Ziegelhoffer, Thomas , Raasch, John A , Austin-Phillips, Sandra .
Cellulases for biomass conversion from transplastomic tobacco.
We have explored the feasibility of utilizing crop plants as a production system for the cellulase enzymes required for biorefining and bioenergy production. One promising approach for recombinant protein production in plants is plastid transformation. Initial experiments in which the endoglucanase E1 of A. cellulolyticus was expressed in transplastomic tobacco yielded levels of enzyme below the levels we obtained using nuclear transformation of chloroplast-targeted enzyme. We tested the effect of N-terminal modification of recombinant proteins with the goal of optimizing translation initiation/elongation. Portions of the 5’ coding sequence of the plastid psbA gene were fused to several target protein ORFs. psbA was chosen because it is the most highly expressed chloroplast protein and appears to have been subjected to evolutionary pressure to facilitate efficient translation. Various lengths of psbA 5’ coding sequence (30-108 bp) were fused to both E1 and cel7D (a cellobiohydrolase gene) of P. chrysosporium. Expression studies in E. coli using the pET vector system demonstrated that as little as 30 nucleotides of the psbA 5’ coding sequence (encoding 10 N-terminal amino acids) significantly increased the expression level of both E1 and Cel7D. In transplastomic tobacco seedlings, 10nE1cd yielded at least 100 times more enzyme than the analogous unmodified construct, with a maximum accumulation of over 12% total soluble protein. Current efforts are directed toward a better understanding of the expression increase seen with psbA 5’ coding sequences and its applicability to other transgenes.
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UW Biotechnology Center
1 - University of Wisconsin-Madison, Biotechnology Center, 425 Henry Mall, Madison, WI, 53706, USA
2 - University of Wisconsin-Madison, Biotechnology Center
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM