Seed Grant: High-level production of multiple cellulolytic and hemicellulolytic enyzmes in plant for the improvement of biomass conversion

Provide a one paragraph summary of the project for a general audience. Identify the main participants and give a brief narrative summarizing the project's importance, objectives and potential impact on Kentucky. This summary will be used in press release announcements. The biorefinery-mediated production of bio-ethanol and other valuable chemicals is primarily fueled by the fermentation of sugars derived from the enzymatic processing of plant biomass. The current system I'or ethanol production in the United States converts starch derived from corn kernels into sugars that fuel the fermentation process to produce ethanol. One of the major biomass components of corn is its stover. Although stover is largely composed of sugar polymers, currently it is not economical to process these carbohydrates into the component monomers useful as feedstocks for fermentation. Biological process of biomass requires a number of cellulolytic and hemicel1ulolytic enzymes. To develop such enzyme-based system, two possible approaches offer attractive potential. Solid substrate cultivation (SSC) utilizes hydrolase-producing microbes to directly convert solid biomass to fermentable sugars. Another strategy is large-scale production of cellulolytic and hemicellulolytic enzymes by industrial fermentation or transgenic plants. The use of transgenic plants is particularly interesting because this approach can contribute to biomass conversion in multiple ways. For example. plant enzyme extracts can be used to substitute industrial enzymes currently used in the conversion process. In addition, plant tissues containing high-levels of hydro lases can be mixed with the biomass as an enzyme source to facilitate the sugar conversion. Alternatively, the targeted biomass (e.g. corn stover and switchgrass) can be engineered to produce these hydrolases. We propose to produce five hydrolytic enzymes at high-levels in a single transgenic plant. These enzymes are key hydrolases in biomass conversion. Individual gene transformation and crossing methods are both impractical for the introduction of five or more genes into a single plant. In addition. large numbers of trans genes inserted in different chromosomal locations will lead to difficulty in subsequent plant breeding. To circumvent these problems, we plan to use chimeric genes that encode two functional enzymes to reduce the number of trans genes required to introduce all enzymes into a single transgenic plant. Once functional hydro lases are produced by the plant, these plantmade enzymes and the tissues producing them will be characterized in enzyme-assisted pretreatment and sse using corn stover.