Cell-free production of isobutanol: A completely immobilized system. (December 2019)

• Record Type: Journal Article
• Authors: Wong, Matthew; Zha, Jian; Sorci, Mirco; Gasparis, Christopher; Belfort, Georges; Koffas, Mattheos
• Is Part Of: Bioresource technology. Volume 294(2019)
• Publication Date: 2019-12
• Subjects: In vitro production -- Biofuels -- Immobilized enzymes -- Isobutanol -- Mutagenesis; Biomass -- Periodicals; Biomass energy -- Periodicals; Bioremediation -- Periodicals; Agricultural wastes -- Periodicals; Factory and trade waste -- Periodicals; Organic wastes -- Periodicals; Bioénergie -- Périodiques; Déchets agricoles -- Périodiques; Déchets industriels -- Périodiques; Déchets organiques -- Périodiques; Déchets (Combustible) -- Périodiques; 662.88
• DOI: 10.1016/j.biortech.2019.122104
• Languages: English
• ISSNs: 0960-8524

Abstract

Highlights: Avoided toxicity limits of cells with cell-free production. Immobilized active mutant enzymes of the ketoisovaleric acid pathway on resin. Screened and identified enzymes for optimal thermal, pH and isobutanol stability. Utilized immobilized mutant variants for the first time to produce isobutanol. Abstract: 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.