KR20010084864A - Amorpha-4,11-diene synthase derived from artemisia annua, gene encoding same, expression vector containing said gene and, e. coli and plant transformed with said vector - Google Patents

Abstract

PURPOSE: An armopha-4,11-diene synthease of Artemisia annua is provided, which mediates the synthesis of armopha-4,11-diene in the biosynthesis pathway of artemisinine useful as an antimalaria agent. And its coding gene, an expression vector containing the gene, and E. coli and plant transformants transformed with the vector are also provided. Therefore, the transformants mass-produce artemisinine by increasing its biosynthesizability using metabolic engineering. CONSTITUTION: The armopha-4,11-diene synthease of Artemisia annua has the amino acid sequence represented by SEQ ID NO: 2. Its coding gene has the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence having over 80% homology with the nucleotide sequence of SEQ ID NO: 1. An expression vector pkcs12 contains the gene of SEQ ID NO: 1. E. coli BL21(DE3)/pLysS (KCTC 0739BP) is produced by transforming E. coli with the expression vector pkcs12. Also, plants can be transformed with the same expression vector.

Description

Amopa-4,11-diene synthase of firefly, gene encoding the same, expression vector containing the gene, and E. coli and plant transformed with the expression vector {AMORPHA-4,11-DIENE SYNTHASE DERIVED FROM ARTEMISIA ANNUA, GENE ENCODING SAME, EXPRESSION VECTOR CONTAINING SAID GENE AND, E. COLI AND PLANT TRANSFORMED WITH SAID VECTOR}

The present invention relates to amopa-4,11-diene synthase of Artemisia annua , a gene encoding the same, an expression vector comprising the gene, and E. coli and plants transformed with the expression vector.

Artemisinin is a powerful antimalarial substance isolated from plants such as Artemisia annua , and is sesquiterpene endoperoxide in the form of amorphane. In particular, the antimalarial activity of atemisinin is very useful because it acts on the more lethal malaria parasite ( Plasmodium falciparum ) when treatment with other antimalarial agents is impossible.

Atemisinin having such a utility has been mainly extracted from plants such as firewood, which produces it. This plant is native only to Asia, so mass extraction of atemisinin has been very limited.

In order to solve this problem, mass production of atemisinin using cell culture of firefly was attempted, but even this method failed to produce commercially sufficient levels of atemisinin.

As a new alternative for the mass production of atemisinin, a method for mass production of atemisinin using biosynthetic pathways and metabolic mechanisms of atemisinin is expected.

The biosynthetic pathway of atemisin has not yet been accurately defined, but the first step in this pathway is that farnesyl diphosphate (FDP) is cyclized by sesquiterpene cyclase as shown in FIG. It is known to react to form amopa-4,11-diene which is a kind of sesquiterpene. Recently, amorpha-4,11-diene synthase was partially purified from firewood as a sesquiterpene cyclase that mediates the cyclization of FDP (Bouwmeester, HJ et al. , Phytochemistry 52 , 843-854 (1999)), the gene of this enzyme has not yet been identified.

Meanwhile, various sesquiterpene cyclases have been reported so far, for example, 5- epi- aristolochene synthase from Nicotian tabacum and Capsicum annuum . (Facchini, PJ et al., Proc. Natl. Acad. Sci. USA. 89 , 11088-11092 (1992); and Back, K. et al., Plant Cell Physiol. 39 , 899-904 (1998)) , Vetispiradiene synthase from Hyoscyamus muticus and potato ( Solanum tuberosum ) (Back, K. et al., J. Biol. Chem. 270 , 7375-7381 (1995); and Yoshioka, H. et al., Plant Cell Physiol. 40 , 993-998 (1999)), (+)-δ-cardinene synthase ((+)-δ-cadinene synthase) derived from Cossypium arboreum (Chen, XY et al., Arch. Biochem. Biophys. 324 , 255-266 (1995)), germacrene C synthase (Colby, SM et al) derived from Lycopersicon esculentum cv.VFNT Cherry tomato . Proc. Natl. Acad. Sci . USA. 95, 2216-222 1 (1998)). These enzymes show a relatively high consensus of about 40% on the amino acid sequence.

Recently, (3R) -linalool synthase, one of the monoterpene cyclases, is a terpene synthase different from sesquiterpene synthase in J., Jia, J.-W. et al. Arch.Biochem.Biophys. 372 , 143-149 (1999)) and epi- cedrol cycleases, one of the sesquiterpene cycleases (Mercke, P. et al., Arch) . Biochem Biophys. 369 , 213-222 (1999); and Hua, L. et al., Arch. Biochem. Biophys. 369 , 208-212 (1999).

Accordingly, an object of the present invention is to provide amopa-4,11-diene synthase of firewood.

Another object of the present invention to provide a gene encoding the Amopa-4,11-diene synthase.

It is another object of the present invention to provide an expression vector comprising the gene.

Still another object of the present invention is to provide an E. coli and plant transformed with the vector.

1 is a schematic diagram of a synthesis reaction of amopa-4,11-diene.

Figure 2 shows the results of electrophoresis on a 1.0% agarose gel of the reaction of the PCR to amplify the partial gene of amopa-4,11-diene synthase.

3 shows Northern blot results of partial genes of amopa-4,11-diene synthase.

4 shows Southern blot results of partial genes of amopa-4,11-diene synthase.

5A and 5B show electrophoresis of the product of 3'-RACE and the product of 5'-RACE on 1.0% agarose gel to amplify the full length gene of amopa-4,11-diene synthase, respectively. The result is.

Figure 6 shows the results of comparing and arranging the amino acid sequence of amopa-4,11-diene synthase with other sesquiterpene cycles.

Figure 7 shows the results of electrophoresis of the reaction product of PCR to amplify the full-length gene of amopa-4,11-diene synthase.

FIG. 8A is a gas chromatography spectrum of the enzyme reactant, FIG. 8B is a mass spectrum of the enzyme reactant obtained at 9.3 minutes of retention time, and 8c is a mass spectrum of standard Amopa-4,11-diene.

In accordance with the above object, in the present invention, Amopha-4,11-diene synthase of Artemisia annua having the amino acid sequence of SEQ ID NO: 2 and substantially the enzyme To provide a polypeptide having the same enzymatic activity.

According to another object, the present invention provides a gene encoding the amopa-4,11-diene synthase and a polypeptide having substantially the same activity.

According to another object, the present invention provides an expression vector comprising the gene.

According to another object, the present invention provides E. coli transformed with the expression vector.

In accordance with another object, the present invention provides a plant transformed with the expression vector.

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

The amopa-4,11-diene synthase of the present invention synthesizes amopa-4,11-diene by cyclization using FDP as a substrate. This enzyme has the amino acid sequence of SEQ ID NO: 2 and can appropriately modify its amino acid sequence within a range having substantially the same enzyme activity. The polypeptide of SEQ ID NO: 2 consists of 546 amino acid residues, has a pi value of 5.59 and a Mw value of 63.9 kDa. In particular, the open reading frame of this polypeptide does not include a plastid targeting sequence, suggesting that this enzyme is widely distributed at the plasma site (Bohlmann, J. et al., Proc. Natl. Acad). . Sci USA 95, 4126-4133 (1998 );... and McGarvey, DJ et al, Plant Cell 7, 1015-1026 (1995)). A polypeptide having an enzymatic activity substantially identical to SEQ ID NO: 2 preferably has at least 80% enzymatic activity and more preferably at least 90% enzymatic activity.

In particular, the active site helix loops in tobacco epi-aristologen synthase (Starks, CM et al., Science 277, 1815-1820 (1997)), whose structure is known by X-ray crystallography. Trp ²⁷³ and Tyr ⁵²⁷ , which hold the lid of the loop, are also conserved as Trp ²⁷¹ and Phe ⁵²⁶ in the enzyme of the present invention. Thus, the nucleophilic sikimyeo of the indole ring of Trp ²⁷¹ to stabilize the intermediate carbonyl cation of the cyclization, the substrate ¹⁰ with Arg and the assistance of Mg ²⁺ has a high density positive charge of Arg ⁴⁴⁰ combines the DDXXD metal binding motif when the ring is closed It is thought to help the ionization of FDP. In addition, the Arg ¹⁰ -Pro ¹¹ pair motif, which is common in sesquiterpene synthase, is well preserved, and the amino acid residues responsible for the catalyst are also preserved as Asp ⁴⁴⁴ , Tyr ⁵²⁰ , and Asp ⁵²⁴ .

The amino acid sequence of the enzyme shows high concordance with other sesquiterpene synthetases, particularly 36% with tobacco 5-epi-aristologen synthase and 41% with cotton (+)-δ-cardinene synthase. Seems. In addition, the amino acid sequence of the enzyme showed 51% concordance with other terpene synthase, Epi-cedrol cyclase, isolated from firefly, and with (3R) -linalul synthase, one of the monoterpene synthase. Low agreement of%.

In addition, the gene encoding the amopa-4,11-diene synthetase of the present invention and a polypeptide having substantially the same activity, for example, has a nucleotide sequence of SEQ ID NO: 1 and is within a range encoding a substantially identical enzyme. The base sequence can be appropriately modified. The gene of SEQ ID NO: 1 is the full length cDNA gene of amopa-4,11-diene synthase isolated from firefly. The gene of SEQ ID NO: 1 consists of an open reading frame of 1641 bp, a 5'-untranslated end of 33 bp, and a 3'-untranslated end of 432 bp. There is also a conserved base sequence GAAAATCATGT in the region from 27 to 37 in the 5'-untranslated terminus, and the typical polyadenine signal AATAAT / A in the region from 1778 to 1786 at the 3'-untranslated terminus. Is repeated twice. There is also an end codon TGA on the open reading frame. The gene encoding the enzyme substantially identical to SEQ ID NO: 1 preferably has at least 80% homology with the nucleotide sequence of SEQ ID NO: 1 and more preferably at least 90% homology.

The amino acid sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 1 were registered in the GenBank / EMBL database under accession number AJ251751.

The gene of the present invention can be obtained by screening and cloning according to a conventional method based on the nucleotide sequence information of SEQ ID NO: 1, for example, after making cDNA using mRNA extracted from young leaves of firefly as a template, the primer START ( Easily obtained by performing a polymerase chain reaction (PCR) using SEQ ID NO: 3) and STOP (SEQ ID NO: 4).

The vector of the present invention can be obtained by inserting the gene into an appropriate expression vector according to a conventional method. As such a vector, the gene may be expressed in a bacterium or a plant, and representative examples thereof include a vector pkcs12, but are not limited thereto.

The transformed E. coli of the present invention can be obtained by transforming the expression vector according to a conventional method. Representative examples of the transformed Escherichia coli include E. coli BL21 (DE3) / pLysS containing the expression vector pkcs12, deposited with the KCTC 0739BP dated February 18, 2000, to the Gene Bank of Biotechnology Research Institute. It doesn't work.

The transgenic plant of the present invention can be obtained by introducing the expression vector into a plant cell according to a conventional method, and then cultivating the plant by inducing differentiation of the cell as appropriate. Examples of gene transduction into representative plant cells include Agrobacterium tumefaciens and appropriate Ti vectors, polyethylene glycol (PEG), biolistic, microinjection ) Method, etc., but is not limited thereto. Representative methods for inducing differentiation of cells include, but are not limited to, treatment of plant hormones 2,4-D and benzyladenin.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

The leaves of Artemisia annua used in the following Reference Examples and Examples were harvested in March 1999 from cultivation at the College of Agricultural and Life Sciences, Seoul National University.

Reference Example 1: Isolation of Genomic DNA from Firefly

In order to obtain genomic DNA of Artemisia annua , 1 g of young leaves of M. Fructus were extracted in 5 ml of extraction buffer (Shure, M. et al., Cell 35, 225-233 (1983)). Genomic DNA was isolated by homogenization in 7 M urea, 0.35 M NaCl, 50 mM Tris / pH 8.0, 20 mM EDTA, 1% sarkosyl, 0.6% SDS).

Reference Example 2: mRNA Isolation from the Leaves

To obtain mRNA of Artemisia annua leaves, guanidine isocyanate was prepared according to the method of Sambrook, J. et al., Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. (1989). After total RNA was extracted using the polytrack mRNA isolation system II (PolyATract mRNA Isolation system III, Promega, Madison, WI) to polyadenylation mRNA was isolated.

Reference Example 3 Synthesis of Amopa-4,11-diene

According to the method of Kim, SU et al. J. Nat. Prod. 56 , 859-863 (1993), 1.5 g of artemisinic acid was isolated from the methanol extract of Gadfly (500 g of dry mass). . Artemisinic acid was then reduced to alcohol using diisobutylaluminum hydride according to a known method (Laemmli, UK Nature 227 , 680-685 (1970)). This material was tosylated with p -toluenesulfonyl chloride according to known methods (Daniewski, AR et al., J. Org. Chem. 47 , 2993-2995 (1982)), followed by lithium aluminum hydride. Reduced to amopa-4,11-diene.

The product was separated by thin layer chromatography (preparative argentated TLC, 15% AgNO ₃ , developing solvent; hexane: benzene: diethyl ether = 50: 50: 1, R _f = 0.75), and NMR and MS were performed. The structure of the product was confirmed.

NMR spectra were LA400 (400 MHz for ¹ H, 100 MHz for ¹³ C) and NMR spectrometer (JEOL, Japan), and AX505WA mass spectrometer (JEOL, Japan) was used for electron impact mass spectrometry (EIMS). The analysis results are as follows.

^OneH-NMR (400 MHz, CDCl₃) δ 0.88 (3H,d, 6.3 Hz, H-15), 0.97 (1H,dddd, 3.4, 10.8, 12.1, 12.7 Hz, H-9ax), 1.27 (1H,dddd, 3.4, 11.0, 12.1, 12.7 Hz, H-8ax), 1.32 (1H,m, H-2), 1.40 (1H,ddd, 3.9, 6.3, 10.8 Hz, H-10), 1.52 (1H,dddd, 1.4, 3.4, 3.4, 12.7 Hz, H-8eq), 1.56 (1H,m, H-2ax), 1.60 (3H,s, H-14), 1.67 (1 H,dddd, 3.4, 3.4, 3.6, 12.7 Hz, H-9eq), 1.74 (3H,s, H-12), 1.79 (1H,m, H-1), 1.87 (1H,m, H-3), 1.95 (1H,m, H-7), 1.97 (1 H,m, H-3), 1.99 (1H,m, H-7), 2.55 (1H,br. s, H-6), 4.64 (1H,s, H-13), 4.87 (1 H,s, H-13), 5.06 (1H,s, H-5)

¹³ C-NMR (100 MHz, CDCl ₃ ) δ 19.8 (C-15), 22.6 (C-14), 23.6 (C-12), 25.8 (C-8), 26.1 (C-2), 26.5 (C -3), 27.9 (C-10), 35.4 (C-9), 37.6 (C-6), 41.8 (C-1), 47.6 (C-7), 109.8 (C-13), 120.9 (C- 5), 134.6 (C-4), 148.0 (C-11)

EI-MS (70 eV) m / z 204 ([M ⁺ ], 83), 119 (100)

Example 1: Cloning of partial genes of firefly amopa-4,11-diene synthase

(Step 1) PCR

To clone a partial gene of amopa-4,11-diene synthase of Artemisia annua , PCR was performed as follows.

First, four kinds of plant sesquiterpene synthase, that is, 5-epi-aristoloken synthase of tobacco ( N. tabacum ) (Facchini, PJ et al., Proc. Natl. Acad. Sci. USA. 89 , 11088-11092 (1992)), Betispyradiene synthase from H. muticus (Back, K. et al., J. Biol. Chem. 270 , 7375-7381 (1995)), tomato (L.esculentum (+)- Δ-cardinene synthesis of Gemacrene C synthase (Colby, SM et al. Proc. Natl. Acad. Sci. USA. 95 , 2216-2221 (1998) and G. arboreum ) Referencing the known base sequence of the enzyme (Chen, XY et al., Arch. Biochem. Biophys. 324 , 255-266 (1995)) selects a conserved region between base sequences and the corresponding primer F7 (SEQ ID NO: 5) and B9 (SEQ ID NO: 6) were prepared, and the 5 'end of each primer was designed to include the cleavage site of restriction enzyme Nco I.

PCR was carried out in Reference Example 1 100 ng of genomic DNA DNA, primers F7 and B9 at 0.8 mM, Taq DNA polymerase 2 units (Promega) and reaction buffer (10 mM Tris / pH 9.0, 50 mM KCl, 2.5 mM MgCl ₂ , 100 μl of a mixture of 0.2 mM dNTP and 0.1% Triton X-100). The number of PCR amplifications was 40, and each amplification step was performed at 94 ° C. for 2 minutes of denaturation (4 minutes at first), 50 ° C. for annealing 0.5 minutes, and 72 ° C. for 1.5 minutes (last 7 minutes). In order.

The obtained PCR product was electrophoresed on 1.0% agarose gel, and the results are shown in FIG. 2. In Figure 2, column M is the molecular weight label, column 2 is the PCR product, and column 1, column 3 and column 4 are the genomes of the genome, genome B9 (SEQ ID NO: 6) and primer, respectively, for comparison. PCR resulted in the same manner without using F7 (SEQ ID NO: 5).

This PCR product is a partial gene of amopa-4,11-diene synthase that is 184 bp in size. This PCR product was cloned into pGEM-T Easy vector (Promega) and the vector obtained was named vector p76.

Subsequently, the nucleotide sequence of the PCR product was determined using an ABI PRISM Dye Terminator Cycle Sequencing Kit, Applied Biosystems, Perkin-Elmer, and DNA Sequencing System, ABI model 373A, Applied Biosystems, Perkin-Elmer. Decided.

As a result, the nucleotide sequence of the PCR product and the amino acid sequence deduced therefrom were 56.4% and 52.3%, respectively, when compared with the nucleotide sequence and amino acid sequence of the 5-epi-aristoloken synthase of known tobacco ( N. tabacum ). The homology of

(Step 2) Northern Blot

To confirm the expression of the partial gene obtained in step 1, Northern blot according to the method of Sambrook, J. et al., Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. (1989). Was carried out as follows. 5 μg of the mRNA obtained in Reference Example 2 was electrophoresed on a 1.2% agarose gel containing formaldehyde and then transferred to a nylon membrane, and washed with 2 × SSC (300 mM NaCl, 30 mM Na-citrate / pH 7.0). Irradiated with UV light for 15 seconds and then pretreated with DIG Easy Hyb solution (Boehringer-Mannheim) at 50 ° C. for 1 hour, and random primers with [α- ³² P] dATP on vector p76 obtained in step 1 Hybridization overnight at 50 ° C. with DIG Easy Hyb solution (Boehringer-Mannheim) containing probes synthesized by a random primer extension, then 15 minutes at 50 ° C. with 2 × SSC with 0.1% SDS Washed 4 times, then dried and exposed to X-ray film for 2 days.

3 shows Northern blot results. In Figure 3, column M is the molecular weight label, column 1 is mRNA. As shown in Figure 3, a 2.2 kb mRNA band was confirmed. Therefore, it can be seen that the partial gene obtained in step 1 is expressed.

(Step 3) Southern Blot

To confirm the genomic structure of the partial gene obtained in step 1, see the method of Sambrook, J. et al., Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. (1989). The genomic DNA obtained in Example 1 was digested with Bam HI, Eco RI or Hind III and Southern blot was performed using the vector p76 obtained in step 1 as a probe.

4 shows Southern blot results. In Figure 4, column M is a molecular weight label, column B is genomic DNA cleaved with Bam HI, column E is genomic DNA cleaved with Eco RI, and column H is genomic DNA cleaved with Hind III. As shown in FIG. 4, partial genes showed single bands of 13 kb, 9 kb and 8 kb for Bam HI, Eco RI and Hind III fragments of genomic DNA, respectively. From this it can be seen that the partial gene obtained in step 1 exists in a single copy in the genome.

Example 2 Cloning of Full-length cDNA of Firefly Amopa-4,11-diene Synthetase

To clone the full-length cDNA of the firefly Amopa-4,11-diene synthase, the rapid cDNA terminus of McPherson, MJ et al., PCR2: A practical approach , p89-118, IRL Press (1995) According to the amplification (Rapid amplification of cDNA ends, RACE) method was performed as follows.

First, to obtain a double-stranded cDNA to be used for 3'-RACE, an AP primer (SEQ ID NO: 8) containing a Universal RiboClone cDNA synthesis system (Promega), UAP (SEQ ID NO: 7) and poly- (dT) ₁₇ The mRNA obtained in Reference Example 2 was reverse transcribed to obtain a double stranded cDNA template. The obtained double-stranded cDNA was used as a template and PCR was performed using a nested primer IPF (SEQ ID NO: 9) and a UAP (SEQ ID NO: 7). At this time, the PCR amplification number was 35 times, and each amplification step was performed in the order of denaturation process 1 minute at 94 ° C, annealing process 1 minute at 50 ° C, and extension process 2 minutes at 72 ° C.

In addition, in order to obtain a single stranded cDNA to be used for 5'-RACE, using the same RiboClone cDNA synthesis system and poly- (dT) ₁₅ primer, the mRNA obtained in Reference Example 2 was reverse transcribed to obtain a single stranded cDNA, followed by the single stranded cDNA. The 5'-NV primer (SEQ ID NO: 10) was linked to obtain a cDNA template to be used for 5'-RACE. At this time, the 3 'end of the 5'-NV primer was blocked by amination (amination), and the 5' end was used by phosphorylation. 5'-RACE was a template of the obtained cDNA and PCR was performed in the same manner as in 3'-RACE using the internal primers IPB (SEQ ID NO: 11) and 5'-3 (SEQ ID NO: 12).

The obtained 3'-RACE product and 5'-RACE product were electrophoresed on 1.0% agarose gel, and the results are shown in FIGS. 5A and 5B, respectively. In Figures 5a and 5b the first row is the molecular weight label and the second row is the 3'-RACE and 5'-RACE products, respectively. In addition, the base sequences of the 3'-RACE and 5'-RACE products were determined.

As a result, the 3'-RACE product is 1,291 bp in size and the 5'-RACE product is 878 bp in size, and the nucleotide sequences of these products overlap by 31 nucleotides, and the base of the partial gene (p76) obtained in step 1 of Example 1 is obtained. All sequences were included. The base sequence information of the 3'-RACE product and the 5'-RACE product was synthesized to obtain a 2,106 bp full-length cDNA sequence. The result is shown in SEQ ID NO: 1. As shown in SEQ ID NO: 1, the conserved nucleotide sequence GAAAATCATGT for translation initiation in plants is in the region from 27 to 37, has an open reading frame of 1641 bp, an end codon on this frame, and a 33 bp size. Has a 5'-untranslated terminus and a 3'-untranslated terminus of 432 bp. Also, the typical polyadenine signal AATAAT / A appears twice in the region from 1778 to 1786. Primer F7, 17 nucleotides in size, differed only by 3 bases from that region of the genome, and 22 nucleotides primer B9 differed only by 4 bases from the corresponding region of the genome. The amino acid sequence of the protein inferred from the open reading frame is as shown in SEQ ID NO: 2, and consists of 546 amino acid residues as shown in SEQ ID NO: 2, a pI value of 5.59 and a Mw value of 63.9 kDa. In particular, proteins do not contain plastid targeting sequences, suggesting that the enzymes encoded from these genes are widely distributed at the plasma sites (Bohlmann, J. et al., Proc. Natl. Acad. Sci USA 95, 4126-4133 (1998) ;... and McGarvey, DJ et al, Plant Cell 7, 1015-1026 (1995)).

This base sequence and amino acid sequence were registered under Gen. AJ251751 in the GenBank / EMBL database.

Comparing the amino acid sequence of the inferred protein with that of other sesquiterpene synthetases, it showed a very high agreement as shown in FIG. 6, in particular 36% with that of the tobacco 5-epi-aristoloken synthase. , 41% concordance with cotton (+)-δ-cardinene synthase.

In addition, the amino acid sequence of the inferred protein was compared with the case of two different terpene synthase epi-cedrol cyclase and (3R) -linalul synthase isolated from firefly, as shown in FIG. It showed a 51% agreement with cedrol cyclase and a low 27% agreement with (3R) -linalul synthase, one of the monoterpene synthetases.

Example 3: Cloning and Expression of Firefly Amopa-4,11-diene Synthase Gene

(Step 1) Cloning of Genes

To obtain the Amopa-4,11-diene synthase cDNA gene containing all of the open reading frames, based on the nucleotide sequence obtained in Example 2, a pair of primers START (SEQ ID NO: : 3) and STOP (SEQ ID NO: 4) were produced. Primers START and STOP were designed to include cleavage sites of restriction enzymes BamH I and EcoR I, respectively. The mRNA obtained in Reference Example 2 was used as a template, and PCR was performed using the primers START and STOP.

The obtained PCR product was electrophoresed, and the results are shown in FIG. In Figure 7, column 1 is the molecular weight label and column 2 is the PCR product. The resulting PCR product is 1641 bp in size and contains full length cDNA.

This PCR product was cloned into BamH I and EcoR I positions of the expression vector pET-5a (Promega) and the resulting expression vector was named pkcs12. The expression vector pkcs12 was transformed into E. coli BL21 (DE3) pLysS cells (Promega). The obtained transformed Escherichia coli BL31 (DE3) / pLysS was deposited in the gene bank affiliated with the Biotechnology Research Institute with accession number KCTC 0739BP dated February 18, 2000.

(Step 2) Expression and Analysis of Full Length Genes

The transformed E. coli kcs 12 obtained in step 1 was incubated overnight in LB medium containing 100 μg / ml ampicillin. 0.5 ml of the culture was inoculated into 50 ml of the same fresh medium and incubated at 37 ° C. with vigorous stirring until absorbance (A ₆₀₀ ) reached 0.5, followed by addition of 1 mM IPTG and at 37 ° C. Incubated for 3 hours.

The cultures were centrifuged at 1,000 x g for 15 minutes at 4 ° C to precipitate the cells, followed by 0.5 ml of lysis buffer (50 mM Tris / pH 8.0, 1 mM EDTA, 100 mM NaCl, and 1 mM DTT). ) Was added and lysed, and the cells were disrupted using ultrasound. Protein concentrations were determined using bovine plasma albumin as a standard according to the Bradford method (Bradford, M. Anal. Biochem. 72 , 248-254 (1976)).

The resulting cell lysate was combined with the reaction solution (200 mM Tris / pH 7.5, 40 mM MgCl ₂ , 0.2 μM [1- ³ H] FDP (108 mCi / mmol. NEN, Boston, Mass.) And 36 μM FDP (Sigma)). The reaction was carried out at 35 ° C. for 1 hour. The obtained reaction product was extracted with hexane and the hexane layer was passed through a small silica gel column made of Pasteur pipette to remove farnesol produced by FDP or phosphatase activity. Radioactivity present in the hexane layer was measured using a scintillation counter to determine enzymatic activity. Enzyme activity is expressed as nmol product / h / mg protein. SDS-PAGE was performed on a 10% gel following Lamley's method (Laemmli, UK Nature 227, 680-685 (1970)) and stained with Coomassie Brilliant Blue. At this time, as a control, the culture and enzyme activity were measured in the same manner using E. coli untransformed.

As a result, a 61 kDa protein band was identified in the insoluble fraction of transformed E. coli. In addition, the expressed protein from the transformed E. coli kcs 12 whereas which reacts with the amorphous FDP generated wave -4,11- diene in the control was not amorphous wave -4,11- diene detected. From these results, it was confirmed that the gene obtained in step 1 is a gene of amopa-4,11-diene synthase.

(Step 3) Analysis of Gene Expressions

In order to identify the chemical identity of the reaction product by the enzyme expressed in step 2, after the large-scale cultivation in 50 ml of the medium, the crude extract was extracted, and the crude crude extract was reacted with 36 M FDP at 37 ° C. overnight. Hexane extraction and silica gel columns were passed through the same method in 3. The obtained eluate was concentrated and then gas chromatography-mass spectroscopy (GM-MS) analysis was performed as follows.

HP5890 series II system (Hewlett Packard, Germany) with AX505WA mass spectrometer (JEOL, Japan) running in electron impact mode

Injector Temperature: 250 ℃

Flow rate: 2 ml / min

Split ratio: 5: 1

Oven temperature: 100 ° C. for 2 minutes, then elevated to 150 ° C. at a rate of 5 ° C./min, then elevated to 250 ° C. at a rate of 10 ° C./min, followed by 2 minutes at 250 ° C.

Carrier gas: N ₂

Column: DB-5 column (30m × 0.25㎛ × 0.25mm)

At this time, Amopa-4,11-diene synthesized in Reference Example 3 was used as a standard, and the reaction product was identified by comparing with an elapsed time and a mass spectrum of the standard.

8A is a gas chromatography spectrum of an enzyme reactant, FIG. 8B is a mass spectrum of an enzyme reactant obtained at 9.3 minutes of retention time, and 8c is a standard amopa-4,11-diene obtained from Reference Example 3. Time: 9.3 min). As shown in FIGS. 8A to 8C, the enzyme reactant showed a retention time of 9.3 minutes and a mass unit of m / z 204, which is also the result of atemisinin synthesized in Reference Example 3. Matches. Selective ion monitoring at m / z 204 showed a single peak representing retention time. It can be seen that the enzyme expressed therefrom is a synthetic enzyme of amopa-4,11-diene. As in step 2, the enzyme activity was 0.212 nmol / h / mg protein as a result of reaction with ³ H-FDP.

The enzyme of the present invention is an amopa-4,11-diene synthetase which mediates the synthesis reaction of amopa-4,11-diene in the biosynthetic pathway of atemisinin, which is useful as an antimalarial agent. Plants can be useful for mass production of atemisinin by increasing the biosynthetic efficiency of atemisinin using metabolic engineering.

Claims (8)

Amopha-4,11-diene synthase of Artemisia annua , having the amino acid sequence of SEQ ID NO: 2. A gene encoding the Amopa-4,11-diene synthase of claim 1. The method of claim 2, An amopa-4,11-diene synthase gene of Artemisia annua having a nucleotide sequence of SEQ ID NO: 1 or more than 80% homology thereto. An expression vector comprising the gene of claim 2 or 3. The method of claim 4, wherein Expression vector pkcs12. E. coli transformed with the expression vector of claim 4. The method of claim 6, Escherichia coli BL21 (DE3) / pLysS transformed with the expression vector pkcs12 (Accession No. KCTC0739BP). Plant transformed with the expression vector of claim 4.