Progress Toward the Characterization of Key Steps in the Synthesis and Degradation of Polyhydroxyalkanoate Biopolymers

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable polymeric materials produced by various microorganisms from sugar and fatty acid feedstocks and are sought out as competing green alternatives to petroleum-based plastics. This project pertains to the characterization of key metabolic reactions involved in the biosynthesis and degradation of PHAs in the Gram-negative soil bacterium Pseudomonas putida KT2440. Our first contribution involves PhaZ (PP_5004), the organism’s native PHA polymer biodegradation enzyme. Specific rates of breakdown and molecular determinants have remained elusive due to challenges in producing PHA oligomers of known length and size. Here we describe our initial steps in cloning phaZ from P. putida KT2440 into the pET-15b vector, a step that will allow for the expression and purification of active PhaZ in Escherichia coli BL21(DE3). Our second contribution relates to the role of the PP_0763, a protein hypothesized to be part of an important metabolic network converting fatty acids into PHAs. In order to gain insights into the protein’s substrate specificity, we have successfully expressed and purified 1.5 mg of our target protein PP_0763 from E. coli BL21(DE3), setting the stage for assay design with 3-hydroxy fatty acids of various lengths and clarification of the protein’s role in the biosynthesis of PHAs