A phasin with extra talents: a polyhydroxyalkanoate granule‐associated protein has chaperone activity

Abstract

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers.

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…Last, the chaperone-like activity observed for PhaP Az (23), together with evidence that other phasins, such as PhaP Ac (42), could have similar properties, suggests that phasins could be used to enhance the production of heterologous proteins in E. coli. These findings expand the already-wide field of potential applications for phasins.…”

“…GA14 from R. ruber contains two hydrophobic domains in the C terminus that were proposed to be interaction domains with PHA based on experimental results (4). A later study that analyzed PhaP1 to PhaP4 from R. eutropha revealed that these phasins do not have (12,18,23). Furthermore, PhaP from B. megaterium (PhaP Bm ) was observed to be an extremely hydrophilic phasin that can effectively bind to the granules in spite of the lack of hydrophobic domains (8).…”

A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

“…Although PhaP is a polyhydroxyalkanoate granule-associated protein, previous work performed in our laboratory showed that it has a general protective effect in E. coli that was not only associated with polymer metabolism, as expression of phaP was observed to protect cells that do not synthesize any polymer against heat and oxidative stress agents (44). Further work showed that PhaP has both in vivo and in vitro chaperone-like activity (45). Since expression of known chaperones and other HSPs was observed to increase tolerance of different bacteria to solvents and other chemical agents (29,(41)(42)(43), the effect of PhaP production on tolerance to different chemicals was tested.…”

“…The mechanism through which HSPs have been proposed to protect cells from stress caused by solvents and other chemicals, thus increasing tolerance, is by stabilizing or refolding proteins important for cell metabolism and survival that are affected by these compounds (33). Since PhaP was demonstrated to have in vitro and in vivo chaperone-like activity (45), it is possible that the increase in tolerance to the chemicals tested is related to its capability to aid in protein stabilization and refolding.…”

“…Overexpression of PhaP in poly(3-hydroxybutyrate)-producing E. coli strains was observed to enhance growth and polymer accumulation and resulted in a dramatic reduction in the expression of stressrelated genes such as ibpA (encoding a small HSP) and dnaK (encoding a chaperone) compared to the case for a strain that does not synthesize the phasin. This protein also has a protective effect against stress in cells that do not accumulate poly(3-hydroxybutyrate) (44) and was observed to exert a beneficial effect in E. coli cells producing heterologous proteins, playing an active role in protein folding and/or unfolding prevention that reduced the number and size of inclusion bodies (45).…”