title: Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.
abstract: Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.
authors.affiliation: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Artur
authors.initials: A
authors.lastname: Wlodarczyk
authors.affiliation: Copenhagen Plant Science Center, 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: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", 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: Georg-August-University, Albrecht-von-Haller Institute, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.
authors.forename: Nodumo Nokolunga
authors.initials: NN
authors.lastname: Zulu
authors.affiliation: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Silas Busck
authors.initials: SB
authors.lastname: Mellor
authors.affiliation: Biozentrum der Ludwig-Maximillians-Universität München, 82152 Planegg-Martinsried, Germany.
authors.forename: Manja
authors.initials: M
authors.lastname: Luckner
authors.affiliation: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Jens Frederik Bang
authors.initials: JFB
authors.lastname: Thøfner
authors.affiliation: Copenhagen Plant Science Center, 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 Center, 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: Mottawie
authors.affiliation: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; DNRF Center DynaMo, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Meike
authors.initials: M
authors.lastname: Burow
authors.affiliation: Copenhagen Plant Science Center, 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: Georg-August-University, Albrecht-von-Haller Institute, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.
authors.forename: Ivo
authors.initials: I
authors.lastname: Feussner
authors.affiliation: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", 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: Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. Electronic address: peje@plen.ku.dk.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D008027:Light; D060847:Metabolic Engineering / Q000379:methods*; D010788:Photosynthesis / Q000502:physiology / Q000528:radiation effects; D011829:Radiation Dosage; D015398:Signal Transduction / Q000502:physiology*; D046939:Synechocystis / Q000502:physiology* / Q000528:radiation effects
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: Cyanobacteria; Cyanogenic glucosides; Cytochrome P450; Light-driven biosynthesis; Metabolic engineering; Synechocystis