Cellulosic ethanol is a desirable transportation fuel for environmental and economic reasons. One of the issues limiting the commercialization is the utilization of xylose that is not fermented to ethanol by conventional brewing yeasts. Trillium FiberFuels is pioneering the use of xylose isomerase to convert xylose to fermentable xylulose. While this can be accomplished with existing industrial xylose isomerase products, there is a mismatch between their optimum pH (7.5) and temperature (55°C) and those for ethanol fermentation (pH 5, 30°C). During Phase I of this proposal, we will demonstrate the feasibility of using xylose isomerase derived from Candida boidinii for biomass to ethanol applications. Of the many organisms known to produce isomerase, the isomerase from this yeast is unique with a pH optimum of 4.5 and temperature optimum of 37°C. During Phase I , the yeast will be cultivated and the isomerase harvested. The isomerase will be characterized for biochemical and economic performance. C. boidinii isomerase will be immobilized on sub-micron scale glass fiber and tested for productivity (mass of product produced/mass of enzyme consumed) with a specialized test fixture. Enzyme yield in the native host will get a preliminary optimization, but it is likely that higher yields and lower costs can be achieved by expressing the xylose isomerase gene (xylA) in a high productivity host such as Pichia pastoris. As a first step in this process, the gene responsible for producing the isomerase will be cloned and sequenced for further development in Phase II. Finally, a one-liter scale demonstration of biomass-derived xylose from straw hydrolyzate will be converted to ethanol using xylose isomerase from Candida boidinii. The Phase II project will be to create a low-cost source of the enzyme and demonstrate its use on a larger scale. The putative C. boidinii xylA will be PCR amplified and cloned into suitable expression vectors for production of xylose isomerase in Pichia pastoris. Practical advantages of the Pichia expression system include extreme protein production and commercially available vectors. Another key parameter is that a eukaryotic host increases the probability of a properly folded and fully active enzyme. Demonstration of the improved recombinant enzyme using Trilliums 200-liter scale system will provide the stepping stone needed for development of commercial applications
