WO2006009334A1

WO2006009334A1 - Cytochrome p450 enzyme and the gene encoding the same

Info

Publication number: WO2006009334A1
Application number: PCT/KR2004/002125
Authority: WO
Inventors: Jin Suk Woo; Jae Kyung Sohng; Hei Chan Lee; Kwang Kyoung Liou; Young Soo Jung; Sun Youp Kang; Dae Hee Kim; Wun Min Seo; Ji Young Yang
Original assignee: GENECHEM Inc
Current assignee: GENECHEM Inc
Priority date: 2004-07-23
Filing date: 2004-08-24
Publication date: 2006-01-26
Anticipated expiration: 2007-01-23
Also published as: KR20060008195A; KR100564158B1

Abstract

The present invention relates to a cytochrome P450 enzyme (CYP105P2) with function of introducing hydroxy groups into aromatic ring compounds, a gene encoding the enzyme, a recombination vector containing the gene, a microorganism transformed with the recombination vector and a method for introducing hydroxy groups into aromatic ring compounds, the method being characterized by using the enzyme or the transformed microorganism. The CYP 105P2 enzyme according to the present invention is useful in preparing hydroxy group-introduced aromatic ring compound for enzymatic ability to introduce hydroxy group into aromatic ring compound, especially, to introduce pentahydroxy group into benzene ring compound.

Description

CYTOCHROMEP450ENZYMEANDTHEGENEENCODING

THESAME

TECHNICAL FIELD

The present invention relates to a cytochrome P450 enzyme (CYP105P2) with function of introducing hydroxy groups into aromatic ring compounds, a gene encoding the enzyme, a recombination vector containing the gene, a microorganism transformed by the recombination vector and a method introducing hydroxy groups into aromatic ring compounds, the method being characterized by using the enzyme or the transformed microorganism.

BACKGROUND ART

Cytochrome P450 (CYP) enzymes are the superfamily of protein which exists in all eukaryotic organisms, plants, animals, fungi, and microorganisms, and they are found in 45 different species of the genus Streptomyces. As apposed to the state that CYPs found in eukaryotes are bound to membrane, most of the CYPs found in the genus Streptomyces are expressed in the phase of aqueous solution, and only a few of them have been characterized.

CYPs do not seem to be essential for the metabolism of most prokaryotes, and some of them are involved in catabolism of carbohydrates, terpenes and the like, such that they provide the sole carbon and energy sources for bacteria. (Omura, T., Biochem. Biophys. Res. Commun., 266:690, 1999).

They are also involved in the oxidative, peroxidative, and reductive metabolisms of l various endogenous compounds such as steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines, and other secondary metabolites.

The main function of CYP is the monooxygenation of various substrates. This reaction requires oxygen molecule and NADPH or NADH. Most of the bacterial

CYPs receive necessary electrons from NADH. They are able to bind oxygen atom to allylic positions, double bonds, or even to non-activated C-H bonds.

CYPs encode enzymes containing heme-thiolate and they are often located in macrolide antibiotic biosynthetic gene clusters, such that they catalyze stereo- and region-specific oxidation of precursors leading to structural diversity of macrolide

(Lamb, D.C. et al, Biochem. Biophys. Res. Commun., 307:610, 2003).

For example, CYP 107Al derived from Streptomyces erythraea participates in hydroxylation of 6-deoxyerythronolide B to erythronolide B in the biosynthesis of erythromycin (Weber, J.M. et al, Science, 252:114, 1991).

It has been reported that most of the CYP450 derived from the genus Streptomyces produces 7-hydroxycoumarin or 6,7-dihydroxycoumarin from 7-ethoxycoumarin, and oxidizes benzo(α)pyrene. However, there has not been any report yet, on bacterial P450 which binds more than two hydroxy groups to benzene ring.

Accordingly, the present inventors found 5.5 kb gene clusters from S. peucetius, which shows significant similarity to gene site participating in biosynthetic pathway of filipin and polyene which is an antibiotic substance in S. avermitilis, identified genes encoding CYP450 among the gene clusters above, and then confirmed that hydroxy group can be introduced into aromatic ring compound by the proteins encoded with the genes above, thereby completing the present invention. DISCLOSURE OF INVENTION

It is an object of the present invention to provide a CYP105P2 enzyme with the function of introducing hydroxy groups into aromatic ring compounds and a gene encoding the same.

Another object of the present invention is to provide a recombination vector containing the gene and a microorganism transformed with the recombination vector.

Still another object of the present invention is to provide a method for introducing hydroxy groups into aromatic ring compounds, the method being characterized by using the enzymes or the transformed microorganisms.

In order to accomplish the above objects, the present invention provides CYP 105P2 enzyme having amino acid sequence of SEQ ID NO: 2. The enzyme is characterized by having the function of introducing hydroxy groups into aromatic ring compounds, especially, the function of introducing pentahydroxy groups into benzene ring.

The present invention also provides a gene encoding the CYP105P2 enzyme. The CYP105P2 gene preferably has DNA sequence of SEQ ID NO: 1 and is preferably derived from Streptomyces peucetius.

The present invention also provides a recombination vector containing the gene and a microorganism transformed with the recombination vector.

The present invention also provides a method for preparing the CYP105P2 enzyme, the method being characterized by culturing of the transformed microorganism. The present invention also provides a method for introducing hydroxy groups into aromatic ring compounds, the method being characterized by using the CYP105P2 enzyme, the transformed microorganism or lysate thereof.

In the present invention, the aromatic ring compound is preferably 7- hydroxycoumarin, 7-ethoxycoumarin or cinnamic acid, and a compound obtained by introducing hydroxy groups into aromatic ring compounds is preferably cis- 5,6,7,8,9-pentahydroxycinnamic acid.

In the present invention, the aromatic ring compound is preferably benzene ring compound, pentahydroxy groups are preferably introduced into the benzene ring compounds, the benzene ring compound is preferably benzoic acid or salicylic acid, and a compound obtained by introducing pentahydroxy group into benzene ring compound is preferably pentahydroxybenzoic acid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing identification of the 5.5 kb regions of conserved operon in S. peucetius and S. avermitilis and cloning strategy of CYP105P2 from S. peucetius for expression in E. coli using pET32a.

FIG. 2 is the comparison of the amino acid alignment of CYP105P2 from S. peucetius, CYP 105Pl (PetD) from S. avermitilis, CYP1O5D6 (PikC) from S. venezulae. Shadow boxes indicate identical amino acid residues according to each amino acid. Black boxes represent conserved amino acid residues for all sequences.

FIG. 3 is a photograph of SDS-PAGE of CYP105P2 expressed at a large amount in E. coli. Lane 1 represents cell extracts of E. coli BL21 (DE3)/ρNP105P2. Lane 2 represents molecular weight markers.

FIG. 4 represents the bioconversion mechanism of substrates by CYP105P2 according to the present invention (7-ethoxycoumarin -> 7- hydroxycoumarin -> cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid).

FIG. 5 is the result of HPLC analysis showing the conversion of 7-ethoxycoumarin to cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid by CYP105P2 according to the present invention.

FIG. 6 is the result of HPLC analysis showing the conversion of cinnamic acid to cis-5, 6, 7, 8, 9-pentahydroxy cinnamic acid by CYP105P2 according to the present invention.

FIG. 7 is the result of HPLC analysis showing the conversion of salicylic acid to pentahydroxy benzoic acid by CYP105P2 according to the present invention.

FIG. 8 is the result of HPLC analysis showing the conversion of benzoic acid to pentahydroxy benzoic acid.

FIG. 9 represents the substrate binding and the releasing activity of reaction product for CYP 105P2.

FIG. 10 is the result of ¹H-NMR analysis of pentahydroxy cinnamic acid.

FIG. 11 is the result of DEPT-NMR analysis of pentahydroxy cinnamic acid.

FIG. 12 is the result of ¹³C-NMR analysis of pentahydroxy cinnamic acid.

FIG. 13 is the result of FAB-MASS analysis of pentahydroxy cinnamic acid. FIG. 14 is the schematic diagram showing the conversion mechanism of substrates by CYP105P2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will hereinafter be described in further detail by following examples. It will however be obvious to a person skilled in the art that these examples are given for illustrative purpose only, and the scope of the present invention is not limited to or by these examples.

Example 1: Construction of cosmid library

S. peucetius ATCC 27952 was inoculated in TSB medium and cultured at 28 ^°C for 2 days, and chromosomal DNA was isolated by the method of Sambrook after collection of S. peucetius by centrifugation. Isolated chromosomal DNA of S. peucetius was cut and fragmented with restriction enzyme Sau3AI, and then gene library of chromosomal DNA was constructed using pSuperCosl vector (Stratagene, USA).

Recombination DNA was packaged using Gigapack IH XL packing extract (Stratagene Inc., USA), in vitro. The chromosomal DNA was screened using dnrF of SEQ ID NOs: 3 and 4 and dpsY of SEQ ID NOs: 5 and 6, thus cosmid containing the sequences was identified.

SEQ ID NO: 3: 5'-AGG TTT GAG GTG GCC TTG ACG-3' SEQ ID NO: 4: 5'-TCC GCG TCA GTT CGC CGG AGG-3¹ SEQ ID NO: 5: 5'-GGA CTG CCG GTG TGC TGT GGT-3¹ SEQ ID NO: 6: 5¹CCG GAA CGT TCA TTC GTC GAC-3¹ Example 2: Sequencing and analysis of S. peucetius genome

To determine the base sequence of the cosmid constructed in Example 1, first, shot gun library was constructed by sonication of chromosomal DNA of S. peucetius.

Whole genomic sequence of S. peucetius was determined using the several cosmids constructed in Example 1 and the 2-4kb genomic fragments prepared by the shot gun method. The all non-redundant fragments were then assembled by PHRED

(Ewing, B. and Green, P., Genome Res., 8: 186, 1998) and PHRAP (http://www.phrap.org).

Whole genomic sequence was analyzed by BLAST searches of the Genebank, by which the most homologous sequence in other organisms is identified, using recent non-redundant protein database from NCBI .

Example 3; Identification and analysis of CYPs

Genome databases of S. peucetius were established by the present inventors for the first time (^"http://203.247.223.80). Cytochrome P450 was primarily screened based on the heme binding domain signature, GXXXCXG. The open reading frames (ORFs) of all CYPs obtained by using Glimmer 2.0 (http://tigr.org/software/glimmer/) were analyzed by BLAST. The open reading frames containing the GXXXCXG motifs were secondarily screened using ORF having a highly conserved threonine in the putative I-helix region and EXXR motif conserved in the K-helix. The genes including all the three motifs were used as queries for the BLAST searches of the Genebank and then analyzed. Amino acid sequence of each gene was predicted by GeneDoc program.

The DNA sequence of CYP (CYP105P2) from S. peucetius screened according to said method was deposited in the EMBL GeneBank international nucleotide sequence database under the accession number, AJ605540 (SEQ ID NO: 1).

The inventive sequence of CYPl 05 P2 was clustered with modular PKS and dehydrogenase in S. avermitilis and had 89% homology with CYP105Pl(PteC), involved in biosynthesis of antifungal polyene, filipin (FIG. 1). Also, the same PKS and dehydrogenase homologues were arranged with CYP105P2 in S. peucetius.

CYP105P2 with ATC start codon and TGA stop codon, is oriented in forward direction with ribosome-binding site and has distinct I-helix (²³⁷AAHDT²⁴¹), K- helix (²⁷⁶ELLR²⁷⁹) and heme-binding motif (³⁴¹FGFGAHQCIG³⁵⁰), thereby making it possible to confirm that it is cytochrome P450 (FIG. 2). Modified amino acid sequence by the base sequence of CYP105P2 is shown in SEQ ID NO: 2.

Example 4; Construction of recombinant plasmids and transformation

Two oligonucleotides, one primer as shown in SEQ ID NO: 7 and another primer as shown in SEQ ID NO: 8, were synthesized with Ndel and Kpnl sites respectively, and used to amplify the CYP105P2 from S. peucetius. The PCR conditions were as follows: denaturation at 94⁰C, annealing at 65⁰C and polymerization at 74⁰C, and the DNA amplification was performed in a total volume of 50 μl with 2.5 units of Taq-UNA polymerase, 10% dimethylsulfoxide, 2.5 pmol of primers, 0.2 mM dNTP and 0.1 μg genomic DNA dissolved in appropriate buffer.

SEQIDNO: 7: 5'-AGACATATGTCCCAGCCCACCG-3 SEQIDNO: 8: 5-TCAGGTACCAAGGAGCACCGTCGG-3

As a result of PCR, 1200bp of CYP105P2 was amplified, the amplified CYP105P2 (1200bp) was cloned into Ndel and Kpnl sites of pET32a expression vector (Novagen, USA) to produce plasmid pNP105P2. pNP105P2 prepared above was introduced into E.coli BL21(DE3) to obtain transformed E.coli BL21(DE3)/ pNP105P2.

Example 5: Expression of the CYP105P2 in E. coli

Recombinant CYP105P2 expressed by E. coli BL21 (DEi)/ pNP105P2 was expressed in a water-soluble form, and it was expressed at a large amount by induction with 0.4 mM IPTG. E. coli BL21(DE3) transformed with the plasmid pNP105P2 was inoculated in 3ml of LB medium containing 3μl/ml of ampicillin and incubated for overnight. After the cultured cells were transferred into 50ml fresh LB medium supplemented with 50μg of ampicillin and allowed to grow at 37⁰C to an optical density of 0.6 at 600 nm, 0.4mM IPTG was added into the medium, and then the culture was re-incubated at 20 ⁰C for 37 hours to induce the expression of CYP105P2. E. coli cells were harvested by centrifugation, and washed twice with cold 5OmM Tris/HCl (pH7.5). The cells were disrupted ultrasonically and centrifuged, and the sample of supernatant was isolated and electrophoresed. As a result of electrophoresis with standard molecular weight marker (Novagen Co., USA), molecular weight of expressed CYP105P2 was determined as 44 kDa (FIG. 3).

Example 6: Activity determination of the CYP105P2 enzyme

In order to determine the activity of cytochrome P450 (CYP105P2), lOOμl of reaction buffer [5mM Tris/HCl (pH7.5)], ImM of substrate (7-ethoxycoumarin or 7-hydroxycoumarin), 1.25mM of NADH and 30μl of cell extract and 50μl of distilled water] were allowed to react at 37 ⁰C for 24hours. The reaction was finished by ImI of ethyl acetate, the reaction mixture was centrifuged at 33,172xg, at 4°C for 20 minutes to remove supernatant, and then the pellet was dissolved in DMSO. Similarly, in order to observe the biotransformation in vivo system, the expression of CYP105P2 was induced by culturing E.coli BL21(DE3)/pNP105P2 in the same method as Example 3, ImM of substrate was added to 50ml of culture broth, after 10 minutes of induction with IPTG and then it was further incubated at 20⁰C for 48 hours. The reaction was finished by adding ImI of ethyl acetate into lOOμl of culture supernatant, and the post-treatment was performed as mentioned above.

To isolate reaction products from the reaction mixture, column packed with silica gel 60 (70-230 mesh ASTM) was used. The column chromatography make it possible to acquire various fractions using hexane and ethyl acetate as developing solvents, and TLC was performed using 3:2/ Hexane: Ethyl acetate solvents as a mobile phase. Biochemical conversion of substrate was also analyzed in

Mightysil, RP- 18 column by performing inverse HPLC with detection at 254 nm. Substrate and product were separated by mixture of methanol, acetonitrile and water (3.5: 3: 3.5) as a mobile phase at flow rate of 1 ml/min.

As a result of HPLC analysis, 7-ethoxycoumarin and 7-hydroxycoumarin produced the same product, cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid, under in vitro system (FIG. 4 & FIG. 5). No peak corresponding to 7-hydroxycoumarion was noticed when 7-ethoxycoumarin is considered as a substrate in vivo assay, although many literatures reported deethylation of 7-ethoxycoumarin (Sarialani F. S. et ah, Appl. Environ. Micrbiol, 46:468, 1983). However, formation of 7- hydroxycoumarin by deethylation was observed under in vitro assay.

On the other hand, it has been found that the both substrates (7-ethoxycoumarin and 7-hydroxycoumarin) were bound to enzyme effectively when reacted for more than 4 hours at 37°C . Therefore, no apparent formation of reaction product was observed up to 4hours of incubation and substrate was conversed completely after 24hours of incubation. Also, NADH played an important role in enzyme activity of CYP105P2. Namely, NADH was essential in complete conversion of substrate by CYP105P2.

As a result of the same reaction using cinnamic acid as substrate, three different products were produced, and major product among these products was cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid which is the same as reaction product of 7- ethoxycoumarin (FIG. 6).

Also, production of pentahydroxy benzoic acid was confirmed by performing the same reaction using benzoic acid and salicylic acid as substrates (FIG. 7 & FIG. 8).

Finally, it has been found that the cytochrome P450 (CYP105P2) according to the present invention has an activity to convert 7-ethoxycoumarin, 7-hydroxycoumarin and cinnamic acid into cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid and benzoic acid and salicylic acid into pentahydroxybenzoic acid.

Example 7; Substrate binding spectrum of the CYPl 05P2 enzyme

Cytochrome P450 included in bacteria, cytoplasm and membrane fraction was quantified by the method of Omura using extinction coefficient (91mM/cm) employing spectrophotometer (Omura, T. et al., Biochem. Biophys. Res. Commun., 266:690, 1999). Substrate binding spectrum was obtained by measuring with the split cell method after dividing CYP105P2 expressed in cytoplasm into test cuvette. Both control group and test group contained reaction buffer [glycerol:50mM Tris/HCl(ρH7.5)=l :8, 0.5mM~5mM of substrates(7-ethoxycoumarin, erythromycin, oleandomycin and chloramphenicol)]. lOOμl of CYP105P2 cell extract was added into test cuvette, and then the spectrum was measured at 350nm and 500nm. It is possible to analyze the role of CYP105P2 in drug metabolism, based on the measured spectrum. lOOμl of reaction buffer [20OmM of Tris/HCl(ρH7.5), 5mM of substrate, 1.25mM NADH, 5μl of ferredoxin, 5μl of ferredoxin reductase and 30μl of CYP105P2] was used to determine enzyme activity using erythromycin and oleandomycin.

The bioconversion level of substrate was measured by TLC and LC-Mass. TLC was performed using EtOAC: MeOH: ammonia (1 :2:0.1) solution as a mobile phase.

As a result, 2.5mM of oleandomycin and chloramphenicol was metabolized by 138μg/ml of CYP105P2 (FIG. 9). 2.7mM of 7-ethoxycoumarin and 1.5mM of erythromycin were also metabolized by the same concentration (138μg/ml) of CYP105P2.

Example 8: Structure analysis of the reaction products

NMR and mass spectroscopy were performed to analyze the structure of cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid and pentahydroxybenzoic acid. Fast atom bombardment mass (FAB-MASS) spectroscopy with high-resolution were also performed and IR spectra(Bio-Rad model FT3000MX FT-IR) was obtained from pure sample of KBr disk. All NMR data was recorded using DMSO-d_ό solution. The ¹H-NMR result of sample showed 5.5ρρm (d, 1H-H2) and 7.4ppm (d, IH, H-3) in a DMSO-Cl₆ solution (FIG. 10), which indicates that the existence of two carbon atoms and two protons and also, the existence of two hydrogen atoms was confirmed in DEPT-NMR (FIG. 11). The number and position of carbon atoms was confirmed by ¹³C-NMR in a DMSO-d₆ solution [C(I) 165.2ppm, C(2) 100.9ppm, C(3) 142.9ppm, C(5)(9) 138.4ppm, C(7) 129.1ppm, C(6)(8) 126.7ppm](FIG. 12). These results indicate that nine carbon atoms exist in the sample. FAB-MASS spectrum showed the fragmentation pattern of component of reaction product. FT-IR spectrum revealed the existence of double bond between carbon atoms (1415cm^"1), phenolic hydroxy group, carboxyl group (2955-3554Cm^"1 of wide band) and carbonyl group (1563cm ¹).

Finally, it was confirmed that the product produced by conversion of 7- ethoxycoumarin and 7-hydroxycoumarin using CYP105P2 was cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid. As shown in FIG. 14, it was confirmed that 7- ethoxycoumarin was converted into 7-hydroxycoumarin, which was again converted into cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid, as the last product.

While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.

INDUSTRIAL APPLICABILITY

The present invention has an effect to provide a cytochrome P450 enzyme (CYP105P2) with function of introducing hydroxy groups into aromatic ring compounds, a gene encoding the enzyme, a recombination vector containing the gene, a microorganism transformed by the recombination vector and a method introducing hydroxy groups into aromatic ring compounds, the method being characterized by using the enzyme or the transformed microorganism.

The CYP105P2 enzyme according to the present invention is useful in converting 7-ethoxycoumarin, 7-hydroxycoumarin and cinnamic acid into cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid, converting benzoic acid and salicylic acid into pentahydroxy benzoic acid or introducing hydroxy group into aromatic ring compound. The present invention is also useful in preparing variants such as filipin, polyene, etc., or increasing the conventional yield of filipin, polyene, etc., by modifying partial or whole genomic sequence provided in the present invention.

Claims

THE CLAIMSWhat is Claimed is:

1. A CYP105P2 enzyme having amino acid sequence of SEQ ID NO: 2.

2. The CYP105P2 enzyme according to claim 1, wherein the enzyme has a function of introducing hydroxy group into aromatic ring compound.

3. The CYP105P2 enzyme according to claim 2, wherein the enzyme has a function of introducing pentahydroxy group into benzene ring.

4. A gene encoding CYP 105P2 enzyme of claim 1.

5. The gene according to claim 4, wherein the gene has DNA sequence of SEQ ID NO: 1.

6. The gene according to claim 4, wherein the gene is derived from Streptomyces peucetius.

7. A recombination vector containing the gene of claim 4.

8. A microorganism transformed with the recombination vector of claim 7.

9. The transformed microorganism according to claim 8, wherein the microorganism is E. coli.

10. A method for preparing CYP105P2 enzyme according to claim 1, wherein the method comprises culturing the transformed microorganism of claim 8.

11. A method for introducing a hydroxy group into an aromatic ring compound, the method is characterized by using the CYP105P2 enzyme of claim 1, the transformed microorganism of claim 8 or cell extract thereof.

12. The method according to claim 11, wherein said aromatic ring compound is 7-hydroxycoumarin, 7-ethoxycournarin or cinnamic acid, and a compound obtained by introducing hydroxy group into aromatic ring compound is cis-5, 6, 7, 8, 9- pentahydroxy cinnamic acid.

13. The method according to claim 11, wherein said aromatic ring compound is benzene ring compound and pentahydroxy group is introduced into the benzene ring compound.

14. The method according to claim 13, wherein said benzene ring compound is benzoic acid or salicylic acid, and a compound obtained by introducing pentahydroxy group into benzene ring compound is pentahydroxy benzoic acid.

15. The method according to claim 11, which further comprises a step of adding NADH.