Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

Silas Busck Mellor; Marcos Hamborg Vinde; Agnieszka Zygadlo Nielsen; Guy Thomas Hanke; Kaltum Abdiaziz; Maxie M. Roessler; Meike Burow; Mohammed Saddik Motawia; Birger Lindberg Møller; Poul Erik Jensen

doi:10.1016/j.ymben.2019.05.003

Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

Silas Busck Mellor, Marcos Hamborg Vinde, Agnieszka Zygadlo Nielsen, Guy Thomas Hanke, Kaltum Abdiaziz, Maxie M. Roessler, Meike Burow, Mohammed Saddik Motawia, Birger Lindberg Møller, Poul Erik Jensen^*

^*Corresponding author for this work

8 Citations (Scopus)

Abstract

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.

Original language	English
Journal	Metabolic Engineering
Volume	55
Pages (from-to)	33-43
ISSN	1096-7176
DOIs	https://doi.org/10.1016/j.ymben.2019.05.003
Publication status	Published - 1 Sept 2019

Keywords

CYP79A1
Cytochrome P450
Electron transfer
Enzyme engineering
Ferredoxin
Flavodoxin
Photosynthesis
Redox

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10.1016/j.ymben.2019.05.003

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title = "Defining optimal electron transfer partners for light-driven cytochrome P450 reactions",

abstract = "Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.",

keywords = "CYP79A1, Cytochrome P450, Electron transfer, Enzyme engineering, Ferredoxin, Flavodoxin, Photosynthesis, Redox",

author = "Mellor, {Silas Busck} and Vinde, {Marcos Hamborg} and Nielsen, {Agnieszka Zygadlo} and Hanke, {Guy Thomas} and Kaltum Abdiaziz and Roessler, {Maxie M.} and Meike Burow and Motawia, {Mohammed Saddik} and M{\o}ller, {Birger Lindberg} and Jensen, {Poul Erik}",

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T1 - Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

AU - Mellor, Silas Busck

AU - Vinde, Marcos Hamborg

AU - Nielsen, Agnieszka Zygadlo

AU - Hanke, Guy Thomas

AU - Abdiaziz, Kaltum

AU - Roessler, Maxie M.

AU - Burow, Meike

AU - Motawia, Mohammed Saddik

AU - Møller, Birger Lindberg

AU - Jensen, Poul Erik

PY - 2019/9/1

Y1 - 2019/9/1

N2 - Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.

AB - Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.

KW - CYP79A1

KW - Cytochrome P450

KW - Electron transfer

KW - Enzyme engineering

KW - Ferredoxin

KW - Flavodoxin

KW - Photosynthesis

KW - Redox

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U2 - 10.1016/j.ymben.2019.05.003

DO - 10.1016/j.ymben.2019.05.003

M3 - Journal article

C2 - 31091467

AN - SCOPUS:85067431797

SN - 1096-7176

VL - 55

SP - 33

EP - 43

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -

Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

Abstract

Keywords

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