Plastid Targeting of Polyhydroxybutyrate Biosynthetic Pathway in Tobacco

Abstract

Poly[(R)-(−)-3-hydroxyalkanoate] (PHA) synthase from Aeromonas caviae FA440 and β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha H16 were targeted into tobacco plastid by utilizing a plastid-targeting signal peptide derived from the tomato ribulose-bisphosphate carboxylase (Rubisco) small subunit. The resulting tansgenic tobacco plant expressed all of the introduced genes, and GC-MS analysis of chloroform extract of tobacco leaves demonstrated that the transgenic plant produced poly[(R)-(−)-3-hydroxybutyrate] (PHB). The productivity of PHB in plastids was about 100-fold greater than that in cytoplasm without any apparent deleterious effects on growth and seed production. Intracellular localization of PHB in the leaf of the transgenic plant was examined under epifluorescence microscopy after Nile blue A staining, and it was proven that PHB is formed in chloroplasts around the inside of cell walls.