Chloroplast Transformation for High-Level Expression of Useful Proteins

Transgenic plants have potential as an industrial source of enzymes due to easier scalability and lower cost compared to other systems, but the protein production of plants grown in field conditions has not been examined. Two transplastomic lines were created that express Cel6A, a cellulase from Thermobifida fusca, with one of two different downstream boxes (DBs), TetC and NPTII. The DB is a fourteen amino acid element located immediately between the start codon and the remainder of the gene. TetC-Cel6A accumulated protein to the greatest extent and to levels higher than previously reported. TetC-cel6A plants accumulated Cel6A to an average abundance of 17% of total soluble protein (TSP) in the field and 35% of TSP in growth chambers. In comparison, plants containing the gene for NPTII-Cel6A production accumulated Cel6A to only 3% of TSP in the field and approximately 6% in the chamber. These results confirm that the DB influences foreign protein abundance via an important regulatory interaction. Furthermore, while transformed plants showed no reduction in Rubisco or total soluble protein concentration. In fact, some transformed field plants marginally increased TSP accumulation per gram dry tissue to accommodate production of the foreign protein. Aboveground biomass was not reduced and protein mass per unit ground area was as high as 8 g / m² in some lines in the field. This suggests that the added burden of producing the foreign proteins does not compromise carbon fixation via a reduction in Rubisco concentration, the critically important carbon-fixing enzyme in plants.