온도조절형 발현 Vector 를 함유한 Phenylalanine 생산균의 분자육종

정동효(Dong Hyo Chung),심상국(Sang Kook Shim),이영춘(Young Chun Lee),정호권(Ho Kwon Chung) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.1

In order to produce phenylalanine without tyrosine co-production, we constructed various temperature-controllable expression vectors by insertion of lower expression of the tyrA gene into the plasmid pSY130-14. And tyrosine revertant to cultivate without addition of tyrosine, was selected from Escherichia coli strain AT2471[tyrA , thi ] by spontaneous mutation. The strain AT2471 harbouring plasmid pSY146A and the tyrosine revertant 5 harbouring plasmid pSY111-14 produced 12 g/ℓ and 15 g/ℓ of phenylalanine respectively in a 2.5ℓ jar fermenter at a constant temperature of 39℃ after 55 hours cultivation.

효모 유전자 발현용 (發現用) Promoter 개발에 관한 연구

정동효,정호권,박준희,심상국 ( Dong Hyo Chung,Ho Kwon Chung,Joon Hee Park,Sang Kook Shim ) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.1

The purpose of this study was the development of promoter for the lacZ` gene. Two heterologous promoter Ⅰ and Ⅱ of lacZ` gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoter Ⅰ was estimated to be 2.5 kb and β-galacrosidase activity was 124.6 U/㎎ protein, and the size of the promoter Ⅱ was 4.0 kb and its β-galactosidase activity was 168.8 U/㎎ protein, respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. YIp plasmid was constructed from YEp plasmid, and expressed both in E. coli and yeast. The promoter Ⅰ aid Ⅱ iso-laced from yeast chromosomal DNA can be used for promoter of plasmid YEp and YIp.

遺傳子 發現用 Promoter 開發에 關한 硏究

정동효,정호권,박준희,심상국 中央大學校 遺傳工學硏究所 1988 遺傳工學硏究論集 Vol.1 No.1

본 연구는 lac'z gene의 promoter개발을 위하여 착수하였다. lac'z gene의 promoterⅠ과 Ⅱ를 효모의 염색체 Bam HI DNA 단편에서 발견하였다. PromoterⅠ의 크기의 2.5kb정도이고 β- galactosidase 활성은 124.6 U/㎎ protein이었으나 promoterⅡ의 크기와 효소활성은 4.0kb 와 168.8 U/㎎이었다. 형질전환체에서 YEp plasmid 안정성은 52.7%에서 67.4%정도였다. YEp plasmid에서 YIp plasmid를 구출할 수 있었고 이 YIp plasmid는 대장균에서나 효모에서도 발현되었고 특히 HIS5 gene이 promoter로서 가능함을 알 수 있었다. It was attempted to observe the development of promoter on lac'z gene. Two promoterⅠand Ⅱ of lac'z gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoterⅠwas estimated to be 2.5kb and β- galactosidase activity was 124.6 U/㎎ protein but the size of the promoterⅡ was 4.0kb and its β- galactosidase activity was 168.8 U/㎎ protein respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. And YIp plasmid was constructed by YEp plasmid. This YIp plasmid both in E.coli and yeast. The HIS5 gene, coding gene of histidiolphosphate aminotransferase functioned as the promoter of YIp plasmid .

溫度調節型 發現 Vector를 함유한 Phenylalanine 生産菌의 分子有種

田口久治,이영춘,정동효,정호권,심상국 中央大學校 遺傳工學硏究所 1988 遺傳工學硏究論集 Vol.1 No.1

In order to produce phenylalanine without tyrosine co-prodction, we constructed various temperature-controllable expression vectors by insertion of lower expression of the tyrA gene into plasmid pSY 130-14. And tyrosine revertant to cultivate without addition of tyrosine, was selected from Escherichia coli strain AT2471[tyrA­,thi­]by spontaneous mutation, The strain AT2471 harbouring plasmid pSY 146A and the tyrosine revertant 5 harbouring plasmid pSY 111-14 produced 12g/ℓand 15g/ℓof phenylalanine respectively in 2.5ℓjar fermenter at a constant temperature of 39℃ after 55 hours cultivation.

효모의 발현 Vector 계의 개발

황용일,심상국,오시마 야스지,정동효 中央大學校 遺傳工學硏究所 1990 遺傳工學硏究論集 Vol.3 No.1

The cloned gene of glyceraldehyde-3-phosphate dehydrogenase(GAP) of Saccharomyces cerevisiae (Holland et al., 1983) has been characterized. Based on the communication, we have also cloned 2.1kb GAP DNA fragment and modified that fragment as a portable promoter. Two yeast exression vectors, one is a YCp type vector being maintained at low copy number(1 or 2) and the other is a YEp type vector at high copy number, have been constructed with the GAP promoter PH05' gene as an indicator gene. Our plasmids were introduced into Saccharomyces cerevisiae. HU-1, which has been improved. The Trp+ transformants expressed APase activity efficiently and showed the high level of PH05' transcripts.

Development of Host - Temperature Controllable Vector System in Streptomyces

Chung,Dong Hyo,Shim,Sang Kook,Tatsuji,Seki 中央大學校 遺傳工學硏究所 1989 遺傳工學硏究論集 Vol.2 No.1

Streptomyces에서 온도에 의하여 유전자 발현이 조절되는 시스템을 개발하기 위하여 Streptomyces-E.coli에서 발효되는 온도조절형 발현 shuttle vector를 작제하였다. 작제된 plasmid에 존재하는 lacZ로 encoded된 β-galactosidase와 그 mRNA의 생산이 온도에 의하여 엄밀히 조절되었다. Stirred fermentor와 PHE type fementor에 의한 배양액의 순환발효에 있어서도 β-galactosidase의 생산이 적절히 조절되었다. 이러한 결과로 부터 작제된 온도조절형 발현 벡터를 이용하면 limiting enzyme의 양을 온도에 의하여 조절할 수 있으므로 Streptomyces로 부터 중간 대사산물을 생산하고져 할 때 이용할 수 있음을 알 수 있었다. To develop a gene expression system which could be regulated by temperature manipulation in Streptomyces, We constructed bifunctional Streptomyces-E. coli temperature-controllable shuttle vectors harbouring lacZgene of which expression was directed by promoter P? and P? and was controlled by cI? repressor. The synthesis of β-galactosidase as well as its specific mRNA was strictly controlled by temperature in Streptomyces. Repetitive switch on and off of the gene expression were also perfomed successfully. By recycling the culture broth between a conventional stirred fermentor and a plate heat exchange type fermentor, the amount of β-galactosidase was controlled at a desired level.

Bacillus 속이 생산하는 생전분 가수분해효소 유전자의 클로닝과 발현

정동효,심상국,김연계,김철호 中央大學校 遺傳工學硏究所 1989 遺傳工學硏究論集 Vol.2 No.1

생전분 분해 α-amylase를 강력하게 생산하는 Bacillus sp.를 분리하였다. E.coli-B. subtilis shuttle vector인 pYEJ001과 pHY300PLK를 사용하여 생전분 분해α-amylase 유전자를 E.coli와 Bacillus subtilis에 형질전환하였다. 클로닝된 α-amylase gene은 5.4kb HindⅢ 와 3.3kb EcoR V 단편의 크기였으며, E.coli내에서 이들 유전자는 비교적 안정하게 유지되었고 발현되었지만 Bacillus subtilis에서는 불안정하였으며 발현이 낮았다. 대장균내에서 생성된 생전분 분해 α-amylase는 그 효소의 생성량의 80%가 periplasmic space에 존재하였다. Bacillus sp which produces the raw starch digesting α-amylase screened. 5.4kb HindⅢ and EcoR V fragment of raw starch digesting α-amylase gene were cloned in the plasmid pYEJ001 or pHY300PLK which are E.coli-Bacillus subtilis vector using E.coli and Bacillus subtilis as a host,respectively. The cloned gene was certificated to hybridize to the Bacillus sp chromosomal DNA. The cloned gene was stably maintained and expressed in E.coli C 600,but was very unstable and low expressed in Bacillus subtilis 207-25. Analysis of α-amylase expressed in the transformants indicated that about 80% of α-amylase produced by the constructed plasmid of E.coli was localized in the periplasmic space and can be released by an cold osmatic shock.

대두유 응고효소 생산에 관한 연구

정동효,이기성,한면수,심상국 한국농화학회 1990 Applied Biological Chemistry (Appl Biol Chem) Vol.33 No.2

A bacterial strain which was capable of producing extracellular soymilk-clotting enzyme was isolated from soil samples during the course of screening test. The characteristics of the isolated strain K-324-7, indicated that the strain belonged to species of Bacillus cereus. The crude purification of this enzyme was precipitated by salting out with ammonium sulfate of 0.8 saturation. The optimal pH for the enzyme activity was at 6.l ∼7.0 and below 50℃. The optimal culture medium for the production of soymilk dotting enzyme were consisted of 0.2% glucose, 0.2% peptone, and 0.5% KH₂PO₄ with initial pH value of 6.5. The activity of enzyme was maximum when the microbe was cultured for 3 days at 35℃.

응집성 Saccharomyces cerevisiae CA-1에 의한 에탄올 연속발효

이용범,심상국,한면수,정동효 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6

침전도가 부착된 air-lift reactor를 제작하여 응집성 Saccharomyces cerevisiae CA-1의 에탄올 생산성을 검토하였다. 회분식 발효에서의 최적 aeration rate는 0.5vvm이었고 각 유입 기질농도에서 희석속도가 증가됨에 따라 반응기 내부의 균체가 반응산물과 분리되지 못하고 유출되어 잔당이 증가되었으며 기질소모비, 에탄올함량, 유출 균체량이 감소되었다. 에탄올 생산성은 10% glucose 농도에서 희석속도 0.7h^-1에서 20.44g/l·h로 최대값이었으며 에탄올 생산성과 기질 소모비로 복합시킨 최적 작동조건은 10% glucose 농도에서 희석속도 0.5h^-1이었고 이때 에탄올 생산성은 19.7 g/l·h, 기질소모비는 0.98 g/g이었다. Using a flocculating Saccharomyces cerevisiae CA-1, an air-lift reactor equipped with a modified settler was used for ethanol fermentation. The effects of conditions such as aeration rate, initial glucose concentration, and dilution rate were studied using the air-lift reactor. In batch fermentation, optimum aeration rate was 0.5 vvm. In continuous fermentation, aeration rate and initial pH were fixed 0.5 vvm and 4.5, substrate concentration and dillution rate were changed 10∼15% and 0.1∼1.3. The maximum ethanol productivity was shown to be 20.4 g/l·h in 10% glucose and 0.7 h^-1 dilution rate., and optimum operation condition considering the ethanol productivity and glucose utilization ratio was 0.5 h^-1 dilution rate in 10% glucose concentration.

효모, Saccharomyces cervisiae의 GAP 유전자를 이용한 발현 벡터계의 개발

황용일,서애란,심상국,정동효 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.6

효모에서 대량의 물질생산계를 구축하기 위하여 먼저 여러 종류의 벡터의 이용이 가능한 다양한 영양요구성 marker를 지니며 형질전환율이 향상된 효모숙주를 선별 개량하였다. 벡터의 제작에 사용되는 프로모터로는, 효모의 여러 유전자 중에서 그 활성이 매우 높은 해당계의 효소 GAP-DH의 구조 유전자 GAP를 이용하기로 하여, 효모염색체 DNA 중에서 GAP 프로모터를 분리하여 이용하기 쉽게 변형하였다. 분리된 GAP promoter의 기능을 검토하기 위하여, reporter로 APase의 구조유전자 PHO5'를 이용하여 세포내의 copy수가 상이한 발현 벡터를 제작하여 GAP 프로모터에 의한 APase의 활성 및 전사산물을 측정한 결과, 정상적인 전사가 이루어졌으며, 효소활성도 높게 나타났으며, 벡터의 copy수에 의한 효소활성의 차이도 감지되었다. 이러한 결과로부터 GAP 프로모터를 이용한 발현계의 구축에 의하여 다량으로 이종 유전자 산물의 생산이 가능한 물질 생산계를 구축할 수 있었다. The cloned glyceraldehyde-3-phosphate dehydrogenase(GAP) gene of Saccharomyces cerevisiae(Holland et al., 1983) has been characterized. Based on the communication, we have also cloned 2.1kb GAP DNA fragment and modified this fragment as a portable promoter. Two yeast expression vectors, one is YCp type vector being maintained at low copy number (1 or 2) and the other is YEp type vector at high copy number, have been constructed with the GAP promoter and the PHO5' gene as a reporter. Our plasmids were introduced into S. cerevisiae HY-1, which has been improved. The Trp transformants expressed APase activity efficiently and showed high level of PHO5' transcripts.