Genetic Engineering of Schizosaccharomyces pombe to Produce Bacterial Polyhydroxyalkanotes

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

Genetic Engineering of Schizosaccharomyces pombe to Produce Bacterial Polyhydroxyalkanotes

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

Biosynthesis Of Polyhydroxyalkanotes In Wildtype Yeasts

Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different Egyptian ecosystems for their ability to accumulate PHAs. Identification of the isolates and characterization of PHAs produced by the positive strains in the Nile-red staining assay was envisaged. One positive isolates which was identified using the API 20C yeast identification system as Rhodotorula minuta strain RY4 produced 2% of PHA in biomass, in glucose, oleic acid and tween 60 containing medium, over a growth period of 96 h. The nature of the PHA thus produced was analyzed by infrared spectroscopy and nuclear magnetic resonance (1H and 13C NMR) and found to contain polyhydroxybutyrate and polyhydroxyvalerate. This study shows for the first time monomer detection by 1H and/or 13C NMR of PHA polymers synthesized in wild type yeasts