Cell free production of isobutanol

Abstract

With a need for greener fuels, research into production of biofuels is essential. Isobutanol out preforms ethanol in key metrics such as engine compatibility, energy density, and gasoline blending. Current biofuel strategies of fermentation are constrained by the inherent toxicity of alcohol on microbial cells. While work has been performed on engineering these strains for higher tolerance, cell-free production with enzymes offers a novel approach to bypass the toxicity limitations altogether. These enzymes can also be immobilized to retain enzyme activity and facilitate separations. Based on previous work in the Belfort laboratory, the ketoisovaleric acid pathway was chosen for production of the biofuel, isobutanol. High preforming and stable enzymes were selected from the literature, cloned, expressed, and purified and tested for activity, kinetics, and stability. They were utilized in a novel in vivo to in vitro system, resulting isobutanol titer of 1.78 g/L and yield of 93%. An epoxy immobilized reaction scheme resulted in a titer of 2 g/L and 43% yield. The pathway enzymes were then fused to dockerins, which bound to a cohesin scaffold on cellulose. The reaction utilizing this immobilization scheme resulted in a titer of 5.92 g/L and 78.4% yield. Further work can be done to optimize this reaction, as well as to expand the pathway or scaffold, and incorporate separation of the isobutanol for eventual scaleup.