• Journal of Applied Biological Chemistry
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• 제42권2호
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• pp.92-96
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• 1999

To study the regulatory mechanism of isoprenoid (carotenoid) biosynthesis, we have compared the expression patterns of nine isoprenoid biosynthetic genes in Korean red pepper (Capsicum. annuum cv. NocKaung). The expression of geranylgeranyl pyrophosphate synthase gene was initially induced at early ripening stage (I1) and was rather slightly decreased during pepper fruit ripening. The ex-pression of phytoene synthase gene was strongly induced at semi-ripening stage (I2) and the phytoene desaturase transcript was maximally induced at the fully ripened stage (R). Our results suggest that genes encoding two 3-hydroxy-3-methylglutaryl-CoA reductase isozymes (HMGR1 and HMGR2) and farnesyl pyrophosphate synthase might be not so critical in pepper carotenoid biosynthesis but three genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase were induced in a sequential manner and coordinately regulated during the ripening of pepper fruit.

• 생명과학회지
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• 제18권12호
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• pp.1764-1770
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• 2008

이 연구의 목적은 생물공학적으로 이소프레노이드 생합성 유전자를 클로닝하여 이들을 형질전환시킨 대장균을 제조하여 이들을 숙주로 사용하여 아스타잔틴의 생산을 증가시키는 것이다. 본 연구진은 선행연구에서 Paracoccus haeundaensis로부터 아스타잔틴 생산에 관여하는 6개의 아스타잔틴 생합성 유전자군을 보고하였고, 이들 유전자들을 발현 벡타(pCR-XL-TOPO-Crt)에 재조합한 후 이 벡터를 대장균에 형질 전환시켜서 건조중량으로 400 ${\mu}g$/g의 아스타잔틴을 생산하였다. 아스타잔틴의 생산성을 증가시키기 위해서 대장균으로부터 이소프레노이드 생합성 경로에 관여하는 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), isopentenyl (IPP) diphossphate isomerase (idi) 유전자들을 클로닝하였고, 이들 유전자를 (pCR-XL-TOPOCrt-full)와 같이 대장균에 각각 공발현시켰다. idi 유전자와 아스타잔틴 생산에 관여하는 아스타잔틴 생합성 유전자군이 함께 형질 전환된 BL21(DE3) Codon Plus RIL 대장균를 배양하였을때, 건조중량으로 1,200 ${\mu}g$/g의 아스타잔틴을 생산하였다. 따라서 본 연구 결과, 이소프레노이드 생합성 유전자와 아스타잔틴 생합성 유전자군을 공발현 시킬 때 아스타잔틴의 생산이 3배 증가하였다.

• 생명과학회지
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• 제13권6호
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• pp.933-942
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• 2003

Coenzyme Q은 긴 isoprenoid 사슬을 갖는 quinone의 유도체이다. Coenzyme Q는 진핵생명체의 미토콘드리아의 내막과 원핵생명체의 세포막에 위치하는 전자전달계에 존재하는 지용성 물질이며, 또한 항산화제로의 기능도 갖는다. Coenzyme Q는 Saccharomyces cerevisiae의 호기적 성장에 필수적이며, coq 돌연변이체는 발효가 불가능한 탄소 원에서의 성장이 불가능하다. S. cerevisiae의 $coq^7$p 효소들과 유사성을 나타내는 단백질을 암호화하는 Neurcspora crassa cDNA를 효모의 발현 벡터에 삽입하였다. N. crassa COQ7의 예상 서열은 S. cerevisiae의 효소와 58% homology를 보였다. N. crassa $coq^{-7}$ 유전자의 S. cerevisiae $coq^7$ 형질전환체는 야생형 균주와 유사한 성장률을 보였다. 형질전환 균주들은 발효가 불가능한 탄소원인 글리세롤을 유일한 탄소원으로 배양하였을 경우에도 정상적인 성장을 나타냈다. 또한 불포화지방산인 linolenic acid를 성장 배지에 첨가하여도 야생형 균주와 유사한 생존율이 관찰되었다.

• Journal of Plant Biotechnology
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• 제39권3호
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• pp.189-195
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• 2012

In plants, the fifth step of the plastidial 2-Cmethyl-D-erythritol 4-phosphate (MEP) pathway is catalyzed by 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECP; EC: 4. 6. 1. 12), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 823 bp Brassica rapa MECP (Brmecp) cDNA encoding a deduced polypeptide of 230 amino acid residues. Transcription levels of Brmecp were two-fold higher in petal compared to leaves. In addition, Brmecp expression in cabbage seedlings treated with ABA, $H_2O_2$ and drought was higher than control seedlings. These results were consistent with changes in chlorophyll contents in transgenic Arabidopsis. Thus, the Brmecp may contribute to the production of primary (chlorophylls and carotenoids) isoprenoid end-products in chloroplasts.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.114-119
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• 2018

Astaxanthin is one of the major carotenoids used in pigment has a great economical value in pharmaceutical markets, feeding, nutraceutical and food industries. This study was to increase the production of astaxanthin by co-expression with transformed Escherichia coli using six genes involved in the non-mevalonate pathway. Involved in the non-mevalonate biosynthetic pathway of the strain Kocuria gwangalliensis were cloned dxs, ispC, ispD, ispE, ispF, ispG, ispH and idi genes in order to increase astaxanthin production from the transformed E. coli. And co-expression with the genes to compared the amount of astaxanthin production. This engineered E. coli, containing both the non-mevalonate pathway gene and the astaxanthin biosynthesis gene cluster, produced astaxanthin at $1,100{\mu}g/g$ DCW (dry cell weight), resulting in approximately three times the production of astaxanthin.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.145.3-146
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• 2003

Transgenic Eleutherococcus senticosus plants were prepared by introducing the genes for squalene synthase (SQS), hygromycin phosphotransferase (HPT) and green fluorescent Protein (GFP) through Agrobacterium-mediated transformation. The enzyme, SQS, represents a putative branch point in the isoprenoid pathway capable of diverting carbon flow specifically to the biosynthesis of phytosterol and oleanolic acid. The full SQS gene was isolated from P. ginseng roots. Early globular embryo clusters developed from embryogenic callus were used as the explant source. (omitted)

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 1995년도 Proceedings of special lectures on Molecular Biological Approaches to Plant Disease National Agricultural Science and Technology Institute Suwon, Korea
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• pp.55-75
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• 1995

Induction of HMG-Co A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, three classes of cDNAs for HMGR (hmg1, hmg2, and hmg3) were isolated from a potato tuber library. The potato cDNAs had extensive homology in open reading frames but had low homology in the 3'-untranslated regions. RNA gel blot analysis using gene-specific probes revealed that hmg1 is induced by wounding but wound induction is strongly suppressed by arachidonic acid or by inoculation with Phytophthora infestants. In contrast, hmg2 and hmg3 are slightly induced by wounding and strongly enhanced by arachidonic acid or inoculation. The induction and suppression of HMGR genes parallel the suppression of steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. Treatment of the tuber disks with a low concentration of methyl-jasmonate doubled the wound induced accumulation of hmg1 transcripts and steroid-glycoalkaloid accumulation, but did not affect the abundance of transcripts for hmg2 or hmg3 nor induce phytoalexins. High concentration of methyl-jasmonate suppressed hmg1 mRNA and steroid-glycoalkaloid accumulation, induced hmg3 mRNA, and did not elicit phytoalexins. Lipoxygenase inhibitors suppressed the accumulation of of hmg1 transcripts and steroid-glycoalkaloids, which were restored by exogeneous methyl-jasmonate. Methyl-jasmonate applied together with arachidonic acid enhanced the elicitor induced accumulation of sesquiterpenes and sustained steroid-glycoalkaloid levels with transcript levels for the various HMGR mRNAs equal to or greater than wound-only treatment. These results domonstrate that the consequences of wound- and pathogen-responses of plants are different at the levels of gene expression and associated secondary metabolism.

• Journal of Ginseng Research
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• 제34권2호
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• pp.98-103
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• 2010

The medicinal plant Panax ginseng (P. ginseng) contains various phytosterols and bioactive triterpene saponins (ginsenosides). Squalene synthase catalyzes the first committed step in ginsenoside biosynthesis. Transgenic plants of P. ginseng were generated by introducing the squalene synthase gene derived from P. ginseng. Adventitious roots of the transgenic ginseng grew best in B5 medium, and 2 g of inoculum secured an optimal growth rate. Two phytohormones, indolebutyric acid and 1-naphtalene acetic acid, increased root growth and decreased ginsenoside production. Treatment with two selected elicitors, chitosan and jasmonic acid, and a precursor of the isoprenoid pathway, mevalonic acid, enhanced ginsenoside production and retarded ginseng growth rate.

• Journal of Ginseng Research
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• 제34권1호
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• pp.17-22
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• 2010

Sesquiterpenes are found naturally in plants and insects as defensive agents or pheromones. They are produced in the cytosolic acetate/mevalonate pathway for isoprenoid biosynthesis. The inducible sesquiterpene synthases (STS), which are responsible for the transformation of the precursor farnesyl diphosphate, appear to generate very few olefinic products that are converted to biologically active metabolites. In this study, we isolated the STS gene from Panax ginseng C.A. Meyer, designated PgSTS, and investigated the correlation between its expression and various abiotic stresses using real-time PCR. PgSTS cDNA was observed to be 1,883 nucleotides long with an open reading frame of 1,707 bp, encoding a protein of 568 amino acids. The molecular mass of the mature protein was determined to be 65.5 kDa, with a predicted isoelectric point of 5.98. A GenBank BlastX search revealed the deduced amino acid sequence of PgSTS to be homologous to STS from other plants, with the highest similarity to an STS from Lycopersicon hirsutum (55% identity, 51% similarity). Real-time PCR analysis showed that different abiotic stresses triggered significant induction of PgSTS expression at different time points.

• BMB Reports
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• 제40권6호
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• pp.911-920
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• 2007

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-Derythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and $Mg^{2+}$. The turnover number of MtIspD is $3.4 s^{-1}$. The Km for MEP and CTP are 43 and $92{\mu}M$, respectively. Furthermore, MtIspD shows thermal instable above $50^{\circ}C$. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.