This Small Business Innovation Research (SBIR) Phase I project focuses on the development of a high-yield fermentation route for the production of levulinic acid (LA). LA is one of the best-suited C5 building blocks for bio-refinery production due to higher value, broad applications, and likely quick adoption by the chemical industry. To date, no bioprocess for LA exists, and known chemical processes have not reached commercial stage due to high cost and lower yield. Arzeda, the world leader in computational enzyme engineering, has invented a new biochemical method to convert sugars to LA. The objective of this Phase I project is to demonstrate the feasibility of the concept by validating the proposed biochemical conversion in vitro. Arzeda will use its enzyme engineering platform to design the biocatalyst(s) needed, including computational modeling and design, gene assembly, and enzyme production. <br/>The broader/commercial impacts of this research are the advancement of a U.S. ?green? chemistry industry, and strengthening, economically and environmentally, of a sustainable United States bio-refinery industry. The lack of a high-yield alternative to costly thermochemical processes has been preventing a widespread adoption of levulinic acid. Because LA can be converted, chemically or biochemically, to synthetic rubber (through isoprene and butenes), bio-fuels (such as kerosene and HMF), polymers (for instance, nylons) and polymer additives (for changing polymer characteristics), the addressable market is in excess of $20B annually. When considered as the end product, LA trades at a considerable higher price than ethanol, the current product of most commercial bio-refineries, and thus can help diversify their product offering and considerably increase their margins. Application of Arzeda?s proven technology of computational enzyme design to bring to the world a high-yield fermentation route for LA will considerably advance