Using Protein Scaffolds to Redirect Photosynthetic Reducing Power for Biosynthesis of Natural Products

Photosynthesis in plants provides ATP and NADPH as well as carbon sources for primary metabolism. Cytochrome P450 monooxygenases (P450s) in the plant endoplasmic reticulum (ER) are essential in the synthesis of many bioactive natural products, powered by single electron transfers from NADPH. We have recently demonstrated that it is possible to break the evolutionary compartmentalization of energy generation and P450-catalysed biosynthesis, by relocating an entire P450 dependent pathway to the chloroplast and driving the pathway by direct use of the reducing power generated by photosystem I in a light-dependent manner ^[1]. This demonstrates the potential of transferring pathways for structurally complex high-value natural products and directly tapping into the reducing power generated by photosynthesis to drive the P450s using water as the primary electron donor.

Current work is directed towards exploring different strategies to optimize channeling of product formation. One approach to ensure co-localization of the enzymes in the thylakoid membrane is the fusion of the enzymes to components of the Twin-arginine translocation pathway – TatB and TatC. These are membrane anchored and have inherent self-organizing properties which will allow us to recruit the enzymes into close proximity and thereby reduce metabolic load. An alternative approach is scaffolding which aims at more efficient channeling of the substrate and intermediates. For this we utilize the protein-protein binding properties of PDZ domains for building a modular synthetic scaffold. These are around 80-90 amino acids and bind to the C-terminus of their target proteins (~10 aa) with different specificity. By making fusion proteins of the required enzymes with the ligand peptides, we can spatially recruit them in a desirable manner. We expect that both strategies should reduce formation of intermediaries, limit cross-talk between signaling pathways, improve substrate channeling and consequently increase product yields.

References

[1]Zygadlo-Nielsen, A. et al. Redirecting Photosynthetic Reducing Power toward Bioactive Natural Product Synthesis. ACS Synth. Biol., 2013, 2 (6), pp 308–315, DOI: 10.1021/sb300128r

Funding from UNIK Center for Synthetic Biology, Interdisciplinary Research Center “bioSYNergy”, and the VILLUM Research Center “Plant Plasticity”, is gratefully acknowledged.