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

doi:10.1016/bs.mie.2022.03.003

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 proceeding › Chapter › peer-review

5 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 language	English (US)
Title of host publication	Carotenoids
Subtitle of host publication	Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology
Editors	Eleanore T. Wurtzel
Publisher	Academic Press Inc.
Pages	527-552
Number of pages	26
ISBN (Print)	9780323913539
DOIs	https://doi.org/10.1016/bs.mie.2022.03.003
State	Published - Jan 2022

Publication series

Name	Methods in Enzymology
Volume	671
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

Access to Document

10.1016/bs.mie.2022.03.003

Cite this

Alagoz, Y., Mi, J., Balakrishna, A., Almarwaey, L., & Al-Babili, S. (2022). Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system. In E. T. Wurtzel (Ed.), Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology (pp. 527-552). (Methods in Enzymology; Vol. 671). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2022.03.003

Alagoz, Yagiz ; Mi, Jianing ; Balakrishna, Aparna et al. / Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system. Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology. editor / Eleanore T. Wurtzel. Academic Press Inc., 2022. pp. 527-552 (Methods in Enzymology).

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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",

author = "Yagiz Alagoz and Jianing Mi and Aparna Balakrishna and Lamyaa Almarwaey and Salim Al-Babili",

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: {\textcopyright} 2022 Elsevier Inc.",

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language = "English (US)",

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Alagoz, Y, Mi, J, Balakrishna, A, Almarwaey, L & Al-Babili, S 2022, Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system. in ET Wurtzel (ed.), Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology. Methods in Enzymology, vol. 671, Academic Press Inc., pp. 527-552. https://doi.org/10.1016/bs.mie.2022.03.003

Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system. / Alagoz, Yagiz; Mi, Jianing; Balakrishna, Aparna et al.
Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology. ed. / Eleanore T. Wurtzel. Academic Press Inc., 2022. p. 527-552 (Methods in Enzymology; Vol. 671).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

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AU - Mi, Jianing

AU - Balakrishna, Aparna

AU - Almarwaey, Lamyaa

AU - Al-Babili, Salim

N1 - 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.

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N2 - 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.

AB - 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.

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SN - 9780323913539

T3 - Methods in Enzymology

SP - 527

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BT - Carotenoids

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PB - Academic Press Inc.

ER -

Alagoz Y, Mi J, Balakrishna A, Almarwaey L, Al-Babili S. Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system. In Wurtzel ET, editor, Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology. Academic Press Inc. 2022. p. 527-552. (Methods in Enzymology). doi: 10.1016/bs.mie.2022.03.003

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

Abstract

Publication series

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

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