효모 Saccharomyces cerevisiae에서의 재조합 단백질 분비생산과 분비신호

남수완 한국산업미생물학회 1994 생물산업 Vol.7 No.3

연속배양에서 고핵산 효모 변이주의 배양생리학적 특성

남수완 동의대학교 산업기술개발연구소 2005 産業技術硏究誌 Vol.19 No.-

In order to investigate the physiological characteristics for RNA accumulation, continuous cultures of S. cerevisiae MTY62 under the carbon-limitation or carbon and phosphate-limitation were performed. In the carbon-limited chemostat, the higher RNA concentration of 550 g-DCW/L and the RNA content of 144 mg-RNA/g-DCW cell was observed at the dilution rate of 0.30 hr^(-1). In the carbon- and phosphate-limited chemostat, the culture condition with the dilution rate of 0.25 hr^(-1) gave the maximal RNA content of 209 mg-RNA/g-DCW. The accumulation rate of RNA (mg-RNA/L·hr) was decreased at higher dilution rate in the carbon and phosphate-limited chemostat.

재조합 Saccharomyces cerevisiae에서 Inulinase와 Invertase의 발현과 분비에 미치는 배양조건의 영향

남수완,신동하,김연희 동의대학교 기초과학연구소 1998 基礎科學硏究論文集 Vol.8 No.1

The effects of medium pH and culture temperature on the expression and secretion and secretion of inulinase and invertase were investigated with recombinant Saccharomyces cerevisiae cells. These cells were obtained by transformation of 2μ-based plasmids pYI10 and pYS10 which contain Kluyveromyces marxianus inulinase gene (INU1A) and S. cerevisiae invertase gene (SUC2), respectively, in the downstream of GAL1 promoter. The expression level and localization of inulinase and invertase were not affected significantly by the initial medium pH: secretion efficiencies of inulinase and invertase into the medium were about 90% and 60%, respectively, in the pH ranges of 4.0 to 6.5. However, the expression and secretion of both enzymes were strongly dependent on the culture temperature. The highest expression (7.7 units/mL) and secretion (6.7 units/mL) of inulinase were observed at 28℃ and 30℃. As a consequence of decreased localization of inulinase in the periplasmic space, the secretion efficiency increased from 68% at 20℃ to 95% at 35℃. The total expression level and secretion efficiency of invertase increased from 19 units/mL and 55% at 20℃ to 25 units/mL and 68% at 35℃, respectively. Irrespective of the culture temperature, the invertase activity in the cellular fraction (periplasmic space and cytoplasmic fractions) was kept constant at around 33∼45%.

Saccharomyces cerevisiae에서 GAL 또는 GAP 프로모터 조절에 의한 재조합 Inulinase의 발현 및 분비

남수완,임현정,정봉현,장용근 동의대학교 기초과학연구소 1997 基礎科學硏究論文集 Vol.7 No.1

To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP was constructed to contain the inulinase gene (INU1) as a reporter under the control of GAL10, GAL7, GAL1 and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD_600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarily : they dropped from 0.24 h^-1 during the glucose-consuming period to 0.04-0.10 h^-1 during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7, and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indication that the secretion efficiency of inulinase is independent on the type of promoter.

Saccharomyces cerevisiae에서 Trichoderma Endoglucanase의 발현과 분비

남수완,김병우,신동하,김재범,신지원,정대균,정춘수 동의대학교 기초과학연구소 1999 基礎科學硏究論文集 Vol.9 No.1

The endoglucanase gene, egl6, of Trichoderma sp. was connected with the yeast ADH1 promoter, and the resultant plasmid, pVT-C4, was introduced into three S. cerevisiae host strains (YNN27, 2805, and SEY2102). Among each 80 transformants, the cell growth and expression level of endoglucanase were compared in test-tube cultivation, and three respective transformants for each host cells showing the highest expression level and cell growth were selected. When three recombinant yeast cells were batchwise cultivated for 48 hr in flask, the total activities of endoglucanase expressed were about 1140 unit/1 with 2805/pVT-C4, 1020 unit/l with SEY2102/pVT-C4, and 590 unit/l with (YNN27/pVT-C4. Irrespective of host strain, about 80% of the expressed endoglucanase was detected in the extracellular medium. In addition, it was also found that the recombinant enzyme was secreted into the culture medium as two major forms of lightly and heavily glycosylated proteins.

대사공학을 이용한 효모균주의 육종

남수완,Nam, Su-Wan 한국주류산업협회 2002 주류산업 Vol.22 No.1

효모의 구성적 Promoter들에 의한 Inulinase 유전자의 발현

남수완,김연희 동의대학교 기초과학연구소 2000 基礎科學硏究論文集 Vol.10 No.1

To express constitutively the inulinase gene (INU1) of Kluyveromyces marxianus in Saccharomyces, cerevisiae, three yeast promoters such as GAPDH, ADH1 and ENO1 were connected upstream of INU1. The resulting plasmids, pYIGP, pADH1-INU, and pENO-INU were introduced to S. cerevisiae SEY2102 host strain, respectively, and then each transformants were selected by staining of colonies on sucrose-agar plate. When the yeast transformants were cultivated on 2% dextrose media, the total expression levels of inulinase reached to 1.11 unit/mL, 0.88 unit/mL, and 0.69 unit/mL for respective GAPDH, ADH1, and ENO1 promoter systems. On 4% dextrose media, however, the inulinase activities were observed at 2.00 unit/mL for pYIGP, 0.71 unit/mL for pADH1-INU, and 1.40 unit/mL for pENO-INU. This result indicates that the constitutive expression of INU1 was significantly affected by the initial concentration of dextrose and the promoter strength was in the order GAPDH, ENO1, and ADH1 promoter at high dextrose concentration. Taking into account the plasmid stability, however, it is suggested that the ENO1 promoter system is more suitable for the INU1 expression on high dextrose medium or in the fed-batch cultivation accumulating ethanol at high level.

재조합 효모세포로부터 의약용 단백질의 분비생산

남수완 동의대학교 기초과학연구소 1996 基礎科學硏究論文集 Vol.6 No.1

Saccharomyces cerevisiae에서 Clostridium thermocellum Endoglucanase 유전자의 구성적 발현

남수완,정대균,정봉현 동의대학교 기초과학연구소 1998 基礎科學硏究論文集 Vol.8 No.1

To develop an effective and powerful yeast probiotics, Saccharomyces cerevisiae strains producing cellulolytic enzymes were genetically engineered. We constructed two plasmids in which the endoglucanase A gene, celA, of Clostridium thermocellum was connected in frame with ADH1 or GAPDH promoter. These plasmids were transformed into various S. cerevisiae host strains, and then the cell growth, expression level and plasmid stability between the transformed yeast cells were examined in the flask culture. The difference in genetic background of host strains did affect significantly the cell growth and expression level of endoglucanase. Based on the higher levels of cell growth(5.4∼5.9 g-DCW/L) and plasmid stability(79∼85%), three host cells(YNN27, 2805 and SEY2102) and ADH1 promoter were selected as optimal host-vector systems for the constitutive expression of celA. The recombinant yeast produced about 200 unit/L of endoglucanase as a growth-associated manner in the batch fermentation, due to the increased cell growth of 11∼13 g-DCW/L. In addition, 25∼38% and 53∼64% of the endoglucanase activity were detected in the culture medium and periplasmic space, respectively. These results indicate that the signal sequence of celA functioned well in S. cerevisiae cells and the recombinant yeast cells can be employed for the production of probiotics.

Ashbya gossypii로부터 riboflavin 대량생산을 위한 배지 최적화와 유가식 배양

남수완,장형욱,반재구,김익환,민태익 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

본 연구에서는 Ashbya gossypii를 이용한 비타민 B2(riboflavin) 생산에 있어서 발효배지의 최적화를 위해 탄소원(포도당과 대두유), 유기 질소원(CSL외 8종), 전구체(glycine 및 glutamate)의 영향을 검토하였다. 또한 생성균주의 고농도 배양을 통한 riboflavin 생산성 향상을 위해 유가식 배양을 수행하였다. 최적 발효배지의 탄소원은 기질 저해를 피할 수 있는 3%의 포도당과 0.5%의 대두유를 사용함으로써 riboflavin 생산수율을 증가시킬 수 있었다. 유기 질소원으로는 corn steep liquor(CSL)가 가장 좋았으며, 최적 CSL의 농도는 1%였다. Riboflavin 생합성의 전구체인 glycine과 glutamate는 배양 초기에 0.5%로 첨가하는 것이 riboflavin 생산에 가장 좋았다. 세포의 riboflavin 생합성 활성을 장기간 유지시키고 균체의 균체의 고농도 배양을 위해 포도당과 yeast extract로 구성된 복합배지를 연속적으로 공급한 유가식 발효 결과, 플라스크 배양(3.5g/ℓ)보다 약 100%, 발효조를 이용한 회분식발효(5.4 g/ℓ)보다 약 20% 향상된 6.8 g/ℓ의 최종 riboflavine 생산농도를 얻었다. 이상의 결과로부터 탄소원, 전구체 및 필수 영양소(특히 비타민) 공급원인 CSL의 최적화된 공급 전략을 통한 유가식 발효와 고농도 균체 배양 기술의 확립으로 riboflavin 생산수율을 더욱 향상시킬 수 있을 것으로 생각된다. In order to maximize the riboflavin production by a mutant strain Ashbya gosspyii, the optimization of medium and fed-batch fermentation were performed. As carbon sources, glucose and soybean oil were necessary for the riboflavin overproduction. Optimal concentrations of glucose and soybean oil in the flask cultures were found to be 3.0% and 0.5%, respectively, in a complex medium containing corn steep liquor (CSL) 1%. Among the various organic nitrogen sources tested, CSL as the most effective one both for the cell growth and riboflavin overproduction. More than 1% concentration of CSL, however, was inhibitory to the cell growth and riboflavin production. Glycine and glutamate at each 0.5% concentration stimulated the riboflavin production. The riboflavin concentration was reached to 5.4 g/ℓ in the batch fermentation with the optimized medium. The riboflavin production could be enhanced upto 6.8 g/ℓ through the fed-batch fermentation employing continuous feeding of glucose and yeast extract.