Polyhydroxyalkanoate‐associated phasins as phylogenetically heterogeneous, multipurpose proteins

Summary Polyhydroxyalkanoates (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.


Introduction
Polyhydroxyalkanoates (PHAs) are natural polyesters produced and accumulated by diverse organisms from the Bacteria and Archaea kingdoms as energy and carbon storage compounds under nutrient limitation conditions (i.e. nitrogen, oxygen or phosphorus) but in the presence of an excess of carbon sources (Anderson and Dawes, 1990;Lee, 1996). These polymers have acquired notoriety in recent years because they display plastic properties similar to their oil-derived counterparts, but show biodegradability and biocompatibility features which results in a versatile and eco-friendly alternative (Madison and Huisman, 1999;Potter and Steinbuchel, 2006;Keshavarz and Roy, 2010). PHAs were first described by M. Lemoigne in France, who in the 1920s reported the presence of poly(3-hydroxybutyrate) [P (3HB)], in the cytoplasm of Bacillus megaterium (Lemoigne, 1926). Since then, over 300 species, including both Gram-positive and Gram-negative bacteria, have been described with the metabolic ability to synthesize PHAs (Steinbuchel and Fuchtenbusch, 1998;Zinn et al., 2001;Suriyamongkol et al., 2007;Chanprateep, 2010;Keshavarz and Roy, 2010).
Chemically, PHAs are polyoxoesters of R-hydroxyalkanoic acid monomers. They are usually classified depending on the number of carbon atoms of the alkyl groups: small chain length PHAs (scl-PHAs) contain 3-5 carbon atoms [as poly(3-hydroxybutyrate) -P(3HB)-or poly(4hydroxybutyrate) -P(4HB)], whereas medium chain length PHAs (mcl-PHAs) possess 6-14 carbon atoms [e.g. poly (3-hydroxyhexanoate), -P(3HHx) or poly(3-hydroxyoctanoate) -P(3HO)]. Long-chain-length PHAs (lcl-PHAs) consisting of hydroxyacids with more than 14 carbon atoms are more scarcely found (Rutherford et al., 1995;Singh and Mallick, 2009). These differences are mainly due to the substrate specificity of the PHA synthases from the particular microorganism (Park et al., 2012). Moreover, the incorporation of different monomer units in the same chain gives rise to heteropolymers with new properties. The properties and functionalities of the PHAs depend on their monomer composition: whereas scl-PHAs show thermoplastic properties similar to polypropylene, mcl-PHAs display elastic features similar to rubber or elastomer (Keshavarz and Roy, 2010;Park et al., 2012). Applications of PHAs in the industry are widespread, ranging from the manufacturing of packages and covers to the generation of enantiomeric pure chemicals (Philip et al., 2007) or as protein immobilization supports (Draper and Rehm, 2012;Dinjaski and Prieto, 2015;Hay et al., 2015). Of significant relevance is the implementation of PHAs in the biomedical discipline, especially supported by the recent FDA approval for P(4HB) to be used as suture material (Tepha Inc., MA, USA). The utility of PHAs in this field arises from their biocompatibility characteristics and has found its application in a variety of processes such as drug delivery, development of medical devices and construction of tissue engineering scaffolds (Misra et al., 2006;Wu et al., 2009;Wang et al., 2010;Xiong et al., 2010;Brigham and Sinskey, 2012;Martinez-Donato et al., 2016;Rubio Reyes et al., 2016).
The PHA polymer accumulates in the cytoplasm in the form of water-insoluble granules (Fig. 1), the number per cell and size of which depend on the different species and the culture conditions (Jendrossek and Pfeiffer, 2014). Early studies carried out by Merrick 0 s group showed that these inclusions were constituted by approximately 98% (w/w) PHA, 2% granule-associated proteins (GAPs) and 0.5% phospholipids (Griebel et al., 1968). Since then, several studies have confirmed the presence of a phospholipid layer in PHA preparations (Parlane et al., 2016, and references therein). However, some data have put into question the actual presence of the lipid coat in vivo (Potter and Steinbuchel, 2006;Beeby et al., 2012;Jendrossek and Pfeiffer, 2014), especially from electron cryotomography (Wahl et al., 2012) and fluorescence microscopy (Bresan et al., 2016) results, according to which the presence of the lipid layer might arise from an experimental artefact on PHA extraction and preparation.
Four different types of GAPs have been identified so far, namely PHA synthases, PHA depolymerases, phasins and other proteins , the latter including transcriptional regulators as well as hydrolases, reductases and other enzymes involved in the synthesis of PHA monomers (Jendrossek and Pfeiffer, 2014;Sznajder et al., 2015). Among them, phasins, which received their name in analogy to oleosins [proteins on the surface of oil globules found in oleaceous plants ], are the most abundant polypeptides in the PHA carbonosome (Mayer et al., 1996). These low molecular weight proteins normally contain a hydrophobic domain, associated with the PHA, and a hydrophilic/amphiphilic domain exposed to the cytoplasm (Potter and Steinbuchel, 2005). On the basis of their sequence, phasins are distributed in four families according to the Pfam database (http://pfam.xfam.org/), namely PF05597, PF09602, PF09650 and PF09361. A recent survey showed that a high percentage of phasins and phasin-like proteins contains a leucine-zipper motif in their amino acid sequences, suggesting that oligomerization is a common organization mechanism in these polypeptides (Maestro et al., 2013). In the recent years, a large number of phasins have been identified, constituting a phylogenetically heterogeneous group of proteins. We will review the current knowledge on the most representative phasins participating in important biological functions (summarized in Table 1) such as the formation of network-like covers on the PHA granule surface (Dennis et al., 2003(Dennis et al., , 2008 or the regulation of the synthesis, morphology, distribution during cell division and degradation of the storage granules (Mezzina and Pettinari, 2016). Finally, the biotechnological potential of this group of proteins will be discussed.

A0A0K2QTP6
Phasin bound to P(3HB-co-3HA) copolymers solely when granules are enriched in 3HB in more than 87 mol% (Matsumoto et al., 2002;Hokamura et al., 2015) ª  (Pieper and Steinbuchel, 1992;Pieper-Furst et al., 1994;Pieper-Furst et al., 1995) Azotobacter sp.  (Sznajder et al., 2015) covering an estimated 27-54% of surface of the PHA granules (Tian et al., 2005a), and representing around 5% of the total cell protein fraction (Wieczorek et al., 1995). PhaP1 Reu is only synthesized in PHA-producing cells in levels correlating well with the PHA accumulation, and it is never found in soluble form but only attached to the granules (Wieczorek et al., 1995;York et al., 2001aYork et al., ,b, 2002Tian et al., 2005a). Besides PhaP1 Reu , six additional and minoritaire phasins have also been identified in R. eutropha (PhaP2 Reu -PhaP7 Reu ). Phasins PhaP2 Reu -Pha-P4 Reu are homologous to PhaP1 and are only synthesized under permissive conditions for PHB accumulation, although in much lower amounts (Potter et al., 2004;Pfeiffer and Jendrossek, 2012). On the other hand, the PhaP5 Reu -PhaP7 Reu proteins are not homologous to PhaP1 Reu and probably represent an independent subgroup of phasin-like proteins. Despite much effort dedicated to this task, the elucidation of the exact role of R. eutropha phasins other than PhaP1 Reu in PHB homoeostasis remains elusive Jendrossek, 2011, 2012).
Regarding the major phasin PhaP1 Reu , this polypeptide appears strongly bound to the hydrophobic surface of the PHB polymer as soon as its accumulation starts (York et al., 2001b;Cho et al., 2012), ensuring the dispersion of the granules and preventing the non-specific binding of other proteins. PhaP1 Reu plays a crucial role in the amount (York et al., 2001a,b), size and number of granules Wieczorek et al., 1995;Kuchta et al., 2007) and probably prevents PHB crystallization (Horowitz and Sanders, 1994). It has been demonstrated that PhaP1 Reu deletion mutants exhibit less PHB production as compared to the wild-type strain (Wieczorek et al., 1995;York et al., 2001b;Kuchta et al., 2007), indicating that it is important but not crucial for PHB synthesis, and suggesting that other minor phasins may also contribute to its accumulation. In fact, the expression level of PhaP3 Reu significantly increases in PhaP1-negative mutants (Potter et al., 2004). Nevertheless, in the presence of PhaP1 the relative importance of the other phasins must be lower, as the individual deletion of any of them does not induce any appreciable effect on polymer synthesis (Kuchta et al., 2007). Moreover, Pha-P1 Reu deletion mutants only produce a large, single granule per cell unlike wild-type cells, which usually contain between 6 and 15 disperse, medium-size granules (Wieczorek et al., 1995;Kuchta et al., 2007). In contrast, Pha-P1 Reu overexpression leads to the generation of a high number of small granules (Potter et al., 2002).
Ralstonia eutropha phasins also play a role in the stability and mobilization of PHB inclusions. Lack of Pha-P1 Reu in a single deletion mutant causes a certain degree of PHB autodegradation in vivo, an event that is dramatically augmented when combined with the multiple deletion of other phasins (Kuchta et al., 2007), suggesting that phasins are essential to stabilize the granule. Paradoxically, phasins are also critical for the mobilization of PHB induced by CoA thiolysis as catalysed by the PhaZ depolymerase. While PHB devoid of phasins is unable to be degraded by PhaZ, PhaP1 Reu alone is sufficient to assist the depolymerase in PHB degradation (Uchino et al., 2007;Eggers and Steinbuchel, 2013). On the other hand, in the absence of PhaP1 Reu , the other minor phasins may also participate in PHB mobilization to a variable extent (Kuchta et al., 2007;Uchino et al., 2007;Eggers and Steinbuchel, 2013).
Expression of PhaP1 Reu is strictly regulated at the transcription level by PhaR (Potter et al., 2002;York et al., 2002), thus ensuring that the phasin is produced only when conditions are permissive for PHB accumulation and PhaC is present (York et al., 2001a), and in enough quantity to cover all the biopolymer surface, but without inducing a protein stock in the cytoplasm (Wieczorek et al., 1995).
It has been proposed that PhaP1 Reu possesses a modulatory action on PHB synthesis in vitro on a PhaCdependent manner. Addition of pure recombinant Pha-P1 Reu increases the lag phase in the polymer formation for the R. eutropha PhaC1 synthase . A two-hybrid assay did not detect any interaction between the two proteins . A similar decrease in activity has also been detected for the synthase from Delftia acidovorans (PhaC Da ) (Ushimaru et al., 2014) although no mechanism was proposed in this case. On the contrary, Pha-P1 Reu increases the activity of the synthases from Aeromonas caviae (Ushimaru et al., 2014) and Pseudomonas aeruginosa (Qi et al., 2000), this time by reducing the enzymatic lag phase, while it does not affect the activity of PhaC from Chromatium vinosum (Jossek et al., 1998).
Secondary structure analysis of the PhaP1 Reu sequence predicts a highly a-helical conformation that is characteristic of phasins (Neumann et al., 2008). The phasin has been shown to acquire a planar, triangularshaped homotrimeric conformation as revealed by smallangle X-ray scattering analysis (Neumann et al., 2008). First sequence analyses did not unveil a clear, predicted PHA-binding motif such as long hydrophobic patches (Neumann et al., 2008).

Pseudomonas species
Most members of the Pseudomonas species are able to accumulate only mcl-PHA granules based on a well-conserved gene cluster containing two operons that are transcribed in opposite direction: (i) the phaC1ZC2D operon, encoding two type-II polymerases (PhaC1 and PhaC2), a depolymerase (PhaZ) and the PhaD protein described as a putative transcriptional regulator (Huisman et al., 1991;Klinke et al., 2000;Steinbuchel and Hein, 2001); and (ii) the phaFI operon, located downstream and coding for the PhaF and PhaI phasins (Prieto et al., 1999;Sandoval et al., 2007). The mcl-PHA granules in Pseudomonas are covered by a protein layer that contains the PhaF and PhaI phasins, together with PhaC, PhaZ and the acyl-CoA synthetase ACS1 (Prieto et al., 1999;Moldes et al., 2004;Peters and Rehm, 2005;de Eugenio et al., 2007;Sandoval et al., 2007;Ruth et al., 2008).
PhaF is the major phasin in Pseudomonas species, and it is structurally organized in two well-defined domains (Prieto et al., 1999;Moldes et al., 2004), (i) the N-terminal, PHA-binding domain, (referred to as BioF in the case of P. putida GPo1), which shares sequence similarity with PhaI, and (ii) the C-terminal moiety, a highly positively charged, histone-like domain, containing eight AAKP-like tandem repeats, and responsible for non-specific binding to DNA (Prieto et al., 1999;Moldes et al., 2004;Galan et al., 2011). Biophysical studies carried out on PhaF, supported by a three-dimensional structural model, suggest an elongated disposition in which the PHA-binding domain acquires an amphipathic helix conformation suitable to recognize the surface of the polymer granule and that is separated from the DNA-binding domain by a short leucine zipper presumably involved in the protein tetramerization (Maestro et al., 2013) (Fig. 2). Remarkably, similar coiled-coil sequences were found in the majority of phasins included in the UniProtKB database, suggesting that oligomerization might constitute a common feature of these proteins (Maestro et al., 2013). Moreover, the protein might be intrinsically disordered in its majority unless bound to its ligands (PHA and DNA), a trait that is also probably shared by many other phasins (Maestro et al., 2013).
The functionality of PhaF is not only ascribed to a mere role in PHA intracellular stabilization, but it also plays a critical role in the localization of the granule in the cell centre, ensuring an equal distribution between daughter cells during cell division by a simultaneous attachment to the PHA polymer and to nucleoid DNA (Galan et al., 2011;Maestro et al., 2013). In this sense, lack of PhaF induces in vivo a considerable reduction in total PHA content as the defects caused in granule segregation gives rise to population heterogeneity (Galan et al., 2011;Dinjaski and Prieto, 2013). Interestingly, a similar function has been detected for the PhaM protein in R. eutropha, a phasin-like polypeptide responsible for attachment of PHB granules to the bacterial nucleoid, ensuring an almost equal number of PHB granules to that both daughter cells after cell division Wahl et al., 2012). Finally, it has been demonstrated that PhaF is also involved in the in the control of expression of the phaC1 synthase and phaI phasin genes (Prieto et al., 1999;Galan et al., 2011).
The PhaI phasin displays a high sequence similarity with the PHA-binding domain of PhaF, including the probable Leu-zipper sequence. Together with PhaF, it has been demonstrated to be essential for optimal PHA biosynthesis and accumulation in P. putida KT2442 and P. putida U (Ren et al., 2010;Dinjaski and Prieto, 2013) although it can be replaced by the homologous PHAbinding domain of PhaF (Dinjaski and Prieto, 2013).

Paracoccus denitrificans
Paracoccus denitrificans is a facultative methylotrophic bacterium capable of synthesizing scl-PHAs from several alcohols (Yamane et al., 1996). The major phasin associated with PHA granules in P. denitrificans is PhaP Pde (GA-16) (Maehara et al., 1999). The expression of the phaP gene is negatively controlled by the auto-regulated repressor PhaR (Maehara et al., 2002), and a positive correlation between the accumulation of PhaP Pde protein and production of PHA has been demonstrated (Maehara et al., 1999). PhaP Pde plays a structural role in the PHA granule formation, constituting an amphipathic layer, preventing the coalescence of the granules and ensuring the correct number and size of granules. Besides, it is also involved in the distribution of the granules throughout the cytoplasm (Maehara et al., 1999).

Rhodococcus ruber
The coryneform bacterium Rhodococcus ruber NCIMB 40126 accumulates a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from single, unrelated carbon sources (Haywood et al., 1991). The GA14 protein has been identified as the major phasin bound to the surface of the PHA granules, showing a direct correlation between the amount of protein and the level of PHA synthesis in the cells (Pieper and Steinbuchel, 1992;Pieper-Furst et al., 1994). The C-terminal region of the protein, containing two hydrophobic patches, has been demonstrated as responsible for the granule anchoring (Pieper-Furst et al., 1995). This protein has also been isolated from lipid inclusions in this bacterium (Kalscheuer et al., 2001).

Azotobacter genus
PhaP Az is the most abundant PHB granule-associated protein observed in Azotobacter sp. FA-8 (Pettinari et al., 2003;Mezzina et al., 2015). This protein displays a growth-promoting effect, also enhancing the polymer production in recombinant PHB-producing Escherichia coli (de Almeida et al., 2007(de Almeida et al., , 2011. Moreover, it exerts a stress-reduction action, both in PHB and non-PHB synthesizing bacteria, decreasing the induction of heat shock-related genes in E. coli (de Almeida et al., 2011) and promoting protein folding through a chaperone-like mechanism, which suggests an in vivo general protective role of this phasin (Mezzina et al., 2015). PhaP Az has been suggested to conform a coiled-coil tetramer when it is not bound to any target. Secondary structure analysis predicts the existence of a-helices and disordered regions, with two amphipathic helices probably responsible for protein-protein or PHB interactions. Spectroscopical studies suggest that hydrophobic environments, such as those provided by PHB, can induce phasin structuration (Mezzina et al., 2014).

Aeromonas genus
Aeromonas caviae FA440 is a Gram-negative bacterium isolated from soil that is capable of producing copolyesters consisting of scl-and mcl-PHA from alkanoates or oils (Doi et al., 1995). This organism possesses a biotechnological potential as the films made of the random copolymer of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] produced by this bacteria have demonstrated very good soft and flexible properties, and better biocompatibility when compared to a P(3HB) homo-polymer, making them suitable for more practical applications (Doi et al., 1995;Yang et al., 2002). The PHA biosynthetic operon in A. caviae consists on phaP-phaC-phaJ genes, which encode the PHA granule-associated protein phasin (PhaP Ac ) (Fukui et al., 2001), as well as the PhaC Ac synthase (Fukui and Doi, 1997), and the R-specific enoyl-CoA hydratase (PhaJ Ac ) .
The PHA granules isolated from A. caviae are relatively simple in terms of its GAPs composition, as their protein cover only comprises the PHA synthase and the PhaP Ac phasin (Fukui et al., 2001). PhaP Ac (also referred to as GA13) is a 13-kDa protein, which shows an appreciable similarity with the PhaP phasin from Acinetobacter sp. (Fukui et al., 2001). Moreover, no hydrophobic or amphiphilic regions are evident in the primary structure of this protein (Fukui et al., 2001).
PhaP Ac plays an important role in the biosynthesis and metabolism of PHAs. A high level activity of PHA synthase has been documented when overexpression of phaC Ac takes place together with phaP Ac , and the PHA copolymer composition is enriched in the 3HHx fraction when compared to overexpression of phaC Ac alone, although the substrate specificity of PhaC Ac is not affected in this conditions (Fukui et al., 2001). Besides, in a recombinant strain of R. eutropha which is capable of synthesizing P(3HB-co-3HHx), the replacement of the PhaP1 Reu phasin by PhaP Ac resulted in an increase in 3HHx proportion in the copolymer (Kawashima et al., 2015). Moreover, the activity of PhaC Ac synthase in vitro is activated by the presence of PhaP Ac both in the prepolymerization and the polymer-elongation states, and the in vivo P(3HB) accumulation in a recombinant E. coli strain expressing PhaP Ac increased 2.3-fold when compared with the corresponding PhaP Ac -free strain (Ushimaru et al., 2014). This effect is not due to a mere increase in the amount of soluble PhaC Ac , but probably arises from the phasin assisting the withdrawal of the growing PHA polymer chain from PhaC Ac (Ushimaru et al., 2014). In contrast, the prepolymerization activities of PhaC Re and PhaC Da synthases decrease by the presence of PhaP Ac , whereas the activity of polymer-elongating PhaC Re is not affected. Interestingly, the in vivo accumulation of P(3HB) increases 1.2-fold in a recombinant E. coli strain when PhaP Ac is expressed together with PhaC Re , compared to the phasin-free strain. As the amount of PhaC Re in the soluble fraction increases approximately threefold by PhaP Ac coexpression, this has led to postulate that this enhanced PHA accumulation could be attributed to a chaperone-like role of Pha-P Ac in the folding of PhaC Re (Ushimaru et al., 2014).
Finally, an enhancement in the in vivo PHA accumulation has been observed in E. coli harbouring the phaPCJ operon from A. caviae when a single nucleotide mutation is present in the phaP Ac gene (PhaP Ac D4N) (Saika et al., 2014). The mutation does not induce an increase in the activity of the PHA synthase, but a higher expression level of phaP Ac gene was demonstrated, suggesting that this effect could be attributed to the enhanced expression of the whole phaPCJ operon (Ushimaru et al., 2015).
Another Aeromonas species, A. hydrophila 4AK4, is a Gram-negative bacterium initially isolated from raw sewage samples that is able to accumulate 35-50 wt. % copolymer [P(3HB-co-3HHx)] (Lee et al., 2000) reaching 70 wt. % in a metabolic engineered strain (Qiu et al., 2006;Liu et al., 2011), so this microorganism has been used for the industrial-scale production of this PHA (Chen et al., 2001). A pha operon similar to A. caviae has been found in this species (Qiu et al., 2006). The phasin produced by this microorganism (PhaP Ah ) is a 13-kDa protein whose overexpression leads to a higher number and a decrease in size of P(3HB-co-3HHx) granules, as well as to an increase in phaC Ah gene transcription and to an increment of 3HHx fraction on the P(3HBco-3HHx) accumulated copolymer, concomitantly with a reduced molecular weight of the polyester (Tian et al., 2005b). The 3-D structure of PhaP Ah has been recently elucidated by X-ray crystallography (Zhao et al., 2016). The protein folds in solution into a brick-like tetramer built from the packing of four amphipathic a-helical monomers through their corresponding hydrophobic faces. On the basis of several biophysical and mutational studies, it has been suggested that in the presence of hydrophobic entities such as PHB surfaces, the tetramer dissociates and individual monomers are able then to interact with the non-polar compound (Zhao et al., 2016).
ApdA (activator of polymer degradation) is a 17.5-kDa phasin that is bound to the PHB granules in vivo in R. rubrum (Handrick et al., 2004a). It is absolutely required for the efficient hydrolysis in vitro of the native PHB (nPHB) granules by the PhaZ1 depolymerase, a role that is not affected by several physical and chemical stresses, such as high temperatures, extreme pH's or 5 M guanidinium, but that can be mimicked by the pretreatment of the granules with trypsin or other proteases, although no protease activity has been found for this phasin (Handrick et al., 2004a,b). On the other hand, ApdA presents a 55% identity with Mms16, a magnetosome-associated protein in Magnetospirillum that has also been shown, in turn, to act as a phasin-like protein bound to the PHB granules produced by this bacteria (Handrick et al., 2004a;Schultheiss et al., 2005). In fact, it has been shown that Mms16 is able to functionally replace the activating role of ApdA in R. rubrum (Handrick et al., 2004a).

Bradyrhizobium diazoefficiens
Bradyrhizobium diazoefficiens is a Gram-negative soil bacterium that accumulates a large amount of PHB, a process that competes with the fixation of atmospheric N 2 in symbiosis with soybean plants (Romanov et al., 1980). Four phasins have been identified in PHA granules from B. diazoefficiens, namely PhaP1 Bd -PhaP4 Bd (Yoshida et al., 2013). None of them are involved in the bacterial growth kinetics (Quelas et al., 2016), but they are all expressed in levels that correlate with the accumulated PHA (Yoshida et al., 2013). In any case, expression of PhaP4 Bd is favoured when using yeast extract-mannitol (YM) medium, and it presents the highest affinity to PHA granules in vitro (Yoshida et al., 2013). Transcription of phaP3 seems to be low and constant during growth, suggesting that this phasin does not have a relevant role in PHA metabolism (Yoshida et al., 2013). On the other hand, the study of single and double mutants has revealed that the combined role of PhaP1 Bd and PhaP4 Bd must be crucial in determining the number and size of the granules (Quelas et al., 2016).
Structurally, PhaP1 Bd -PhaP4 Bd are predicted to be predominantly alpha-helical but only PhaP4 Bd contains additionally a C-terminal region very rich in alanine residues (13 out of 34 amino acids) (Yoshida et al., 2013). Besides, they are all proposed to oligomerize (Quelas et al., 2016).

Other phasins
Several other phasin proteins have been identified in other organisms such as Sinorhizobium meliloti, Haloferax mediterranii or Herbaspirillum seropedicae, but there is little information about them other than their involvement in PHA accumulation (Wang et al., 2007;Cai et al., 2012;Tirapelle et al., 2013;Alves et al., 2016).

Binding of phasins to PHA
Little is known about the molecular details of phasin-PHA interaction. In the absence of deeper biophysical analyses, some speculations can be made on the basis of the scarce protein structural data and secondary structure predictions. As described above, it has been suggested for the P. putida KT2442 PhaF phasin a nonspecific interaction through an amphipathic a-helix, so that the hydrophobic side of the helix faces the polymer whereas the hydrophilic side is exposed to the solvent. Such statement is based on the fact that the granulebinding sequence also interacts strongly with hydrophobic compounds (oleic acid) and chromatographic resins (phenyl-sepharose) (Maestro et al., 2013). This idea receives considerable support after the elucidation of the PhaP Ac three-dimensional structure (Zhao et al., 2016), which confirms the widespread presence of amphipathic sequences along this protein. In addition, selected mutants of PhaP Ac designed to increase the amphipathic character of the helices concomitantly led to a stronger binding to P(3HB-co-3HHx) films (Zhao et al., 2016). With the aim of checking whether this proposed mechanism might represent a common procedure used by phasins to interact with the PHB granule, we have carried out a theoretical study of secondary structure and amphipathicity prediction for each of the four Pfam phasin families. Due to the high number of phasin sequences to be analysed, we generated a consensus sequence for each family using the Jalview utility (Waterhouse et al., 2009). Then, a secondary structure prediction was carried out for each consensus sequence using Jpred4 (Drozdetskiy et al., 2015), and finally, all predicted a-helical sequences were analysed for their amphipathicity with HeliQuest (Gautier et al., 2008). The results show phasins (belonging to the four Pfam families) as generally predicted, highly helical proteins with appreciable amphipathic stretches (See Fig. S1 and Fig. 3 for the specific case of PhaP1 Reu from R. eutropha). This simple theoretical model, in the absence of more experimental confirmation, would explain some observations such as the lack of a defined PHA-binding region in PhaP1 Reu , as the PHA-binding ability seems distributed throughout the protein (Neumann et al., 2008).

Biotechnological application of phasins
The amphiphilic character of phasins makes them suitable to be used as natural biosurfactants. In this sense, pure recombinant PhaP Ah from A. hydrophila 4AK4 shows a strong effect to form emulsions with lubricating oil, diesel and soybean oil when compared with bovine serum albumin, sodium dodecylsulfate, Tween 20 or sodium oleate, even retaining its activity after heat-treatment of the protein or the emulsions themselves (Wei et al., 2011). In any case, the most widely studied application of phasins arises from their PHA-binding capacity. In this regard, the N-terminal, PHA-binding domain of PhaF from Pseudomonas putida GPo1 (referred to as BioF sequence) has shown to be very effective as an affinity tag to immobilize in vivo fusion proteins using mcl-PHA as support (Moldes et al., 2004(Moldes et al., , 2006. Polyester granules carrying BioF-tagged fusion proteins can be easily isolated by centrifugation and used directly or, if required, the purification of the adsorbed protein can be achieved by gentle elution with detergents, keeping their full activity in both cases (Moldes et al., 2004). This system has been demonstrated to be an eco-friendly way to deliver active proteins to the environment such as the Cry1Ab toxin with insecticidal activity (Moldes et al., 2006). Similar in vivo immobilization procedures have also been developed for PhaP1 Reu using E. coli as heterologous host for the PHA synthesis (Chen et al., 2014). In this case, the gene coding for the D-hydantoinase (D-HDT) (enzyme involved in the generation of D-amino acids of commercial values such as one of the precursors required for the synthesis of semi-synthetic antibiotics) was fused to phaP1. The recombinant fusion protein, PhaP1 Reu -HDT, resulted to be effectively attached to the granules, and the enzyme showed to be active and stable (Chen et al., 2014). In a further development, Wood 0 s group also used the PhaP1 Reu phasin and E. coli or R. eutropha as expression and immobilization hosts, but in this case they incorporated a selfcleaving intein sequence between the affinity tag and the protein of interest, allowing the easy removal of the tag and the subsequent purification of the native product by a simple pH change (Banki et al., 2005;Barnard et al., 2005). The advantage of these procedures comes from the fact that both protein and support are easily and effectively produced by the same bacterial host, leading to cost reduction in the downstream process. In any case, binding and purification can also be carried out in vitro, allowing protein production in a continuous way as demonstrated by Wang and coworkers for Pha-P Ac .
The specific immobilization of fusion proteins to PHA via phasins is starting to be employed in medicine, both in diagnostic and drug delivery applications. In the first case, two hybrid genes encoding either the mouse interleukin-2 (IL2) or the myelinoligodendrocyte glycoprotein (MOG) fused to PhaP1 Reu were constructed and expressed in a recombinant, PHA-accumulating E. coli strain. The PHA beads obtained from this system displayed the eukaryotic proteins correctly folded, and they were subsequently implemented for specific and sensitive antibody detection using the fluorescence-activated cell sorting (FACS) technology (Backstrom et al., 2007). In another example, two recombinant fusion proteins with PhaP1 Reu were generated to achieve specific delivery: mannosylated human a1-acid glycoprotein (hAGP), that is able to bind to the mannose receptor of macrophages, and a human epidermal growth factor (hEGF), able to recognize EGF receptors on carcinoma cells. The resulting proteins (rhAGP-PhaP1 Reu and rhEGF-PhaP1 Reu ) were self-assembled on P(3HB-co-3HHx) nanoparticles, achieving the specific delivery of the payload both in vitro and in vivo . On the other hand, the sequence coding for a peptide containing the amino acids Arg-Gly-Asp, the most effective peptide sequence used to improve cell adhesion on artificial surfaces, was fused to PhaP Ac (Dong et al., 2010). Different polyesters, such as P(3HB-co-3HHx) or P(3HB-co-3HV), were coated with purified PhaP-RGD hybrid protein, and the complex proved effective in adhesion and improvement of cell growth on two different fibroblast cellular lines, suggesting viable applications on implant biomaterials (Dong et al., 2010).

Concluding remarks
The generic name of 'phasin' denotes a set of proteins which indeed share the ability to recognize and adsorb to PHA polyesters. They play an essential contribution in the physical stabilization of the PHA granule within the cell, ensure the correct distribution of the polyester upon cell division and assist other proteins (synthases and depolymerases) on PHA metabolism. Nevertheless, their specific role is highly dependent both on the microbial strain and on the metabolic state of the cell. Their versatility is such that they may even participate in opposite events (e.g. synthesis and degradation of the PHA polymer). Besides, their strong affinity to PHA allows their use as protein affinity tags for polymer functionalization and therefore constitutes an opportunity to develop valuable applications in biotechnology and biomedicine. Although little structural data are still available, phasins are predicted to acquire relatively simple, amphipathic, three-dimensional structures and to bind to PHA via nonspecific hydrophobic interactions. This makes them amenable to be easily engineered to produce recombinant variants that display a modulated affinity to PHA, that may be useful both for in vivo PHA production and in vitro biotechnological and biomedical applications. supported by grants BIO2013-47684-R and BIO2016-79323-R (Spanish Ministry of Economy and Competitiveness).