Cloning, expression and characterization of P450 monooxygenase CYP102H1 from Nocardia farcinica

Abstract

The cytochrome P450 oxygenases (CYPs) are heme-containing enzymes that catalyze a wide range of reactions such as aliphatic hydroxylation, epoxidation, oxidative phenolic coupling, heteroatom oxidation, and dealkylations. One of the well-characterized CYPs is the CYP102 subfamily, which catalyze hydroxylation of medium- to long-chain fatty acids at their ω-1, ω-2 and/or ω-3 positions in the presence of molecular oxygen and nicotinamide cofactors. With an aim to search for a new P450 oxygenase belonging to CYP102 subfamily, we cloned, expressed, and characterized CYP102H1 from Nocardia farcinica. The CYP102H1 gene was amplified from the pNF1 of N. farcinica and the DNA fragment was cloned into the pTrc99A and pET28(+) vectors, resulting in plasmid pTrc-CYP102H1 and pET-CYP102H1, respectively. Escherichia coli BL21 was transformed with the pTrc-CYP102H1 or pET-CYP102H1 vector. After the transformants were selected on the LB agar medium containing antibiotics, expression of CYP102H1 in the recombinant cells was investigated with SDS-PAGE and western blot analysis. When the recombinant cells harboring pET-CYP102H1 were induced with 1.0 mM IPTG in the LB medium at 30oC, the CYP102H1 gene was expressed in the soluble form. The recombinant cells were subjected to in vitro CYP102H1 activity assay. When the enzyme was purified and complemented with the redox partner proteins of Pseudomonas putida (i.e., CamAB), the enzyme showed oxygenation activity of linoleic acid and relatively slight oxygenation activity of arachidonic acid but little activity towards decanoic acid and dodecanoic acid. The reaction products were identified with GC/MS, while the structure of products remained to be investigated. The biotransformation of linoleic acid with the recombinant E. coli expressing CYP102H1 and camAB was also carried out. Hydroxy linoleic acid was produced to a concentration of 70.9 μM from 400 μM linoleic acid at the Tris-HCl (pH 7.5) buffer containing 5 g/L glucose.;cytochrome P450 oxygenases (CYPs) 은 heme을 포함하는 효소로 aliphatic hydroxylation와 epoxidation, oxidative phenolic coupling, heteroatom oxidation, dealkylations 같은 넓은 범위의 반응을 촉매한다. CYPs중 가장 잘 알려진 CYP102 계열은 산소와 nicotinamide cofactors 존재 하에, 중간 또는 긴 사슬의 지방산의 ω-1와 ω-2 , ω-3 위치를 hydroxylation 시킨다. CYP102 계열에 속하는 새로운 P450 oxygenase를 찾을 목적으로 Nocardia farcinica 유래의 CYP102H1을 재조합 및 발현하여 그 특성에 대해 알아보았다. CYP102H1 유전자를 Nocardia farcinica 의 pNF1으로부터 분리하고 DNA 단편을 발현 벡터인 pTrc99A와 pET28(+) vector에 각각 발현시켰다 (pTrc-CYP102H1 and pET-CYP102H1). Escherichia coli BL21에 pTrc-CYP102H1와 pET-CYP102H1를 transform하였다. transformants들을 선별하기 위해 각각 항생제가 포함된 LB agar 배지를 이용하였고, CYP102H1의 발현을 SDS-PAGE 와 western blot 으로 확인하였다. pET-CYP102H1를 가진 재조합 균주들은 LB 배지에서 30 oC에서 배양한 후, 1.0 mM IPTG로 유전자를 발현시켰다. 재조합 균주로 in vitro CYP102H1 활성 측정을 진행하였다. 효소 반응을 위해 CYP102H1과 Pseudomonas putida 유래의 CamAB 단백질을 redox partner 단백질로 각각 정제하여 산소 존재하에 지방산을 반응시켰다. 불포화 지방산인 linoleic acid와 arachidonic acid는 반응이 일어났지만 포화지방산인 decanoic acid와 dodecanoic acid에서는 반응이 거의 일어나지 않았다. 반응물은 GC/MS로 분석하였다. CYP102H1과 camAB 를 E. coli 에 동시발현시켜 linoleic acid를 생물전환시켰다. Tris-HCl (pH 7.5)와 5 g/L glucose 존재 하에서, 400 uM linoleic acid로부터 hydroxy linoleic acid 70.9 μM가 생산되었다.