• Title/Summary/Keyword: Metabolic engineering

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Heat Shock Causes Oxidative Stress and Induces a Variety of Cell Rescue Proteins in Saccharomyces cerevisiae KNU5377

  • Kim, Il-Sup;Moon, Hye-Youn;Yun, Hae-Sun;Jin, Ing-Nyol
    • Journal of Microbiology
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    • v.44 no.5
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    • pp.492-501
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    • 2006
  • In this study, we attempted to characterize the physiological response to oxidative stress by heat shock in Saccharomyces cerevisiae KNU5377 (KNU5377) that ferments at a temperature of $40^{\circ}C$. The KNU5377 strain evidenced a very similar growth rate at $40^{\circ}C$ as was recorded under normal conditions. Unlike the laboratory strains of S. cerevisiae, the cell viability of KNU5377 was affected slightly under 2 hours of heat stress conditions at $43^{\circ}C$. KNU5377 evidenced a time-dependent increase in hydroperoxide levels, carbonyl contents, and malondialdehyde (MDA), which increased in the expression of a variety of cell rescue proteins containing Hsp104p, Ssap, Hsp30p, Sod1p, catalase, glutathione reductase, G6PDH, thioredoxin, thioredoxin peroxidase (Tsa1p), Adhp, Aldp, trehalose and glycogen at high temperature. Pma1/2p, Hsp90p and $H^+$-ATPase expression levels were reduced as the result of exposure to heat shock. With regard to cellular fatty acid composition, levels of unsaturated fatty acids (USFAs) were increased significantly at high temperatures ($43^{\circ}C$), and this was particularly true of oleic acid (C18:1). The results of this study indicated that oxidative stress as the result of heat shock may induce a more profound stimulation of trehalose, antioxidant enzymes, and heat shock proteins, as well as an increase in the USFAs ratios. This might contribute to cellular protective functions for the maintenance of cellular homeostasis, and may also contribute to membrane fluidity.

Transiently Experessed Salt-Stress Protection of Rice by Transfer of a Bacterial Gene, mtlD

  • Lee, Eun-A;Kim, Jung-Dae;Cha, Yoo-Kyung;Woo, Dong-Ho;Han, In-Seob
    • Journal of Microbiology and Biotechnology
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    • v.10 no.3
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    • pp.415-418
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    • 2000
  • Productivity of a rice plant is greatly influenced by salt stress. One of the ways to achieve tolerance to salinity is to transfer genes encoding protective enzymes from other organisms, such as microorganisms. The bacterial gene, mtlD, which encodes mannitol-1-phosphate dehydrogenase (Mtl-DH), was introduced to the cytosol of a rice plant by an imbibition technique to overproduce mannitol. The germination and survival rate of the imbibed rice seeds were markedly increased by transferring the mtlD gene when it was delivered in either a pBIN19 or pBmin binary vector. When a polymerase chain reaction was performed with the genomic DNAs of the imbibed rice leaves as a template and with mtlD-specific primers, several lines were shown to contain an exogenous mtlD DNA. However, a reverse transcription (RT)-PCR analysis revealed that not all of them showed an expression of this foreign gene. This paper demonstrates that the growth and germination of rice plants transiently transformed with the bacterial gene, mtlD, are enhanced and these enhancements may have resulted from the experssion of the mtlD gene. The imbibition method empolyed in this study fulfills the requirements for testing the function of such a putative gene in vivo prior to the production of a stable transgenic plant.

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Evaluation of the Effect of Exercise on Nonalcoholic Fatty Liver By Sonography (초음파 검사를 이용한 비알코올성 지방간에 운동이 미치는 영향 평가)

  • Kim, Kyoung-Yeon;Lim, Hyun-Soo
    • Journal of radiological science and technology
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    • v.35 no.1
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    • pp.17-23
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    • 2012
  • Nonalcoholic fatty liver disease (NAFLD) is accumulation state of fat in liver cells without excessive alcohol intake, and it has been studied that is closely related to obesity. The purpose of this study is to identify risk factors for NAFLD and may prevent or to manage risk factors. This study was in progress for six months (2011 May 1 to October 31), of the 83 people who underwent abdominal ultrasound 11 people eventually were selected. Research results was as follows : First, the decreased body weight and body mass index (BMI), and the second, a decrease of the deepening of fatty liver in ultrasound diagnosis, and the third, steady movement reduces the deepening of fatty liver regardless of calories. Thus, the implication of this research is that long-term exercise programs have positive effects in the treatment of fatty liver.

Importance of culture history on 17α-ethinylestradiol cometabolism by nitrifying sludge

  • Jantanaprasartporn, Angkana;Maneerat, Suppasil;Rongsayamanont, Chaiwat
    • Environmental Engineering Research
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    • v.23 no.1
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    • pp.28-35
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    • 2018
  • $17{\alpha}-ethinylestradiol$ (EE2), a synthetic estrogen which interfere the endocrine and reproductive function in living organisms, has been found extensively to be deposited into municipal wastewater treatment plants and the environment via human excretion. EE2 has long been known to be efficiently cometabolized by ammonia-oxidizing bacteria (AOB) during ammonia ($NH_3$) oxidation. Current study aims to investigate the effect of culture history on the biotransformation of EE2 by nitrifying sludge which was enriched under different ammonia loading rates in continuous flow reactors. Result showed that past growth condition largely affected not only the metabolic rate of $NH_3$ oxidation but also EE2 cometabolism. Sludge previously acclimated with higher $NH_3$ loads as well as sludge dominated with AOB belong to high growth cluster (Nitrosomonas europaea-Nitrosococcus mobilis) showed higher rate of EE2 biotransformation than those one being acclimated with lower $NH_3$ loads because of its ability to provide more reducing power from $NH_3$ oxidation. Moreover, the correlation between the degradation rates of $NH_3$ and EE2 was higher in sludge being acclimated with higher load of $NH_3$ in comparison with other sludge. Implication of the findings emphasized the role of volumetric $NH_3$ loading rate in determining EE2 removal in wastewater treatment system.

Biosynthesis of Polyunsaturated Fatty Acids: Metabolic Engineering in Plants (고도불포화지방산 생합성: 식물에서의 대사공학적 응용)

  • Kim, Sun-Hee;Kim, So-Yun;Kim, Jong-Bum;Roh, Kyung-Hee;Kim, Young-Mi;Park, Jong-Sug
    • Journal of Applied Biological Chemistry
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    • v.52 no.3
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    • pp.93-102
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    • 2009
  • Polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have significantly beneficial effects on health in relation to cardiovascular, immune, and inflammatory conditions and they are involved in determining the biophysical properties of membranes as well as being precursors for signaling molecules. PUFA biosynthesis is catalyzed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of PUFA is now known to contain analogous polyketide synthases (PKS). Certain fish oil can be a rich source of PUFA although processed marine oil is generally undesirable as food ingredients because of the associated objectionable flavors that are difficult and cost-prohibitive to remove. Oil-seed plants contain only the 18-carbon polyunsaturated fatty acid alpha-linolenic acid, which is not converted in the human body to EPA and DHA. It is now possible to engineer common oilseeds which can produce EPA and DHA and this has been the focus of a number of academic and industrial research groups. Recent advances and future prospects in the production of EPA and DHA in oilseed crops are discussed here.

Production of tropane alkaloids by metabolic engineering of Hyoscyamus niger H6H(hyoscyamine $6{\beta}-hydroxylase$) gene introduced Scopolia parviflora hairy root

  • Kang, Young-Min;Lee, Ok-Sun;Jung, Hee-Young;Kim, Won-Jung;Kang, Seung-Mi;Min, Ji-Yun;Bahk, Dong-Jin;Yun, Dae-Jin;Bahk, Jung-Dong;Choi, Myung-Suk
    • 한국생물공학회:학술대회논문집
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    • 2003.04a
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    • pp.568-570
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    • 2003
  • The Hyoscyamus niger hyoscyamine $6{\beta}-hydroxylase$ (H6H, EC 1.14.11.11) gene was introduced into the genome of a Scopolia parviflora by the binary vector system using the disarmed Agrobacterium rhizogenes strain KCTC 2703. Expression of H6H enzyme which are involved in alkaloids pathway by western blot analysis using proteins extracted from leaf, stem flower, branch root and main root were examined The enzyme expression was found only in the roots, with no expression in leaf, stem and flower. The alkaloids contents were the most higher in root and then leaf and stem has very small amount of alkaloid contents were analyzed by HPLC. The expression level of H6H in transgenic plants were two or more times than wild type plants. In transgenic plant which constitutively expresses H6H enzyme, high concentration of scopolamine was accumulated.

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Screening of Crude Drugs for the Inhibitory Effect on $NF-{\kappa}B$ Activation in Transfectant HaCaT Cells (형질전환된 각질형성세포에서 생약추출물에 의한 $NF-{\kappa}B$ 활성화 억제효과 탐색)

  • Ahn, Kwang-Seok;Kim, Seong-Kie;Moon, Ki-Young;Hahn, Bum-Soo;Kang, Sam-Sik;Kim, Yeong-Shik
    • Korean Journal of Pharmacognosy
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    • v.34 no.2 s.133
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    • pp.156-160
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    • 2003
  • $NF-{\kappa}B$ (nuclear factor-kappa B) plays a particularly central role in epidermal biology. It has been established that ultraviolet radiation (UVR) is one of the mechanisms to induce the activation of $NF-{\kappa}B$ in human skin. We previously demonstrated that melanogenic inhibitors may act through the inhibition of $NF-{\kappa}B$ activation in keratinocytes. In order to find another type of melanogenic inhibitors of $NF-{\kappa}B$ activation, various kinds of the extracts from crude drugs $(30\;{\mu}g/ml)$ were preincubated with transfectant HaCaT cells for 3 hrs and then UVR $(60\;mj/cm^2)$ was irradiated. UVR-exposed cells were incubated for another 6 hrs to measure the $NF-{\kappa}B$ activity. $NF-{\kappa}B$ activation was measured with the secreatory alkaline phosphates (SEAP) reporter gene assay using a fluorescence detection method. Among natural products, Lycium chinense, Acanthopanax senticosus, Angelica koreana, Kalopanax pictus and Asparagus cochinchinensis were the most potent inhibitors of $NF-{\kappa}B$ activation by UVR. These observations suggest that some crude drugs might act partially through the modulation of the synthesis of melanotrophic factors to decrease melanogenesis in keratinocytes.

Estimation of Antibacterial Properties of Chlorophyta, Rhodophyta and Haptophyta Microalgae Species

  • Imran Bashir, Khawaja Muhammad;Lee, Jae-Hyeon;Petermann, Maike Julia;Shah, Abid Ali;Jeong, Su-Jin;Kim, Moo-Sang;Park, Nam-Gyu;Cho, Man-Gi
    • Microbiology and Biotechnology Letters
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    • v.46 no.3
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    • pp.225-233
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    • 2018
  • In this exploratory study, eight types of microalgae from different phyla (Chlamydomonas reinhardtii, Chlorella species, Haematococcus pluvialis, Porphyridium purpureum, Porphyridium cruentum, Isochrysis species, Isochrysis galbana, and Pavlova lutheri) were tested for their antibacterial activities against eight target pathogenic bacterial strains. The agar well diffusion method and broth micro dilution assay were conducted to estimate the antibacterial activity. Microalgae cell-free supernatants, exopolysaccharides (EPS), water, and organic solvent extracts were used for inhibition analysis. EPS extracted from P. lutheri showed activity against Bacillus subtilis and Pseudomonas aeruginosa. Inhibition zone diameters of 14-20 mm were recorded on agar plates, while the minimum inhibitory concentrations in the broth micro dilution assay were $0.39-25mg\;ml^{-1}$. During this study, haptophyte microalgae, Isochrysis species, and P. lutheri extracts showed the highest activity against most of the tested pathogenic bacterial strains, while most of the extracts were active against the important foodborne pathogen P. aeruginosa. This study showed promising results regarding important microalgae phyla, which will further aid research related to extracts and exploitation of bioactive metabolic compounds in the food and pharmaceutical industries.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT

  • Ghosh, Suparna;Dhanasingh, Immanuel;Ryu, Jaewon;Kim, Si Wouk;Lee, Sung Haeng
    • Journal of Microbiology and Biotechnology
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    • v.30 no.8
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    • pp.1261-1271
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    • 2020
  • Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+, Ca+, and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

Anti-obesity Effects of Genistein in Female Ovariectomy-induced Obese Mice (난소절제로 비만이 유도된 암컷 쥐에서 제니스테인의 항비만 효과)

  • Jeong, Sun-Hyo
    • Journal of the Korean Applied Science and Technology
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    • v.34 no.3
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    • pp.427-435
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    • 2017
  • To investigate whether genistein regulates menopause-induced obesity, it was studied the effects of genistein on anti-obesity effects in female ovariectomized (OVX) mice, an animal model of postmenopausal women. 7-week-old female mice (C57BL/6J) were randomly divided into three groups. All the animals received a high fat diet or a high fat diet supplemented with genistein for 8 weeks and variables and determinants of obesity were measured. The OVX mice had significantly higher body weight and adipose tissue mass than sham mice. However, genistein supplementation reduced body weight, adipose tissue mass, and adipocyte size of OVX mice. The OVX mice treated with genistein had significantly lower levels of serum triglycerides and total cholesterol than the vehicle-treated OVX mice. Lipid accumulation in liver was also markedly decreased by genistein in OVX mice. The results suggest that genistein can effectively prevent adiposity, adipocyte phertrophy, and llipid disorders caused by ovariectomy. Moreover, this study may contribute to the alleviation of metabolic syndrome, including obesity and hyerlipidemia in postmenopausal women.