Chloroplast vector systems for recombinant protein production in marker-free plants

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Abstract

Plant chloroplasts provide an attractive platform for expressing recombinant proteins. The main advantages are high yield accumulation of recombinant proteins, relatively low cost, scalability, biological containment of transgenes and the reduced risk of contamination with animal pathogens. These features of the chloroplast technology are particularly attractive to pharmaceutical companies. However, some major issues need to be addressed before the technology can move from laboratory and pilot testing to large-scale manufacturing. The high alkaloid content in the model plant tobacco and the presence of an amplified antibiotic-resistant marker gene in transplastomic plants raises biosafety concerns. Hence, in this work, an alternative non-antibiotic-based marker and marker-free methods were investigated, and plastid transformation of a commercial cultivar of lettuce was developed. In the first part of this thesis, an algal phytoene desaturase gene (pds) with a point mutation (F128V) that confers resistance in algae to norflurazon (an inhibitor of the carotenoid biosynthetic pathway) was transformed into tobacco chloroplasts. The primary goal of this study was to test if the F128V algal PDS in plant chloroplasts makes tobacco plants resistant to the bleaching herbicide norflurazon and therefore pds could be used as a non-antibiotic-based selectable marker gene for chloroplast transformation in higher plants. Seedlings of isolated transplastomic lines were only slightly more resistant to norflurazon compared to wild-type seedlings. In the second part of the thesis, I used a tobacco vector system, pEMA1, to express a laccase from Aspergillus fumigatus. pEMA1 system has a two-gene operon comprised of the aadA gene and the dao gene and allows stepwise selection on spectinomycin and D-alanine to identify plastid transformants. Flanking the marker cassette with direct repeats promotes spontaneous marker excision. The release of selection allows the accumulation of marker-free transplastomic cells. Marker-free shoots regenerate on D-valine medium, which suppresses by negative selection growth of cells containing the dao gene. Laccase protein accumulation in transplastomic plants was undetectable despite the accumulation of laccase mRNA. Furthermore, marker-free transplastomic plants were isolated from second- generation seedlings. Accordingly, I designed pEMA2.1, an updated version of pEMA1 that allows isolation of marker-free plants in the T0 generation. The pEMA2.1 system has the aadA-dao marker cassette flanked by incomplete rbcL gene sequences with 953 bp overlap. Full-length rbcL and hence a functional large subunit of RuBisCO is restored following recombination between the overlaps and marker excision. Marker-excision and reconstitution of wild-type pigmentation can be visually monitored on a rbcL knockout tobacco mutant (chlorotic). Regeneration from dark green sectors generates T0 marker-free transplastomic plants. In the last part, I designed a new chloroplast vector system (pBSLS) and a transformation protocol that allows isolation of marker-free transplastomic Lactuca sativa L. cv. Iceberg 2 (lettuce) in the T0 generation. The aadA spectinomycin-resistant marker gene was placed in the backbone of the chloroplast transformation vector (CTV). This prevents stable integration of the aadA-gene into the chloroplast genome due to active recombination between two large direct repeats. The gene of interest in the pBSLS system was associated with dao as a secondary selection marker gene. Therefore, selection on D-alanine after spectinomycin ensures that the isolated plastid transformants contain the gene of interest. Excision of the dao gene by recombination between engineered direct repeats that flank the dao gene is promoted by selection on D-valine. Regenerating lettuce shoots on D-valine are marker-free.

Keyword(s)

Chloroplast transformation; Chloroplast vector system; Co-integrate vector systems; D-amino acid oxidase; Lettuce cv. Iceberg2 chloroplast transformation; Marker-free transplastomic plants; Phytoene desaturase; Plant-made recombinant proteins; Plastid transformation

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