Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice

Isoflavonoids are distributed predominantly in leguminous plants and play c ritical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisofl avanone synthase, is the key enzyme in their biosynthesis. In cultured lico rice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an i soflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin ) is rapidly induced upon elicitation. In this study, we obtained a full-le ngth P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. ec hinata cells. When the flavanones liquiritigenin and naringenin were incuba ted with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and geniste in by acid treatment. The chemical structures of the products from liquirit igenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectr ometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, wh ich catalyzes the hydroxylation associated with 1,2-aryl migration of flava nones. Northern-blot analysis revealed that transcripts of CYP93C2, in addi tion to those of other P450s involved in phenylpropanoid/flavonoid pathways , transiently accumulate upon elicitation.