(188bd) Cell-Free Production of Isobutanol

Cell culture biofuel production is a promising alternative to fossil fuels, but currently limited by problems such as cell toxicity and product yield. One way to solve this problem is to develop, express and purify the enzymes that are part of the pathway of interest, allowing for production of the biofuel in vitro or outside of the cell. This allows for easier removal of the biofuel, but enzyme stability is still a concern for long term production. A previous study in our laboratory examined immobilization of enzymes to increase stability for production of the biofuel isobutanol. Building on these previous results, two more stable variant enzymes that can convert 2-ketoisovaleric acid to isobutanol, (2-ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH)), were immobilized on epoxy functionalized methacrylate resin provided by Resindion (Rome, Italy). A third enzyme, (formate dehydrogenase (FDH)), which recycles a cofactor, was also immobilized, to facilitate a continuous system. We also examined the efficacy of synthesizing 2-ketoisovaleric acid in vivo and then moving to in vitro, using the previously mentioned enzymes to produce isobutanol. The biofuel produced was syphoned from the reaction broth utilizing a membrane separation step. Future work will examine optimization of the system and use of an electrochemical bioreactor that can recycle the cofactor without FDH.