A.B. Chemical and Physical Biology, 2009
Harvard University

Evolving for Synergy—Directed Evolution of Cellulase Enzymes for Biofuels in the Presence of Complementary Cellulases

Second-generation biofuels can offer sustainable, low-carbon, renewable alternatives to fossil fuels. Lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like miscanthus, contain lignin and the polysaccharides hemicellulose and cellulose. Cellulase enzymes catalyze the depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism, thus completing the transformation of sunlight energy into chemical energy that can replace non-renewable transportation fuels.

Due to strong intermolecular interactions, cellulose is significantly more difficult to break down than starch, another polymer of glucose. While most mammals cannot digest cellulose (dietary fiber), certain bacteria and fungi produce cellulase enzymes and are thus able to hydrolyze it. These organisms secrete a wide variety of such glycoside hydrolase enzymes which all work in concert; while a minimum of three is required for effective cellulose depolymerization, often 10 to 60 of these enzymes are employed.

Because cellulase enzymes are slow-acting and expensive to produce, our aim is to improve properties of cellulases, such as specific activity, using directed evolution. This protein engineering technique allows us to evolve proteins with desirable characteristics using a process inspired by natural selection. Directed evolution is most powerful when the experimental conditions accurately mimic those of the application. Cellulase enzymes are often evolved individually; however, they work in combination on cellulose substrates and are known to exhibit synergistic effects—that is to say that more cellulose may be degraded by two enzymes together than the sum of the degradation by those enzymes individually.

We will be evolving cellulase enzymes in the presence of complementary cellulases on solid biomass substrates. By avoiding enzyme library screens in isolation and instead using more realistic conditions (multiple enzymes), we hope to capture—if not evolve for—inter-enzyme synergy. We anticipate that this directed 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.