Cell-Free Optimized Production of Protoporphyrin IX

Background: The asymmetric and aromatic structures of porphyrins enable semiconducting properties allowing them to absorb light to initiate complex photocatalytic activity. These properties coupled with biological origins have garnered these molecules and their derivatives wide interest as a biotechnological platform. However, porphyrin production in cells is challenging due to limited titers, long production timescales, and difficult purification. Results: A cell-free metabolic engineering platform was constructed to produce protoporphyrin IX (PPIX) from E. coli crude extracts. Using acoustic liquid-handling, design of experiments for high-throughput buffer optimization and co-culturing techniques for extract production, our cell-free reactions effectively produced 0.109 mg/mL quantities of porphyrins. Conclusions: The use of cell-free metabolic engineering as a bioproduction platform could improve the production of toxic or inefficient biomolecules such as PPIX. The engineering strategies applied in this study provide a roadmap towards increasing the scale of cell-free metabolic engineering by elucidating batch to batch variability, as well as the need for batch specific optimization of reaction conditions.