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Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO₂ by a Recombinant Cupriavidusnecator.

ABSTRACT: The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO₂ as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H₂/O₂/CO₂ = 8:1:1). The results of GC and ¹H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO₂ with high cellular content. When 1.0 g/L (NH₄)₂SO₄ was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccr_MeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccr_MeJ_Ac-emd harboring short-chain-length-specific PhaJ.

SUBMITTER: Tanaka K

PROVIDER: S-EPMC8615203 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

Project description:A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.

| S-EPMC179330 | biostudies-other

Effect of Graphene Oxide on the Properties of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate).

Project description:The main shortcomings of polyhydroxybutyrate (PHB), which is a biodegradable and biocompatible polymer used for biomedical and food packaging applications, are its low thermal stability, poor impact resistance and lack of antibacterial activity. This issue can be improved by blending with other biodegradable polymers such as polyhydroxyhexanoate to form poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which is a copolymer with better impact strength and lower melting point. However, PHBHHx shows reduced stiffness than PHB and poorer barrier properties against moisture and gases, which is a drawback for use in the food industry. In this regard, novel biodegradable PHBHHx/graphene oxide (GO) nanocomposites have been prepared via a simple, cheap and environmentally friendly solvent casting method to enhance the mechanical properties and antimicrobial activity. The morphology, mechanical, thermal, barrier and antibacterial properties of the nanocomposites were assessed via several characterization methods to show the enhancement in the biopolymer properties. The stiffness and strength of the biopolymer were enhanced up to 40% and 28%, respectively, related to the strong matrix-nanofiller interfacial adhesion attained via hydrogen bonding interactions. Moreover, the nanocomposites showed superior thermal stability (as far as 40 °C), lower water uptake (up to 70%) and better gas and vapour barrier properties (about 45 and 35% reduction) than neat PHBHHx. They also displayed strong biocide action against Gram positive and Gram negative bacteria. These bio-based nanocomposites with antimicrobial activity offer new perspectives for the replacement of traditional petroleum-based synthetic polymers currently used for food packaging.

| S-EPMC8309387 | biostudies-literature

Blends of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Fruit Pulp Biowaste Derived Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) for Organic Recycling Food Packaging.

Project description:In the present study, a new poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) [P(3HB-co-3HV-co-3HHx)] terpolyester with approximately 68 mol% of 3-hydroxybutyrate (3HB), 17 mol% of 3-hydroxyvalerate (3HV), and 15 mol% of 3-hydroxyhexanoate (3HHx) was obtained via the mixed microbial culture (MMC) technology using fruit pulps as feedstock, a processing by-product of the juice industry. After extraction and purification performed in a single step, the P(3HB-co-3HV-co-3HHx) powder was melt-mixed, for the first time, in contents of 10, 25, and 50 wt% with commercial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Thereafter, the resultant doughs were thermo-compressed to obtain highly miscible films with good optical properties, which can be of interest in rigid and semirigid organic recyclable food packaging applications. The results showed that the developed blends exhibited a progressively lower melting enthalpy with increasing the incorporation of P(3HB-co-3HV-co-3HHx), but retained the PHB crystalline morphology, albeit with an inferred lower crystalline density. Moreover, all the melt-mixed blends were thermally stable up to nearly 240 °C. As the content of terpolymer increased in the blends, the mechanical response of their films showed a brittle-to-ductile transition. On the other hand, the permeabilities to water vapor, oxygen, and, more notably, limonene were seen to increase. On the overall, this study demonstrates the value of using industrial biowaste derived P(3HB-co-3HV-co-3HHx) terpolyesters as potentially cost-effective and sustainable plasticizing additives to balance the physical properties of organic recyclable polyhydroxyalkanoate (PHA)-based food packaging materials.

| S-EPMC8038484 | biostudies-literature

Fabrication of Porous Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Monoliths via Thermally Induced Phase Separation.

Project description:This study deals with the fabrication of biodegradable porous materials from bacterial polyester, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HHx), via thermally induced phase separation. P3HB3HHx monoliths with topological porous structure were prepared by dissolution of P3HB3HHx in dimethyl sulfoxide (DMSO) at 85 °C and subsequent quenching. The microstructure of the resulting P3HB3HHx monoliths was changed by the P3HB3HHx concentration of the polymer solution. Differential scanning calorimetry and polarized optical microscope analysis revealed that the P3HB3HHx monoliths crystallized during phase separation and the subsequent aging. The mechanical properties, such as compression modulus and stress, of the monoliths depended on the 3-hydroxyhexanoate content of P3HB3HHx. Furthermore, the P3HB3HHx monolith absorbed linseed oil in preference to water in a plant oil⁻water mixture. In combination with the biodegradable character of P3HB3HHx, the present study is expected to contribute to the development of bio-based materials.

| S-EPMC6432561 | biostudies-literature

Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds.

Project description:BackgroundPoly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial polyester with high biodegradability, even in marine environments. Ralstonia eutropha has been engineered for the biosynthesis of P(3HB-co-3HHx) from vegetable oils, but its production from structurally unrelated carbon sources remains unsatisfactory.ResultsRalstonia eutropha strains capable of synthesizing P(3HB-co-3HHx) from not only fructose but also glucose and glycerol were constructed by integrating previously established engineering strategies. Further modifications were made at the acetoacetyl-CoA reduction step determining flux distribution responsible for the copolymer composition. When the major acetoacetyl-CoA reductase (PhaB1) was replaced by a low-activity paralog (PhaB2) or enzymes for reverse β-oxidation, copolyesters with high 3HHx composition were efficiently synthesized from glucose, possibly due to enhanced formation of butyryl-CoA from acetoacetyl-CoA via (S)-3HB-CoA. P(3HB-co-3HHx) composed of 7.0 mol% and 12.1 mol% 3HHx fractions, adequate for practical applications, were produced at cellular contents of 71.4 wt% and 75.3 wt%, respectively. The replacement by low-affinity mutants of PhaB1 had little impact on the PHA biosynthesis on glucose, but slightly affected those on fructose, suggesting altered metabolic regulation depending on the sugar-transport machinery. PhaB1 mostly acted in the conversion of acetoacetyl-CoA when the cells were grown on glycerol, as copolyester biosynthesis was severely impaired by the lack of phaB1.ConclusionsThe present results indicate the importance of flux distribution at the acetoacetyl-CoA node in R. eutropha for the biosynthesis of the PHA copolyesters with regulated composition from structurally unrelated compounds.

| S-EPMC6716841 | biostudies-literature

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains.

Project description:The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

| S-EPMC3126409 | biostudies-literature

Microbial Degradation Behavior in Seawater of Polyester Blends Containing Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx).

Project description:The microbial degradation behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and its compound with several polyesters such as poly(butylene adipate-co-telephtharate) (PBAT), poly(butylene succinate) (PBS), and polylactic acid (PLA) in seawater was tested by a biological oxygen demand (BOD) method. PHBHHx showed excellent biodegradation in seawater in this study. In addition, the biodegradation rate of several blends was much influenced by the weight ratio of PHBHHx in their blends and decreased in accordance with the decrement of PHBHHX ratio. The surface morphology of the sheet was important factor for controlling the biodegradation rate of PHBHHx-containing blends in seawater.

| S-EPMC5793082 | biostudies-literature

Studies on Chemical IR Images of Poly(hydroxybutyrate?co?hydroxyhexanoate)/Poly(ethylene glycol) Blends and Two-Dimensional Correlation Spectroscopy.

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| S-EPMC6473784 | biostudies-literature

Effect of Different Plasticizers on Thermal, Crystalline, and Permeability Properties of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Films.

Project description:Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) films were prepared using a cast film technique. Dioxane was chosen over other polymer solvents as it resulted in homogenous films with better morphology. Several plasticizers with different molecular weights and concentrations were added to the biopolymer solution prior to casting. Thermal, crystalline, and permeability properties were analyzed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and both water vapor and oxygen transmission rate analysis. In general, the addition of plasticizers decreased the glass transition temperature (Tg), cold crystallization temperatures (Tcc), melting temperatures, as well as crystallinity degrees and increased the crystallite sizes and water vapor and oxygen transmission rates. The use of isosorbide and low-molecular-weight poly(ethylene glycol) (PEG) lowered the Tg around 30 °C at the highest used concentration, also being the most effective in increasing the crystallite size. When considering isosorbide and low-molecular-weight poly(ethylene glycol) (PEG) as very good plasticizers for PHBH, the question of which plasticizer to use strongly relies on the desired PHBH application.

| S-EPMC9460745 | biostudies-literature

Molecular Characterization of the Bacterial Community in Biofilms for Degradation of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Films in Seawater.

Project description:Microplastics are fragmented pieces of plastic in marine environments, and have become a serious environmental issue. However, the dynamics of the biodegradation of plastic in marine environments have not yet been elucidated in detail. Polyhydroxyalkanoates (PHAs) are biodegradable polymers that are synthesized by a wide range of microorganisms. One of the PHA derivatives, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has flexible material properties and a low melting temperature. After an incubation in seawater samples, a significant amount of biofilms were observed on the surfaces of PHBH films, and some PHBH films were mostly or partially degraded. In the biofilms that formed on the surfaces of unbroken PHBH films, the most dominant operational taxonomic units (OTUs) showed high similarity with the genus Glaciecola in the family Alteromonadaceae. On the other hand, the dominant OTUs in the biofilms that formed on the surfaces of broken PHBH films were assigned to the families Rhodobacteraceae, Rhodospirillaceae, and Oceanospirillaceae, and the genus Glaciecola mostly disappeared. The bacterial community in the biofilms on PHBH films was assumed to have dynamically changed according to the progression of degradation. Approximately 50 colonies were isolated from the biofilm samples that formed on the PHBH films and their PHBH-degrading activities were assessed. Two out of three PHBH-degrading isolates showed high similarities to Glaciecola lipolytica and Aestuariibacter halophilus in the family Alteromonadaceae. These results suggest that bacterial strains belonging to the family Alteromonadaceae function as the principal PHBH-degrading bacteria in these biofilms.

| S-EPMC5877338 | biostudies-literature