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

Abstract

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.

Stabilization and scale‐up of photosynthesis‐driven ω‐hydroxylation of nonanoic acid methyl ester by two‐liquid phase whole‐cell biocatalysis

“…Functional replacement of the NADH‐dependent rubredoxin reductase AlkT by an NADPH‐dependent spinach ferredoxin reductase has been shown in in vitro studies (McKenna & Coon, ). The same was reported for recombinant CYP450 enzymes actively coupling with ferredoxins/ferredoxin reductases originating from phototrophic species in vitro and in vivo (Hara et al, ; Jensen, Johnston, de Montellano, & Møller, ; Mellor et al, ). Identification and overexpression of enzymes involved in this electron transfer might be a powerful strategy to improve the coupling of oxyfunctionalization and photosynthetic metabolism.…”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…Likewise, biosynthesis of phycobilisome bilin pigments in cyanobacteria requires ferredoxin-dependent heme oxygenase and bilin reductase enzymes (Dammeyer and Frankenberg-Dinkel 2008). Such high abundance of enzymes that compete for reduced ferredoxin seriously limits the amount of reducing power that can be redirected (Yacoby et al 2011;Nielsen et al 2013;Mellor et al 2016). Therefore, optimization of light-driven biosynthesis requires improving the competitiveness of Scheibe and Dietz (2012), dashed arrows indicate pathways of cyclic electron transport.…”

Determinants of thermostability in the cytochrome P450 fold

“…One promising opportunity for the identification of novel thermostable redox systems could be BLAST searches using the sequences of mesophilic redox partners that are known to interact with a thermostable P450 from a given thermophilic P450 organism. Alternative options, such as the linking of a thermostable P450 to a Fd and photosystem I [146] might provide future opportunities, considering that photosystem I complexes from thermophilic organisms are available [147][148][149][150] and have already successfully been applied in other fields, such as the light-induced generation of hydrogen in artificial systems [148].…”