Combining Parts: Coupling Photosynthetic Electron Transport to Metabolic Engineering

Photosynthesis drives the production of ATP and NADPH mainly used to fix CO₂. Excess of redox power can be exploited for production of high-value compounds. Important natural products are often synthesized in low quantities by their host organism and can be difficult to produce by chemical synthesis because of their complex chemical structures. The cytochromes P450 (P450s) situated in the endoplasmic reticulum play key roles in natural product biosynthesis, and are powered by electron transfers from NADPH. We have shown that plant P450s expressed in chloroplasts and cyanobacteria are directed to the thylakoid membrane^1,2. Here, the photosynthetic electron transport will support P450 catalytic activity independent of NADPH and dedicated reductases. In order to route reducing power more efficiently to P450s, we have fused them with ferredoxin (Fd) or flavodoxin-like FMN domains³. These fusions allow the P450s to obtain electrons for catalysis directly from the photosynthetic electron transport chain by interacting with photosystem I and make them competitive with the natural occurring ferredoxin requiring enzymes. Further dedicated redirection of reducing power can be obtained by scaffolding all the enzymes of a pathway on the thylakoid membrane. In a novel strategy, we have fused enzymes with transmembrane domains of TatB and TatC from the chloroplast twin arginine translocation system⁴. This reduced the accumulation of unwanted intermediates, side products and increased the accumulation of the end product fivefold. This work shows that chloroplasts and cyanobacteria are attractive for metabolic engineering, and suggests unexplored potential for engineering of photosynthetic electron transfer chains to accommodate heterologous enzymes.

[1] Gnanasekaran, T et al. (2016) J. Exp. Bot. 67(8):2495-506.

[2] Wlodarczyka, A et al. (2016) Met. Engineering 33: 1-11.

[3] Mellor, S et al. (2016) ACS Chem. Biol. 11(7):1862-9.

[4] Henriques de Jesus et al. (2017). Met. Engineering 44: 108-116.