Heterologous Co-expression of CYP6B7 and NADPH-Dependent Cytochrome P450 Reductase From Helicoverpa armigera (Lepidoptera: Noctuidae) in Pichia pastoris.

Abstract

As important metabolic enzymes, the function of cytochrome P450 monooxygenases (CYPs) has been demonstrated repeatedly through various means, including heterologous expression systems. Unfortunately, most model systems typically lack expression of a conspecific NADPH-dependent cytochrome P450 reductase (CPR), which is the electron transfer partner of CYPs. As a result, the activities of heterologously expressed insect CYPs may not accurately reflect detoxification activities in vivo. Previously, CYP6B7 from Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) (HaCYP6B7) has been expressed in the Pichia pastoris GS115 strain and shown to detoxify bifenthrin, fenvalerate and chlorpyrifos. However, it remains to be determined if co-expression of HaCYP6B7 with HaCPR will enhance the detoxification ability of the expression system. In the present study, HaCYP6B7 and HaCPR genes were co-expressed in P. pastoris using a reconstituted expression vector, pPICZA-HaCYP6B7-HaCPR. Protein expression was confirmed by Western blot, and the detoxification activities of microsomal fractions to p-nitroanisole O-demethylation (PNOD), 7-ethoxycoumarin O-deethylation (ECOD), fenvalerate and chlorpyrifos were measured. Co-expression of HaCYP6B7 with HaCPR resulted in PNOD and ECOD activities of 1.90 nmol/min/mg·protein and 12.39 pmol/min/mg·protein, which were 1.6- and 1.5-fold of that catalyzed by HaCYP6B7 expressed alone, respectively. Furthermore, microsomes of pPICZA-HaCYP6B7-HaCPR-GS115 had higher detoxification activity than that of pPICZA-HaCYP6B7-GS115 to fenvalerate, but not chlorpyrifos. The results indicated that co-expression of HaCYP6B7 with conspecific CPR could enhance the detoxification activities to some substrates comparing with expression of HaCYP6B7 alone.