Granule-Associated Proteins - American Chemical Society

Biomacromolecules 2005, 6, 552-560

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Poly(3-hydroxybutyrate) Granule-Associated Proteins: Impacts on Poly(3-hydroxybutyrate) Synthesis and Degradation† Markus Po¨tter and Alexander Steinbu¨chel* Institut fu¨r Molekulare Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 3, 48149 Mu¨nster, Germany Received September 22, 2004; Revised Manuscript Received November 8, 2004

Polyhydroxyalkanoates (PHAs) represent a group of biopolymers that are synthesized by many bacteria as storage compounds and deposited as insoluble cytoplasmic inclusions. Because they have many putative technical and medical applications, PHAs may play an important role in human life in the future. Therefore, for academic interest the bacterial PHA metabolism has been studied in much detail. In the past decade much new and unexpected information about the metabolism of PHA in bacteria became available. Aspects of the biogenesis of PHA granules in bacteria become more and more important in the literature. Several enzymes, proteins, and mechanisms of regulation are involved in PHA biosynthesis and PHA granule biogenesis. The intention of this review is to give an overview about our current knowledge of the structure of the PHA granule surface and the PHA granule-associated proteins involved in biogenesis and degradation. The focus is on the PHA synthases, the intracellular PHA depolymerases, the phasins, and the transcriptional regulator PhaR, which are the main actors in biosynthesis and intracellular degradation of PHAs and formation of PHA granules. In addition, putative applications of PHA granules and PHA granule-associated proteins in nanotechnology are discussed. Introduction Living systems are capable of synthesis of a wide range of different biopolymers. Biopolymers occur in any organism and constitute by far the major fraction of cellular dry matter. According to their chemical structure biopolymers belong to eight classes: (1) nucleic acids such as DNA and RNA, (2) polyamides such as proteins and poly(amino acids), (3) polysaccharides such as cellulose, dextran, and starch, (4) polythioesters (PTEs), which were reported very recently, such as poly(3-mercaptopropionate), (5) polyanhydrides with polyphosphate as the only example, (6) polyisoprenoids such as natural rubber or Gutta Percha, (7) polyphenols such as lignin, and (8) organic polyoxoesters such as polyhydroxyalkanoates (PHAs), polymalate, and cutin. Many biopolymers are reserve compounds and are often stored in the cytoplasm occurring as insoluble inclusions.1 The best-studied storage compounds in bacteria are PHAs. In most bacteria, accumulation of PHAs in bacteria is implemented in the presence of an excess of the carbon source with concomitant limitation of growth by another essential nutrient like nitrogen or oxygen.2 Therefore, PHAs serve as storage the compound for energy and carbon under starvation conditions. Recently, a new class of structurally related biopolymers, PTEs, were discovered which consist of mercaptoalkanoic acids.3,4 The key enzyme of PHA synthesis, the PHA synthase (PhaC), synthesizes both PHAs * To whom correspondence should be addressed. Phone: +49-2518339821. Fax: +49-251-8338388. E-mail: [email protected]. † This paper was presented at the ISBP 2004 (International Symposium on Biological Polyesters), held in Beijing, China, August 22-28, 2004.

and PTEs. Cyanophycin is another transiently accumulated storage compound, which is synthesized by cyanobacteria and some heterotrophic bacteria. Cyanophycin plays an important role in the conservation of nitrogen, carbon, and energy, and it is nonribosomally synthesized by the cyanophycin synthetase.5 Beside polymers, other carbon and energy storage compounds in bacteria are triacylglycerols and wax esters. Although only few examples of substantial triacylglycerol accumulation have been reported in bacteria,6 biosynthesis of wax esters (oxoesters of long-chain primary fatty alcohols and long-chain fatty acids) has been frequently shown for members of the genus Acinetobacter.7 Approximately 150 different PHA constituents have been characterized representing a very miscellaneous class of bacterial polymers.8 Besides 3-, 4-, 5-, and 6-hydroxyalkanoates, different functionalized hydroxyalkanoates such as those with halogenated and aromatic side chains have been described as constituents of PHAs.9 In the cytoplasm of bacteria, PHA granules occur as waterinsoluble inclusions. PHAs can contribute up to 80% (w/w) of the cell dry weight matter of cells or even more. The resulting granules are coated with a layer of phospholipids and proteins.10 The granule-associated proteins play a major role in the synthesis and degradation of PHAs and in the formation of PHA granules. Research done in the past decade gained interesting and unexpected insights into the structure of PHA granules, their granule-associated proteins, and their biogenesis. This review summarizes our current knowledge of prokaryotic PHA inclusions. It will focus in particular on the PHASCL granules and their associated proteins involved in biogenesis. A hypothetical model for PHB mobilization

10.1021/bm049401n CCC: $30.25 © 2005 American Chemical Society Published on Web 01/11/2005

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PHB Granule-Associated Proteins Table 1. Granule-Associated Compounds of PHA Granules Reported in the Literature representative species

classes of PHA synthase

PHA synthase

PHA depolymerase or 3HB-oligomer hydroxylase

regulator

phasin

R. eutropha P. oleovorans A. vinosum B. megaterium

I II III IV

PhaC PhaC1/PhaC2 PhaE/PhaC PhaC/PhaR

PhaZ1, PhaZ2, PhaZ3, PhaZ4, PhaZ5, PhaY PhaZ unknown unknown

PhaR PhaF/PhaI ORF4 unknown

PhaP1, PhaP2, PhaP3, PhaP4 unknown unknown PhaP

is presented, and biotechnological applications for PHA granules and associated proteins in the field of nanotechnology are discussed. Occurrence of PHAs in Bacteria The capability for biosynthesis of inclusion bodies is widely distributed in nature and is found in animals and plants as well as microorganisms. In prokaryotes, the most abundant class of inclusions are PHAs serving as an intracellular carbon and energy storage compound.11 Among bacterial PHAs the best known example is the polyhydroxybutyric acid, also referred to as poly(3HB) or PHB. The occurrence PHB as intracellular inclusions was first reported for Bacillus megaterium by Maurice Lemoigne in 1925,12 who reported the presence of 3-hydroxybutyric acid (3HB) in the autolysate of B. megaterium. One year later, he discovered a polyester from B. megaterium with the empirical formula of (C4H6O2)n, which was referred as PHB.13 By the end of the 1950s, sufficient evidence had accumulated from physiological studies to suggest that PHB functions as an intracellular reserve for carbon and energy. Hitherto the presence of PHA in members of the family Halobacteriaceae of the Archaea14 and the cyanobacteria15,16 has also been shown. Meanwhile, PHB synthesis has been shown to occur in members of all phyla of prokaryotes. Accordingly, the majority of bacteria accumulate PHB in response to physiological stress (e.g., oxygen or nitrogen deficiency) and the notice on PHB in bacteria suggests that it is a primary product of carbon assimilation like glycogen. Merrick and Doudoroff17 concluded that PHB granules are surrounded by a membrane containing both the PHA synthase and the PHA depolymerase. PHB granules are, therefore, subcellular structures of the bacterial cells, capable of responding by synthesis or degradation to various physiological stresses. The physical properties of PHB granules are very different, and two physical states can be distinguished represented by intracellular native PHB granules and partially crystalline PHB granules.18 Intracellular native PHB granules are in the amorphous rubbery state, and the surface is surrounded with a layer consisting of phospholipids and granule-associated proteins.19-23 During extraction of PHB granules from the cell, the phospholipid and protein layer is damaged or lost.10,24,25 Scandola and co-workers26 described for extracellular PHB granules 50-60% of crystallinity. This crystalline fraction melts in the range of 170-180 °C. In contrast, PHB in the amorphous fraction is characterized by the same glass transition temperature as PHB in native granules (Tg ≈ 0 °C). Molecular weights as high as 106 Da have been reported for PHB.

Structure and Dimensions of PHA Granules Recent studies on the structure of PHA granules and on granule-associated proteins have been especially carried out in Pseudomonas oleoVorans by Witholt and co-workers27 and Lenz et al.,28-30 in Paracoccus denitrificans by Yamane with co-workers,31,32 and also with R. eutropha in the laboratories of Steinbu¨chel,33-35 Sinskey,36-38 and Dennis.39 PHA synthesis occurs intracellularly in multiple inclusions which are surrounded by a membrane to which proteins are bound. Four types of granule-associated proteins are found within bacterial genera producing PHAs (Table 1): (i) PHA synthase, (ii) PHA depolymerases and 3HB-oligomer hydroxylase, (iii) phasins (PhaPs), which are thought to be the major structural proteins of the membrane surrounding the inclusion, and (iv) the regulator of phasin expresssion PhaR. First investigations of purified PHA granules were done by Williamson and Wilkinson40 and also by Griebel and coworkers10 who demonstrated that the granules contained proteins and lipids besides the PHB. Bacterial PHA granules are mostly between 200 and 500 nm in diameter.41 Electron microscopic studies by Lundgren et al.19 with Bacillus cereus and B. megaterium revealed a dense membrane of a thickness of 15-20 nm at the surface of PHB granules. It was also assumed that the PHA granules were covered with a layer of the PHB synthase protein.42,43 Chemical analyses have shown that inclusion bodies contain approximately 97.5% PHA, 2% protein, and 0.5% lipid,10 although some estimates of the lipid contents are considerably higher.22 The granuleassociated proteins exhibited polymerase activity and also depolymerase activity.25 De Koning and Maxwell44 proposed a model with a phospholipid monolayer at the surface of the PHA granules. Later it has also been recognized that these inclusions contain proteins at their surface that are responsible for structure and function.22 The question of whether the boundary layer is protein or membrane has been clarified by Mayer and Hoppert45 and Boatman.46 They have determined that the thickness of the layer is 4 nm; this excludes a lipid bilayer, which has a thickness of approximately 8 nm. Very recently atomic force microscopy (AFM) studies on PHB granules in Comamonas acidoVorans47 and Ralstonia eutropha39 provided further evidence for the existence of an envelope surrounding the PHB granules. PHB inclusions from sonicated cells of R. eutropha revealed on one hand a rough and ovoid and on the other hand a smooth and spherical surface structure and shape. Furthermore, Dennis and co-workers39 discovered globular structures connected by a network of linear structures of 4-nm width. Also splits and fissures were identified at the surface of rough PHB granules; measurements of the splits indicated a thickness

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Table 2. Overview of the Various Granule-Associated Proteins in Representative Species year

strain

1964 1968 1989 1991 1993

B. cereus and B. megaterium B. megaterium in general prokaryotes P. putida R. eutropha in general prokaryotes R. ruber in general prokaryotes P. oleovorans

1994

1995 1999

2001 2002

2003 2004

R. eutropha P. oleovorans P. oleovorans B. megaterium Pa. denitrificans B. megaterium R. eutropha Pa. denitrificans R. eutropha R. eutropha R. eutropha B. megaterium R. eutropha

granule-associated compounds discovered in the cited source

reference

unknown membrane of 15-20 nm exclusive PHA synthase exclusive PHA synthase two PHA synthases and phospholipids exclusive PHA synthase PHA synthase and phospholipids PHA synthase, GA14 protein, phospholipid PHA synthase, phospholipids, and other proteins two PHA synthases, PHA depolymerase, 18- and 43-kDa proteins and phospholipids PHA synthase, PhaP, unknown proteins 43-, 55-, and 59-kDa proteins PhaF and PhaI PhaC and PhaP GA16 PhaR PhaZ1 PhaR D-(-)-3-hydroxybutyrate-oligomer hydrolase PhaR globular structures with central pore and phospholipids PhaQ PhaP2, PhaP3, PhaP4

of the boundary layer of 4 nm. AFM investigations at higher resolution clearly demonstrated that the rough inclusions show globular structures with 35 nm in diameter, and a central pore could be identified.39 These data agree with the model of Mayer and Hoppert45 with regard to the lipid monolayer, but the granule model presented by Dennis et al.39 is much more complete than previous models. The evolving AFM technique seems to be very useful to analyze the surface structures and shapes of PHB granules, and its high resolution allows also analysis of the binding of single proteins to the surface. To investigate the mechanisms which are essential for in vivo PHA granule formation computer simulation could help to develop strategies to optimize bio-engineering pathways and fermentation processes. A computer program simulating the formation of PHA could also suggest approaches to control granule size, molecular weight, and number of granule-associated proteins. First attempts to explain PHB granule formation in vitro by a computer program were done by Marchessault and co-workers.48,49 This computer program was then extended to a more complex system, including phasins, to quantify their anticipated effect on the granule properties. This actualized simulation program appears to be a useful tool for analyzing hypothetical options for the mechanism of the effects of phasins on properties of PHB granules in vivo.50 PHB granule formation relating to R. eutropha could also be represented by a mechanistic simulation based on computer program logic.51 This latter computer program captures the dynamics of all important variables, such as cell dimensions, granule size, number of granuleassociated molecules, and PHA yield as well as degree of polymerization of the PHB molecules. Previous studies on Pseudomonas PHA granules have suggested a controversially discussed model for the structure

Lundgren et al.19 Ellar et al.42 Masamune et al.43 Huismann86 Gerngross et al.58 de Koning and Maxwell44 Pieper-Fu¨rst et al.87 Hocking and Marchessault21 Foster et al.88 Wieczorek et al.33 Stuart et al.52 Prieto et al.89 McCool and Cannon90 Maehara et al.31 McCool and Cannon64 Saegusa et al.73 Maehara et al.32 Saegusa et al.76 Po¨tter et al.34 Dennis et al.39 Lee et al.91 Po¨tter et al.35

of the granule layer.52 This model suggests that PHA synthesis, containment, and degradation of polyester are carried out in association with a complex membrane assembly at the PHAMCL granule surface. This model is in contradiction to the model of Mayer and Hoppert,45 who discussed a lipid monolayer surrounding the PHA granules. Also they showed a drawing of the boundary layer, reconcilable with the phospholipid monolayer model. They assumed that the micelles in statu nascendi are covered by PHA synthase protein. During the micelle growth, their surface is expanded, and phasin and phospholipids were inserted in the spaces emerging between the PHA synthase.1 The various compounds associated to PHA granules are summarized in Table 2. PHA Synthase (PhaC) Biosynthesis of the most frequently occurring type of PHA, PHB, proceeds in three steps and starts from the central intermediate acetyl-coenzyme A. Two molecules of acetylCoA are condensed to acetoacetyl-CoA, catalyzed by a β-ketothiolase (PhaA); acetoacetyl-CoA is subsequently reduced by a stereospecific acetoacetyl-CoA-reductase (PhaB) to R-(-)-3-hydroxybutyryl-CoA.53-55 The final step is catalyzed by the PHA synthase and polymerizes the acyl moieties of 3-hydroxybutyryl-CoA to PHB with concomitant release of coenzyme A. Because PHA synthases represent the key enzymes of PHA biosynthesis, these enzymes were investigated and characterized in great detail.56 Immunogold labeling of PHB granules and enzymatic studies clearly demonstrated association of the PHA synthase from R. eutropha and Allochromatium Vinosum to the surface of the PHB granules.57-59 However, the PHA synthase becomes only PHB-granule-bound during PHB biosynthesis, when the growing hydrophobic polyester molecules, which are co-

PHB Granule-Associated Proteins

valently linked to the enzymes during polymerization, confer amphiphilicity to the enzyme-polyester complex. In cells of R. eutropha not accumulating PHB, the PHA synthase is soluble in the cytoplasm.57 Whereas PhaC is essential for storage PHA biosynthesis in R. eutropha, PhaA and PhaB can be replaced by other isoenzymes. Today, many additional genes involved in PHA metabolism are known; however, the functions of several genes or proteins, respectively, remain to be elucidated. Since the cloning of the PHA biosynthesis operon from R. eutropha H16 about 15 years ago,60-62 more than 60 different PHA synthase genes have been sequenced from different bacteria.56 According to the substrate specificities and sequence homologies, four different classes of PHA synthases were distinguished. Class I PHA synthases synthesize PHAs of hydroxyalkanoates of short-chain-length (PHASCL). Class II PHA synthases prefer coenzyme A thioesters of hydroxyalkanoates of medium-chain-length (HAMCL) comprising 6-14 carbon atoms as substrates and occur in strains belonging to the genus Pseudomonas of the rRNA homology group I. The third class of PHA synthases exhibit also substrate specificities with HASCL. However, in contrast to class I and class II PHA synthases, these PHA synthases are composed of two different subunits, which are designated as PhaE and PhaC. The first class III synthase investigated in detail was the enzyme of A. Vinosum.63 Class IV PHA synthases are also composed of two different types of subunits and occur in species belonging to the genus Bacillus.64 Although the PhaC protein of class IV PHA synthases revealed high homologies to PhaC of class III PHA synthases, an activator, PhaR, is required for PHA synthase activity. The class I and class III PHA synthases of the β-proteobacterium R. eutropha and of the γ-proteobacterium A. Vinosum, respectively, are regarded as model enzymes for studying PHASCL biosynthesis in bacteria, whereas the PHAMCL synthases of P. oleoVorans and Pseudomonas putida represent the most detailed studied class II PHA synthases. The molecular weight of PHB synthesized by PHA synthases depends on several factors. Metabolic factors are the intracellular concentration of the PHA synthases, the enzyme-to-substrate ratio, and probably also enzymes capable to hydrolyze PHAs, such as intracellular PHA depolymerases18 or esterases and lipases.65,66 The absence of the latter enzymes results in the formation of PHAs exhibiting a higher molecular mass as shown for recombinant strains of Escherichia coli expressing the PHA operon from R. eutropha.67 Also the level of the PhaC expression has a significant influence on the molecular mass of the synthesized polyester. A higher concentration of active PHA synthase protein resulted in a lower molecular mass of the resulting PHA molecules.68,69 PHA Depolymerase (PhaZi) Several bacteria express extracellular depolymerases, which are secreted into the environment to degrade PHA released from dead bacteria. The unmatured extracellular depolymerases (PhaZe) contain in general N-terminal signal peptides of 25-58 amino acids, a large catalytic domain at the N-terminal region, a substrate binding domain localized


at the C-terminal region, and a linking domain connecting the catalytic domain with the substrate-binding domain.70 In contrast to the well-studied extracellular PHA depolymerases, intracellular PHA depolymerases (PhaZi) have been far less investigated although they play an important role for the overall PHA metabolism. In B. megaterium, Zooglea ramigera, Sinorhizobium melioti, and R. eutropha PHA hydrolyzing activity has been demonstrated.17,71-73 The first nucleotide sequence of an intracellular PHA depolymerase of R. eutropha was published in 2001,73 and the enzyme was designated PhaZ1. Very recently, four additional intracellular PHA depolymerases were identified in R. eutropha by York and co-workers (PhaZ2 and PhaZ3),74 Schwartz et al. (PhaZ4),75 and Po¨tter et al. (PhaZ5).35 Western blot experiments employing polyclonal antibody raised against PhaZ1 demonstrated that this PHB depolymerase is expressed in R. eutropha in a nitrogen-starved and carbon-rich medium, whereas in cells grown in a nutrient-rich medium for 2 days an immunostained band could not be detected. In addition, if purified native PHB granules were subjected to Western blots, a highly specific band was obtained. No immunostained band was observed in the supernatant fraction of PHB-rich cells.73 It is worth mentioning that an intracellular D-(-)-3-hydroxybutyrate-oligomer hydrolase of R. eutropha is also granule-associated.76 To avoid confusion with the nomenclature of PHA depolymerases, which are referred to as PhaZ, the 3HB-oligomer hydrolase, which was previously designated PhaZ2, should be assigned as PhaY. Also in Pa. denitrificans an intracellular PHA depolymerase was annotated and characterized in much detail.77 Phasins (PhaP) Phasins represent a class of most probably noncatalytic proteins consisting of a hydrophobic domain, which associates with the surface of the PHB granules, and of a predominantly hydrophilic/amphiphilic domain exposed to the cytoplasm of the cell. These proteins are synthesized in very large quantities under storage conditions, representing as much as 5% of the total protein.33,78 This layer of phasins stabilizes PHA granules and prevents coalescence of separated granules.22,33 PhaP adheres very tightly to native as well as to artificial PHB granules. In the PHASCL accumulating bacteria Rhodococcus ruber, the anchoring region was located at the carboxy terminus, and it was demonstrated that phasin molecules truncated at the carboxy-terminal region lost their capability to bind to PHA granules.79 PHA granules can also be generated in vitro simply by incubating purified PHA synthase and a suitable substrate. The addition of phasins accelerated the PHA synthesis rate. Furthermore, the size of these inclusions can be regulated by the addition of PhaP, and electron microscopic studies revealed that the in vitro generated granules do not have the same surface structure as native inclusions.80 It was also shown that addition of PhaP from R. eutropha increased the activity of class II PHA synthase from Pseudomonas aeruginosa by approximately 50%.81 In recent years, phasins gained more significance because several laboratories have shown that defective or lacking phasins have substantial effects on PHA synthesis. PhaP from

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R. eutropha is the best studied phasin. Several hypotheses for its function have been suggested. Further insights into the role of phasins were provided by genetic studies. Tn5induced PhaP mutants with defective phasin biosynthesis were still able to synthesize PHB; however, they synthesized PHB at a significantly lower rate, and almost the entire PHB was present in only one single large granule in the cell.33 Overexpression of PhaP, on the other hand, resulted in formation of many small granules in the cell.34 Gene deletion experiments of PhaP demonstrated that the amount of PHB under a defined set of growth conditions was reduced by 50% with respect to wild-type R. eutropha.38 It should be mentioned that R. eutropha synthesized only those amounts of phasin protein that could be bound to the granule surface, because soluble phasin protein was not detectable.82 The occurrence of four genes for phasin homologues in R. eutropha raised the question whether also the three additionally detected genes are intact as it was previously shown for PhaP1 or whether some of them are silent genes, which are not transcribed and translated into functionally active proteins. However, reverse transcription and polymerase chain reaction (RT-PCR) analysis clearly demonstrated that PhaP2, PhaP3, and PhaP4 were also transcribed under conditions permissive for PHB biosynthesis and accumulation.35 When one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis was used and followed by N-terminal amino acid sequence and matrixassisted laser desorption ionization time-of-flight analysis, PhaP3 and PhaP4 could be identified.35 PhaP2 was identified in crude cellular extracts of R. eutropha by Gerngross and co-workers.83 Although PhaP2 could not be localized in vivo at the PHB granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules.35 These new and unexpected findings will affect our current models of the structures of PHA granules and on the mobilization of PHA in vivo and may also have severe impacts on the establishment of heterologous production systems for PHAs. Transcriptional Repressor (PhaR) Further insights into the role of PhaP1 were provided by the detection of the transcriptional repressor PhaR in R. eutropha that regulates the expression of PhaP1.34,38 PhaR interacts in this bacterium with upstream regulatory sequences of both PhaP1 and PhaR. Homologues to PhaR were identified in various bacteria, for example, S. melioti, Pa. denitrificans, A. Vinosum, and several other PHASCL-producing bacteria. A number of R. eutropha deletion strains were constructed to examine the role of PhaR in PHB accumulation. PhaR is proposed to be a repressor protein of transcription that binds to the PhaP1 upstream region in R. eutropha, thereby repressing expression of PhaP1. Derepression of PhaR requires biosynthesis and accumulation of PHB.34 PhaR has the capability to bind to at least three different targets in cells of R. eutropha: (i) the promoter region of PhaP1, (ii) the promoter region of PhaR, and (iii) the surface of PHB granules. All these data support the following simple but elegant and efficient model for the regulation of PhaP1 expression in R. eutropha with PhaR functioning as a transcriptional repressor protein as shown in Figure 1.


Situation A: If the cells are cultivated under conditions not permissive for PHB biosynthesis, PhaR cannot bind to PHB granules because they do not exist in the cells. The cytoplasmic concentration of PhaR is sufficiently high to repress transcription of PhaP1. No PhaP1 protein is formed and detectable in the cytoplasm. Situation B: If conditions permissive for PHB biosynthesis start, the constitutively expressed PHA synthase (PhaC) starts to synthesize PHB molecules that remain covalently linked to the enzyme. At the beginning small micelles are formed which become larger and constitute the nascent PHB granules. PhaC no longer covers the PHB granule surface entirely, and proteins with a binding capacity to the hydrophobic surface like PhaR bind to the granules. This lowers the cytoplasmic concentration of PhaR. Situation C: From a certain point the cytoplasmic concentration of PhaR becomes so low that it can no longer repress transcription of PhaP1. PhaP1 is then synthesized and binds subsequently to the PHB granules. The concentration of soluble PhaP1 in the cytoplasm remains beyond a detectable level. Situation D: The PHB granules grow and reach their maximum size; PhaP protein is being continuously synthesized in sufficient amounts. Situation E: When the PHB granules have reached the maximum possible size according to the physiological conditions, almost the entire surface will be covered by PhaP1 protein, and the latter is displacing PhaR protein from the PHB granules. Consequently, the cytoplasmic concentration of PhaR increases, and it will exceed the threshold concentration required to repress again transcription of PhaP1. As a consequence, PhaP1 protein is no longer synthesized. This mode of regulation ensures that PhaP1 is not produced in higher amounts than required to cover the surface of PHB granules. In addition, the binding capacity of PhaR to the promoter region of its own gene prevents overexpression of this repressor protein that is, therefore, under autocontrol. In the PhaR deletion mutant strain, PhaP1 is constitutively expressed at high levels. If PhaC is deleted, then no PhaP1 is produced;33,36,37 PhaR is proposed to remain bound to the promoter region of PhaP1. Importantly, deletion of both PhaR and PhaC results in high levels of PhaP1 expression, even though the cells cannot produce PHB. Evidence for binding of PhaR to the PHB granules is based on Western blots, using antibodies raised against PhaR and proteins that co-purify with the granules. On the other hand, in vitro studies also revealed that Pa. denitrificans PhaR was able to cause a gel shift of a sequence of DNA to which it is proposed to bind and that this gel shift was reversed in the presence of PHB.32 All these studies suggested that PhaR has a role in addition to regulation of PhaP. E. coli strains have been engineered to contain PhaA, PhaB, and PhaC in the absence of PhaP1 or PhaR. More detailed analysis of the concentration of each protein at various stages in PHB production should provide insights into the role of the regulation of PhaP1 production required for effective granule formation. PHB Granules as Nanoparticles Surface coating of nanoparticles is an important area in nanomaterials synthesis.84 Because of their special composition, these coatings possess a unique combination of proper-



Figure 1. Sequence of events occurring during PHA granule formation that ensure appropriate expression of phasin PhaP1 in R. eutropha through regulation by the transcriptional regulator protein PhaR. For an explanation, see the text. (The PHA synthases and PHA depolymerases are not shown for reasons of simplicity.)

ties of the inorganic and organic components, for instance, hydrophobic, hydrophilic, anti-adhesive, and Teflon-like properties. The current trend of developing nanophase materials has created an increased need for nanometer-scale structures in a variety of applications.

PHB granules are interesting cores for surface coatings, because the PHB core is biodegradable and nontoxic. In addition, size and number of the PHB granules in R. eutropha and other bacteria can be defined in vivo by overexpression or repression of the PhaP1 protein. The noncovalently

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Figure 2. Potential configurations in PHB nanoparticles applied to medical or technical applications. The oval form represents the phasin part of the various fusion proteins.

attached phasins are predestined for coating of PHB granules. The high amount of PhaP1 in the cells and the knowledge about its regulation made this protein an excellent candidate for fusions with pharmaceutically and chemically relevant tags to be used in medical or technical applications. In the case of fusion proteins, which are produced in a recombinant organism accumulating PHA granules, these decorated nanoparticles can be produced also in vivo. In principle, these fusions can be generated by recombinant DNA technology, enzymatically, or by chemical reactions. The tag itself may be a protein or another organic molecule obtained biochemically or chemically. However, fusion proteins and PHA granules could also be produced separately in two different organisms and subsequently combined in vitro after isolation. All other fusions as well as enzymatically produced fusion proteins must be coupled in vitro to isolated PHA granules. Putative approaches used in constructing nanobiomaterials are schematically presented in Figure 2. Very appropriate for this system of biodegradable matrixes carrying protein complexes are, for example, hormones such as insulin, antibodies, antitumor components, and also tags, which allow simple in vitro modifications after harvesting of the PHB granules. The latter could be done enzymatically or chemically. Very recently, a protein immobilization and purification


system based on PHAMCL granules was developed. The N-terminal domain of the PHAMCL granule-associated protein PhaF from P. putida strain Gpo1 was used as a polypeptide tag to anchor fusion proteins to PHAMCL granules.85 The socalled BioF fusion proteins could be isolated simply by centrifugation. The use of PHB in drug delivery has been evaluated in a number of studies.92 Toxicity tests according to ISO 10993 revealed that PHB is suitable for use as nanoparticles in animals. In early studies, Korsatko et al.93 evaluated PHB for use as the matrix in retard tablets and reported also on the in vivo degradation, which occurred at a rate directly proportional to the elapsed time. Many subsequent studies have confirmed that PHB does degrade in vivo. However, in vivo degradation occurred very slowly, and PHB is completely resorbed after only 24-30 months.94,95 While PHB is generally degraded slowly in vivo, the homopolymer poly(4HB) behaves different and might, therefore, be more suitable for pharmaceutical and medical applications. Martin et al.96 reported that in vivo degradation of this polymer is relatively rapid. This might be advantageous in nanoparticle applications, where a sudden loss of the matrixes is desirable. Further studies on treatment of PHB-coated granules with the beta-blocker Celiprolol-HCl were also performed.92 Outlook In the last 10 years, our knowledge about the metabolism of PHB and also about the biogenesis and structure of PHB granules has increased significantly. The occurrence of three additional phasin homologues in R. eutropha does not at all disturb previous models on the structure of the PHB granules and on the regulation of PhaP1 expression. This is because any of these other phasin proteins are expressed at a much lower level than PhaP1 and because they occur in the cells only at very low concentrations. This is also because none of the other phasin proteins can really substitute PhaP1 in a PhaP1 mutant. These new and unexpected findings extend our current knowledge on the structure of PHA granules and possibly also on the in vivo mobilization of PHA. PhaP2 and PhaP4 may not be considered as phasins sensu strictu and may, therefore, have a different function requiring only low concentrations of these proteins. In the future, we will investigate whether the functions of these two proteins are related with mobilization of PHB and whether they interact with one of the five PHA depolymerases in R. eutropha. A

Figure 3. Model of the putative role of PhaP2 or PhaP4 in mobilization of PHB granules. Blue globes, phasins (PhaP1 or PhaP3); orange globes, PhaP2 or PhaP3; red globes, PHA synthase; yellow globes, PHA depolymerase, green globes, PhaR; and magenta globes, phospholipids.


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Granule-Associated Proteins - American Chemical Society

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