Karen Bohmert-Tatarev, Susan McAvoy, Sean Daughtry, Oliver P. Peoples, Kristi D. Snell
Metabolix, Inc. 21 Erie St., Cambridge, MA, USA 02139.
An optimized genetic construct for plastid transformation of Nicotiana tabacum for production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5’ end by the host plant’s psbA coding sequence and at the 3’ end by the host plant’s 3’ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded to plants that were capable of producing up to 18.8% dwt PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dwt PHB in samples of leaf tissue and 8.8% dwt PHB in the total biomass of the plant were also isolated.