Directed evolution of carbon–hydrogen bond activating enzymes

Frey, Raphael; Hayashi, Takahiro; Buller, Rebecca

doi:10.1016/j.copbio.2018.12.004

Cited by 21 publications

(14 citation statements)

References 79 publications

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“…A specific P450 isoform is referred to by the prefix 'CYP' (CYtochrome P450) followed by a numerical designation based on homology (Nelson, 2004). P450s have a reputation for being challenging enzymes to express in heterologous systems because they are membrane bound and require dedicated reducing systems, such as the ER membrane-localized NADPH-dependent cytochrome P450 reductases (CPR) (Frey et al, 2019;Jung et al, 2011). Both of these difficulties may be partly overcome by targeting P450s to chloroplasts or cyanobacteria.…”

Section: Introductionmentioning

confidence: 99%

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

Mellor

Vinde

Nielsen

et al. 2019

Metabolic Engineering

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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 NADPHdependent 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 photosynthesisdriven 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 comparisonnearly 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 an engineering strategy useful for metabolic engineering aimed at producing valuable natural products.

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Section: Introductionmentioning

confidence: 99%

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

Mellor

Vinde

Nielsen

et al. 2019

Metabolic Engineering

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“…Already today, 2-oxoglutarate-dependent oxygenases are employed for a variety of industrially and academically relevant transformations, ranging from the production of amino acid derivatives to the chemo-enzymatic total synthesis of complex natural products (vide supra). As attested by recent enzyme engineering studies on 2-oxoglutarate-dependent oxygenases [25,99,140], the progress made in the field of protein design and evolution over the last few decades [147,148] facilitates the development of robust and active C-H functionalization biocatalysts [149]. As the integration of automation and the use of advanced computer algorithms continue to facilitate enzyme engineering, the industrial implementation of 2OGX-driven processes will be further accelerated.…”

Section: Discussionmentioning

confidence: 99%

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

Peters

Buller

2019

Catalysts

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C–H functionalization is a chemically challenging but highly desirable transformation. 2-oxoglutarate-dependent oxygenases (2OGXs) are remarkably versatile biocatalysts for the activation of C–H bonds. In nature, they have been shown to accept both small and large molecules carrying out a plethora of reactions, including hydroxylations, demethylations, ring formations, rearrangements, desaturations, and halogenations, making them promising candidates for industrial manufacture. In this review, we describe the current status of 2OGX use in biocatalytic applications concentrating on 2OGX-catalyzed oxyfunctionalization of amino acids and synthesis of antibiotics. Looking forward, continued bioinformatic sourcing will help identify additional, practical useful members of this intriguing enzyme family, while enzyme engineering will pave the way to enhance 2OGX reactivity for non-native substrates.

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“…Many of the biocatalysts described here and in previous works 255,256 look ready to be incorporated into screening kits for use in drug discovery laboratories. The challenges to be overcome before they are more widely used in industry have been identified for each enzyme class, and these are being tackled by numerous research groups.…”

Section: Late-stage C–h Functionalization Of Drug Leads Using Engineered Enzymesmentioning

confidence: 92%

A Perspective on Synthetic Biology in Drug Discovery and Development—Current Impact and Future Opportunities

et al. 2021

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Directed evolution of carbon–hydrogen bond activating enzymes

Cited by 21 publications

References 79 publications

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

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

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

A Perspective on Synthetic Biology in Drug Discovery and Development—Current Impact and Future Opportunities

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