Polyhydroxyalkanoate‐associated phasins as phylogenetically heterogeneous, multipurpose proteins

Abstract

SummaryPolyhydroxyalkanoates (PHAs) are natural polyesters of increasing biotechnological importance that are synthesized by many prokaryotic organisms as carbon and energy storage compounds in limiting growth conditions. PHAs accumulate intracellularly in form of inclusion bodies that are covered with a proteinaceous surface layer (granule‐associated proteins or GAPs) conforming a network‐like surface of structural, metabolic and regulatory polypeptides, and configuring the PHA granules as complex and well‐organized subcellular structures that have been designated as ‘carbonosomes’. GAPs include several enzymes related to PHA metabolism (synthases, depolymerases and hydroxylases) together with the so‐called phasins, an heterogeneous group of small‐size proteins that cover most of the PHA granule and that are devoid of catalytic functions but nevertheless play an essential role in granule structure and PHA metabolism. Structurally, phasins are amphiphilic proteins that shield the hydrophobic polymer from the cytoplasm. Here, we summarize the characteristics of the different phasins identified so far from PHA producer organisms and highlight the diverse opportunities that they offer in the Biotechnology field.

Coordinated Regulation of the Size and Number of Polyhydroxybutyrate Granules by Core and Accessory Phasins in the Facultative Microsymbiont Sinorhizobium fredii NGR234

“…Indeed, the ΔphaP2 mutant showed the most drastic change in PHB granule size among individual mutants of phasin genes. This is similar to the finding in R. eutropha H16 and B. diazoefficiens USDA110, in which PhaP1 Reu and PhaP4 Bdi , respectively, were the major phasin proteins (1,30,45). In contrast, PhaP1 and PhaP2 in S. meliloti Rm1021 made roughly equal contributions, as no obvious difference in either the size or number of PHB granules was found between individual phaP1 and phaP2 mutants (27).…”

“…PHB granules are accumulated under nutrient-limiting conditions (nitrogen, oxygen, phosphorus, etc.) but with an excess of carbon sources (1) and are considered a store of intracellular carbon and energy. Therefore, the PHB synthesis is tightly connected with the tricarboxylic acid (TCA) cycle, though the molecular mechanisms of acetyl-CoA partition between two processes remain elusive.…”

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

“…Microbial PHA preparations usually contain adsorbed phospholipids as well (19), although the actual occurrence of this coating in vivo has been recently questioned and might represent a purification artifact from the cell extracts (20)(21)(22). Phasins, the main component of GAPs, have a relevant role in the PHA intracellular metabolism, participating in events that include the control of the size, number, and localization of granules in the cell (23,24). The high affinity of phasins for PHA suggests the application of such polypeptides as affinity tags for the immobilization of proteins on polyester granules.…”

“…Therefore, we decided to characterize the binding of several BioF fusions to scl-PHA particles such as PHB. Although several phasins naturally bound to PHB inclusions have been described (23,24), our approach considered the aim of expanding the applications of the BioF tag (a naturally mcl-PHA-binding polypeptide) rather than finding a strong tag strictly recognizing a particular PHA. Furthermore, we have assessed the functional performance of a BioF-␤-galactosidase hybrid protein immobilized on PHB as a proof of concept for the development of PHA-based enzymatic bioreactors.…”

Domain-centric dissection and classification of prokaryotic poly(3-hydroxyalkanoate) synthases

“…There are another two subunits, PhaE and PhaR, for Poly(3-hydroxyalkanoate) polymerase, which integrate with subunit PhaC to form active PHA synthase, respectively [4]. A heterogeneous group of small-sized proteins with no catalytic functions, Phasin (PhaP), was also tightly involved in granule structure formation and PHA metabolism [5]. As for PHA utilization, two enzymes, intracellular PhaZ and extracellular PhaZ, play important roles in this process [4].…”