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.

Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum

Seo, S.,Jeon, H.,Hwang, S.,Jin, E.,Chang, K.S. Elsevier B.V 2015 Algal research Vol.11 No.-

Phaeodactylum tricornutum is a pennate model diatom whose entire genome has been sequenced and an expressed sequence tag database established. This study aimed at developing a genetic tool kit to modify the genetic traits of P. tricornutum. A new expression system containing the elongation factor 2 (EF2) promoter fused to the luciferase gene was constructed, resulting in the pPhaT-EF2-Luc vector. This new vector was compared to the pPhaT-BP-Luc which harbors the fcpB promoter of P. tricornutum. After P. tricornutum transformation with these vectors, the presence of the exogenous luciferase gene inserted into the genomic DNA of the selected transformants was confirmed by Southern blot analysis. In the transformants, the fcpB promoter was activated dramatically upon 5h of light exposure and its activity was reduced in the dark. However, the expression level of the Luc gene regulated by the EF2 promoter was not significantly affected by light and was higher than that of the Luc gene regulated by the fcpB promoter. Transformants harboring the pEF2::Luc cassette had a high level of luciferase protein expression and constant luciferase activity throughout the light/dark cycle. In addition, the EF2 promoter of P. tricornutum was able to drive the expression of a heterologous gene in the green alga Chlamydomonas reinhardtii. These results indicate that the P. tricornutum EF2 promoter can be used for the expression of target genes in genetically transformed P. tricornutum and C. reinhardtii.

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.

바이오디젤 생산을 위한 규조류 Phaeodactylum tricornutum의 지질 추출 전처리 공정 최적화

김근호,김시욱 한국생물공학회 2019 KSBB Journal Vol.34 No.4

Phaeodactylum tricornutum is an emerging diatom in the microalgal biotechnology field for biofuel application. P. tricornutum B2089 was incubated for 13 days in a 3 L flask containing f/2 medium mixed with oceanic sediment extracts. To select the most effective pretreatment process (cell destruction method) of P. tricornutum B2089 for lipid extraction, various mechanical methods including autoclave, bead beating, sonication, French-press, and microwave were tested. Of these, enhanced lipid extraction was obtained through microwave treatment (1200W for 20 minutes at 2450 MHz, 150oC). Under this condition, the lipid content from P. tricornutum B2089 was 53.3% (w/w) of dry cell weight or 3.4 g/L and the lipid contained palmitoleic acid (C16:1) and oleic acid (C18: 1) as major fatty acids.

LED의 파장 및 광도, 공기주입이 Pavlova lutheri와 Phaeodactylum tricornutum의 최적 성장에 미치는 영향

최보람(Bo-Ram Choi),김동수(Dong-Soo Kim),이태윤(Tae-Yoon Lee) 한국생물공학회 2013 KSBB Journal Vol.28 No.3

The purpose of this study was to determine optimum condition of Pavlova lutheri and Phaeodactylum tricornutum. Detailed studies were carried out on the effects of various wavelengths of light-emitting diodes (LEDs), light intensities and air flow rations. For the Pa. lutheri, cell growth rates and maximum cell concentrations were similar regardless of wavelengths and air flow rates. Among the different light intensities, cell concentration increased when light intensity of red LED increased. For Ph. tricornutum, red LED was found to be the most effective light source, and light intensity of 3,100 Lux resulted in the most effective for the cultivation of Ph. tricornutum. Different air flow rates were tested to overcome shading effects due to denser cell concentration in the solution. Aeration of 0.8 vvm was determined to be the optimum aeration rate for the cultivation of Ph. tricornutum. Especially, five and two times greater cell concentrations of Pa. lutheri and Ph. tricornutum, respectively, were observed when air was applied.

Phaeodactylum tricornutum의 in vivo 항비만 효과 연구

김정화 ( Jeong Hwa Kima ),김영훈 ( Young Hoon Kima ),김상민 ( Sang Min Kimb ),판철호 ( Cheol Ho Panb ),이재권 ( Jae Kwon Lee ) 충북대학교 과학교육연구소 2013 과학교육연구논총 Vol.29 No.1

Marine microalga has been considered as an important primary production of marine environments and plays a critical role in supporting aquatic animals. Marine microalga has already been identified physiological and nutritional value as primary producers in the food chain. Phaeodactylum tricornutum (PT) is one of classical model diatoms most widely studied for its ecology, physiology, biochemistry and molecular biology. In this study, we evaluated anti-obesity activities of PT with in vivo study. Anti-obesity activity of PT was evaluated with freeze dried powder on a number of metabolic parameters in a model of diet-induced obesity. C57BL/6 mice were fed on a high-fat diet supplemented with PT powder (20 or 5% w/w) for 8 weeks. Energy intake, weight loss and lipid profile were determined, and these results were evaluated. PT powder significantly reduced body weight gain and epididymal adipose tissue weights. However, in fasted serum level of total cholesterol, triglyceride and glucose were not changed by PT powder. Our results indicate that PT has anti-obesity activities, and suggesting that PT could be a potential candidate for a new compound for anti-obesity drug.

Preparation of Fucoxanthin-Loaded Nanoparticles Composed of Casein and Chitosan with Improved Fucoxanthin Bioavailability

Koo, Song Yi,Mok, Il-Kyoon,Pan, Cheol-Ho,Kim, Sang Min American Chemical Society 2016 Journal of agricultural and food chemistry Vol.64 No.49

<P>To facilitate the utilization of fucoxanthin (FX), a valuable marine carotenoid, in the food industry, FX-loaded casein nanoparticles (FX-CN) and chitosan-coated FX-CN (FX-CS-CN) were developed using the FX-enriched fraction from Phaeodactylum tricornutum. Two nanoscale particles (237 +/- 13 nm for FX-CN and 277 +/- 26 nm for FX-CN-CN) with spherical and smooth surfaces showed over 71% encapsulation efficiency and polydispersity index (PDI) value of 0.31-0.39 in water. Owing to the chitosan coating, FX-CS-CN showed a positive zeta potential (24.00 mV), whereas that of FX-CN was negative (-12.87 mV). In vitro simulated digestion demonstrated better FX bioaccessibility from the nanoparticles versus P. tricornutum powder (Pt-powder) and from FX-CN versus FX-CS-CN. However, in C57BL/6 mice, fucoxanthinol absorption to the blood circulation was two times higher for FX-CS-CN versus FX-CN, possibly due to increased retention or adsorption to mucin by the cationic biopolymer in the chitosan-coated particles. These results demonstrate that FX-CS-CN can enable the application of FX, with improved bioavailability and water dispersibility, in the food industry.</P>

Fucoxanthin bioavailability from fucoxanthin-fortified milk: <i>In vivo</i> and <i>in vitro</i> study

Mok, Il-Kyoon,Lee, Jae Kwon,Kim, Jeong Hwa,Pan, Cheol-Ho,Kim, Sang Min Elsevier 2018 Food chemistry Vol.258 No.-

<P><B>Abstract</B></P> <P>Our previous study reported the improved stability of fucoxanthin (FX) fortified in whole milk (WM) and skimmed milk (SM). In this study, <I>in vivo</I> and <I>in vitro</I> FX bioavailability were investigated using FX-fortified milk (FX-SM and FX-WM) and microalga <I>Phaeodactylum tricornutum</I> biomass (Pt-powder). Organ tissue accumulation of FX and its metabolites (FXOH: fucoxanthinol, AXA: amarouciaxanthin A) after repeated oral administration was in the following order: FX-SM > FX-WM > Pt-powder. <I>In vivo</I> pharmacokinetic study with a single oral administration also demonstrated that the absorption of FXOH and AXA was the highest for FX-SM. To reinforce the <I>in vivo</I> results, <I>in vitro</I>-simulated digestion and Caco-2 cell uptake assays were performed, which revealed that FX-SM showed the highest FX bioaccessibility (release from food matrices) and cellular uptake efficiency of FX and FXOH. In conclusion, skimmed milk was validated as an excellent food matrix for FX application in terms of stability and bioavailability.</P> <P><B>Highlights</B></P> <P> <UL> <LI> FX-SM showed the highest organ tissue accumulation and plasma absorption. </LI> <LI> Higher bioaccessibility and cellular uptake efficiency of FX-SM increased FX bioavailability. </LI> <LI> Higher protein and Ca<SUP>2+</SUP> ion and lower fat accounted for the higher bioaccessibility and cellular uptake efficiency of FX-SM. </LI> </UL> </P>

Antioxidant Effect of Phaeodactylum tricornutum in Mice Fed High-fat Diet

강민정,김상무,정수미,최하늘,장양희,김정인 한국식품과학회 2013 Food Science and Biotechnology Vol.22 No.1

High-fat diet (HFD)-induced obesity is associated with oxidative stress. The purpose of this study was to examine the antioxidant effect of Phaeodactylum tricornutum extract in mice with diet-induced obesity. Four-week-old C57BL/6J mice were fed a normal diet or HFD with and without 0.7% P. tricornutum lipid extract corresponding to 0.2% fucoxanthin for 8 weeks. P. tricornutum significantly decreased body weight and epidydimal white adipose tissue in mice fed the HFD. Serum triglyceride, glucose,insulin, and leptin levels, as well as homeostasis model assessment for insulin resistance (HOMA-IR) values, were significantly lower in the P. tricornutum group than in the HFD group. P. tricornutum significantly decreased thiobarbituric acid reactive substances (TBARS) and increased glutathione and the activities of superoxide dismutase,catalase, and glutathione peroxidase in the liver compared with the HFD group. Thus, P. tricornutum could exert antiobesity and antioxidant effects in mice fed a HFD.

Overexpression of PtVDL1 in Phaeodactylum tricornutum Increases Fucoxanthin Content under Red Light

Seo Seungbeom,Chang Kwang Suk,Choi Min Sun,Jin EonSeon 한국미생물·생명공학회 2024 Journal of microbiology and biotechnology Vol.34 No.1

Phaeodactylum tricornutum is a model diatom with significant biotechnological applications, including enhancing biomass, biofuel, and carotenoid production. Specifically, owing to the capacity of this organism to serve as a valuable source of essential raw materials for pharmaceuticals and nutraceuticals, ongoing research is actively focused on enhancing its productivity. One of the genes involved in various stages of fucoxanthin (Fx) biosynthesis, violaxanthin de-epoxidase like 1 (VDL1), has recently been identified. To validate the intracellular function of this gene and boost Fx production through overexpression, we established and examined three transgenic P. tricornutum lines characterized by elevated P. tricortunum VDL1 ( PtVDL1) expression and evaluate their cell growth and Fx productivity. These transgenic lines exhibited substantially increased PtVDL1 mRNA and protein levels compared to the wild type (WT). Notably, the enzyme substrate violaxanthin was entirely depleted and could not be detected in the transformants, whereas it remained at constant levels in the WT. Interestingly, under standard white light conditions, Fx productivity in the transformants remained unchanged; however, but after 48 h of exposure to red light, it increased by up to 15%. These results indicate that PtVDL1-overexpressing P. tricornutum has industrial potential, particularly for enhancing Fx production under red light conditions.