Cell-Free Production of Isobutanol

Limitations on biofuel production using cell culture include low product concentrations (<few vol%) due to product feedback inhibition, instability of cells, and lack of economic product recovery. In previously published studies, a novel cell-free immobilized enzyme system was developed in our group to overproduce the biofuel, isobutanol. Two enzymes, (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) were immobilized and one enzyme, (formate dehydrogenase (FDH)), was free in solution, producing isobutanol titers much higher than previously reported with S. cerevisiae. Building on these previous studies, we have utilized mutagenesis to increase enzyme activity while immobilized. One enzyme (KdcA) was supplemented with the higher preforming 2-ketoisovalerate decarboxylase (KivD), and site-directed mutagenesis was conducted on two other enzymes (ADH, FDH) from previous publications to increase enzyme activity. We also immobilized FDH previously free in solution, creating a truly cell free system. Future work will combine the reaction system with a separation method.