This Small Business Innovation Research Phase I project will test the feasibility of a proposed byproduct-free butanol-production host developed using molecular means to engineer specific enzymes important to secondary processing of the alcohols. Biofuels have the potential to not only reduce the United States' dependency on foreign oil imports vital to homeland security but to also dramatically decrease greenhouse gas emissions associated with global warming. Biofuels, such as ethanol, can be obtained from the conversion of carbon based feedstock. While ethanol is increasingly being used as an oxygenate additive for standard gasoline, butanol has several advantages over ethanol for fuel. It can be made from the same feedstocks as ethanol but, unlike ethanol, butanol has the potential of being stored, transported and used in the same manner as gasoline. Butanol can be produced using Clostridium strains that naturally produce butanol but this process is relatively inefficient and produces large amounts of byproducts. This project will aid in developing microorganisms capable of converting biomass to butanol with greater efficiency than Clostridium and without byproducts. Phase I will evaluate the feasibility of the proposed byproduct-free butanol-production host. Specifically, enzymes that catalyze one of the key steps within the glucose-to-butanol metabolic pathway will be expressed in a heterologous host to identify the one the functions best. Phase II will optimize the metabolic pathway within a suitable production host for increased space-time-yields. The broader benefits of the technology developed by this project will be the potential of providing a second generation renewable transportation fuel, reducing dependence on imported oil, lowering greenhouse gas emissions, and expanding markets for agricultural products worldwide.