This Small Business Innovation Research (SBIR) Phase II project will develop the xylose isomerase (XI) enzyme from a marine bacterium as part of a process to convert biomass to ethanol. Xylose isomerase converts xylose, the second most common sugar in biomass, into xylulose. Xylose is not fermented to ethanol by brewing yeast, but xylulose is. Previous XI enzymes are unable to work in conjunction with fermentation due to incompatibilities in pH and inhibiting compounds. The successful Phase 1 proposal identified this marine XI as capable of performing Simultaneous Isomerization and Fermentation (SIF) of xylose to ethanol. High efficiency conversion of xylose to ethanol can improve overall process yield by 20-40%. The Phase II project will optimize the production of the enzyme in native and high-productivity heterologous hosts leading to a low cost source of the new enzyme. The optimized enzyme will be further characterized and then produced in a 200 liter bioreactor to demonstrate scalability leading to commercialization.
The broader impact/commercial potential of this project is to enable the cost-effective commercial production of cellulosic ethanol. Ethanol from local cellulosic biomass is a sustainable transportation fuel that reduces greenhouse gas emissions by an average of 87% according to the Argonne National Lab?s GREET model. The toxicity of tailpipe emissions are also reduced relative to petroleum-based fuels. As a domestic source of fuel, cellulosic ethanol adds to U.S. energy security and strengthens our economy. By creating jobs and recycling dollars into the U.S. economy, cellulosic ethanol improves the trade deficit and lessens the dependence on foreign petroleum. By developing a low-cost enzyme that is added directly to the fermentation, difficulties with genetically modified fermentation organisms are avoided. This not only simplifies the ethanol production process, but also reduces the GMO content of co-products that may enter the food chain.
The goal of this NSF project was to develop a robust, cost-effective technology for converting the common 5-carbon sugar xylose into its isomer xylulose, which can be directly fermented to ethanol by conventional yeast. Converting xylose to ethanol is a critical element of a commercially-viable cellulosic ethanol process. Trillium FiberFuels technology uses a unique low-pH xylose isomerase (XI) enzyme, produced by the marine bacterium Fulvimarina pelagi (Fp), to convert xylose to xylulose under the acidic conditions present in traditional yeast fermentations. Trillium optimized production of F. pelagi and the native XI enzyme in both batch and fed-batch bioreactor systems, but enzyme production rates were too low to support commercial production from the native host. To develop a low-cost source of active enzyme, in all nine different recombinant host organisms were created, sequences validated, and enzyme expression tested. Three E. coli hosts produced an active recombinant enzyme (rFpXI), and after further testing a single host was selected as the most promising for rFpXI production. Production of rFpXI by this host and enzyme purification techniques were developed and the purified enzyme was characterized. The genetic engineering portion of the project was completed by researchers at Oregon State University. In a supplemental Research Experiences for Undergraduates (REU) project, two OSU students developed techniques to purify and thoroughly characterize the native XI enzyme from F. pelagi. This provided properties of the purified native enzyme to use as a benchmark for comparison with the recombinant enzyme as well as valuable hands-on research experience for the undergraduate interns. Another student project team gained valuable research experience characterizing propagation of the E. coli host and gene expression in a fed-batch bioreactor system. The native and recombinant enzymes were characterized for activity and stability at different conditions of pH (4-7.8) and temperature (5-70 degrees-C), in the presence of promoters (magnesium, manganese) or inhibitors (calcium, xylitol). Kinetic parameters were determined for the purified enzymes. The rFpXI showed maximum activity 55 degrees-C, but stability deteriorated above 30 degrees. It showed less inhibition by calcium and xylitol than many other XI enzymes. It retained significant activity at pH 6, but was not as active at lower pH as the native enzyme. Expression and activity were not sufficient to support high-volume, low-cost commercial production. Concurrent with this project, domestic petroleum production experienced unprecedented growth which substantially undermined economic and policy support for renewable fuels development. As prospects for rapid growth of a cellulosic ethanol industry waned, Trillium investigated alternate applications for the FpXI enzyme and its gene. Several promising applications were identified in food processing or specialty chemical production, but none showed immediate commercialization potential.
