Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system

Methods Enzymol. 2022:671:527-552. doi: 10.1016/bs.mie.2022.03.003. Epub 2022 Mar 26.


Cytochrome P450 enzymes (CYPs) are involved in metabolic steps that provide structural diversity during the biosynthesis of carotenoids and their oxidative cleavage products called apocarotenoids. Recent studies on bioactive apocarotenoids in plants revealed the necessity of performing further research to uncover the function of novel CYP enzymes that might be involved in apocarotenoid metabolism. We describe a series of in-vitro methods to characterize plant CYPs that metabolize apocarotenoids, using a specific Saccharomyces cerevisiae strain, WAT11, engineered to express a CYP redox partner, Arabidopsis thaliana NADPH-P450 reductase 1 (ATR1). This chapter provides protocols for construction and transformation of plasmids that express CYPs in yeast, isolation of yeast microsomes, and in-vitro enzymatic assays to validate the final metabolic products using LC-MS.

Keywords: Apocarotenoids; Carotenoids; Cytochrome P450; WAT11; Yeast.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis* / genetics
  • Arabidopsis* / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Microsomes / metabolism
  • NADPH-Ferrihemoprotein Reductase / genetics
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism


  • Cytochrome P-450 Enzyme System
  • NADPH-Ferrihemoprotein Reductase