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

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

by Yagiz Alagoz, Jianing Mi, Aparna Balakrishna, Lamyaa Almarwaey, Salim Al-Babili
Scientific Year: 2022 DOI: 10.1016/bs.mie.2022.03.003

Abstract

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
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