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

Yagiz Alagoz, Jianing Mi, Aparna Balakrishna, Lamyaa Almarwaey, Salim Al-Babili*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationCarotenoids
Subtitle of host publicationCarotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology
EditorsEleanore T. Wurtzel
PublisherAcademic Press Inc.
Pages527-552
Number of pages26
ISBN (Print)9780323913539
DOIs
StatePublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume671
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
This work was financially supported by King Abdullah University of Science and Technology (KAUST) baseline funding, and a Competitive Research Grant (CRG4) given to S.A.B. Fig. 2 was prepared using an online-available software ( https://biorender.com ). We would like to express our sincere gratitude to the editor, Prof. Eleanore Wurtzel, for her valuable comments and time in revising this manuscript.

Publisher Copyright:
© 2022 Elsevier Inc.

Keywords

  • Apocarotenoids
  • Carotenoids
  • Cytochrome P450
  • WAT11
  • Yeast

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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