| Polyhydroxyalkanoates (PHAs) have recently received considerable interest as renewable resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. Cost barriers to widespread use of PHA may be overcome by application of biotechnology; one approach is to introduce PHA pathways into new host cells. Saccharomyces cerevisiae that natively doesn't produce PHA was genetically modified into a PHA producer.; I have engineered the synthesis of PHA polymers comprised of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of S. cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical beta-oxidation chemistries, which are natively found in peroxisomes. This platform was utilized to supply medium chain (C6--C14) PHA precursors, from both fatty acid degradation and synthesis, to a cytosolically expressed medium chain length (mcl) polymerase (PhaC1) from Pseudomonas oleovorans.{09}This yeast system synthesized PHA comprised of C12, C10, C8 and C6 monomers when fed C12 fatty acids, and it synthesized PHA comprised of C13, C11, C9 and C7 monomers when fed an odd number C13 fatty acid. Feeding substrates like pyruvate, acetate, or formate in addition to C12 fatty acids resulted in the accumulation of PHAs containing C14 monomers, indicating that intermediates from fatty acid biosynthesis were also utilized in cytosolic PHA synthesis.; The synthesis of short chain length (scl)(C3--C5) PHAs from fatty acid beta-oxidation intermediates was established in the peroxisome of a wild-type S. cerevisiae by targeting the Ralstonia eutropha scl polymerase (PhbC) to the peroxisome. This stain harboring a peroxisomally targeted scl-PHA synthase accumulated PHA up to approximately 7% of the cell dry weight. An untargeted scl-PHA synthase strain accumulated less than 0.05% PHA in the cytosol. These results indicated that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from the fatty acid metabolism to synthesize PHAs. Because different fatty acids can be offered in the culture medium using controlled strategies, it is possible to manipulate the synthesis and the composition of PHA polymers with potentially useful material properties. |
