Directed Evolution of Hemicellulosic Hydrolases for Conversio nof Biomass for Production of Biofuels and Bioproducts

Biofuels derived from cellulose/hemicellulose, due to their high-level sustainability and origin from nonfood portions of renewable feedstocks, will have a large-scale impact on our state's and nation's agriculture. Corn fiber, which is a byproduct of existing corn milling processes, represents a promising value-added substrate for production of biofuels. As such, the technology for producing ethanol from corn fiber was recognized as early as the 1990's by R&D magazine as one of the 100 most important technological innovation targets. The enzymatic saccharification of corn fiber as well as other cellulosic biomass is dependent on a number of critical enzymes. None of the required enzymes have been optimized to date, and the available natural enzymes perform only poorly in enzymatic saccharification. Although not the only enzymes required for the process, xylosidase and arabinofuranosidase activities play important and synergistic roles in hemicellulose degradation. Thus it is desirable to improve the performance of these enzymes in particular, and to reduce the number of enzymes required in the process. Directed evolution is an emerging technology for improvement of proteins through recursive creation of mutations and selection of desired progeny. We propose applying directed evolution technologies to improve the temperature optima of these enzymes, and also to engineer a single enzyme to possess dual xylosidase-arabinofuranosidase activities. The target enzymes are the glycoside hydrolase (GH) 39 xylosidase and the GH51 arabinofurnaosidase, two structurally and mechanistically similar enzymes \vith different substrate specificities. In the case of bioconversion, the ability to optimize enzymatic activities to work at 100vertemperatures will lead to a significant reduction in the input energy required for production of biofuels. In addition, the development of an enzyme with dual-specificity will result in significant cost savings for the large-scale production of the hydrolase enzymes. Ultimately, these approaches, if successful, will be readily applicable to the other enzymes required for the bioconversion of biomass. This project emphasizes and stimulates interactions and collaborations between the PD and a team of USDA scientists (co-PDs). This grant will support the first phase of our research and allow us to generate data supporting the scientific and economic validity of these approaches for use in subsequent research grant proposals. In the longer term, our objectives include broadened enzyme targets and more in-depth understanding of enzymes for biomass saccharification which will allow improved design and engineering of the enzyme systems required for the efficient production ofbiofuels.