In the 2007 State of the Union address, President Bush called for 35 billion gallons of alternative fuel use in the US by 2017 which would replace 20% of the nation's petroleum consumption. It is apparent that this goal cannot be achieved with existing technology and existing feedstocks alone. New technology is needed to improve the conversion of non-grain (cellulosic) feedstocks. In the US, total fuel ethanol production in 2006 reached 4.86 billion gallons - a 24% increase over 2005. Domestic demand for fuel ethanol rose 33% over 2005 to 5.4 billion gallons (Renewable Fuels Association, March 2007.) There are 114 ethanol biorefineries in operation in the US with an aggregate capacity of 5.6 billion gallons. Nearly all fuel ethanol is produced by fermentation of corn glucose (from corn grain) in the US. Presently, there are no commercial cellulosic ethanol biorefineries in operation (Renewable Fuels Association, February 2007.) Commercialization of ETI's proprietary technology will address present and future needs for the growing biofuels market. In the short-term, ETI's biocatalyst will be developed as an accessory protein used in conjunction with the most advanced cellulase technologies. Successful implementation of the proposed Phase I and subsequent research and development effort will result in the use of an ETI protein as a separate, plant-derived additive to cellulase during the hydrolysis of recalcitrant cellulose to glucose. Longer term protein development will shift from use in biomass conversion to functionality in a dedicated, genetically modified biofuel crop. Since expansin proteins are ubiquitous in plant cell walls, it is possible that, through genetic engineering, expression of a select expansin protein will enhance the accessibility of plant cellulose to enzymatic attack. Developments in this arena could lead to proprietary plant crops which encode the ETI protein. The present Phase I application will provide important fundamental insight into the technical feasibility of this approach. OBJECTIVES: The proposed research will develop a process for the production and use of a plant protein to improve the performance of enzymes used in the conversion of recalcitrant cellulose to fuel ethanol. In research conducted at Penn State University, a novel class of cell wall-loosening proteins, named expansins, was discovered. Expansin itself lacks cellulytic activity, but it was found to enhance the hydrolytic digestion of pure cellulose by cellulases. We believe that expansins could prove to be potent and economical synergists for the industrial deconstruction of cellulose into sugars and then by fermentation into liquid fuel for transportation purposes. To make its way onto the proving ground of industrial scale testing, two important milestones for commercial feasibility need to be met: (a) a scaleable system for expansin production and delivery must be established and (b) the effectiveness of expansin as a cellulase synergist needs to be tested with real-world samples of cellulosic biomass, such as corn stover, switchgrass, and forestry residues. These goals in fact are intertwined because goal (b) requires substantial amounts of expansin, necessitating goal (a). This proposal outlines a research program to accomplish these goals, which if successful will constitute a major step towards commercialization of this potential method for reducing the cost of cellulosic conversion to biofuel. The technical objectives include: 1) Use the virus-based plant expression system to produce g-scale quantities of expansin (four different types) and assess the activity and stability of the proteins. 2) Test expansin-cellulase synergy with corn stover, switchgrass and poplar wood, as representatives of the real-world cellulosic biomass. 3) Develop protocols for storage and if necessary purification. APPROACH: The proposed research will be carried out by Expansyn Technologies, Inc. Dr. Nuwan Sella Kapu, will be the project principal investigator. The proposed work for phase I is as follows: A. Clone expansin genes into the appropriate magnifection viral expression systems. B. Infect host plants (Nicotiana benthamiana and Nicotiana excelsior) with Agrobacterium tumefaciens carrying the expansin-expression replicon. C. Assess the time course of protein expression with antibody. D. Assess expansin activity by wall extension assays and cellulase synergism. E. Produce g level amounts of expansin and test for cellulase-synergy with real world forms of biomass. F. Assess stability and storage requirements (wet, dry, refrigeration, freezing); G. If necessary, develop purification protocols
