• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• 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.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.307-313
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• 2008

Bacillus sp. endoxylanase 유전자(xynB, 642 bp)의 효모 표면발현계 pCTXYN(6.8 kb)를 구축하고 Saccharomyces cerevisiae EBY100에 형질전환시켜 형질전환체 EBY100/pCTXYN를 얻었다. 형질전환체들을 xylan이 포함된 YPDG 배지에서 배양 후 활성염색을 통하여 고찰성의 형질전환체를 최종 선별하였다. 갈락토스 배지에서 자란 효모 형질전환체로부터 xynB는 성공적으로 표면발현되었고, xylan으로 부터 xylooligosaccharides를 효율적으로 생성함도 화인하였다. Endoxylanase 활성은 세포분획에서만 검출되었고 배양 48시간에 최종 1.9 unit/mL의 활성을 보였다. Xylooligosaccharides 생산을 위한 치적 반응 조건으로, 기질과 농도는 oat spelt xylan 6%, 효모 생촉매 농도는 5 unit/mL, 반응온도는 $50^{\circ}C$, 반응시간은 $2{\sim}4$시간이었다 효모 생촉매를 oat spelt xylan과 corncob xylan에 처리한 결과, xylotriose가 주성분이었다.

• Journal of Microbiology and Biotechnology
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• 제24권3호
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• pp.408-420
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• 2014

Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and $V{\kappa}1$-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than $10^9$ by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ${\sim}10^7$. The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

• 미생물학회지
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• 제55권3호
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• pp.199-205
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• 2019

Yeast two-hybrid는 특정 단백질에 대한 상호작용 파트너 단백질의 선별을 위한 방법으로 개발되었다. 하지만 대규모 단백질 상호작용체 분석을 수행하기에 요구되는 노동과 대량의 한천배지 사용에 따른 문제에 의해 널리 사용되지 못하고 있다. 따라서 본 연구에서는 새로운 리포터 시스템을 yeast two-hybrid 방법에 도입하여 fluorescence-activated cell sorting (FACS) 또는 magnetic-activated cell sorting (MACS)를 이용하여 상호작용 파트너 단백질을 포함하는 효모 클론을 손쉽게 선별할 수 있도록 하였다. 새로운 리포터 시스템은 c-myc 항원 결정기가 총 10번 반복되는 형태로 효모 표면에 발현되도록 하였으며, p53과 SV40 T항원을 이용한 실험을 통하여 리포터 단백질의 정상적인 발현을 flow cytometry 분석을 통하여 확인하였다. 따라서, 새로운 리포터 시스템을 도입한 yeast two-hybrid 방법은 대규모 상호작용체 분석을 위해 필요한 노력을 현저히 줄일 수 있을 것으로 기대한다.

• 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.

• 한국가시화정보학회지
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• 제21권3호
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• pp.93-100
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• 2023

The yeast Saccharomyces cerevisiae (S. cerevisiae) is considered an ideal eukaryotic model and has long been recognized for its pivotal role in numerous industrial production processes. Depending on the cell cycle phases, microenvironment, and species, S. cerevisiae varies in shape and has different sizes of each shape such as singlets, doublets, and clusters. Obtaining high-purity populations of uniformly shaped S. cerevisiae cells is crucial in fundamental biological research and industrial operations. In this study, we propose an acoustofluidic method for separating S. cerevisiae cells based on their size using surface acoustic wave (SAW)-induced acoustic radiation force (ARF). The SAW-induced ARF increased with cell diameter, which enabled a successful size-based separation of S. cerevisiae cells using an acoustofluidics device. We anticipate that the proposed acoustofluidics approach for yeast cell separation will provide new opportunities in industrial applications.

• KSBB Journal
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• 제21권6호
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• pp.479-483
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• 2006

본 연구는 효모의 세포벽에 존재하는 본 연구는 효모의 세포벽에 존재하는 ${\beta}-Glucan$을 대량생산하기 위해 Saccharomyces cerevisiae JUL3의 균체량을 증가시키기 위한 연구를 수행하였다. S. cerevisiae JUL3의 배양학적 특성을 알아보기 위해 kinetic parameter를 조사하였을 때 specific growth rate (${\mu}$)는 $0.145\;h^{-1}$, yield ($Y_{x/s}$)는 0.332 g/g, glucose 소모속도($q_{s}$)는 $0.437\;h^{-1}$이며 productivity (P)는 $0.4827\;g/{\ell}{\cdot}h$을 나타내었다. 균체량이 가장 높게 나타난 탄소원과 질소원은 고과당 (high fructose syrup)과 yeast extract이었다. 효모균체 대량생산 및 scale up을 위하여 반응표면 분석법을 통하여 고과당과 yeast extract에 대한 최적농도를 조사하였다. 균체량이 가장 많이 생산되어질 수 있는 조건으로 제시된 고과당과 yeast extract의 최적 농도는 각각 8.0 %와 5.2 %였으며, 이 때 예측되는 균체량은 $16.95\;g/{\ell}$ 이었다.

• 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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• 제10권3호
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• pp.333-337
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• 2000

An oil-degrading yeast, Yarrowia lipolytica 180, exhibits interesting cell surface characteristics under the growth on hydrocarbons. An electron microscopic study revealed that the cells grown on crude oil showed protrusions on the cell surface, and thicker periplasmic space and cell wall than the cell surface, and thicker periplasmic space and cell wall than the cells grown on glucose. Y. lipolytica cells lost its cell hydrophobicity after pronase(0.1 mg/ml) treatment. The strain produced two types of emulsifying materials during the growth on hydrocarbons; one was water-soluble extracellular materials and the other was cell wall-associated materials. Both emulsifying materials at lower concentration (0.12%) enhanced the oil-degrading activity of Moraxella sp. K12-7, which had medium emulsifying activity and negative cell hydrophobicity; however, it inhibited the oil-degrading activity of Pseudomunas sp. K12-5, which had medium emulsifying activity and cell hydrophobicity. These results suggest that the oil-degrading activity of Y. lipolytica 180 is closely associated with cell surface structure, and that a finely controlled application of Y.lipolytica 180 in combination with other oil-degrading microorganisms showed a possible enhancing efficiency of oil degradation.