효모 Saccharomyces cerevisiae에서의 재조합 단백질 분비생산과 분비신호

남수완 한국산업미생물학회 1994 생물산업 Vol.7 No.3

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.

대장균에서 분자 chaperone에 의한 alginate lyase의 가용성 발현 증대

남수완,신은정,이재형,박소림,김형락 한국생명과학회 2007 생명과학회지 Vol.17 No.2

When alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was produced as aggregated insoluble particles known as inclusion bodies. In order to produce a soluble and active form of alginate lyase, E. coli cells were cotransformed with the plasmids designed to permit coexpression of aly together with molecular chaperones such as DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results revealed that the coexpression of aly together with DnaK/DnaJ/GrpE chaperone had a marked effect on the production of this protein as a soluble and active form, presumably through facilitating correct folding of alginate lyase protein. The optimal concentration of L-arabinose for the induction of DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/ml. When DnaK/DnaJ/GrpE chaperone was coexpressed, about 34% in the total alginate lyase was produced in the soluble fraction. By addition of 10% cetylpyridinium chloride, a clear zone around the colony coexpressing aly and DnaK/DnaJ/GrpE chaperone was formed, indicating that the alginate in the medium was hydrolyzed by active alginate lyase enzyme. E. coli에서 Pseudoalteromonas elyakovii 유래의 alginate lyase 유전자(aly)를 발현시킬 때, 대부분의 단백질이 불용성 내포체 형태로 발현됨을 확인하였다. Alginate lyase를 가용성 활성형으로 생산하기 위해 aly와 DnaK/DnaJ/GrpE 또는 aly와 GroEL/ES을 공발현하는 형질전환체를 얻었다. 공발현 결과, 단백질의 올바른 접힘을 도와주는 DnaK/DnaJ/GrpE chaperone이 가용성 및 활성형의 alginate lyase 생산에 매우 효과적임을 알 수 있었다. DnaK/DnaJ/GrpE chaperone의 발현에 유도제인 L-arabinose 최적 농도는 0.05 mg/ml이었으며, 이러한 공발현에 의해 약 34%의 alginate lyase가 가용성 분획에서 생산되었다. 또한 10%의 cetylpyridinium chloride를 첨가함으로써, 공발현 콜로니 주위에 투명환이 형성됨을 확인할 수 있었고, 이는 활성형 alginate lyase 효소에 의해 alginate가 분해되었음을 시사하였다.

Sacharomyces cerevisiae에서 GAL또는 GAP 프로모터 조절에 의한 재조합 Inulinase의 발현 및 분비

남수완,임현정정봉현장용근 한국생물공학회 1996 KSBB Journal Vol.11 No.4

본 연구에서는 GALl, GALl, GALlO 및 GAP promoter 하류에 reporter 유전자인 K. marxianus의 inulinase 유전자(lNUl)를 연결하여 각각의 재조합 plasmid들을 구축하고, 이들로 형질전환된 S. cerevrswe를 회분배양(YPOG 배지 )하여 외래 유전자 발현에 미치는 promoter의 영향을 비교.검토하 였다. 재조합 효모의 최종 균체농도는 36-39 00600 값을 보여 promoter에 따른 큰 차이를 보이지 않았으나, 포도당 소모기간 동안 비증식속도는 평균 $0.24 h^{-1}$로 유지되다가 galactose 소모기간 동안에 GAL promoter 함유 효모배양의 경우 $0.04-0.06 h^{-1}$, pYIGP 함유 재조합 효모배양은 $0.10 h^{-1}$로 감소하였다. 포도당 고갈 후 inulinase 발현은 시작되었고 균체외 inulinase의 발현 수준은 배양 72시간에 4.3 (GALl promoter), 4.0 (GAL7 promoter), 3.8 (GAL10 promoter) 및 1.6 (GAP promoter) unit/mL에 도달하였다. 평판배지상에서의 활성염색과 회분배양의 결과(최종발현양 및 초기 inulinase 말현속도), inulinase 발현에 미치는 promoter 세기 는 GALl > GALlO > GAL7 > GAP 순임을 알 수 있었다. GAL promoter가 배양말기까지 78 % 이상의 높은 plasmid 안정성을 보인 반면에, GAP promoter의 경우 55%의 낮은 plasmid 안정성을 보였다. 또한, 재조합 inulinase는 promoter 종류에 상관없이 98% 이상 배양액으로 분비되였다. To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) 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 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : 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, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

Effect of Molecular Chaperones on the Soluble Expression of Alginate Lyase in E. coli

남수완,김영태,신은정,이진우,So-Lim Park,전숭종,Yeon-Hee Kim 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.5

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasmids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

대사공학을 이용한 효모균주의 육종

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

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.