SACCHAROMYCES-CEREVISIAE EXPRESSING BACTERIAL POLYHYDROXYBUTYRATE SYNTHASE PRODUCES POLY-3-HYDROXYBUTYRATE

The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligen es eutrophus was used to construct a yeast plasmid which enabled expre ssion of the functional synthase enzyme in Saccharomyces cerevisiae. C ells transformed with the synthase plasmid accumulated up to 0.5% of c ell dry weight as PHB, with accumulation occurring in the stationary p hase of batch growth, The identity of PHB in recombinant yeast cells w as confirmed with H-1-NMR spectra of chloroform-extracted cell materia l. In addition, freeze-fracture electron microscopy revealed cytoplasm ic granules exhibiting plastic deformations characteristic for PHB, CC results indicated a low background level of PHB in the wild-type stra in, but intact polymer could not be detected by H-1-NMR. Formation of PHB in the recombinant strain implies the participation of native yeas t enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA). Inhib ition studies with cerulenin indicated that the fatty acid synthesis p athway is not involved in PHB precursor formation, Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity [150-200 nmol min(- 1) (mg protein)(-1)] and acetoacetyl-CoA thiolase activity [10-20 nmol min(-1) (mg protein)(-1)], which together could synthesize monomer fr om acetyl-CoA. PHB accumulation was simultaneous with ethanol producti on, suggesting that PHB can act as an alternate electron sink in ferme ntative metabolism. We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native beta-oxidation protein possessing D-3-HB-CoA dehydrogenase activity a nd heterologous PHB synthase.