Overexpression of an insect cellulase in switchgrass for improved sugar release

Renewed interest in biofuels research has paved the way to explore alternative sources of novel biocatalysts for improved cellulosic ethanol yields. Insect species who predominately feed on lignocellulosic biomass serve as potential source of catalysts due to their functionality under stringent pH and reducing conditions found in their digestive system, which could be suitable for use in bioreactors. We selected a cellulase from a coleopteran species with unique biochemical properties for overexpression into switchgrass as biofuel crop model, and characterized transformants for improved cellulosic ethanol production. The insect cellulase was sub-cloned into the proven pANIC10-A overexpression switchgrass transformation vector and used to transform switchgrass var ‘Performer’ seed derived callus.   Hygromycin resistance was used as a selectable marker for transgene confirmation of callus and the pporRFP visual marker was used to select against natural hygromycin resistant escape plants. Gene expression was confirmed by RT-PCR with primers targeting the 3’ end of the transgene. Protein expression was confirmed with antibody to the AcV5 epitope tag at the C terminus.  Qualitative confirmation of production of functional cellulase protein was performed using visual degradation of the model cellulose substrate carboxymethyl cellulose infused in agar plates.  . Of the 10 transgenic events produced, only one showed a lodging phenotype compared to wildtype plants.  Biomass was collected at R1 growth stage, dried at room temperature, and analyzed for sugar release.   Our data support that the aforementioned insect cellulase is active in planta and further experiments are in place to ascertain its biochemical efficacy in cellulose degradation.