Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis.
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.
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
Silas Busck
SB
Mellor
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
Agnieszka Zygadlo
AZ
Nielsen
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|DynaMo Center of Excellence, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Meike
M
Burow
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
Mohammed Saddik
MS
Motawia
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
Dainius
D
Jakubauskas
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.|Villum Research Center of Excellence "Plant Plasticity", University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Birger Lindberg
BL
Møller
Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.|Center for Synthetic Biology "bioSYNergy" , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.|Villum Research Center of Excellence "Plant Plasticity", University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Poul Erik
PE
Jensen
D001688:Biological Products / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D008027:Light; D009994:Osmolar Concentration; D014026:Nicotiana / Q000378:metabolism
D001688:Biological Products; D005288:Ferredoxins; D003577:Cytochrome P-450 Enzyme System