| The United States has mandated that 16 billion gallons of cellulosic biofuels be produced annually by 2022. This production process includes the saccharification of the plant cell wall into sugars by cell wall degrading (CWD) enzymes for fermentation. Together, the pretreatment and saccharification steps are a significant cost of the process. Expressing the cell wall degrading enzymes recombinantly in the biomass substrate could potentially reduce the need for physical pretreatment and decrease the need for exogenously added enzymes. In recent years, it has been determined that transient expression in plants can produce high yields of heterologous proteins.;A combination of synergistic enzymes are required to degrade cellulose and hemicellulose in the plant cell wall. Here, thermostable cellobiohydrolase I (CBHI) from Thermoascus aurantiacus, and beta-glucosidase (BglB) and beta-xylosidase (BXyl) from Thermatoga maritima were transiently expressed in N. benthamiana leaves through agroinfiltration. The production of these enzymes under several vector systems were compared. For BglB and BXyl, the highest expression was found using the Tobacco mosaic virus-based TRBO system, resulting in 28 and 206 mg/kg fresh weight, assuming specific activities of 7.7 U/mg BglB and 1.7 U/mg BXyl. For CBHI, the highest expression levels were found using Cucumber mosaic virus-based CMVar system at 338 mg/kg fresh weight, assuming a specific activity of 0.11 U/mg CBHI. The pH and temperature profiles were determined for each enzyme, concluding that all enzymes have >80% activity at pH 5.0 and 70C.;Previous studies have shown that constitutive expression of endoglucanase I (E1) from Acidothermus cellulolyticus increases hydrolysis rates in transgenic Nicotiana tabacum plants. Here, the autohydrolysis potential of transiently expressed E1 in wild-type N. benthamiana leaves was evaluated. Biomass composition analysis was performed to determine the effects of agroinfiltration and the transient expression of E1. It was found that agroinfiltration decreases structural glucan content by 60%. The transient expression of E1 did not alter structural glucan content, indicating that it was not active at plant growth or incubation temperatures. Hydrolysis studies were performed with an exogenously added commercial cellulase cocktail on 1) wild type, 2) wild type with additional exogenously added E1, and 3) agroinfiltrated with transiently expressed E1 in N. benthamiana leaves. Transient expression of E1 increased released sugars over 24 hours at 50C. Together these studies indicate that while transient expression of E1 increases hydrolysis rates, total potential glucose yields are significantly decreased. |
