title: Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.
abstract: Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Thiyagarajan
authors.initials: T
authors.lastname: Gnanasekaran
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Karcher
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Agnieszka Zygadlo
authors.initials: AZ
authors.lastname: Nielsen
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Helle Juel
authors.initials: HJ
authors.lastname: Martens
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ruf
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: Xenia
authors.initials: X
authors.lastname: Kroop
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Mohammed Saddik
authors.initials: MS
authors.lastname: Motawie
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Mathias
authors.initials: M
authors.lastname: Pribil
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Bock
authors.affiliation: Copenhagen Plant Science Centre, Center for Synthetic Biology bioSYNergy, Villum Research Center "Plant Plasticity", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark peje@plen.ku.dk.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
mesh_terms: D018533:Biomass; D053898:Biosynthetic Pathways / Q000235:genetics / Q000528:radiation effects; D002736:Chloroplasts / Q000378:metabolism* / Q000528:radiation effects* / Q000648:ultrastructure; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects; D054628:Genome, Chloroplast; D018745:Genome, Plant; D005960:Glucosides / Q000378:metabolism; D008027:Light; D013058:Mass Spectrometry; D009570:Nitriles / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D010641:Phenotype; D010788:Photosynthesis / Q000528:radiation effects; D030821:Plants, Genetically Modified; D021122:Protein Subunits / Q000378:metabolism; D045868:Sorghum / Q000201:enzymology*; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic / Q000528:radiation effects
chemical_list: D005960:Glucosides; D009570:Nitriles; D021122:Protein Subunits; D003577:Cytochrome P-450 Enzyme System; C011220:dhurrin
keywords: Cytochrome P450; chloroplast; dhurrin pathway; light-driven; photosynthesis; thylakoids.