The molecular cloning and structural analysis of a cytochrome P450 (CYP71D500) encoding gene from ajowan (Trachyspermum ammi L.) medicinal plant

Plant phenolic monoterpenes, such as thymol and carvacrol, have a wide range of applications in medicinal, pharmaceutical and other industries. Ajowan is an aromatic medicinal plant from the Apiaceae family with thymol as an active component of its seeds. The seeds of ajowan are valuable for medicinal purposes because their essential oil contains active substances of thymol, carvacrol, γ-terpinene, and p-cymene. Cytochrome P450 (CYP) –related genes have an indispensable role in the biosynthetic pathway of thymol and other substances in ajowan. A cytochrome P450 gene (CYP71D500) with a high expression level was isolated from the ajowan, cloned and sequenced. The sequencing information from the previous RNA-Seq study confirmed that the isolated gene belongs to the plant CYP71 clan, with a 1654 bp length containing two exons and a 115 bp intron. The full-length cDNA of CYP71D500 was also cloned. The sequencing of CYP71D500 cDNA showed the complete homology of CYP71D500 cDNA and exon regions of the CYP71D500 genomic sequence. The sequencing analysis of CYP71D500 cDNA also revealed a mutation that changed isoleucine to valine amino acid. The 3D structural analysis of the enzyme showed that the modified amino acid is not located in the enzyme’s active site. Therefore, this cannot probably affect the synthesis of thymol. The isolated gene and cDNA could be used for the metabolic engineering of ajowan and other medicinal plants with active phenolic monoterpenes. It is also applicable for identifying the different functions of the cloned CYP gene in ajowan or other medicinal plants.