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Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

[Image: see text] Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form,...

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Autores principales: Mellor, Silas Busck, Nielsen, Agnieszka Zygadlo, Burow, Meike, Motawia, Mohammed Saddik, Jakubauskas, Dainius, Møller, Birger Lindberg, Jensen, Poul Erik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2016
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949584/
https://www.ncbi.nlm.nih.gov/pubmed/27119279
http://dx.doi.org/10.1021/acschembio.6b00190
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author Mellor, Silas Busck
Nielsen, Agnieszka Zygadlo
Burow, Meike
Motawia, Mohammed Saddik
Jakubauskas, Dainius
Møller, Birger Lindberg
Jensen, Poul Erik
author_facet Mellor, Silas Busck
Nielsen, Agnieszka Zygadlo
Burow, Meike
Motawia, Mohammed Saddik
Jakubauskas, Dainius
Møller, Birger Lindberg
Jensen, Poul Erik
author_sort Mellor, Silas Busck
collection PubMed
description [Image: see text] Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.
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spelling pubmed-49495842016-07-21 Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis Mellor, Silas Busck Nielsen, Agnieszka Zygadlo Burow, Meike Motawia, Mohammed Saddik Jakubauskas, Dainius Møller, Birger Lindberg Jensen, Poul Erik ACS Chem Biol [Image: see text] Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. American Chemical Society 2016-04-27 2016-07-15 /pmc/articles/PMC4949584/ /pubmed/27119279 http://dx.doi.org/10.1021/acschembio.6b00190 Text en Copyright © 2016 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Mellor, Silas Busck
Nielsen, Agnieszka Zygadlo
Burow, Meike
Motawia, Mohammed Saddik
Jakubauskas, Dainius
Møller, Birger Lindberg
Jensen, Poul Erik
Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title_full Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title_fullStr Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title_full_unstemmed Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title_short Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis
title_sort fusion of ferredoxin and cytochrome p450 enables direct light-driven biosynthesis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949584/
https://www.ncbi.nlm.nih.gov/pubmed/27119279
http://dx.doi.org/10.1021/acschembio.6b00190
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