| Lignocellulose is an abundant feedstock for the production of fuel ethanol. It consists of approximately 70% carbohydrate in the form of hemicellulose and cellulose. Both of these polymers must be solubilized prior to microbial conversion to ethanol. Although hemicellulose can be hydrolyzed to monomers by dilute acids, stronger acids or expensive fungal enzymes are necessary for the solubilization of cellulose. The simultaneous saccharification-fermentation (SSF) process developed by the Gulf Oil Co. in 1976 combines cellulose hydrolysis with microbial fermentation. This dissertation focuses on the genetic engineering of Erwinia for hydrolysis of cellulose and fermentation to ethanol using a modified SSF process. Erwinia species offer potential advantages over other organisms for the bioconversion of cellulose. These include the secretion of endoglucanases and the ability to metabolize cellobiose, a potent feedback inhibitor of fungal cellulases.; Two laboratory strains of Erwinia and a number of field isolates were engineered to produce ethanol by expressing the Zymomonas mobilis pdc and adhB genes on plasmids. Recombinant E. chrysanthemi and E. carotovora produced over 45 g liter{dollar}sp{lcub}-1{rcub}{dollar} ethanol from glucose, xylose, and cellobiose, approaching the theoretical maximum for each. The rates of ethanol production from xylose and glucose were greater than 2.0 g liter{dollar}rmsp{lcub}-1{rcub} hsp{lcub}-1{rcub}{dollar} for both strains. The rate of cellobiose conversion to ethanol, 1.7 g liter{dollar}rmsp{lcub}-1{rcub} h{lcub}-1{rcub},{dollar} was higher than previously reported for any previous ethanologenic microorganism.; Several approaches have been evaluated to reduce the amount of fungal cellulase required for SSF. One method is to produce supplemental Erwinia endoglucanase during the pentose fermentation. E. coli strain KO11, which rapidly ferments corn hydrolysates to ethanol (38 g liter{dollar}sp{lcub}-1{rcub}{dollar} in 48 h), was used to express cloned celZ of E. chrysanthemi PI. Expression of celZ by E. coli KO11 during the fermentation of xylose at a concentration of 90 g liter{dollar}sp{lcub}-1{rcub}{dollar} produced 44 g liter{dollar}sp{lcub}-1{rcub}{dollar} ethanol and 36,000 IU liter{dollar}sp{lcub}-1{rcub}{dollar} of endoglucanase activity. By adding this recombinant endoglucanase to cellulose fermentations with Klebsiella oxytoca strain P2, the requirement for fungal cellulase was reduced to half the level needed in previous investigations. Under these conditions over 39 g liter{dollar}sp{lcub}-1{rcub}{dollar} ethanol (70% theoretical yield) was produced in 168 hours by K. oxytoca P2. |
