earticle

영어

Cytochrome P450 monooxygenases (CYP) are a superfamily of ubiquitous heme proteins and performs various oxidative reactions such as C-H bond hydroxylation, N-dealkylation, N- hydroxylation, O-dealkylation, S-oxidation and epoxidation of numerous endogenous and exogenous compounds. Indeed, the P450 enzyme plays a crucial role in fungi for the adaption of diverse ecological niches by its hydrophobic conversion of the primary and secondary metabolites and in the degradation of ecological pollutants. Genome sequencing projects has revealed the presence of several thousands of putative P450 genes from various families in the fungal kingdom. Bioinformatic annotations showcase the presence of 169 putative P450 genes and 2 putative Cytochrome P450 reducatase (CPR) genes from the whole genome sequence of the filamentous fungus Fusarium oxysporum f. sp. lycopersici. To elucidate the molecular diversity and functional capabilities of the Cyp P450 monooxygenases from F.oxysporum, we initiated the functional genomic research by comprehensive screening of the putative novel P450s by using Bioinformatic approaches and by analyzing the catalytic potential of P450s against a wide variety of compounds. The functional targets includes the Sterol 14α-Lanosterol demethylase, Flavonoid Hydroxylase, ω-Fattyacid Hydroxylase, Benzoate Hydroxylase, Alkalene Hydroxylase, Polycyclic aromatic hydrocarbons (PAH) oxidase, Phenyl Acetate Hydroxylase, and Steroid 11α-Hydroxylase, etc. For the construction of P450 gene library various parameters such as mRNA expression, cDNA synthesis, gene amplification and cloning followed by transformation into E.coli cells as well as Yeast expression system are been optimized. The functional characterization includes the isolation of Microsomes from the Saccharomyces cerevisiae cells harboring the recombinant P450 co- expressed with F.oxyxporum NADPH-P450 oxidoreductase and the enzymatical analysis with various substrates. Hence, the FoCYP P450 gene library will drive us towards an intensive approach for the functional characterization of various novel P450s and facilitates the comprehension of metabolic diversity in ascomycetes and has future biotechnology applications.