Genetic engineering of to produce Bacterial Polyhydroxyalkanotes

Abstract

A commercial use of microbial produced products, like polyhydroxyalkanotes (PHAs), in the sense of an environmental precaution appears meaningful and necessary. In order to more economically produce microbial products, this investigation was focused on suitable producers, like the yeast Schizosaccharomyces pombe. Since it is not capable of the PHA synthesis, easily cultured and they must be modified genetically. Therefore, the genes of the
PHB biosynthesis pathway of Ralstonia eutropha [beta-ketothiolase (phbARe); acetoacetyl-CoA reductase (phbBRe); as well as PHB synthase (phbCRe), located onto the plasmid pBHR68 were cloned into the cohesive ended pYIplac128 integrated vector that transformed into the chromosome of the yeast Schizosaccharomyces pombe strain Q01. Under the optimized cultivation conditions, the transgenic yeast S. pombe strain Q01/PHB was able to produce PHB and accumulated up to 9.018 % PHB. The presence of heterologous DNA in the transgenic yeast was examined by means of Western blot analysis. In addition, both PHA synthase activity and kinetics were determined. The UV/Vis, 1H and 13C NMR spectral analysis have confirmed that the polymer produced by the yeast S. pombe strain Q01/PHB is a pure homopolymer of 3-hydroxybutyric acid