A cytochrome P450 monooxygenase responsible for the C-22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway

Polyphyllins are the major steroidal saponin components of Paris polyphylla, the main source plant of the common Chinese herbal medicine Paridis Rhizoma with strong pharmacological activity and extremely high economic value and great market prospects. However, the production of polyphyllins in plants is limited, and their biosynthesis pathway has not been reported. The downstream hydroxylation step was particularly unclear. To clarify the enzymes and intermediates involved in the downstream steps of polyphyllin biosynthesis, we performed a comparative transcriptome analysis and discovered a cytochrome P450 gene that encodes a protein with monooxygenase activity. Heterologous expression in Saccharomyces cerevisiae demonstrated that it encodes an enzyme that catalyzes the formation of 22(R)-hydroxycholesterol from cholesterol. The relative gene expression measured by RT-PCR and polyphyllin contents measured by HPLC in P. polyphylla roots at different ages confirmed that this gene is involved in polyphyllin biosynthesis. To our best knowledge, this is the first report on the cloning of a CYP450 enzyme gene from the steroidal saponin pathway of higher plants.