Effects Of The Downstream Box Region On High-Level Expression Of Foreign Proteins In Plastid-Transformed Tobacco

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The potential for significant environmental benefits from lignocellulosic ethanol production via enzymatic cellulose hydrolysis have not yet been realized, in large part because of high enzyme production costs when using microbial protein production systems. Enzyme production in planta may lower these production costs, and the potential for high protein yields from plastid transformation makes this an attractive platform for cellulolytic enzyme production. The Thermobifida fusca cel6A and bglC genes, encoding an endoglucanase and a Beta-glucosidase, respectively, were inserted into the Nicotiana tabacum chloroplast for expression with various 14-amino acid downstream box (DB) fusions added to the N-terminus of each protein. The DB region, comprised of the 10-15 codons immediately downstream of the start codon, has previously been shown to be an important factor in determining foreign protein accumulation in chloroplasts and in other prokaryotic systems through an unknown mechanism. Chloroplast expression of cel6A and bglC with the various DB fusions resulted in the accumulation of active protein varying over more than 2 orders of magnitude, from less than 0.1% of total soluble protein (%TSP) to over 10%TSP. Analysis of cel6A and bglC transcripts revealed differences in RNA processing, suggesting a feedback between the DB region and RNA degradation rates. Transcript abundance, however, did not appear to be the main driver of protein accumulation in the transgenic these plant lines. Instead efficient translation would appear to stabilize properly processed transcripts. Analysis of codon usage within the downstream box regions tested showed that high-level protein accumulation correlated with frequently used plastid codons. Moreover, an analysis of codon usage within highly expressed plastid ORFs revealed differential codon usage within the DB regions of highly expressed genes as compared with the overall codon usage within the chloroplast, similar to observations in E. coli. These differences in codon usage preferences were exploited to design codonoptimized DB regions for high-level foreign protein production in chloroplasts and other prokaryotic hosts.
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