Production of polyhydroxybutyrate in sugarcane

We report here the production of the bacterial polyester, polyhydroxybutyrate (PHB), in the crop species sugarcane (Saccharum spp. hybrids). The PHB biosynthesis enzymes of Ralstonia eutropha [beta-ketothiolase (PHAA), acetoacetyl-reductase (PHAB) and PHB synthase (PHAC)] were expressed in the cytosol or targeted to mitochondria or plastids. PHB accumulated in cytosolic lines at trace amounts, but was not detected in mitochondrial lines. In plastidic lines, PHB accumulated in leaves to a maximum of 1.88% of dry weight without obvious deleterious effects. Epifluorescence and electron microscopy of leaf sections from these lines revealed that PHB granules were visible in plastids of most cell types, except mesophyll cells. The concentration of PHB in culm internodes of plastidic lines was substantially lower than in leaves. Western blot analysis of these lines indicated that expression of the PHB biosynthesis proteins was not limiting in culm internodes. Epifluorescence microscopy of culm internode sections from plastidic lines showed that PHB granules were visible in most cell types, except photosynthetic cortical cells in the rind, and that the lower PHB concentration in culm internodes was probably a result of dilution of PHB-containing cells by the large number of cells with little or no PHB. We discuss strategies for producing PHB in mitochondria and mesophyll cell plastids, and for increasing PHB yields in culms.