Meeting the world's rapidly growing energy needs while protecting Earth's increasingly threatened climate and ecological balance is one of the greatest challenges facing society. This project will explore a new route to producing an advanced liquid fuel from biomass by constructing a thermophilic microbial platform to convert cellulose to isobutanol. Compatible with current engines and with higher energy density, isobutanol is superior to ethanol as a fuel. Isobutanol can replace dwindling and politically unstable petroleum supplies without contributing to global warming and, unlike ethanol, can be incorporated into the energy economy using current technology and infrastructure. To target the fundamental hurdle in advanced biofuel research of utilizing cellulose as the feedstock, the goal will be to develop a thermophilic and (partially) cellulolytic Geobacillus host organism to produce cellulase(s) to augment the native cellulolytic activity. The ability of this organism to grow at the moderately elevated temperatures where cellulases are highly active and to utilize the sugar products of cellulose degradation that normally inhibit the cellulases will enhance performance and minimize the need for additional expensive enzyme addition during fermentation to generate biofuels. Specific objectives of this project will be to generate stable, active bacterial cellulases and express those new enzymes in Geobacillus. Simultaneously, the isobutanol pathway will be established in this moderately thermophilic host. The final goal will be to integrate and optimize cellulose degradation and isobutanol production pathways.
Broader impacts: This research effort will train some of the most talented students and postdoctoral researchers in the world in protein and metabolic engineering, two technologies that are in very strong demand in the biotechnology industry, particularly the rapidly-growing biofuels/chemicals industry. This project will support extensive undergraduate research while interfacing with numerous outreach programs. The educational plan includes joint research meetings and co-advising of graduate students as well as the integration of parts of the research project into modules for laboratory courses. This project proposes a novel approach to addressing one of society's greatest challenges, developing a renewable source of transportation fuels and chemicals.
We are developing new approaches to producing fuels and chemicals from renewable resources using microbial systems. Our goal in this project was to investigate the feasibility of producing the important chemical isobutanol from cellulose in a thermophilic Geobacillus spp. This organism grows optimally at moderately elevated temperatures (55-60 oC). From a process standpoint, an organism that produces isobutanol efficiently at these temperatures could represent a significant advance and dramatically lower the overall cost of producing isobutanol from cellulosic feedstocks. Biofuel production at high temperatures has several advantages, including reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lowered cost for product separation. However, ethanol is the only biofuel that has been produced in thermophilic organisms. High temperature alcohol production, in general, is limited by enzyme stability and the lack of suitable expression systems in the thermophilic hosts. The problem is further complicated when a volatile aldehyde intermediate is involved. Here we engineered Geobacillus thermoglucosidasius to produce isobutanol at 50 oC. We mined databases to identify various enzymes in the isobutanol synthesis pathway that are thermostable. We also constructed an enzyme expression system based on the lactate dehydrogenase promoter from G. thermodenitrificans. This system allowed us to test the feasibility of advanced biofuel production using the thermophilic organism. With the best enzyme combination, the system produced 3.31 g/L of isobutanol from glucose in G. thermoglucosidasius within 48 h at 50 oC which represents the first demonstration of isobutanol production in a thermophile. Further engineering and optimization will increase both the productivity and the temperature range. We also investigated mechanisms of cellulase inactivation at elevated temperatures. Numerous protein engineering studies have focused on increasing the thermostability of fungal cellulases to improve production of fuels and chemicals from lignocellulosic feedstocks. However, the engineered enzymes still undergo thermal inactivation at temperatures well below the inactivation temperatures of hyperthermophilic cellulases. We found that free cysteines play a key role in the thermal inactivation of wild-type and engineered fungal family 6 cellobiohydrolases (Cel6A), via disulfide bond degradation and thiol-disulfide exchange. Whereas conserved disulfide bonds are essential for retaining activity after heat treatment, free cysteines contribute to irreversible thermal inactivation in wild-type and engineered thermostable Cel6A and should be removed by protein engineering. This project provided training to two master’s students, a postdoctoral researcher, a PhD student, several undergraduate researchers, and an inner-city high school science teacher.
