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AMB Express. 2012; 2: 26.
Published online Apr 26, 2012. doi:  10.1186/2191-0855-2-26
PMCID: PMC3430594
Examination of PHB Depolymerases in Ralstonia eutropha: Further Elucidation of the Roles of Enzymes in PHB Homeostasis
Christopher J Brigham,1 Esther N Reimer,2 ChoKyun Rha,3 and Anthony J Sinskeycorresponding author1,4,5
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
2RWTH Aachen University, 52062, Aachen, Germany
3Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
4Division of Health Sciences Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
5Engineering Systems Division, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
corresponding authorCorresponding author.
Christopher J Brigham: cbrigham/at/mit.edu; Esther N Reimer: esther.reimer/at/rwth-aachen.de; ChoKyun Rha: ckrha/at/mit.edu; Anthony J Sinskey: asinskey/at/mit.edu
Received January 12, 2012; Accepted April 26, 2012.
Abstract
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.
Keywords: Ralstonia eutropha, Polyhydroxyalkanoates, Polyhydroxybutyrate, Biomaterials, Depolymerase, Granules, Carbon utilization, Electron microscopy, Stereology
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