JP4606604B2 - Isomultiphlorenol synthase and the enzyme gene - Google Patents

Description

【図面の簡単な説明】
【図１】標準品イソマルチフロレノールとpYES2-OSC3形質転換酵母抽出物のHPLCクロマトグラムおよびLC-MS/MSフラグメントを比較した図である。
【図２】CAS1、OSC2、IMS1のアミノ酸配列の比較した図である。相同なアミノ酸を白文字反転で示した。
【図３】高等植物由来オキシドスクワレン環化酵素のアミノ酸配列から作成した系統樹である。方法：UPGMA (Genetyx software）。bAS,β-アミリンシンターゼ；LUS,ルペオールシンターゼ；IMS,イソマルチフロレノールシンターゼ；CAS,シクロアルテノールシンターゼ；CDS,ククルビタジエノールシンターゼ；OSC,オキシドスクワレン環化酵素。
【図４】CAS1,OSC2,IMS1の制限酵素サイトおよびDIGラベルRNAプローブ部位を示す図である。
【図５】細胞増殖およびブリオノール酸生産のタイムコースを示す図である。
【図６】ヘチマ細胞培養時のIMS1 mRNA、CAS1 mRNA、OSC2 mRNA量の変化を示した電気泳動図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to isomultiflorenol synthase which is a biosynthetic enzyme of isomultiflorenol, DNA encoding the enzyme, and isomultiflorenol and / or a method for producing various terpenoids biosynthesized from this isomultiflorenol. .
[0002]
[Prior art]
Bryonolic acid is a type of plant secondary metabolite classified as a triterpenoid. It has been found that it is contained in the roots of cucurbitaceae plants such as watermelon (Citrullus lanatus) and Bryonia dioica (Bryonia dioica). Bryonolic acid and its derivatives have a structure different from that of other plant-derived triterpenoids, and are expected to have a physiological activity not found elsewhere, and are also attracting attention as raw materials for pharmaceuticals.
[0003]
However, when producing bryonolic acid and its derivatives, when a plant body is used as a raw material, it costs a great deal of cultivation and its productivity must be extremely low. Therefore, studies have been conducted on the production of triterpenoids by cell culture of loofah, and it has been reported that loofah callus produces bryonolic acid and its derivatives [Plant and Cell Physiology 25,1571-1574 (1984); Organic Magnetic Resonance 22,93-100 (1984)].
Several studies have also been conducted on the biosynthetic pathway of bryonolic acid, and isomultiflorenol produced by cyclization of 2,3-oxide squalene produced by the mevalonic acid pathway is considered to be a biosynthetic precursor. It is considered that bryonolic acid is produced by oxidation of the produced isomultiphlorenol. It is said that more than 90 different types of triterpene skeleton exist in nature, and several triterpene synthases such as β-amylin synthase and lupeol synthase that cyclize 2,3-oxide squalene are known. [Eur. J. Biochem. 256, 238-244 (1998); Eur. J. Biochem. 266, 302-307 (1999)]. However, there has been no research on isomultiphlorenol synthase that catalyzes the production of isomultiphlorenol, which is a precursor of brionolic acid, from 2,3-oxide squalene.
[0004]
[Problems to be solved by the invention]
The present invention clones DNA encoding isomultiphlorenol synthase, determines its nucleotide sequence, and further encodes isomultiphlorenol synthase and the enzyme using a cell expressing the cloned recombinant DNA. The object is to produce DNA and isomultiflorenol and / or various terpenoids biosynthesized from this isomultiflorenol.
[0005]
[Means for Solving the Problems]
As a result of diligent research, the present inventors screened cDNA of loofah cultured cells using licorice β-amylin synthase cDNA as a probe, and cloned one kind of oxidosqualene cyclase cDNA. The full-length ORF of this cDNA was incorporated into a yeast expression vector, transformed into yeast lacking lanosterol synthase, and as a result of functional analysis, production of isomultiflorenol that was not originally biosynthesized in the yeast was observed. It was confirmed that the cDNA encodes isomultiphlorenol synthase.
[0006]
That is, the first aspect of the present invention is a polypeptide having the enzyme activity of isomultiphlorenol synthase and / or a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. The second aspect is a DNA comprising a nucleotide sequence encoding a polypeptide having isomultiphlorenol synthase enzyme activity and / or a DNA comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. And a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2. A third aspect of the present invention is a vector containing the DNA described in the first aspect. A fourth aspect of the present invention is a cell transformed with the above vector. Moreover, the 5th aspect of this invention is a method of manufacturing a plant secondary metabolite using the said transformed cell. This plant secondary metabolite may be a terpenoid biosynthesized from isomultiflorenol.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The present inventors prepared a cDNA library from cultured hechima cells with high productivity of bryonolic acid, screened using licorice β-amylin synthase (GgbAS1) cDNA as a probe, and one kind of oxidosqualene cyclase cDNA. Was cloned. The full-length ORF of the resulting cDNA was incorporated into a yeast expression vector (pYES2), transformed into yeast lacking lanosterol synthase (GIL77), and functional analysis revealed that this cDNA encodes isomultiflorenol synthase. Was confirmed. The amino acid sequence of the newly obtained isomultiphlorenol synthase was 67% homologous to the licorice β-amylin synthase used as a probe, but some of the known clones have higher homology. I was not able to admit.
[0008]
The isomultiphlorenol synthase according to the present invention refers to an enzyme that catalyzes a reaction for producing isomultiphlorenol from 2,3-oxide squalene, and its origin is not particularly limited, but preferably is derived from a plant. Examples of such a material include those described in the following examples.
[0009]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
Seeds of creation and culture loofah Example 1 loofah cell line (Luffa cylindrica) was sterilized, cut sections of the cotyledons and stems from seedlings germinated under aseptic conditions, which the 2,4-D and 10 ^-6 M It was transplanted to an agar medium of Rinsmeier Skoog (LS) added as described above and cultured in the dark at 27 ° C. After 5-7 weeks, the callus of loofah obtained was transferred to LS liquid medium supplemented with 2,4-D at 10 ^-7 M, and cultured at 27 ° C in the dark for 3 years. After subculturing as described above, the cells were subcultured in a liquid medium of LS in which 2,4-D was changed to NAA at 27 ° C. in the dark.
[0010]
Example 2 Preparation of cDNA Library Total RNA was extracted by the guanidine thiocyanate / hot phenol method from the thus obtained 10-day cultured hechima cells, and an mRNA purification kit (Pharmacia) was used. Poly (A) ⁺ RNA was purified according to the instructions in A purified poly (A) ⁺ RNA was used to prepare a loofah cDNA library using a ZAP-cDNA synthesis kit (Stratagene) according to the manufacturer's instructions.
[0011]
Example 3 Preparation of Probe A 1227-bp dioxygenin (DIG) -labeled DNA probe corresponding to amino acid residues 353-729 of GgbAS1β-amylin synthase derived from G.glabra was used as a probe for cloning of isomultiphlorenol synthase. . This probe was prepared by the PCR method using GgbAS1 as a template and using the two primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, Taq DNA polymerase, and DIG-dNTP mixture according to the manufacturer's instructions.
[0012]
Example 4 Screening of cDNA Library As a result of screening the loofah cDNA library under low stringency conditions using the DIG-labeled probe thus prepared, one positive clone was obtained from 200,000 plaques of the cDNA library, Subcloned into pBluescript SK (-) vector. As a result of sequencing this clone for both strands, this clone was assumed to be a translation product polypeptide consisting of 2551 bases comprising an open reading frame (ORF) consisting of 2277 bases and a molecular weight of 87.7 kD and consisting of 759 amino acids. It is pointed out that the cDNA sequence is the active site of the QW motif [Trends Biochem. Sci. 19, 157-158 (1994)] and oxidosqualene cyclase that are repeatedly observed in known oxidosqualene cyclase sequences. DCTAE motif [J. Biol. Chem. 269, 802-804 (1994)] existed.
[0013]
Example 5 Function Confirmation In order to clarify the function of the cDNA obtained in Example 4, the ORF of the obtained cDNA was amplified by PCR and incorporated into the expression plasmid pYES2 (Invitrogen) in yeast. PCR was performed using the obtained cDNA as a template, using primers of the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, 94 ° C for 40 seconds, 50 ° C for 40 seconds, and 72 ° C for 2 minutes. The extension reaction was 1 cycle, and 25 cycles were allowed to react. The obtained PCR product was treated with restriction enzymes KpnI and XbaI and incorporated into the KpnI and XbaI sites of the expression plasmid pYES2, which was designated as pYES2-LcOSC3. As a result, LcOSC3 is introduced in the sense direction downstream of the GAL1 promoter. Further, the obtained pYES2-LcOSC3 plasmid was introduced into the yeast mutant GIL77, which is a lanosterol synthetase-deficient strain, by the lithium acetate method. Culture conditions, expression induction by lactose, and preparation of triterpene monoalcohol fraction were carried out in the same manner as described in Kushiro, T. et al. [Eur. J. Biochem. 256, 238-244 ( 1998)]. After induction of expression by galactose, the product was extracted from the transformed yeast, purified by TLC, and analyzed for the triterpene monoalcohol fraction by LC-MS / MS. LC-MS / MS was measured by LCQ (Thermo Quest) under the following HPLC conditions.
(HPLC conditions)
Column: SUPER-ODS (diameter: 4.6mm, length: 200mm; manufactured by Tosoh)
Solvent system: 95% CH ₃ CN aq., Flow rate: 1ml / min, Column temp .: 40 ℃, Detection: UV205nm, Retention time for isomultiflorenol: 23.9min.
The peak obtained from the triterpene monoalcohol fraction identified by LC-MS / MS based on comparison with the MS / MS fragment pattern of the standard isomultiflorenol is isomultiflorenol, and the compound obtained from this fraction is It was only isomultiflorenol (FIG. 1). As a result of MS and MS / MS analysis of isomultiflorenol, MS m / z 409 [M + HH ₂ O] ⁺ , MS / MS (precursor ion at m / z 409) 353 (10%), 313 ( 22%), 299 (57%), 245 (65%), 231 (91%), 217 (100%), 191 (71%) were obtained. Therefore, it was concluded that this cDNA encodes isomultiphlorenol synthase (IMS1), which is one of the oxide squalene cyclases obtained from higher plants.
[0014]
Three types of oxide squalene cyclase, cycloartenol synthase (CAS1) [Plant Physiol. 121,1384 (1999)], oxide squalene cyclase (OSC2) [Plant Physiol. 122,1457 (Unknown function) 2000)], comparing the amino acid sequence of isomultiphlorenol synthase (IMS1), the amino acid sequence of IMS1 showed 51% homology with CAS1 and 50% with OSC2 (FIG. 2). Furthermore, a phylogenetic tree was created from the amino acid sequence of IMS1 and the amino acid sequence of oxidosqualene cyclase cloned from a higher plant (FIG. 3). IMS1 showed 67-66% homology with β-amylin synthase and 61-57% homology with lupeol synthase. Since the amino acid sequence of IMS1 is 67-57% less homologous with other triterpene synthases, isomultiphlorenol synthase is classified as a new class of triterpene synthases found in cucurbitaceae plants.
[0015]
Example 6 Northern blot analysis
The DIG-labeled RNA probe was prepared from BamHI-treated CAS1, EcoRI-treated OSC2, and BamHI-treated IMS1 using T7 RNA polymerase and DIG RNA Labeling Mix (Roche Diagnostic) according to the manufacturer's instructions. Total RNA was extracted using Extract-A-Plant ^™ RNA Isolation Kit (Clontech). 5 μg of total RNA per lane was separated on a 1% agarose gel containing formaldehyde and blotted onto a positively charged nylon membrane (Roche Diagnostic). RNA on the membrane in hybridization buffer containing 50 mM Na-Pi buffer (pH 7.0), 50% formamide, 0.1% N-lauryl sarcosine, 7% SDS, 5xSSC, 1 mg / ml tRNA (yeast), blocking reagent Hybridization was performed using a DIG-labeled RNA probe at 50 ° C., and detection was performed using DIG Nucleic Acid Detection Kit Mix (Roche Diagnostic) according to the manufacturer's instructions (FIG. 4).
[0016]
Example 7 Time course of cell growth and bryonolic acid production The time course of cell growth and bryonolic acid production on days 4, 8, 12, 16, 20, and 24 after the start of culture was shown (FIG. 5). The qualitative analysis of bryonolic acid was performed by the following method. After completion of the culture, the cells were freeze-dried, and 50 mg of the obtained freeze-dried cells were extracted twice with stirring at 60 ° C. for 1 hour using 2 ml of ethyl acetate. After adding 1 mg of cholesterol as an internal standard to the obtained extract and flushing off the solvent using a rotary evaporator, the dried sample was added to pyridine 20 (l, N, O-bistrimethylsilylacetamide 20 (l mixed solvent). It was redissolved and incubated for 1 hour at 50 ° C. After the reaction, liquid 1 (l was analyzed by gas chromatography under the conditions shown below, and bryonolic acid was quantified. Calculated from the peak area of the derivative.
(Gas chromatography analysis conditions)
Column: Ultra Alloy ⁺ -17 capillary column (15m x 0.5mm id, film thickness: 1 (l, manufactured by Frontier Lab Ltd.), Column temp .: 295 ° C (isothermal), injector and detector temp .: 320 ° C, Carrier gas: He 3.5ml / min.
Production of bryonolic acid by loofah cultured cells rises later in the logarithmic growth phase. Comparison of the time course of bryonolic acid production with the mRNA level of IMS1, the mRNA level of CAS1, and the mRNA level of OSC2, the mRNA levels of the three oxidosqualene cyclases were regulated differently in loofah cells ( FIG. 6). Isomultiphlorenol synthase mRNA levels are highest on the 16th and 20th days of culture, and at this point the bryonolic acid synthesis activity is very high. However, on days 4, 8, and 24, isomultiphlorenol synthase mRNA levels were low, and bryonolic acid production was not so noticeable at this point. On the other hand, the cycloartenol synthase mRNA level is high on the 4th and 8th days, and at this point, the proliferation of the cells is remarkably observed. And in the late phase of the logarithmic growth phase, the mRNA level is lower. In addition, the mRNA level of OSC2, which is presumed to be oxidosqualene cyclase, is high on the 20th and 24th days, and at this point, the cells are already in the stable phase.
[0017]
【The invention's effect】
As described above, the first successful cloning of DNA encoding isomultiflorenol synthase, and further using the cells expressing the cloned recombinant DNA, isomultiflorenol synthase, DNA encoding the enzyme and It is possible to produce isomultiflorenol and / or various terpenoids biosynthesized from this isomultiflorenol.
[0018]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram comparing HPLC chromatograms and LC-MS / MS fragments of standard isomultiflorenol and pYES2-OSC3 transformed yeast extract.
FIG. 2 is a comparison of the amino acid sequences of CAS1, OSC2, and IMS1. Homologous amino acids are shown in white letters reversed.
FIG. 3 is a phylogenetic tree created from amino acid sequences of higher plant-derived oxide squalene cyclase. Method: UPGMA (Genetyx software). LAS, lupeol synthase; IMS, isomultiphlorenol synthase; CAS, cycloartenol synthase; CDS, cucurbitadienol synthase; OSC, oxide squalene cyclase.
FIG. 4 is a view showing restriction enzyme sites and DIG-labeled RNA probe sites of CAS1, OSC2, and IMS1.
FIG. 5 is a diagram showing a time course of cell growth and bryonolic acid production.
FIG. 6 is an electrophoretogram showing changes in IMS1 mRNA, CAS1 mRNA, and OSC2 mRNA levels during culture of loofah cells.

Claims (11)

Isomaltitol flow have a les synthase activity, and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. Polypeptide comprising an amino acid sequence shown in SEQ ID NO: 1. A DNA comprising a nucleotide sequence encoding the polypeptide of claim 1. DNA consisting of a nucleotide sequence encoding a polypeptide of claim 2 wherein. DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1. DNA consisting of 130 to 2409 th nucleotide sequence of SEQ ID No. 1. A vector comprising the DNA according to any one of claims 3 to 6. A cell transformed with the vector according to claim 7. A method for producing a polypeptide having the enzyme activity of isomultiphlorenol synthase using the cell according to claim 8. A method for producing bryonolic acid using the cell according to claim 8. The method according to claim 10, wherein the brionolic acid is a terpenoid biosynthesized from isomultiflorenol.

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