PHA granule

Abstract: Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).

Abstract: Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I-III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.

Abstract: Summary The metabolism of polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) has been investigated by many groups for about three decades, and good progress was obtained in understanding the mechanisms of biosynthesis and biodegradation of this class of storage molecules. However, the molecular events that happen at the onset of PHB synthesis and the details of the initiation of PHB/PHA granule formation, as well as the complex composition of the proteinaceous surface layer of PHB/PHA granules, have only recently come into the focus of research and were not reviewed yet. In this contribution, we summarize the progress in understanding the initiation and formation of the PHA granule complex at the example of Ralstonia eutropha H16 (model organism of PHB-accumulating bacteria). Where appropriate, we include information on PHA granules of Pseudomonas putida as a representative species for medium-chain-length PHA-accumulating bacteria. We suggest to replace the previous micelle mode of PHB granule formation by the Scaffold Model in which the PHB synthase initiation complex is bound to the bacterial nucleoid. In the second part, we highlight data on other forms of PHB: oligo-PHB with ≈100 to 200 3-hydroxybutyrate (3HB) units and covalently bound PHB (cPHB) are unrelated in function to storage PHB but are presumably present in all living organisms, and therefore must be of fundamental importance.

Abstract: Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the −10 region and a region immediately upstream of the −35 region of the σ70 promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the σ54-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaRΩKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed. In addition to this, phaR expression is subject to autoregulation. Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.