KR102022242B1 - Terpene synthase gene from Polyporus brumalis, Shizosaccharomyces pombe mutant transformants containing the gene - Google Patents

Abstract

The present invention relates to pobTPS, a terpene synthetase, and more particularly, to a pobTPS gene and a vector comprising the same, a transformant transformed through the vector, and a gene of pobTPS , a terpene synthetase derived from winter mushrooms. It can be provided to provide a terpene synthetase having better activity than the wild type strain.

Description

Terpene synthase gene from Polyporus brumalis, Shizosaccharomyces pombe mutant transformants containing the gene}

The present invention relates to a terpene synthase, pobTPS, and more particularly, to a pobTPS gene, a vector comprising the same, and a transformant transformed through the vector.

In addition to carbohydrates, proteins, nucleic acids, and lipids, primary metabolites that are directly involved in cell function, such as those produced by plants and microorganisms, are secondary metabolites that are not essential for survival but are secondary. Mushrooms also produce a variety of secondary metabolites, and there are a variety of bioactive materials that can be useful in humans, and despite the variety, there is a lack of information on related biosynthetic pathways. Current research trends show that genome sequences have been decoded in many fungi due to the development of next generation sequencing (NGS) technology, and studies are actively underway to explore gene clusters related to secondary metabolite biosynthesis. Representative compounds among secondary metabolites are terpene compounds. Natural terpenes are mainly biosynthesized by plants or microorganisms, and about 50,000 structures have been identified to date, and structures with useful physiological activities such as anticancer, antibacterial, and anti-inflammatory properties have been identified.

Polyurus brumalis mycelium was confirmed to produce eudesmol (C ₁₅ H ₂₄ O) substance of sesquiterpenes in liquid medium containing glucose and magnesium (MgSO ₄ ) inorganic elements. Eudesmol has been found to be a useful material that exhibits anti-inflammatory and anti-microbial effects. Profiling of mRNA and protein expression levels of P. brumalis mycelium was performed to identify gene and enzyme information that plays a direct role in eudesmol production . Secondary metabolite biosynthesis pathways of microorganisms are classified according to precursors, and terpene components are known to be synthesized through the mevalonate pathway metabolic pathway. The mevalonate synthesis pathway produces a farnesyl diphosphate structure, a sesquiterpene precursor by eight enzymes. Sesquiterpene structures of various structures are produced by additional enzymatic reactions to the resulting precursors. An important enzyme that determines these various structures is called a terpene synthase. In studies of existing terpene synthase, terpene substances have relatively higher contents in plant tissues than microorganisms.

In order to confirm the function of the original gene, the most common method is to remove the target gene using a homologous host system and then look at the changes in the essentiality and shape of the gene. There is a limitation in the use as a matter of time and stability established in the present invention was confirmed using a heterologous yeast (yeast). Used yeast Schizosaccaharomyces pombe For the strain, genome wide deletion mutant library was developed by Korea Research Institute of Bioscience and Biotechnology. In addition, S. pombe has not been found in terpene synthase which has similar function to winter mushroom in the existing gene level, so it is advantageous for pobTPS gene function and mevalonate pathway for terpene precursor generation.

On the other hand, Patent Publication No. 10-2014-0033002 discloses a yeast cell for producing a terpene. In particular, it contains a functional gene encoding a soluble hydroxymethylglutaryl-coenzyme-A (HMG-CoA) reductase and increased terpene production by eliminating one or more genes encoding steril acyltransferase. Technical characteristics differ from the present invention regarding the specific gene of silver terpene synthase.

Korean Patent Publication No. 10-2014-0033002

Therefore, an object of the present invention is to isolate a terpene synthase gene derived from a winter umbrella mushroom and to provide a vector comprising the gene and a mutant yeast transformant using the vector.

In order to achieve the object of the present invention, the present invention is to provide a gene of pobTPS which is a terpene synthase derived from the winter umbrella.

In addition, the present invention is to isolate the gene of the pobTPS in the winter Umbrella strain, insert the gene of the isolated pobTPS into a vector to produce a recombinant vector, transformed to yeast using the recombinant vector transformed mutant yeast Provided are methods for preparing a sieve.

The present invention also provides a recombinant vector comprising the pobTPS gene produced by the above method.

The present invention also provides a mutant yeast transformant transformed by the above method.

Specifically, there is provided a pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the gene is a gene encoding a terpene synthase pobTPS, provides pobTPS gene, characterized in that derived from winter mushroom umbrella.

The present invention also provides a recombinant vector into which the gene is introduced, and more specifically, a pSLF273-pobTPS recombinant vector having a cleavage map disclosed in FIG. 5.

In addition, the recombinant vector is provided a transformant, wherein the transformant is a transformant, characterized in that the transformed yeast, the yeast is Shizosaccharomyces pombe It provides a transformant characterized by.

Hereinafter, the present invention will be described in more detail.

The present invention is to isolate a terpene synthase ( pobTPS ) gene derived from the winter umbrella mushroom ( Polyporus brumalis ), inject the gene into yeast using a plasmid, and to prepare a transformant capable of expressing the gene.

As used herein, the term “yeast cell” is in the broadest sense, and the yeast cell may be an ascomycota. The aseptic fungus may be saccharomycetaceae. The Saccharomycesettas include Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, and Debaryomyces. ), Genus Zygosaccharomyces, Genus Shizosaccharomyces or Saccharomycopsis. Examples of genus Saccharomyces include S. cerevisiae, Saccharomyces S. bayanus, Saccharomyces S. boulardii, Saccharomyces S. bulderi, S. cariocanus, S. cariocans, S. cariocus, S. romealis, S. chevalieri, Saccharomyces S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, Saccharo S. florentinus, S. kluyveri, S. martiniae, S. romacensis, Saccharin S. norbensis, S. paradoxus, S. romanis Pastorianus (S. pastori) anus, S. spencerorum, S. turicensis, Saccharomyces unisporus, Saccharomyces uvarum .uvarum), or S. zonatus. The genus Kluyveromyces lactis can be Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans. Candida genus is Candida glabrata, Candida boidinii, Candida magnolia, Candida methanosorbosa, Candida sonorensis, or Candida utility It may be Candida utilis. The genus Pichia may be Pichia stipitis. The genus Issatchenkia may be Issatchenkia orientalis. The genus Debaryomyces may be Debaryomyces hansenii. The genus Zaygo Saccharomyces may be Zygosaccharomyces bailli or Zygosaccharomyces rouxii. In the genus Schizocaromyosis S.cryophilus, S. japonicus, Schizocaromyces S.octosporus or Schizozo It may be S. pombe. Preferably may be S. pombe (S. pombe)

In the present invention, the term "nutrition" can be understood in the broadest sense that yeast is unable to synthesize certain organic compounds required for its growth. In the context of the present invention, the yeast cells may be nutriotrophic for histidine, leucine and / or uracil. Therefore, the cells cannot synthesize histidine, leucine and / or uracil on their own, and therefore in histidine-, leucine- and / or uracil-free medium and histidine-, leucine- and / or uracil-free cells. It cannot grow on culture plates. The trophogenic yeast cells need to be supplemented with histidine, leucine and / or uracil, respectively, in the culture medium. Yeast cells may also be nutritional for other nutritional factors in the group consisting of, but not limited to, vitamins, amino acids, sugars and fatty acids.

The term "vector" as used in the present invention may refer to any composition that transfers genetic information to yeast cells. The genetic information is as deoxyribonucleic acid (DNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), ribonucleic acid (RNA) (single-ray RNA (ssRNA), double-stranded RNA (dsRNA)), or For example, it may be encoded with a DNA analog such as peptide nucleic acid (PNA), morpholino, glycol nucleic acid (GNA), threose nucleic acid (TNA) or methylated DNA. Preferably, the genetic information is encoded in DNA. The vector can be any vector known in the art, such as, for example, a linear vector, a cyclic vector, a viral vector or a bacterial vector. Preferably, the vector comprises linear DNA or cyclic DNA, more preferably cyclic DNA, in particular plasmid. The vector may be inserted into cells by methods known in the art and may be transfectant (s) (e.g., lithium acetate, polyethylene imine (PEI), fugene, LT-1, JetPEI, transfectamine , Lipofectamine, UptiFectin, PromoFectin, GenePORTER, Hilymax, carbon nanofibers, carbon nanotube cell-penetrating peptides (CPPs), protein transduction domains (PTDs), liposomes, DEAE-dextran, dendrimers, electrophoresis ), Or with or without gene gun, optical transfection, gene electrotransmission, impalefection, magnetofection and / or magnet-assisted transfection. Preferably, the vector is a cyclic DNA vector, more preferably the vector is a plasmid. The plasmid can be any plasmid known in the art, such as, for example, a YEpH2 or pUC19 plasmid. The vector may also be a linear expression cassette, which may or may not be integrated into the genome of the target cell. However, it can be understood that all other vectors besides these may also be used.

The term "gene insertion" refers to any means of incorporating a gene into yeast cells. The gene can be, for example, DNA (double-stranded DNA (dsDNA), single-stranded DNA (ssDNA)), RNA (single-threaded RNA (ssRNA), double-stranded RNA (dsRNA)), or, for example, peptide nucleic acid (PNA). ), DNA analogs such as morpholino, glycol nucleic acids (GNA), threose nucleic acids (TNA) or methylated DNA, or hybrids thereof, or any carrier of genetic information. Preferably, the gene is encoded on DNA, more preferably on duplex DNA, in particular cyclic duplex DNA strands, in particular on plasmids. The plasmid can be any plasmid known in the art.

The genetic information can be inserted into the cell as a vector. As described in detail above, the vector may be any vector known in the art, such as, for example, a linear vector, a cyclic vector, a viral vector or a bacterial vector. Preferably, the vector comprises linear DNA or cyclic DNA, more preferably cyclic DNA, in particular plasmid. The vector can be inserted into the cells by methods known in the art and may be transfectant (s), fugene, LT-1, JetPEI, transfectamine, lipofectamine, UptiFectin, PromoFectin, GenePORTER, Hilymax, carbon nanofibers, carbon nanotube cell-penetrating peptides (CPPs), protein transduction domains (PTDs), liposomes, DEAE-dextran, dendrimers), electrophoration, or gene guns, optical transfection, It may be used with or without gene electrophoresis, impalefection, magnetofection and / or magnet-assisted transfection. Preferably, the vector is a cyclic DNA vector, more preferably the vector is a plasmid and may be any plasmid known in the art. However, it can be understood that all other vectors can also be used.

The inserted gene may be active or passive in the cell. Preferably, the inserted gene is actively transcribed and expressed in the cell. The gene can be actively transcribed and expressed as an extranuclear plasmid, as an intranuclear plasmid, as an extranuclear linear vector, as an intranuclear linear vector, or inserted into the genome of a yeast cell.

By providing a gene of pobTPS which is a terpene synthetase derived from a winter umbrella mushroom according to the present invention, it is possible to provide a terpene synthetase having better activity than a wild type strain.

Figure 1 shows a schematic diagram of eudesmol production (FPP, farnesyl pyrophosphate) by Terpene synthase.
2 shows the mevalonate pathway.
Figure 3 shows the pSLF vector map for transformation into S. pombe .
4 shows the cloning and confirmation of pCR2.1-TOPO vector.
5 shows the cloning and confirmation of the pSLF 273 vector (for S. pombe expression).
Fig. 6 shows a cleavage map of pSLF273-pobTPS into which the pobTPS gene was introduced.
Figure 7 is a gel electrophoresis picture by performing the RACE PCR (a. GSPs PCR: 5'-1300bp, 3'-800bp, b. Nested GSPs PCR: 5'-1,000bp, 3'-600 bp, c. Ladder) It is shown.
FIG. 8 is a comparison of pobTPS amino acid sequences and shows high homology genes (Gene bank accession number XP_007269573: Fomitiporia mefiterranea; Gene bank accession number KZT23360: Neopentinus lepideus; Gene bank accession number XP_007868748: Gloeophyllum trabeum).
Figure 9 shows the growth pattern of wild-type strains and transformants in solid YE medium.
Figure 10 shows the gene resequencing results of WT and WT + TPS.
Figure 11 shows malachite assay.
Figure 12 shows the results of the enzyme activity test using malachite.
Figure 13 shows the results of the gene expression pattern analysis after insertion of pobTPS in the mutant strain for each gene of the eight genes present in the mevalonic acid pathway.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> pobTPS  Sequence identification of genes

1-1. RNA extraction and gene acquisition

Winter Umbrella was a strain isolated by the National Forest Research Institute. The test strain was deposited in Korean Collection for Type culture (KCTC46459). Total RNA was isolated from mycelium cultured in Potato dextrose agar medium. RNA isolation was extracted using Gene all kit (Korea). cDNA synthesis. PCR reaction conditions were premodified at 94 ° C. for 5 minutes, then denatured at 94 ° C. for 1 minute, unrolled at 55 ° C. for 1 minute, and extended at 72 ° C. for 1 minute, and finally extended at 72 ° C. for 10 minutes. Specific primers were used for the PCR reaction and the forward used (5'-TGTACTATGCTCACTTCCACC-3 ') primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) were (5'-CTCATCCCACGGTTACAG-3'). PCR products were identified by gel electrophoresis on 1% agarose gel. Final PCR products were separated by gel elution kit (bioneer) and the final concentration was measured by Nanodrop (USA).

1-2. Rapid Amplification of cDNA ends-PCR (RACE-PCR)

RACE-PCR is a PCR method that cloning the unknown region to the cDNA terminal based on the nucleotide sequence information of the known region when the nucleotide sequence of cDNA (Clonetech, Mountain View CA, USA) is partially known. RT reaction was carried out using smart RACE cDNA amplification kit (Clontech) as a template of total RNA, and 5 ′ and 3′cDNA were synthesized through this. Gene specific primer (pobTPS: 5′-GAT TAC GCC AAG CTT ACC ATT CGC ACC ATC GAC AGC TAC C-3 ′ () based on the sequence of the gene reference (gene ID: 00005412-RA) obtained from previous transcript NGS analysis. SEQ ID NO: 5; forward), 5'-GAT TAC GCC AAG CTT GCT CAT CCC ACG GTT ACA GGG CTC-3 '(SEQ ID NO: 6; reverse) nested gene specific primer (5'-CAT TAC GCC AAG CTT AAC AAG GAA CAA GCA ACG GGA GAC G-3 '(SEQ ID NO: 7; forward), 5'-GAT TAC GCC AAG CTT GAG GAA CCG CAT CTC GCC TTC TTT G-3' (SEQ ID NO: 8) Unknown portions at both ends were amplified.

Example 2 Preparation of Transgenics

2-1. Used strain and plasmid

As a yeast host cell for pobTPS expression, Shizosaccharomyces pombe (ura4-D18, diploid, bioneer), which is an 8 gene deletion strain and a uracil nutritional requirement strain in the mevalonate pathway, were used (Table 1). . For smooth growth of S. pombe , 0.3g / L of adenine, uracil, and leucine were added to yeast extract broth medium.

Hosts and Genes Used enzyme Serial number Deletion gene Name One BG_D3965 Acetyl-CoA C-acetyltransferase erg10 2 BG_D4401 3-hydroxy-3-methylglutaryl-CoA synthase hcs1 3 BG_D1464 3-hydroxy-3-methylglutaryl-CoA reductase hmg1 4 BG_D3443 Mevalonate kinase Erg12 erg12 5 BG_D0087 Phosphomevalonate kinase erg8 6 BG_D3691 Diphosphomevalonate decarboxylase mvd1 7 BG_D0163 Isopentenyl-diphosphate delta-isomerase idi1 8 BG_D2055 Farnesyl pyrophosphate synthetase fps BG_0000 Wild type WT

The pobTPS gene donor plasmid for transformation into yeast was used pSLF vector (ATCC, USA) in which the promotor nmt1 (no message in thiamine 1) region exists (FIG. 3). pSLF vector is a plasmid that is frequently used as an expression vector of S. pombe . pSLF 273 vector and insert ( pobTPS ) were constructed by sub-cloning with pCR2.1-TOPO vector (ThermoFisher, USA), respectively (FIG. 4). As a sub-cloning strain, E. coli Escherichia coli (DH-5a) strain was used, and Luria-Bertani (LB: 0.5% yeast extract, 1% bacto-tryton, 1% sodium chloride) liquid commonly used for the culture of E. coli. Medium was used and 100 μg / ml of ampicillin was added as a selective marker.

2-2. Sub-cloning and clone acquisition

A vector containing a target gene was transformed into competent cell E. coli DH5a (MacCell ^TM -DH5a 10 ⁹ , iNtRON Biotechnology, Inc.). 50 ul of competent cells were mixed with 5 ul of the target gene and ligation vector, and the mixture was left to rest on ice for 15 minutes. Immediately after transferring to ice for 5 minutes, LB broth 450 ul was added and incubated for 1 hour at 37 ° C for regeneration. After 1 hour, 100 ul were plated on LB agar plates containing ampicillin (50 ug / ml) and X-Gal (40 ug / ml). The plate was incubated at 37 ° C. for one day. After plasmid extraction using plasmid extraction kit (bioneer), each clone was obtained by enzyme cutting. Afterwards, the pSLF vector and insert ( pobTPS ) were subjected to digesion reaction at 36 ° C. for 90 minutes with Not1 and BamH1 as restriction enzymes. Since both enzymes have blunt end characteristics, the ligation kit (DNA ligation kit long, takara 6024) was used to produce an over night reaction at 16 ° C. in the form of a circle. In order to confirm the well-known ligation, cut with the same enzyme (Not1, bamH1) to confirm the sequence of terpene synthase through the target band (pSLF 273 vector: 8.5 kb, pobTPS insert: 1.2 kb) and PCR and finally the pSLF273 which is the ligation product -pobTPS clone (9.7 kb) was obtained (FIG. 6).

2-3. Transformation with Yeast

The constructed recombinant clone (pSLF273-TPS) is a mutant in which each of the eight genes in the wild type (bioneer) and mevalonic acid pathways of the yeast Shizosaccharomyces pombe is deleted by chemical methods. (mutant 1-8) (see Table 1) strains were transformed (Table 2).

List of Yeast Transgenics Host Deletion gene
in host Clone Transformation Transgenics wild type None pSLF273-TPS Chemical method WT-pSLF273-TPS mutant 1 erg10 pSLF273-TPS Chemical method M1-pSLF273-TPS mutant 2 hcs1 pSLF273-TPS Chemical method M2-pSLF273-TPS mutant 3 hmg1 pSLF273-TPS Chemical method M3-pSLF273-TPS mutant 4 erg12 pSLF273-TPS Chemical method M4-pSLF273-TPS mutant 5 erg8 pSLF273-TPS Chemical method M5-pSLF273-TPS mutant 6 mvd1 pSLF273-TPS Chemical method M6-pSLF273-TPS mutant 7 idi1 pSLF273-TPS Chemical method M7-pSLF273-TPS mutant 8 fps pSLF273-TPS Chemical method M8-pSLF273-TPS

As a selective marker of the TPS gene transformant, a trophic marker uracil was used. Chemical transformation method is as follows. First, S. pombe was incubated in yeast extract (YE) medium, colonized to an appropriate concentration of 1 × 10 ⁹ cells / ml, and harvested in LiAc-TE buffer. After the reaction for 5 minutes at room temperature with the cloned pSLF273-TPS 5ul secured by the addition of 260ul of 40% LiAc-TE buffer for 60 minutes at 30 degrees. After adding 43 ul of DMSO, heat shock was applied at 42 ° C. for 5 minutes, and finally spreading to selective medium to secure transformants after 4-6 days. EMM (Edinburgh Minimal Media) + L (leucine), a uracil deficient medium, was used as a medium for selection of yeast S. pombe transformants (Table 3).

Chemical Composition of EMM + L Medium Composition g / L L-leucine 0.3 KH phthalate 3 Na ₂ HPO ₄ 2.2 NH ₄ Cl ₂ 5 Glucose 20

Experimental Example 1 pobTPS Complementaion confirmation of genes

1-1. genomic DNA extraction

Genomic DNA was extracted from WT and transformants using liquid nitrogen. About 20 ml of sub-culture was centrifuged to separate only the cells. The isolated cell was immediately lysed with 300 μl of beads and DNA extraction buffer (1M Tris-HCL pH 8.0, 5M NaCl, 0.5M EDTA pH 8.0, 20% SDS). Precipitate the cells for 15 minutes using a centrifuge, transfer 250 µl of the supernatant to a new ep-tube, add 250 µl of phenol-chloroform-isoamyl alcohol (25: 24: 1) (Bioneer), and mix well. Protein was separated using a separator. The supernatant was transferred to a new tube again and the DNA was precipitated by adding 500 μl of 95% ethanol. After washing twice with 70% ethanol, the ethanol was completely evaporated, and the pellet precipitated DNA was dissolved in sterile distilled water containing 10 ug / ml RNase.

1-2. Illumina paired-end library construction and sequencing

After extracting genomic DNA from yeast, RNA quality check was performed using Agilent Technologies 2100 Bioanalyzer. The quality of Total RNA extracted from two species of wood fungi was confirmed using Hiseq2000. From the selected total RNA, phosphate was present only at the 5 'end of intact mRNA in total RNA by Bacterial Alkaline Phosphatase (BAP) and Tobbaco Acid Pyrophosphatase (TAP) reaction, and then mRNA was separated from total RNA using oligo dT column. . The isolated mRNA was fragmented through Covaris (Adaptive Focused Acoustics ™ (AFA) technology), and two cDNA libraries of high-efficiency wood fungus were prepared by PCR based oligocapping using random primers. Only fragments corresponding to ˜400 bp were selected and amplified to the appropriate concentration by PCR amplification, and confirmed by Agilent Bioanalyzer, and high quality reads were extracted from each sample by filtering the sequence redad using Sickle (vl.33). The acquired reads were mapped to S. pombe genome using EquCab2.0 using BWA ver.0.7.12 program, and after realignment with GATK (ver. 2.3-9) program after mapping, SnpEff (v.4.1) The modified annotation result was confirmed using.

Since pobTPS was identified through de novo sequencing as a gene identified through mass genome analysis (NGS) of P. brumalis , accurate gene sequence information including an open reading frame (ORF) was needed. PCR was performed using a gene specific primer and a nested gene specific primer for RACE PCR (FIG. 7), and the sequence information of the entire sequence information of the pobTPS protein including the ORF region and the entire sequence information of the pobTPS protein including the ORF region. As a result of performing alinement (web program: Clustal Omega) on the gene, total sequence sequence information of about 1.2 kb was obtained.

In addition, the ORF length was estimated by using the ORF finder program using the isolated terpene synthase gene information. The 1,185 bp, 394 amino acid, MW and PI values were 45.74 kDa and 5.07, respectively. The pobTPS gene sequence information (SEQ ID NO: 1) and the complete sequence information of the pobTPS protein (SEQ ID NO: 2) are registered in the gene bank library of the NCBI (National Institute of Biological Information) (accession number: KY063082).

As a result of blast searching for the obtained genetic information, gene similarity of 60-70% was shown with the heterologous mushrooms Fomitiporia mefiterranea, Neopentinus lepideus, and Gloeophyllum trabeum (FIG. 8). As described above, it seems that the gene homology is low due to the rapid mutation of the TPS gene, and it is expected that the result of the genome analysis is a relatively small mushroom. In the case of the previously known TPS gene, conserved domains are common in plant, animal, and human-derived genes other than microorganisms, and there are two characteristic motifs, DDXXD and NSE regions, aspartate-rich regions. do. For the pobTPS gene, the conserved domain confirmed that the DEYTD region was identified in the DDXXD motif, and the NDIASYNKE region was identified in the NSE motif. In the case of pobTPS , the first motif has an amino acid sequence of DEXXD rather than DDXXD. Another mushroom species, Armillaria gallica, has also been reported to have a DEXXD motif in the TPS region. Synthesize It is also known that this motif is characterized by being activated by magnesium ions. Therefore, the pobTPS enzyme is activated in the culture medium containing magnesium and may be related to sesquiterpene biosynthesis.

Experimental Example 2 Transformant Selection and Confirmation

Transgenics were selected by confirming the growth pattern in the solid YE medium of the transformants in which the pSLF273-pobTPS vector obtained in Example 2-2 was introduced into the Uracil nutritionally demanding mutant strain (S. pombe).

S. pobme strain shows pink colony in yeast extract medium, which is a solid medium without amino acids. On the other hand, the transformants show white colony because of the presence of nutritional requirements. For transformant selection, EMM + Leucine medium containing uracil deficient and amino acid leucine was used. As a result, 39 colonies were identified from the uracil deficient medium (FIG. 9).

Re-sequencing of gDNA of WT and WT + pobTPS samples was performed to confirm the transformation of pobTPS into yeast. Re-sequencing was performed using Illumina Hiseq. As a result, it was confirmed that the sample covering the pobTPS gene was WT + pobTPS. Coverage rate shows homology with pobTPS gene sequence. In case of WT homology, 0%, WT + TPS showed 100% coverage, indicating that the overexpression of the pobFPS gene and the pobTPS gene were complementaioned to S. pombe . In addition, it could be confirmed by reverse-transcription PCR. Total RNA was extracted from the yeast transformants to confirm the expression of TPS, but expression was not confirmed in WT, but expression intensity was measured in the WT + TPS sample (FIG. 10).

Experimental Example 3 Transformant Characteristic Evaluation

3-1. Enzyme activity test

Enzyme activity was measured to confirm that the inserted gene is actually expressed at the protein level. Enzyme activity test was performed using a malachite green assay (FIG. 11). Malachite minerals have a property of changing to blue green color when combined with phosphate groups. At this time, yellowing results in the yellow reaction solution changing to blue green color when the phosphate group and malachite react. Therefore, it is an experiment to measure the degree of change from light yellow to blue when combined with phosphate (pyrophosphate) released from the precursor by TPS.

As an experimental method, cells are obtained from 20 ml sub-culture as in genomic DNA extraction samples. Mix the obtained cell, protein extraction buffer, and beads and vortax the solution at room temperature and 4 ℃ for 5 minutes. Thereafter, only the supernatant separated through centrifugation was separated and the amount of protein was quantified by bradford solution. 80 ul of the extracted protein was immediately mixed with 10 ul of malachite solution and 0.1 M FPP as a substrate and reacted at room temperature for 30 minutes. Since the absorbance at 600 nm was measured by measuring the amount of color change.

As a result, the change in absorbance and the rate of change were increased in the strain transformed with pobTPS , and the amount of free pyrophosphate produced by the reaction was increased by about 3 times when quantitatively indicated (FIG. 12).

3-2. Metabolite analysis of transformants

Since the metabolite analysis of which reaction product is produced through the phosphate (farnesyl pyrophosphate) desorption reaction should be performed together, the metabolites generated from each strain were analyzed using two strains of the pobTPS transformed strain in the wild type strain.

First, the culture medium in 20ml seed culture was transferred to 100ml sub-culture and precultured for 24 hours. After 24 hours, farnesyl pyrophosphate ammonia salt, a precursor of sesqui terpene, was added. Thereafter, after reacting at 160 rpm at 28 ° C., after 32 hours, 100 ml of the culture solution was extracted three times using 100 ml of organic solvent, ethyl acetate. The extracted solution was subjected to gas chromatography analysis by completely concentrating the organic solvent using an evaporator. The analysis method of GC MS is as follows. The analytical conditions were injector 260 ℃ and detector 280 ℃ and the mass patterns of the detected peaks were compared to the National Institute of Standards Technology 2005 library.

A total of three samples were used for analysis using WT and WT + TPS. The analysis was confirmed by scale up of the culture. The scale up was performed from the existing 20 ml culture to 300 ml culture. The metabolite was analyzed in the same way as the metabolite analysis of winter mushrooms. The culture was extracted with ethyl acetate and analyzed by gas chromatograph-mass spectrometer (GC MS).

Each metabolite was analyzed at 24h, 48h, 72h for 24 hours and metabolites were identified at 72h, respectively. In particular, the metabolite pattern was different according to the sample type. The sample that showed the most meaningful metabolite was the sample detected at 72h of WT + TPS. In this sample, 5B, 7B and 10a-eudesm-11-en-1α-ol containing eudesmane backbone were detected. In this structure, alcohol is not bonded to the side chain as in eudesmol, but alcohol is bonded at position 1. The results deduced from these results suggest that the eudesmol-like structure targeted in this study was generated due to the overexpression of FPS, deletion of downstream genes, and the presence of TPS. As a result, it was confirmed that pobTPS gene may be directly related to terpene production.

Experimental Example 4 Correlation with UP-stream Gene

In this experiment, we tried to confirm the association of the pobTPS gene with the top 8 genes using a mutant strain with 8 genes deleted. PobTPS was transformed into each mutant by electrophoresis. The transformants were then cultured in yeast extract medium medium. After culturing in 20 ml medium, RNA was extracted from mycelia and subjected to reverse transcription PCR targeting the TPS gene. As a result, the expression levels of pobTPS inserted into enzyme 2 (HMG-CoA synthase), 3 (isopentenyl-isomerase), and 8 (farnesyl pyrophosphate synthase) deficient strains tended to decrease. This indicates that the expression of TPS decreases when such a gene is deleted, and it is expected that such higher genes affect TPS gene expression most. In particular, HMG-CoA synthase, isopentenyl pyrophosphate isomerase, and farnesyl pyrophosphate are known as genes and enzymes that play an important role in intermediate skeletal formation. In particular, the FPS gene is known as a type of transferase that generates GPP and FPP, which are substrates, and plays a key role in the generation of various types of structures. In the case of plants, the results of increasing the concentration of artemisin, a sesquiterpene having antimalarial activity, in the transformant overexpressed FPS (FIG. 13).

<110> National Institute of Forest Science <120> Terpene synthase gene from Polyporus brumalis, Shizosaccharomyces          pombe mutant transformants containing the gene <130> 18-11008 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1185 <212> RNA <213> Artificial Sequence <220> <223> pobTPS mRNA <400> 1 atgctcactt ccaccacctc cacctccatg tttgagccca ggcaatccgt cgtcgtccgc 60 accgaagacg gccgcgagca gaggtacctc tacttgccag acacgatggc caactggccg 120 tggccccgca agatcaaccc gtactacgag gaggtcaccg ccgagtccaa tgcatggttc 180 aagaccttca aggctttcac gcccgaatca cagtatgcgt acgacaagtg cgacttcggc 240 cggctggcat ccctcgcata cccggacgtc tctcgagagg cgctccgcac cggggtcgat 300 ctcatgaacg tcttattcct cgtggacgag tacacggatt tccagcccgc tcctgtcgtc 360 cgggagataa ccggggttgt gatcgacgcc atactgaacc ccgacaaggt ccgtccggag 420 ggcgagagca tcctcggcga ggtcacccga cagttctggg cgcgcggacg gaccaccgct 480 actccggagg ctgcgaagca cttcgtcgag actctcaagg cgtatttgaa ctccgtgatt 540 gtccaggccg aggaccggga caatgatacc attcgcacca tcgacagcta cctcgagata 600 cgccgtgaga acgtcggtgt acgctcggcc tacatgcctg gggagctgca cctctcgatt 660 cccgacgagg ccttctacca ccctgtcatc aaggaactcg aatacctcgc catagacctg 720 atcatccttg acaatgacat cgcatcctac aacaaggaac aagcaacggg agacgacagg 780 cacaatattc ttacaatcgc catgcaccag tttaataccg acttcgacag cgccatggac 840 tgggtcgcaa actaccacaa agaaggcgag atgcggttcc tcgacgcgct caagcgcgtg 900 ccctcctggg gcccggaggt cgacaagcag gtcgcggtgt acatcgagca ccttgcgaac 960 tggccgcggt gcaacgactg ctggaacttc gagtcgtggc ggtactttgg gcacaagggc 1020 ctcgagtacc agaagacgcg cctcgtgccg atgctttcga agcgtccgca gaatctgacc 1080 catcgtcggg agaacgtaga agtgccattt gtagacaagt tcgaggagat gatgtccggc 1140 attacgaacc cgctaggcca tcaggtagaa gacagagccc tgtaa 1185 <210> 2 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> pobTPS Protein <400> 2 Met Leu Thr Ser Thr Thr Ser Ser Thr Met Met Phe Glu Pro Arg Gln Ser   1 5 10 15 Val Val Val Arg Thr Glu Asp Gly Arg Glu Gln Arg Tyr Leu Tyr Leu              20 25 30 Pro Asp Thr Met Ala Asn Trp Pro Trp Pro Arg Lys Ile Asn Pro Tyr          35 40 45 Tyr Glu Glu Val Thr Ala Glu Ser Asn Ala Trp Phe Lys Thr Phe Lys      50 55 60 Ala Phe Thr Pro Glu Ser Gln Tyr Ala Tyr Asp Lys Cys Asp Phe Gly  65 70 75 80 Arg Leu Ala Ser Leu Ala Tyr Pro Asp Val Ser Arg Glu Ala Leu Arg                  85 90 95 Thr Gly Val Asp Leu Met Asn Val Leu Phe Leu Val Asp Glu Tyr Thr             100 105 110 Asp Phe Gln Pro Ala Pro Val Val Arg Glu Ile Thr Gly Val Val Ile         115 120 125 Asp Ala Ile Leu Asn Pro Asp Lys Val Arg Pro Glu Gly Glu Ser Ile     130 135 140 Leu Gly Glu Val Thr Arg Gln Phe Trp Ala Arg Gly Arg Thr Thr Ala 145 150 155 160 Thr Pro Glu Ala Ala Lys His Phe Val Glu Thr Leu Lys Ala Tyr Leu                 165 170 175 Asn Ser Val Ile Val Gln Ala Glu Asp Arg Asp Asn Asp Thr Ile Arg             180 185 190 Thr Ile Asp Ser Tyr Leu Glu Ile Arg Arg Glu Asn Val Gly Val Arg         195 200 205 Ser Ala Tyr Met Pro Gly Glu Leu His Leu Ser Ile Pro Asp Glu Ala     210 215 220 Phe Tyr His Pro Val Ile Lys Glu Leu Glu Tyr Leu Ala Ile Asp Leu 225 230 235 240 Ile Ile Leu Asp Asn Asp Ile Ala Ser Tyr Asn Lys Glu Gln Ala Thr                 245 250 255 Gly Asp Asp Arg His Asn Ile Leu Thr Ile Ala Met His Gln Phe Asn             260 265 270 Thr Asp Phe Asp Ser Ala Met Asp Trp Val Ala Asn Tyr His Lys Glu         275 280 285 Gly Glu Met Arg Phe Leu Asp Ala Leu Lys Arg Val Pro Ser Trp Gly     290 295 300 Pro Glu Val Asp Lys Gln Val Ala Val Tyr Ile Glu His Leu Ala Asn 305 310 315 320 Trp Pro Arg Cys Asn Asp Cys Trp Asn Phe Glu Ser Trp Arg Tyr Phe                 325 330 335 Gly His Lys Gly Leu Glu Tyr Gln Lys Thr Arg Leu Val Pro Met Leu             340 345 350 Ser Lys Arg Pro Gln Asn Leu Thr His Arg Arg Glu Asn Val Glu Val         355 360 365 Pro Phe Val Asp Lys Phe Glu Glu Met Met Ser Gly Ile Thr Asn Pro     370 375 380 Leu Gly His Gln Val Glu Asp Arg Ala Leu 385 390 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 tgtactatgc tcacttccac c 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 ctcatcccac ggttacag 18 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> gene specific primer (forward) <400> 5 gattacgcca agcttaccat tcgcaccatc gacagctacc 40 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> gene specific primer (reverse) <400> 6 gattacgcca agcttgctca tcccacggtt acagggctc 39 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> nested gene specific primer (forward) <400> 7 cattacgcca agcttaacaa ggaacaagca acgggagacg 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> nested gene specific primer (reverse) <400> 8 gattacgcca agcttgagga accgcatctc gccttctttg 40

Claims (18)

Shizosaccharomyces pombe transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into the Shizosaccharomyces pombe , which has deleted the gene encoding acetyl-CoA C-acetyltransferase (erg10). Shizosaccharomyces pombe ) . delete Shizocara transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into Shizosaccharomyces pombe , which lacked the gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase (hcs1). Shizosaccharomyces pombe . delete Shizocarro transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into Shizosaccharomyces pombe , which lacks the gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase (hmg1). Shizosaccharomyces pombe . delete Gene encoding the Mevalonate kinase Erg12 (erg12) the rest research Caro My process pombe (Shizosaccharomyces pombe) the rest research Caro My process pombe (Shizosaccharomyces pombe) switches the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1, transfection is inserted into the deleted . delete Shizosaccharomyces pombe transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into the Shizosaccharomyces pombe , which is a gene encoding the phosphomevalonate kinase ( erg8 ) . delete Shizosaccharomyces pombe transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into the Shizosaccharomyces pombe , which is a gene encoding diphosphomevalonate decarboxylase ( mvd1 ) . delete Shizosaccharomyces pombe transformed by inserting the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1 into the Shizosaccharomyces pombe , which is a gene encoding isopentenyl-diphosphate delta-isomerase (idi1). Shizosaccharomyces pombe ) . delete Farnesyl pyrophosphate synthetase gene (fps) the rest research Caro My process pombe (Shizosaccharomyces pombe) the rest research Caro My process pombe (Shizosaccharomyces pombe) switches the pobTPS gene consisting of the nucleotide sequence of SEQ ID NO: 1, transfection is inserted into the deletion encoding the . delete The method according to any one of claims 1, 3, 5, 7, 9, 11, 13, and 15, wherein the pobTPS gene is derived from a winter umbrella. Shizosaccharomyces pombe transformed into. The method of claim 1, 3, 5, 7, 9, 11, 13, or 15, wherein the pobTPS gene encodes an enzyme involved in terpene synthesis. Shizosaccharomyces pombe , characterized in that the transformed Shizosaccharomyces pombe .

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