• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.725-730
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• 2003

Three recombinant Saccharomyces cerevisiae strains showing different levels of xylose reductase activity were constructed to investigate the effects of xylose reductase activity and glucose feed rate on xylitol production. Conversion of xylose to xylitol is catalyzed by xylose reductase of Pichia stipitis with cofactor NAD(P)H. A two-substrate fermentation strategy has been employed where glucose is used as an energy source for NADPH regeneration and xylose as substrate for xylitol production. All recombinant S. cerevisiae strains Yielded similar specific xylitol productivity, indicating that xylitol production in the recombinant S. cerevisiae was more profoundly affected by the glucose supply and concomitant It generation of cofactor than the xylose reductase activity itself. It was confirmed in a continuous culture that the elevation of the glucose feeding level in the xylose-conversion period enhanced the xylitol productivity in the recombinant S. cerevisiae.

• Journal of Microbiology
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• 제38권1호
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• pp.48-51
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• 2000

In liquid cultures using sucrose media, the coculture of Monascus with recombinant Saccharomyces cerevisiae expressing the glucoamylase gene from Aspergillus niger enhanced red pigment production by approx. 19％, compared with the coculture of wild type S. cerevisiae. Coculture with recombinant S. cerevisiae was more effective than with wild type S. cerevisiae for Monascus red pigment production. Cocultures of Monascus with commercial amylases of Aspergillus also induced high production of pigment and morphological changes in a solid culture using sucrose media.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.384-388
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• 2001

The metabolic pathway synthesis algorithm was applied to estimate the maximum ethanol yield from xylose in a model recombinant Saccharomyces cerevisiae strain containing the genes involved in xylose metabolism. The stoichiometrically independent pathways were identified by constructing a biochemical reaction network for conversion of xylose to ethanol in the recombinant S. cerevisiae. Two independent pathways were obtained in xylose-assimilating recombinant S. cerevisiae as opposed to six independent pathways for conversion of glucose to ethanol. The maximum ethanol yield from xylose was estimated to be 0.46 g/g, which was lower than the known value of 0.51 g/g for glucose-fermenting and wild-type xylose-fermenting yeasts.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1529-1533
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• 2010

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 was overexpressed by using the GAL10 promotor in a ${\Delta}ga180$ strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to a mating factor ${\alpha}$ signal sequence for secretory expression. Use of the ${\Delta}ga180$ strain allowed for the galactose-free induction of inulinase expression using a glucose-only medium. Shake-flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the ${\Delta}ga180$ strain improved the expression of inulinase in the recombinant S. cerevisiae in both aerobic and anaerobic conditions by about 2.9- and 1.7-fold, respectively. A 5-l fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at the $OD_{600}$ of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5-l scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and with the pH controlled at 5.0. The temperature was maintained at $30^{\circ}C$ and $37^{\circ}C$, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/l, respectively.

• KSBB Journal
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• 제26권5호
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• pp.427-432
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• 2011

In this study, a EXGA gene code for exo-β-1,3-glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3-glucanase was successfully expressed and secreted into the medium and the β--1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3-glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.652-658
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• 1995

We attempted simultaneous expression of genes coding for endoglucanase and $\beta $-glucosidase from Pseudomonas sp. by using a synthetic two-cistron svstem in Escherichia coli and Saccharomyces cerevisiae. Two-cistron system, 5'--tac promoter-endoglucanase gene--$\beta $-glucosidase gene-- 3', 5'-tac promoter--$\beta $-glucosidase gene--endoglucanase gene--3' and 5'-tac promoter--endoglucanase gene--SD sequence--$\beta $-glucosidase gene--3, were constructed, and expressed in E. coli and S. cerevisiae. The E. coli and S. cerevisiae contained two-cistron system produced simultaneously endoglucanase and $\beta $-glucosidase. The recombinant genes contained the bacterial signal peptide sequence produced low level of endoglucanase and $\beta $-glucosidase in S. cerevisiae transformants: Approximately above 44% of two enzymes was localized in the intracellular fraction. The production of endoglucanase and $\beta $-glucosidase in veast was not repressed in the presence of glucose or cellobiose. The veast strain contained recombinant DNA with two genes hydrolyzed carboxvmethyl cellulose, and these endoglucanase and $\beta $-glucosidase degraded CMC synergistically to glucose, cellobiose and oligosaccharide. This result suggests the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.

• KSBB Journal
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• 제15권2호
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• pp.181-187
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• 2000

본 연구에서 갈락토즈를 거의 사용하지 않고 glucose repression 정도가 줄어든 변이주를 이용하여 fed-batch를 통한 발현최적화를 수행하였다. 두 균주에서의 GAL promoter에 의한 외래단백질 생산시 glucose repression 정도에 대해 조사하였는데 대조구 Y2805는 글루코즈가 다 소비된 후 2∼3시간 지난 후 발현이 시작되나 변이주 Y334는 약 0.5 g/L 글루코즈 농도에서 25%정도의 발현이 이루어짐에 따라 변이주 Y334는 GAL promoter에 미치는 glucose repression 정도가 매우 약한 장점을 확인하였다. 배양 중 재조합 미생물인 두균주 변이주 Y334와 대조구 효모 Y2805의 plasmid stability에 대해 안정한 균주임을 알 수 있었으며 대조구 Y2805의 경우도 plasmid stability는 90%로 유지됨을 알 수 있었다. GAL promoter에 의한 외래 단백질 생산시 글루코즈와 갈락토즈, 에탄올의 소비속도를 조사하였는데, 글루코즈와 에탄올의 소비속도는 거의 비슷하였으나 갈락토즈 소비속도는 Y2805는 0.1232 g/L/hr/O.D.이고 변이주 Y334는 0.0131 g/L/hr/O.D. 이다. 또한 mutant Y334와 대조구 효모 Y2805의 Fed-batch 시의 올바른 feeding 방법을 구하기위한 실험을 수행하여 각 균주의 Fed-batch 실험에서의 최적 발현 방법을 획득하였다.

• 미생물학회지
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• 제28권3호
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• pp.181-187
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• 1990

전분 분해능력을 갖는 알콜생산용 효모를 만들기 위해 Saccharomyces cerevisiae에 glucoamylase 유전자인 STA를 도입하였다. 도입된 형질의 발현증대를 위해 STA 유전자의 promoter 부위를 alcohol dehydrogenase isoenzyme I 유전자의 promoter 부위와 치환 시켜준 재조합 플라스미드를 재조하였으며 안정성을 증진시키기 위해 centrometer 부위를 치환시킨 결과 glucoamylase의 발현이 증가하였으며, STA 유전자와 centromere를 갖고 있는 재조합 플라스미드는 여러세대가 거듭되어도 비교적 안정하게 유지되었으나 낮은 copy 수로 인해 형질전환체의 효소 역가와 형질전환 빈도는 낮아졌다. STA 유전자가 도입되어 형질전환된 다배체 산업용 효모는 액화 과정만을 거친 주정생산 배지(액화액)에서 원래의 알콜 생산용 효모에 비해 훨씬 많은 양의 알콜을 생산해 내었다. 그러나 centromere를 보유하는 플라스미드에의한 산업용 효모의 형질전환에는 실패하였다.

• KSBB Journal
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• 제17권1호
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• pp.38-43
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• 2002

중금속 결합 단백질인 metallothionein (MT)를 발현하는 CUP1 유전자를 다중으로 함유하는 재조합 Saccharomyces cerevisiae의 균체성장과 중금속 제거특성을 조사하였다. CUP1 유전자를 다중으로 함유하는 재조합 효모는 중금속을 포함한 배지에서의 균체성장과 중금속 흡착능이 중금속에 대하여 내성을 가지고 있는 야생효모나 숙주세포에 비하여 우수하였다. 서로 다른 플라스미드를 함유하는 중금속을 함유하는 배지에서의 균체성장과 중금속 흡착능의 차이는 중금속에 대한 내성과 중금속 흡착에 MT 단백질의 발현 수준이 중요한 영향을 미치는 것으로 추정되었다. 구리를 함유하지 않고 고농도의 아연과 망간을 함유하는 배지에서 재조합 효모는 높은 균체 농도와 중금속 제거능을 나타내었는데, 이는 MT 단백질을 발현하는 CUP1 promoter가 아연과 망간에 의해서도 발현이 유도된다는 것을 알 수 있었다. MT 단백질을 효율적으로 발현하여 재조합 효모에 의한 중금속 흡착을 최대화 할 수 있는 최적의 구리농도는 0.31 mM로 결정되었으며, 비이온계 계면활성제인 Triton X-100은 재조합 효모의 균체성장과 중금속 흡착을 증가시켰지만, 대사 저해제는 균체성장과 중금속 흡착을 모두 저해하였다.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.411-416
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• 2002

Bacillus macerans cyclodextrin glucanotransferase (CGTase) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein, Aga1p. The surface display of CGTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form ${\alpha}$-cyclodextrin from starch. The maximum surface-display of CGTase was obtained by growing recombinant S. cerevisiae at $20^{\circ}C$ and pH 6.0. S. cerevisiae cells displaying CGTase on their surface consumed glucose and maltose, inhibitory byproducts of the CGTase reaction, to enhance the purity of produced cyclodextrins. Accordingly, the experimental results described herein suggest a possibility of using the recombinant S.cerevisiae anchored with bacterial CGTase on the cell surface as a whole-cell biocatalyst for the production of cyclodextrin.