An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts
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Production of enzymes for lignocellulose hydrolysis in planta has been proposed as a lower-cost alternative to microbial production, with plastid transformation as a preferred method due to high foreign protein yields. An important regulator of chloroplast protein production is the downstream box (DB) region, located immediately downstream of the start codon. Protein accumulation can vary over several orders of magnitude by altering the DB region. Experiments in bacteria have suggested that these differences in protein accumulation may result from changes in translation efficiency, though the precise mechanism of DB function is not known. In this study, three DB regions were fused to the bglC ORF encoding a β-glucosidase from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. More than a two order of magnitude of difference in BglC protein accumulation was observed, dependent on the identity of the DB fusion. Differential transcript accumulation explained some the observed differences in protein accumulation, but in addition, less 3′ degradation of bglC transcripts was observed in transgenic plants that accumulated the most BglC enzyme. Chloroplast-produced BglC was active against both pure cellobiose and against tobacco lignocellulose. These experiments demonstrate the potential utility of transplastomic plants as a vehicle for heterologous β-glucosidase production for the cellulosic ethanol industry.
KeywordsTransplastomic Beta-glucosidase Tobacco Thermobifida fusca Cellulosic ethanol Downstream box
We thank David Wilson and Diana Irwin for their kind donation of anti-BglC antibodies and the pNS6 plasmid for cloning the bglC gene, and Deborah Sills for her kindly donated Spezyme CP for tobacco hydrolysis. BNG was the recipient of an NSF Graduate Research Fellowship. This work was supported by a USDA Grant (USDA NRI 2007-02133) to BAA and MRH. This research was also supported in part by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-165425 received from Cooperative State Research, Education and Extension Service, US Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture.
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