Cell-free production of isobutanol: A completely immobilized system

A completely immobilized cell-free enzyme reaction system was used to convert ketoisovaleric acid to isobutanol, 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. The selected enzyme variants include keto-acid decarboxylase 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.