title: Functional expression of Carassius auratus cytochrome P4501A in a novel Shewanella oneidensis expression system and application for the degradation of benzo[a]pyrene.
abstract: Cytochrome P4501A (CYP1A) in fish has attracted an increasing interest because of its important roles in the metabolic activation of certain xenobiotics such as aromatic hydrocarbons. CYPs are reported to be expressed in yeast, insect cells and Escherichia coli, but are critical for high-level expression. Besides, this study found that the purification of recombinant goldfish CYP1A would result in a loss of enzyme activity. Because large quantities of functional CYP1A are required, it is necessary to find a suitable host with high-level expression. In the present study, a novel expression system using Shewanella oneidensis was established successfully for the production of goldfish CYP1A. A signal peptide in the expression vector leads to the high-level expression in the periplasmic space of Shewanella. The recombinant CYP1A in Shewanella reached up to 1μmol per liter of culture, and showed the typical P450 hemoprotein spectra. An ethoxyresorufin O-deethylase assay revealed the amount of functional proteins in Shewanella to be almost ten times more than those in other expression systems. Furthermore, the CYP1A-mediated degradation of benzo[a]pyrene was also observed by (1)H NMR spectroscopy. These results indicate the possible application of periplasmic fractions to obtain sufficient quantities of functional CYP1A for further studies.
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, South Korea.
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Chang
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, South Korea.
authors.forename: Ming
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, South Korea.
authors.forename: Kwang-Jae
authors.initials: KJ
authors.lastname: Shon
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, South Korea. Electronic address: jaspark@pusan.ac.kr.
authors.forename: Jang-Su
authors.initials: JS
authors.lastname: Park
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics; D006054:Goldfish / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D019897:Periplasm / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020592:Shewanella / Q000145:classification / Q000201:enzymology / Q000235:genetics*
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1
keywords: Benzo[a]pyrene; Cytochrome P4501A; Degradation; NMR; Shewanella oneidensis