• 제목/요약/키워드: sesquiterpene cyclase

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Hyoscyamus muticus의 모상근배양으로부터 Sesquiterpene 화합물의 Intergration 추출시 Sesquiterpene Cyclase의 유도 (Induction of Sesquiterpene Cyclase During Integrated Extraction of Sesquiterpenes from Hairy Root Cultures of Hyoscyamus muticus)

  • BACK, Kyoungwhan;SHIN, Dong Hyun;KIM, Kil Ung;De HAAS, Cynthia R.;CHAPPELL, Joseph;CURTIS Wayne R.
    • 식물조직배양학회지
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    • 제24권5호
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    • pp.273-277
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    • 1997
  • Rhizoctonia solani 추출물(elicitor)을 Hyoscyamus muticus 모상근배양에 처리하였을 때 sesquiterpene cyclase (SC)가 유도됨과 동시에 sesquiterpene화합물이 합성되어 배양액속에 축적하는 것으로 나타났다. 24시간 배양기간 동안 배양액으로부터 sesquiterpene을 추출ㆍ분리하여 배양할 경우, 추출ㆍ분리하지 않은 모상근배양보다 거의 두배의 함량이 생성되었다. Cyclase monoclonal antibody을 이용하여 immunoblot을 시도한 결과, 추출한 모상근배양과 추출하지 많은 모상근배양에서의 SC 절대량은 동일한 것으로 나타났으며, 효소활성도 현저한 차이가 없는 것으로 측정되었다. 이러한 결과는 terpenoid pathway에서 sesquiterpene 생합성이 이용가능한 기질의 량에 의해 조절되는 것으로 사료되며, feedback 조절은 sesquiterpene cyclase 효소에 앞서 일어나는 것으로 추정된다.

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담배 (Nicotiana tabacum L.) 현탁배양세포의 Elicitor 유도성 5-epi-Aristolochene Hydroxylase (Elicitor-Inducible 5-epi-Aristolochene Hydroxylase in Suspension Cultures of Tobacco (Nicotiana tabacum L.))

  • KWON, Soon-Tae;CHAPPELL, Joseph
    • 식물조직배양학회지
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    • 제25권3호
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    • pp.141-146
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    • 1998
  • [$^3$H] 5-epi-aristolochene (5-EAS)를 담배 현탁배양세포에 투여하여 elicitor에 의해 유도된 세포가 생합성하여 배지로 방출하는 [$^3$H]-capsidiol의 량을 측정함으로써 5-EAS hydroxylase의 활성을 검정하였고, 이 반응의 전 단계에 관여하는 효소인 sesquiterpene cyclase의 발현특성과 비교하였다. 5-EAS hydroxylase는 정상세포에는 전혀 활성을 보이지 않으나 elicitor로써 cellulase를 처리한 세포는 9시간 후부터 유도를 시작하여 18시간 후에 최대 활성을 보였는데 동일한 세포내에서 유도되는 cyclase와 유사한 패턴을 보였다. Cyt P450계 효소의 특이적 억제제로 알려진 ancymidol과 ketoconazole에 의해 5-EAS hydroxylase의 활성은 강한 억제를 보인 반면 cyclase의 활성은 억제를 보이지 않아 5-EAS hydroxylase가 P450계 효소임이 시사되었다.

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Molecular Cloning and Characterization of Sesquiterpene Cyclase cDNAs from Pepper Plant Infected with Phytophthora capsici

  • Kim, Jong-Bum;Lee, Sung-Gon;Ha, Sun-Hwa;Lee, Myung-Chul;Ye, Wan-Hye;Lee, Jang-Yong;Lee, Shin-Woo;Kim, Jung-Bong;Cho, Kang-Jin;Hwang, Young-Soo
    • Journal of Applied Biological Chemistry
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    • 제44권2호
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    • pp.59-64
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    • 2001
  • Pepper plants (Nogkwang, 60-day old) were inoculated with Phytophthora capsici to induce sesquiterpene cyclase associated with the biosynthesis of phytoalexin (capsidiol), a substance related to the defense against pathogens in plants. One day after inoculation, mRNA was isolated from the root, cDNA synthesized, and a library constructed in a ZAP express XR vector. The efficiency was $2{\times}10^6pfu/{\mu}g$. Sesquiterpene cyclase cDNA from Hyoscyamus muticus was labeled with $^{32}P$ and used as a probe for screening the cDNA library. After the third screening, 25 positive clones were selected. Through restrictive digestion and DNA gel-blot analysis, six different cyclase gene expressions were identified. PSC1B sequences of the six clones were determined, which were 1966 base pairs encoded 556 amino acids with an expected molecular weight of 63.8 kDa. Response against the pathogen was different between the resistant and susceptible peppers. After the infection of the pathogen, the expression of PSC genes continued in the resistant peppers while the plants were alive. The expression in the susceptible peppers lasted for only 4 days.

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고추의 sesquiterpene cyclase promoter-cinnamic acid 4-hydroxylase chimeric gene의 담배에서 발현 (Expression of Cinnamic Acid 4-Hydroxylase Chimeric Gene fused with Sesquiterpene Cyclase Promoter from Hot Pepper in Tobacco)

  • 이경민;윤용휘;김길웅;이인중;신동현
    • 생명과학회지
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    • 제14권4호
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    • pp.657-663
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    • 2004
  • 고추에서 클로닝된 sesquiterpene cyclase (CASC) promoter에 cinnamic acid 4-hydroxylase (C4H)유전자를 클로닝하여 담배에 형질전환하였다. 형질전환체의 분석은 PCR, Southern blot분석으로 하였으며, CASC promoter의 작동여부를 조사하기 위하여 GUS histochemical assay를 하였다. 스트레스에 반응한다고 알려진 CASC promoter를 C4H gene과 fusion하여 C4H activity를 조사하여 스트레스에 반응하는 정도를 조사하였다. 최종적인 형질전환체로 확인된 개체는 평균 16.4% 정도였으며, 각 promoter별로 약간의 편차를 보였다. C4H activity 조사시 스트레스를 주지 않았을 때 대조구가 비슷한 양상을 나타내었다. 스트레스를 주었을 때 대조구와 비교하여 promoter 3번은 1.3배 activity가 높아졌으며 4번은 1.4배, 6은 2배, cyc 600은 1.1배 높아졌다. 이런 결과로 보아 이 promoter들은 UV에 반응하는 promoter이며 앞으로 stress에 저항하는 유전자의 적당한 promoter로, 타작물에 적용하여 stress저항성 작물의 육성 시 매우 유용하게 사용될 것으로 사료된다.

Cloning and Functional Characterization of the Germacradienol Synthase (spterp13) from Streptomyces peucetius ATCC 27952

  • Ghimire, Gopal Prasad;Oh, Tae-Jin;Lee, Hei-Chan;Kim, Byung-Gee;Sohng, Jae-Kyung
    • Journal of Microbiology and Biotechnology
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    • 제18권7호
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    • pp.1216-1220
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    • 2008
  • Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The full-length recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the $Mg^{2+}$-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

방사선과 자외선에 대한 고추 식물체 및 배양세포의 생장반응과 Capsidiol 생합성 유전자의 발현 차이 (Differential Growth Response and Gene Expression in Relation to Capsidiol Biosynthesis of Red Pepper Plant and Cultured Cells by γ-Ray and UV Stress)

  • 안정희;김재성;정정학;오세명;권순태
    • Journal of Plant Biotechnology
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    • 제30권2호
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    • pp.201-206
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    • 2003
  • Differential responses of red pepper plant and cultured cells to enhanced ${\gamma}$-ray($^{60}$ Co) and ultraviolet(UV) stress were investigated. In seed treatment, 1 Gy of ${\gamma}$-ray increased seedling dry weight up to 19.1%, but 50 Gy treatment markedly ingibited seed germination and subsequent growth of seedling. UV treatment to seed did not change the germination ability of seeds and the growth of seedlings regardless of duration of UV treatment until 24 hrs. In case of UV treatment to seedlings, plant injury was seriously progressed even after the seedlings were returned to no UV condition, and eventually all the leaves showed chlorosis by the stress. However, progress of plant injury by ${\gamma}$-ray stress slower than that caused by UV stress, and even at the high dose of ${\gamma}$-ray 50 Gy, did not caused the cholrosis of stressed plant leaf. Amount of electrolytes leakage from plant leaf by UV treatment for 24hrs was increased up to 28.8 folds in comparison with untreated control, whereas that of 50 Gy of ${\gamma}$-ray was increased only 1.2 folds. UV stress induced the production of capsidiol, antimicrobial phytoalexin, by activation of gene expression involved in capsidiol biosynthesis, such as sesquiterpene cyclase and cyclase and cytochrome P450 hydroxylase in the leaf and cultured cell, but ${\gamma}$-ray stress induced neither the production of capsidiol nor expression of the genes.

Molecular Cloning of a Partial Cadinane Synthase Gene from Artemisia annua

  • Song, Seung-hwan;Chang, Yung-jin;Kim, Jeong-gu;Kim, Soo-Un
    • 한국응용약물학회:학술대회논문집
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    • 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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    • pp.121-121
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    • 1998
  • Artemisia annua, an indigenous plant in Korea, contains a clinically important potent antimalarial principle, artemisinin. Artemisinin is a cadinane-type sesquiterpene endoperoxide. Cadinane synthase catalyzes the first committed step in artemisinin biosynthesis by cyclizing farnesyldiphosphate. In hopes of finding a cadinane synthase gene involved in artemisinin biosynthesis, oligonucleotides were synthesized on. the basis of the consensus nucleotide sequences and Nco I restriction sites for convenience in cloning. Specifically, nucleotide sequences of two highly conserved regions were deduced from the genes of similar function of Hyoscyamus muticus, Nicotiana tabacum, Abies grandis, Lycopersicon esculentum, and Gossypium hirsutum to construct a set of primers for polymerase chain reation (PCR). A 184 bp fragment was found to be amplified by PCR, and subsequently cloned. The gene revealed 62.8% identity in nucleotide and 55.6% in amino acid sequence to correspondent gene of N. tabacum. The gene was different from another sesquiterpene cyclase gene of A. annua, germacranadiene synthase gene, recently reported by Mercke and Bordelius (1998).

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2016년도 춘계학술대회 및 임시총회
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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