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

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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 languageEnglish
JournalMetabolic Engineering
Pages (from-to)33-43
Publication statusPublished - 1 Sep 2019

    Research areas

  • CYP79A1, Cytochrome P450, Electron transfer, Enzyme engineering, Ferredoxin, Flavodoxin, Photosynthesis, Redox

ID: 223676245