Transient expression and co-expression of cell wall degrading enzymes in N. benthamiana for biofuels applications

Cell wall degrading enzymes for cellulosic biofuel production are currently produced in fungal fermentations in large stainless steel bioreactors. To improve the economic feasibility of the overall cellulosic biofuel process, these enzymes need to be produced at lower costs and in larger quantities. In planta production, offers a potentially more scalable and cost effective production platform. In addition, in planta production offers the unique opportunity to utilize the production host as the biomass substrate. Transient expression via agroinfiltration additionally allows non-transgenic, wild-type plants to be used. Co-expression of multiple synergistic cell wall degrading enzymes has the potential to reduce pretreatment or volume of exogenously added enzymes in enzymatic hydrolysis.

Gene constructs were developed, codon optimized, and synthesized for thermostable cell wall degrading enzymes, beta-glucosidase from Themotoga maritima and cellobiohydrolase I from Thermoascus aurantiacus. Both genes were inserted into 35S, TRBO, and CMVar vectors, which were inserted into binary plasmids and transformed into Agrobacterium tumefaciens. The enzymes were then transiently expressed in tobacco relative Nicotiana benthamiana via a vacuum infiltration method. Enzymes were purified and optimal assay conditions were determined. Resulting yields from expression vectors 35S, TRBO, and CMVar were compared with and without co-expression of gene silencing suppressor protein p19 from Tomato bushy stunt virus. Finally, co-expression of multiple cell wall degrading enzymes was achieved, although at lower yields than individually expressed enzymes.