• Bulletin of the Korean Chemical Society
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• 제33권8호
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• pp.2475-2476
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• 2012

• Journal of Microbiology
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• 제43권3호
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• pp.313-318
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• 2005

In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of $C_7N$ aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of $C_7N$ aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 $C_7N$ aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of $C_7N$ aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.

• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1867-1876
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• 2017

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

• Nutrition Research and Practice
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• 제5권5호
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• pp.396-403
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• 2011

Ceramides (Cer) comprise the major constituent of sphingolipids in the epidermis and are known to play diverse roles in the outermost layers of the skin including water retention and provision of a physical barrier. In addition, they can be hydrolyzed into free sphingoid bases such as $C_{18}$ sphingosine (SO) and $C_{18}$ sphinganine (SA) or can be further metabolized to $C_{18}$ So-1-phosphate (S1P) and $C_{18}$ Sa-1-phosphate (Sa1P) in keratinocytes. The significance of ceramide metabolites emerged from studies reporting altered levels of SO and SA in skin disorders and the role of S1P and Sa1P as signaling lipids. However, the overall metabolism of sphingoid bases and their phosphates during keratinocyte differentiation remains not fully understood. Therefore, in this study, we analyzed these Cer metabolites in the process of keratinocyte differentiation. Three distinct keratinocyte differentiation stages were prepared using 0.07 mM calcium (Ca$^{2+}$) (proliferation stage), 1.2 mM Ca$^{2+}$ (early differentiation stage) in serum-free medium, or serum-containing medium with vitamin C (50 ${\mu}L$/mL) (late differentiation stage). Serum-containing medium was also used to determine whether vitamin C increases the concentrations of sphingoid bases and their phosphates. The production of sphingoid bases and their phosphates after hydrolysis by alkaline phosphatase was determined using high-performance liquid chromatography. Compared to cells treated with 0.07 mM Ca$^{2+}$, levels of SO, SA, S1P, and SA1P were not altered after treatment with 1.2 mM Ca$^{2+}$. However, in keratinocytes cultured in serum-containing medium with vitamin C, levels of SO, SA, S1P, and SA1P were dramatically higher than those in 0.07- and l.2-mM Ca$^{2+}$-treated cells; however, compared to serum-containing medium alone, vitamin C did not significantly enhance their production. Taken together, we demonstrate that late differentiation induced by vitamin C and serum was accompanied by dramatic increases in the concentration of sphingoid bases and their phosphates, although vitamin C alone had no effect on their production.

• 농업과학연구
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• 제45권3호
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• pp.455-461
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• 2018

Deoxynivalenol (DON), a Fusarium mycotoxin, causes health hazards for both humans and livestock. Therefore, the aim of this study was to investigate the metabolic profiles of the liver, serum, and urine of piglets fed DON using proton nuclear magnetic resonance ($^1H-NMR$) spectroscopy. The $^1H-NMR$ spectra of the liver, serum, and urine samples of the piglets provided with feed containing 8 mg DON/kg for 4 weeks were aligned and identified using the icoshift algorithm of MATLAB $R^2013b$. The data were analyzed by multivariate analysis and by MetaboAnalyst 4.0. The DON-treated groups exhibited discriminating metabolites in the three different sample types. Metabolic profiling by $^1H-NMR$ spectroscopy revealed potential metabolites including lactate, glucose, taurine, alanine, glycine, glutamate, creatine, and glutamine upon mycotoxin exposure (variable importance in the projection, VIP > 1). Forty-six metabolites selected from the principal component analysis (PCA) helped to predict sixty-five pathways in the DON-treated piglets using metabolite sets containing at least two compounds. The DON treatment catalyzed the citrate synthase reactions which led to an increase in the acetate and a decrease in the glucose concentrations. Therefore, our findings suggest that glyceraldehyde-3-phosphate dehydrogenase, citrate synthase, ATP synthase, and pyruvate carboxylase should be considered important in piglets fed DON contaminated feed. Metabolomics analysis could be a powerful method for the discovery of novel indicators underlying mycotoxin treatments.

• 한국산림과학회지
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• 제81권2호
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• pp.183-190
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• 1992

기내배양된 현사시나무의 조직배양세포로부터 안토시아닌 생성과 세포생장에 적합한 요인을 구명하여 차후 기내배양을 통한 2차대사산물생산의 기초자료를 제공하고자 연구를 수행하였다. Callus는 0.5mg/l 2, 4-D, 0.1mg/l BAP을 첨가한 MS배지에서 유발시켜 같은 배지에서 증식하였다. 안토지아닌 생산은 MS 기본배지를 기준으로 하여 질산염은 12.5% 감량시키고 인산염은 400%로 증가시켜 5% sucrose와 1.0mg/l IAA 및 1.0mg/l BAP를 첨가한후 7,000 lux의 연속광 하에서 배양했을 때 가장 높게 나타났다. 그러나 세포의 생장은 MS 기본배지를 기준으로 하여 질산염은 50% 감량시키고 인산염은 400%로 증가시켜 5% sucrose와 0.5mg/l 2, 4-D를 첨가한 배지에서 가장 양호한 결과를 얻었다. 안토시아닌의 동정은 1% 염산-메탄올의 유기용매로 추출하여 정제 후 TLC와 UV spectrophotometer로 확인한 결과 pelargonidin 3-rhamnoside-5-glucose로 추정되었다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.208-213
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• 2000

Penicillium fellutanum produces a phosphorylated, choline-containing extracellular peptido-polysaccharide, peptidophosphogalactomannan (pPxGM) (8). The $\^$13/C-methyl labeled pPxGM ([methyl-$\^$13/C]pPxGM) was prepared from the cultures supplemented with L-[methyl-$\^$13/C]methionine or [2-$\^$13/C]glycine and was used as a probe to monitor the fate of phosphocholine in this polymer. Addition of purified [methyl-$\^$l3/C]pPxGM to growing cultures in low phosphate medium resulted in the disappearance of [methyl-$\^$13/C]phosphocholine and -N,N'-dimethyl-phosphoethanolamine from the added [methyl-$\^$13/C]pPxGM. Two $\^$l3/C-methyl-enriched cytoplasmic solutes, choline-O-sulfate and glycine betaine, were found in mycelial extracts, suggesting that phosphocholine-containing extracellular pPxGM of P.fellutanum is a precursor of intracellular choline-O-sulfate and glycine betaine and thus of phosphatydilcholine (l0). $\^$13/C-Methyl-labeled cells grown in 3 M NaCl-containing medium showed 2.6- and 22-fold more accumulation of $\^$13/C-methyl labeled choline-O-sulfate and glycine betaine, respectively, originated from the extracellular [$\^$13/C-methyl]pPxGM than those grown without added NaCl. The results suggest that, in addition to glycerol and erythritol, glycine betaine and choline-O-sulfate and thus choline are also osmoprotectants and hence that pPxGM is involved in osmotolerance of this fungus (11). Taken collectively, the $\^$l3/C- and $\^$31/P-NMR analyses of cytosolic solute pools and structural modulation of extracellular pPxGM corresponding to environmental stimuli in P. fellutanum, provided evidence that pPxGM is involved in cellular choline metabolism, osmotolerance, and recycling of metabolites.

• 한국환경농학회지
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• 제20권1호
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• pp.1-7
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• 2001

토양에서 분리된 인산가용화 사상균 Penicillium sp. GL-101 균주를 PDB-인광석 배지에서 액침배양했을 때 유리인산을 배양액속에 다량 방출함으로써 높은 인산가용화능을 보였다. 일반적으로 미생물에 의한 인산가용화 기작은 산성화, 킬레이트 대사산물의 생성, 산화환원 활성 등이 알려져 있는데 본 연구에서는 GL-101 균주의 유리인산 생성기작을 밝히기 위하여 균체를 PDB-인광석 배지에 키우면서 유리인산 생성능을 분광학적인 방법으로 정량분석하였다. 또한 균체의 액침배양중의 pH 변화를 측정한 결과 pH의 급격한 감소 즉 배지의 산성화가 주된 인산가용화 기작임을 확인하였다 즉 이 균주는 배양 4일이 경과하면 pH가 4.0 이하로 떨어지며, 특히 1.0%(w/v)의 황토를 첨가할 경우 pH가 3.2까지 떨어졌다. 이때 pH 감소에 영향을 주는 주 원인물질을 HPLC로 분석한 결과 citric acid 임을 확인하였다. 또한 이 균주는 균체의 생장중에 배지속으로 phosphatase를 생성 ${\cdot}$ 분비하였으며, 특히 황토를 1.0% 첨가했을 때 최대 1.3 unit의 효소활성을 보였다. 그러나 이 균주는 2-ketogluconic acid와 같은 킬레이트 물질은 거의 생성하지 않았기 때문에 이와 같은 기작에 의한 유리인산 생성은 거의 없을 것으로 생각된다. 따라서 Penicillium sp. GL-101 균주의 유리인산 생성기작은 citric acid 생성에 의한 산성화 및 phosphatase 활성의 두 가지 기작에 의한 것으로 결론지었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.70-70
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• 2002

Sphingosine-1-phosphate(S1P) is one of the sphingolipid metabolites which affect a variety of cellular processes including the proliferation, differentiation, growth, survival, migration and gene expression. The present study was undertaken to investigate the effect of SIP on nuclear maturation of porcine oocytes. In vitro maturation frequency of porcine oocytes were compared in three different media; group Ⅰ: NCSU23＋0.1% PVA, group Ⅱ: NCSU23＋10% PFF(porcine follicular fluid), and group Ⅲ: NCSU23＋10% PFF＋10 ng/㎖ EGF＋2.5 mM β-mercaptoethanol. Each group containing 0.1 ㎎/㎖ cysteine was divided into 4 sub-groups of SIP concentration(0, 50, 500 and 5000nM). Porcine oocytes were incubated in each maturation medium supplemented with hormones(10 IU/㎖ PMSG and 10 IU/㎖ hCG) for 22h and then further cultured in the same medium without the hormones for 22h. After completion of in vitro maturation, the oocytes were fixed and stained to examine nuclear maturation by using a rapid stain method. In the group Ⅰ, the proportions of metaphase Ⅱ stage among oocytes cultured in 0nM(control), 50 nM, 500nM and 5000nM S1P were 45.5%, 66.7%, 56.6% and 48.7%, respectively. (omitted)

• 분석과학
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• 제12권4호
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• pp.326-331
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• 1999

방향족 탄화수소인 trimethylbenzene (TMB)은 그 사용량이 늘어갈 뿐 아니라 직업적으로 폭로되는 양도 증가하고 있으므로 생물학적 모니터링 및 흡수, 대사, 배설에 관한 연구가 중요시되고 있다. 일반적으로 TMB는 간의 산화효소에 의해 하나의 메틸기가 산화되고 이것이 glycine과 포합되어 배설되는 것으로 알려져 있다. 본 연구에서는 1,2,4-TMB의 대사체를 합성하고, 모세관 전기영동법으로 분석할 수 있는 방법을 개발하였다. 모세관 전기영동법으로 흰쥐의 뇨 중에서 1,2,4-TMB의 대사체인 3,4-, 2,4-, 2,5-dimethylbenzoic acid 및 3,4-, 2,4-, 2,5-dimethylhippuric acid를 분석하기 위하여 내경 $75{\mu}m$, 총길이 36cm (검출기까지 29cm)인 용융실리카 모세관을 $15^{\circ}C$로 유지하면서 양단에 10㎸의 전압을 걸어주고, 전해질로는 15mM ${\beta}-CD$, 3% 2-프로판올을 포함하는 0.1M 인산완충액 (pH 7)을 사용하였으며, 검출신호는 UV 210nm와 254nm에서 동시에 모니터링하였다. 뇨 시료의 분석 결과 배설된 1,2,4-TMB의 대사체의 상대량은 3,4-이성질체가 56.7%, 2,4-이성질체가 30.5%, 2,5-이성질체가 12.8%였다. 이 방법은 노동자의 뇨 분석에도 적용될 수 있을 것으로 생각된다.