Enhanced Conversion of Lignocellulose to Biofuels.

Our program will address several of the major bottlenecks impeding the practical production of biofuels, such as ethanol and butanol, from cellulosic feedstocks. The scope of the program spans the discovery and application of new thermophilic organisms as enzyme sources and/or for biofuel production, protein engineering and kinetic modeling of improved cellulases, cellular engineering for improved solvent tolerance, and bioprocess engineering to optimize fermentation. The specific components of the program are summarized below.

Cellulase Engineering

Cellulose, a polymer of glucose, is the most abundant and renewable biopolymer on earth. Converting cellulosic biomass to biofuels is a renewable alternative to fossil fuels. A major impediment to the commercial conversion of cellulose to biofuels is the difficulty of breaking the cellulose down into glucose, which can then be used in fermentation or chemical processes to produce fuels. Cellulases, enzymes that degrade cellulose, are limited by high production costs, low hydrolysis efficiency, and poor stability. We are therefore pursuing multiple strategies to understand and improve these enzymes with the end goal of making biofuel production practical. 

Increasing the Stability of Cellulases by Directed Evolution

Protein engineering has proven to be a powerful tool in creating enzymes with new and improved properties; however, designing and employing methods to screen or select cellulase mutants using solid cellulosic substrates remains a largely unmet challenge. Our research seeks to overcome this challenge, as well as that of developing more cost-effective cellulases, by developing high-throughput solid substrate assays and applying them in the directed evolution of cellulases. The methodology developed will be applicable to the generation and study of improved cellulases that can be used in various process configurations for the production of biofuels from cellulosic biomass.

Elucidation of Mechanistic and Kinetic Detail of Multi-Enzyme Systems

We are attempting to develop a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose. We are pursuing this objective in the following manner: 

  • Capture all solution phase and surface reactions with a set of differential equations where each parameter has a physically relevant step.
  • Use these equations to assess the relative importance of system properties or the kinetic impact of particular phenomena.
  • Pursue novel experimental strategies to investigate potential rate-limiting processes at the molecular level. 
For example, we have made photoactivatable carbohydrate-binding constructs that can be pinpointed to 1nm precision with knowledge of the microscope point spread function. We are using these to observe cellulose complexation with an insoluble substrate (shown on right).

Evolving for Cellulase Enzyme Synergy

Cellulase enzymes work together to depolymerize cellulose into glucose, which can be converted to renewable liquid fuels. Cellulases are synergistic—more cellulose is degraded by two enzymes together than the sum individually. We will be evolving cellulase enzymes in the presence of complementary cellulases. By using realistic conditions (multiple enzymes), we hope to capture inter-enzyme synergy. We anticipate that this evolution strategy will generate fungal cellulase cocktails with improved hydrolytic activities on industrially-relevant substrates. This should reduce the amount of enzyme needed to break down cellulose to glucose and thus the price of the resulting biofuel, making it more cost-competitive with fossil fuels.

Engineering for Simultaneous Saccharification and Fermentation

The native Trichoderma reesei cellulase cocktail works best at 50°C. However, the yeast that would do the fermentation work best at 30°C. We are trying to combine these two important steps of cellulosic biofuel production by making these two processes function at the same temperature. Specifically, we are trying to improve the activity and stability of each major cellulase, and the cellulase mixture as a whole, at the optimal temperature for the fermentation.

Bioprospecting for High-Temperature Conversion of Lignocellulose to Ethanol

Lignocellulose degradation systems from extremely thermophilic microorganisms are ideal candidates for the development of more active, cost-effective enzymes for cellulose processing. Elevated operating temperatures would also be beneficial in fermentations to produce biofuels. In addition to lower risk of microbial contamination, a higher temperature would reduce cooling costs and facilitate ethanol (or, for example, butanol) removal and recovery. To enable translation of these advantages to practice, we propose to isolate and characterize multi-subunit extracellular and periplasmic glycolytic enzymes in several extremely thermophilic bacterial strains specifically adapted for cellulose and hemicellulose degradation. We will also isolate novel, extreme thermophiles that produce ethanol and/or butanol from enrichments of hot spring samples previously collected in Eastern Russia and the continental US. Prospecting for cellulose/hemicellulose degradation systems will be assisted by whole genome sequencing of novel isolates. Another component of the proposed effort is the development of a simultaneous saccharification and fermentation process for operation near the boiling point of ethanol. Ethanol production during saccharification of cellulose/hemicellulose at 75°C will increase process efficiency, minimize contamination, and facilitate evaporative removal of the fuel product. Individual thermophilic bacterial strains with high rates of specific lignocellulose digestion will be sequenced and the cellulose and xylanase genes will be expressed in productive recombinant host strains.

Alleviating Product Toxicity in Biofuel Production

The development of new microbes with greater tolerance toward the final fuel product, e.g., butanol, could lead to substantial improvements in the cost effectiveness of producing biofuels from cellulosic biomass. We propose to engineer enhanced tolerance toward butanol into E. coli and solventogenic organisms, including yeast and Clostridia sp. By building upon previous studies in our laboratory showing that the effects of product inhibition during acetone-butanol fermentations can be reduced by extractive fermentation, an extractive fermentation system was set up and used to optimize in situ product removal in fed-batch fermentations by the high solvent-producing strains. Higher intrinsic butanol tolerance combined with extractive fermentation is expected to result in extremely high production rates and volumetric productivities of biobutanol.