Pathway Development and Enzyme Characterization for Microbial Production of Isobutanol and n-Butanol at Elevated Temperatures

Long-term energy stability and environmental concerns have driven development toward the production of transportation fuels from renewable feedstocks such as sugars or lignocellulosic biomass. n-Butanol and isobutanol have emerged as prominent advanced transportation biofuels because of their favorable fuel properties, compatibility with current infrastructure, and ability to serve as chemical feedstocks. Cellulolytic Clostridium species are among the most promising organisms to serve as hosts for consolidated bioprocessing (CBP) of cellulolytic butanol production. Here, the benefits of cellulolytic Clostridia, pathways for microbial n-butanol and isobutanol production, and strategies for achieving and improving cellulosic C-4 alcohol production will be described. In addition, a strategy for improving isobutanol production in Clostridium thermocellum was investigated. Although isobtuanol production has been realized in the thermophile C. thermocellum, 2-ketoisovalerate (KIV) decarboxylase, or Kivd, is a limiting enzyme due to its poor thermostability. There are two promising strategies for improving thermophilic KIVD activity: 1) improving the thermostability of Lactococcus lactis Kivd or 2) improving the KIVD activity of a thermostable decarboxylase. Here, the latter strategy was investigated by identifying and characterizing the KIVD activity of enzymes similar to L. lactis Kivd from thermophilic hosts. Ultimately, the specific activities of the native thermophilic enzymes were found to be substantially lower than L. lactis Kivd and are not recommended for use in thermophilic isobutanol production. However, this work characterizes the KIVD activity for thermophilic decarboxylases have not been previously characterized