The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Abstract

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

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“…Whereas P. oleovorans is estimated to have about one or two large granules (128). The PHA granules are mostly spherical and surrounded by a phospholipids membrane (129) separating two crystalline protein layers (130,131) composed of the PHA polymerases (132), the intracellular PHA depolymerase (133), amphipathic phasing proteins (117,134), PHAspecific regulator proteins (135)(136)(137) and additional proteins with unknown function ( Figure 5) (128). Granules membrane coat is about 2 nm thick, containing 0.5% lipid and 2% protein, of the granule weight (138).…”

“…Simulation of the selfassembly process showed that phasins might impact on the kinetics of granule formation by reducing the lag phase (147). Various PHA-specific regulators such as PhbR from C. necator (137,142). PhaF from Pseudomonads (135,148,149) and PhaR from Paracoccus denitrificans (136,150) were found to bind non-covalently to the PHA granules.…”

A comparative perspective on lipid storage in animals

“…Phasins are the most abundant proteins in these intracellular inclusions, contributing up to 5% of the total cellular protein in bacteria (Steinbuchel et al, 1995;Wieczorek et al, 1995). In prokaryotes, phasin expression and lipid accumulation are tightly coupled and regulated by the cellular concentration of PhaR, a transcriptional repressor that can simultaneously bind to lipids and DNA elements (Maehara et al, 2002;Pötter et al, 2002;York et al, 2002;Yamada et al, 2007). Phasins stabilize lipid granules and prevent their fusion, a role that later in evolution is assumed by lipid droplet (LD)-associated proteins in eukaryotes.…”

A Novel Method for Producing Transgenic Enzymes and Peptides

“…By looking at the entire proteome before and after changing specific environmental conditions, we identified an inducible promoter in R. eutropha that could be regulated with a simple process parameter (i.e., phosphate limitation). Previous work using shake flasks has shown that phaPp is induced under PHB production conditions and that expression is controlled by a DNA binding protein, PhaR (43).…”