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Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing.  Ralstonia eutropha  is regarded as the model organism for PHA biosynthesis. We examined the effects of PHA depolymerase (PhaZ) expression on PHA homeostasis in  R. eutropha  strains. In order to analyze the impact of PhaZs on  R. eutropha  granule architecture, we performed electron microscopy on several  phaZ  knockout strains and the wild type strain grown under PHA production conditions. Analysis of the acquired micrographs was based on stereology: the ratio of granule area and cell area was determined, along with total granule count per full-size cell image. Cells bearing a  phaZ2  knockout mutation alone or in conjunction with a  phaZ1  mutation were found to have a high granule volume per cell volume and a higher granule count compared to wild type. A  phaZ  quadruple knockout strain appeared to have a low granule volume per cell volume and a low granule count per cell. Cells bearing a  phaZ3  knockout were found to have a higher granule count than the wild type, whereas granule volume per cell volume was similar. Accordingly, we hypothesize that PhaZs have not only an impact on PHA degradation but also on the 3-dimensional granule architecture. Based on our data, PhaZ2 is postulated to affect granule density. This work increased our knowledge about PHA depolymerases in  R. eutropha , including enzymes that had previously been uncharacterized.

Examination of PHB Depolymerases in Ralstonia eutropha: Further Elucidation of the Roles of Enzymes in PHB Homeostasis