A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts
Abstract
We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions. Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance me...Continue Reading
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