Minimal & Adapted Carbon Sequestration

Based on current rates of carbon emission and the state of carbon content in the atmosphere, it is essential to find highly efficient methods of carbon sequestration. The primary method of sequestration on earth occurs naturally in the form of the Calvin Cycle in photosynthetic organisms. Though it is highly advantageous to have carbon sequestration be the responsibility of a naturally propagating and self-replicating system such as living cells, this cycle is highly inefficient. To take the place of this cycle, more efficient, synthetic carbon sequestration cycles are being explored. One recent study demonstrated the capability and efficiency of a minimal, synthetic enzymatic cycle for carbon sequestration that involves only four enzymes, titled the POAP pathway, in vitro. Here we aim to demonstrate the functionality of the POAP pathway in vivo, in the JCVI Syn 3.0b minimal cell. To implement the POAP Pathway in vivo, we developed a 2-Enzyme per Plasmid Design, allowing us to create minimal cell strains that encode half of the POAP pathway each. Ongoing work focuses on testing the efficacy of these strains in processing either acetate or pyruvate and outputting the opposite using gas chromatography-mass spectrometry. In order to increase the feasibility of our project in the long-run, we began development on a defined media called C5 Media. Cells were determined to not be viable without a supplement called CMRL, and work to eliminate this costly supplement is ongoing. We anticipate our findings will be a starting point for using the minimal cell as a screening platform for metabolic processes such as a minimal carbon sequestration system. Our results lay the foundation for future work optimizing the POAP pathway in vivo and present a novel understanding of enzymatic carbon sequestration systems.