Abstract
The P450 enzyme CYP701A is known as ent-kaurene oxidase (KO) in higher plants and it catalyzes the oxidation reaction of ent-kaurene to form ent-kaurenoic acid. We found a CYP701B1 gene instead of CYP701A in PHYSCObase database of Physcomitrella patens. We obtained the full-length cDNA of CYP701B1 from Riken BRC and tried heterologous expression for analysis KO activity. The CYP701B1 ORF in pPICZ vector was used to transformation of Pichia pastoris harbouring Arabidopsis NADPH cytochrome P450 reductase 1 (ATR1). A transformant was incubated and induced the recombinant protein by addition of methanol. The microsome fraction was incubated with ent-kaurene and NADPH, and metabolite was analyzed by GC-MS. From the GC-MS analysis, the recombinant CYP701B1 catalyzes the oxidation of ent-kaurene to produce ent-kaurenoic acid. These results indicated that the GYP701B1 gene encodes ent-kaurene oxidase in P. patens (PpKO). We tested tolerance activity against uniconazole-P of PpKO. PpKO showed higher resistant activity rather than AtKO at 50μM of uniconazole-P.
