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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.

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.

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).

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.

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Innovative approaches for recycling and upcycling of textile waste are required to sustainably treat the tremendous, discarded amounts. Due to their high specificity and mild reaction conditions, enzymes offer an environmentally friendly alternative for separation of blended materials. Glucose was recovered from cellulose (viscose) and polyamide blends and utilized as carbon source for biosynthesis of two biodegradable polymers. Polyhydroxybutyrate (PHB) was produced by Cupriavidus necator reaching an intracellular accumulation of 60 % (w/w) and 5.2 g/L of PHB from 20 g/L of glucose-rich hydrolysate. Bacterial cellulose (BC) was produced by Komagataeibacter sucrofermentans exhibiting equal properties to BC produced in standard media as confirmed by FTIR spectroscopy, SEM, and XRD. The recovered polyamide fibers were thermo-mechanically reprocessed and characterized by tensile testing showing comparable properties to virgin material. Thus, a combined bioconversion and mechanical reprocessing approach for textile waste is demonstrated allowing for recycling and valorization of each fraction of blended materials.

Photosynthesis (PS) in purple non-Sulfur bacteria (PNSB) is strictly controlled to avoid energy consumption and to prevent oxidative stress when oxygen is present. PpsR, a transcriptional repressor encoded in the photosynthesis gene cluster (PGC), is one of the main players in the regulation of PS-related genes. In some cases, PpsR does not sense the environmental cue by itself but delegates this task to other proteins like PpaA/AerR anti-repressors, which can ultimately affect PpsR affinity to some promoter regions. In this work, the effects of locus Rru_A0625 product (HP1) on PS regulation of Rhodospirillum rubrum were studied by mutation/complementation and transcriptomic strategies. Rru_A0625 is located next to ppsR gene, just like other PpaA/AerR members, and its deletion annuls pigment synthesis in dark micro/anaerobic growth conditions. HP1 shows similarity to PpaA/AerR anti-repressors family, although it does not possess their typical cobalamin binding domain. A transcriptomic analysis of Rru_A0625 deletion mutant showed that HP1 not only has effects on bacteriochlorophyll and carotenoid biosynthesis, but also many other biological processes in the cell. The most notorious is the impact on the transcription of the nitrogenase complex components and accessory proteins. Our results suggest that this new member of the PpaA/AerR family has evolved losing the canonical cobalamin binding domain, but not the redox sensing capability, conserving a not fully understood mechanism of PS regulation.

Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain à la carte materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, P. putida KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a “deaf” PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from Rhodospirillum rubrum, Cupriavidus necator and Pseudomonas pseudoalcaligenes, demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from C. necator resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaCCn synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in P. putida KT2440.

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.

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.

Background: Increasing antibiotic resistance is depleting the available arsenal of these conventional antimicrobials, thus making the development of alternative antibacterial agents a priority for biomedical research. This is the case for Streptococcus penumoniae, a severe respiratory pathogen which, upon colonization of the lung alveoli below the lung surfactant layer (LS), causes community-acquired pneumonia. One of the alternative approaches is the use of enzybiotics, phage-encoded peptidoglycan hydrolases that degrade the bacterial cell wall, thus leading to their death by osmotic shock. To meet therapeutic parameters such as longer in vivo half-life or targeted activity release, the design of enzybiotic formulations is required. Polyhydroxyalkanoates (PHAs) nanoparticles (NPs), present some ideal properties as biomedical nanocarriers such as their inherent biocompatibility, biodegradability, and ability to be vehiculized through hydrophobic barriers, including the lung surfactant (LS). Here, we develop PHA NPs as a platform for the immobilization of enzybiotics against S. pneumoniae via a minimal PHA affinity tag., Results In this study, we tagged the Cpl-711 enzybiotic, which specifically targets S. pneumoniae, with the minimal PHA affinity peptide MinP, resulting in the M711 protein. Then, a PHA nanoparticulate suspension with adequate physicochemical properties for pulmonary delivery was formulated, and M711 was immobilized on their surface. Finally, we assessed the antipneumococcal activity of the nanosystem against planktonic and sessile forms of the pathogen. The resulting pioneer nanosystem displayed sustained antimicrobial activity against free cells, and effectively disaggregated S. pneumoniae biofilms., Conclusions Our findings indicate tag-mediated immobilization of enzybiotics as an effective method for the antimicrobial functionalization of PHA NPs. This straightforward approach may be extrapolated to other enzybiotics (or cargo proteins) with other specificities, highlighting the versatility of the system.

The emergence of new technologies, such as synthetic biology, enables the creation of nextgeneration- advanced materials presenting smart functional properties. Among these group, biohybrid materials have recently grown in attention due to the enormous range of possibilities presented by a material that has the functionalities of living cells or simply enzymes supported on a scaffold. Bacterial biopolymers such as bacterial cellulose (BC) and polyhydroxyalkanotes (PHAs) have stood out in thisfield as their properties can be engineered by microbial biotechnology tools through sustainable bioprocesses combined with materials science. Moreover, the fabrication of new formulations based on BC and PHA polymers with complementary characteristics provides them with highly diverse properties, conferring them non-native features [1]. BC, an extracellular biopolymer produced by bacteria of the genus Komagataeibacter spp., shows excellent mechanical performance with a high capacity of retaining water in its nanofibrillar structure that can be controlled by bioprocess engineering. PHAs, intracellular biopolyesters synthetized by bacteria of many genus, highlight by theirhydrophobic character which triggers this material has excellent barrier properties. Designing cell factories for the production of novel PHAs via smart metabolic engineering is key to obtain à la carte materials with tailored monomer composition and thus, customized mechanical and thermal properties. In this work, we used the model mcl-PHA producing bacterium, Pseudomonas putida KT2440 as a chassis, to assemble heterologous PHA biosynthetic modules to implement an orthogonal PHA switch in a chassis specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. The final goal is to design customized materials with compartments of different functionalities involving the complementary use of BC and PHA and carrying active cargos (i.e. enzymes and/or microorganisms). Se

Bio-based materials are a sustainable alternative for the substitution of oil derived plastics. Within this group we can highlight the polyhydroxyalkanoates (PHAs), biodegradable polymers with similar properties to conventional plastics. The physicochemical properties of the PHA can be tuneable and strongly depends on its hydroxyacids’ monomeric composition1. The synthesis of these PHAs is triggered in many bacteria by a complex regulatory network driven by many factors such as the nutritional imbalance, where the cells experience an excess of carbon coupled with a limitation of other nutrients such as nitrogen. In this study we focus on the model bacterium Pseudomonas putida KT2440 for the production of à la carte PHAs due to its versatile metabolism, resistance to stress, and that naturally produces medium-chain-length-PHA. In order to avoid the complexity of the native regulatory system in this chassis, the entire pha operon was deleted generating the P. putida ∆pha strain, named as PP05_01. Using the Golden Gate technology, a battery of synthetic operons was constructed bearing the minimum set of genes needed for tuneable PHA production in PP05_01. For this, we explored a range of enzymatic set scenarios from different microorganisms (i.e. Rhodospirillum rubrum, Cupriavidus necator, P. putida) producing a panel of PHA with different monomer composition. The most promising candidates were expressed in a multicopy system in PP05_01 strain chassis and the amount and composition of PHA synthesized from various substrates was determined. This study provides P. putida optimized cell factories for the production of tailored PHAs.

The environmental challenge associated with conventional plastics has created a need for alternative solutions, placing bio-based polymers like polyhydroxyalkanoates (PHAs) in a promising position.These polyesters are produced under nutritional stress conditions by several microorganisms including the model bacterium Pseudomonas putida. Carbon sources for PHA synthesis can be classified into two categories: i) PHA related substrates, primarily fatty acids, which provide high production yields, and ii) non-PHA related substrates, such as sugars, whose conversion into PHA precursors through de novo fatty acid synthesis route has been associated with low production yields [1]. Significant efforts have been devoted for the use of carbohydrates as carbon sources within a circular economy framework. To address this challenge, systems biology approaches, like genomescale metabolic models, offer valuable insights into the metabolic features of PHA cycle in P. putida KT2440 [2]. These approaches enable the prediction of the key reaction to be deleted in order to redirect the metabolic routes of interest, and represent powerful tools for optimizing and enhancing the utilization of non-PHA related carbon sources in a sustainable manner. In order to construct an optimized PHA producer P. putida KT2440 strain when using sugars as feedstock, we employed the iJN1411 genome-scale metabolic model to predict those genes to be deleted for eliminating potential competing pathways and redirecting carbon flux from acetyl-CoA towards PHA biosynthesis [3], and those genes to be added for using sucrose as carbon source. We obtained the engineered MT9 strain which was tested to produce PHA from sucrose. Additionally, the synthesis of the antimicrobial biopolymer PHACOS was explored by co-feeding the chemical precursor 6-acetylthiohexanoic acid (6-ATH) and glucose or sucrose [4]. The results revealed that MT9 cultures grown with sucrose as carbon sources achieved a PHA accumulation of 35% per

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.

Annually, about 760 million tons of rice are produced worldwide. This results in agricultural residues such as rice straw. For 1 kg of rice grain, about 1.5 kg of rice straw is produced.Therefore, some management strategies have been studied to extract value-added compounds from this abundant residue and convert it into commodities. In this context, cellulose is one of the compounds studied that can be used for various applications, such as the development of materials and food packaging. Recently, cellulose-based aerogels have become the focus of research as a substitute for absorbent materials. However, using cellulose to absorb aqueous solutions is challenging because the material usually loses its integrity due to its hydrophilic nature. Therefore, stabilization methods should be proposed to maintain the integrity of the material under high humidity conditions to solve this problem., The aim of this work was to produce water stable cellulose-based aerogels from rice straw. Two strategies were investigated: first, TEMPO mediated oxidation followed by high pressure homogenization (HPH) and second, regeneration of cellulose by solubilization with ionic liquids.Briefly, rice straw cellulose was suspended in water by sonicating it at high intensity. Then, the suspension was oxidized and passed through HPH 2, 4 and 8 cycles at 600 bar. On the other hand, BMIM-Cl ionic liquid (IL) was used to dissolve the cellulose at 90 ºC. Then, the suspensions were poured off and freeze-dried. Both methods allowed the preparation of cellulose aerogels with improved water resistance. In addition, this work proposes a novel method to study the internal structure of the porous aerogel using confocal laser scanning microscopy (CLSM).The images acquired by CLSM showed differences in the size and distribution of microfibers in the aerogels at the different HPH cycles tested. In addition, the materials obtained by the regeneration method had different porosity/density than those produced by HPH. Therefore, HPH and regeneration by IL are valuable tools in the fabrication of materials with stable aerogel structure from rice straw. These advances mark a starting point for the development of cellulose- based aerogels without the need for compatibilization with other polymers.

Materials science in the field of oral implantology is focused on the development of bioactive surfaces able to promote both osteogenic and antibacterial mechanisms. In this work, we propose an affordable and reproducible methodology for the fabrication of bioactive SAMs containing Sr/Zn phytate complexes. Phytate SAMs were fabricated by condensation reaction (pH 4) promoted by casting evaporation, and physicochemical features were studied by XPS, SEM, AFM and profilometry. In vitro biological studies performed with hObs cultures demonstrated the ability of modified surfaces for sustaining the cellular colonization and stimulating the ALP activity and the mineralization degree. Moreover, antibacterial assays performed with S. mutans cultures seeded over modified substrates corroborated the potential application of phytate-SAMs as bioactive coatings for titanium dental implants.

La bacteria depredadora Bdellovibrio bacteriovorus ha despertado gran interés en los últimos años debido a su potencial uso como antibiótico vivo, ya que puede lisar otras bacterias Gram negativas incluyendo patógenos humanos. Su ciclo de vida está compuesto por una fase libre móvil o de ataque, en la que nada a gran velocidad hasta localizar a la presa, y una fase intraperiplásmica o de crecimiento, en la que consume los componentes celulares de la presa como paso previo a la lisis de ésta y liberación de la progenie. Además,en nuestro laboratorio se ha puesto de manifiesto la aplicación de B. bacteriovorus en el campo industrial para la purificación de productos intracelulares como los bioplásticos. Para llevar a cabo el procesado de bioproductos a escala industrial es necesario disponer de biomasa suficiente del depredador. En este trabajo,presentamos la optimización de la producción de B. bacteriovorus en biorreactores con un volumen de cultivo de 1 litro, empleando diferentes modos de operación y concentración de bacteria presa. La tasa de crecimiento y la biomasa final de B. bacteriovorus fueron los parámetros de optimización del escalado.Debido a la complejidad que dicha bacteria presenta para el seguimiento de su crecimiento, en este proceso se ha empleado una cepa modificada del depredador que es fluorescente y que permite la monitorización del número de células viables mediante citometría de flujo. Por último, con el fin de facilitar su conservación,transporte y comercialización, se ha puesto a punto un proceso de liofilización en diferentes condiciones.

This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator–prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain.

Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications.

Bdellovibrio bacteriovorus, depredador de otras bacterias Gram-negativas, ha despertado un enorme interés en terapia antimicrobiana y como herramienta lítica para la liberación de productos intracelulares como los bioplásticos o polihidroxialcanoatos. La reciente publicación de su modelo metabólico a escala genómica (iCH455) abre nuevas posibilidades en el estudio de su particular ciclo celular, compuesto por una fase natatoria de ataque a su presa, y una fase intraperiplásmica en la que degrada los componentes celulares de la presa para replicarse. Este modelo permite predecir requerimientos y auxotrofías nutricionales, así como rutas metabólicas incompletas cuya reconstrucción podría reducir dichos requisitos nutricionales o su dependencia de la presa. Sin embargo, a pesar del potencial de B. bacteriovorus, carecemos de herramientas genéticas que nos permitan su domesticación, limitándose principalmente a la interrupción de genes mediante recombinación homóloga y su complementación mediante plásmidos replicativos inestables. En este trabajo, hemos desarrollado plásmidos replicativos en B. bacteriovorus compatibles con el sistema de ensamblado modular GoldenGate-ModularCloning, que nos permite generar librerías genéticas intercambiables para controlar los niveles de expresión de proteínas heterólogas. Debido a las limitaciones de la expresión de proteínas en plásmidos replicativos, por su variable número de copias en B.bacteriovorus, hemos puesto a punto la integración cromosómica mediada por el transposón Tn7, caracterizada por localizarse en un punto concreto del genoma. La inserción en esta región de una proteína fluorescente fusionada a diferentes componentes de una unidad génica (promotores, RBS o terminadores) nos permite determinar rápidamente su influencia en los niveles de expresión y depredación.

Environmental pollution due to the widespread consumption of petroleum-based polymers has hastened the development of biodegradable and environmentally friendly materials. Bacterial biopolymers have attracted much attention over the last decade due to their sustainable production,biodegradability and to the fact that their properties can be altered by new technologies such as synthetic and systems biology combined with advanced materials approaches, providing pathways to diversify their structure and functional complexity. This allows expanding the catalogue of available biomaterials beyond that which exists in nature, as well as extending their potential applications. In the context of the Circular Economy, bacteria are able to grow and produce materials of interest from complex carbon sources such as industrial and municipal wastes. It entails developing bioprocesses in order to up-cycle waste into added-value materials with application in numerous industrial sectors (e.g. packaging, biomedicine). Bacteria produce a broad range of polymers as part of their inherent physiology. An example is the family of polyhydroxyalkanoates (PHAs), that are linear, intracellular polyesters of R-3-hydroxyalkanoate units that accumulate in the cytoplasm as hydrophobic inclusions or granules (100-500 nm) coated with a series of granule-associated proteins (GAPs) involved in the PHA metabolism and granule formation. Bacterial cell factories for PHAs production have been extensively reported in the literature over the last three decades.Among them, Pseudomonas putida constitutes an excellent model to study and manipulate PHA biosynthesis from a wide variety of substrates. Different approaches to engineering P. putida for the production of functional PHAs will be discussed.

La bacteria púrpura no del azufre Rhodospirillum rubrum, ha sido ampliamente estudiada por su gran versatilidad metabólica, pudiendo consumir una amplia gama de sustratos como el gas de síntesis (syngas) y ácidos grasos volátiles (VGA), como el acetato. Además, su complejo metabolismo les permite sintetizar bioplásticos (PHB), derivados del licopeno y combustibles (hidrógeno). En el presente trabajo se estudia el crecimiento anaeróbico de esta bacteria en diferentes concentraciones de monóxido de carbono (CO, principal componente del syngas) con acetato como fuente secundaria de carbono, en presencia o ausencia de luz. Como resultado, se observó un crecimiento en luz óptimo entre presiones parciales de CO (ppCO)= 0,2-0,6 bar. Sin embargo, una vez que el CO se consumió completamente, las células entraron en lisis después de unos días. Este fenómeno no se observó a altas concentraciones de CO en las que el mismo no se consumió completamente (ppCOinicial > 0,8 bar). La duración de la fase de adaptación aumentó a mayor ppCO inicial, demostrando los efectos tóxicos del CO. Si bien el acetato se consumió completamente en luz, en oscuridad su concentración final fue inversamente proporcional al CO consumido, demostrando la dependencia de CO como fuente de energía en ausencia de luz y con fuentes de carbono cuyo nivel de reducción es similar al de la biomasa. Estos resultados sugieren que es posible el crecimiento de R. rubrum (y concomitantemente la producción de PHB o licopeno) a altas concentraciones de CO, aunque mejorar la adaptación inicial es crucial para acelerar el proceso.

Superadas las primeras generaciones de biofactorias alimentadas por fuentes de carbono fácilmente hidrolizables, las nuevas generaciones requieren de materias primas que no compitan con procesos agrícolas. En este sentido, la posibilidad de utilizar polímeros naturales como la lignina o sintéticos como plásticos derivados del petróleo, no solo cumple este requerimiento, sino que posibilita la sostenibilidad del proceso a la vez que resuelve problemas medioambientales a escala global. En este trabajo, abordamos este reto mediante la revalorización de metabolitos derivados de la lignina y de tereftalato de polietileno (PET) y el poliuretano (PU) en bioplásticos (Polihidroxialcanoatos, PHA) usando a la bacteria modelo en biotecnología Pseudomonas putida KT2440 usando aproximaciones de ingeniería metabólica de sistemas. En primer lugar, se utilizó el modelo metabólico de P. putida, iJN1411, como plataforma computacional para diseñar una colección de cepas in sílico superproductoras de PHA a partir de metabolitos derivados de la hidrólisis de estos polímeros.Posteriormente, los diseños más prometedores fueron implementados utilizando tecnologías de vanguardia de edición genética y mutagénesis en esta bacteria. Las cepas resultantes mostraron una producción de PHA muy prometedora. Finalmente, la contextualización fenotípica de estas cepas usando el modelo iJN1411 no sólo identificó los cambios metabólicos claves responsables de esta alta producción, sino que sugirió nuevas intervenciones genéticas que incrementaría aún más los rendimientos obtenidos.

Pseudomonas putida KT2440 is a model environmental bacterium in the production of medium-chain-length polyhydroxyalkanoates (PHAs). Its metabolism has been extensively characterized and a high-quality metabolic model is available (iJN1441), allowing for the systems evaluation of its metabolic capabilities. The use of plastic wastes, like polyethylene terephthalate (PET) and polyurethane (PU), has been proposed as a novel second-generation carbon source for biotechnology. In this work, by using the iJN1411 as a computational platform, we present a set of in silico and in vivo validated growth-coupled PHA overproducer P. putida strains, using monomers derived from the hydrolysis of these polymers, as carbon sources. Cutting edge synthetic biology approaches were used to generate the high number of gene knockouts and knockins required to systematically remove all potential competing pathways and to reroute the carbon flux towards PHA biosynthesis. The effect on PHA production of the iterative rounds of model-guided genetic modifications was subsequently evaluated by means of high-throughput physiological experiments.

Biopolymers such as polyhydroxyalkanoates (PHAs) are considered as an alternative to conventional plastics, but their production can only be economically feasible by waste valorization approaches. The project Afterlife1, funded by the Bio-Based Industries Joint Undertaking under the European Union's Horizon 2020 research and innovation program, aims a flexible, cost- and resource-efficient process for recovering and valorizing the relevant fractions from wastewater (WW). The AFTERLIFE process will isolate the different components of value using a cascade of membrane filtration units that will separate all the solids in the WW. These will then be treated to obtain highly-pure extracts and metabolites or, alternatively, to be converted into PHA. Volatile fatty acid (VFA) fractions with different compositions were obtained by the acidogenic fermentation of the WW from three food industries. In this study, we present a strategy to revalorize those VFAs for the production of PHA using pure cultures. The model strain Cupriavidus necator H16 is a non-pathogenic bacterium that has been the subject of intensive research due to its metabolical versatility and its ability to store up to 90 % of its cell dry weight as polyhydroxybutyrate (PHB). The tolerance studies of H16 strain to VFAs and the determination of key physiological parameters have been applied to optimize the fermentation conditions using three fermented industrial WW on a lab-scale up to 5 liters. The highest PHA concentration (5 g L-1 ), PHA productivity (0.1 g L-1 h-1), and PHA content (80 %) have been achieved by modulating the carbon pulses in a fed-batch system, based on the specific consumption and biomass/product produced. 1https://www.bbi.europa.eu/projects/afterlife.

Background: Rhodospirillum rubrum is a purple non-sulphur bacterium that produces H2 by photofermentation of several organic compounds or by water gas-shift reaction during CO fermentation. Successful strategies for both processes have been developed in light-dependent systems. This work explores a dark fermentation bioprocess for H2 production from water using CO as the electron donor., Results: The study of the influence of the stirring and the initial CO partial pressure (pCO) demonstrated that the process was inhibited at pCO of 1.00 atm. Optimal pCO value was established in 0.60 atm. CO dose adaptation to bacterial growth in fed-batch fermentations increased the global rate of H2 production, yielding 27.2 mmol H2 l−1 h−1 and reduced by 50% the operation time. A kinetic model was proposed to describe the evolution of the molecular species involved in gas and liquid phases in a wide range of pCO conditions from 0.10 to 1.00 atm., Conclusions: Dark fermentation in R. rubrum expands the ways to produce biohydrogen from CO. This work optimizes this bioprocess at lab-bioreactor scale studying the influence of the stirring speed, the initial CO partial pressure and the operation in batch and fed-batch regimes. Dynamic CO supply adapted to the biomass growth enhances the productivity reached in darkness by other strategies described in the literature, being similar to that obtained under light continuous syngas fermentations. The kinetic model proposed describes all the conditions tested.

Polyhydroxyalkanoates (PHA) are polyesters produced intracellularly by many bacterial species as energy storage materials, which are used in biomedical applications, including drug delivery systems, due to their biocompatibility and biodegradability. In this study, we evaluated the potential application of this nanomaterial as a basis of inhaled drug delivery systems. To that end, we assessed the possible interaction between PHA nanoparticles (NPs) and pulmonary surfactant using dynamic light scattering, Langmuir balances, and epifluorescence microscopy. Our results demonstrate that NPs deposited onto preformed monolayers of DPPC or DPPC/POPG bind these surfactant lipids. This interaction facilitated the translocation of the nanomaterial towards the aqueous subphase, with the subsequent loss of lipid from the interface. NPs that remained at the interface associated with liquid expanded (LE)/tilted condensed (TC) phase boundaries, decreasing the size of condensed domains and promoting the intermixing of TC and LE phases at submicroscopic scale. This provided the stability necessary for attaining high surface pressures upon compression, countering the destabilization induced by lipid loss. These effects were observed only for high NP loads, suggesting a limit for the use of these NPs in pulmonary drug delivery.

Bacterial biopolymers such as bacterial cellulose (BC), alginate or polyhydroxyalkanotes (PHAs) have aroused the interest of researchers in many fields, for instance biomedicine and packaging, due to their being biodegradable, biocompatible and renewable. Their properties can easily be tuned by means of microbial biotechnology strategies combined with materials science. This provides them with highly diverse properties, conferring them non-native features. Herein we highlight the enormous structural diversity of these macromolecules, how are they produced, as well as their wide range of potential applications in our daily lives. The emergence of new technologies, such as synthetic biology, enables the creation of next-generation-advanced materials presenting smart functional properties, for example the ability to sense and respond to stimuli as well as the capacity for self-repair. All this has given rise to the recent emergence of biohybrid materials, in which a synthetic component is brought to life with living organisms. Two different subfields have recently garnered particular attention: hybrid living materials (HLMs), such as encapsulation or bioprinting, and engineered living materials (ELMs), in which the material is created bottom-up with the use of microbial biotechnology tools. Early studies showed the strong potential of alginate and PHAs as HLMs, whilst BC constituted the most currently promising material for the creation of ELMs.

[EN] The invention relates to recombinant Pseudomonas putida strains derived from P. putida KT2440 which harbor genetic modifications that make them able to produce significant amounts of polyhydroxyalkanoate (PHA) under balanced nutritional conditions, even by using fatty acid-unrelated carbon sources. Genetic modifications comprised in these recombinant strains focus on avoiding nitrogen limitation and overproducing PHA. The invention also refers to a method for the production of PHA in which the strains of the invention are used, [FR] L'invention concerne des souches de Pseudomonas putida recombinantes dérivées de P. putida KT2440 qui portent des modifications génétiques qui les rendent aptes à produire des quantités significatives de polyhydroxyalcanoate (PHA ) dans des conditions nutritionnelles équilibrées, même en utilisant des sources de carbone non liées aux acides gras. Des modifications génétiques comprises dans ces souches recombinantes se concentrent sur la prévention de la limitation de l'azote et de la surproduction de PHA. L'invention concerne également un procédé de production de PHA dans lequel les souches de l'invention sont utiliséesL'invention concerne des souches de Pseudomonas putida recombinantes dérivées de P. putida KT2440 qui portent des modifications génétiques qui les rendent aptes à produire des quantités significatives de polyhydroxyalcanoate (PHA ) dans des conditions nutritionnelles équilibrées, même en utilisant des sources de carbone non liées aux acides gras. Des modifications génétiques comprises dans ces souches recombinantes se concentrent sur la prévention de la limitation de l'azote et de la surproduction de PHA. L'invention concerne également un procédé de production de PHA dans lequel les souches de l'invention sont utilisées

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese–European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

BACKGROUND: Growing environmental concern sparked renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point towards renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs., PURPOSE AND SCOPE: The surge of synthetic biology and metabolic engineering approaches in recent years now offers the possibility of exploiting the untapped potential of Pseudomonas-based cell factories for the production of tailored PHAs. In this article, we provide an overview of the metabolic and regulatory circuits that rule PHA accumulation in P. putida, and we discuss approaches leading to the biosynthesis of novel polymers (e.g. PHAs including non-biological chemical elements in their structures)., CONCLUSION: The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. We conclude the review by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and we propose strategies towards programmable polymer biosynthesis from alternative substrates in engineered P. putida strains.

Polymeric hydrogels from bacterial cellulose (BC) have been widely used for the development of wound dressings due to its water holding capacity, its high tensile strength and flexibility, its permeability to gases and liquids, but lacks antibacterial activity. In this work, we have developed novel antimicrobial hydrogels composed of BC and the antimicrobial poly(3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate) (PHACOS). Hydrogels based on different PHACOS contents (20 and 50 wt%) were generated and analysed through different techniques (IR, DSC, TGA, rheology, SEM and EDX) and their bactericidal activity was studied against Staphylococcus aureus. PHACOS20 (BC 80%-PHACOS 20%) hydrogel shows mechanical and thermal properties in the range of human skin and anti-staphylococcal activity (kills 1.8 logs) demonstrating a huge potential for wound healing applications. Furthermore, the cytotoxicity assay using fibroblast cells showed that it keeps cell viability over 85% in all the cases after seven days.

[ENG]Biology-based plastics or bioplastics, are becoming an alternative to plastics derived from the petrochemical industry due to their potential biodegradability and their sustainable origin. Nowadays, bioplastics are mostly produced from urban and industrial residues by biotechnological processes, following the principles of circular economy., [ES]Los plásticos de base biológica o bioplásticos son una alternativa a los plásticos derivados de la industria petroquímica debido a su potencial biodegradabilidad y su origen renovable. En la actualidad se producen mayoritariamente a partir de residuos industriales y urbanos mediante procesos biotecnológicos siguiendo los principios de la economía circular.

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.

This chapter reviews the potential of the predatory bacteria Bdellovibrio bacteriovorus, an obligate predator of other gram-negative bacteria, as a biotechnological tool. Due to the unique lifestyle and the different applications, predatory bacteria have awakened interest to be developed as a lytic tool. The lack of physiological and metabolic information makes difficult this development. However, in the last years, different approaches have been described in order to understand the physiology, morphology, and metabolism of the predators, as well as the population dynamics of the prey-predator interactions. Besides its potential of “living antibiotic”, predatory bacteria have been proposed as a biocontrol agent in the food industry or aquaculture. A recent work using B. bacteriovorus as a biological lytic tool for the recovery of intracellular bioproducts highlighted the potential use of predators in industrial bioprocesses. The bottlenecks of using other Bdellovibrio and like organisms (BALOs) have been also considered and discussed during this chapter.

Bacterial cellulose (BC) is an excellent wound dressing device due to its characteristics, such as its biocompatibility, non-toxicity, mechanical stability and high moisture content. However, it lacks antimicrobial activity, which is a critical function of the skin-barrier that is compromised during wound healing. One of the leading candidates with potential applications in the medical field is the group of naturally produced bacterial polyesters or poly([R]-3-hydroxyalkanoates) (PHAs) that constitute a large class of biodegradable biopolymers that show minimal tissue toxicity. The physical and mechanical properties of PHAs are significantly influenced by their monomer composition and chemical structure, which can be tailored by metabolic engineering approaches. In this work, antimicrobial BC membranes were obtained by chemical blending of BC with PHACOS, a second generation polymer containing thioester groups in the side chain with demonstrated antibacterial properties against Methicillin-resistant Staphylococcus aureus (MRSA) (1). MRSA are especially troublesome in hospitals, prisons, schools, and nursing homes where patients with open wounds, invasive devices, and/or weakened immune systems are at high risk of infection. BC was produced in the strain Komagataeibacter medellinenesis ID13488 (2). PHACOS was produced in the model bacterium Pseudomonas putida KT2440 by metabolic engineering using decanoic acid as inducer of the growth and polymer synthesis, and 6-acetylthiohexanoic acid as PHA precursor in a twostage strategy (1). Homogeneous mixtures of BC and PHA or PHACOS were obtained using the ionic liquid (IL) BMIMCl as solvent. Hydroxyl groups of BC and carbonyl groups of PHA form new hydrogen bonds as demonstrated by the FTIR experiments. TGA analysis showed that not only the blends show Td (decomposition temperatures) values in between the individual components, but also the Td increases with the cellulose content in the mixture. The SEM images show the morp

Background Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHAs). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins., Methods Structure prediction analyses were used to characterize the coiled-coil motifs of phasins PhaF and PhaI –produced by the model bacterium Pseudomonas putida KT2440-. Their oligomerization was evaluated by biolayer interferometry and the in vivo two-hybrid (BACTH) system. The interaction ability of a series of coiled-coil mutated derivatives was also measured., Results The formation of PhaF and PhaI complexes was detected. A predicted short leucine zipper-like coiled-coil (ZIP), containing “ideal” residues located within the hydrophobic core, was shown responsible for the oligomers stability. The substitution of key residues (leucines or valines) in PhaI ZIP (ZIPI) for alanine reduced by four fold the oligomerization efficiency., Conclusions These results indicate that coiled-coil motifs are essential for phasin interactions. Correct oligomerization requires the formation of a stable hydrophobic interface between both phasins., General Significance Our findings elucidate the oligomerization motif of PhaF and PhaI. This motif is present in most phasins from PHA-accumulating bacteria and offers a potentially important target for modulating the PHA granules stability.

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.

Polyhydroxyalkanoates (PHAs) are polyesters of microbial origin that can be synthesized by prokaryotes from noble sugars or lipids and from complex renewable substrates. They are an attractive alternative to conventional plastics because they are biodegradable and can be produced from renewable resources, such as the surplus of whey from dairy companies. After an in silico screening to search for SS-galactosidase and PHA polymerase genes, several bacteria were identified as potential PHA producers from whey based on their ability to hydrolyse lactose. Among them, Caulobacter segnis DSM 29236 was selected as a suitable strain to develop a process for whey surplus valorization. This microorganism accumulated 31.5% of cell dry weight (CDW) of poly(3-hydroxybutyrate) (PHB) with a titre of 1.5gl-1 in batch assays. Moreover, the strain accumulated 37% of CDW of PHB and 9.3gl-1 in fed-batch mode of operation. This study reveals this species as a PHA producer and experimentally validates the in silico bioprospecting strategy for selecting microorganisms for waste re-valorization.

Shoe patterns and a sole made of BC, baby shoe made of BC and, on the right BC material dyed in red. (prototype made by www.patent-shoes.com).

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biotechnologically useful natural products found in many bacteria. This biopolymer functions as a carbon and energy storage reservoir in cells but has physical and mechanical properties that make it a promising bioplastic with applications ranging from adhesives to medical implants. Therefore, there is much interest in understanding the biology of mcl-PHA synthesis and metabolism. Increased knowledge of PHA biology serves as a foundation for the bioengineering of PHA and its eventual use as a biologically derived product. This chapter covers the state of knowledge on mcl-PHA, including its synthesis and its central role in cellular metabolism. Moreover, this chapter discusses methods for bioengineering mcl-PHA production in bacteria as well as synthetic biology methods for its study and production in the natural mcl-PHA producer, 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.

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF– -galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

The regulation of the Pg promoter, which controls the expression of the meta operon of the 4-hydroxyphenylacetic acid (4-HPA) catabolic pathway of Escherichia coli W, has been examined through in vivo and in vitro experiments. By using Pg-lacZ fusions we have demonstrated that Pg is a promoter only inducible in the stationary phase when cells are grown on glucose as the sole carbon and energy source. This strict catabolite repression control is mediated by the cAMP receptor protein (CRP). This event does not require the presence of the specific HpaR repressor or the 4-HPA permease (HpaX), excluding the involvement of a typical inducer exclusion mechanism. However, the acetic acid excreted in the stationary phase by the cells growing in glucose acts as an overflow metabolite, which can provide the energy to produce cAMP and to adapt the cells rapidly to the utilization of a new less preferred carbon source such as the aromatic compounds. Although Pg is not a final sigma(38)-dependent promoter, it is activated by the global regulator integration host factor (IHF) in the stationary phase of growth. Gel retardation assays have demonstrated that both CRP and IHF simultaneously bind to the Pg upstream region. DNase I footprint experiments showed that cAMP-CRP and IHF binding sites are centered at -61.5 and -103, respectively, with respect to the transcription start site +1 of the Pg promoter.

The nucleotide sequences of the hpaB and hpaC genes encoding the 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli W ATCC 11105 have been determined. These genes appear to be part of an operon and encode two proteins of 58,781 and 18,679 Da, respectively, that are required for hydroxylase activity. This aromatic hydroxylase is NADH-dependent and uses FAD as the redox chromophore. The largest component (HpaB) has been purified by affinity chromatography in Cibacron blue. E. coli cells that express exclusively hpaB showed only a very low hydroxylase activity that was enhanced in the presence of extracts containing the smallest protein HpaC. This behavior resembles that of the coupling protein of the 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, and it might prevent the wasteful oxidation of NADH in the absence of substrate. Using a promoter-probe plasmid we have demonstrated that the hpaBC operon is expressed by a promoter inducible by 4-hydroxyphenylacetic acid. A gene, named hpaA, encoding a protein homologous to the XylS/AraC family of regulators, was identified upstream of the hydroxylase operon. The role played by HpaA in the regulation of the hpaBC operon remains to be elucidated. Since HpaB is not homologous to other aromatic hydroxylases, we suggest that the E. coli 4-hydroxyphenylacetate 3-hydroxylase is the first member of a new family of two-component aromatic hydroxylases sequenced so far.