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. In previous work, thermostable endoglucanase E1 and endo-xylanase from Acidothermus cellulolyticus were transiently expressed in Nicotiana benthamiana through agroinfiltration. Here, thermostable cellobiohydrolase I (CBHI) from Thermoascus aurantiacus, and beta-glucosidase (BglB) and beta-xylosidase (BXyl) from Thermatoga maritima are transiently expressed in N. benthamiana through agroinfiltration. The production of these enzymes under several vector systems were compared. For CBHI, BglB, and BXyl, the highest expression was found using the Tobacco mosaic virus-based TRBO system, resulting in 20, 3, and 64 mg/kg fresh weight respectively. The pH and temperature profiles were determined for each enzyme, concluding that all enzymes have &gt;80% activity at pH 5.0 and 70C. In the literature, previous studies have shown that constitutive expression of E1 increases hydrolysis rates in transgenic Nicotiana tabacum. Here, we evaluate the autohydrolysis potential of transiently expressed E1 in wild-type N. benthamiana. 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 N. benthamiana. In addition, biomass composition analysis was performed to determine the effects that agroinfiltration and transient expression of E1 may have on the cell wall composition.

Evaluation of In planta transient expression of cell wall degrading enzymes as a biological pretreatment for cellulosic biomass

251st National Meeting (2016)

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