Formulation of biogenic fluorescent pigmented PHB nanoparticles from Rhodanobacter sp. for drug delivery.

Biogenic nanoparticles (NPs) have emerged as promising therapeutic formulations in effective drug delivery. Despite of various positive attributes, these NPs are often conjugated with various cytotoxic organic fluorophores for bioimaging, thereby reducing its effectiveness as a potential carrier. Herein, we aim to formulate biogenic fluorescent pigmented polyhydroxybutyrate (PHB) NPs from Rhodanobacter sp. strain KT31 (OK001852) for drug delivery. The bacterial strain produced 0.5 g L-1 of polyhydroxyalkanoates (PHAs) from 2.04 g L-1 of dry cell weight (DCW) under optimised conditions via submerged fermentation. Further, structural, thermal, and morphological charactersiation of the extracted PHAs was conducted using advance analytical technologies. IR spectra at 1719.25 cm-1 confirmed presence of C = O functional group PHB. NMR and XRD analysis validated the chemical structure and crystallinity of PHB. TG-DTA revealed Tm (168 °C), Td (292 °C), and Xc (35%) of the PHB. FE-SEM imaging indicated rough surface of the PHB film and the biodegradability was confirmed from open windro composting. WST1 assay showed no significant cell death (> 50%) from 100 to 500 µg/mL, endorsing non-cytotoxic nature of PHB. PHB NPs were uniform, smooth and spherical with size distribution and mean zeta potential 44.73 nm and 0.5 mV. IR and XRD peaks obtained at 1721.75 cm-1 and 48.42 Å denoted C = O and crystalline nature of PHB. Cell proliferation rate of PHB NPs was quite significant at 50 µg/mL, establishing the non-cytotoxic nature of NPs. Further, in vitro efficacy of the PHB NPs needs to be evaluated prior to the biomedical applications.

Coastal and deep-sea biodegradation of polyhydroxyalkanoate microbeads.

Microbeads find widespread usage in personal care items and cosmetics, serving as exfoliants or scrubbing agents. Their micro-scale size poses challenges in effective drainage capture and given their origin from non-biodegradable oil-based plastics, this contributes substantially to marine pollution. In this study, microbeads were prepared by a simple yet scalable melt homogenization method using four types of polyhydroxyalkanoates (PHA), namely poly[(R)-3-hydroxybutyrate] (P(3HB)), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (P(3HB-co-3HV)), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (P(3HB-co-3HHx)) and poly[(R)-3-hydroxybutyrate-co-(R)-4-hydroxyvalerate] (P(3HB-co-4HB)). Microbeads with different surface smoothness, compressive strength (6.2-13.3 MPa) and diameter (from 1 ~ 150 µm) could be produced. The microbeads were subjected to a comprehensive degradation analysis using three techniques: enzymatic, Biochemical Oxygen Demand (BOD) evaluations, and in situ degradation tests in the deep-sea off Misaki Port in the northern Pacific Ocean (depth of 757 m). Qualitatively, results from enzymatic and in situ degradation demonstrated significant degradation within one week and five months, respectively. Quantitatively, BOD findings indicated that all PHA microbeads degraded similarly to cellulose (~ 85% biodegradability in 25 days). In conclusion, PHA microbeads from this study exhibit promising potential as alternatives to conventional non-biodegradable microbeads.

The effect of additives on the biodegradation of polyhydroxyalkanoate (PHA) in marine field trials.

The persistence of conventional fossil fuel-derived plastics in marine ecosystems has raised significant environmental concerns. Biodegradable plastics are being explored as an alternative. This study investigates the biodegradation behaviour in two marine environments of melt-extruded sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) bioplastic as well as blends of PHBV with a non-toxic plasticiser (triethyl citrate, TEC) and composites of PHBV with wood flour. Samples were submerged for up to 35 weeks in two subtropical marine conditions: on the sandy seabed in the sublittoral benthic zone and the sandy seabed of an open air mesocosm with pumped seawater. Rates of biodegradation, lag times and times to 95 % mass loss (T95) were determined through mass loss data and Gompertz modelling. Mechanisms of biodegradation were studied through changes in molecular weight, mechanical properties and surface features. Results reveal a rapid biodegradation rate for all PHBV samples, demonstrating a range of specific biodegradation rates relative to exposed surface area of 0.03 ± 0.01 to 0.09 ± 0.04 mg.d-1.cm-2. This rapid rate of biodegradation meant that the subtle variations in biodegradation mechanisms across different sample thicknesses and additive compositions had little effect on overall lifetimes, with the T95 for most samples being around 250-350 days, regardless of site, highlighting the robust biodegradability of PHBV in seawater. It was only the PHBV-wood flour composite that showed faster biodegradation, and that was only in the exposed ocean site. The mesocosm site was otherwise shown to be a good model for the open ocean, with very comparable biodegradation rates and changes in mechanical properties over time.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production.

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Green alternatives to petroleum-based plastics: production of bioplastic from Pseudomonas neustonica strain NGB15 using waste carbon source.

Polyhydroxyalkanoates have attracted great interest as a suitable alternative to petrochemical based plastics due to their outstanding properties such as biodegradability and biocompatibility. However, the biggest problem in the production of microbial polyhydroxyalkanoates is low cost-effectiveness. In this study, polyhydroxyalkanoate production was carried out using waste substrates with local isolates. Culture conditions were optimized to increase the polyhydroxyalkanoate production potential. The produced polyhydroxyalkanoate was characterized by FTIR analyses, and its metabolic pathway was determined by real-time PCR. According to the results, the best polyhydroxyalkanoate producer bacteria was characterized as Pseudomonas neustonica NGB15. The optimal culture conditions were detected as 30 g/L banana peel powder, 25 °C temperature, pH 8, and 4-day incubation time. Under the optimized conditions, 3.34 g/L PHA production was achieved. As a result of FTIR analyses, major peaks were obtained at 1723, 1277, 1261, 1097, 1054, and 993 cm-1. These peaks represent that the type of produced polyhydroxyalkanoate was poly-ß-hydroxybutyrate. According to gene expression profile of NGB15, it was determined that Pseudomonas neustonica NGB15 produces PHA using the de novo fatty acid synthesis metabolic pathway. In conclusion, poly-ß-hydroxybutyrate production by Pseudomonas neustonica NGB15 using a low-cost fermentation medium has been shown to be biotechnologically promising.

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.

Biological carbon capture from biogas streams: Insights into Cupriavidus necator autotrophic growth and transcriptional profile.

Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator, known for converting carbon dioxide (CO2) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H2 and O2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO2-adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications.

Polyhydroxyalkanoate involvement in stress-survival of two psychrophilic bacterial strains from the High Arctic.

An ever-growing body of literature evidences the protective role of polyhydroxyalkanoates (PHAs) against a plethora of mostly physical stressors in prokaryotic cells. To date, most of the research done involved bacterial strains isolated from habitats not considered to be life-challenging or extremely impacted by abiotic environmental factors. Polar region microorganisms experience a multitude of damaging factors in combinations rarely seen in other of Earth's environments. Therefore, the main objective of this investigation was to examine the role of PHAs in the adaptation of psychrophilic, Arctic-derived bacteria to stress conditions. Arctic PHA producers: Acidovorax sp. A1169 and Collimonas sp. A2191, were chosen and their genes involved in PHB metabolism were deactivated making them unable to accumulate PHAs (ΔphaC) or to utilize them (Δi-phaZ) as a carbon source. Varying stressors were applied to the wild-type and the prepared mutant strains and their survival rates were assessed based on CFU count. Wild-type strains with a functional PHA metabolism were best suited to survive the freeze-thaw cycle - a common feature of polar region habitats. However, the majority of stresses were best survived by the ΔphaC mutants, suggesting that the biochemical imbalance caused by the lack of PHAs induced a permanent cell-wide stress response thus causing them to better withstand the stressor application. Δi-phaZ mutants were superior in surviving UV irradiation, hinting that PHA granule presence in bacterial cells is beneficial despite it being biologically inaccessible. Obtained data suggests that the ability to metabolize PHA although important for survival, probably is not the most crucial mechanism in the stress-resistance strategies arsenal of cold-loving bacteria. KEY POINTS: • PHA metabolism helps psychrophiles survive freezing • PHA-lacking psychrophile mutants cope better with oxidative and heat stresses • PHA granule presence enhances the UV resistance of psychrophiles.

Pseudomonas putida as a platform for medium-chain length α,ω-diol production: Opportunities and challenges.

Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Evaluation of the production and extraction of polyhydroxybutyrate from volatile fatty acids by means of mixed cultures and B. cepacia.

The global consumption of plastics generates accelerated environmental pollution in landfills and marine ecosystems. Biopolymers are the materials with the greatest potential to replace synthetic polymers in the market due to their good biodegradability, however, there are still several disadvantages, mainly related to their production cost. Considering the above, the generation of biodegradable and biocompatible bioplastics stands out as an alternative solution, some of which are made from renewable raw materials, including polyhydroxyalkanoates PHAs. Although much research has been done on bacteria with the capacity for intracellular accumulation of PHAs, among others, it is also possible to produce PHAs using mixed microbial cultures instead of a single microorganism, using natural microbial consortia that have the capacity to store high amounts of PHAs. In this contribution, three methods for the extraction and purification of PHAs produced by fermentation using volatile fatty acids as a carbon source at different concentrations were evaluated, using the pure strain Burkholderia cepacia 2G-57 and the mixed cultures of the activated sludge from the El Salitre WWTP, in order to select the best method from the point of view of environmental sustainability as this will contribute to the scalability of the process. The mixed cultures were identified by sequencing of the 16S gene. A yield of 89% was obtained from the extraction and purification of PHA using acetic acid as a solvent, which according to its properties is "greener" than chloroform. The polymer obtained was identified as polyhydroxybutylated PHB.

Investigating the efficiency of a two-stage anaerobic-aerobic process for the treatment of confectionery industry wastewaters with simultaneous production of biohydrogen and polyhydroxyalkanoates.

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h-30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m3 H2/m3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m3 initial CIW.

Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Marine biodegradation of plastic films by Alcanivorax under various ambient temperatures: Bacterial enrichment, morphology alteration, and release of degradation products.

The global ocean has been receiving massive amounts of plastic wastes. Marine biodegradation, influenced by global climate, naturally breaks down these wastes. In this study, we systematically compared the biodegradation performance of petroleum- and bio-based plastic films, i.e., low-density polyethylene (LDPE), polylactic acid (PLA), and polyhydroxyalkanoates (PHAs) under three ambient temperatures (4, 15, and 22 °C). We deployed the our previously isolated cold-tolerant plastic-degrading Alcanivorax to simulate the accelerated marine biodegradation process and evaluated the alteration of bacterial growth, plastic films, and released degradation products. Notably, we found that marine biodegradation of PHA films enriched more bacterial amounts, induced more conspicuous morphological damage, and released more microplastics (MPs) and dissolved organic carbon (DOC) under all temperatures compared to LDPE and PLA. Particularly, MPs were released from film edges and cracks with a mean size of 2.8 µm under all temperatures. In addition, the degradation products released by biodegradation of PHA under 22 °C induced the highest acute toxicity to Vibrio fischeri. Our results highlighted that: (1) marine biodegradation of plastics would release millions of MPs per cm2 exposed surface area even in cold environments within 60 days; (2) different marine biodegradation scenarios of these plastics may raise disparate impacts and mitigation-related studies.

PHA is not just a bioplastic!

Polyhydroxyalkanoates (PHA) have evolved into versatile biopolymers, transcending their origins as mere bioplastics. This extensive review delves into the multifaceted landscape of PHA applications, shedding light on the diverse industries that have harnessed their potential. PHA has proven to be an invaluable eco-conscious option for packaging materials, finding use in films foams, paper coatings and even straws. In the textile industry, PHA offers a sustainable alternative, while its application as a carbon source for denitrification in wastewater treatment showcases its versatility in environmental remediation. In addition, PHA has made notable contributions to the medical and consumer sectors, with various roles ranging from 3D printing, tissue engineering implants, and cell growth matrices to drug delivery carriers, and cosmetic products. Through metabolic engineering efforts, PHA can be fine-tuned to align with the specific requirements of each industry, enabling the customization of material properties such as ductility, elasticity, thermal conductivity, and transparency. To unleash PHA's full potential, bridging the gap between research and commercial viability is paramount. Successful PHA production scale-up hinges on establishing direct supply chains to specific application domains, including packaging, food and beverage materials, medical devices, and agriculture. This review underscores that PHA's future rests on ongoing exploration across these industries and more, paving the way for PHA to supplant conventional plastics and foster a circular economy.

Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Application of PHA surface binding proteins of alkali-tolerant Bacillus as surfactants.

Amphiphilic protein has lipophilic and hydrophilic domains, displaying the potential for development as a biosurfactant. The polyhydroxyalkanoate (PHA) surface binding protein derived from Bacillus is a type of protein that has not been studied for its emulsifying properties. In this study, PHA granule-associated protein (PhaP), PHA regulatory protein (PhaQ), and PHA synthase subunit (PhaR) derived from an alkali-tolerant PHA-producing Bacillus cereus HBL-AI were found and heterologously expressed in E. coli and purified to investigate their application as biosurfactants. It showed that the emulsification ability and stability of three amphiphilic proteins were higher than those of widely used chemical surfactants in diesel oil, vegetable oil, and lubricating oil. In particular, the PhaQ protein studied for the first time can form a stable emulsion layer in vegetable oil at a lower concentration (50 µg/mL), which greatly reduced the amount of protein used in emulsification. This clearly demonstrated that the PHA-binding protein of HBL-AI can be well applied as an environmentally friendly biosurfactants.