Abstract
Artemisia annua produces an anti-malarial sesquiterpenoid, artemisinin. A cytochrome P450 monooxygenase CYP71AV1 was identified from A. annua. The CYP71AV1 is a multifunctional enzyme capable of oxidizing amorpha-4,11-diene at the C12 position to generate artemisinic acid via artemisinic alcohol and artemisinic aldehyde in artemisinin biosynthetic pathway.
In this study, we revealed that CYP71AV1 homologous genes (more than 94% identity at amino acid level) were expressed in artemisinin non-producing species; A. afra and A. absinthium, inconsistent with the absence of amorpha-4,11-diene and transcripts for amorpha-4,11-diene synthase (ADS).
To analyze amorpha-4,11-diene oxidizing activity of these CYP71AV1 homologs, ADS and each of CYP71AV1 homologs were coexpressed in yeast, and the ethyl acetate extracts of the cultures were analyze by GC-MS. Putative sesquiterpene alcohol, with retention time different from artemisinic alcohol (C12 hydroxylated form), was detected from yeast expressing ADS and each of CYP71AV1 homologs. These results indicate that these homologs have amorpha-4,11-diene oxidizing activity with different regiospecificity from CYP71AV1.
