TECHNICAL FIELD

The present invention relates to a method for high-throughput screening useful gene products by metagenomics analysis and a use thereof.

BACKGROUND ART

A metagenome is defined as a set of genomes of all microorganisms present in a specific natural environment. Although microorganisms are the largest portion of all organisms, it is difficult to provide conditions similar to the original natural environment in which microorganisms are present. Thus, more than 99% of the microorganisms present in the environment cannot be cultured in a laboratory. When using the metagenome, DNA can be directly extracted from various microorganisms existing in natural environment regardless of whether it can be cultured in a laboratory, thereby enabling gene screening, identification, and function analysis.

Through the analysis of such metagenome samples, the role of the microorganisms in specific environments can be revealed so that microorganisms are used to help all fields related to human life such as industry, environment, energy, agriculture, and medical care, for example, wastewater treatment, chemical compound production, pharmaceutical production, bioenergy production, and industrial process. Recent metagenomic studies are attractive because they are useful for the search of novel materials or new enzymes as an applicable field as mentioned above. In particular, enzymes are biocatalysts prepared by living organisms, and enzymes derived from microorganisms and metagenomes have been attempted to use in catalytic reactions required in chemical synthesis processes. Enzymes are very useful in various industrial fields because they have very high precision, specificity, selectivity, and high efficiency compared to chemical catalysts. The catalytic efficiency of enzymes is 108to 1014times higher than that of the non-enzymatic reaction under similar reaction conditions, and the selectivity of the reaction thereof is very high compared to that of the chemical catalytic reaction. In addition, if an enzyme capable of reacting exists under even special conditions such as reaction under high temperature, high pressure, and organic solvent, it is an item having a very high utility value as a product for the future industry.

Because the economic feasibility of the enzyme was so low in the beginning, it was difficult to use it. However, the remarkable development of novel technologies including gene manipulation technology, biotechnology, and molecular evolution technology results in high-throughput screening and its improvement for developing new functional enzymes, thereby expanding the application to new application field.

The method of measuring the presence or activity of enzymes for screening such enzymes includes screening methods using a microorganism and utilizing a non-culturable metagenome. In the case of the former, various strains collection is mandatory, and selection of candidate strains should be preceded by direct cultivation and activity comparison of these strains. In addition, there is a drawback that it requires a lot of labor and research expenses and takes a long time. In order to address these issues, a high-throughput screening system has been developed and utilized. However, in order to identify the desired useful enzyme, it is necessary to purify genomic DNA (gDNA) from a candidate strain, to construct a DNA library, to reconfirm the activity of enzymes utilizing expression system, and to reanalyze nucleotide sequence of selected library strain, thereby ensuring the desired useful enzyme resources. In the latter case, the DNA library is constructed using a metagenome purified from gDNA directly from an environmental sample. This method can also be classified as functional-based enzyme screening in which the activity thereof is compared using the expression system or DNA sequence-based enzyme screening in which DNA of the desired useful enzyme resources is secured using degeneracy primers. In the case of an enzyme screening method based on its activity, a library is prepared by direct cloning of the metagenome and then it is directly confirmed that an enzyme which reacts with the substrate through expression to convert it into a desired substance exists. Although this method has the advantage of surely confirming the existence of gene resource, a considerable amount of labor and time is invested in the process of preparing and expressing the library, andEscherichia colisystem is mainly used for the expression system, thereby having a limitation of the heterologous gene expression system. In the case of a bacterial gene that has a far evolutionary relationship from the same, in which the construction or use of its promoter or codon does not match withEscherichia coli, there are disadvantages that the expression of the gene may be affected, and the efficiency of screening is lower compared to its effort and investment. In the case of an enzyme screening method based on a nucleotide sequence, the nucleotide sequence of the target enzyme is confirmed by a polymerase chain reaction using a primer constructed based on the specific motif of the target enzyme. However, since this method also uses a primer based on the motif of a specific enzyme, there are issues that the obtained nucleotide sequence is biased to anyone enzyme to be amplified, and the nucleotide sequence should be confirmed by directly sequencing the DNA library including the target enzyme.

Polymerase chain reaction (PCR) is a gene detection method in which a specific deoxyribonucleic acid region is amplified by a polymerase in vitro. The polymerase chain reaction is an enzymatic reaction for several hours, thereby resulting in the explosive mass production of the gene and has become a critical technique for the highly-applicable deoxyribonucleic acid detection so that it has become an essential technology in all life science fields requiring molecular biology approaches. Meanwhile, the term of “degeneracy” refers to a phenomenon in which one kind of amino acid corresponds to a plurality of codons. The degeneracy does not lead to determine the nucleotide sequence of a gene encoding any particular amino acid sequence but allows to synthesize oligonucleotides including all possible nucleotide sequences. When a polymerase chain reaction is performed using a degenerate oligonucleotide as a primer, a DNA fragment of a gene encoding the protein may be obtained based on amino acid sequence information. Primers designed for such purpose refer to degenerate primers, and its polymerase chain reaction refers to degenerate polymerase chain reaction. This method is a technique for cloning a gene from an amino acid sequence of a substrate but is now widely used for searching gene families based on information of the primary structure (nucleotide sequence) of an abundantly accumulated nucleic acid. Despite a disadvantage in that the actual presence or activity of the enzyme is unknown, and it must be ultimately confirmed through expression, it has an advantage in that the target DNA can be screened from the metagenome departing from the labor-intensive method.

PRIOR ART DOCUMENT

Korean Patent Application No.: 10-2009-0130204

DISCLOSURE

Technical Problem

Thus, the inventors of the present invention have developed a method for quickly and accurately determining the utility value of a metagenome sample in the screening poly-peptides such as industrially useful enzymes. Further, the inventors have extensively amplified genes of poly-peptides such as various enzymes and have designed and prepared a degenerate primer which is superfamily-specific to a target peptide, which is capable of being utilized as bar-code sequence for efficient and quick analysis of massive nucleotide sequences of the amplified fragments. Thus, the method was used for the polymerization chain reaction so that DNA fragments extensively amplified from the metagenome sample were analyzed by a next-generation sequencing, thereby confirming the massive nucleotide sequences of the useful peptides to complete the present invention.

Accordingly, one aspect of the present invention relates to a method for analyzing metagenomic information using a superfamily-specific degenerate primer set to a target gene.

Further, another aspect of the present invention relates to a superfamily-specific degenerate primer set to the target gene.

Technical Solution

One aspect of the present invention relates to a method of analyzing metagenomic information including:

(1) amplifying a gene in a metagenome sample, which is a template, with a superfamily-specific degenerate primer set to a target gene; and

(2) analyzing sequence of the amplificatied product of the gene.

The superfamily-specific degenerate primer set to the target gene may be such that

i) the primer set is specific for a gene encoding a target protein exhibiting amino acid identity of 35% or more;

ii) sequence of the binding site between the primer and the target gene has identity of 80% or more;

iii) a degeneracy ratio is 1,500 or less;

iv) a temperature of annealing is 70° C. or less;

v) a length of the primer is 30 bases or less;

vi) identity of an inside of the primer binding site is 40% or less; and

vii) a size of the amplified fragment is 1,000 bp or less.

More preferably, the degeneracy ratio may be 800 or less;

the length of the primer may be 25 bases or less; and

the size of the amplified fragment may be 500 bp or less.

The metagenome sample may be a metagenomic library, a vector which the metagenomic library is introduced, or a cell transformed with the vector.

Further, the metagenome sample may be a soil, a wetland, a volcano, a tidal mudflat, a salt pan, fresh water, seawater, a body fluid, urine, an agricultural product, an aquatic product, or feces.

Further, the target gene encodes a polypeptide, an oligopeptide, an enzyme, an antibiotic resistance gene, an antimicrobial peptide, an antifungal peptide, an oligopeptide, or a marker.

Further, the step (2) of analyzing the sequence may use

one or more processes selected from the group consisting of single-molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis (SBS), sequencing by ligation (SBL), and chain termination.

Further, another aspect of the present invention provides a superfamily-specific degenerate primer set to a target gene for analyzing metagenomic information, in which

i) the primer set is specific for a gene encoding a target protein exhibiting amino acid identity of 35% or more;

ii) sequence of the binding site between the primer and the target gene has identity of 80% or more

iii) a degeneracy ratio is 1,500 or less;

iv) a temperature of annealing is 70° C. or less;

v) a length of the primer is 30 bases or less;

vi) identity of an inside of the primer binding site is 40% or less; and

vii) a size of the amplified fragment is 1,000 bp or less.

The degeneracy ratio may be 800 or less;

the length of the primer is 25 bases or less; and

the size of the amplified fragment is 500 bp or less.

Advantageous Effects

The method for the metagenomic information analysis and the primer set used for the method according to the present invention can be applied for quickly determining the utility value of a massive amount of metagenome samples. In particular, the superfamily-specific degenerate primers of the present invention are used to quickly detect the presence or absence of the genetic information of the target peptides in the metagenome by a simple method, thereby collecting a large amount of useful peptide resource information from various metagenome samples at high speed. Further, the present invention may be used for screening new peptide coding genes by designing and producing superfamily-specific degenerate primers of new target peptides based on the method of the present invention. In addition, the method of the present invention can be applied not only to enzymes but also to studies related to polypeptides, oligopeptides, antibiotic resistance genes, antimicrobial peptides, antifungal peptides, oligopeptides, markers or single-nucleotide polymorphism (SNP).

