Abstract

Vascular plant biomass or lignocellulosic biomass is the world's most abundant renewable carbon source. Ethanol production from lignocellulosic biomass has the potential to reduce the use of fossil fuels and the production of greenhouse gasses. Currently, pre-treated lignocellulosic biomass is converted to ethanol in a four-step process: (1) production of glycosylhydrolase enzymes for polysaccharide hydrolysis; (2) hydrolysis of the polysaccharide component of lignocellulosic biomass into fermentable sugars; (3) fermentation of hexose sugars; and (4) fermentation of pentose sugars. A major contributor to the high cost of cellulosic ethanol is the cellulase enzymes produced in step 1 and used in step 2 to hydrolyze cellulose into fermentable sugars. Consolidated bioprocessing (CBP) is a process that uses an organism or organisms that can both produce the enzymes for hydrolysis of the cellulosic component of plant biomass into fermentable glucose, and ferment the glucose into ethanol. CBP-competent Saccharomyces cerevisiae should dramatically reduce the cost of cellulosic ethanol production by bypassing the need for separate steps for cellulase enzyme production, enzymatic hydrolysis and fermentation. The goal of my research is to contribute to the development of S. cerevisiae strains for application in the production of renewable cellulosic ethanol using the one-step CBP process. This work describes: the construction of recombinant S. cerevisiae strains that are capable of efficiently expressing a fungal β-glucosidase and are therefore capable of using cellobiose as their sole carbon source; the identification and cloning of a library of 25 heterologous fungal endoglucanases; screening S. cerevisiae transformants expressing the library of cloned endoglucanases for transformants that could produced functional secreted endoglucanase activity; coding region optimization of 5 endoglucanase genes that were able to produce functional endoglucanase in S. cerevisiae; the construction of recombinant S. cerevisiae strains that are capable of producing both functional β-glucosidase and endoglucanase at levels sufficient for using the soluble cellulosic polymer carboxymethylcellulose (CMC-4M) as a sole carbon source; and, the identification of several heterologous fungal cellobiohydrolase ORFs that can express functional secreted cellobiohydrolase in S. cerevisiae.