1.1.1.383 biofuel production a cytosolically located, cofactor-balanced isobutanol pathway, consisting of a mosaic of bacterial enzymes is expressed in Sacchaormyces cerevisiae. In aerobic cultures, the pathway intermediate isobutyraldehyde is oxidized to isobutyrate rather than reduced to isobutanol. Significant concentrations of the pathway intermediates 2,3-dihydroxyisovalerate and alpha-ketoisovalerate, as well as diacetyl and acetoin, accumulate extracellularly. While the engineered strain cannot grow anaerobically, micro-aerobic cultivation results in isobutanol formation at a yield of 0.018 mol/mol glucose. Simultaneously, 2,3-butanediol is produced at a yield of 0.64 mol/molglucose 738975 1.1.1.383 biofuel production combination of high activity alcohol dehydrogenase YqhD mutants with IlvC mutants, both accepting NADH as a redox cofactor, in an engineered Escherichia coli strain, enabling comprehensive utilization of the biomass for biofuel applications. The refined strain, shows an increased fusel alcohol yield of about 60% compared to wild type under anaerobic fermentation on amino acid mixtures.When applied to real algal protein hydrolysates, the strain produces 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively 737411 1.1.1.383 biofuel production Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain 738976