• Mycobiology
• /
• v.47 no.1
• /
• pp.59-65
• /
• 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
• /
• 2003.10a
• /
• pp.468-472
• /
• 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.

• Journal of Life Science
• /
• v.19 no.12
• /
• pp.1685-1689
• /
• 2009

Phytochelatins (PCs) are small polypeptides synthesized by PC synthase (PCS). They are present in various living organisms including plants, fission yeast, and some animals. The presumed function of PCs is the sequestration of cytosolic toxic heavy metals like cadmium (Cd) into the vacuoles via vacuolar membrane localized heavy metal tolerance factor 1 (HMT-1). HMT-1 was first identified in fission yeast (SpHMT-1), and later in Caenorhabdtis (CeHMT-1). Recently, its homolog has also been found in PC-deficient Drosophila (DmHMT-1), and this homolog has been shown to be involved in Cd detoxification, as confirmed by the heterologous expression of DmHMT-1 in fission yeast. Therefore, the dependence of HMT-1 on PC in Cd detoxification should be re-evaluated. I heterologously expressed SpHMT-1 in cytosolic PC-deficient yeast, Saccharomycea cerevisiae, to understand the dependence of HMT-1 on PC. Yeast cells expressing SpHMT-1 showed increased tolerance to Cd compared with control cells. This result indicates that SpHMT-1 is not strictly correlated with PC production on its function. Moreover, yeast cells expressing SpHMT-1 showed increased tolerance to exogenously applied glutathione (GSH) compared with control cells, and the tolerance to Cd was further increased by exogenously applied GSH, while tolerance in control cells was not. These results indicate that the function of SpHMT-1 in Cd detoxification does not depend on PCs only, and suggest that SpHMT-1 may sequester cytosolic GSH-Cd complexes into the vacuole.

• Journal of Microbiology
• /
• v.44 no.3
• /
• pp.311-319
• /
• 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
• /
• v.17 no.6
• /
• pp.555-559
• /
• 2002

Three different strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities of enzyme production. It was found that specific enzyme activity was the highest with strain ATCC 9348 and the enzyme production was closely coupled to growth. Studies on morphology during the growth of A. pullulans revealed that mycelia cells were dominant at the initial stages of growth. However, yeast-like cells and chlamydospores were dominant in the latter stages of batch culture. The pattern of morphological changes during the growth period was not affected by pH. However, it appears that the ratio of intra- to extracellular enzyme activity tended to increase with fermentation time irrespective of the pH employed, suggesting that the secretion efficiency of intracellular enzyme to broth likely depends on cell morphology Using molasses as a cheap source of sucrose, enzymatic production of fructo-oligosaccharides as a feed additive with A. pullulans cells could be achieved successfully at 55$\^{C}$ and pH 5.5.

• Journal of Veterinary Science
• /
• v.24 no.1
• /
• pp.15.1-15.13
• /
• 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
• /
• v.31 no.6
• /
• pp.607-610
• /
• 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
• /
• v.41 no.1
• /
• pp.35-44
• /
• 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.

• Korean Journal of Microbiology
• /
• v.47 no.2
• /
• pp.163-166
• /
• 2011

For reduction of side effects by anti-fungal agents, a less toxic anti-fungal substance or a synergistic substance with a new mechanism is needed. The anti-yeast substance (AYS) originated from Rahnella aquatilis strain AY2000 is like to be a heterogeneous protein. The AYS inhibited the growth of Candida albicans in culture broth, and AYS-treated cells were arrested in each phase during cell cycle. Among AYS-treated cells, the population of the cells belonging to sub-G1 phase was not increased during cell cycle. Therefore, AYS has rather yeaststatic than yeastcidal effect to C. albicans. Moreover, with combination of itraconazole or fluconazole, AYS had a synergistic anti-yeast activity against Saccharomyces cerevisiae based on the analysis of fractional inhibitory concentration index.

• Korean Journal of Microbiology
• /
• v.22 no.1
• /
• pp.1-18
• /
• 1984

With 156 water samples collected from 39 locations in the Yeong San River estuary during the 12month period from March 1976 to February 1977, the seasonal distribution of yeast and the distributional pattern of yeast on salinity gradient have been investigated. An overall average number of yeast ranged from 52 to 487 viable cells (c.f.u.) per 100ml water sample. The highest count of yeast was obtained in spring while the lowest value came in summer. 933 yeast and one yeast-like fungus pertaining to 14 genera and 83 species were recovered, of which Candida were 29%, Debaryomyces 17.3%, Rhodotorula Glutinis were dominant forms in all locations as well as throughout the year. The population size of total aerobic bacteria, the amount of terrestrial imputs, and some of geographical and/or climatic factor appear to reflect the seasonal distribution of yeast as well as the composition of yeast species in an estuarine environ. Average number of yeast, species diversity, and particularly the number of fermentative and pseudomycelium-producing yeasts increased with decreasing salinity whereas nitrate-utilizing yeasts showed opposite trend, suggesting that salinity gradient can be used as a feasible detector for the distributional pattern of yeast in estuarine habitat.