A holistic view of polyhydroxyalkanoate metabolism in Pseudomonas putida

Summary

Polyhydroxyalkanoate (PHA) metabolism has been traditionally considered as a futile cycle involved in carbon and energy storage. The use of cutting-edge technologies linked to systems biology has improved our understanding of the interaction between bacterial physiology, PHA metabolism and other cell functions in model bacteria such as Pseudomonas putida KT2440. PHA granules or carbonosomes are supramolecular complexes of biopolyester and proteins that are essential for granule segregation during cell division, and for the functioning of the PHA metabolic route as a continuous cycle. The simultaneous activities of PHA synthase and depolymerase ensure the carbon flow to the transient demand for metabolic intermediates to balance the storage and use of carbon and energy. PHA cycle also determines the number and size of bacterial cells. The importance of PHAs as nutrients for members of the microbial community different to those that produce them is illustrated here via examples of bacterial predators such as Bdellovibrio bacteriovorus that prey on PHA producers and produces specific extra-cellular depolymerases. PHA hydrolysis confers Bdellovibrio ecological advantages in terms of motility and predation efficiency, demonstrating the importance of PHA producers predation in population dynamics. Metabolic modulation strategies for broadening the portfolio of PHAs are summarized and their properties are compiled.

Introduction

Advances in systems biology and high-throughput ‘omic’ techniques are providing new opportunities for studying bacterial physiology from a holistic standpoint. In addition, computational tools now allow in silico genome-scale metabolic network analysis to be combined with quantitative omic data to generate rational designs for metabolic pathways. This allows information to be gathered on bacterial physiology and metabolism, and allows pathways to be engineered for industrial purposes (Chubukov et al., 2014; Monk et al., 2014). This is of particular interest with respect to model bacterial strains such as Pseudomonas putida KT2440, an outstanding candidate for the development of microbial chassis with specialized phenotypes (Nikel et al., 2014). P. putida KT2440 is a TOL plasmid-cured, spontaneous restriction-deficient derivative of P. putida mt-2 (Regenhardt et al., 2002). As non-pathogen, it colonizes many different environments, and it is well known for its broad metabolic versatility and genetic plasticity (Nikel et al., 2014). It is a prototype microorganism for environmental and industrial applications based on its ‘Generally Recognized As Safe’ credentials (Federal Register, 1982), its stress resistance and its amenability to genetic modification. Since the publication of the P. putida KT2440 genome (∼ 6.2 Mb) (Nelson et al., 2002), much effort has been invested in exploiting these capacities in biotechnological applications, which include the production of bioplastics [such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs)], the bioremediation of contaminated areas, fossil fuel quality improvement, the biocatalytic production of fine chemicals, the promotion of plant growth and plant pest control (Prieto et al., 2007; Poblete-Castro et al., 2012a; Tripathi et al., 2013). The availability of P. putida KT2440 genome-scale metabolic models (Nogales et al., 2008; Puchałka et al., 2008; Poblete-Castro et al., 2012b), together with the widespread use of new omic techniques, provide a basis for understanding, for example, the physiology of mcl-PHA metabolic and regulatory networks.

PHAs offer an alternative to petroleum-based plastics. They are produced (usually as a response to excess carbon), stored as intracellular energy granules and degraded by strains of P. putida and many other microorganisms, and are attracting industrial interest as technical-grade polymers, a consequence of their unique properties. For example: (i) they show the potential to substitute industrial thermoplastics such as polypropylene, polyethylene, polyvinylchloride and polyethylene terephthalate; (ii) they are biodegradable, both under aerobic and anaerobic conditions (including in aquatic environments); (iii) their biological source makes them renewable; (iv) they show biocompatibility with cells and tissues and (v) they show structural diversity.

The physiology of PHA production has aroused much interest since the ability to accumulate these compounds is widespread among bacteria and PHA metabolism influences many cell activities. PHAs have been generally classified according to their monomer size as either short-chain-length PHAs (scl-PHAs) (e.g. poly-3-hydroxybutyrate or PHB) with C4-C5 monomers, or as mcl-PHAs with C6-C14 monomers. Many bacterial species can produce PHB, but mcl-PHAs are mainly, though not exclusively, produced by fluorescent pseudomonads such as P. putida (Prieto et al., 2007). The above-mentioned division is based on differences in the in vivo substrate specificity of PHA polymerase or synthase, the enzyme responsible for the assembly of PHA monomeric precursors [(R)-3-hydroxyacyl-CoAs] and the specialization of metabolic and regulatory networks in each specie. To date, the most widely studied reference mcl-PHA producers are P. putida KT2440 (and its rifampicin-resistant mutant KT2442) and Pseudomonas oleovorans GPo1 (ATCC 29347), reclassified and referred to herein as P. putida GPo1 (van Beilen et al., 2001).

This paper reviews PHA metabolism in P. putida, describes the metabolic and regulatory networks associated with it, and discusses its physiological role as well as the impact of the PHA-producing phenotype on the surrounding microbial environment. It also highlights studies that have aimed to tailor PHA synthesis.

The carbonosome as an intracellular pseudo-organelle

PHAs accumulate as intracellular inclusions or granules (50–500 nm) possibly covered by phospholipid monolayers (Jendrossek and Pfeiffer, 2014), the surface of which contains protein components of the PHA metabolic machinery. The considerable number of proteins on the surface of PHA granules suggests that they represent supramolecular complexes with specific functions, rather than being simple carbon and energy stockpiling systems produced during periods of nutrient imbalance (Fig. 1). The idea of this multifunctionality is captured by the term ‘carbonosomes’ (Jendrossek, 2009). PHA synthases (PhaC), depolymerase (PhaZ_KT), phasins (PhaF, PhaI) and acyl-CoA synthase – together referred to as granule-associated proteins (GAPs) – have been identified as carbonosome components in P. putida, and are all involved in PHA metabolism and granule formation (Galán et al., 2011). Three granule formation scenarios have been proposed, known as the micelle, budding and scaffold models (Jendrossek, 2009; Pfeiffer and Jendrossek, 2014). In contrast to the micelle model, in which PHA granules are assumed to be randomly distributed in the cytoplasm, the budding and scaffold models suggest them to have defined locations and to involve granule–cell membrane interactions and PhaC–scaffold molecule interplay, respectively. Cooperation between PhaC and phasins has recently been proposed within the scaffold model for Ralstonia eutropha H16, and phasins suggested as the main components of the network interconnecting granules, DNA and the enzymes involved in PHA metabolism (Pfeiffer et al., 2011; Jendrossek and Pfeiffer, 2014). This network may mediate granule localization within the cell. In mcl-PHA granules of pseudomonads, phasins PhaF and PhaI have been identified as the major GAPs (Prieto et al., 1999). PhaF has been shown involved in the organization of the granules into a needle array along the long axis of the cell, and to organize granule sharing during cell division (see below). However, PhaF might bind directly to other cytoskeletal proteins, facilitating the formation of the needle array structure by direct or indirect interaction with the cell nucleoid, although the precise mechanism by which PHA granules are positioned by PhaF remains elusive (Galán et al., 2011).

Figure 1

PHA granules location and its segregation during cell division in P. putida KT2440. Homogeneity of the population in terms of PHA granule content.

A. The PHA granule or carbonosome is a multifunctional complex formed by all enzymes and proteins involved on the PHA machinery.

B. The PHA granules are located lengthwise the cell, forming the characteristic needle array structure in the wild type P. putida KT2440.

C. The lack of PhaF phasin results in a different phenotype; the PHA granules in P. putida KT40F mutant strain remained accumulated in one of the cell poles.

D. Transmission electron microscopy (TEM) image of wild type strain population. The segregation is homogeneous in the wild type strain and the previously formed PHA granules are distributed to the daughter cells keeping the needle array organization.

E. TEM image of PhaF mutant cell population. The granules in the mutant strain remain agglomerated in one of the daughter cells resulting in a heterogeneous population.

F. The impact of cells containing PHA granules in the microbial community due to extracellular depolymerase activities. The extracellular PHA consumption implies an advantage for other microorganisms in the niche providing them an extra source of carbon and energy.

Synchronous with PHA production, phasins are synthesized to protect the host cell by contributing to the coating of the hydrophobic polymer surface. All PHA-producing bacteria [e.g. R. eutropha (Wieczorek et al., 1995), Bacillus megaterium (McCool and Cannon, 1999), Rhodococcus ruber (Pieper-Fürst et al., 1994; 1995), Paracoccus denitrificans (Maehara et al., 1999), P. putida (Prieto et al., 1999; Sandoval et al., 2007; Galán et al., 2011), Acinetobacter sp. (Schembri et al., 1995)] synthesize these proteins. According to its three-dimensional model (validated by thermodynamic, hydrodynamic and spectroscopic techniques), PhaF is a tetramer made up of monomers elongated (Maestro et al., 2013). These monomers are in turn composed of a long, amphipathic N-terminal helix, followed by a short leucine zipper, a proposed signature motif for protein–protein interaction, and a superhelical C-terminal domain (Maestro et al., 2013). Interestingly, PhaI shares considerable sequence similarity (57%) with the N-terminal region of PhaF containing the putative oligomerization linker (Moldes et al., 2004). Thus, the structural redundancy of PhaF/PhaI autonomous modular units could be crucial for granule formation and segregation. The structural and functional independence of the PhaF N- and C-terminal domains has been reported, and both domains were shown essential for optimal PHA biosynthesis and accumulation in P. putida. When one of the modules is missing, PHA production decreases, falling to 7% of cell dry weight when phasins are completely absent (Galán et al., 2011; Dinjaski and Prieto, 2013).

The structural model suggests a partially disordered state for the amphipathic α-helical N-terminal domain of PhaF in the absence of PHAs (Galán et al., 2011; Maestro et al., 2013). In turn, when the polymer is produced, the hydrophobic face of the N-terminal helices becomes associated with the granule, either through its phospholipid coating or naked polyester, whereas the hydrophilic side faces the solvent. The resulting polarity prevents granule coalescence and non-specific protein association through hydrophobic interactions (Maestro et al., 2013).

PhaF also regulates the expression of the pha genes, and indeed the entire transcriptomic profile. This effect is derived from its DNA binding abilities and putative histone-like functionality (Galán et al., 2011). The highly positively charged PhaF C-terminal domain carries eight AAKP-like tandem repeats characteristic of the histone H1 family. The strong similarity of the PhaF C-terminal to histones able to bind to a variety of DNA sequences led to the discovery that PhaF can bind DNA through the C-terminal domain in a non-specific fashion. It can therefore be classified as a nucleoid-associated protein. This explains how PhaF functions as a pleiotropic transcriptional regulator; it determines the accessibility of nucleosomal DNA to the transcription machinery. The C-terminal domain is natively unfolded in solution in the absence of DNA, rather like histones, which are intrinsically disordered proteins. The in vivo cellular localization of the PhaF N- and C-terminal domains confirmed their biological role as coupling agents between PHAs and the bacterial genetic material (Galán et al., 2011; Dinjaski and Prieto, 2013). In relation to this, PhaF has been demonstrated a key player in PHA granule localization within the cell, directing them towards the centre where the needle array forms (Fig. 1). In addition, the role of PhaF in the balanced distribution of PHA granules during cell division has been demonstrated via its interaction with the bacterial chromosome (a possible granule carrier during cell division). Importantly, a strain lacking PhaF with an unbalanced granule distribution during cell division shows two markedly different cell populations: those that have and those that do not have PHA granules (Galán et al., 2011) (Fig. 1). Since PHA production provides important advantages in competitive settings (see below), an intact PhaF protein might be defined as a crucial factor for survival. In this sense, similar characteristics has been described for the protein PhaM of R. eutropha H16, that is responsible for anchoring PHB granules to the bacterial nucleoid via binding to the PHB synthase and DNA (Pfeiffer et al., 2011; Wahl et al., 2012; Pfeiffer and Jendrossek, 2014).

The influence on, and possible cooperative function of PhaI in determining the homogeneity of the cell population (in terms of granule content and segregation) remains poorly understood. Importantly, only minimal amounts of natural phasins are needed to achieve optimal PHA production and segregation (Dinjaski and Prieto, 2013). Further, the fact that the PhaI of P. putida KT2440 has a predicted coiled-coil sequence within its primary structure, similar to that possessed by PhaF, suggests that heterodimers/heterotetramers might be formed (Maestro et al., 2013). It has been experimentally shown that the function of PhaI in terms of PHA production can be substituted by PhaF or its N-terminal domain. The PhaF C-terminal domain is, however, essential. The above findings have led to the production of an array of synthetic biology tools for configuring the mentioned phasin modules into peptide tags, and have opened new avenues of research into polymer peptide functionalization and new possible applications (Grage et al., 2009; Dinjaski and Prieto, 2013).

Metabolic and regulatory networks wiring the PHA cycle, a critical pathway for synchronizing global carbon metabolism

The systemic nature of the PHA metabolic machinery suggests the existence of intricate regulatory and metabolic networks consisting of interactions between polyesters, metabolites, enzymes (including GAPs), the bacterial nucleoid and regulators (Fig. 2). PHA accumulation involves: (i) indirect PHA precursor routes that link the catabolism of other carbon sources (non-PHA related) to fatty acid and PHA metabolism; (ii) central pathways such as fatty acid ß-oxidation and de novo fatty acid synthesis; these provide direct PHA precursor routes that convert fatty acid or acetyl-CoA from non-PHA-related intermediates, respectively, into different (R)-3-hydroxyalkanoyl-CoAs, the substrates of PHA synthases associated with the carbonosome; and (iii) specific PHA metabolism (the PHA cycle) encoded by the pha cluster (Fig. 2). In P. putida KT2440 and other pseudomonads (Prieto et al., 2007), the pha cluster is organized into two main operons, phaC1ZC2D and phaIF, and is controlled by the transcriptional activator PhaD in response to ß-oxidation metabolites (Fig. 2) (de Eugenio et al., 2010a). Structurally related substrates, such as fatty acids, are processed by pseudomonads through the ß-oxidation cycle. The resulting acyl-CoAs are sequentially oxidized into enoyl-CoA, (S)-3-hydroxyacyl-CoA and (R)-3-ketoacyl-CoA. All of these intermediates are then converted into (R)-3-hydroxyacyl-CoA by a stereospecific trans-enoyl-CoA hydratase (PhaJ), an epimerase, and a specific (R)-ketoacyl-CoA reductase (FabG) respectively. Although most mcl-PHA intermediates are obtained through the β-oxidation of fatty acids, non-PHA-related carbon sources such as acetate, ethanol, fructose, glucose, gluconate and glycerol can be oxidized to acetyl-CoA and channelled towards PHA formation via the fatty acid de novo synthesis pathway. In this process, malonyl-CoA and its precursor acetyl-CoA are activated by transacylation to acyl-carrier protein (ACP). Malonyl- and acyl-ACP derivatives are condensed by ketoacyl-ACP synthetase, reduced (losing a ketone group), dehydrated and saturated to the corresponding (R)-3-hydroxyacyl-ACP. This may then be further elongated by successive two-carbon units. Acyl-ACP intermediates can then be re-transformed into (R)-3-hydroxyacyl-CoAs by the specific transacylase PhaG, which is present in most pseudomonads. Only P. putida GPo1 is unable to synthesize mcl-PHAs out of non-fatty acid substrates such as gluconate, apparently due to deficiencies in PhaG transcription (Hoffmann and Rehm, 2004).

Figure 2

Metabolic and regulatory network of PHA metabolism in P. putida KT2440. In P. putida, synthesis and degradation of PHAs were found to operate as a continuous cycle with 3-hydroxy-fatty acids released from PHA granules by PhaZ depolymerase and activated to 3-hydroxyacyl-CoAs by ACS1 acyl synthetase with the concomitant consumption of one ATP molecule (green). The PHA metabolic machinery is highly regulated and is closely related to fatty acid metabolism, since it is the direct precursor route (red). These paths provide the interconnection between external fatty acid sources and non-fatty acid related carbon sources with the activated 3-hydroxyacyl-CoAs. Acetyl-CoA is a crucial metabolite that connects carbohydrate metabolism (blue) with PHA cycle (green) through the fatty acid metabolism (red). The dynamic PHA cycle is a highly controlled route, from allosteric enzyme regulation and metabolic flux to DNA topology. Thereby, the regulatory network of PHA metabolism operates a sensitive mechanism that is able to balance the use of available energy and distribution of carbon resources.

Understanding the function of PHA metabolism within the system network requires an analysis of its regulatory logic, i.e. the relationship between the input and output of the route. However, PHA metabolism is not a unidirectional metabolic process in which the PHAs are either polymerized or depolymerized, but a bidirectional dynamic process in which there is a continuous cycle of synthesis and degradation (Ruth et al., 2008; Ren et al., 2009; de Eugenio et al., 2010b) (Fig. 2). This helps to adapt the carbon flow to the transient demand for metabolic intermediates, thus balancing carbon resources, ensuring optimal growth under changing environmental conditions. PHAs fuel other cell activities with building blocks (ß-oxidation metabolites such as (R)-3-hydroxyalkanoyl-CoAs) and energy (reduction equivalents via the TCA cycle) (Escapa et al., 2012). The peripheral supplier routes and regulatory networks ensure the funnelling of monomeric precursors towards the polymer. PHA synthesis and degradation are linked to the metabolic network thanks to a continuous cycle in which PHA synthase and depolymerase are simultaneously active, thus ensuring PHA turnover (Ren et al., 2009; de Eugenio et al., 2010b; Arias et al., 2013). Under PHA-producing conditions, PhaZ_KT depolymerase continuously releases 3-hydroxy fatty acids from PHA granules. ACS1 acyl synthetase then uses ATP to activate these metabolites to 3-hydroxyacyl-CoAs, which are then either catabolized via the β-oxidation pathway or re-incorporated into the PHA granule by PHA polymerases.

The maintenance of an ongoing PHA cycle in P. putida ensures a more robust metabolism and plays a key physiological role. PHAs act as carbon and energy reservoirs that allow bacteria to better fend off carbon starvation. However, PHAs also provide a metabolic link between carbon and nitrogen metabolism. Biochemical studies on PHB production in R. eutropha indicate that this polymer acts as a sink for reducing equivalents, and that PHB production is controlled by intracellular levels of NADH, NADPH and acetyl-CoA (Schlegel and Gottschalk, 1962; Schubert et al., 1988; Budde et al., 2010). Transcriptomic and proteomic studies involving recombinant E. coli strains expressing phb genes have confirmed that the inhibition of the TCA cycle during PHB production facilitates the metabolic flux of acetyl-CoA to the PHB synthetic pathway (Brigham et al., 2010; Peplinski et al., 2010; Raberg et al., 2011). In P. putida acetyl-CoA/free CoA and NADH/NAD ratios regulate the PHA storage/mobilization equilibrium to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand (Klinke et al., 2000; Ruth et al., 2008; de Eugenio et al., 2010b). Transcriptomic and metabolic flux analyses have recently shown the PHA cycle to play an important buffering role; it balances global biomass (including PHA carbon/energy storage), cell division and energy spillage (Escapa et al., 2012). The deletion of the PHA cycle in a PHA-impaired P. putida mutant causes an increase in acetyl-CoA synthesis when a fatty acid is used as a carbon source. The surplus acetyl-CoA overflows through the TCA cycle and glyoxylate shunt leading to the formation of additional reducing power that, instead of generating more biomass, results in increased respiration and cell division rates.

In P. putida, the nature of the substrate used as carbon source and PHA precursor has an impact on PHA metabolism. Fatty acids, which are structurally related to PHAs, are incorporated into them during the exponential phase of growth, driven by the global regulator PsrA (Fonseca et al., 2014). In contrast, a biomass build-up phase is required before structurally unrelated compounds such as carbohydrates, glycerol and aromatic molecules become involved in PHA accumulation. Although a nutritional imbalance (i.e. an excess of carbon or the limitation of an inorganic nutrient such as nitrogen) has been traditionally considered a prerequisite for PHA synthesis (Madison and Huisman, 1999), this might only be true when non-related substrates are used as precursors in biomass and PHA production (Sun et al., 2007; Wang and Nomura, 2010; Follonier et al., 2011). Nitrogen limitation, albeit not been strictly necessary for PHA synthesis from fatty acids in P. putida, can improve the polymer production yield and is therefore used in industrial biopolymer production. Multi-omic analysis of octanoate-grown P. putida KT2440 cells has revealed its physiological state in terms of biomass and PHA production under different nutrient conditions (strict nitrogen or carbon limitations, and carbon + nitrogen limitation). Poblete-Castro and colleagues (2012b) detected PHA production under all the conditions assayed. The PHA content was highest under nitrogen limitation, followed by carbon + nitrogen limitation, and then carbon limitation. Other proteomic analyses of P. putida CA-3, using styrene as a precursor, have confirmed the production of PHA metabolism-associated proteins only under a nitrogen limitation regimen (Nikodinovic-Runic et al., 2009). Indeed, it has been described that the 3-hydroxy-ACP-CoA transacylase (PhaG), which is responsible for the conversion of de novo intermediates into PHA monomers, is overexpressed under nitrogen starvation in P. putida (Hoffmann and Rehm, 2004; Hervás et al., 2008). It should be noted that under these conditions, and unlike in scl-PHA-producing bacteria, the accumulation of polyphosphate (polyP) and mcl-PHA is not interdependent in P. putida KT2440 (Casey et al., 2013). Follonier and colleagues (2013) analysed the growth rate and production of PHA in P. putida KT2440 and KT2442 when consuming different carbon sources. The strain KT2442 seems to be altered in such a way that it has difficulties coping with nitrogen starvation. Hence, its growth and PHA production are impaired in gluconate, but not when growing in octanoic acid (Follonier et al., 2013).

P. putida cell size depends on the ability to accumulate PHA and on the C/N ratio. Under high C/N growth conditions, the PHA-deficient mutant P. putida produces considerably less total biomass (PHA plus other cellular components) than the wild type (de Eugenio et al., 2010b). This is due to the difference in PHA content between the strains: in the wild type, the PHA-free (residual) biomass is nearly identical to that of the mutant strain. However, at similar residual biomass levels, the cell number of the P. putida PHA mutant reaches 10-fold that of the wild type, although the cell size is considerably smaller (de Eugenio et al., 2010b; Escapa et al., 2012). Thus, the number and size of the cells directly relate to the ability to accumulate PHAs. Furthermore, the cell size of the P. putida mutant unable to accumulate PHA is inversely correlated to the C/N ratio of the culture medium (Martínez et al., 2013) (Fig. 3). These findings correlate with the upregulation of genes linked to peptidoglycan and lipopolysaccharide synthesis in a mutant unable to accumulate PHAs and cultured under a high C/N ratio. Although the molecular basis needs further experimental demonstration, transcriptome analysis suggests the simultaneous activation of the TCA cycle and the cell division machinery (Escapa et al., 2012).

Figure 3

PHA-related features influencing bacterial fitness.

A. The PHAs have been described in several biological and physiological processes and suppose an ecological edge to microbial community. Soil bacteria face to different and fluctuating stresses, examples of PHA impact on bacterial physiology are: Azospirillum, and other rhizosphere bacteria in keeping plant and microorganism symbiosis; the formation of spores or cysts is linked to the PHA cycle in Bacillus cereus and Azotobacter vinelandii; microbial biofilm formation in Pseudomonas aeruginosa (Kadouri et al., 2005; Castro-Sowinski et al., 2010); promotion of predator fitness in B. bacteriovorus (Martínez et al., 2013).

B, C. The synthesis of PHA granules determines the cellular size due to the control of the biomass distribution. Scanning electron microscopy (SEM) images of P. putida mutant strain KT40C1ZC2, which lacks the enzyme coding genes of the PHA cluster. The mean length of the cells cultured on balanced medium (C/N ratio = 1.25) is 2 ± 0.5 μm at 24 h (B). The mean length of the cells grown on unbalanced medium (C/N ratio, 12.5) is 1.1 ± 0.2 μm at 24 h (C).

Different pieces of evidence suggest that the expression of the pha genes in Pseudomonas species is under the influence of global regulators, including the so-called catabolite repression response that facilitates the hierarchical and sequential use of carbon sources when the growth medium contains several at non-growth-limiting concentrations (Prieto et al., 2007; Velázquez et al., 2007; de Eugenio, 2009; de Eugenio et al., 2010b; Ryan et al., 2013; La Rosa et al., 2014). Under balanced C/N conditions, the expression of the pha genes is negatively modulated by the global regulator Crc (La Rosa et al., 2014). Although sequences resembling the canonical CA motifs indicative of Crc regulation can be predicted close to the translation initiation regions of phaC1, phaI and phaF mRNAs, only the site at phaC1 appears to be functional. Inactivation of the crc gene further increased PHA production by 42–57%. Crc activity is influenced not only by the carbon source but also by the C/N ratio of the culture medium; high concentrations of octanoic acid provoke a Crc-dependent repression response only if the C/N ratio is properly balanced. The regulatory response is suppressed if the nitrogen source becomes limiting. This extends the role of the Crc protein in the optimization of metabolism beyond that of the hierarchical utilization of carbon sources, a facet that has not been studied in other regulatory circuits (La Rosa et al., 2014). Other global transcriptional factors such as RpoS, PsrA and the GacS/GacA system are involved in the regulation of PHA production (Ruiz et al., 2004; de Eugenio, 2009; Fonseca et al., 2014). A reduction in PHA content linked to GacS sensor kinase disruption has been seen in the CA-3 and KT2442 strains of P. putida (de Eugenio, 2009; Ryan et al., 2013). Although this phenotype was restored by in trans complementation with the gacS gene in both strains, the mechanism directly involved in reduced PHA synthesis remains to be determined. For the CA-3 strain, Ryan and colleagues suggested a post-transcriptional mechanism of regulation of the phaC1 gene, but transcriptomic experiments recently performed at our laboratory involving DNA arrays of P. putida KT2440 showed a reduction in the rate of transcription of the full pha cluster, including that of the phaD transcription activator (see Table S1). Moreover, reduced pha transcription is restored by producing PhaD protein in the gacS mutant of P. putida suggesting that, very likely, the role of GacS in the PHA machinery is more complex than previously imagined (P. Fonseca, unpubl. results).

PHAs as carbon and energy sources for the microbial community

PHAs have attracted interest given their importance in understanding microbial physiology and metabolism. Since Lemoigne discovered the production of PHB in B. megaterium (Lemoigne, 1926), the ability to synthesize or catabolize PHAs in species of the three major kingdoms – Archaea, Bacteria and Eukaryotes – has been described. These include organisms present in a wide variety of habitats, including free-living species, parasites, symbionts and predators, and they might be aerobics or anaerobics, and chemotrophs or phototrophs. This widespread ability to metabolize PHAs, plus the fact that pha genes have been horizontally transferred between different phylogenetic groups, indicates that PHAs afford some advantage to the microorganisms that synthesize them (Kadouri et al., 2005). Figure 3 summarizes the main implications of PHA metabolism with respect to the biological fitness of microorganisms. PHAs would therefore appear to be valuable nutrients for PHA producers as well as other members of the microbial community. They can be degraded in biologically active environments such as soil, sludge, compost or water, and provide a potential substrate for heterotrophic microorganisms. Two types of enzymes are actively involved in the biological degradation of PHAs: intracellular and extracellular PHA depolymerases. The degradation products are then incorporated into microbial metabolism to be used as carbon and energy sources (Jendrossek and Handrick, 2002). Over the last decade, many PHA depolymerases have been characterized in depth. Most of these have been extracellular scl-PHA depolymerases (Behrends et al., 1996; Abe and Doi, 1999; Handrick et al., 2001; 2004; Jendrossek and Handrick, 2002; Braaz et al., 2003; Numata et al., 2006; Knoll et al., 2009) although novel mcl-PHA depolymerases have recently been identified (Schirmer and Jendrossek, 1994; de Eugenio et al., 2008; Gangoiti et al., 2012; Martínez et al., 2012; Santos et al., 2012).

From a biotechnological point of view, PHA depolymerases are interesting biocatalysts of use in the production of a great diversity of enantiopure hydroxyalkanoic acids (HAs), the monomers of PHAs and valuable starting materials for the pharmaceutical and biomaterial industries (Ren et al., 2005; 2010; Wang et al., 2007; O'Connor et al., 2013). For example, P. putida degrades PHAs with its own intracellular depolymerase, PhaZ_KT (de Eugenio et al., 2007), and as mentioned before constantly recycles the biopolymers as part of an ongoing PHA cycle (de Eugenio et al., 2010b). Permanently active PhaZ_KT is attached to the granules and attends to the demand for HAs as carbon and energy precursors. This prototype enzyme behaves like a serine hydrolase. When complexed with 3-hydroxyoctanoate, the three-dimensional structure of the PhaZ_KT dimer reveals an α/β-hydrolase core capped by a lid structure. This suggests that interfacial activation may occur. The contact of PhaZ_KT with the hydrophobic granule would induce the movement of the lid, exposing the active site to the polymer (de Eugenio et al., 2007; 2008). Similar catalytic model has been demonstrated for the PHB depolymerase PhaZ7 of Paucimonas lemoignei (Jendrossek et al., 2013).

Extracellular mcl-PHA degradation takes place in the environment of PHA producer microorganisms. The rhizospheric bacterium P. putida KT2440 was first isolated from garden soil based on its ability to use 3-methylbenzoate. It was later found to occupy niches in soils and sediments (including those with high concentrations of heavy metals or organic contaminants), highlighting its exceptional nutritional versatility (Regenhardt et al., 2002). The presence of a wide variety of bacterial strains sharing the same habitats as this mcl-PHA producer, including extracellular mcl-PHA-degrading bacteria, might be expected. After the lysis of P. putida, the accumulated PHA granules are released to spread throughout the extracellular medium (Fig. 1). These granules can be degraded and used as a carbon source by bacteria that produce extracellular depolymerases, regardless of their ability to accumulate PHA in their cytoplasm. To date, most of the mcl-PHA depolymerases reported belong to Gram-negative bacteria, mainly Pseudomonas species (Schirmer et al., 1995; Jendrossek et al., 1996). The most extensively characterized prototype depolymerase involved in extracellular mcl-PHA degradation is that of Pseudomonas fluorescens GK13 (PhaZ_GK13 depolymerase) (Schirmer and Jendrossek, 1994; Gangoiti et al., 2010). Very recently, new mcl-PHA depolymerases produced by Actinobacteria have been characterized, such as those of Streptomyces roseolus SL3 (Gangoiti et al., 2012) or Streptomyces venezuelae SO1 (Santos et al., 2012). These Gram-positive strains can be isolated from habitats similar to those in which P. putida is found (soil, sludge or water), as well as many others.

Interestingly, an extracellular-like mcl-PHA depolymerase has been identified in the obligate predator Bdellovibrio bacteriovorus HD100 (Martínez et al., 2012). A highly motile delta proteobacterium, B. bacteriovorus, is ubiquitously distributed in terrestrial and aquatic environments. It grows and reproduces in the periplasm of other Gram-negative prey bacteria, including P. putida KT2440 (Rendulic et al., 2004; Martínez et al., 2013). Its mcl-PHA depolymerase (PhaZ_Bd, ORF Bd3709), part of the hydrolytic arsenal needed for its predatory life cycle, shows significant amino acid sequence similarity to the prototype extracellular mcl-PHA depolymerase PhaZ_GK13. Remarkably, P. fluorescens GK13 is a potential prey bacterium for Bdellovibrio, but there is no evidence of the latter's acquisition of the depolymerase-encoding gene from the prey bacterium via lateral gene transfer (LGT). This agrees with previously published LGT studies of the predator bacterium which report no cases of recent prey-derived LGT (Gophna et al., 2006).

These findings prompted the investigation of the ability of these predators to prey upon PHA-accumulating strains and the impact of this on their fitness (Martínez et al., 2013). In P. putida and other PHA producers, PHAs play an important role in bacterial fitness, improving survival under environmental stress conditions in water and soil, and providing a means of reacting to changing carbon and energy availability conditions (Kadouri et al., 2005; Zhao et al., 2007). However, PHA accumulation does not protect the cells from predation by B. bacteriovorus HD100, which can enter and develop inside mcl-PHA-accumulating P. putida cells (Fig. 4). During the predatory life cycle, Bdellovibrio secretes its PhaZ_Bd depolymerase (very likely) into the prey's periplasm and establishes there to consume the intracellular content of the prey, degrading part of the previously accumulated biopolymer. The degradation of this extra carbon and energy source confers Bdellovibrio ecological advantages in terms of motility and predation efficiency, but does not increase the biomass or number of predator cells. It is puzzling why B. bacteriovorus hydrolyses and consumes part, but not all, of its prey's PHA. Indeed, significant quantities of PHA granules and free HA oligomers (54% and 25% of the prey's initially accumulated PHA, respectively) remain untouched by the predator and can be found in the prey's extracellular environment after the predation event (Martínez et al., 2013). Bdellovibrio motility and predation efficiency (in terms of time to progeny release) are greater when growth occurs inside PHA-accumulating P. putida. High predator velocities have been correlated with high ATP levels; these therefore increase the fitness of Bdellovibrio.

Figure 4

Bacterial predator preying on P. putida KT2440 containing PHA granules as an example of PHA catabolism in the non-PHA producers members of the microbial community.

A. SEM image of Bdellovibrio attached to the prey cell prior occupying its periplasm.

B. TEM image of bdelloplast, a rounded structure of the prey P. putida KT2440 containing PHA granules where the predator is established in the periplasm of the prey. Bdellovibrio grows in the periplasm, replicates its DNA and develops using the prey as a source of nutrients. During the intraperiplasmic growth of Bdellovibrio on P. putida KT2440, the polymer plays a key role as energy source (Martínez et al., 2013).

C. The progeny of the predator lyses the cell wall of the bdelloplast forming lysis plaques growing on a lawn of P. putida KT2440 accumulating PHA.

Predation has been much studied in higher animals, but much less investigated at the microbial level, even though bacterial predation may significantly affect the structure and function of bacterial communities. Predation may favour or suppress particular bacterial species and promote adaptations aimed at reducing mortality. Martínez and colleagues (2013) reported the potential importance of the predation of PHA producers: after preying on PHA-accumulating P. putida cells, many PHA granules and HAs are released into the environment to become ‘public goods’ of benefit to many bacteria, fungi and yeast (Fig. 1). PHAs released by prey cells might act as a substrate for secretable PHA depolymerases produced by members of the microbial community other than the predator. Bdellovibrio and Bdellovibrio-like organisms are ubiquitously distributed in terrestrial and aquatic environments, sharing niches with many PHB producers. B. bacteriovorus HD100 also produces a putative PHB depolymerase and is able to prey on a wide range of Gram-negative bacteria, further demonstrating the importance of PHA producer predation in population dynamics.

Systemic metabolic modulation: broadening the PHA application portfolio

Over the last decade, different biotechnological strategies have been developed to increase the P. putida biopolymer product portfolio. The carbon flux towards mcl-PHA production/accumulation can be precisely controlled by tailored fermentation procedures, metabolic engineering strategies and their combination. Scl-PHAs, such as poly-3-hydroxybutyrate (PHB) and its copolymers with poly-3-hydroxyvalerate (PHB-co-HV), are now produced on a commercial scale in species such as R. eutropha H16, and have extensive applications in packaging, molding, fibers and other commodities. Mcl-PHAs are currently considered promising candidates for special bioplastic applications, the consequence of properties derived from their longer side-chains, e.g. lower crystallinity, elasticity, hydrophobicity, low oxygen permeability and biodegradability (Chen, 2009). These products can be moulded and processed into compostable packaging and resorbable materials for medical applications. They have also been used as food coatings, pressure-sensitive adhesives, paint binders and biodegradable rubbers (Wang et al., 2014).

Chemically, PHAs are polyesters composed of (R)-3-hydroxyalkanoic acids or HA monomers. More than 150 such monomers can be combined to form materials with very different properties (Steinbüchel and Valentin, 1995). The specific chemical monomer composition and molecular structure determines the biological, thermal and mechanical properties (elasticity, crystallinity or rigidness) of the resulting polymer. PHAs can be classified in terms of their monomer size (scl- and mcl-PHAs), but also in terms of other criteria concerning the macrostructure of the polymer (homogenous, random or block copolymers), and the functional substituent(s) in the radical chain (such as double bonds or aromatic groups) (Olivera et al., 2010). The incorporation of different functional moieties into the polymer side-chains gave rise to the unconventional mcl-PHAs mentioned above and allowed the physical and chemical properties of the polymer to be engineered. Functional mcl-PHAs typically contain unsaturated or other highly reactive groups (e.g. halogens, hydroxyl, epoxy, phenoxy, cyanophenoxy, nitrophenoxy, thiophenoxy and methylester groups) (Tortajada et al., 2013). Table 1 provides a summary of the mcl-PHAs produced in P. putida KT2440 (or KT2442) along with their properties.

Table 1. Properties of PHA produced from P. putida KT2442 and derived species

Polymer structure	Monomer composition	Strain	Mw (KDa)	Tg (°C)	Tm (°C)	ΔHm (J/g)	εt (%)	σt (MPa)	PDI (Mw/Mn)	References
Homopolymers	PHB	KT2442	1635	−3.08	163.30	70.64	3.0	18.0	1.93	(Li et al., 2011; Wang et al., 2011)
	P4HB	KTHH06a, b	854	−45.67	50.12	29.06	ND	ND	1.75	(Wang et al., 2011)
	PHV	KTHH08a, c	1056	−15.09	112.27	73.31	3.5	6.6	1.30	(Li et al., 2011; Wang et al., 2011)
	PHHx	KTHH03a, d	272	−28.19	ND	ND	ND	ND	1.32	(Wang et al., 2011)
	PHHp	KTHH01a, e KTHH03a, d KTOY08a, f KTOY06a, g	455	−32.13	ND	ND	ND	ND	1.81	(Wang et al., 2009; 2011)
	PHO	KTOY08a, f KTHH03a, d	180	−38.38	66.06	30.23	ND	ND	1.22	(Wang et al., 2011)
	PHD	KTQQ20a, h	361	−37.21	72.20	11	312.86	11.96	1.45	(Liu et al., 2011)
Heteropolymers	P(3HN-co-3HHp) (HN: 70–95%; HHp: 30-5%)	KT2440	ND	−45/−48	46/63	12/27	1222/1327	6.6/15.7	ND	(Jiang et al., 2012)
	P(HO-co-HHx) (HO:88–94%; HHX:12-2%)	KT2440	ND	−40	54/58	9/12	1267/1384	9.6/11.7	ND	(Jiang et al., 2012)
	P(HHx-co-HO-co-HD-co-HDD) (HHx:5%; HO:35%; HD:32%; HDD:28%)	KTOY06a, g	134	−45	58	22	180	12.6	1.41	(Ouyang et al., 2007)
	P(HHx-co-HO-co-HD-co-HDD) (HHx: 4%; HO:32%; HD:28%; HDD:39%)	KTOY06a, g	157	−43	65	28	125	11.3/16.3	1.45	(Liu and Chen, 2007; Ouyang et al., 2007)
	P(HTD-co-HDD-co-HD-co-HO-co-HHx) (HTD:31–49%;HDD:15–19%; HD:21–27%; HO: 11–18%; HHx:2%)	KTOY06a, g	80/95	−40/−42	58/66	25/30	107/275	2.97/7.57	1.4/1.8	(Liu and Chen, 2007)
	P(HD-co-HDD) (HD:15%; HDD:84%)	KTQQ20a, h	155	−32.49	77.62	40.85	88.3	5.23	1.30	(Liu et al., 2011)
	P(HHx-co-HO-co-HD-co-HDD) (HH:15%; HO:40%; HD:30%; HDD:15%)	KT2442	100	−44	53	18	188.7	8.7	1.25	(Liu and Chen, 2007; Ouyang et al., 2007; Liu et al., 2011)
	P(HB-co-HHx) (21%HHx)	ND	ND	−18.1	55.4	ND	75.29	1.84	ND	(Tripathi et al., 2012)
	P(HB-co-HV-co-HHp) (HB:73%; HV:13%; HHp:14%)	KTOYO6ΔCa, i	370	−7.3	ND	ND	462	7.0	2.9	(Li et al., 2011)
Block	58%PHB-b-42%PHHx	KTOYO6ΔCa, i	ND	−16.14, 2.7	172.1	ND	270.31	1.42	2.5	(Tripathi et al., 2012)
	73%PHB-b-27%PHVHHp	KTOYO6ΔCa, i	450	−23.6, 3.5	170.6	ND	63.4	7.5	8.7	(Li et al., 2011)
	49%P3HHx-b-P(15%3HD-co-35%3HDD)	KTQQ20a, h	158.5	−43.08	33.45/66.08	16.43/8.92	369.13	15.91	1.9	(Tripathi et al., 2013)
Functionalized	PHACOS (OH-alkyl 16.5–65.0)	KT2442FadBa, j	315	−39	48.5	ND	ND	ND	3.3	(Escapa et al., 2011)
	PHACOS (OH-alkyl 77.0)	KT2442	393	−5.0	ND	ND	ND	ND	4.6	(Escapa et al., 2011)
	Thioether functional groups (6.02% w/w S)	KT2440	ND	ND	ND	ND	ND	ND	ND	(Ewering et al., 2002)
	Cyano functional group (19.6% 3HCPH)	KT2440	ND	−37.5	53.5	15.0	ND	ND	ND	(Kim et al., 1995; Gross et al., 1996)
	Nitro functional group	KT2440	ND	ND	ND	ND	ND	ND	ND	(Kim et al., 1995)
	Fluoro functional group (12.4%)	KT2442	ND	−41	51	ND	ND	ND	ND	(Kim et al., 1996)

• ^a Mutants of the strain KT2442.
• ^b KTHH06: KTHH03 ΔphaC1ZC2; harbouring PHA synthase phbC of R. eutropha and orfZ genes from C. kluyveri.
• ^c KTHH08: KTHH03 ΔphaC1ZC2; harbouring PHA biosynthesis phaPCJ operon of A. hydrohila 4AK4.
• ^d KTHH03: ΔfadB2x ΔfadAx ΔfadB ΔfadA ΔphaG.
• ^e KTHH01: ΔfadB ΔfadA ΔphaG.
• ^f KTOY08: ΔfadB2x ΔfadAx ΔfadB ΔfadA.
• ^g KTOY06: ΔfadB ΔfadA.
• ^h KTQQ20: ΔfadAB ΔfadAx ΔfadB2x ΔphaG ΔPP2047 ΔPP2048.
• ⁱ KTOYO6ΔC: KTOY06 phaPCJ_A.C.
• ^j KT2442FadB: ΔfadB.
• M_w, weight average molecular weight; T_g, glass transition temperature; T_m, melting temperature; ΔH_m, heat of melting fusion; ε_t, elongation at break; σ_t, tensile strength; PDI, polydispersity index. HB, 3-hydroxybutyrate; 4HB, 4-hydroxybutyrate; HV, 3-hydroxyvalerate; HHx, 3-hydroxyhexanoate; HHp, 3-hydroxyheptanoate; HO, 3-hydroxyoctanoate; HD, 3-hydroxydecanoate; HDD, 3-hydroxydodecanoate; HTD, 3-hydroxytetradecanoate; PHACOS, poly-3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate.

As mentioned earlier, the β-oxidation pathway plays an important role in providing intermediates to PHA synthesis when fatty acids are used as the sole carbon source. Mcl-PHA monomer heterogeneity occurs since, even if a pure homogeneous substrate is used, β-oxidation produces heterogeneous substrates (showing two carbons fewer per round) for PHA synthase (Olivera et al., 2001). Further, by altering the ratio of co-substrates, the molar amounts of specific monomers including those with functional groups, can be modified. P. putida KT2440 has a large array of β-oxidation enzymes. The FadAB protein complex has at least two sets of fadAB genes, fadB and fadA (PP_2136 and PP_2137) and fadBx and fadAx (PP_2214 and PP_2215) (Liu and Chen, 2007). The latter set apparently plays a role in fatty acid degradation since fadA and fadB deletion mutants still show β-oxidation activity. However, these mutants produce PHAs with more long chains of monomers, possibly due to the β-oxidation pathway being somewhat defective. Modification of this pathway has proved to be a good strategy for tailoring PHAs (Olivera et al., 2001; Escapa et al., 2011; Wang et al., 2011). By controlling the carbon flow through the β-oxidation pathway, PHAs can be rationally designed to have shorter or longer monomer chain lengths [e.g. by deleting the repressor PsrA (Fonseca et al., 2014) and by slowing down the β-oxidation route respectively] (Olivera et al., 2001; Liu et al., 2011). A β-oxidation-impaired mutant, with deletions concerning the last two steps of this pathway (i.e. in fadA and fadB), overaccumulates mcl-PHAs. The mcl-PHA content and composition can be as well controlled by using a rational co-feeding strategy (Jiang et al., 2012). This could yield new biopolymers with significantly improved mechanical properties (degree of crystallization, tensile strength, stress at breakage, elongation at breakage). Engineering fadA and fadB mutant strain via the substitution of the natural pha operon by phaPCJ encoding a phasin, a PHA synthase and a (R)-specific enoyl-CoA hydratase – all from Aeromonas caviae – results in the accumulation of the scl-mcl block polymer of poly-3-hydroxybutyrate, poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate) and poly-(3-hydroxybutyrate-block-poly-3-hydroxyhexanoate) (PHB-b-PHVHHp and P3HB-b-P3HHx) (Li et al., 2011). Depending on the substrates, the monomer ratios can be adjusted within the copolymer and the block copolymer, allowing the formation of PHAs with tailor-made properties. This novel strain produces copolymers consisting of 3HHx fractions with an adjustable monomer composition, as well as mcl-PHA diblock copolymer poly-3-hydroxyhexanoate-block-poly-(3-hydroxydecanoate-co-3-hydroxydodecanoate) [P3HHx-b-P(3HD-co-3HDD)] (Tripathi et al., 2012; 2013). β-oxidation-weakened mutants of P. putida KT2442 are also a good platform for producing homopolymers with chain lengths varying from C4 to C7. For example, by disrupting the carbon flow through the β-oxidation pathway and de novo fatty acid synthesis, different homopolymers can be synthesized, e.g. poly-4-hydroxybutyrate (Wang et al., 2011). This interesting Food and Drug Administration-approved polymer shows high elasticity, low glass transition (T_g) and melting (T_m) temperatures, and a high thermal degradation temperature.

Another PHA modification strategy relies on feeding the producing strain with structurally related fatty acid poorly processed by the β-oxidation pathway. Such substrates would be incorporated into PHAs without being totally catabolized to acetyl-CoA. For example, different PHA compositions bearing acetyl-thioester groups in the side-chains have been produced using wild type P. putida and P. putida KT42FadB (a strain mutated in fadB). A family – named PHACOS – of novel PHAs can be produced using decanoic acid as an inducer of growth and PHA synthesis, and 6-acetylthiohexanoic acid as a PHA precursor, following either one- or two-stage cultivation strategies. These polymers have low T_g and T_m temperatures, and show thermal stability up to 200 °C. In addition, at room temperature, these amorphous polymers are relatively soft and deformable, a consequence of their low T_g values (Escapa et al., 2011). Moreover, the PHACOS polymers show excellent biocompatibility properties as well as antimicrobial activity against methicillin-resistant Staphylococcus aureus strains (Dinjaski et al., 2014). Another important feature of this polymer is its susceptibility to post-biosynthetic chemical modification via the reactive thioester group in the side-chains (Tortajada et al., 2013). This characteristic is shared with other functionalized polymers (also known as second generation PHAs) produced by P. putida, e.g. those containing unsaturated monomers such as 3-hydroxy-5-dodecanoate and 3-hydroxy-7-tetradecanoate generated from carbohydrates and other non-fatty acid precursors via de novo fatty acid synthesis (Hartmann et al., 2006; Escapa et al., 2013). Double bonds are introduced into acyl-CoA or acyl-ACP intermediates as a means of regulating membrane fatty acid composition and thus membrane fluidity in response to changes in ambient temperature. The peripheral supplier route regulators of P. putida (del Castillo et al., 2008) have been contemplated as targets for designing metabolic engineering strategies that would favour the assimilation of non-fatty acids precursors such low-cost substrates as glycerol, to optimize the production of mcl-PHAs bearing unsaturated monomers. P. putida KT2440 has evolved a tightly regulated system for metabolizing glycerol. This involves a prolonged lag phase of growth, but this can be avoided by the addition of small amounts of growth precursors such as fatty acids as a co-feeder. This completely eliminates the lag phase, shortening the time to grow and accumulate PHA. The key factor in this improvement is the repressor GlpR, which controls the utilization of glycerol as a growth precursor in P. putida KT2440. A glpR knockout mutant of P. putida KT2440 showed no lag phase when cultured on glycerol in the absence of a co-feeder. In addition, the production of PHA in this strain was almost doubled (Escapa et al., 2013).

New system metabolic engineering strategies might be designed based on data from transcriptomic, proteomic and metabolomic studies and making use of the power of computational predictions based on accurate genome-scale network reconstructions (Hervás et al., 2008; Nogales et al., 2008; Puchałka et al., 2008; Brigham et al., 2010; Peplinski et al., 2010; Rojo, 2010; Raberg et al., 2011). This might lead to the design of new strains and fermentation strategies aimed at increasing PHA production, enabling the use of alternative carbon sources and boosting PHA synthesis. In silico genome-scale metabolic reconstruction of P. putida (iJN746) has allowed the identification of a large set of non-glycolytic substrates, such as aromatic compounds, with a high potential for the production of new PHA monomers (Nogales et al., 2008). Poblete-Castro and colleagues (2013; 2014) made use of the metabolic versatility of P. putida to re-engineer its metabolic network towards more efficient PHA production, using glucose as the carbon source. Based on computational flux analysis, a glucose dehydrogenase mutant was designed, which, when used following a finely tuned fed-batch strategy, accumulated 67% of its dry weight as PHAs, with no compromise of its growth rate or by-product production.

Concluding remarks and present trends

The use of cutting-edge technologies such as transcriptomics, proteomics and metabolic flux analyses has improved our understanding of the interplay between PHA metabolism, which is extremely context dependent, and other cell functions.

PHA granules represent supramolecular complexes that carry GAPs, essential protein components of the PHA metabolic machinery. Aside from the carbon and energy stockpiling function ascribed to the polymers themselves, GAPs play a critical physiological role in PHA metabolism and granule formation. Phasins form part of the mediation element network responsible for granule localization and segregation. PHA synthase and depolymerase are also attached to the granules, and operate a continuous metabolic cycle that ensures the turnover of the accumulated polymer and play a key role in the maintenance of metabolism homeostasis.

PHAs, usually considered as simple bacterial reservoirs of carbon and energy, are now recognized as having impact in many physiological roles (Fig. 3). In P. putida, PHA production ensures a more robust metabolism and improves bacterial fitness. Although traditionally considered as a futile cycle, PHA metabolism balances the storage versus spillage equilibrium of both carbon and energy, determining cell number and size. The accumulation of PHAs depends on the carbon source used as growth and PHA precursors, and on the C/N ratio. While N limitation is necessary for PHA production from non-PHA-related compounds, it is not essential, though it is favourable when the substrate is a fatty acid. Thus, PHA metabolism is driven by the network of local and global regulators controlling the pathways involved in carbon and nitrogen assimilation.

PHAs are valuable nutrients for producer strains, but since they can be extracellularly degraded, they are also useful to the wider microbial community. The predation of PHA producers by bacterial predators such as B. bacteriovorus has an important effect on microbial population dynamics since PHA granules and free HA oligomers are released into the environment. These can be then incorporated into the microbial metabolism of the non-PHA producer members of the population enhancing their biological fitness.

The availability of high-throughput experimental tools and quantitative analysis techniques now allow the design of more robust metabolic engineering strategies to enhance and/or tailor PHA production. The integration of the information and data generated by genomics, transcriptomics, proteomics, metabolomics and flux analysis at the systems level is changing the biotechnological production of these biopolymers. Perfect examples are metabolic engineering strategies to control PHA monomer production by re-channelling the fatty acid β-oxidation route (Table 1). Moreover, synthetic biology strategies based on the concepts of systems biology in P. putida KT2440 should lead to shaped chassis designs (Nikel et al., 2014). In this context, strategies for peptide polymer functionalization based on the use of natural intrinsic P. putida PHA granules as scaffold, to immobilize fusion proteins in vivo via the granule binding module of PhaF phasin (BioF tag), illustrates the wide variety of applications that can be envisioned from the study and control of this metabolic system, beyond the production of polyester in itself (Dinjaski and Prieto, 2013).

Acknowledgements

This work was supported by the Ministerio of Economía y Competitividad, España (BIO2010-21049, BIO2013-44878-R, 201120E092, 201120E050). Natalia Tarazona is a PhD student granted by the Department of Science Technology and Innovation-Colciencias, Colombia.