Phaeodactylum tricornutum의 (E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate Reductase 유전자의 형질전환

신복규 ( Bok Kyu Shin ),정유진 ( Yu Jin Jung ),김상민 ( Sang Min Kim ),판철호 ( Cheol Ho Pan ) 한국응용생명화학회 2015 Journal of Applied Biological Chemistry (J. Appl. Vol.58 No.3

해양 미세조류인 Phaeodactylum tricornutum은 게놈 염기서열이 완전히 밝혀진 규조류로서, 형질전환 방법이 개발되어 있고, 여러 가지 분자생물학적 연구 기술이 개발되어 규조류 연구에서 모델 종으로 여겨지고 있다. 본 연구의 목적은 methylerythritol phosphate (MEP) 대사경로의 마지막 효소인 (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR)를 코딩하는 P.tricornutum의 Pthdr 유전자를 P. tricornutum에 도입하여 형질전환체를 확보하는 것이다. 유전자 도입 방법은 gold microcarrier 를 사용한 bombardment 방법을 사용하였고, 형질전환 유무 및 목적 유전자의 전사체 확인에는 각각 genomic DNA-PCR 및 cDNA-PCR 방법을 사용하였다. 양성대조군으로 egfp 유전자를 P. tricornutum에 도입하여 최종적으로 eGFP 단백질이 발현되는 것을 형광 공초점 현미경을 통해 확인하였다. 이를 바탕으로, 확보된 Pthdr 형질전환체에서도 도입한 Pthdr 유전자로부터 발현된 PtHDR 효소도 잘 발현될 것으로 추측할 수 있었다. 이렇게 준비된 Pthdr 형질전환체는 추후 연구를 통해, P. tricornum 의 유용물질인 카로티노이드의 생합성 과정 연구 및 고부가가치 카로티노이드 과발현 균주 개발 등에 유용한 정보를 제공할 것으로 기대된다. Phaeodactylum tricornutum is a model diatom that its genomic information and biological tools are well established. In this study, a gene encoding (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (PtHDR), a terminal enzyme of the methylerythritol phosphate pathway regulating chlorophyll and carotenoid biosynthesis, was isolated from P. tricornutum. The isolated gene was cloned into pPha-T1 vector containing fcpA promoter to prepare pPha-T1-HDR plasmid. As a positive control, pPha-T1-eGFP plasmid was constructed with egfp gene. Stable nuclear transformation was carried out with these plasmids by particle bombardment method and zeocin resistant colonies of P. tricornutum were selected on f/2 agar plate. In result, transformation efficiency was evaluated according to the amount of plasmid DNA coated with gold particles. Integration of introduced plasmids was confirmed with genomic DNA of each transformant by polymerase chain reaction. The eGFP fluorescence was visible in the cytoplasm, indicating that eGFP was successively expressed in P. tricornutum system. The transcript level of exogenous Pthdr gene was evaluated with the obtained transformants. The results presented here demonstrated that introduction of Pthdr gene into P. tricornutum chromosome succeeded and expression of PtHDR was enhanced under the fcpA promoter.

Characterization of Burkholderia glumae BGR1 4- Hydroxy-3-methylbut-2-enyl Diphosphate Reductase (HDR), the Terminal Enzyme in 2-C-Methyl-Derythritol 4-Phosphate (MEP) Pathway

권문혁,신복규,Jaekyoung Lee,한재홍,김수언 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.1

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the ultimate enzyme in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway converting (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Burkholderia glumae, a Gram-negative rice-pathogenic bacterium, harbors 2hdr genes and lacks isopentenyl diphosphate isomerase (idi). Both HDR enzymes could complement E. coli hdr deletion mutant (DYTL1). Both of the recombinant HDR proteins, BgHDR1 and BgHDR2, catalyzed reduction of HMBPP into IPP and DMAPP at a ratio of 2:1, in contrast to 5:1 ratio of other bacterial HDRs so far characterized. The kcat and Km values of BgHDR1 and BgHDR2 were 187.0 min−1 and 6.0 μM and 66.6 min−1 and 21.2μM, respectively. Physiological significance of the kinetic properties was discussed.

High-Throughput HDR Inhibitor Screening

김혜선,신복규,한재홍 한국응용생명화학회 2014 Applied Biological Chemistry (Appl Biol Chem) Vol.57 No.1

Isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP), biochemical precursors of the isoprenoids,are biosynthesized by mevalonate pathway and methylerythritolphosphate (MEP) pathway. Because these two pathways aremutually exclusive in most organisms, inhibition of MEP pathwayhas become a target for the development of new bioactivematerials, including antibiotics, antimalarial drugs, and herbicides. In the final step of MEP pathway, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HMBPP reductase, HDR) catalyzesreduction of HMBPP to IPP and DMAPP. HDR requires electrontransfers to the Fe/S cluster in the active site for the catalysis, andmethyl viologen has been used as a common redox mediator forin vitro studies. We developed a high throughput colorimetricHDR inhibitor screening method by utilizing microtiter-platescreening method. This method was applied to various plantextracts to measure HDR inhibition activity, and potent HDRinhibitor was successfully screened.

Synthesis and the Absolute Configurations of Isoflavanone Enantiomers

원동호,신복규,한재홍 한국응용생명화학회 2008 Journal of Applied Biological Chemistry (J. Appl. Vol.51 No.1

Isoflavanone has been synthesized from the reduction of isoflavone in nearly quantitative yield. Isoflavone with seven equivalents of ammonium formate in the presence of Pd/C in ethanol under N2 atmosphere exclusively produced the two-electron reduced product in two hours. It was characterized by various spectroscopic methods, including UV-VIS, EI-MS, 1H-NMR, 13C-NMR and 1H,1H-COSY. The racemic mixture was separated by Sumi-Chiral column chromatography and the absolute configurations of the enantiomers were characterized by circular dichroism spectroscopy. Isoflavanone has been synthesized from the reduction of isoflavone in nearly quantitative yield. Isoflavone with seven equivalents of ammonium formate in the presence of Pd/C in ethanol under N2 atmosphere exclusively produced the two-electron reduced product in two hours. It was characterized by various spectroscopic methods, including UV-VIS, EI-MS, 1H-NMR, 13C-NMR and 1H,1H-COSY. The racemic mixture was separated by Sumi-Chiral column chromatography and the absolute configurations of the enantiomers were characterized by circular dichroism spectroscopy.

Axial Conformation of 3-Methyl-2-butenoyl Group in Pyranocoumarin Ring Endows Biological Activity of (+)-Decursin

한재홍,신복규,김혜민,이상현 대한화학회 2008 Bulletin of the Korean Chemical Society Vol.29 No.8

Mechanistic Study of Hydrogen Peroxide Disproportionation by Dimanganese Complex

한재홍,신복규,김유진,이정봉,노동이 한국공업화학회 2008 한국공업화학회 연구논문 초록집 Vol.2008 No.-

Anti-obesity Effect of Carbon Dioxide Supercritical Fluid Extracts of Panax Ginseng C. A. Meyer

우현철,신복규,조인자,김미향,한재홍 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.5

The root of Korean ginseng, Panax ginseng C. A. Meyer, has been known worldwide as a panacea for many centuries. Contradictory weight gain and loss effects in Korean ginseng led to the adoption of supercritical carbon dioxide extraction to assure the quality of food-grade anti-obesity ginseng products. Among different extraction conditions of CO2 pressure (200 and 300 bars) and temperature (40, 50, and 60oC), the highest extraction yield of 0.66% was obtained with 300 bars at 40oC. To test anti-obesity effect, each supercritical fluid extract (SFE) was supplemented to the high-fat diet of male obese-prone C57BL/6J strain mice. All carbon dioxide SFEs of ginseng showed significant weight-loss effects in the tested mice. The average weights of normal and high fat diet mice were 28.7±2.0 and 35.0±1.6 g, respectively, after 10 weeks. Average weights of mice on SFE-supplemented high fat diets were between 24.5±1.4 and 25.9±1.3 g. SFEs obtained at 50oC, and at both 200 and 300 bars, showed the most profound anti-obesity effect. Both SFEs showed ~15and ~30% weight loss for the normal and high-fat diet mice, respectively. The major component of the SFE was analyzed as polyacetylene compounds, and panaxydol and panaxynol were identified as major polyacetylene compounds. However, the anti-obesity effect of the ginseng SFEs was not correlated with contents of these two polyacetylene compounds in SFEs. No side-effects of ginseng SFEs were observed, and the anti-obesity effect was reversible.

FT-IR and XRD Analyses of Commercial Methionine-Mineral Chelates

한재홍,치용석,신복규,김성규,백인기 한국응용생명화학회 2006 Journal of Applied Biological Chemistry (J. Appl. Vol.49 No.1

Compositions of methionine-metal chelates have been investigated by FT-IR and XRD studies to elucidate their molecular structures. It was concluded that Copamin and Zincamin contain a high percentage of crystalline products, presumably 2:1 Methionine-Cu or Zn complexes. On the contrary, FT-IR and XRD spectra of Ferramin didn’t show any characteristics of the chelate and it was concluded to contain major components of starting FeSO4 and methionine without chelation. Compositions of methionine-metal chelates have been investigated by FT-IR and XRD studies to elucidate their molecular structures. It was concluded that Copamin and Zincamin contain a high percentage of crystalline products, presumably 2:1 Methionine-Cu or Zn complexes. On the contrary, FT-IR and XRD spectra of Ferramin didn’t show any characteristics of the chelate and it was concluded to contain major components of starting FeSO4 and methionine without chelation.

Cloning of a novel endogenous promoter for foreign gene expression in Phaeodactylum tricornutum

ERDENE OCHIR ERDENEDOLGOR,신복규,HUDA MD NAZMUL,김다혜,이은하,송대근,김윤미,김상민,판철호 한국응용생명화학회 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.6

Phaeodactylum tricornutum is a model diatom, and its genomic sequence data and expressed sequence tag databases are available. This study was to discover a new endogenous promoter that drives strong constitutive expression of a protein of interest in P. tricornutum. To find promoter candidates, the intracellular proteins of P. tricornutum grown to stationary phase were extracted and identified by LC–MS/MS. Glutamine synthetase (GLNA) was one of the most abundantly expressed proteins during the stationary phase. Promoter is usually located on 5′ upstream region of open reading frame of the gene. Thus, two fragments of 5′ upstream region of the GLNA gene as putative promoters, 501 and 997 bp long, were amplified and cloned into enhanced Green Fluorescent Protein (eGFP) reporter systems. The constructed reporter systems were transformed into P. tricornutum and the eGFP expression levels were compared to those of reporter systems using the promoters of fcpA (fucoxanthin chlorophyll a/c binding protein A) and CIP1 (putative replication-associated proteins of a Chaetoceros lorenzianus-infecting DNA virus) as controls. The expression of eGFP driven by either GLNA promoter (501 and 997 bp) was linearly related to cell density, and eGFP was expressed constitutively regardless of the cultivation phase. The eGFP expression level driven by the GLNA promoters was at least 4 times higher than the fcpA-driven eGFP expression level at the stationary phase. The 501 and 997 bp regions of the GLNA promoter had similar activity patterns for transcribing the downstream gene. These results indicate that at least 501-bp region of the GLNA promoter can be used as a strong constitutive promoter in genetic engineering of P. tricornutum.