(107g) In Planta Transient Expression of Cell Wall Degrading Enzymes for Biofuel Applications

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 enzymes for fermentation. Currently, these enzymes contribute a significant cost to the total process. Expressing cell wall degrading enzymes recombinantly in the biomass substrate could potentially reduce the costs in this step. In recent years, it has been determined that transient expression in plants can produce high yields of heterologous proteins.

Thermostable endoglucanase E1 and endo-xylanase from Acidothermus cellulolyticus were transiently expressed in Nicotiana benthamiana utilizing several vectors and a gene silencing suppressor. Gene constructs were designed to target the enzymes to the apoplast, codon optimized for high level expression in the plant host, and synthesized. Genes were inserted into binary vector systems which incorporate the gene into the T-DNA region in Agrobacterium tumefaciens. Production of the enzymes using the CaMV 35S, Tobacco mosaic virus-based TRBO, and Cucumber mosaic virus-based CMVar expression systems were compared. The use of the gene silencing suppressor p19 from Tomato bushy stunt virus was shown to improve yields for all systems with expression levels of functional endoglucanase and xylanase reaching levels of 15.2 and 4.3 mg/kg fresh weight, respectively.

Co-expression of E1 and xylanase using the Cucumber mosaic virus-based CMVar system was evaluated. E1 and xylanase were co-expressed with simple mixing of A. tumefaciens carrying separate expression cassettes.  E1 and xylanase were also co-expressed with both genes carried on the same expression cassette. Proteins expressed from the same CMVar cassette were produced in the same cell, whereas proteins expressed with mixed cassettes are produced in distinct cells. This has implications for the thermally activated hydrolysis of the enzyme-producing host tissue for cellulosic biofuel production.

Efficient transient production and thermal activation of multiple lignocellulosic enzymes within the same plant cells may be a cost-effective saccharification route for biofuel production.