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재조합 Alcaligenes eutrophus 균주 개발 및 Poly(3-hydroxybutyrate) 생산에 관한 연구

Development of Recombinant Alcaligenes eutrophus for the Production of Poly(3-hydroxybutyrate)

최종일 이상엽

Choi JI Lee SY

HWAHAK KONGHAK, October 1997, 35(5), 684-689(6), NONE

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Abstract

미생물에 의해 생산되는 환경친화 고분자물질 poly(3-hydroxybutyrate)[P(3HB)]의 생산성 향상을 위하여, 생산균주인 Alcaligenes eutrophus에 대해서 재조합 유전자 기술을 이용한 P(3HB) 생합성 효소들의 과다 발현에 의한 균주 개량과 개발된 재조합 A. eutrophus에서의 P(3HB) 합성능 향상을 연구하였다. P(3HB) 생합성 효소들이 과다발현된 재조합 A. eutrophus의 제작을 위하여 broad-host-range plasmid pVK101에 P(3HB) 생합성 유전자들을 결합하여 vector system을 제작하였고, electroporation에 의한 방법으로 10³ transformant/㎍ 유전자의 높은 형질 전환 기술을 확립하였다. 재조합 A. eutrophus와 야생형 A. eutrophus의 P(3HB) 합성능을 비교연구한 결과, 재조합 A. eutrophus의 경우 야생형보다 높은 최종 P(3HB) 농도, P(3HB) 함량, 그리고 P(3HB) 합성속도를 보였다.

In order to improve the productivity of poly(3-hydroxybutyrate)[P(3HB)], Alcaligenes eutrophus was metabolically engineered to amplify the activities of the three enzymes involved in the synthesis of P(3HB). The A. eutrophus P(3HB) biosynthesis genes coding for P(3HB) synthase, β-ketothiolase, and reductase were cloned into a broad-host-range plasmid pVK101. Recombinant A. eutrophus strain was developed by transforming with this plasmid by electroporation. The efficiency of transformation was in an order of 10³ transformants/μg DNA. In flask cultures, the final cell concentration of recombinant A. eutrophus decreased with increasing carbon/nitrogen(C/N) molar ratio. On the other hand, P(3HB) concentration was highest at the medium C/N molar ratio. For the fixed nitrogen concentration, the concentrations of cell and P(3HB) increased with increasing glucose concentration. Comparison of cell growth and P(3HB) production by recombinant and wild type A. eutrophus in batch culture showed that the final P(3HB) concentration, P(3HB) content, and P(3HB) synthesis rate were all higher in the recombinant strain compared with the wild type.

Keywords

Poly(3-hydroxybutyrate) Recombinant Alcaligenes eutrophus P(3HB) Synthesis Genes Enzyme Amplification

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