(550f) Cell-Free Production of Isobutanol: A Completely Immobilized System

Our long-term goal is to synthesize fuels efficiently and economically using in vitro (i.e. cell free) technology by minimizing diffusion limitations (i.e. at high Damkohler number) with an immobilized multi-enzyme system. A completely immobilized cell-free enzyme reaction system was used to convert ketoisovaleric acid to iso- butanol, a desirable biofuel, with a molar yield of 43% and a titer of 2 g/L, which are comparable to high performing in vivo systems (e.g. 41% and 5.4 g/L, respectively, for Clostridium thermocellum). The approach utilizes, for the first time, a series of previously reported enzyme mutants that either overproduce the product or are more stable when compared with their wild type (Wong, M., Sorci, M., Gasparis, G., Belfort,G., Koffas, M. (2020) “Cell-Free Production of Isobutanol- A completely immobilized system” Bioresource Technol., 294, 122104. ). The selected enzyme variants include keto-acid dec- arboxylase attached to a maltose binding protein, alcohol dehydrogenase, and formate dehydrogenase. These enzymes were screened for thermal, pH, and product stability to choose optima for this system which were pH 7.4 and 35 °C. This system is designed to address well-known limitations of in vivo systems such as low product concentrations due to product feedback inhibition, instability of cells, and lack of economic product recovery.