A holistic view of polyhydroxyalkanoate metabolism in Pseudomonas putida.

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.

Cell system engineering to produce extracellular polyhydroxyalkanoate depolymerase with targeted applications.

Novel platforms based on the application of bacterial cell systems as factories for production of new bioproducts open avenues and dramatically expand the catalogue of existing biomaterials. Herein, we designed the strategy based on in vivo production of extracellular Pseudomonas fluorescens GK13 (PhaZGK13) depolymerase to degrade previously biosynthesized polyhydroxyalkanotes (PHAs) or to obtain 3-hydroxyalkanoic acids (HAs). With this aim, extracellular PhaZGK13 was produced in recombinant strains and the optimal conditions for controlled release of HAs and oligomers by growing cells were set up with a particle suspension of (14)C-labelled PHA, being maximal after 24h of incubation. Genetic modification of key factors involved in fatty acids metabolism revealed the influence of an active ß-oxidation pathway on the extracellular degradation of PHA and subsequent HAs isolation. The highest HAs production was obtained using Pseudomonas putida KT2442 fadB mutant (0.27mg/mL) due to the reduced ability of this strain to metabolize the degradation products. The system was applied to produce new added value HAs harboring thioester groups in the side chain from the functionalized mcl-PHA, PHACOS. Remarkably, hydrolyzed PHACOS showed greater potential to inhibit Staphylococcus aureus(T) growth when compared to that of degradation products of non functionalized polyhydroxyoctanoate-co-hexanoate P(HO-co-HH).

A role for the regulator PsrA in the polyhydroxyalkanoate metabolism of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a Gram-negative bacterium capable of producing medium-chain-length-polyhydroxyalkanoates (mcl-PHA). When fatty acids are used as growth and polymer precursors, the biosynthesis is linked to fatty acid metabolism via ß-oxidation route. In the close-related Pseudomonas aeruginosa, the transcriptional repressor PsrA regulates the ß-oxidation, but little is known about the regulatory system in P. putida. To analyze the effect of the absence of psrA gene on the growth and PHA production in P. putida, a set of different carbon sources were assayed in the wild type strain and in a generated psrA deficient strain (KT40P). The growth rates were in all cases, lower for the mutant. The amount of PHA produced by the mutant strain is lower than the wild type. Moreover, the monomeric composition seems to be different among the strains, as there is enrichment in monomers with shorter carbon length in the mutant strain. To understand the role of the psrA gene on the metabolism of fatty acids, we have determined the expression profile of several genes related to fatty acid metabolism in the wild type and in the mutant strain. The results indicated that PsrA mostly negatively regulate genes related to fatty acid metabolism.

The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow.

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the α/ß-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ(Bd) has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ(Bd) is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.