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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.
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.
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.