• 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.

• 식물조직배양학회지
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• 제24권5호
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• pp.279-284
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• 1997

Hyoscyamus muticus의 phytoalexin으로 알려진 solavetivone, lubimin, rishitin 생합성에 결정적 역할을 담당하는 isoprenoid pathway의 첫 분지효소인 vetispiradiene synthase의 유도특성과 solavetivone, lubimin phytoalexin 생합성과의 관련성을 구명하기 위하여 H. muticus 현탁세포배양 및 모상근배양에 Rhizoctonia solani 추출액을 elicitor로 처리하였다. Elicitor을 처리하지 않은 현탁세포 및 모상근배양에서는 효소활성, 효소단백질 및 solavetivone, lubimin등이 전혀 검출되지 않았으나, elicitor을 처리한 세포에서는 효소활성이 급속히 유도되어 처리 12시간 후에 최고의 활성을 보였으며 그 후 점점 감소하였다. 효소활성의 정도는 면역반응을 이용하여 측정한 효소 단백질의 함량과 밀접한 상관관계를 보였으며, phytoalexin 총 함량은 모상근배양에서 2배이상 높았다. 특히 solavetivone 및 lubimin 생합성은 현탁 및 모상근배양에서 서로 다른 특이성을 보였는데, 현탁배양에서는 lubimin을, 모상근배양에서는 solavetivone을 선택적으로 다량 생산하였다.

• 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)

• Applied Biological Chemistry
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• 제41권1호
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• pp.47-52
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• 1998

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 환원효소는 식물의 병원균 방어물질 (phytoalexin), 광합성 색소 (the phytol of chlorophyll), 성장 호르몬 (abscisic acid와 gibberellin 등) 및 스테롤 (phytosterol) 등의 생합성에 관여하는 주효소이다. 암조건 하에서 발아 후 4일째의 벼 유묘 microsome을 재료로 비이온성 detergent 인 Brij W-1 (final 0.4%)을 사용하여 가용화 시킨 후, DEAE-Sephadex A-50 크로마토그래피 칼럼과 hexaneagarose를 matrix로 기질인 HMG-CoA를 결합시켜 제조한 친화성 크로마토그래피 칼럼을 이용하여 세포막 결합효소인 HMG-CoA 환원효소를 정제하였다. 정제된 HMG-CoA 환원효소의 최종 회수율은 7.14% 였고, 분자량은 10% SDS-PAGE에서 55 kDa이였다 HMG-CoA 환원효소의 최적 반응 온도는 $37^{\circ}C$, 최적 반응 pH는 6.9였고, 기질인 HMG- CoA에 대한 HMG-CoA 환원효소의 $K_m$ 및 $V_{max}$ 값은 $180\;{\mu}M$과 107 pmol/mg, 수소공여체인 NADPH에 대한 $K_m$과 $V_{max}$값은 $810\;{\mu}M$과 32.1 pmol/min/mg이였다.

• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1206-1212
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• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.

• 한국식품영양학회지
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• 제23권4호
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• pp.526-530
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• 2010

Synechocystis sp. PCC6803의 type II Isopentenyl diphosphate isomerase gene(sll1556, Syidi2)의 특성을 살펴보기 위하여 ${\Delta}idi$인 E. coli $DH5{\alpha}$를 제작하고, 이 균주에서 cloning하고 발현시켰다. 라이코펜 합성 유전자들(crtE, crtB, and crtI)과 mevalonate pathway 유전자들(MvK1, MvK2, Mvd)를 함유한 ${\Delta}idi$ E. coli $DH5{\alpha}$ 균주를 mevalonate가 함유된 LB 배지에서 배양하면 mevalonate pathway 유전자들을 함유한 E. coli $DH5{\alpha}$균주는 mevalonate에 의해 생성된 isopentenyl diphosphate의 독성에 의해 매우 느린 성장을 보였다. 라이코펜 합성유전자들과 mevalonate 합성유전자들을 함유한 ${\Delta}idi$ E. coli $DH5{\alpha}$ 균주에 Syidi2를 도입한 결과, mevalonate가 함유된 LB배지에서 균체의 성장이 완전히 회복되었으며, 라이코펜이 합성되었음을 나타내는 붉은 균락이 형성되었다. 이에 따라, SyIdi1과 ECidi를 도입하여 비교한 결과, 라이코펜 합성 유전자들과 mevalonate pathway 유전자들을 함유한 ${\Delta}idi$ E. coli $DH5{\alpha}$ 균주 자체와 SyIdi1을 도입한 라이코펜 합성 유전자들과 mevalonate pathway 유전자들을 함유한 ${\Delta}idi$ E. coli $DH5{\alpha}$ 균주는 IPP의 독성에 의해 성장이 매우 느렸으나, SyIdi2, RSidi, HPidi, 및 ECidi를 함유하고 있는 ${\Delta}idi$ E. coli $DH5{\alpha}$ 균주는 균체의 성장과 라이코펜의 합성을 완전히 회복하였으며, 그중 가장 우수한 라이코펜 생합성 결과를 나타낸 것은 pSUP-LYCSyIdi2로서 균체당 라이코펜 생성량이 control 대비 2.8배이었다.

• 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 Plant Biotechnology
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• 제36권4호
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• pp.352-359
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• 2009

Panax ginseng roots produce triterpene saponins called ginsenosides, which are high value secondary metabolites and has been used as drugs, detergents, sweeteners, and cosmetics. In the recent years plant cell, tissue and organ cultures have developed as important alternative sources for the saponin production in Panax ginseng. Adventitious roots and hairy roots have been successfully induced and cultured for the improvement of saponin contents. Genetic and metabolic engineering to regulate saponin biosynthesis in P. ginseng might be important way to improve the medicinal values of P. ginseng. Here we introduced the protocol of genetic transformation and recent progress of functional characterization of genes involved in saponin biosynthesis in P. ginseng.

• Journal of Ginseng Research
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• 제33권3호
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• pp.212-218
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• 2009

인삼으로부터 뽑은 PgCYP 유전자와 표지유전자인 NPTII 유전자를 함유하고 있는 Agrobacterium tumefaciens GV3101균주를 이용하여 담배 잎절편과 공동배양한 후 MS 기본배지에 kanamycin 100 $\mu$g/ml, cefotaxime 500 $\mu$g/l, BA 2 mg/l와 NAA 0.2 mg/l가 첨가된 선발배지에 치상하여 4주 후 항생제가 첨가된 기본배지에서 발근시켰다. 생존한 선발체의 잎을 이용하여 PCR 반응으로 도입유전자의 삽입여부를 확인하였다. 또한 선발된 형질전환체를 이용하여 RT-PCR을 실시하여 PgCYP 유전자가 담배식물에 안정적으로 도입되어 전사되고 있음을 확인하였다.

• 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.