The utilization of biomass as a petroleum alternative faces many challenges, including the design of biocatalysts that cheaply and efficiently produce ethanol. A combination of directed engineering and mutational selection of Escherichia coli has resulted in a recombinant biocatalyst, LY168, that produces up to 4.5 g L-1 ethanol in 48 hours in a simple xylose-based mineral salts medium and a non-recombinant biocatalyst, SE2378, that converts glucose and xylose to ethanol through a novel pathway. The ethanol biocatalyst design approach has been applied to other commodity chemicals, such as optically pure lactic acid. Identification and analysis of spontaneous mutations contributing to increased ethanol production in mineral salts medium will also be discussed.