Light-Driven Energy Production for Cell-Free Metabolic Systems

• Author / Creator

  Minor, Kyle

• Industrial biotechnology is used to produce biofuels, pharmaceuticals, chemicals, and other products and for wastewater treatment and soil remediation. Over the last several years, the use of whole-cell biocatalysts (WCB) for sequestering carbon dioxide has gained considerable attention. Although these technologies have been shown to have a significant reduction in CO2 lifecycle emissions, there are inefficiencies associated with using whole cell biocatalysts. The mechanisms for cellular growth and repair waste energy and nutrients that could otherwise be used for the production of targeted compounds.
  Ideally, isolated enzymes responsible for the sequestration of CO2 in living systems would be advantageous over WCB. Isolated enzymes or cell-free metabolic systems (CFMS) would allow for all carbon and energy to be directed to the formation of the desired product. Before cell-free metabolic systems can become a disruptive technology, providing and adjusting energy at the nanoscale must be addressed. Enzymatic cofactors are the most cost prohibitive component of cell-free metabolic systems and are responsible for providing the energy for enzymes to proceed in otherwise thermodynamically unfavorable reactions. Due to the cost and stability of enzymatic cofactors like nicotinamide (NADH) recycling coenzymes is essential. The focus of this work was to engineer biotic/abiotic organelle capable of reducing NADH using light and water. This device pairs two membrane protein complexes that do not interact naturally: NADH: Ubiquinone oxidoreductase (CMI) and Photosystem II (PSII). Through the controlled directional assembly into 180 nm liposomes, these two proteins function collectively to reduce NAD+. The significant findings of this work include, the explicit demonstration of the reversibility of CMI, the inability of Peiricidin A to inhibit NAD+ reduction by CMI, determined the optimal PSII to CMI ratio and show the rate of NADH production is proportional to quantum flux and has coupling efficiencies near unity. Applying this technology to cell-free metabolic systems will permit control over NADH balance and eliminate constraints on the design of new biosynthetic pathways.

• Subjects / Keywords

  • NADH
  • Complex I
  • Enzymatic Cofactor Recycling
  • Bacteriorhodopsin
  • Photosystem II
  • Artificial Organelles
  • Artificial Photosynthesis

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3WH2DW9T

• License

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