• 농약과학회지
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• 제4권3호
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• pp.52-59
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• 2000

Saccharomyces cerevisiae를 이용하여 효율적인 호흡저해 스크리닝 방법을 개발하고자 실험하였다. S. cerevisiae균을 glucose 발효와 미토콘드리아 호흡이 가능한 yeast extract-peptone-dextrose (YPD) 배지와 단지 미토콘드리아 호흡만이 가능한 non-fermentable carbon source-yeast extract (NFY) 배지로 수확하였다. 96-well plate의 각 well에 균 현탁액을 분주한 다음 다양한 작용기작의 46개 살균제를 여러 가지 농도로 처리하였다. NFY배지에서의 non-fermentable carbon source로는 ethanol (NFY-E배지) 및 glycerol (NFY-G배지), lactate (NFY-L배지)를 이용하였다. 접종 후 $1{\sim}3$일 동안 배양한 다음 최소억제농도 (minimum inhibitory concentration, MIC)를 결정한 결과, 4개의 호흡억제 살균제인 azoxystrobin, kresoxim-methyl, metominostrobin, trifloxystrobin은 YPD 배지에서 균의 생육을 전혀 억제하지 못하였으나, 세가지 NFY배지에서는 높은 항균활성을 보였다. 이와는 반대로 5개의 N-trihalomethylthio계 살균제는 NFY배지보다 YPD 배지에서 높은 활성을 보였다. 그리고 11개 살균제는 두 배지 모두에서 같은 항균활성을 나타내었고, 나머지 26개의 살균제는 모든 배지에서 전혀 항균활성을 보이지 않았다. 그러므로 S. cerevisiae와 96-well plate를 이용한 호흡저해제 검정법은 신속하고 편리하게 호흡저해제를 스크리닝 할 수 있는 방법으로 여겨진다.

• BMB Reports
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• 제38권5호
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• pp.609-618
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• 2005

$\gamma$-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the $\gamma$-glutamyl moiety from $\gamma$-glutamyl containing ompounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wild type yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in wild type KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of $\beta$-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

• Journal of Microbiology
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• 제44권6호
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• pp.689-693
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• 2006

Pbh1, from the fission yeast Schizosaccharomyces pombe, is a baculoviral inhibitor of apoptosis (IAP) repeat (BIR) domain-containing protein. Its unique encoding gene was previously found to be regulated by nitric oxide and nitrogen starvation. In the current work, the Pbh1-lacZ fusion gene was used to elucidate the transcriptional regulation of the pbh1 gene under various carbon sources. When fermentable carbon sources, such as glucose (at a low concentration of 0.2 %), sucrose (2.0 %) and lactose (2.0 %), were the sole carbon source, the synthesis of $\beta$-galactosidase from the Pbh1-lacZ fusion gene was reasonably enhanced. However, the induction by these fermentable carbon sources was abolished in the Pap1-negative S. pombe cells, implying that this type of induction of the pbh1 gene is mediated by Pap1. Ethanol (2.0%), a nonfermentable carbon source, was also able to enhance the synthesis of $\beta$-galactosidase from the fusion gene in wild-type cells but not in Pap1-negative cells. The results indicate that the S. pombe pbh1 gene is up-regulated under metabolic oxidative stress in a Pap1-dependent manner.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.84-91
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• 2006

A Saccharomyces cerevisiae pgs1 nulI mutant, which is deficient with phosphatidyl glycerol (PG) and cardiolipin (CL) biosynthesis, grows well on most fermentable carbon sources, but fails to grow on non-fermentable carbon sources such as glycerol, ethanol, and lactate. This mutant also cannot grow on galactose medium as the sole carbon source. We found that the incorporation of $[^{14}C]-galactose$, which is the first step of the galactose metabolic pathway (Leloir pathway), into the pgs 1 null mutant cell was extremely repressed. Exogenously expressed PGS1 (YCpPGS1) under indigenous promoter could completely restore the pgs1 growth defect on non-fermentable carbon sources, and dramatically recovered $[^{14}C]-galactose$ incorporation into the pgs1 mutant cell. However, PGS1 expression under the GALl promoter $(YEpP_{GAL1}-PGS1myc)$ could not complement pgs1 mutation, and the GAL2-lacZ fusion gene $(YEpP_{GAL2}-lacZ)$ also did not exhibit its $\beta-galactosidase$ activity in the pgs1 mutant. In wild-type yeast, antimycin $A(1\;{\mu}g/ml)$, which inhibits mitochondrial complex III, severely repressed not only the expression of the GAL2-lacZ fusion gene, but also uptake of $[^{14}C]-galactose$. However, exogenously expressed PGS1 partially relieved these inhibitory effects of antimycin A in both the pgs1 mutant and wild-type yeast, although it could not basically restore the growth defect on galactose by antimycin A. These results suggest that the PGSI gene product has an important role in utilization of galactose by Gal genes, and that intact mitochondrial function with PGS1 should be required for galactose incorporation into the Leloir pathway. The PGS1 gene might provide a clue to resolve the historic issue about the incapability of galactose with deteriorated mitochondrial function.

• Mycobiology
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• 제48권4호
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• pp.326-329
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• 2020

Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.

• 한국미생물·생명공학회지
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• 제51권4호
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• pp.449-456
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• 2023

Sugar or dextrose increases the cost of production of xanthan gum by Xanthomonas campestris. Brewers' Spent Grain (BSG) was chosen as a source of fermentable sugars. BSG is a significant industrial by-product generated in large quantities from the breweries. Primarily used as animal feed due to its high fiber and protein content, BSG holds great potential as an economically and ecologically sustainable substrate for fermenting biomolecules. This study explores BSG's potential as a cost-effective carbon source for producing xanthan, utilizing Xanthomonas campestris NCIM 2961. An aqueous extract was prepared from BSG and inoculated with the bacterium under standard fermentation conditions. After fermentation, xanthan gum was purified using a standard protocol. The xanthan yield from BSG media was compared to that from MGYP media (control). The fermentation parameters, including pH, temperature, agitation and duration were optimized for maximum xanthan gum yield by varying them at different levels. Following fermentation, the xanthan gum was purified from the broth by alcoholic precipitation and then dried. The weight of the dried gum was measured. The obtained xanthan from BSG under standard conditions and commercial food-grade xanthan were characterized using FTIR. The highest xanthan yields were achieved at 32 ℃, pH 6.0, and 72 h of fermentation at 200 rpm using BSG media. The FTIR spectra of xanthan from BSG media closely resembled that of commercial food-grade xanthan. The results confirm the potential of BSG as a cost-effective alternative carbon source for xanthan production, thereby reducing production costs and solid waste.

• 한국미생물·생명공학회지
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• 제51권3호
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• pp.257-267
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• 2023

The potential polyhydroxyalkanoates (PHA)-producing bacteria, Bacillus megaterium PP-10, was successfully isolated and studied its feasibility for utilization of pineapple peel waste (PPW) as a cheap carbon substrate. The PPW was pretreated with 1% (v/v) H₂SO₄ under steam sterilization and about 26.4 g/l of total reducing sugar (TRS) in pineapple peel hydrolysate (PPH) was generated and main fermentable sugars were glucose and fructose. A maximum cell growth and PHA concentration of 3.63 ± 0.07 g/l and 1.98 ± 0.09 g/l (about 54.58 ± 2.39%DCW) were received in only 12 h when grown in PPH. Interestingly, PHA productivity and biomass yield (Y_x/s) in PPH was about 4 times and 1.5 times higher than in glucose. To achieve the highest DCW and PHA production, the optimal culture conditions e.g. carbon to nitrogen ratios of 40 mole/mole, incubation temperature at 35℃ and shaking speed of 200 rpm were performed and a maximum DCW up to 4.24 ± 0.04 g/l and PHA concentration of 2.68 ± 0.02 g/l (61% DCW) were obtained. The produced PHA was further examined its monomer composition and found to contain only 3-hydroxybutyrate (3HB). This finding corresponded with the presence of class IV PHA synthase gene. Finally, certain thermal properties of the produced PHA i.e. the melting temperature (T_m) and the glass transition temperature (T_g) were about 176℃ and -4℃, respectively whereas the M_w was about 1.07 KDa ; therefore, the newly isolated B. megaterium PP-10 is a promising bacterial candidate for the efficient conversion of low-cost PPH to PHA.

• 생명과학회지
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• 제13권6호
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• pp.933-942
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• 2003

Coenzyme Q은 긴 isoprenoid 사슬을 갖는 quinone의 유도체이다. Coenzyme Q는 진핵생명체의 미토콘드리아의 내막과 원핵생명체의 세포막에 위치하는 전자전달계에 존재하는 지용성 물질이며, 또한 항산화제로의 기능도 갖는다. Coenzyme Q는 Saccharomyces cerevisiae의 호기적 성장에 필수적이며, coq 돌연변이체는 발효가 불가능한 탄소 원에서의 성장이 불가능하다. S. cerevisiae의 $coq^7$p 효소들과 유사성을 나타내는 단백질을 암호화하는 Neurcspora crassa cDNA를 효모의 발현 벡터에 삽입하였다. N. crassa COQ7의 예상 서열은 S. cerevisiae의 효소와 58% homology를 보였다. N. crassa $coq^{-7}$ 유전자의 S. cerevisiae $coq^7$ 형질전환체는 야생형 균주와 유사한 성장률을 보였다. 형질전환 균주들은 발효가 불가능한 탄소원인 글리세롤을 유일한 탄소원으로 배양하였을 경우에도 정상적인 성장을 나타냈다. 또한 불포화지방산인 linolenic acid를 성장 배지에 첨가하여도 야생형 균주와 유사한 생존율이 관찰되었다.

• 한국축산시설환경학회지
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• 제19권1호
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• pp.1-8
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• 2013

Slurry treatments included peanut shell, palm golden fiber, almond hull, which was added 2% of the amount of slurry, and non-treatment control (n=4 each group). Levels of odorous compounds were measured from the liquid slurry incubated in $20^{\circ}C$ for 2 wk in chamber whose structure is similar to slurry pit. Concentration of phenols and indoles was higher (p<0.05) in control (48.4, 4.0 ppm) compared to almond hull (31.5, 1.4 ppm) or palm golden fiber (29.1, 1.6 ppm) group. Short chain fatty acid (SCFA) level was lowest (p<0.05) in control (2,121 ppm) but highest in peanut shell group (3,640 ppm). Branched chain fatty acid (BCFA) concentration was highest (p<0.05) in peanut shell (296 ppm), but lowest in almond hull (90 ppm). Taken together, concentration of odorous compounds was decreased by addition of almond hull in pig slurry by which crude fiber and non-digestible fiber (NDF) may act as a carbon source.