| For several reasons, ethanol derived from cornstarch cannot meet the domestic demand for liquid transportation fuel. Cellulosic biomass is an untapped source of the sugars used to produce ethanol and more complex biofuels. Compared to starch, cellulosic biomass requires a higher loading and wider variety of enzymes for hydrolysis into simple sugars. This research explores in planta production of cellulosic biomass-degrading enzymes.;Genes for an endoglucanase and xylanase from the thermophilic bacteria Acidothermus cellulolyticus were codon optimized for expression in the tobacco relative Nicotiana benthamiana. Transient expression was induced by infiltrating N. benthamiana plants with Agrobacterium tumefaciens containing the gene(s) of interest. Three different expression systems were tested, including a 35S-driven system, a Cucumber Mosiac Virus replicase-based system (CMVva), and a Tobacco Mosaic Virus replicase-based system (TRBO). Co-expression of the gene-silencing suppressor p19 from Tomato Bushy Stunt Virus improved the expression of 35S-driven endoglucanase from 5.4 +/- 0.4 to 15.2 +/- 2.0 mg/kg fresh weight of plant tissue. Co-expression of p19 with the replicase-based systems was necessary for endoglucanase expression, resulting in 6.3 +/- 0.9 from CMVva and 7.0 +/- 0.9 from TRBO. For the xylanase gene, only the 35S-driven system was tested, and co-expression with p19 or an alternative gene silencing suppressor HcPro from Tobacco Etch Virus improved expression up to 6.9 +/- 1.5 mg/kg fresh weight of plant tissue.;The codon-optimized endoglucanase gene was expressed in the methylotrophic yeast Pichia pastoris and secreted into culture media at 550 mg/L culture supernatant. The Pichia and N. benthamiana-produced endoglucanases had the same optimal temperature and pH, as well as the same Michaelis-Menten constant (KM = 105 &mgr;M). The highest-yielding strain of Pichia is available to produce endoglucanase as a reference for future research.;A commercial scale process for in planta production of cellulosic biomass-degrading enzymes was modeled in SuperPro. It was determined that a transient expression process was not economically feasible. An economically feasible process using stably transformed plants expressing a battery of cellulosic biomass-degrading enzymes and incorporating up-to-date enzyme loading assumptions from industrial enzyme manufacturers was modeled and sensitivity analyses were conducted. |
