Plants produce a diverse range of secondary metabolites, including sesquiterpenoids. Their various roles include antimicrobial defense against pathogens, mediation of interspecific communication, and (a)biotic stress protection. Sesquiterpenoids are common substrates of cytochrome P450 enzymes (CYP), which are responsible for the diversity of sesquiterpenoids in nature. The goal of this project is to investigate the regiospecificity of two CYPs that catalyze the oxidation of himachalol, a sesquiterpenoid produced by the model legume Medicago truncatula. CYP71D81 and CYP71D82 are two tandem duplicated cytochromes P450 with a high identity of ninety percent. Coexpression of these CYPs with their native substrate, himachalol, resulted in the formation of oxygenated intermediates in a regiospecific manner. Select amino acids will be mutated through site-directed mutagenesis and subsequently coexpressed with himachalol in a modular metabolic engineering system. Using gas chromatography-mass spectrometry (GC-MS), the mutated CYP’s reaction with himachalol is then compared with that of a wild type, revealing the effect of the mutational change on CYP function. 

Elucidating the molecular basis by which sesquiterpenoid-derived products are formed can deepen understanding of CYP molecular and plant evolution and aid in crop optimization in agricultural settings.