TY - JOUR

T1 - Fusion of ferredoxin and cytochrome P450 enables direct light-driven biosynthesis

AU - Mellor, Silas Busck

AU - Nielsen, Agnieszka Janina Zygadlo

AU - Burow, Meike

AU - Motawie, Mohammed Saddik

AU - Jakubauskas, Dainius

AU - Møller, Birger Lindberg

AU - Jensen, Poul Erik

PY - 2016

Y1 - 2016

N2 - 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 towards 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.

AB - 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 towards 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.

U2 - 10.1021/acschembio.6b00190

DO - 10.1021/acschembio.6b00190

M3 - Journal article

C2 - 27119279

VL - 11

SP - 1862

EP - 1869

JO - A C S Chemical Biology

JF - A C S Chemical Biology

SN - 1554-8929

IS - 7

ER -