Akira Maehara

A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

Surface structure, morphology and stability of polyhydroxyalkanoate inclusions characterised by atomic force microscopy

Atomic force microscopy was used to analyse the surface structure, morphology and stability of polyhydroxyalkanoate (PHA) inclusions isolated from various microorganisms. The isolated PHA inclusions adsorbed firmly onto positively charged polyelectrolyte film on glass cover slip. Freshly isolated inclusions appeared spherical, smooth surfaced and without any structural features. With time, the adsorbed inclusions showed drastic morphological changes. PHA inclusions containing short-chain-length monomers (C4, C5) isolated from Comamonas acidovorans showed the presence of membrane-like layers on the surface, to which globular particles of about 30 nm in apparent size were attached. Repeated centrifugation during inclusion isolation resulted in the disappearance of the membrane-like layers. Prolonged storage of the adsorbed inclusions at ambient conditions resulted in the formation of lamella stacks, with a uniform thickness of about 5 nm. PHA inclusions isolated from Pseudomonas oleovorans containing predominantly C8 monomers showed a complex surface structure that appeared as a network of microfibril-like structures.

Direct observation of polyhydroxyalkanoate granule-associated-proteins on native granules and on poly(3-hydroxybutyrate) single crystals by atomic force microscopy

Polyhydroxyalkanoate (PHA) granules isolated from Comamonas acidovorans revealed the presence of globular particles on the granule surface when imaged using atomic force microscopy (AFM). The globular particles appeared to form a monolayer on the granule surface. Repeated washing with pure water resulted in granules with smooth surface without any globular particles. Purified granule-associated-proteins and PHA single crystals were used to construct a model representing native granule surface for AFM imaging. For this purpose, PhaR and PhaP proteins, purified from Paracoccus denitrificans were adsorbed onto poly(3-hydroxybutyrate) [P(3HB)] single crystals. PhaR is a 22 kDa protein involved in the regulation of PhaP (phasin) protein (16 kDa). Both purified proteins were morphologically different but adsorbed uniformly on the single crystal surface forming a monolayer. Immunogold-labeling was used to further confirm the adsorption and identity of PhaR protein on P(3HB) single crystals. The study shows for the first time, that proteins associated with PHA granules can be imaged directly and characterised using AFM. In addition, AFM images obtained in this study provide direct evidence for the binding of PhaR and PhaP proteins to the hydrophobic PHA surface.

Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans.

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.