American energy and chemical industries are currently almost entirely dependent on oil (most of the it from foreign origin). In particular, the chemical industry has been optimizing, for at least 6 decades, most of its processes to be able to produce all the widely used chemicals (fuels,solvents,plastics) from petroleum. Growing environmental as well as geopolitical concerns demand alternatives to this development. Arzeda's innovative technology can play a pivotal role in this, and this proposal represent the first step to prove that using Arzeda's technology, it is possible to produce existing chemicals cheaply from plant material as opposed to petroleum. Arzeda uses computational models of biological macromolecules called proteins to alter their biological function and "redesign" them for industrial or pharmaceutical purposes. Under this proposal, Arzeda will modify existing proteins to make them produce a valuable solvent, methyl ethyl ketone (MEK), from sugars from agricultural waste as opposed to petroleum. MEK is a solvent that is used in paint coatings and is, for instance, used widely in the auto industry. Doing so will provide several major benefits. First, it will be the first proof-of-concept that a valuable chemical can be produced economically from biomass using computationally engineered enzymes. In addition, it will allow the reduction of the nation dependence on oil while opening new avenues to value US-based agricultural production. Finally, MEK producers have been suffering from cost-cutting competitors in Asia. Providing a "bio"-MEK can provide a competitive edge to local producers, especially agricultural biorefineries and pulp and paper mills that can extend their offering using the process that will be developed through the funding of this proposal. OBJECTIVES: This project focuses on research to be able to ferment 2,3-butandiol (BDO) into methyl ethyl ketone (MEK). 2,3-BDO itself can be produced through fermentation of biomass, namely sugars and lignocellulosic biomass. The proposal focuses on the design of one enzyme (a novel dehydratase) using computational techniques (see background) to convert 2,3-BDO into MEK in high yield. Later research will integrate the designed enzyme into a metabolic pathway to directly ferment sugars from biofeedstock into MEK. The project specific aims for the Phase I are the following: 1. Obtain and Active Site Model for Dehydration 2,3-BDO. The active site will contain a model of the transition state for the reaction as well the choice of metal cofactor and position of other supporting catalytic residues. The different reaction mechanisms will be studied and modeled. 2. Design 20 Enzyme Candidate that Incorporate such an Active Site. The active site from aim 1 will be used as an input for Arzeda's computational enzyme design process. The process will lead to a set of around 20 candidate protein sequences. 3. Obtain at least One Designed Enzyme with Detectable Activity. Each design will be tested in vitro using a Gas Chromatography assay. The active designs will be optimized further in Phase II.
