each having a gene deleted were prepared, and the expression level of hydroxylated cyclosporin A (4- (γ-hydroxy) -CyA) expressed from each of these strains was measured, and then the 4- (γ-hydroxy) -CyA was measured. As a result of selecting a strain whose expression level was reduced compared to that of the wild type strain, it was confirmed that only the CYP008 gene can express a protein exhibiting an activity capable of mediating the hydroxylation of cyclosporin A.

Example  2: 4- (γ- hydroxy ) - CyA Construction of Expression Vector to Produce

A CYP008 gene confirmed to encode a protein exhibiting hydroxyl activity against cyclosporin A and a CYP506 gene (NCBI accession No .: DI139785.1), which have already been demonstrated to exhibit hydroxyl activity, encode a protein exhibiting electron transfer activity. An expression vector containing a gene was prepared. At this time, the electron transfer to the gene encoding a protein showing the activity were used for the three groups, 1 group was used camA gene, putidaredoxin gene camB and putidaredoxin reductase of P. putida origin, is one group 2 using was used as the ferredoxin gene of FD506 and S. coelicolor A3 (2) derived from the ferredoxin reductase gene, SCO0681 present in the 3'-downstream of CYP506 genes in S. benihana, No. 3 group of flavodoxin fldA gene of E. coli-derived And the fpr gene, ferredoxin (flavodoxin) reductase, were used.

The genes were obtained by performing a polymerase chain reaction (RPC) to include a ribosome binding site (RBS) and nucleotide sequences from start codon to stop codon. Tag polymerase (Genotech) was used for PCR, and primers used were primers of SEQ ID NOs: 3 and 4 for amplifying the CYP008 gene, primers of SEQ ID NOs: 5 and 6 for amplifying the CYP506 gene, and sequences for amplifying the camA gene. No. 7 and 8 of the primer, SEQ ID NO: for amplifying camB genes 9 and 10 primers, SEQ ID NO: for amplifying FD506 genes 11 and 12 primers, SEQ ID NO: 13 and 14 primers for amplifying SCO0681 gene, fldA Primers of SEQ ID NOs: 15 and 16 for amplifying genes, and primers of SEQ ID NOs: 17 and 18 for amplifying fpr genes were used, and the base sequences of the respective primers are shown in Table 2. Each gene fragment obtained by PCR amplification was cloned into pLitmus28 vector by treatment with restriction enzymes of EcoRI (or PacI) and XbaI.

PCR primer sequence for each gene gene Type of primer Sequence (5'-3 ') SEQ ID NO: CYP008 Forward TACGTAGAATTCACTAGTCCTCGCGCTGGTTTCATA 3 Reverse TCTAGAGGTTCAGCCGAGGGTGAC 4 CYP506 Forward TACGTAGAATTCACTAGTAGCGGAACGCGTAATATC 5 Reverse TCTAGAATCCACTTCGAGTTCCAT 6 camA Forward TTAATTAAACTAGTATCGATACACATGGGAGTGCGTGCTAAGTG 7 Reverse TCTAGATTCAGGCACTACTCAGTTCAGCTT 8 camB Forward TTAATTAAACTAGTCATAGCGTGTGAGGATAAACAGAT 9 Reverse TCTAGATTTACCATTGCCTATCGGGAACA 10 FD506 Forward GAATTCTACGTAACTAGTATGGAACTCGAAGTGGAT 11 Reverse TCTAGATCATGAAGGGTCCGCAGGCGA 12 SCO0681 Forward GAATTCTACGTAACTAGTGATCTGTCACCGCTCGAA 13 Reverse TCTAGATCAGGCGCCGCTCTCGCGGAG 14 fldA Forward GAATTCTACGTAACTAGTTTCAATAAGTTTCAAGAG 15 Reverse TCTAGATCAGGCATTGAGAATTTCGTC 16 fpr Forward GAATTCTACGTAACTAGTAAGGCAAGTCAATCAAAA 17 Reverse TCTAGATTACCAGTAATGCTCCGCTGT 18

Each expression vector was prepared comprising a combination of the genes encoding the two hydroxyl activity proteins and the gene encoding the three groups of electron transfer activity (FIG. 1). 1 is a schematic diagram showing the configuration of an expression vector for producing 4- (γ-hydroxy) -CyA. As shown in FIG. 1, ermE is used as a promoter in each of three expression vectors, and a gene connecting the CYP008 gene and the CYP506 gene is commonly used as a gene encoding a protein showing hydroxyl activity. The expression vector for cyclosporin A was prepared by combining the genes represented by the genes of each group in the pLitmus28 vector and cloning them into the integration vector pSET152. At this time, the expression vector containing group 1 was named "pYJ1621", the expression vector containing group 2 was named "pYJ1623", and the expression vector containing group 3 was named "pYJ1625".

In addition, as a negative control group, an expression vector containing only the gene encoding the protein showing two hydroxyl activities and the gene encoding the protein showing the electron transfer activity was prepared and named "pYJ1627".

Example  3: 4- (γ- hydroxy ) - CyA Preparation of a transformant to produce

First, each of the expression vectors prepared in Example 2 was introduced into E. coli ET12567 / pUZ8002 to obtain respective transformants, and 25 µg / ml of apramycin was added to the LB liquid medium. After 12 hours of incubation at 37 ° C., 1 ml of the culture was transferred to 50 ml of LB liquid medium and incubated at 37 ° C. until the optical density (OD600) was 0.4-0.5. The culture was centrifuged, washed twice with LB broth without antibiotics, and concentrated to 500 μl of LB.

Meanwhile, 500 µl of 2xTY (trptone 0.16%, yeast extract 0.1%, NaCl 0.05%) was mixed with 80 µl of S. benihana spore, followed by centrifugation and resuspension, followed by heat-shock for 10 minutes at 50 ° C. In addition, mycelia were obtained.

500 μl of each E. coli concentrate and 500 μl of mycelia were mixed, and each of these mixtures was applied to a mISP4 solid medium (Difco ISP4 medium 3.7%, yeast extract 0.05% and tryptone 0.15%), and then at 30 ° C. Incubate for 18-20 hours. The cultured solid medium was plated with 30 μl / ml nalidixic acid and 30 μl / ml apramycin, further incubated for 15-20 days, and then each colony showing antibiotic resistance. Selected (FIG. 2). Figure 2 is a schematic diagram showing a transformation method through conjugation of S. benihana .

The transformant into which the expression vector pYJ1621 was introduced was named "YJ405", and the transformant into which the expression vector pYJ1623 was introduced was named "YJ407", and the transformant into which the expression vector pYJ1625 was introduced was "YJ409". The transformant into which the expression vector pYJ1627, which is a negative control group, was introduced was named "YJ410".

Example  4: Assay of transformants in which Group 1 was introduced

Among the transformants produced by the above method, PCR method was used to determine whether the target genes (CYP506, CYP008, camA and camB ) were normally introduced into the transformant YJ405 into which the expression vector pYJ1621 containing the first group was introduced. And southern blot hybridization methods, respectively.

First, in the case of the PCR method, genomic DNA of YJ405 was used as a template, and PCR was performed using each primer described in Table 5 above to obtain PCR fragments, and the obtained PCR fragments were electrophoresed (FIGS. 3A and 3B). ). Figure 3a is a schematic diagram showing the desired genes (CYP506, CYP008, camA and camB ) contained in the expression vector pYJ1621, Figure 3b is an electrophoresis picture showing the results of PCR amplification of genomic DNA of YJ405. As shown in Figure 3b, it was confirmed that the genomic DNA of YJ405 contained 1.3kb CYP8 gene, 1.3kb CYP506 gene, 1.3kb camA gene and 0.3kb camB gene.

Next, in the southern blot hybridization method, probe 1 (SEQ ID NO: 19) and a kit (DIG labeling and detection kit) capable of detecting a gene region of about 2.8kb, which is the CYP008 gene and the CYP506 gene, were detected on genomic DNA of YJ405. Southern blot hybridization method using (Roche) (Fig. 3c). Figure 3c is an electrophoresis picture showing the results of analyzing the genomic DNA of YJ405 by southern blot hybridization method. As shown in FIG. 3c, a band of 2.8 kb, which is the size of the DNA fragment used as a probe, was confirmed to confirm that the hydroxyl gene and the electron transfer gene were inserted into the recombinant strain YJ405.

Example  5: Assay of transformants in which Group 2 was introduced

Whether or not the desired genes (CYP506, CYP008, FD506 and SCO0681 ) were normally introduced into the transformant YJ407 into which the expression vector pYJ1623 containing the second group among the transformants prepared by the above method was introduced was determined by the PCR method And southern blot hybridization, respectively.

First, in the case of the PCR method, genomic DNA of YJ407 was used as a template, and PCR was performed using each of the primers described in Table 5 above to obtain PCR fragments, and the obtained PCR fragments were electrophoresed (FIGS. 4A and 4B). ). Figure 4a is a schematic diagram showing the desired genes (CYP506, CYP008, FD506 and SCO0681 ) contained in the expression vector pYJ1623, Figure 4b is an electrophoresis picture showing the result of PCR amplification of genomic DNA of YJ407. As shown in Figure 4b, it was confirmed that the genomic DNA of YJ407 contained 1.3kb CYP8 gene, 1.3kb CYP506 gene, 0.2kb FD506 gene and 1.4kb SCO0681 gene.

In the southern blot hybridization method, approximately 2.5 kb of genomic DNA of YJ407 (after restriction AscI cleavage site), CYP506 gene and FD506 gene (before restriction AscI cleavage site) were obtained. Southern blot hybridization was performed using probe 2 (SEQ ID NO: 20) and a kit (DIG labeling and detection kit, Roche) capable of detecting a gene region (FIG. 4C). 4C is an electrophoresis image showing the result of analysis of the genomic DNA of YJ407 by Southern blot hybridization. As shown in FIG. 4c, a band of 2.5 kb, which is the size of the DNA fragment used as the probe, was confirmed to confirm that the hydroxyl gene and the electron transfer gene were inserted into the recombinant strain YJ407.

Example  6: Assay of transformants in which Group 3 was introduced

Among the transformants produced by the above method, PCR method was used to determine whether the target genes (CYP506, CYP008, fldA, and fpr ) were normally introduced into transformant YJ409 into which the expression vector pYJ1625 containing group 3 was introduced. And southern blot hybridization methods, respectively.

First, in the case of the PCR method, using genomic DNA of YJ409 as a template and performing PCR using each primer described in Table 5 above, PCR fragments were obtained, and the obtained PCR fragments were electrophoresed (FIGS. 5A and 5B). ). Figure 5a is a schematic diagram showing the desired genes (CYP506, CYP008, fldA and fpr ) contained in the expression vector pYJ1625, Figure 5b is an electrophoresis picture showing the result of PCR amplification of genomic DNA of YJ409. As shown in Figure 5b, it was confirmed that the genomic DNA of YJ409 contains 1.3kb CYP8 gene, 1.3kb CYP506 gene, 0.5kb fldA gene and 0.7kb fpr gene.

Next, in the southern blot hybridization method, a gene region of about 1.9 kb, which is a part of the CYP506 gene (after the restriction enzyme NruI cleavage site) and a part of the fldA gene (before the restriction enzyme SspI cleavage site), was selected for genomic DNA of YJ409. Southern blot hybridization using a probe 3 (SEQ ID NO: 21) and a kit (DIG labeling and detection kit, Roche) was performed (FIG. 5C). Figure 5c is an electrophoresis picture showing the results of analyzing the genomic DNA of YJ409 by southern blot hybridization method. As shown in Figure 5c, the band of 1.9kb, the size of the DNA fragment used as a probe was confirmed that the hydroxyl gene and the electron transfer gene was inserted into the recombinant strain YJ409.

Example  7: Production of 4- (γ- hydroxy ) - CyA Production of

Each of the transformants prepared above was cultured in a liquid culture method to produce 4- (γ-hydroxy) -CyA, and the content of 4- (γ-hydroxy) -CyA produced was measured.

Specifically, each of the prepared transformants was a 5 ml GSMY liquid medium (glucose 0.7%, soluble starch 0.75%, yeast extract 0.45%, malt extract 0.5%, calcium carbonate 0.005 containing apramycin 25㎍ / ㎖ %), And incubated for 3 days with shaking at 230 rpm at 30 ℃, each culture was centrifuged to obtain the respective cells. 50 mg of each obtained cell was inoculated into 50 ml of GSMY liquid medium containing 25 µg / ml of apramycin, and incubated for 2 days with shaking at 230 rpm at 30 ° C. 1.25 mg was added and then further incubated for 3 days under the same conditions. At the end of the culture, the cultures were centrifuged to obtain respective cells, which were then left for 12 hours by adding 2 times the volume of ethyl acetate to the culture volume, thereby obtaining respective ethyl acetate layers. The solvent was removed from the obtained ethyl acetate layer to obtain each sample, which was dissolved in 200 µl of methanol and used as a sample for HPLC and HPLC-ESI-MS analysis. At this time, a wild type strain was used as a control group.

High performance liquid chromatography (HPLC) analysis was performed on instruments (waters (Milford, MA) model 2690 separation module), columns (Gemini 5u C18 110A from Phenomenex, 250 x 4.60 mm, 5 micron), absorption wavelength (210 nm), pump flow rate (1.00 mL / min), pump pressure (50.0 psi), column temperature (50 ° C.), solvent A (25% methanol), solvent B (100% acetonitrile) and analytical conditions (0-4 minutes) Silver solvent B 40%, solvent B 61% for 5 to 20 minutes, solvent B 100% for 21 to 40 minutes, solvent B 40% to 45 to 60 minutes).

HPLC-ESI-MS (HPLC-Electro Spray Ionization-mass spectrometry) analysis was performed on the instrument (waters (Milford, MA) model 2690 separation module), column (XTetra® MS C18 3.5 μm, 2.1 × 50 mm), solvent A (5 mM) Ammonium acetate and 0.05% acetic acid aqueous solution), solvent B (80% acetonitrile with 5 mM ammonium acetate and 0.05% acetic acid), flow rate (0.22 mL / min), analytical conditions (40% solvent A for 0-40 minutes, 5% solvent A for 41-47 minutes, 40% solvent A for 48-60 minutes). Samples passed through the column were passed through a micromass ((Baverly, MA) Quattro LC triple-tandem quadrupole mass spectrometer) and applied to a detector in cationic mode (FIG. 6). Figure 6 shows the results of the analysis of 4- (γ-hydroxy) -CyA produced in the strain of the control by HPLC-ESI-MS, A shows the HPLC-ESI-MS chromatogram, B is the ESI-MS spectra Indicates. As shown in FIG. 6, not only 4- (γ-hydroxy) -CyA) is produced by the control S. benihana wild type strain, but also the hydroxylated derivative 4 methyl leucine and 6 methyl leucine are hydroxy derivatives (Di -4,6- (γ-hydroxy) -CyA) was also produced in very small amounts.

As described above, the results of HPLC and HPLC-ESI-MS analysis are shown in FIG. 7. 7 is a result of analyzing the hydroxylation rate of 4- (γ-hydroxy) -CyA produced by the liquid culture method, A shows the HPLC chromatogram of 4- (γ-hydroxy) -CyA, B is 4- ( γ-hydroxy) -CyA is a graph showing the result of comparing the production rate and the hydroxyl ratio of each transformant, a represents 4- (γ-hydroxy) -CyA, b represents CyA.

As shown in FIG. 7, when the production amount of 4- (γ-hydroxy) -CyA is compared, 0.55 mg (44% of bioconversion rate) was produced in the control group, and 0.56 mg (45% of bioconversion rate) in the transformant YJ410, a negative control group. %) Was produced, 0.63 mg (50% live conversion rate) was produced in transformant YJ405, 0.61 mg (49% live conversion rate) was produced in transformant YJ407, and 0.84 mg (birth conversion rate) in transformant YJ409. 67%) was produced.

As such, when comparing the control group and the negative control group (YJ410) overexpressed only the hydroxyl enzyme, there was no difference in the production of 4- (γ-hydroxy) -CyA, whereas each transformant was further overexpressed by the electron transfer enzyme. (YJ405, YJ407 and YJ409) was confirmed to produce 4- (γ-hydroxy) -CyA with 1.1 to 1.5-fold improved production.

Therefore, in order to increase the production of 4- (γ-hydroxy) -CyA it was found that the electron transfer enzyme must be overexpressed alone or in combination with the hydroxylase.

Example  8: 4- (γ- using solid culture method hydroxy ) - CyA Production of

Each transformant prepared above was cultured by a solid culture method to produce 4- (γ-hydroxy) -CyA, and the content of 4- (γ-hydroxy) -CyA produced was measured.

Specifically, the prepared transformants were inoculated in 5 ml GSMY liquid medium containing 25 µg / ml of apramycin, and cultured for 3 days while shaking at 230 rpm at 30 ° C., and 20 µl of each culture. Was smeared in 50 ml of GSMY solid medium to which 1.25 mg of cyclosporine A was added and incubated at 30 ° C. for 5 days. When the incubation was completed, the solid medium was cut into a size of 0.5-0.8 mm, methanol was added thereto, extracted, and then concentrated in a vacuum dryer. A mixed solvent (water: ethyl acetate = 1: 3, v / v) was added to the methanol concentrate and left to stand for 12 hours, thereby obtaining respective ethyl acetate layers. The solvent was removed from the obtained ethyl acetate layer to obtain each sample, which was dissolved in 200 µl of methanol and used as a sample for HPLC and HPLC-ESI-MS analysis. At this time, a wild type strain was used as a control group.

HPLC and HPLC-ESI-MS analysis using the sample were performed in the same manner as in Example 7, and the results are shown in FIG. 8.

8 is a result of analyzing the hydroxylation rate of 4- (γ-hydroxy) -CyA produced by the solid culture method, A shows the HPLC chromatogram of 4- (γ-hydroxy) -CyA, B is 4- ( γ-hydroxy) -CyA is a graph showing the result of comparing the production rate and the hydroxyl ratio of each transformant, a represents 4- (γ-hydroxy) -CyA, b represents CyA.

As shown in FIG. 8, when the production amount of 4- (γ-hydroxy) -CyA is compared, 0.55 mg (44% of bioconversion rate) was produced in the control group, and 0.56 mg (conversion rate of 45) in the transformant YJ410, a negative control group. %) Was produced, 0.69 mg (55% live conversion rate) was produced in transformant YJ405, 0.68 mg (54% conversion rate) was produced in transformant YJ407, and 1.01 mg (birth conversion rate) in transformant YJ409. 81%) was produced.

As in the result of Example 7, the negative control group (YJ410) overexpressing only the control and the hydroxyl enzyme showed no difference in the production of 4- (γ-hydroxy) -CyA, whereas the electron transfer enzyme In addition, it was confirmed that each of the transformants overexpressed (YJ405, YJ407 and YJ409) produced 4- (γ-hydroxy) -CyA with an improved yield of 1.2 to 1.8 times.

Therefore, in order to increase the production of 4- (γ-hydroxy) -CyA, it can be seen that the electron transfer enzyme must be overexpressed alone or together with the hydroxylase.

<110> Ewha University-Industry Collaboration Foundation          SNU R & DB FOUNDATION <120> A novel Cytochrome P450 hydroxylase and process for preparing          hydroxylated Cyclosporin A employing the same <130> PA111000 / KR <160> 21 <170> Kopatentin 2.0 <210> 1 <211> 394 <212> PRT <213> CYP008 <400> 1 Val Asn Ile Asp Leu Val Asp Gln Asp His Tyr Ala Thr Phe Gly Pro   1 5 10 15 Pro His Glu Gln Met Arg Trp Leu Arg Glu His Ala Pro Val Tyr Trp              20 25 30 His Glu Gly Glu Pro Gly Phe Trp Ala Val Thr Arg His Glu Asp Val          35 40 45 Val His Val Ser Arg His Ser Asp Leu Phe Ser Ser Ala Arg Arg Leu      50 55 60 Ala Leu Phe Asn Glu Met Pro Glu Glu Gln Arg Glu Leu Gln Arg Met  65 70 75 80 Met Met Leu Asn Gln Asp Pro Pro Glu His Thr Arg Arg Arg Ser Leu                  85 90 95 Val Asn Arg Gly Phe Thr Pro Arg Thr Ile Arg Ala Leu Glu Gln His             100 105 110 Ile Arg Asp Ile Cys Asp Asp Leu Leu Asp Gln Cys Ser Gly Glu Gly         115 120 125 Asp Phe Val Thr Asp Leu Ala Ala Pro Leu Pro Leu Tyr Val Ile Cys     130 135 140 Glu Leu Leu Gly Ala Pro Val Ala Asp Arg Asp Lys Ile Phe Ala Trp 145 150 155 160 Ser Asn Arg Met Ile Gly Ala Gln Asp Pro Asp Tyr Ala Ala Ser Pro                 165 170 175 Glu Glu Gly Gly Ala Ala Ala Met Glu Val Tyr Ala Tyr Ala Ser Glu             180 185 190 Leu Ala Ala Gln Arg Arg Ala Ala Pro Arg Asp Asp Ile Val Thr Lys         195 200 205 Leu Leu Gln Ser Asp Glu Asn Gly Glu Ser Leu Thr Glu Asn Glu Phe     210 215 220 Glu Leu Phe Val Leu Leu Leu Val Val Ala Gly Asn Glu Thr Thr Arg 225 230 235 240 Asn Ala Ala Ser Gly Gly Met Leu Thr Leu Phe Glu His Pro Asp Gln                 245 250 255 Trp Asp Arg Leu Val Ala Asp Pro Ser Leu Ala Ala Thr Ala Ala Asp             260 265 270 Glu Ile Val Arg Trp Val Ser Pro Val Asn Leu Phe Arg Arg Thr Ala         275 280 285 Thr Ala Asp Leu Thr Leu Gly Gly Gln Gln Val Lys Ala Asp Asp Lys     290 295 300 Val Val Val Phe Tyr Ser Ser Ala Asn Arg Asp Ala Ser Val Phe Ser 305 310 315 320 Asp Pro Glu Val Phe Asp Ile Gly Arg Ser Pro Asn Pro His Ile Gly                 325 330 335 Phe Gly Gly Gly Gly Ala His Phe Cys Leu Gly Asn His Leu Ala Lys             340 345 350 Leu Glu Leu Arg Val Leu Phe Glu Gln Leu Ala Arg Arg Phe Pro Arg         355 360 365 Met Arg Gln Thr Gly Glu Ala Arg Arg Leu Arg Ser Asn Phe Ile Asn     370 375 380 Gly Ile Lys Thr Leu Pro Val Thr Leu Gly 385 390 <210> 2 <211> 1185 <212> DNA <213> CYP008 <400> 2 gtgaacatcg acctcgtcga tcaggaccac tacgcgacct tcggcccccc gcacgagcag 60 atgcgctggc tgagagagca cgctcccgtc tactggcacg agggcgagcc gggattctgg 120 gccgtcaccc gccacgagga cgtcgtccac gtctcccgcc actccgacct gttctcctcc 180 gcgcgcaggc tcgccctgtt caacgagatg ccggaggagc agcgggagct gcagcggatg 240 atgatgctca accaggaccc gcccgagcac accagaaggc gctccctggt caaccgcggt 300 ttcaccccgc gcaccatccg cgcgctggag cagcacatcc gcgacatctg cgacgacctg 360 ctcgaccagt gctcaggcga aggagacttc gtcaccgacc tcgccgcgcc cctcccgctc 420 tacgtgatct gtgagctgct cggcgcgccc gtcgccgacc gcgacaagat cttcgcctgg 480 tccaaccgca tgatcggcgc ccaggacccc gactacgccg cctcaccgga ggagggcggc 540 gccgcggcca tggaggtcta cgcctacgcc tccgaactgg ccgcccagcg tcgcgccgcc 600 ccgcgcgacg acatcgtcac caagctcctc cagtccgacg agaacggcga gagcctgacg 660 gagaacgagt tcgagctctt cgtgctgctc ctcgtggtcg cgggcaacga gaccacccgt 720 aacgccgcct ccggcggcat gctcaccctc ttcgagcatc cggaccagtg ggacaggctg 780 gtcgccgacc cctccctcgc cgccacggcc gccgacgaga tcgtccgctg ggtctcgccg 840 gtgaacctct tccgccgtac ggcgacagcc gacctcaccc tgggcggaca gcaggtcaag 900 gccgacgaca aggtcgtggt cttctactcc tctgccaaca gggacgcgtc ggtcttctcc 960 gatccggagg tcttcgacat cggacggtct cccaacccgc acatcgggtt cggaggcgga 1020 ggcgcgcact tctgcctggg caaccatctg gccaagctcg agctgcgcgt actcttcgag 1080 cagctggcca ggaggttccc gcggatgcgg cagaccggcg aggcccgccg gttgcgctcg 1140 aacttcatca acggcatcaa gaccctcccc gtcaccctcg gctga 1185 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacgtagaat tcactagtcc tcgcgctggt ttcata 36 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tctagaggtt cagccgaggg tgac 24 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tacgtagaat tcactagtag cggaacgcgt aatatc 36 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tctagaatcc acttcgagtt ccat 24 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ttaattaaac tagtatcgat acacatggga gtgcgtgcta agtg 44 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tctagattca ggcactactc agttcagctt 30 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ttaattaaac tagtcatagc gtgtgaggat aaacagat 38 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tctagattta ccattgccta tcgggaaca 29 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gaattctacg taactagtat ggaactcgaa gtggat 36 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tctagatcat gaagggtccg caggcga 27 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gaattctacg taactagtga tctgtcaccg ctcgaa 36 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tctagatcag gcgccgctct cgcggag 27 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gaattctacg taactagttt caataagttt caagag 36 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tctagatcag gcattgagaa tttcgtc 27 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gaattctacg taactagtaa ggcaagtcaa tcaaaa 36 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tctagattac cagtaatgct ccgctgt 27 <210> 19 <211> 2822 <212> DNA <213> Artificial Sequence <220> <223> probe 1 <400> 19 ggatcctcta gtcctcgcgc tggtttcata ccctggcacc taccaagcgc ttgaccaagc 60 ccgaggtgac ggctcgtgaa catcgacctc gtcgatcagg accactacgc gaccttcggc 120 cccccgcacg agcagatgcg ctggctgaga gagcacgctc ccgtctactg gcacgagggc 180 gagccgggat tctgggccgt cacccgccac gaggacgtcg tccacgtctc ccgccactcc 240 gacctgttct cctccgcgcg caggctcgcc ctgttcaacg agatgccgga ggagcagcgg 300 gagctgcagc ggatgatgat gctcaaccag gacccgcccg agcacaccag aaggcgctcc 360 ctggtcaacc gcggtttcac cccgcgcacc atccgcgcgc tggagcagca catccgcgac 420 atctgcgacg acctgctcga ccagtgctca ggcgaaggag acttcgtcac cgacctcgcc 480 gcgcccctcc cgctctacgt gatctgtgag ctgctcggcg cgcccgtcgc cgaccgcgac 540 aagatcttcg cctggtccaa ccgcatgatc ggcgcccagg accccgacta cgccgcctca 600 ccggaggagg gcggcgccgc ggccatggag gtctacgcct acgcctccga actggccgcc 660 cagcgtcgcg ccgccccgcg cgacgacatc gtcaccaagc tcctccagtc cgacgagaac 720 ggcgagagcc tgacggagaa cgagttcgag ctcttcgtgc tgctcctcgt ggtcgcgggc 780 aacgagacca cccgtaacgc cgcctccggc ggcatgctca ccctcttcga gcatccggac 840 cagtgggaca ggctggtcgc cgacccctcc ctcgccgcca cggccgccga cgagatcgtc 900 cgctgggtct cgccggtgaa cctcttccgc cgtacggcga cagccgacct caccctgggc 960 ggacagcagg tcaaggccga cgacaaggtc gtggtcttct actcctctgc caacagggac 1020 gcgtcggtct tctccgatcc ggaggtcttc gacatcggac ggtctcccaa cccgcacatc 1080 gggttcggag gcggaggcgc gcacttctgc ctgggcaacc atctggccaa gctcgagctg 1140 cgcgtactct tcgagcagct ggccaggagg ttcccgcgga tgcggcagac cggcgaggcc 1200 cgccggttgc gctcgaactt catcaacggc atcaagaccc tccccgtcac cctcggctga 1260 acctctagta gcggaacgcg taatatcgcg actaaggagc tgacgtgacg gagcccccgt 1320 acaccgtgac cgcgctgccc accatgcgcc ctcccggctg cccgttcgac ccgcccgagg 1380 agttggcgca gctgcgcgag cagcacccac tcagcaggat ggtcttcccc gacgggcacg 1440 tcggctggct ggtgaccagt cacgccctgg ctcgcgcggt cgtggccgac ccccgcttca 1500 gctcgcggca cgagctcatg cactcgccgt tccctggggc catctcggcg gagaggccgc 1560 cgccggccgc gcccggcatg ttcatcggcg tcgatccgcc cgagcacacc cgctaccgca 1620 agctgctcac ggggaagttc accgtccgca ggatgcgcag tctcgccgcc cgcgtcgagg 1680 agatcaccgc cgagcacctg gacgccatgg agcggcaggg cccgcctgtg gacctggtgc 1740 aggcctacgc ccagcccgtc cccgcgctga tgatctgcga gctgctcggc gttccctacg 1800 ccgacaggga gttcttccag cgtcacgcgc tggcgctgaa caacttcgac gccgtccccg 1860 aggagcagta cgccgcctac gtcgcgctgc aggagtacct gcacgcgctg gtcctggcca 1920 aacgcgccac ccccaccgac gacctgctcg gcgatctcgc gcgcgaggag ctcaccgacg 1980 aggagctcac caacatcggg gttctgctgc tgggcgccgg gctcgacacc accgccaaca 2040 tgctcgcgct gggcaccttc gccctgctga gccaccccgg ccagctcgcc gccctgcgcg 2100 ccgacccggg cctcgccgac caggccgtcg aggagctgct gcgctacctg agcatcaccc 2160 acaccgggat acgggccgca ctggaggatg tcgagctgaa cggtcacgtg atcaaagcgg 2220 gggagagcgt cgccctctcg gcgcaggcgg ccaaccgcga ccctgagcgc ttcgccgacc 2280 ccgacactct cgacctgcac cgtcaggcca ccggacatct gagcttcggt cacggcgtcc 2340 accagtgcct cggccagcag ctggcccgcg tggagatgcg cgtcgccctt cccgcgctgg 2400 tcacccgttt cccgacgctg cggctggcca tatcctcagc agaggtgccg ctgcgcacca 2460 attccgacat ctacggcgtg caccggctcc cggtcgcgtg ggacaaggag tagccgatgg 2520 aactcgaagt ggattctagt gagctcggta ccagcccgac ccgagcacgc gccggcacgc 2580 ctggtcgatg tcggaccgga gttcgaggta cgcggcttgc aggtccagga aggggacgtc 2640 catgcgagtg tccgttcgag tggcggcttg cgcccgatgc tagtcgcggt tgatcggcga 2700 tcgcaggtgc acgcggtcga tcttgacggc tggcgagagg tgcggggagg atctgaccga 2760 cgcggtccac acgtggcacc gcgatgctgt tgtgggcaca atcgtgccgg ttggtaggat 2820 cc 2822 <210> 20 <211> 2460 <212> DNA <213> Artificial Sequence <220> <223> probe 2 <400> 20 ggcgcgcccg tcgccgaccg cgacaagatc ttcgcctggt ccaaccgcat gatcggcgcc 60 caggaccccg actacgccgc ctcaccggag gagggcggcg ccgcggccat ggaggtctac 120 gcctacgcct ccgaactggc cgcccagcgt cgcgccgccc cgcgcgacga catcgtcacc 180 aagctcctcc agtccgacga gaacggcgag agcctgacgg agaacgagtt cgagctcttc 240 gtgctgctcc tcgtggtcgc gggcaacgag accacccgta acgccgcctc cggcggcatg 300 ctcaccctct tcgagcatcc ggaccagtgg gacaggctgg tcgccgaccc ctccctcgcc 360 gccacggccg ccgacgagat cgtccgctgg gtctcgccgg tgaacctctt ccgccgtacg 420 gcgacagccg acctcaccct gggcggacag caggtcaagg ccgacgacaa ggtcgtggtc 480 ttctactcct ctgccaacag ggacgcgtcg gtcttctccg atccggaggt cttcgacatc 540 ggacggtctc ccaacccgca catcgggttc ggaggcggag gcgcgcactt ctgcctgggc 600 aaccatctgg ccaagctcga gctgcgcgta ctcttcgagc agctggccag gaggttcccg 660 cggatgcggc agaccggcga ggcccgccgg ttgcgctcga acttcatcaa cggcatcaag 720 accctccccg tcaccctcgg ctgaacctct agtagcggaa cgcgtaatat cgcgactaag 780 gagctgacgt gacggagccc ccgtacaccg tgaccgcgct gcccaccatg cgccctcccg 840 gctgcccgtt cgacccgccc gaggagttgg cgcagctgcg cgagcagcac ccactcagca 900 ggatggtctt ccccgacggg cacgtcggct ggctggtgac cagtcacgcc ctggctcgcg 960 cggtcgtggc cgacccccgc ttcagctcgc ggcacgagct catgcactcg ccgttccctg 1020 gggccatctc ggcggagagg ccgccgccgg ccgcgcccgg catgttcatc ggcgtcgatc 1080 cgcccgagca cacccgctac cgcaagctgc tcacggggaa gttcaccgtc cgcaggatgc 1140 gcagtctcgc cgcccgcgtc gaggagatca ccgccgagca cctggacgcc atggagcggc 1200 agggcccgcc tgtggacctg gtgcaggcct acgcccagcc cgtccccgcg ctgatgatct 1260 gcgagctgct cggcgttccc tacgccgaca gggagttctt ccagcgtcac gcgctggcgc 1320 tgaacaactt cgacgccgtc cccgaggagc agtacgccgc ctacgtcgcg ctgcaggagt 1380 acctgcacgc gctggtcctg gccaaacgcg ccacccccac cgacgacctg ctcggcgatc 1440 tcgcgcgcga ggagctcacc gacgaggagc tcaccaacat cggggttctg ctgctgggcg 1500 ccgggctcga caccaccgcc aacatgctcg cgctgggcac cttcgccctg ctgagccacc 1560 ccggccagct cgccgccctg cgcgccgacc cgggcctcgc cgaccaggcc gtcgaggagc 1620 tgctgcgcta cctgagcatc acccacaccg ggatacgggc cgcactggag gatgtcgagc 1680 tgaacggtca cgtgatcaaa gcgggggaga gcgtcgccct ctcggcgcag gcggccaacc 1740 gcgaccctga gcgcttcgcc gaccccgaca ctctcgacct gcaccgtcag gccaccggac 1800 atctgagctt cggtcacggc gtccaccagt gcctcggcca gcagctggcc cgcgtggaga 1860 tgcgcgtcgc ccttcccgcg ctggtcaccc gtttcccgac gctgcggctg gccatatcct 1920 cagcagaggt gccgctgcgc accaattccg acatctacgg cgtgcaccgg ctcccggtcg 1980 cgtgggacaa ggagtagccg atggaactcg aagtggattc tagtgagctc ggtaccagcc 2040 cgacccgagc acgcgccggc acgcctggtc gatgtcggac cggagttcga ggtacgcggc 2100 ttgcaggtcc aggaagggga cgtccatgcg agtgtccgtt cgagtggcgg cttgcgcccg 2160 atgctagtcg cggttgatcg gcgatcgcag gtgcacgcgg tcgatcttga cggctggcga 2220 gaggtgcggg gaggatctga ccgacgcggt ccacacgtgg caccgcgatg ctgttgtggg 2280 cacaatcgtg ccggttggta ggatcctcta gtatggaact cgaagtggat cgcgagcgct 2340 gcatcggggc gggcatgtgc gcgctgaccg cccccgaggt cttcgaccag gacctcgacg 2400 acggcagggt gatgctgctg gattcgtcgc cgccccccgg gcaccaggcg aaggcgcgcc 2460                                                                         2460 <210> 21 <211> 1865 <212> DNA <213> Artificial Sequence <220> <223> probe 3 <400> 21 tcgcgactaa ggagctgacg tgacggagcc cccgtacacc gtgaccgcgc tgcccaccat 60 gcgccctccc ggctgcccgt tcgacccgcc cgaggagttg gcgcagctgc gcgagcagca 120 cccactcagc aggatggtct tccccgacgg gcacgtcggc tggctggtga ccagtcacgc 180 cctggctcgc gcggtcgtgg ccgacccccg cttcagctcg cggcacgagc tcatgcactc 240 gccgttccct ggggccatct cggcggagag gccgccgccg gccgcgcccg gcatgttcat 300 cggcgtcgat ccgcccgagc acacccgcta ccgcaagctg ctcacgggga agttcaccgt 360 ccgcaggatg cgcagtctcg ccgcccgcgt cgaggagatc accgccgagc acctggacgc 420 catggagcgg cagggcccgc ctgtggacct ggtgcaggcc tacgcccagc ccgtccccgc 480 gctgatgatc tgcgagctgc tcggcgttcc ctacgccgac agggagttct tccagcgtca 540 cgcgctggcg ctgaacaact tcgacgccgt ccccgaggag cagtacgccg cctacgtcgc 600 gctgcaggag tacctgcacg cgctggtcct ggccaaacgc gccaccccca ccgacgacct 660 gctcggcgat ctcgcgcgcg aggagctcac cgacgaggag ctcaccaaca tcggggttct 720 gctgctgggc gccgggctcg acaccaccgc caacatgctc gcgctgggca ccttcgccct 780 gctgagccac cccggccagc tcgccgccct gcgcgccgac ccgggcctcg ccgaccaggc 840 cgtcgaggag ctgctgcgct acctgagcat cacccacacc gggatacggg ccgcactgga 900 ggatgtcgag ctgaacggtc acgtgatcaa agcgggggag agcgtcgccc tctcggcgca 960 ggcggccaac cgcgaccctg agcgcttcgc cgaccccgac actctcgacc tgcaccgtca 1020 ggccaccgga catctgagct tcggtcacgg cgtccaccag tgcctcggcc agcagctggc 1080 ccgcgtggag atgcgcgtcg cccttcccgc gctggtcacc cgtttcccga cgctgcggct 1140 ggccatatcc tcagcagagg tgccgctgcg caccaattcc gacatctacg gcgtgcaccg 1200 gctcccggtc gcgtgggaca aggagtagcc gatggaactc gaagtggatt ctagtgagct 1260 cggtaccagc ccgacccgag cacgcgccgg cacgcctggt cgatgtcgga ccggagttcg 1320 aggtacgcgg cttgcaggtc caggaagggg acgtccatgc gagtgtccgt tcgagtggcg 1380 gcttgcgccc gatgctagtc gcggttgatc ggcgatcgca ggtgcacgcg gtcgatcttg 1440 acggctggcg agaggtgcgg ggaggatctg accgacgcgg tccacacgtg gcaccgcgat 1500 gctgttgtgg gcacaatcgt gccggttggt aggatcctct agtttcaata agtttcaaga 1560 ggttatttca ctcatggcta tcactggcat ctttttcggc agcgacaccg gtaataccga 1620 aaatatcgca aaaatgattc aaaaacagct tggtaaagac gttgccgatg tccatgacat 1680 tgcaaaaagc agcaaagaag atctggaagc ttatgacatt ctgctgctgg gcatcccaac 1740 ctggtattac ggcgaagcgc agtgtgactg ggatgacttc ttcccgactc tcgaagagat 1800 tgatttcaac ggcaaactgg ttgcgctgtt tggttgtggt gaccaggaag attacgccga 1860 atatt 1865

Claims (17)

Cytochrome P450 hydroxylase (CYP), comprising the amino acid sequence of SEQ ID NO: 1 and mediating the hydroxylation of Cyclosporin A (CyA).
The method of claim 1,
Cytochrome P450 hydroxylase derived from Sebekia benihana .
A polynucleotide encoding the cytochrome P450 hydroxylase of claim 1.
The method of claim 3,
Polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2.
An expression vector comprising the polynucleotide of claim 3.
A transformant expressing cytochrome P450 hydroxylase having an amino acid sequence of SEQ ID NO: 1 introduced by the expression vector of claim 5.
(Iii) culturing the transformant of claim 6 to obtain a culture; And
(Ii) recovering cytochrome P450 hydroxylase (CYP) from the culture.
A polynucleotide encoding at least one cytochrome P450 hydroxylase (CYP) selected from the group consisting of CYP, CYP501, CYP502, CYP503, CYP504, CYP506, and combinations thereof of SEQ ID NO: 1, and putidaredoxin (putidaredoxin and putidaredoxin reductase set, ferredoxin and ferredoxin reductase set, and flavodoxin and flavodoxin reductase set) An expression vector comprising a polynucleotide encoding at least one electron transfer enzyme selected from the group.
10. The method of claim 9,
An expression vector comprising polynucleotides encoding CYP and CYP506 of SEQ ID NO: 1 as CYP, and polynucleotides encoding putidaredoxin and putidaredoxin reductase as electron transfer enzymes.
10. The method of claim 9,
An expression vector comprising a polynucleotide encoding CYP and CYP506 of SEQ ID NO: 1 as CYP, and a polynucleotide encoding peredoxin and peredoxin reductase as an electron transfer enzyme.
10. The method of claim 9,
An expression vector comprising a polynucleotide encoding CYP and CYP506 of SEQ ID NO: 1 as CYP, and a polynucleotide encoding flavodoxin and flavodoxin reductase as an electron transfer enzyme.
The transformant of claim 9, wherein the expression vector is introduced into a host cell to produce 4- (γ-hydroxy) -CyA.
The method of claim 12,
The host cell is a transformant capable of intrinsically hydroxylating cyclosporin A (CyA).
The method of claim 13,
The microorganism is Sebekia benihana ( Sebekia benihana ) transformants.
(Iii) preparing an expression vector comprising cytochrome P450 hydroxylase (CYP) and each polynucleotide encoding an electron transfer enzyme;
(Ii) preparing a transformant by introducing the prepared expression vector into a host cell;
(Iii) culturing the transformant to obtain a culture; And
(Iii) recovering 4- (γ-hydroxy) -CyA from the culture, method of producing 4- (γ-hydroxy) -CyA.
16. The method of claim 15,
The host cell is a transformant capable of intrinsically hydroxylating cyclosporin A (CyA).
16. The method of claim 15,
Wherein the culturing is carried out by solid culture or liquid culture.

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