From Residues to Added-Value Bacterial Biopolymers as Nanomaterials for Biomedical Applications.

Bacterial biopolymers are naturally occurring materials comprising a wide range of molecules with diverse chemical structures that can be produced from renewable sources following the principles of the circular economy. Over the last decades, they have gained substantial interest in the biomedical field as drug nanocarriers, implantable material coatings, and tissue-regeneration scaffolds or membranes due to their inherent biocompatibility, biodegradability into nonhazardous disintegration products, and their mechanical properties, which are similar to those of human tissues. The present review focuses upon three technologically advanced bacterial biopolymers, namely, bacterial cellulose (BC), polyhydroxyalkanoates (PHA), and γ-polyglutamic acid (PGA), as models of different carbon-backbone structures (polysaccharides, polyesters, and polyamides) produced by bacteria that are suitable for biomedical applications in nanoscale systems. This selection models evidence of the wide versatility of microorganisms to generate biopolymers by diverse metabolic strategies. We highlight the suitability for applied sustainable bioprocesses for the production of BC, PHA, and PGA based on renewable carbon sources and the singularity of each process driven by bacterial machinery. The inherent properties of each polymer can be fine-tuned by means of chemical and biotechnological approaches, such as metabolic engineering and peptide functionalization, to further expand their structural diversity and their applicability as nanomaterials in biomedicine.

Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag.

Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP-ß-galactosidase and MinP-CueO from Escherichia coliIMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.

Interfacial Activity of Phasin PhaF from Pseudomonas putida KT2440 at Hydrophobic-Hydrophilic Biointerfaces.

Phasins, the major proteins coating polyhydroxyalkanoate (PHA) granules, have been proposed as suitable biosurfactants for multiple applications because of their amphiphilic nature. In this work, we analyzed the interfacial activity of the amphiphilic α-helical phasin PhaF from Pseudomonas putida KT2440 at different hydrophobic-hydrophilic interfacial environments. The binding of PhaF to surfaces containing PHA or phospholipids, postulated as structural components of PHA granules, was confirmed in vitro using supported lipid bilayers and confocal microscopy, with polyhydroxyoctanoate- co-hexanoate P(HO- co-HHx) and Escherichia coli lipid extract as model systems. The surfactant-like capabilities of PhaF were determined by measuring changes in surface pressure in Langmuir devices. PhaF spontaneously adsorbed at the air-water interface, reducing the surface tension from 72 mN/m (water surface tension at 25 °C) to 50 mN/m. The differences in the adsorption of the protein in the presence of different phospholipid films showed a marked preference for phosphatidylglycerol species, such as 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol. The PHA-binding domain of PhaF (BioF) conserved a similar surface activity to PhaF, suggesting that it is responsible for the surfactant properties of the whole protein. These new findings not only increase our knowledge about the role of phasins in the PHA machinery but also open new outlooks for the application of these proteins as biosurfactants.