8a: Comprehensive Enzyme Stabilization for Cell-Free Bioproduction

Bioproduction is typically reliant on host-organisms to express the enzymes needed for a synthetic pathway, which suffers from drawbacks like wasted metabolic resources, feedstock toxicity, and purification. Cell-free bioproduction is the logical alternative, but one major drawback is stability of the enzymes in the absence of a cell. For cell-free bioproduction to be feasible, enzymes will need to be reusable over multiple feedstock additions. To address this defined problem, Luna proposes development of a flexible platform technology to preserve enzyme activity for weeks of continuous use. This technology is based on an inorganic amorphous polymer that cages enzymes to aid in biological and physical stability when used in a bioreactor, and also aids in enzyme durability in shipping and storage prior to use. Luna will leverage two enzymatic pathways isolated from P. putida to demonstrate feasibility for enhanced production of high value commodities from lignin derivatives. This Phase I program is designed to demonstrate the universality of Luna’s enzyme preservation system at re-use over many feedstocks, and with the inclusion of heat, mechanical, and contamination stressors a cell-free bioproduction system would have to endure. The stabilizing system will be produced and evaluated as compared to other stabilization systems. Bench-scale bioproduction will be performed to evaluate the performance of the stabilized enzymes in comparison to existing systems related to the challenges of re-use, and also challenged with storage temperature fluctuations, bacterial contamination, and mechanical stress.Cell-free bioproduction is not fully realized in industrial processes because of several technical hurdles. However, once these technical hurdles are solved, current molecular biotechnologies will rapidly allow pathway and enzyme innovation to new levels of efficiencies for biomanufacturing. The benefits of a fully-integrated renewable biomanufacturing process that is cost-competitive with traditional manufacturing would be realized by consumers. There would be a greater diversity of products at lower prices, and consumption would have a lower environmental impact. With Luna’s low-cost universal enzyme preservation system, cell-free bioproduction will be one step closer to an industrial-scale reality. Besides the goal of biomanufacturing, there are many existing applications for enzymes in industrial practices that an enhanced stabilization technology can find use immediately. The companies Novozymes, DuPont, and Royal DSM collectively have $4B of enzyme sales annually.