KR20150049554A - Novel vitamin d_3 specific hydroxylase from sebekia benihana - Google Patents

Abstract

The present invention relates to a novel vitamin D_3-specific (VD_3) hydroxylase derived Sebekia benihana. The present invention provides a vitamin D_3-specific (VD_3) hydroxylase including an amino acid sequence of SEQ NO: 1. Moreover, the present invention provides a vitamin D_3 bioconversion method comprising the steps of: producing a recombinant expression vector by inserting a gene encoding the hydroxylase in an expression vector; transforming a cell through the recombinant expression vector; and hydroxylating vitamin D_3 by culturing the transformed cell.

Description

{Novel vitamin D3specific hydroxylase from Sebekia benihana}, a vitamin D3-

The present invention relates to a process for the preparation of benihana) derived from vitamin _{D 3 (Vitamin D 3; VD} 3) relates to specific novel enzyme hydroxide (hydroxylase).

Actinomycetes, a high G + C gram-positive soil bacterium, has been shown to inhibit the biosynthesis of many important secondary metabolites such as antibiotics and anticancer agents as well as various bioconversion enzymes including cytochrome P450 hydroxylases (CYPs) Clusters. In particular, the rare actinomycetes Sebekia benihana bind hydroxyl groups to specific sites of various natural products such as monensin, nigericin and cyclosporine A It has excellent ability to do. CYP, a heme-containing monooxygenase enzyme, is one of the most important industrial enzymes to promote the position-specific hydroxylation of endogenous and xenobiotic compounds. It is also associated with important steps in a variety of biological processes, including the early stages of steroid hormone synthesis and cholesterol biosynthesis. Generally, CYPs are mostly present in living organisms, and electron transfer partners such as ferredoxin (FD) and ferredoxin reductase (FDR) are required for CYP activation.

Vitamin _{D 3 (Vitamin D 3; VD} 3) is a pro hormone that plays a crucial role in homeostasis of plasma calcium and phosphate, cell differentiation, proliferation and immunological control. This has important effects in the treatment and prevention of various diseases such as hypothyroidism, osteoporosis and chronic renal failure. VD ₃ is synthesized from 7-dehydrocholesterol in skin exposed to UV light and exists in a physiologically inactivated form. In order to perform physiological functions thereof through the binding of the vitamin D receptor VD ₃ is 25-hydroxy vitamin _{D 3 (25-hydroxyvitamin D 3} ; 25 (OH) VD 3) , and 1α, 25-dihydroxy vitamin D ₃ (1?, 25-dihydroxyvitamin D ₃ ; 1 ?, 25 (OH) ₂ VD ₃ ). These two reactions are performed by the respective CYPs in the liver and kidney. Therefore, patients with liver or renal dysfunction should not be able to synthesize 1α, 25 (OH) ₂ VD ₃ , and should externally receive an activated form of VD ₃ .

1α, 25 (OH) ₂ VD ₃ molecules are generally synthesized from cholesterol by chemical methods. However, this process requires a lot of reaction steps, and the yield is as low as less than 1%. On the other hand, biological processes are proposed as effective alternatives because they can produce 1α, 25 (OH) ₂ VD ₃ by only a two-step process. Some researchers have reported on the transition to VD ₃ 25 (OH) VD ₃ and _{1α, 25 (OH) 2 VD} 3 of using a number of microorganisms. The 25 (OH) VD ₃ molecule is a Pseudonocardia In the rare actinomycetes named autotrophica) VD ₃ - it was successfully produced by a specific 25-P450VD25 (CYP105A2) hydroxide enzyme _{(VD 3 -specific 25-hydroxylase)} . In a recent study, no pseudo Arcadia VD ₃ containing CYP105A1 of Vdh and S. griseolus of the auto car trophy (P. autotrophica) - the specific enzyme hydroxide of _{1α, 25 (OH) 2 VD} 3 to make the VD ₃ 25 and 1, but the enzymatic kinetics of these have not yet been clarified. In order to overcome the above problems and increase the VD ₃ -specific bioconversion yield, new strategies such as random mutagenesis, enzyme active site modification and actinomycete heterologous expression have been promoted.

The inventors have in a previous study, three Vecchia Benny a whole genome sequencing and in silico (in the (S. benihana) silico ) analysis, 21 CYPs and their electron transport partners (7 FD and 4 FDRs) genes were identified. In addition, the CYPome genes were inactivated by an E. coli junction-based PCR target gene disruption system. CYP-sb21, a cyclosporine A (CsA) -specific hydroxylase gene, was identified using the S. benihana CYPome mutations, and the fourth N-methyl leucine leucine position of CsA-specific hydroxylase.

Korean Patent Laid-Open Publication No. 10-2013-0081394 discloses a novel cytochrome P450 hydroxylase (CYP) derived from Sebecia bennyana. However, the vitamin D ₃ specific novel hydroxyapatite of the present invention There is no mention of enzymes (hydroxylases).

Thus, the present inventors have found that three Vecchia Benny one (S. benihana) Figure ₃ VD - specifically it was confirmed that it is possible to perform hydroxylation, three Vecchia Benny one (S. benihana) a three Vecchia Benny by screening the mutant CYPome One ( S. benihana ) CYP gene has been found to be involved in the VD- ₃ position-specific hydroxylation reaction. The present inventors also confirmed the function of the newly classified VD ₃ -specific CYP gene through genetic complementation experiments as well as heterologous host expression in a VD ₃ non-hydroxylated Streptomyces host, Completed.

An object of the present invention is vitamin _{D 3 (Vitamin D 3; VD} 3) comprising the amino acid sequence of SEQ ID NO: 1 to provide a specific enzyme hydroxide (hydroxylase).

It is another object of the present invention to provide a method for producing a recombinant expression vector comprising the steps of: preparing a recombinant expression vector by inserting a gene encoding the hydroxylase into an expression vector; Transforming the cells with the recombinant expression vector; And to provide a vitamin D ₃ bioconversion (bioconversion) comprises the step of the plasma vitamin D ₃ hydroxide by culturing the transformed cells (hydroxylation).

In order to achieve the above object, the present invention comprises the amino acid sequence of SEQ ID NO: 1 vitamin _{D 3 (Vitamin D 3; VD} 3) provides specific enzyme hydroxide (hydroxylase) and a functional equivalent thereof with water.

Specifically, the vitamin D ₃ hydroxide specific enzyme vitamin D ₃ for 25-hydroxy-vitamin _{D 3 (25-hydroxyvitamin D 3} ; 25 (OH) VD 3) or 1α, 25-dihydroxy-vitamin D ₃ (1α , 25-dihydroxyvitamin D ₃ ; 1?, 25 (OH) ₂ VD ₃ ).

"Functional equivalent" of the present invention includes all of the amino acid sequence variants in which some or all of the hydroxylases of SEQ ID NO: 1 are substituted, or in which a part of the amino acid is deleted or added maintains the above enzyme function. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the hydroxylase.

The present invention also relates to a method for producing a recombinant expression vector, comprising the steps of: preparing a recombinant expression vector by inserting a gene encoding the hydroxylase into an expression vector; Transforming the cells with the recombinant expression vector; And it provides a vitamin D ₃ bioconversion (bioconversion) comprises the step of the plasma vitamin D ₃ hydroxide by culturing the transformed cells (hydroxylation). In particular, the Vitamin D ₃ bioconversion (bioconversion) is a vitamin D ₃ 25-hydroxy-vitamin _{D 3 (25-hydroxyvitamin D 3} ; 25 (OH) VD 3) or 1α, 25-dihydroxy vitamin D ₃ (1α, 25-dihydroxyvitamin D ₃ ; 1α, 25 (OH) ₂ VD ₃ ).

Preferably, the gene is represented by SEQ ID NO: 2, but is not limited thereto, and provides equivalent nucleic acid sequences to the sequence.

The "equivalent nucleic acid sequence" includes the codon degenerate sequence of the above-mentioned hydroxylase sequence. The term " codon degenerate sequence "means a nucleic acid sequence which differs from the above sequence but encodes a polypeptide having the same sequence as the hydroxylase disclosed in the present invention.

In the present invention, " vector " means a DNA molecule that is replicated by itself, which is used to carry the clone gene (or another fragment of the clone DNA).

In the present invention, an "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as ampicilin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, and apramycin. And is suitably selectable by a person skilled in the art.

The issues related to the genetic engineering techniques used in the present invention are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) Frederick M. Ausubel et al., Current Protocols in Molecular Biology Volume 1,2,3, John Wiley & Sons, Inc. (1994)).

The invention three Vecchia Benny one (Sebekia benihana) derived from vitamin _{D 3 (Vitamin D 3; VD} 3) specific novel relates to a hydroxide enzyme (hydroxylase), VD ₃ to 25-hydroxy vitamin D ₃ (25-hydroxyvitamin D _3; a ₂ VD ₃₎ a position-specific hydroxylation (hydroxylation); 25 (OH) VD 3) , and 1α, 25- dihydroxy-vitamin _{D 3 (1α, 25 (OH} 1α, 25-dihydroxyvitamin D 3) And can be synthesized more effectively than a process generally synthesized from cholesterol by a chemical method.

Figure 1 shows the HPLC profile and structures of VD ₃ and a hydroxide VD ₃ eseo one of three Vecchia Benny (S. benihana). The S. benihana wild type was cultured in GSMY medium for 48 hours, then 100 mg / L VD ₃ as a hydroxylation substrate was added to the culture medium and further cultured for 48 hours. Sampling was performed 2 times, after 0 hr and 48 hr, after addition of substrate and analyzed by HPLC.
FIG. 2 shows conversion yields of 25 (OH) VD ₃ and 1α, 25 (OH) ₂ VD ₃ in (B) FD and FDR destructors of S. benihana (A) CYP fragment and (B) The vertical bar represents the mean standard deviation value for each of the two measurements.
Figure 3 shows the genetic complementation to S. benihana CYP-sb3a. (A) Recombinant plasmid pCYP-sb3a. (B) HPLC analysis of VD ₃ biotransformation in S. benihana wild-type strain, S. benihana CYP-sb3a, S. benihana CYP-sb3a / pCYP-sb3a and S. benihana CYP-sb3a / pCYP- Profile. (C) Bioconversion results from 25 (OH) VD ₃ to 1α, 25 (OH) ₂ VD _{3 in} S. benihana wild type and S. benihana ΔCYP-sb3a.
4 (A) shows a sequence comparison of CYP-sb3a, Vdh from Pseudonocardia autotrophica , and CYP105A1 from Streptomyces griseolus . Boxes represent conserved amino acid residues that make up the CYP-specific motif. (B) Phylogenetic tree of CYP-sb3a and other CYP families. CYP-sb3a (S. benihana), Vdh ( Pseudonocardia autotrophica ), CYP105A1 ( S. griseolus ). CYP51 ( Nocardia farcinica ), CYP101 ( Novosphingobium aromaticivorans ), CYP102 ( Bacillus anthracis ), CYP103 ( Agrobacterium tumefaciens ), CYP105 ( Saccharopolyspora erythraea ), CYP106 ( B. anthracis ), CYP110 ( Nostoc sp ); CYP111 ( N. aromaticivorans ), CYP112 ( Sinorhizobium fredii ), CYP113 ( S. erythraea ), CYP114 ( M. loti ), CYP115 ( M. loti ), CYP116 ( S. erythraea ), CYP117 ( S. fredii ); CYP119 ( Sulfolobus tokodaii ), CYP120 ( Trichodesmium erythraeum ).
Figure 5 (A) shows the pHE-CYP-sb3a plasmid map derived from a pHSEV-1. (B) is S. coelicolor M145 / pHE-CYP-sb3a. Fig. ₃ shows the HPLC profile of the VD ₃ metabolite heterologously expressed in CYP-sb3a.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Sebecia Benny Hana Sebekia Benihana )in VD ₃ - Identification of specific hydroxylation genes

1. Strain, culture condition

Rare actinomycetes Sebecia Bennyana ( Sebekia benihana) KCTC 9610 is Bank of Korea (Korean Collection for Type Cultures cell line; was purchased from _{KCTC, Korea), VD 3 (} glucose 0.7% GSMY medium to determine the hydroxylation, yeast extract 0.45%, malt extract 0.5%, soluble starch 0.75% and calcium carbonate 0.005%). For conjugation experiments, the cells were incubated at 30 ° C in RARE3 medium (glucose 1%, yeast extract 0.4%, malt extract 1%, peptone 0.2%, MgCl ₂ .6H ₂ O 0.359%, glycerol 0.5%). E. coli BL21 (DE3) (Escherichia coli BL21 (DE3)) was cultured in Luria-Bertani (LB) medium and maintained at 37 ° C in LB agar medium supplemented with appropriate antibiotics if necessary. Streptomyces coelicolor ) M145 was cultured in Tryptic Soy Broth (TSB) medium to confirm the VD ₃ hydroxylation reaction and YEME medium (yeast extract 0.3%, peptone 0.5%, malt extract 0.3%, glucose 1 %, sucrose 34% and MgCl ₂ .6H ₂ O 5 mM, separately sterilized glycine 0.5%) at 30 ° C.

2. HPLC analysis for VD ₃ -specific hydroxylation reaction

The selected S. benihana strain was cultured in GSMY medium for 72 hr at 37 ° C, treated with VD ₃ (100 mg / L) as a hydroxylation substrate, and further incubated for 48 hrs. The crude compound was extracted from the culture solution using ethylacetate, ethyl acetate was evaporated and the crude compound was resuspended in methyl alcohol. Samples were analyzed using HPLC equipped with a photodiode array detector under the following conditions: column, YMC-Pack ODS-AM (4.6 x 300 mm) (YMC, Tokyo, Japan); UV detection, 265 nm; Flow rate, 1.5 mL / min; Column temperature, 40 [deg.] C and a two-buffer gradient system; ddH ₂ O (Buffer A) and 100% acetonitrile (Buffer B). One cycle of the buffer B gradient was programmed starting at 50% and proceeding to 100% for 10 minutes followed by elution at 100% and 50% for 25 and 10 minutes, respectively. The injection volume was 20 uL.

3. E. coli - S. benihana Intergeneric conjugation ( E. coli )

10 mL of S. benihana culture, which had been cultivated for 3 days in RARE3 medium (the same medium composition as mentioned above except that 2% MgCl ₂ .6H ₂ O was added), was added to 90 mL of fresh medium , And re-cultured at 30 ° C for 24 hours. The culture was then centrifuged and resuspended in 10 mL of fresh medium, homogenized, and fragmented by ultrasonication (two runs with 3 seconds of operation using an ultrasonic generator and 2 operations of 4 seconds of inactivity) 36%, Ulsso Hitech, Korea). After further incubation at 30 DEG C for 20 hours, the culture was again centrifuged, resuspended in medium, and sonicated as before. The donor E. coli strain ET12567 / pUZ8002 for transferring the disruption construct was prepared according to the standard E. coli - Streptomyces junction. In each mating experiment, donor and recipient cells were mixed and transformed with ISP4 medium (soluble starch 1%, dipotassium phosphate 0.1%, magnesium sulfate USP 0.1%, sodium chloride 0.1%, ammonium sulfate 0.2%, calcium , zinc sulfate 0.0001%, tryptone 0.15%, yeast extract 0.05%, agar 2%). After incubation at 30 ° C for 20 hours, each plate was submerged in 1 mL of sterile water containing a final concentration of 100 ug / mL and 25 ug / mL hygromycin and nalidixic acid, respectively . In addition, a final concentration of 50 ug / mL apramycin was added for the compensation experiments. Colonies of S. benihana exconjugant usually appeared 1-2 weeks after culture.

4. Results

Since S. benihana has been reported to bind hydroxyl groups to specific sites of various natural products, in order to determine if S. benihana can perform the VD ₃ -specific hydroxylation reaction, VD ₃ bioconversion analysis. S. benihana strain was cultured in GSMY medium at 37 ° C for 72 hours and VD ₃ (100 mg / L) was added as a hydroxylation substrate. After an additional 48 hours of incubation, the crude extract was subjected to HPLC analysis. Several deduced VD ₃ derivatives were detected. Through a standard sample spiking and LC-MS analysis, it was identified two known VD ₃ derivative, which is _{25 (OH) VD 3 (m} / z 401.3426 [M + H]) and _{1α, 25 (OH) 2 VD} 3 (m / z 417.3376 [M1-H]) (Fig. 1). The results indicate that S. benihana can perform a VD ₃ -specific hydroxylation reaction.

Previously, we identified a complete CYPome containing 21 CYPs and an electron transfer protein such as 7 FD and 4 FDR genes. GenBank accession numbers for S. benihana CYPs are KC208044 to KC208064. Subsequently, to produce the S. benihana CYPome mutation, each gene was inactivated by PCR-target gene disruption. A unique CYP (designated CYP-sb21) responsible for the CsA-specific hydroxylation reaction was identified by selection of S. benihana CYPome mutants. In order to identify unique CYPs responsible for VD ₃ -specific hydroxylation, the CYPome mutations of S. benihana were similarly screened. The 32 mutants tested, △ CYP-sb6, △ CYP -20 and △ CYP-24 that was observed to be slightly reduced in several other CYP fracture strain, such as, in the VD ₃ 25 (OH) VD ₃ and 1α, 25 ( OH) ₂ VD ₃ were all significantly reduced in the ΔCYP-sb3 and ΔCYP-sb3a mutants (FIG. 2A). The CYP-sb3 mutant strain contains a deletion in two translationally-coupled CYP genes, CYP-sb3a and CYP-sb3b. These results indicate that CYP-sb3a is mainly involved in the VD ₃ -specific hydroxylation reaction. Although small amounts of 25 (OH) VD ₃ and 1α, 25 (OH) ₂ VD ₃ exist in S. benihana ΔCYP-sb3a, this may be the effect of other non-specific CYPs. On the other hand, the majority of S. benihana FD-sb and ΔFDR-sb mutations still have VD ₃ hydroxylation activity (FIG. 2B), suggesting that a single FD or FDR is VD ₃ -specific It does not take up the hydroxylation reaction. Since FD and FDR, which are involved in the electron transfer of NAD (P) H to heme-iron of CYP, are present in low numbers in most actinomycetes species, S. benihana VD ₃ -specific CYP-sb3a appears to have no specific preference for FD or FDR in the VD ₃ -specific hydroxylation reaction.

< Example  2> Sebecia Benny Hana S. mehana )of CYP - sb3a Compensation of complementation ) And characterization

1. Construction of Recombinant Insertion Plasmid pCYPs

Three Vecchia Benny for a functional overexpression experiments and compensation of CYP-sb3 gene in (S. benihana), three Vecchia Benihana 2.4 kb DNA fragment containing the entire gene CYP-sb3 (S. benihana) of genomic DNA derived from it CYP-sb3 forward primer (5'- AGATCT CCCGTCGTCATCAAG-3 ') and reverse primer (5'- TCTAGA AGGGGGCCCCGGCCATAC-3'). The primers were used as templates and amplified by PCR using the following primers. Similarly, the CYP-sb3a gene (1.2 kb) DNA fragment was obtained using a CYP-sb3a forward primer (5'- AGATCT CCCGTCGTCATCAAG-3 ') and a reverse primer (5'- TCTAGA GCGTAGACGGCGTAG-3' The DNA fragment (1.2 kb) was obtained using a CYP-sb3b forward primer (5'- AGATCT CCCGGCTCGGCGACATCA-3 ') and a reverse primer (5'- TCTAGA AGGGGGCCCCGGCCATAC-3'). Each forward and reverse primer contained Bgl II (AGATCT) and Xba I (TCTAGA) restriction enzyme sites. PCR contained 0.25 mM of each primer, 0.4 μM of each dNTPs, 1 μL of extracted DNA (400 ng / μL), 1 U Ex Taq polymerase (TaKaRa, Japan) and 10% dimethyl sulfoxide (DMSO) dissolved in the reaction buffer Lt; RTI ID = 0.0 &gt; uL. &Lt; / RTI &gt; Amplification was performed with a thermal cycler (BioRad, USA) according to the following profile: 60 s at 95 ° C, 60 s at 58 ° C, and 30 s at 75 ° C for 60 s. The amplified PCR products were analyzed by 1% (w / v) agarose gel electrophoresis and purified using a DNA extraction kit (COSMO, Korea). The PCR product was then ligated to pGEM T-easy vector (TaKaRa, Japan). The ligation vector was fully sequenced to confirm integrity (Macrogen, Korea). The PCR product was finally subcloned into the insertion plasmid pSET152, which contains the constitutive promoter ermE * and the hygromycin resistance gene (Fig. 3A). The resulting primers were constructed as pCYP-sb3, pCYP-sb3a and pCYP-sb3b, respectively. This plasmid was introduced into the can through the joint (for over-expression), a three Vecchia Benny (S. benihana) wild-type, one-year-old Vecchia Benny (compensate for) (S. benihana) △ CYPs, and then inserted into the att B site Hygromycin. &Lt; / RTI &gt; PCYP-sb13 (pMMBL301) containing the CYP-sb13 (CYP506) gene was introduced as S. benihana △ CYP-sb3a as a negative control.

2. Results

To confirm the role of CYP-sb3a in the VD ₃ -specific hydroxylation reaction, gene-compensation experiments of CYP-sb3a were performed in S. benihana △ CYP-sb3a. The coding sequence of CYP-sb3a was cloned into a chromosomally inserted plasmid that always contained the constitutive ermE * promoter. pCYP-sb3a was introduced by the junction into three Vecchia Benny one of dielectric (S. benihana) △ CYP-sb3a , three Vecchia Benny one (S. benihana) △ CYP-sb3a / pCYP-sb3a to genomic DNA was PCR amplified in Lt; / RTI &gt; HPLC showed that the VD ₃ hydroxylation reaction to 25 (OH) VD ₃ and 1α, 25 (OH) ₂ VD _{3 in} S. benihana ΔCYP-sb3a / pCYP-sb3a was significantly restored (FIG. 3A) . On the other hand, overexpression of CYP-sb13 (negative control) in S. benihana △ CYP-sb3a failed to produce both 25 (OH) VD ₃ and 1α, 25 (OH) ₂ VD ₃ 3B). When 25 (OH) VD ₃ was added to the culture of S. benihana as a hydroxylated substrate, 25 (OH) VD ₃ was not a mutation in S. benihana △ CYP-sb3a It was converted to 1α, 25 (OH) ₂ VD ₃ more in S. benihana wild type (FIG. 3C). These results indicate that the VD ₃ hydroxylation first takes place at position 25, and then the secondary hydroxylation takes place at the 1α position. In the CYP-sb3a mutation, 25 (OH) VD ₃ was not further converted to 1α, 25 (OH) ₂ VD ₃ because S. benihana CYP-sb3a was found in both position 25 and 1α VD ₃ &lt; / RTI &gt;

We analyzed CYP-specific motifs in CYP-sb3a by comparing previously reported VD ₃ -specific CYPs with amino acids (FIG. 4A). Three typical CYP-conserving motifs were present in the CYP amino acid sequence. The GXXXCXG motif is the cysteine heme-iron ligand site, and the EXXR motif within the K-helix site is associated with the interaction between the oxidation-reduction partners and the GXXT motif is the oxygen-binding site. FIG significant sequence homology and the system shown in 4B occurs (phylogenetic) results CYP105 family of VD ₃ pseudo furnace Arcadia auto trophy car (P VD ₃ hydroxide than CYP107 enzyme family CYP105A1 (32% homology) of S. griseolus hydroxide enzyme . autotrophica) showed a high similarity with the amino-derived Vdh (41% homology). These results suggest that S. benihana CYP-sb3a, which causes dual hydroxylation at both the 25 and 1α positions of VD ₃ , is a member of the P. autotrophica Vdh-like bacterial CYP107 family Indicates that it belongs.

< Example  3> CYP - sb3a Functional expression of

1. Construction of heterologous expression plasmid pHE-CYP-sb3a

Streptomyces In order to heterologously express the CYP-sb3a gene in the coelicolor , the 1.2 kb DNA fragment containing the entire CYP-sb3a gene was genomic DNA derived from S. benihana as a template and the CYP-sb3a Hexp Was amplified using a forward primer (5'- CATATG AGCGACAAGCTG-3 ') and a reverse primer (5'- GAATTC TCGAACGGTAGTTTC-3'). The forward and reverse primers contained Nde I (CATATG) and EcoR I (GAATTC) restriction enzyme sites, respectively. PCR was performed with 0.25 mM each of 0.4 M of each primer, 4 μl of dNTPs, 1 μL of extracted DNA (400 ng / μL), a final volume of 20 μL containing 1 U Ex Taq polymerase (TaKaRa, Japan) and 10% DMSO dissolved in reaction buffer. Amplification was performed with a thermal cycler (BioRad, USA) according to the following profile: 60 s at 95 ° C, 60 s at 54 ° C, and 30 s at 75 ° C for 60 s. The amplified PCR products were analyzed by 1% (w / v) agarose gel electrophoresis and purified using a DNA extraction kit (COSMO, Korea). The PCR product was then ligated to a T & A cloning vector (Real Biotech Corporation, Taiwan). The ligation vector was fully sequenced to confirm integrity (Macrogen, Korea). The PCR product was finally subcloned into the pHSEV-1 plasmid containing the inducible tipA promoter and the resulting plasmid was named pHE-CYP-sb3a. The pHE-CYP-sb3a plasmid was transformed by the protoplast transformation method into Streptomyces coelicolor ) and then screened by kanamycin (kan).

2. Streptomyces heterozygous expression of CYP - sb3a gene in coelicolor )

Recombinant plasmid pHE-CYP-sb3a was used for protoplast transformation. To prepare protoplasts, S. coelicolor cultured in YEME medium was treated with lysozyme solution (1 mg / mL). Transformation pHE-CYP _vd1 and P buffer _{(sucrose 10.3%, K 2 SO} 4 0.025%, MgCl ₂ .6H ₂ O 0.202%, trace element solution 0.2%). Transformed Streptomyces Silicate color conversion (Streptomyces coelicolor) is R2YE agar plates _{(sucrose 10.3%, K 2 SO} 4 0.025%, MgCl ₂ .6H ₂ O 1.012%, glucose 1%, casamino acids 0.01%, agar 1.1%, KH ₂ PO ₄ (0.5%), CaCl ₂ .2H ₂ O (3.68%), 0.8%, L-proline (20%) 0.15%, TES buffer (5.73%, adjusted to pH 7.2) 0.05% NaOH (1N) prepared separately) at 30 ° C, and kanamycin (kan) was used for screening the recombinant strains.

3. Results

In vivo of CYP-sb3a in heterozygous hosts ( in vivo function, we used Streptomyces coelicolor M145, which is easy to genetically engineer, as an expression host. More importantly, Streptomyces coelicolor does not perform a VD ₃ -specific hydroxylation reaction. The CYP-sb3a gene was cloned into pHSEV-1 containing a thiostreption-induced tipA promoter (pHE-CYP-sb3a, Fig. 5A) and the recombinant plasmid was transformed into polyethylene glycol (PEG) Through transformation, Streptomyces &lt; RTI ID = 0.0 &gt; coelicolor M145, and then screened with kanamycin. 25 (OH) VD ₃ was clearly detected in the S. coelicolor M145 / pHE-CYP-sb3a strain, but no hydroxylation was observed in the S. coelicolor M145 wild-type strain containing only the vector itself (FIG. 5B). These results indicate that even in heterozygous hosts, CYP-sb3a plays a major role in VD ₃ -specific hydroxylation. However, two interesting in S. coelicolor M145 / pHE-CYP-sb3a strain 1α, 25 (OH) ₂ VD ₃ peaks did not clearly detected, which occurs at the 25 (OH) of VD ₃ 1α position, the two kinds of host system Indicating that the efficiency of the tea hydrolysis reaction is worse than the primary hydroxylation reaction occurring at position 25 of VD ₃ . However, it is not possible to exclude the possibility that some additional factors, other than CYP-sb3a, present in S. benihana may be required for the secondary hydroxylation reaction in S. coelicolor . In conclusion, the inventors have shown for the first time that a new member of the bacterial CYP107 family, S. benihana CYP-sb3a, is the major cytochrome P450 hydroxylase involved in site-specific VD ₃ hydroxylation It turned out.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Novel vitamin D3 specific hydroxylase from Sebekia Benihana <130> ADP-2013-0421 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 397 <212> PRT <213> Sebekia Benihana <400> 1 Val Ser Asp Lys Leu Pro Phe Glu Phe Phe Leu Ser Pro Glu Phe Gly   1 5 10 15 Ala Asp Pro Tyr Ala Val Tyr Ala Gly Leu Arg Ala Glu Gly Pro Val              20 25 30 His Ale Ile Asp Phe Pro Pro Gly Ala Ser Ala Phe Val Val Val Asp          35 40 45 Tyr Glu His Ala Arg Pro Ala Leu Asn Asp Pro Arg Leu Ser Lys Asn      50 55 60 Leu Ala Ser Gly Pro Glu Trp Phe Arg Glu Ala Ile Ser Gln Gln Asn  65 70 75 80 Pro Val Leu Gly Asp Asn Met Leu Met Ser Asp Pro Pro Asp His Thr                  85 90 95 Arg Leu Arg Arg Val Val Ser Lys Ala Phe Thr Pro Arg Arg Ile Glu             100 105 110 Arg Leu Arg Pro Arg Val Gln Ala Ile Thr Asp Glu Leu Ile Asp Ala         115 120 125 Phe Glu Gly Asp Thr Val Glu Leu Ile Asp Ala Phe Ala Phe Pro Leu     130 135 140 Pro Ile Ile Val Ile Cys Glu Leu Leu Gly Val Pro Asp Arg Asp Arg 145 150 155 160 Ala Asp Phe Arg Arg Trp Ser Gly Ala Leu Val Thr Pro Ala Val Asn                 165 170 175 Glu Glu Leu Ala Arg Ser Ser Asp Glu Ala Ser Asp Ala Leu Arg Ala             180 185 190 Tyr Phe Thr Arg Leu Ile Ala Glu Arg Arg Ala Ala Pro Thr Asp Asp         195 200 205 Leu Val Ser Val Leu Val Thr Ser Asp Glu Leu Ser Glu Arg Glu Val     210 215 220 Leu Ser Thr Leu Ala Leu Leu Leu Ile Ala Gly His Glu Thr Thr Val 225 230 235 240 Asn Leu Ile Gly Asn Gly Met Leu Ala Leu Leu Arg Asn Pro Asp Gln                 245 250 255 Leu Asp Leu Leu Arg Ala Lys Pro Glu Leu Leu Pro Asn Ala Ile Glu             260 265 270 Glu Phe Leu Arg Tyr Asp Ala Pro Val Glu Arg Ala Thr Phe Arg Ile         275 280 285 Ala Leu Glu Asp Met Glu Ile Ala Gly Thr Pro Ile Pro Lys Gly Ser     290 295 300 Phe Val His Ile Ser Ile Gly Ala Ala Gly Arg Asp Pro Lys Ala Phe 305 310 315 320 Asp Arg Ala Glu Ser Leu Asp Val Ala Arg Glu Asp Asn Arg His Val                 325 330 335 Gly Phe Gly His Gly Leu His Phe Cys Leu Gly Ala Pro Leu Ala Arg             340 345 350 Met Glu Gly Gln Ile Ala Phe Gly Ser Leu Leu Ala Arg Leu Gly Asp         355 360 365 Ile Arg Leu Ala Cys Pro Glu Glu Gln Leu Ser Trp Arg Phe Ser Gly     370 375 380 Ser Ile Leu Arg Ser Leu Ala Ala Leu Pro Ile Ala Ile 385 390 395 <210> 2 <211> 1194 <212> DNA <213> Sebekia Benihana <400> 2 gtgagcgaca agctgccgtt tgagttcttc ctctccccgg agttcggcgc cgacccctac 60 gcggtctacg ccgggctccg ggccgaagga cccgtccacg ccatcgactt ccctcctgga 120 gcgtccgcgt tcgtggtcgt cgactacgag cacgccaggc ccgcgctgaa cgatccgcgc 180 ctgtccaaga acctcgccag cggacccgag tggttccgcg aggccatctc gcagcagaac 240 cccgtgctcg gcgacaacat gctgatgtcg gatcctcccg accacacccg gctgcggcgg 300 gtggtctcga aggcgttcac gccgcgcagg atcgagcggc tgcgcccgcg cgtccaggcg 360 atcaccgacg agctgatcga cgccttcgag ggcgacaccg tcgagctgat cgacgccttc 420 gccttcccgc tgccgatcat cgtgatctgc gagctgctgg gcgtgcccga ccgtgacagg 480 gccgatttcc gcaggtggtc gggggcgctg gtcacgccgg ccgtcaacga ggagctggcc 540 aggagccgcg acgaggccag cgacgcgctg cgcgcctact tcacccgcct gatcgccgaa 600 cgacgcgccg ctcccaccga cgacctggtg agcgtgctgg tgaccagcga cgagctgagc 660 gagcgggagg tgctctccac cctggcgctg ctgctgatcg ccggccacga gaccaccgtc 720 aacctcatcg gcaacggcat gctggccctg ctgcgcaacc ccgaccagct cgacctgctg 780 cgcgccaagc ccgagctgct gccgaacgcg atcgaggagt tcctgcgtta cgacgcgccc 840 gtcgagcggg cgaccttccg catcgcgctc gaggacatgg agatcgccgg cacccccatc 900 cccaagggca gcttcgtgca catctccatc ggcgcggcgg gccgtgatcc gaaggccttc 960 gaccgggccg agagcctcga cgtcgcccgc gaggacaaca ggcacgtcgg cttcggccac 1020 ggcctccact tctgcctcgg cgcgccgctc gcgcgcatgg agggacagat cgccttcggc 1080 tcactgctcg cccggctcgg cgacatcagg ctggcctgcc ccgaggagca gctcagctgg 1140 cgcttcagcg gctccatcct gagatcgctg gctgcccttc cgatagcgat ctag 1194

Claims (6)

Vitamins comprising the amino acid sequence of SEQ ID NO: _{1 D 3 (Vitamin D 3;} VD 3) specific enzyme hydroxide (hydroxylase). The method of claim 1, wherein the hydroxylase is selected from the group consisting of Sebekia &lt; RTI ID = 0.0 &gt; benhana . &lt; / RTI &gt; The method of claim 1 wherein the vitamin D ₃ hydroxide specific enzyme vitamin D ₃ for 25-hydroxy-vitamin _{D 3 (25-hydroxyvitamin D 3} ; 25 (OH) VD 3) or 1α, 25-dihydroxy vitamin D ₃ (1?, 25-dihydroxyvitamin D ₃ ; 1?, 25 (OH) ₂ VD ₃ ). Preparing a recombinant expression vector by inserting a gene encoding the hydroxylase of claim 1 into an expression vector;
Transforming the cells with the recombinant expression vector; And
How vitamin D ₃ bioconversion (bioconversion), comprising the step of the plasma vitamin D ₃ hydroxide by culturing the transformed cells (hydroxylation). The method of claim 4, wherein the gene is vitamin D ₃ bioconversion (bioconversion) characterized in that as shown in SEQ ID NO: 2. The method of claim 4, wherein the vitamin D ₃ bioconversion (bioconversion) is a vitamin D ₃ 25-hydroxy-vitamin _{D 3 (25-hydroxyvitamin D 3} ; 25 (OH) VD 3) or 1α, 25-dihydroxy-vitamin _{D 3 (1α, 25-dihydroxyvitamin} D 3; 1α, 25 (OH) 2 VD 3) by way of vitamin D ₃ bioconversion (bioconversion), characterized in that the conversion.

Images