title: Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.
abstract: Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system.
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180-3590.
authors.forename: Joseph
authors.initials: J
authors.lastname: Grimaldi
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180-3590.
authors.forename: Cynthia H
authors.initials: CH
authors.lastname: Collins
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180-3590.
authors.forename: Georges
authors.initials: G
authors.lastname: Belfort
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry; D056804:Biofuels; D000440:Butanols / Q000138:chemical synthesis* / Q000737:chemistry; D002262:Carboxy-Lyases / Q000737:chemistry*; D002474:Cell-Free System; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005560:Formate Dehydrogenases / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D056804:Biofuels; D000440:Butanols; D004800:Enzymes, Immobilized; C040507:isobutyl alcohol; D000426:Alcohol Dehydrogenase; D005560:Formate Dehydrogenases; D002262:Carboxy-Lyases
keywords: alcohol production; biofuels; cell free; in vitro; isobutanol