• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.263-268
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• 2003

Recently, we identified a domain, termed MIRC3-4, for the protein-protein interaction between yeast chitin synthase 3 (CHS3) and chitin synthase 4 (CHS4). In this study, the functional roles of MIRC3-4 were examined at the G1 phase and cytokinesis of the cell cycle by Calcofluor staining and FISH. Some mutations in MIRC3-4 resulted in disappearance of the chitin ring in the early G1 phase, but did not affect chitin synthesis in the cell wall at cytokinesis. The chitin distribution in chs4 mutant cells indicated that CHS4 was involved in the synthesis of chitinring in the G1 phase and in the synthesis of cell wall chitin after cytokinesis, suggesting that Chs4p regulates chitin synthase 3 activity differently in G1 and cytokinesis. Absence of the chitin ring could be caused either by delocalization of Chs3p to the bud-neck or by improper interaction with Chs4p. When mutant cells were immunostained with a Chs3p-specific antibody to discriminate between these two alternatives, the mutated Ch3p was found to localize to the neck in all MIRC3-4 mutants. These results strongly irdicate that Chs4p regulates Chs3p as an activator but not a recruiter.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.576-581
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• 2005

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast. Saccharomyces cerevisiae, by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) of Aspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast ${\alpha}-agglutinin$, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced into S. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.547-550
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• 2000

The yeast Williopsis californica was shown to secrete a unique broad-spectrum killer toxin (Wicaltin) with antifungal activity against 14 yeast genera, including yeast-like and mycelial forms of the human pathogens Candida albicans and Sporothrix schenkii. Agar diffusion bioassays indicated that its activity was more pronounced than the antifungal potential of frequently used antimycotics; 0.07 pmol Wicaltin showed the same toxicity as 0.2 pmol miconazole and 29 pmol clotrimazole. Since the toxin's primary target would appear to be the yeast cell wall, Wicaltin may be attractive in combatting clinically relevant yeast and fungal infections.

• 미생물학회지
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• 제29권6호
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• pp.397-401
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• 1991

Yeast-form cells of cadmium ion-tolerant Hansenula anomala B-7 were changed to mycelial cells in medium containing more than $400\mu$g/ml of cadmium. Moreover, the mycelial cells were exchanged into clumped cells in a medium containing more than $1,000\mu$g/ml of cadmium. Optimal conditions of mycelial cell formation were achieved in the presence of .$1,000\mu$g/ml of cadmium with shaking cultivation for 7 days. Glucan and mannan contents of the yeast cell wall frown with $1,000\mu$g/ml of cadmium decreased by 10% compared with those grown without cadmium. However, protein and lipid contents increased about 20% respectively. By cadmium, no significant findings in specific amino acid contents were discovered.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.191-193
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• 2000

B. macerans 유래의 CGTase를 yeast surface display기술을 이용하여 S. cerevisiae의 표면에 발현된 것을 halo-test와 immunofluorescence microscopy와 flow cytometry를 통하여 확인하였다. 재조합 효모는 효소의 cyclization작용을 저해하고 CD의 분해작용을 촉진하는 glucose와 maltose를 제거하는 발효공정과 표면 발현된 CGTase의 cyclization 공정을 동시에 수행할 수 있어 CD의 생산, 분리공정을 효율적으로 개선하였다.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.1977-1988
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• 2015

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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• pp.11-11
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• 2018

Cryphonectria parasitica, chestnut blight fungus, has a characteristic of decreasing pathogenicity when infected with Cryphonectria hypovirus 1. C. parasitica is known to be one of the most representative model systems used to observe the interaction between viruses, plants and fungi. The mitogen-activated protein kinase (MAPK) pathway, which is well conserved in various organisms ranging from yeast to humans, functions in relaying phosphorylation-dependent signals within MAPK cascades to diverse cellular functions involved in the regulation of pheromone, cell wall integrity, and osmotolerance in filamentous fungi. Several genes in the MAPK pathway were revealed to be regulated by hypovirus, or to be involved in pathogenicity in C. parasitica. Among these pathways, the CWI pathway has aroused interest because CpBck1, an ortholog of yeast Bck1 (a CWI MAPKKK), was previously reported to be involved in cell wall integrity and sectorization. Interestingly, sporadic sectorization was observed in the CpBck1 mutant and sectored phenotypes were stably inherited in the progeny that were successively transferred from sectored mycelia. In this study, we analyzed the biological function of CpSlt2, downstream gene of CpBck1, to confirm whether the sectorization phenomenon occurred in the specific single gene or cell wall integrity (CWI) pathway. As results, the CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, abnormal pigmentation, CWI-related phenotypic defects, and dramatically impaired virulence. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.

• Mycobiology
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• 제38권2호
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• pp.108-112
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• 2010

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1+ null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, $\beta$-glucosidase (Psu1), cell surface protein, glucan 1,3-$\beta$-glucosidase (Bgl2), and exo-1,3 $\beta$-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.

• The Plant Pathology Journal
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• 제28권3호
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• pp.227-238
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• 2012

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in the eukaryotic cells. They are involved in many major cell processes in fungi such as stress responses, vegetative growth, pathogenicity, secondary metabolism and cell wall integrity. In this review, we summarized the advances of research on the MAPK signaling pathways in Alternaria species. As major phytopathogenic fungi, Alternaria species reduce crop production. In contrast to the five MAPK pathways known in yeast, only three MAPK pathways as Fus3/Kss1-type, Hog1-type, and Slt2-type have been characterized in Alternaria. The Fus3/Kss1-type MAPK pathway participates in regulation of vegetative growth, conidiation, production of some cell-wall-degrading enzymes and pathogenicity. The Hog1-type pathway is involved in osmotic and oxidative stress, fungicides susceptibility and pathogenicity. The Slt2-type MAP kinases play an important role on maintaining cell wall integrity, pathogenicity and conidiation. Although recent advances on the MAPK pathways in Alternaria spp. reveal many important features on the pathogenicity, there are many unsolved problems regarding to the unknown MAP kinase cascade components and network among other major signal transduction. Considering the economic loss induced by Alternaria spp., more researches on the MAPK pathways will need to control the Alternaria diseases.

• 한국식품과학회지
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• 제34권5호
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• pp.867-872
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• 2002

정미성이 높은 효모 추출물을 얻고자 각종 효소의 사용에 대한 최적 조합 및 공정법을 알아보기 위하여 맥주 폐효모박을 각종 효소로 처리하여 효모추출물 중의 정미성분(IMP, GMP 및 유리아미노산)을 측정하여 비교, 분석하였다. Glucanase(0.5%) 처리에 의한 효모추출물중의 조단백질 함량은 33.6% 이었다. Tunicase(1%) 28.0% 와 무처리구 21.1%에 비해 최고 1.6배의 증가를 보였다. 단백질 분해효소처리에 의한 조단백질의 함량은 bromelin(1%), protamex(1%) 처리에서 각각 30.8%, 29.8%로 무처리구에 비해 최고 1.4배의 증가를 보였다. 효소 복합처리에 의한 상승효과는 glucanase(0.5%)+protamex(1%) 처리구에서 조단백질의 함량이 34.4%로 나타나 glucanase 단독처리구의 33.6%보다 높은 함량을 나타냈다. IMP+GMP 총함량은 glucanase + phophodiesterase + adenyldeaminase (G+P+A) 혼합 처리구에서 1,066 mg/100 g, glucanase + ptotamex + phophodiesterase + adenyldeaminase (G+Pro+P+A) 혼합 처리구에서는 1,047 mg/100 g으로 비슷하였다. 유리아미노산의 함량은 protamex가 첨가된 G+Pro+P+A 혼합처리구에서 2,302 mg/100 g으로 가장 높게 나타났다. 따라서 IMP, GMP 및 유리아미노산의 함량을 모두 고려해 볼 때 세포벽 분해효소 (gulcanase 0.5%, 12시간), 단백질 분해효소 (protamex 1%, 3시간), 핵산 분해효소 (phosphodiesterase 0.1%, 3시간) 및 핵산 전이효소 (adenyldeaminase 1%, 1.5시간)를 순차적으로 적용시켜 가수분해시키는 것이 효모 추출물의 정미성을 높이는 최적의 효소 복합 사용공정으로 판단되었다.