Effect of Expression Level of Archaea Chaperones on Fatty Acid Biotransformation Activity of Recombinant Escherichia coli

Abstract

Molecular chaperones은 외래 단백질의 functional expression을 증가시키고 구조적 안정성을 증가시키기 위해 널리 이용되고 있다. 본 연구에서는, 샤페론의 발현수준이 Micrococcus luteus 유래의 alcohol dehydrogenase와 Pseudomonas putida KT2440 유래의 Baeyer-Villiger monooxygenase를 발현시킨 재조합 대장균의 ricinoleic acid 생물전환 활성에 미치는 효과를 연구하였다. 재조합 대장균에서 archaea유래 샤페론 (예를 들어, Methanocaldococcus jannaschii 유래의 γ-prefoldin, thermosome)의 발현수준은 open reading frames의 5’-UTR (untranslated region)의 변화를 통해 조절될 수 있다. 재조합 대장균의 생물전환 활성은 chaperones의 발현수준에 의해 매우 영향을 받는다. 예를 들어 전세포 생물전환 활성은 γ-prefoldin의 발현수준이 이론적 최대 발현량의 약 46 %일 때 12 μmol/g dry cells/min으로 가장 높았으며, 이 수치는 γ -prefoldin의 발현수준이 0 %일 때와 이론적 최대 발현 수준일 때 보다 약 2배 높은 수치였다. 그러므로 생물전환 활성은 세포 내의 효소 활성보다 chaperones의 발현수준에 의해 영향을 받는 것으로 사료된다.;Molecular chaperones are extensively used to improve functional expression of heterologous proteins as well as to increase their structural stability. In this study, effect of expression level of chaperones on the ricinoleic acid biotransformation activity of recombinant Escherichia coli, expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida KT2440, was investigated. Expression level of the archaea chaperones (i.e., γ-prefoldin, thermosome of Methanocaldococcus jannaschii) in the recombinant E. coli was modulated via changing 5’-UTR (untranslated region) of the open reading frames. The biotransformation activity of the recombinant E. coli was significantly influenced by expression level of the chaperones. For instance, the whole-cell biotransformation activity was the highest to 12 μmol/g dry cells/min when expression level of γ-prefoldin was set to ca. 46% of the theoretical maximum. This value was approximately two-folds greater than the E. coli cells, where expression level of γ-prefoldin was zero or set to the theoretical maximum. The whole-cell biotransformation activity was assumed to be influenced by intracellular enzyme activity, which was in turn affected by expression level of the chaperones.