KR100665316B1 - Novel bifunctional hydroxylase from metagenome library genes and screening method thereof - Google Patents

Abstract

The present invention relates to a novel bifunctional hydroxylase derived from metagenome, a gene group directly extracted from soil, and a gene encoding the same. Specifically, the present invention provides a novel dual active hydroxylase gene derived from a gene clone selected from a metagenome library extracted directly from soil, a recombinant vector comprising the gene, a strain transformed with a recombinant vector, and a transformed vector. It relates to a dual active hydroxylase produced by a strain. The dual active hydroxylase gene of the present invention is a novel gene that has not been reported to date, and can be applied to add hydroxyl groups to various low molecular weight natural products or compounds by mass production of hydroxylases by genetic recombination using the gene.

Bifunctional hydroxylase, metagenome

Description

Novel bifunctional hydroxylase from metagenome library, genes and screening method

1 is a schematic of the direct extraction process of soil DNA using a DNA fragment of more than 20kb in size to prepare a metagenomic library, and shows the Bam H1 fragments of the soil DNA of the representative clones obtained from the figure,

Figure 2a is a view showing the structural characteristics of the dual active hydroxylase,

FIG. 2B is a diagram illustrating a strategy for constructing a probe to find a novel dual active hydroxylase gene from soil DNA using these properties, with a conserved region of the hydroxyl domain represented by a red line. Schematic illustration of the preparation of primers based on the conserved regions of the hydroxylase reduction domain, indicated by blue lines, the dotted line is the heme binding motif of the enzyme, and the dotted line is the expected hydroxylase and hydroxylase reduction. Represents the linker region of the enzyme,

3 is a nucleotide sequence of a novel dual active hydroxylase gene discovered by the strategy of FIGS . 1 and 2b ,

4 is the amino acid sequence of the novel dual active hydroxylase,

5 is a schematic of the expression vector of the novel dual active hydroxylase.

The present invention relates to a novel bifunctional hydroxylase derived from metagenome, a gene group directly extracted from soil, and a gene encoding the same. Specifically, the present invention screens gene clones from a genomic library consisting of metagenome gene groups directly extracted from soil, and includes novel dual active hydroxylase genes derived from the gene clones, recombinant vectors comprising the genes, and the recombinant vectors. By a transformant, a dual active hydroxylase produced by the transformant, a preparation method thereof, and a screening method thereof.

Cytochrome P-450, a hydroxylase, is an enzyme group of oxidases that has an enzymatic activity of reducing oxygen molecules to oxidize organic compounds and to produce one molecule of water. Among the compounds having a very difficult structure to add a functional group in organic synthesis, by selectively adding a hydroxyl group to the alkane chain site of the fatty acid, various functional groups (alcohol, ketone, acid, peroxide, etc.) can be added to the hydroxylase. It is important in the chemical and pharmaceutical fields. Hydroxylase found in microorganisms for this purpose cannot show enzymatic activity alone, but it is active in combination with additional coenzymes such as flavin adenine dinucleotide (FAD), ferredoxin (ferrous sulfide protein) and NADPH (electron transporter). It is. They exist in the cytoplasm as different polypeptides and are responsible for the reduction of hydroxylases when they are oxidized. Due to the characteristics of these microbial hydroxylases, it is difficult to find suitable coenzymes in the activity assay of heterozygous-derived hydroxylases, and it is difficult to confirm the activity. In addition, the use of various coenzymes, which are difficult to circulate, has the disadvantage of time and economic loss.

P-450 _BM3 derived from Bacillus megaterium is an example of a dual active hydroxylase that does not have the defect of the microbial-derived hydroxylase (Richard T. Ruettinger, Long-Ping Wen and Armand). J. Fulco, Journal of Biological chemistry, 1989, July 5, (264): 10987-10995). P-450 _BM3 has the same mole number as FAD and FMN (Flavin Mononucleotide) in the second domain which reduces the function of hydroxylase together with P-450 heme binding hydroxyl domain. have. Thus, a polypeptide has a hydroxylase domain and a hydroxylase reduction domain at the same time, so it is called a dual active hydroxylase. The enzyme is also known to have very good hydroxylation activity compared to conventional microbial derived enzymes. This is thought to be due to the advantage of distance in biochemical reactions because the domain responsible for the reduction of hydroxylase is linked to hydroxylase by one polypeptide.

In spite of this outstanding industrial and academic double activity enzyme, this type of hydroxylase has not been found for more than 10 years after the discovery of P-450 _BM3 . Recently, as the sequencing of the genome of microorganisms is progressed, only several kinds of dual active enzymes have been reported, and the search and development of such dual active hydroxylases have not been made.

Therefore, the inventors of the present invention, in order to discover the double active hydroxylase of the P-450 _BM3 type, to separate the DNA directly in the natural state without culturing the cells to produce a metagenome library, and to screen the double active hydroxylase from these gene groups The present invention was completed by identifying a novel dual active hydroxylase, a gene encoding the same, and an expression thereof.

It is an object of the present invention to provide a method for screening dual active hydroxylases with excellent biochemical properties and the novel dual active hydroxylases and genes thereof discovered as a result.

The present invention provides a gene encoding a double active hydroxylase comprising a hydroxylase domain and a hydroxylase reduction domain derived from soil on one polypeptide.

The present invention also provides dual active hydroxylases and variants thereof encoded by the gene.

In addition, the present invention provides an expression vector comprising the gene.

The present invention also provides a transformant into which the expression vector is introduced.

In addition, the present invention

i) preparing a metagenome library using DNA extracted from soil;

ii) constructing forward and reverse primers using the conserved regions of the hydroxylase domain and the hydroxylase reducing domain;

iii) PCR of the purified DNA from the library of step i) with the primer of step ii) to prepare a DNA fragment corresponding to the dual active hydroxylase domain; And

iv) a method of screening a dual active hydroxylase gene comprising screening a metagenomic library having a dual active hydroxylase function using a DNA fragment prepared in step iii) as a probe.

In addition, the present invention

1) introducing the gene encoding the dual active hydroxylase into an expression vector;

2) transforming the expression vector into which the gene of step 1) is introduced into a host cell; And

3) expressing dual active hydroxylase using the transformant of step 2), and

4) It provides a method for producing a dual active hydroxylase comprising the step of separating and purifying the dual active hydroxylase expressed in step 3) by a conventional method.

The present invention also provides a method for adding a hydroxyl group to a low molecular weight natural product or compound comprising contacting the dual active hydroxylase with a low molecular weight natural product or compound under reaction conditions.

Hereinafter, the present invention will be described in detail.

The present invention provides a gene encoding a double active hydroxylase comprising a hydroxylase domain and a hydroxylase reduction domain derived from soil on one polypeptide.

DNA samples isolated from soil were purified by electrophoresis and nickel (Ni) columns (QiaexII, Qiagen, USA) and cloned into phosphmid (Fosmid, Epicentre, USA) vectors. In this metagenome library, primers were prepared using the conserved regions of the two domains of the dual active hydroxylase, and DNA fragments having both domains were obtained by PCR. The DNA fragment was used as a probe to perform the double active hydroxylase. The clones with the genes were identified and sequenced using an automated sequencing analyzer. The gene was named syk181 , and it was confirmed that an open reading frame (ORF) of 3,240bp was present (see SEQ ID NO: 2 and FIG. 3 ). The ORF encodes a polypeptide exhibiting a molecular weight of 118 kDa consisting of a total of 1,079 amino acids set forth in SEQ ID NO: 1 . The homology between the gene of the present invention and P-450 _BM3 hydroxylase derived from Bacillus megaterium, which is known for its activity, showed 73% similarity.

Since there are many genes encoding one amino acid due to codon degeneracy, it is well known that the DNA sequences encoding the same amino acid sequence may be different.

In the present invention, the 'variant' refers to a mutated gene in which one or two or more bases are changed by a change, for example, substitution, deletion, addition, insertion, etc., but which can produce the hydroxylase or a functional equivalent thereof. Thus, those skilled in the art will readily appreciate that such variants may be included within the scope of the present invention. In general, for the dual active hydroxylase genes of the present invention, variants having at least about 80% homology, preferably variants having at least 90% homology, may be included within the scope of the present invention.

The present invention also provides dual active hydroxylases and variants thereof encoded by the gene.

As shown in FIG . 2A , the dual active hydroxylase contains both a domain having hydroxylase activity and a domain having hydroxylase activity in its polypeptide chain. The hydroxylase SYK181 described in the amino acid sequence of SEQ ID NO: 1 has a size of 118 kDa, consists of 1,079 amino acids, and has an isoelectric point of 6.8, which has an activity of imparting a hydroxyl group to the carbon at the end of the fatty acid.

  As used herein, the term “variant” refers to a dual active hydroxylase in which an amino acid is substituted, added or deleted within a range that does not affect the function of an enzyme. Depending on the purpose, only a part of a protein may be used. Sequences of such modified amino acids are also included within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as said protein, wherein substantially identical polypeptides have at least 80% homology, preferably at least 90%, more preferably at least 95% Means things.

In addition, the present invention provides an expression vector comprising the gene. In the present invention, the syk181 gene of the present invention was inserted into pET22b (+) (Novagen, USA) as one of the preferred embodiments for producing dual active hydroxylase, which was named pJA181. However, in addition to pET22b (+) used for the production of the expression vectors, various prokaryotic cells (pET series, etc.) or eukaryotic cells (pPIC and pPICZ series, etc.) are known, and thus, depending on the purpose of expression, It is clear that various expression vectors can be easily used. In addition, the size and nucleotide sequence of the gene inserted into the foreign gene insertion site of the expression vector can also be variously changed by known techniques.

The present invention also provides a transformant into which the expression vector is introduced. The vector pJA181 obtained above was introduced and transformed into E. coli BL21 (DE3), which was used as an expression host, and was transformed into the gene bank of the Biotechnology Research Institute on May 19, 2005. , Accession number: KCTC 10807BP). The method of introducing a recombinant vector for transformed cells of the present invention can also be introduced through known techniques, ie, heat shock, electroshock, and the like, in addition to the above expression host, depending on the vector depending on the purpose of expression. It is obvious that various E. coli strains ( E. coli BL21 (DE3), DH5α, etc.) and yeast ( Saccharomyces genus, Pichia genus, etc.) are readily available.

In addition, the present invention

 i) preparing a metagenome library using DNA extracted from soil;

ii) constructing forward and reverse primers using the conserved regions of the hydroxylase domain and the hydroxylase reducing domain;

iii) PCR of the purified DNA from the library of step i) with the primer of step ii) to produce a DNA fragment corresponding to the dual active hydroxylase domain; And

iv) a method of screening a dual active hydroxylase gene comprising screening a metagenomic library having a dual active hydroxylase function using the DNA fragment prepared in step iii) as a probe.

The screening methods of the present invention utilize forward and reverse primers from the amino acid sequence conserved regions of the hydroxylase and hydroxylase reducing domains, utilizing the constitutive feature of the presence of two domains of dual active hydroxylase in one polypeptide. Were prepared and are represented by SEQ ID NO: 3 and SEQ ID NO: 4 (see FIG. 2B ). By amplifying a nucleotide sequence including these two domains using the primers isolated from the metagenome library of step i) as a template, and confirming the nucleotide sequences of the resulting 1.2-1.7 kb DNA fragments. Gene fragments of active hydroxylase were obtained. Since these two domains have another conserved region, they can be applied using various conserved regions other than the above. Therefore, it is well known that a screening method based on DNA amplification (PCR) based on the continuity of two domains of dual active hydroxylases present as one polypeptide is identical to the configuration of the present invention.

In addition, the present invention

1) introducing a gene encoding the dual active hydroxylase into an expression vector;

2) transforming the expression vector into which the gene of step 1) is introduced into a host cell; And

3) expressing dual active hydroxylase using the transformant of step 2), and

4) It provides a method for producing a dual active hydroxylase comprising the step of separating and purifying the dual active hydroxylase expressed in step 3) by a conventional method.

Genes encoding dual active hydroxylases, preferably genes encoding double active hydroxylases obtained through the screening method of the present invention, were introduced into the vector to prepare transformants, which were then adapted to vectors such as IPTG. Protein expression was induced by adding an analog to the medium. After culturing the transformant cultured using an ultrasonic crusher and the like, the cell extract was purified through a nickel column to obtain pure protein.

The present invention also provides a method for adding a hydroxyl group to a low molecular weight natural product or compound using a hydroxylase having a double active hydroxylase activity and a variant thereof.

The alkane chain site of fatty acids is a very difficult structure to add functional groups in organic synthesis. By selectively adding a hydroxyl group to such a structure, it is possible to add various functional groups (alcohol, ketone, acid, peroxide, etc.), which has a major meaning in chemical and pharmaceutical fields. Thus, in the present invention, after expressing pSYK181 of the present invention as one of the preferred embodiments, the purified dual active hydroxylase can hydroxylate the carbon at the omega position of palmitic acid without the addition of a coenzyme that reduces the hydroxylase. there was.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Preparation of Metagenomic Library Using DNA Directly Extracted from Soil

DNA was extracted as shown in FIG . 1 using a DNA extraction buffer containing 1% CTAB and 2% SDS for extraction of environmental DNA from soil. Agarose gel electrophoresis of DNA samples isolated from soil was able to separate soil DNA with minimal humic acid removal and minimal physical damage. Electrophoresis picture of the purified DNA as shown in Figure 1 can confirm the high concentration of undecomposed DNA. To increase the recovery of soil DNA, the weight of soil per treatment was increased to 50 g and the resulting humic acid was removed by nickel resin (Ni resin, QiaexII, Qiagen, USA).

The separated and purified DNA was physically fragmented, and these DNA fragments were subjected to electrophoresis to fractionate 20 kb or more of DNA, which was cloned using a Fosmid vector (Epicentre, USA). Metagenomic fragments of representative strains of the metagenome library prepared by the above method were digested with BamHI restriction enzyme (New England BioLab, USA) to confirm the size and diversity of the fragments (see FIG. 1 ).

<Example 2> Screening method of double active hydroxylase

In order to detect dual active hydroxylase from the metagenome library prepared in <Example 1> , degenerative primers were prepared. Using the characteristics of the protein domain composition of the dual active hydroxylase, forward and reverse primers described in SEQ ID NO: 3 and SEQ ID NO: 4 from the amino acid sequence conservation sites of the hydroxylase domain and the hydroxylase reduction domain, respectively, were prepared. Was made (see FIG. 2B ).

SEQ ID NO : 5'-GCKGGRCAYGARACAACA-3 ' (forward primer)

SEQ ID NO : 5'-YGGWGAKSWWGAAATYGAATA-3 ' (reverse primer)

   K: G or T,

   Y: T or C,

   R: G or A,

   W: A or T.

In order to amplify the nucleotide sequence between these two domains using the primer set, the prepared metagenomic library whole phosphide was separated and purified and used as a template for PCR. The resulting 1.2-1.7 kb DNA fragments were cloned into TA vectors (Invitrogen, USA) to confirm the base sequence. This secured a gene fragment of the double active hydroxylase. Using this gene fragment as a probe, we found a metagenomic library clone that had the entire open reading frame (ORF) of the gene. This results in the double active hydroxylase detection using DNA amplification (PCR) based on the continuity of the two domains present in one polypeptide, which is a structural characteristic of the enzyme in the detection of the double active hydroxylase. It was confirmed that the selective search of.

<Example 3> Sequence analysis and homology comparison of dual active hydroxylase gene syk181

A phosmid clone of the metagenomic library containing the dual active hydroxylase gene identified in < Example 2> was confirmed. The inserted metagenome DNA was extracted, digested with Bam HI (New England BioLab, USA) and subcloned. Clones containing fragments of the hydroxylase gene in the subclone were reconfirmed through hybridization, and the nucleotide sequences of the identified DNA fragments were analyzed using Genotech's automatic base sequence analyzer.

As a result, it was confirmed that there exists an ORF of 3,240 bp described in SEQ ID NO: 2 (see FIG. 3 ). The ORF encodes a polypeptide exhibiting a molecular weight of 118 kDa consisting of a total of 1,079 amino acids (see FIG. 4 ).

Analysis of the analyzed base sequence with Genbank's database showed 73% similarity with the known BM3 hydroxylase of Bacillus megaterium, namely P-450 _BM3 (Genebank No: JO4382). The highest homology among the nucleotide sequences listed in the database was the gene of Bradyrazobium japonicum (Genebank No: AP005945), whose enzyme activity was not confirmed but was estimated to be 77% of this gene. Homology was shown.

Example 4 Cloning of Dual Active Hydroxylase syk181 Gene

For expression of the gene syk181 , the DNA fragment containing the ORF was amplified to include NcoI and HindIII (New England BioLab, USA) restriction enzyme sites. This DNA fragment was digested with the restriction enzyme and introduced into the pET22b (+) (Novagen, USA) vector digested with the same enzyme, which was named pJA181 (see FIG. 5 ). Was converted to vector transfected pJA181 obtained above was introduced into E. coli strain E. coli BL21 (DE3) is used as the expression host, it was deposited on May 19, 2005, the Research Institute of Bioscience and Biotechnology Gene Bank (E. coli TG2 / pSYK181 , Accession number: KCTC 10807BP).

<Example 5> Expression and activity confirmation of dual active hydroxylase

E. coli TG2 / pSYK181, the transformant, was suspended in a liquid LB medium to an OD value of 0.6 and protein expression was induced by adding 0.8 mM IPTG to the medium. After culturing the transformant thus cultured, the cell extract was purified through a nickel column. Purified dual active hydroxylase was able to hydroxylate the carbon at the omega position of palmitic acid without the addition of hydroxylase reductase. By selectively adding hydroxyl groups to the alkane chain sites of fatty acids, which are very difficult to add functional groups in organic synthesis, it is possible to add various functional groups (alcohol, ketone, acid, peroxide, etc.). Has meaning.

The present invention relates to a novel bifunctional hydroxylase derived from metagenome, a group of genes extracted directly from soil, and a gene encoding the same. The dual active hydroxylase gene of the present invention is a novel gene that has not been reported to date, and by adding the gene to the recombinant mass-producing the double active hydroxylase of the present invention, by adding a hydroxyl group to various low molecular weight natural products or compounds in the existing method Applicable to

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Claims (13)

A dual active hydroxylase set forth in the amino acid sequence of SEQ ID NO: 1 comprising a hydroxylase domain and a hydroxylase reducing domain on one polypeptide. delete The gene encoding the dual active hydroxylase of claim 1. The gene according to claim 3, wherein the gene is described by the nucleotide sequence of SEQ ID NO. An expression vector comprising the gene of claim 3. The pJA181 expression vector according to claim 5, which is represented by the cleavage map of FIG. A transformant into which the expression vector of claim 5 is introduced. The transformant according to claim 7, which is E. coli or yeast. The transformant according to claim 7, which is E. coli TG2 / pSYK181 deposited with accession number KCTC 10807BP. i) preparing a metagenome library using DNA extracted from soil; ii) constructing forward and reverse primers using the conserved regions of the hydroxylase domain and the hydroxylase reducing domain; iii) PCR of the purified DNA from the library of step i) with the primer of step ii) to prepare a DNA fragment corresponding to the dual active hydroxylase domain; And iv) screening a metagenomic library having dual active hydroxylase function using the DNA fragment prepared in step iii) as a probe. The method of claim 10, wherein the primers comprise SEQ ID NO: 3 and SEQ ID NO: 4 . 1) introducing a gene encoding the dual active hydroxylase of claim 3 into an expression vector; 2) transforming the expression vector into which the gene of step 1) is introduced into a host cell; And 3) expressing dual active hydroxylase using the transformant of step 2), and 4) A method of producing a dual active hydroxylase comprising the step of separating and purifying the dual active hydroxylase expressed in step 3) by a conventional method. A method of adding a hydroxyl group to a low molecular weight natural product or compound comprising contacting the dual active hydroxylase of claim 1 with a low molecular weight natural product or compound under reaction conditions.

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