Poster: Bio-Energy Crops
P03010: Requirement of an ER-directed signal peptide for accumulation of active Trichoderma reesei cellobiohydrolase I in transgenic corn seed
Miles, Stacy Contact Presenter||Authors||Miles, Stacy (A) Arellano, Sergio (B) Krebs, Heather (B) Nelson, Andrea (B) Batie, Chris (B) Betts, Scott (B) |
(A): Syngenta Biotechnology, Inc., Enzyme Traits Group|
(B): Syngenta Biotechnology, Inc.,
Cellobiohydrolase I (CBH1) is a key fungal enzyme used for the hydrolysis of plant cellulosic material to soluble sugars, which can be used to generate Bioethanol. Expression of an active CBH1 in plants offers a convenient and low cost source of this enzyme. We have generated transgenic maize lines transformed with TDNA encoding Trichoderma reesei cellobiohydrolase I (TrCBH1) targeted to specific subcellular compartments of the seed endosperm. The TrCBH1 protein was expressed either directly as the mature protein or as a fusion protein with 1-2 signal sequences. The mature protein was predicted to accumulate in the cytoplasm while fusion proteins were predicted to accumulate either in the apoplast (cell wall), endoplasmic reticulum (ER), or amyloplast. Using enzyme activity analysis and western blotting, we demonstrate that accumulation of the TrCBH1 protein in maize endosperm required an N-terminal ER-directed signal sequence. The highest levels of active TrCBH1 enzyme were measured in seed expressing the fusion protein that combined an N-terminal ER-directed signal sequence and a C-terminal ER retention signal.