• Mycobiology
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• 제47권1호
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• pp.59-65
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• 2019

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (${\leq}0.1%$) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per $10^6$ YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.468-472
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• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• 생명과학회지
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• 제19권12호
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• pp.1685-1689
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• 2009

파이토킬레이트(PC)는 PCS에 의해 생성되는 작은 폴리펩타이드로서 여러 생물에서 발견되고 있다. PC의 역할은 카드뮴과 같은 중금속을 세포질에서 결합하며 이는 액포막에 존재하는 HMT에 의해서 액포 안으로 이동된다. HMT1은 분열효모에서 처음으로 알려졌으며 이후 선충, 초파리 등에서도 발견되었으며 세포 내 역할은 카드뮴 같은 중금속 해독에 관여를 하고 있다. 하지만 액포가 존재하지 않고 PC를 생성하지 않는 초파리에서의 HMT1의 발견은 그 동안 알려진 HMT1의 역할을 재 조명하게 된다. 따라서 PC를 생성하지 못하는 출아효모에 PC를 생성하는 분열효모 유래 SpHMT1을 발현시켜 카드뮴에 대한 저항성을 분석하였다. SpHMT1을 발현하는 출아효모는 카드뮴에 대한 저항성이 현저하게 증가되었고 이는 SpHMT1이 PC가 존재하지 않는 조건에서도 카드뮴에 대한 해독작용을 하는 것을 암시한다. 또한 SpHMT1을 발현하는 출아효모는 GSH에 대한 저항성을 보였고 카드뮴에 대한 저항성도 GHS에 의해서 더 증가되는 결과를 보였다. 이러한 결과는 HMT1이 PC와 결합된 카드뮴을 액포안으로 이동시키는 가능성보다 GSH와 결합된 카드뮴을 액포 안으로 이동시켜 카드뮴에 대한 해독작용을 한다는 것을 암시한다.

• Journal of Microbiology
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• 제44권3호
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• pp.311-319
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• 2006

Bud6p is a component of a polarisome that controls cell polarity in Saccharomyces cerevisiae. In this study, we investigated the role of the Candide albicans Bud6 protein (CaBud6p) in cell polarity and hyphal development. CaBud6p, which consists of 703 amino acids, had 37% amino-acid sequence identity with the Bud6 protein of S. cerevisiae. The homozygous knock-out of CaBUD6 resulted in several abnormal phenotypes, such as a round and enlarged cells, widened bud necks, and a random budding pattern. In hypha-inducing media, the mutant cells had markedly swollen tips and a reduced ability to switch from yeast to hypha. In addition, a yeast two-Hybrid analysis showed a physical interaction between CaBud6p and CaAct1p, which suggests that CaBud6p may be involved in actin cable organization, like Bud6p in S. cerevisiae. Taken together, these results indicate that CaBud6 plays an important role in the polarized growth of C. albicans.

• KSBB Journal
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• 제17권6호
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• pp.555-559
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• 2002

프럭토올리고당 합성효소의 생산능력이 우수한 균주를 선발하기 위하여 Aureobasidium pullulan 3종을 배양하여 비교해본 결과 ATCC 9348이 비효소활성 측면에서 가장 우수했으며, 세 균주 모두 균체성장과 효소생산패턴과는 대체적으로 선형적인 관계가 있었다. 균의 형태학적 측면에서 볼 때 배양초기에는 대부분 mycelia cells 형태이었으나 배양이 점차 진행되면서 yeast-like cells 및 chlamydospores 형태로 바뀌었고, 이러한 형태변화와 더불어 균체외효소에 대한 균체내효소의 활성비율도 변화됨을 알 수 있었다. 2.5-L 발효조를 사용하여 pH 영향을 조사해본 결과 pH 7.0 범위에서 효소생산이 최대로 나타났고 pH 4.0 또는 pH 8.0 조건에서는 균체외효소에 대한 균체내효소 활성이 높게 나타나는 것으로 보아 배양조건에 따라 효소의 배출효율이 달라지는 것을 알 수 있었다. 또한 값싼 원료인 당밀로부터 A. pullulans 효소를 사용하여 사료첨가제로서의 프럭토올리고당 생산이 가능함을 확인하였다.

• Journal of Veterinary Science
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• 제24권1호
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• pp.15.1-15.13
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• 2023

Background: Inactivated vaccines are limited in preventing foot-and-mouth disease (FMD) due to safety problems. Recombinant virus-like particles (VLPs) are an excellent candidate for a novel vaccine for preventing FMD, given that VLPs have similar immunogenicity as natural viruses and are replication- and infection-incompetent. Objectives: The 3C protease and P1 polyprotein of type O FMD virus (FDMV) was expressed in yeast Hansenula polymorpha to generate self-resembling VLPs, and the potential of recombinant VLPs as an FMD vaccine was evaluated. Methods: BALB/c mice were immunized with recombinant purified VLPs using CpG oligodeoxynucleotide and aluminum hydroxide gel as an adjuvant. Cytokines and lymphocytes from serum and spleen were analyzed by enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and flow cytometry. Results: The VLPs of FMD were purified successfully from yeast protein with a diameter of approximately 25 nm. The immunization of mice showed that animals produced high levels of FMDV antibodies and a higher level of antibodies for a longer time. In addition, higher levels of interferon-γ and CD4⁺ T cells were observed in mice immunized with VLPs. Conclusions: The expression of VLPs of FMD in H. polymorpha provides a novel strategy for the generation of the FMDV vaccine.

• BMB Reports
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• 제31권6호
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• pp.607-610
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• 1998

Transcription of mitochondrial DNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mitochondrial RNA polymerase specificity factor, which is a 43 kDa polypeptide encoded by the nuclear MTF1 gene. Mtf1p has only limited amino acid sequence homology to bacterial sigma factors, but functions in many ways like sigma in that it is required for promoter recognition and initiation of transcription. To analyze the corebinding region of Mtf1p, monoclonal antibodies to this protein were prepared. Recombinant Mtf1p overproduced in E. coli was purified to near homogeneity and used to raise monoclonal antibodies (mAbs). From fused cells screened for Mtf1p mAbs by immunodot blot analysis, 19 positive clones were initially isolated. Further analysis of positive clones by Western blotting resulted in 4 mAbs of Mtf1p.

• Molecules and Cells
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• 제41권1호
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• pp.35-44
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• 2018

Mitochondria are responsible for supplying of most of the cell's energy via oxidative phosphorylation. However, mitochondria also can be deleterious for a cell because they are the primary source of reactive oxygen species, which are generated as a byproduct of respiration. Accumulation of mitochondrial and cellular oxidative damage leads to diverse pathologies. Thus, it is important to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Eukaryotes have developed defense mechanisms to cope with aberrant mitochondria. Mitochondria autophagy (known as mitophagy) is thought to be one such process that selectively sequesters dysfunctional or excess mitochondria within double-membrane autophagosomes and carries them into lysosomes/vacuoles for degradation. The power of genetics and conservation of fundamental cellular processes among eukaryotes make yeast an excellent model for understanding the general mechanisms, regulation, and function of mitophagy. In budding yeast, a mitochondrial surface protein, Atg32, serves as a mitochondrial receptor for selective autophagy that interacts with Atg11, an adaptor protein for selective types of autophagy, and Atg8, a ubiquitin-like protein localized to the isolation membrane. Atg32 is regulated transcriptionally and post-translationally to control mitophagy. Moreover, because Atg32 is a mitophagy-specific protein, analysis of its deficient mutant enables investigation of the physiological roles of mitophagy. Here, we review recent progress in the understanding of the molecular mechanisms and functional importance of mitophagy in yeast at multiple levels.

• 미생물학회지
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• 제47권2호
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• pp.163-166
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• 2011

항 진균제의 인체에 대한 부작용을 완화시키는 방법은 부작용이 적은 새로운 항 진균제 개발이나 기존 항 진균제와 혼용하여 상승효과를 나타내는 물질이 필요하다. 이를 위해 Rahnella aquatilis AY2000가 생산하는 항 효모물질은 일종의 단백질성 고분자 물질이란 점에서 기존 항 진균제와 차이가 있다. 이 항 효모물질은 유도기나 대수증식 기에 있는 효모 생육을 억제시켰으며, cell cycle 분석에서 sub-G1기에 속하는 세포수의 증가 없이 세포를 arrest시켰다. 따라서 항 효모물질은 Candida albicans에 정균작용을 나타내었다. 또한 in vitro 실험에서 이 항 효모물질과 itraconazole이나 fluconazole을 병용한 후 fractional inhibitory concentration index 분석을 행한 결과 항 효모활성이 더욱 상승되었다. 결론적으로 이 항 효모물질은 정균 작용을 가질 뿐만 아니라 azole계의 항 진균제와 혼용하면 상승효과를 가짐으로 부작용이 적은 새로운 개념의 항 진균제로 개발할 가치가 있을 것이라고 생각된다.

• 미생물학회지
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• 제22권1호
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• pp.1-18
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• 1984

1976年 3月부터 1977年 2月까지 滿 12個月동안 榮山江 汽水域 39個 場所로부터 採水한 156個 water sample로서 酵母의 季節的 分布와 度包配에 따른 이들 酵母의 分布型을 調査하였다. 酵母의 全體 平均値는 100ml당 52에서 487個의 生菌數를 보여주었고 가장 많은 個體數는 春季에 있었고 가장 적은 個體數는 夏季에 있었다. 14屬 83種에 해당하는 933個의 酵母와 한 種의 酵母樣 곰팡이가 分離되었는데 基中 Candida가 29%, Debaryomyces가 17.3%, Rhodotorula가 16%, Saccharomyces가 14%이었고 나머지 다른 屬들은 10%以內이었다. Debaryomyces hansenii와 Rhodotorula glutinis가 全水域에서는 물론 年中 優點種이었고 총 好氣性細菌集團의 크기, 陸水流入量, 그리고 몇가지 지형적 기후적 요소들이 汽水환경에서 酵母의 季節的 分布는 물론 種의 組成을 反映하는 같다. 酵母의 平均數, 種의 多樣性 特히 醱酵性 및 爲菌系形成 母酵들의 數는 감度가 감소함에 따라 增加한 反面 질산염 利用酵母들은 위와 相反된 경향을 보여주었는데 이는 汽水域에서 酵母의 分布型을 認識하는데 감度勾配가 바람직한 看破 요소로서 利用될 수 ㅣ있음을 시사하고 있다.