Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Plant Biotechnology

Background:

• Cytochrome P450s (P450s) are crucial enzymes for natural product biosynthesis but challenging to express heterologously.
• Photosynthetic organisms like plants and cyanobacteria offer potential for P450 expression, utilizing their native electron transport chains.
• Competition for reducing power from endogenous ferredoxin limits P450 activity when linked to photosynthesis.

Purpose of the Study:

• To compare the efficacy of different electron carriers in enhancing photosynthesis-driven P450 activity in plants.
• To identify electron carriers that can overcome competition for reducing power within the photosynthetic apparatus.
• To establish a new metabolic engineering strategy for improved bio-production of natural products.

Main Methods:

• In vitro assessment of electron transfer from four carriers (three ferredoxins, one engineered flavodoxin-like protein) to a model P450 (CYP79A1).
• In vivo evaluation of these carriers by transiently expressing them fused to CYP79A1 in Nicotiana benthamiana.
• Quantification of photosynthetic reducing power sequestration and P450 activity under competitive conditions.

Main Results:

• In vitro, only the flavodoxin-like carrier effectively delivered reducing power to CYP79A1 despite competition.
• In vivo, fusion proteins with the flavodoxin-like carrier showed the greatest improvement (nearly 25-fold) in P450 activity.
• Optimized synthetic electron transfer pathways with specific redox potentials can mitigate competition for ferredoxin.

Conclusions:

• Engineered electron carriers, particularly those with tailored redox potentials, can significantly enhance photosynthesis-driven P450 activity.
• This approach overcomes limitations imposed by endogenous competition for reducing power in metabolic engineering.
• The study presents a novel strategy for efficient bio-production of valuable natural products using engineered plant systems.