• 한국미생물·생명공학회지
• /
• 제31권1호
• /
• pp.63-68
• /
• 2003

구성적으로 고온내성과 고온발효능을 가지고 있는 Saccharomyces cerevisiae KNU5377 균주는 YPD 배지에 0.5M NaCl을 첨가한 YPDS 배지에서 자란 경우, 일반 YPD에서 성장한 지수성장기 세포에서 약 80%의 생존율을 보이던 고온내성이 20% 생존율을 보이고, 또한 약 8.5%정도의 알코올 생산량을 보이던 것이 6%수준으로 감소하여 대조균주와 유사한 고온발효능과 고온내성도를 보였다. 즉 구성적으로 아주 높은 내성도와 발효능을 가진 이 균주가 대조균주와 동질화되는 현상이 발생한 것이다. 이것은 S. cerevisiae KNU5377의 고온내성과 고온발효능의 원인이 이 균주 특유의 NaCl adaptation 과정과 밀접한 관계가 있음을 암시하는 것이다. 또한 이 균주의 고온발효능은 heat adaptation에 의해서 그 알코올 생산량의 증가폭 또한 대조 균주보다 2배 이상을 보이므로, 이 S. cerevisiae KNU5377가 가지는 생리적 특징이 최소한 대조균주에 비해서는 heat adaptation의 효과를 더 크게 볼 수 있는 시스템을 가지고 있음을 알 수 있었다. 이러한 특성들은 그 균주 특이적 현상의 원인을 밝힐 수 있는 중요한 단서로서 활용이 가능하며, 이 균주가 충분히 고온하에서의 알콜 생산 균주가 될 수 있음을 보여주고 있다.

• 생명과학회지
• /
• 제13권1호
• /
• pp.110-117
• /
• 2003

고온내성을 가진 효모 균주, Saccharomyces cerevisiae KNU5377을 황산, 질산 그리고 염산에 노출시켰다. 스트레스 원으로써 무기산은 물에서 쉽게 해리되어 외부 산도를 떨어뜨린다. 여러 가지 무기산이 첨가된 조건에서 배양한 결과 KNU5377은 0.4%의 황산, 질산 농도에서 생육이 가능한 반면 대조 균주인 S. cerevisiae ATCC24858은 이 보다 낮은 농도인 0.3%가 생육의 한계였다. 더욱이 KNU5377은 0.35%의 염산에서 90분 이상, 0.6%의 황산에서는 30분 이상 생존이 가능한 높은 내성을 나타내었다. 반면에 두 균주 모두 0.45%의 질산에서는 생존하지 못하였다. 0.3%의 황산이 첨가된 조건에서 알코올 발효 시 KNU5377은 이틀 후 3%의 알코올을 생산하였다. 더욱이 0.2%의 황산 첨가와 동시에 $40^{\circ}C$ 고온에서도 4.5%의 높은 알코올 생산이 관찰되었다. 또한 황산 0.2%에 한 시간동안 노출시킨 뒤 세포내에 축적되는 trehalose의 농도를 측정한 결과, KNU5377에서는 30분내에 효과적으로 축적되었으며 동일한 스트레스 조건에서 전자현미경(TEM)을 통한 세포의 형태의 관찰 시 어떠한 변화도 나타나지 않았다.

• Journal of Microbiology and Biotechnology
• /
• 제15권6호
• /
• pp.1240-1249
• /
• 2005

Saccharomyces cerevisiae KNU5377 has potential as an industrial strain that can ferment wasted paper for fuel ethanol at $40^{\circ}C$ [15, 16]. To understand the characteristics of the strain, genome-wide expression was performed using DNA microarray technology. We compared the homology of the DNA microarray between genomic DNAs of S. cerevisiae KNU5377 and a control strain, S. cerevisiae S288C. Approximately $97\%$ of the genes in S. cerevisiae KNU5377 were identified with those of the reference strain. YHR053c (CUP1), YLR155c (ASP3), and YDR038c (ENA5) showed lower homology than those of S. cerevisiae S288C. In particular, the differences in the regions of YHR053c and YLR155c were confirmed by Southern hybridization, but did not with that of the region of YDR038c. The expression level of mRNA in S. cerevisiae KNU5377 and S288C was also compared: the 550 ORFs of S. cerevisiae KNU5377 showed more than two-fold higher intensity than those of S. cerevisiae S288C. Among the 550 ORFs, 59 ORFs belonged to the groups of ribosomal proteins and mitochondrial ribosomal proteins, and 200 ORFs belonged to the group of cellular organization. DIP5 and GAP1 were the most highly expressed genes. These results suggest that upregulated DIP5 and GAP 1 might take the place of ASP3 and, additionally, the sensitivity against copper might be contributable to the lowest expression level of copper-binding metallothioneins encoded by CUP 1a (YHR053c) and CUP1b (YHR055c) in S. cerevisiae KNU5377.

• 생명과학회지
• /
• 제19권4호
• /
• pp.429-435
• /
• 2009

산화적 스트레스는 정상적인 대사 과정뿐만 아니라 외부적인 환경에 노출 되었을 때 일어나는 것으로 잘 알려져 있다. 이러한 스트레스를 극복하기 위해 생물체들은 각자의 시스템에 맞게 다양한 항산화 시스템을 진화 발전시켜 왔다. Saccharomyces cerevisiae KNU5377 균주는 고온뿐만 아니라 다양한 스트레스에 대해 내성을 가짐을 확인하였다. 대부분의 스트레스는 궁극적으로는 산화적 스트레스로 귀결된다. 이러한 측면에서 본 연구는 KNU5377 균주가 어떠한 시스템에 의해서 다른 균주보다 스트레스 내성을 가지는지를 밝히기 위해 접근하였다. 수행된 연구결과에서 KNU5377 균주는 항산화 시스템과 밀접하게 관련된 단백질(superoxide dismutase, thioredoxin system, heat shock proteins)과 항산화 관련 물질(trehalose)을 과발현함을 확인하였다. 그러나 이러한 단백질들이 어떠한 조절 시스템에 의해서 균주 특이적인 발현 양상을 보이는지는 현재까지 확인되지 않고 있다. 본 연구는 KNU5377 균주 그 자체의 중요성과 함께 균주 내의 스트레스 내성과 관련된 유용한 유전자를 탐색하여 더욱 우수한 유전자원을 발굴하는데 기여 할 것으로 보인다.

• 생명과학회지
• /
• 제16권3호
• /
• pp.454-458
• /
• 2006

출아효모인 Sacharomyces cerevisiae S288C균주를 이용한 효모의 게놈이 완성된 후 S. cerevisiae는 다양한 연구 모델로 이용되어져 왔다. 현재까지 효모를 이용한 기능 유전체학 측면에서의 연구는 laboratory strainin인 S288C 균주 또는 그 유래의 균주들이다. 그러나 자연에서 분리된 효모 또는 산업적으로 이용되어지고 있는 S. cerevisiae의 유전학 측면에서의 연구는 낮은 포자형성률 및 형질전환률, 그리고 S288C 균주와의 게놈상의 상이성 때문에 거의 이루어지지 않고 있다. 여기서 우리 연구진은 자연에서 분리된 Saccharomyces cerevisiae KNU5377 균주를 이용하여 random spore analysis를 통해 MATa 및 $MAT{\alpha}$ 타입의 각각의 haploid cell을 분리 후 이미 보고된 KanMX module를 가지고 round PCR기법에 의한 short flanking homology 기법을 이용하여 전사조절인자인 HSF1 유전자가 치환된 변이주를 구축할 수 있었다. 덧붙여, 모든 유전자에 이 기법을 적용할 수는 없다는 것을 확인하였다. 앞으로 이 변이주를 통해 기능 유전체학적인 측면에서 이 유전자의 스트레스와의 관련성을 연구하고자 한다.

• Journal of Microbiology and Biotechnology
• /
• 제13권1호
• /
• pp.85-89
• /
• 2003

The difference in the thermotolerance between Saccharomyces cerevisiae KNU5377 and ATCC24858 was compared by assaying the amounts of trehalose accumulated under growth and heat shock conditions. Both strains exhibited similar trehalose accumulation during the growth period, but an intrinsic thermotolerance was much higher in KNU5377 than in the control strain. This result implied that some strain-specific characteristics of KNU5377, other than trehalose accumulation, primarily were responsible fur its higher intrinsic thermotolerance. Heat shock at $43^{\circ}C$ for 90 min to the exponentially growing cells resulted in the maximum level of trehalose In both strains. Trehalose accumulated at least twice more in KNU5377 by the heat shock than in the control, due to the maintenance of its neutral trehalase activity even after the heat shock. Consequently, the Increase of acquired thermotolerance in both strains correlated with an increase in the trehalose content in each strain. In conclusion, KNU5377 exhibited a well-modulated trehalose-related mechanism to accumulate more trehalose by means of maintaining neutral trehalase activity after heat shock than the control strain, thereby contributing to its acquired thermotolerance.

• Journal of Microbiology
• /
• 제45권4호
• /
• pp.326-332
• /
• 2007

In order to understand the functional role of CPRl in Saccharomyces cerevisiae KNU5377 with regard to its multi-tolerance characteristics against high temperatures, inorganic acids, and oxidative stress conditions, whole cellular proteins were analyzed via liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This procedure was followed by two-dimensional (2-D) gel electrophoresis. Under menadione stress conditions, the 23 upregulated proteins were clearly identified only in the wild- type strain of KNU5377. Among the proteins, Sodl1p Tsa1p, Ahp1, Cpr1p, Cpr3, Ssb2p, and Hsp12p were identified as components of antioxidant systems or protein-folding related systems. The CPR1 protein could not be completely detected in the $cpr1{\Delta}$ mutant of KNU5377 and the other upregulated proteins in the wild-type strain evidenced a clear correlation with the results of immunoblot analysis. Moreover, a reduction in growth patterns (about 50%) could be observed in the $cpr1{\Delta}$ mutant, as compared with that of the wild-type strain under mild MD stress conditions. These results indicate that the upregulation of CPR1 may contribute to tolerance against MD as an inducer of oxidative stress.

• Journal of Microbiology
• /
• 제44권5호
• /
• pp.492-501
• /
• 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.

• Journal of Life Science
• /
• 제12권2호
• /
• pp.47-52
• /
• 2002

Saccharomyces cerevisiae KNU5377 (KNU5377) and S. cerevisiae ATCC24858 (ATCC24858) were exposed to $H_2SO_4$ as a stress, which was added at various concentrations to a YPD media. The growth of KNU5377 was reduced to approximately 60% in the YPD media containing 40 nm sulfuric acid when compared to the non-stressed condition. When their growth was monitored during an overnight culture, two strains, KNU5377 and ATCC24858, could not grow when exposed to over 50 mM of sulfuric acid. After a short exposure to this acid for 1 h, KNU5377 exhibited stronger resistance against $H_2SO_4$ than ATCC24858. The neutral trehalase activity of KNU5377 unchanged despite under various concentrations of $H_2SO_4$. In contrast, It at of ATCC24858 was much low at higher $H_2SO_4$concentrations. Trehalose, a non-reducing disaccharide, was maximally accumulated after a short exposure to 60 nm $H_2SO_4$ for KNU5377, but it was reduced under more severe stressful conditions. These results suggest that KNU5377 should modulate the trehalose concentrations under the severe stress condition of high sulfuric acid concentrations. The most highly induced protein in the KNU5377 exposed to sulfuric acid was found to be an approximately 23 kDa protein, which was revealed to be the 605 large subunit ribosomal protein, Ll3 by FASTA search results.

• Journal of Microbiology and Biotechnology
• /
• 제17권2호
• /
• pp.207-217
• /
• 2007

The Saccharomyces0 cerevisiae KNU5377 strain, which was isolated from spoilage in nature, has the ability to convert biomass to alcohol at high temperatures and it can resist against various stresses [18, 19]. In order to understand the defense mechanisms of the KNU5377 strain under menadione (MD) as oxidative stress, we used several techniques for study: peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) followed by two-dimensional (2D) gel electrophoresis, liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technology. Among the 35 proteins identified by MALDI-TOF MS, 19 proteins including Sod1p, Sod2p, Tsa1p, and Ahp1p were induced under stress condition, while 16 proteins were augmented under normal condition. In particular, five proteins, Sod1p, Sod2p, Ahp1p, Rib3p, Yaf9p, and Mnt1p, were induced in only stressed cells. By LC-ESI-MS/MS analysis, 37 proteins were identified in normal cells and 49 proteins were confirmed in the stressed cells. Among the identified proteins, 32 proteins were found in both cells. Five proteins including Yel047cp and Met6p were only upregulated in the normal cells, whereas 17 proteins including Abp1P and Sam1p were elevated in the stressed cells. It was interesting that highly hypothetical proteins such as Ynl281wp, Ygr279cp, Ypl273wp, Ykl133cp, and Ykr074wp were only expressed in the stressed cells. SELDI-TOF analysis using the SAX2 and WCX2 chips showed that highly multiple-specific protein patterns were reproducibly detected in ranges from 2.9 to 27.0 kDa both under normal and stress conditions. Therefore, induction of antioxidant proteins, hypothetical proteins, and low molecular weight proteins were revealed by different proteomic techniques. These results suggest that comparative analyses using proteomics might contribute to elucidate the defense mechanisms of KNU5377 under MD stress.