Biopolymer Engineering: Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by a wide range of bacteria as energy storage compounds. PHAs have properties ranging from thermoplastics to elastomers, making them useful for the production of bulk and specialty plastics. PHAs are also biodegradable. Given these features, PHAs are interesting in the newly emerging field of biopolymer engineering, a major new application for recombinant DNA technology. The Sinskey lab, in collaboration with Prof. JoAnne Stubbe of the Dept. of Chemistry and Prof. ChoKyun Rha of the Dept. of Biomaterials Science and Engineering, focuses on understanding the production of PHAs as this process occurs in vivo. We are studying the role of three classes of proteins, PHA synthases, phasins, and depolymerases in PHA homeostasis. The long-term goal is to develop rational strategies for the controlled synthesis of PHA biopolymers based on the fundamental understanding of priming, initiation, and elongation reactions of synthesis as well as control of intracellular PHA degradation processes.

PHA synthases play the central catalytic role in PHA synthesis and granule formation by catalyzing the polymerization of hydroxyacyl CoA substrates to yield PHAs, which in turn associate to form PHA granules. Studies of the PHA synthases of Ralstonia eutropha (PhaCRe ) and Chromatium vinosum (PhaECCv) have previously yielded important insights on the mechanism of PHA synthesis, namely that PHA synthesis proceeds via covalent catalyis involving a catalytic dyad (PhaC(Re) C319, H508 and PhaC(Cv) C149, H331) as well as a conserved aspartate (PhaC(Re) D480 and PhaC(Cv) D302) that activates the monomer hydroxyl for ester formation.

In the past year, we have continued to expand on our understanding of the PHA synthase PhaECAv (a heterodimer of PhaE and PhaC) using biochemical methods. Experiments performed with purified PHA synthase have extended on our previous model of the elongation process, and provided insight into the mechanisms of initiation and termination of PHA synthesis. SDS-PAGE autoradiography of products formed during in vitro polymerization of 14C-hydroxybutryl-CoEnzyme A with PHA synthase have identified intermediates covalently linked to the synthase during the elongation process, including an HBn-PhaCAv (where n=3-10), which was shown to be competent for elongation. The time dependent formation of this species was observed after substrate was exhausted, suggesting the synthase is capable of performing termination and regeneration of primed enzyme. In addition, studies with the substrate analog hydroxybutyryl-N-acetylcysteamine showed the formation of polyhydroxybutyryl-N-acetylcysteamine during in vitro polymerization, a finding which suggests the existence of a non-covalently bound enzyme-intermediate complex during the elongation and termination reactions.

In addition, we have undertaken expression studies to unravel the dynamic processes underlying PHA homeostasis, exploiting our expertise in molecular biological and fermentation techniques. Realtime quantitative PCR has been employed on controlled batch fermentations of Ralstonia eutropha under nutrient limitation, shedding light on transcriptional control of the phasin gene (phaP), involved in regulating granule architecture, and pointing out surprising potential roles of PHA depolymerase homologs we previously identified. In particular, phaZ1b, which we have implicated in PHA degradation, is shown to be transcribed during PHA production and repressed during degradation, suggesting that its role may be to maintain PHA in a state where it is competent for degradation by other depolymerases. We have extended these transcriptional studies with detailed quantitative immunoblotting of PHA homeostasis-related proteins during growth on both rich and nutrient-limited media, coupled with analysis of transmission electron micrographs which have together allowed us to develop a quantitative picture of protein levels in the cell during different stages of growth, PHA production, and PHA degradation. This analysis allowed us, among other results, to calculate in vivo activities for the PHA synthase and PHA depolymerases, to demonstrate quantititatively that termination and reintiation of individual PHA chains occur during PHA production, and to estimate granule surface coverage by PhaP. We also revealed a tight correlation of PhaP to PHA levels and a nearly 1:1 stoichiometry of PhaP to individual PHA chains, suggesting that PhaP plays a role in PHA synthesis beyond its apparent role in controlling granule size.