Exploring Rhodospirillum rubrum response to high doses of carbon monoxide under light and dark conditions.

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO2, H2, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO2 and H2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO2/H2 during acetate uptake and inhibits CO2 fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness).

Bdellovibrio's prey-independent lifestyle is fueled by amino acids as a carbon source.

Identifying the nutritional requirements and growth conditions of microorganisms is crucial for determining their applicability in industry and understanding their role in clinical ecology. Predatory bacteria such as Bdellovibrio bacteriovorus have emerged as promising tools for combating infections by human bacterial pathogens due to their natural killing features. Bdellovibrio's lifecycle occurs inside prey cells, using the cytoplasm as a source of nutrients and energy. However, this lifecycle supposes a challenge when determining the specific uptake of metabolites from the prey to complete the growth inside cells, a process that has not been completely elucidated. Here, following a model-based approach, we illuminate the ability of B. bacteriovorus to replicate DNA, increase biomass, and generate adenosine triphosphate (ATP) in an amino acid-based rich media in the absence of prey, keeping intact its predatory capacity. In this culture, we determined the main carbon sources used and their preference, being glutamate, serine, aspartate, isoleucine, and threonine. This study offers new insights into the role of predatory bacteria in natural environments and establishes the basis for developing new Bdellovibrio applications using appropriate metabolic and physiological methodologies. KEY POINTS: • Amino acids support axenic lifestyle of Bdellovibrio bacteriovorus. • B. bacteriovorus preserves its predatory ability when growing in the absence of prey.

Aerobic-anaerobic transition boosts poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Rhodospirillum rubrum: the key role of carbon dioxide.

BACKGROUND: Microbially produced bioplastics are specially promising materials since they can be naturally synthesized and degraded, making its end-of-life management more amenable to the environment. A prominent example of these new materials are polyhydroxyalkanoates. These polyesters serve manly as carbon and energy storage and increase the resistance to stress. Their synthesis can also work as an electron sink for the regeneration of oxidized cofactors. In terms of biotechnological applications, the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV, has interesting biotechnological properties due to its lower stiffness and fragility compared to the homopolymer poly(3-hydroxybutyrate) (P3HB). In this work, we explored the potentiality of Rhodospirillum rubrum as a producer of this co-polymer, exploiting its metabolic versatility when grown in different aeration conditions and photoheterotrophically. RESULTS: When shaken flasks experiments were carried out with limited aeration using fructose as carbon source, PHBV production was triggered reaching 29 ± 2% CDW of polymer accumulation with a 75 ± 1%mol of 3-hydroxyvalerate (3HV) (condition C2). Propionate and acetate were secreted in this condition. The synthesis of PHBV was exclusively carried out by the PHA synthase PhaC2. Interestingly, transcription of cbbM coding RuBisCO, the key enzyme of the Calvin-Benson-Bassham cycle, was similar in aerobic and microaerobic/anaerobic cultures. The maximal PHBV yield (81% CDW with 86%mol 3HV) was achieved when cells were transferred from aerobic to anaerobic conditions and controlling the CO2 concentration by adding bicarbonate to the culture. In these conditions, the cells behaved like resting cells, since polymer accumulation prevailed over residual biomass formation. In the absence of bicarbonate, cells could not adapt to an anaerobic environment in the studied lapse. CONCLUSIONS: We found that two-phase growth (aerobic-anaerobic) significantly improved the previous report of PHBV production in purple nonsulfur bacteria, maximizing the polymer accumulation at the expense of other components of the biomass. The presence of CO2 is key in this process demonstrating the involvement of the Calvin-Benson-Bassham in the adaptation to changes in oxygen availability. These results stand R. rubrum as a promising producer of high-3HV-content PHBV co-polymer from fructose, a PHBV unrelated carbon source.

Providing new insights on the biphasic lifestyle of the predatory bacterium Bdellovibrio bacteriovorus through genome-scale metabolic modeling.

In this study we analyze the growth-phase dependent metabolic states of Bdellovibrio bacteriovorus by constructing a fully compartmented, mass and charge-balanced genome-scale metabolic model of this predatory bacterium (iCH457). Considering the differences between life cycle phases driving the growth of this predator, growth-phase condition-specific models have been generated allowing the systematic study of its metabolic capabilities. Using these computational tools, we have been able to analyze, from a system level, the dynamic metabolism of the predatory bacteria as the life cycle progresses. We provide computational evidences supporting potential axenic growth of B. bacteriovorus's in a rich medium based on its encoded metabolic capabilities. Our systems-level analysis confirms the presence of "energy-saving" mechanisms in this predator as well as an abrupt metabolic shift between the attack and intraperiplasmic growth phases. Our results strongly suggest that predatory bacteria's metabolic networks have low robustness, likely hampering their ability to tackle drastic environmental fluctuations, thus being confined to stable and predictable habitats. Overall, we present here a valuable computational testbed based on predatory bacteria activity for rational design of novel and controlled biocatalysts in biotechnological/clinical applications.

Phasin interactome reveals the interplay of PhaF with the polyhydroxyalkanoate transcriptional regulatory protein PhaD in Pseudomonas putida.

Phasin PhaF, a multifunctional protein associated with the surface of polyhydroxyalkanoate (PHA) granules that also interacts with the nucleoid, contributes significantly to PHA biogenesis in pseudomonads. As a protein present on the surface of PHA granules, PhaF participates in granule stabilization and segregation, whereas its deletion has a notable impact on overall transcriptome, PHA accumulation and cell physiology, suggesting more extensive functions besides solely being a granule structural protein. Here, we followed a systematic approach to detect potential interactions of PhaF with other components of the cell, which could pinpoint unexplored functions of PhaF in the regulation of PHA production. We determined the PhaF interactome in Pseudomonas putida KT2440 via pull-down-mass spectrometry (PD-MS) experiments. PhaF complexed with PHA-related proteins, phasin PhaI and the transcriptional regulator PhaD, interactions that were verified to be direct using in vivo two-hybrid analysis. The determination of the PHA granule proteome showed that PhaI and three other potential PhaF interacting partners, but not PhaD, were granule-associated proteins. Analysis of the interaction of PhaF and PhaD with the phaI promoter by EMSA suggested a new role for PhaF in interacting with PhaD and raises new questions on the regulatory system controlling pha gene expression.

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.

Molecular Insights into the Physical Adsorption of Amphiphilic Protein PhaF onto Copolyester Surfaces.

Phasins are amphiphilic proteins located at the polymer-cytoplasm interface of bacterial polyhydroxyalkanoates (PHA). The immobilization of phasins on biomaterial surfaces is a promising way to enhance the hydrophilicity and supply cell-directing elements in bioinstructing processes. Optimizing the physical adsorption of phasins requires deep insights into molecular processes during polymer-protein interactions to preserve their structural conformation while optimizing surface coverage. Here, the assembly, organization, and stability of phasin PhaF from Pseudomonas putida at interfaces is disclosed. The Langmuir technique, combined with in situ microscopy and spectroscopic methods, revealed that PhaF forms stable and robust monolayers at different temperatures, with an almost flat orientation of its α-helix at the air-water interface. PhaF adsorption onto preformed monolayers of poly[(3-R-hydroxyoctanoate)-co-(3-R-hydroxyhexanoate)] (PHOHHx), yields stable mixed layers below π = ∼15.7 mN/m. Further insertion induces a molecular reorganization. PHOHHx with strong surface hydrophobicity is a more adequate substrate for PhaF adsorption than the less hydrophobic poly[(rac-lactide)-co-glycolide] (PLGA). The observed orientation of the main axis of the protein in relation to copolyester interfaces ensures the best exposure of the hydrophobic residues, providing a suitable coating strategy for polymer functionalization.

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the Pseudomonas putida KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the in vitro functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-ß-galactosidase, containing the choline-binding module C-LytA and the ß-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-ß-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials.IMPORTANCE Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization in vitro of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.

A phasin with extra talents: a polyhydroxyalkanoate granule-associated protein has chaperone activity.

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % ß-sheet, 19.4 % ß-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/ß hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

Smart polyhydroxyalkanoate nanobeads by protein based functionalization.

The development of innovative medicines and personalized biomedical approaches calls for new generation easily tunable biomaterials that can be manufactured applying straightforward and low-priced technologies. Production of functionalized bacterial polyhydroxyalkanoate (PHA) nanobeads by harnessing their natural carbon-storage granule production system is a thrilling recent development. This branch of nanobiotechnology employs proteins intrinsically binding the PHA granules as tags to immobilize recombinant proteins of interest and design functional nanocarriers for wide range of applications. Additionally, the implementation of new methodological platforms regarding production of endotoxin free PHA nanobeads using Gram-positive bacteria opened new avenues for biomedical applications. This prompts serious considerations of possible exploitation of bacterial cell factories as alternatives to traditional chemical synthesis and sources of novel bioproducts that could dramatically expand possible applications of biopolymers. FROM THE CLINICAL EDITOR: In the 21st century, we are coming into the age of personalized medicine. There is a growing use of biomaterials in the clinical setting. In this review article, the authors describe the use of natural polyhydroxyalkanoate (PHA) nanoparticulates, which are formed within bacterial cells and can be easily functionalized. The potential uses would include high-affinity bioseparation, enzyme immobilization, protein delivery, diagnostics etc. The challenges of this approach remain the possible toxicity from endotoxin and the high cost of production.

Can bioplastics always offer a truly sustainable alternative to fossil-based plastics?

Bioplastics, comprised of bio-based and/or biodegradable polymers, have the potential to play a crucial role in the transition towards a sustainable circular economy. The use of biodegradable polymers not only leads to reduced greenhouse gas emissions but also might address the problem of plastic waste persisting in the environment, especially when removal is challenging. Nevertheless, biodegradable plastics should not be considered as substitutes for proper waste management practices, given that their biodegradability strongly depends on environmental conditions. Among the challenges hindering the sustainable implementation of bioplastics in the market, the development of effective downstream recycling routes is imperative, given the increasing production volumes of these materials. Here, we discuss about the most advisable end-of-life scenarios for bioplastics. Various recycling strategies, including mechanical, chemical or biological (both enzymatic and microbial) approaches, should be considered. Employing enzymes as biocatalysts emerges as a more selective and environmentally friendly alternative to chemical recycling, allowing the production of new bioplastics and added value and high-quality products. Other pending concerns for industrial implementation of bioplastics include misinformation among end users, the lack of a standardised bioplastic labelling, unclear life cycle assessment guidelines and the need for higher financial investments. Although further research and development efforts are essential to foster the sustainable and widespread application of bioplastics, significant strides have already been made in this direction.

Controlling the expression of heterologous genes in Bdellovibrio bacteriovorus using synthetic biology strategies.

Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that preys upon Gram-negative bacteria. It has been proposed to be applied as a "living antibiotic" in several fields such as agriculture or even medicine, since it is able to prey upon bacterial pathogens. Its interesting lifestyle makes this bacterium very attractive as a microbial chassis for co-culture systems including two partners. A limitation to this goal is the scarcity of suitable synthetic biology tools for predator domestication. To fill this gap, we have firstly adapted the hierarchical assembly cloning technique Golden Standard (GS) to make it compatible with B. bacteriovorus HD100. The chromosomal integration of the Tn7 transposon's mobile element, in conjunction with the application of the GS technique, has allowed the systematic characterization of a repertoire of constitutive and inducible promoters, facilitating the control of the expression of heterologous genes in this bacterium. PJExD/EliR proved to be an exceptional promoter/regulator system in B. bacteriovorus HD100 when precise regulation is essential, while the synthetic promoter PBG37 showed a constitutive high expression. These genetic tools represent a step forward in the conversion of B. bacteriovorus into an amenable strain for microbial biotechnology approaches.

Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16.

The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC-MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Swapping of phasin modules to optimize the in vivo immobilization of proteins to medium-chain-length polyhydroxyalkanoate granules in Pseudomonas putida.

PhaF is a bimodular protein of Pseudomonas putida KT2442 exhibiting multiple functions within the polyhydroxyalkanoate (PHA) apparatus. It behaves as phasin or PHA granule binding protein (by BioF domain) and also as nucleoid-associated protein involved in granule localization and segregation during cell division (by C-terminal domain). This work addresses the function of the PhaI phasin in the PHA granule formation machinery. Epifluorescence microscopy and flow cytometry studies of P. putida phasin mutant cells producing recombinant phasin domains fused to GFP protein demonstrated a balanced granule distribution after cell division only when low dosage of PhaF, and BioF domain or PhaI, are expressed together, revealing the exchangeability of phasins modules. These findings show the precise combination of phasin module production that leads to the optimal PHA production and granule localization and distribution, concomitantly to in vivo immobilization of recombinant proteins to PHA (22 mg of protein/g PHA).

A singular PpaA/AerR-like protein in Rhodospirillum rubrum rules beyond the boundaries of photosynthesis in response to the intracellular redox state.

IMPORTANCE: Rhodospirillum rubrum vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy.

Enzybiotic-mediated antimicrobial functionalization of polyhydroxyalkanoates.

Polymeric nanoparticles (NPs) present some ideal properties as biomedical nanocarriers for targeted drug delivery such as enhanced translocation through body barriers. Biopolymers, such as polyhydroxyalkanoates (PHAs) are gaining attention as nanocarrier biomaterials due to their inherent biocompatibility, biodegradability, and ability to be vehiculized through hydrophobic media, such as the lung surfactant (LS). Upon colonization of the lung alveoli, below the LS layer, Streptococcus pneumoniae, causes community-acquired pneumonia, a severe respiratory condition. In this work, we convert PHA NPs into an antimicrobial material by the immobilization of an enzybiotic, an antimicrobial enzyme, via a minimal PHA affinity tag. We first produced the fusion protein M711, comprising the minimized PHA affinity tag, MinP, and the enzybiotic Cpl-711, which specifically targets S. pneumoniae. Then, a PHA nanoparticulate suspension with adequate physicochemical properties for pulmonary delivery was formulated, and NPs were decorated with M711. Finally, we assessed the antipneumococcal activity of the nanosystem against planktonic and biofilm forms of S. pneumoniae. The resulting system displayed sustained antimicrobial activity against both, free and sessile cells, confirming that tag-mediated immobilization of enzybiotics on PHAs is a promising platform for bioactive antimicrobial functionalization.

Nature-inspired material binding peptides with versatile polyester affinities and binding strengths.

Biodegradable polyesters, such as polyhydroxyalkanoates (PHAs), are having a tremendous impact on biomedicine. However, these polymers lack functional moieties to impart functions like targeted delivery of molecules. Inspired by native GAPs, such as phasins and their polymer-binding and surfactant properties, we generated small material binding peptides (MBPs) for polyester surface functionalization using a rational approach based on amphiphilicity. Here, two peptides of 48 amino acids derived from phasins PhaF and PhaI from Pseudomonas putida, MinP and the novel-designed MinI, were assessed for their binding towards two types of PHAs, PHB and PHOH. In vivo, fluorescence studies revealed selective binding towards PHOH, whilst in vitro binding experiments using the Langmuir-Blodgett technique coupled to ellipsometry showed KD in the range of nM for all polymers and MBPs. Marked morphological changes of the polymer surface upon peptide adsorption were shown by BAM and AFM for PHOH. Moreover, both MBPs were successfully used to immobilize cargo proteins on the polymer surfaces. Altogether, this work shows that by redesigning the amphiphilicity of phasins, a high affinity but lower specificity to polyesters can be achieved in vitro. Furthermore, the MBPs demonstrated binding to PET, showing potential to bind cargo molecules also to synthetic polyesters.