Synthesis and Characterization of Polyhydroxyalkanoate Organo/Hydrogels.

Synthetic organogels/hydrogels are attracting growing interests due to their potential applications in biomedical fields, organic electronics, and photovoltaics. Photogelation methods for synthesis of organogels/hydrogels have been shown particularly promising because of the high efficiency and simple synthetic procedures. This study synthesized new biodegradable polyhydroxyalkanoates (PHA)-based organogels/hydrogels via UV photo-cross-linking using unsaturated PHA copolymer poly[(R)-3-hydroxyundecanoate-co-(R)-3-hydroxy-10-undecenoate] (PHU10U) with polyethylene glycol dithiol (PDT) as a photo-cross-linker. The PHU10U was synthesized by an engineered Pseudomonas entomophila and characterized via Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), and 13C NMR. With decreasing the molar ratio of PHU10U to PDT, both the swelling ratio and pore size were decreased. Meanwhile, increasing densities of the gel networks resulted in a higher compressive modulus. Cell cytotoxicity studies based on the CCK-8 assay on both the PHU10U precursor and PHU10U/PDT hydrogels showed that the novel PHA-based biodegradables acting as hydrogels possess good biocompatibility.

Microbial Poly-3-Hydroxybutyrate (PHB) as a Feed Additive for Fishes and Piglets.

The large-scale use of petrochemical-based plastics is damaging our environment. Discarded plastics are harmful to both marine and land animals, sometimes causing death when ingested. Biodegradable plastics have gained attentions from the public and the academia to reduce environmental burdens. Poly-3-hydroxybutyrate (PHB), the simplest and the best-studied bioplastic member of the polyhydroxyalkanoate (PHA) family synthesized by many bacteria, has been studied as a feed additive for large yellow croaker fish and weaned piglets. The fish grow faster and gain more weight when 1% and 2% PHB is added as a feed additive, accompanied by increased survival rates. Weaned piglets are found to grow normally and showed no significant change in average daily weight gains, average daily feed intakes, feed efficiency, and organ developments when 0.5% PHB is added to the feed. It can therefore be concluded that biodegradable and biocompatible PHB is not harmful as a feed additive for marine large yellow croakers and sensitive weaned piglets. PHB therefore holds great promise as a plastic that combines biodegradability and biocompatibility with good tolerability as a feed supplement for animals.

Superhydrophobic Polyhydroxyalkanoates: Preparation and Applications.

Poly( R-3-hydroxybutyrate- co- R-3-hydroxyhexanoate) (PHBHHx), a family member of microbial polyhydroxyalkanoates (PHA), is a biodegradable and biocompatible material with some hydrophobicity and reasonable strength for packaging and tissue engineering applications. In this study, superhydrophobic PHBHHx is fabricated via a simple nonsolvent-assisted process. The material can absorb all tested hydrophobic solvents and oil up to 6-fold of the material weights from water, permitting applications for cleaning environmental oil or solvent pollutions with convenience of disposal after the usage due to its biodegradability. With an excellent combination of biodegradability and biocompatibility, superhydrophobic PHBHHx films are evaluated for antibioadhesion properities to exploit possible implant usages. Up to 100% reductions for platelet adhesions on the superhydrophobic PHBHHx surfaces are observed compared with that on the control material surfaces. Superhydrophobic biodegradable and biocompatible PHBHHx films demonstrate promising low value and high volume or high value and low volume applications.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates.

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its ß-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of ß-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

[Application perspectives of polyhydroxyalkanoates].

Polyhydroxyalkanoates are polyesters of hydroxyalkanoates synthesized by many bacteria and haloarchaea as carbon and energy storage materials. There are more than 150 types of polyhydroxyalkanoate monomers reported, resulting in a variety of Polyhydroxyalkanoates with diverse properties. The material variability, nonlinear optical properties, piezoelectric properties, gas barrier properties, thermoplasticity, biodegradability, and biocompatibility allow polyhydroxyalkanoates to be used for plastic packaging, chiral chemicals generation, medicine, agriculture and bio-energy fields. This review introduces the current applications and future development of polyhydroxyalkanoates.

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.

Metabolic engineering of Escherichia coli for the synthesis of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Escherichia coli was metabolically engineered to effectively produce a series of biopolymers consisted of four types of monomers including glycolate, lactate, 3-hydroxybutyrate and 4-hydroxybutyrate from glucose as the carbon source. The biosynthetic route of novel quadripolymers was achieved by the overexpression of a range of homologous and heterologous enzymes including isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, glyoxylate/hydroxypyruvate reductase, propionyl-CoA transferase, ß-ketothiolase, acetoacetyl-CoA reductase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, CoA transferase and PHA synthase. In shake flask cultures using Luria-Bertani medium supplemented with glucose, the recombinant E. coli reached 7.10g/l cell dry weight with 52.60wt% biopolymer content. In bioreactor study, the final cell dry weight was 19.61g/l, containing 14.29g/l biopolymer. The structure of the produced polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the quadripolymer possessed decreased crystallinity and improved toughness, in comparison to poly-3-hydroxybutyrate homopolymer. This is the first study reporting efficient microbial production of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), short as P(3HB-co-4HB), was successfully produced by engineered Halomonas bluephagenesis TD01 grown in glucose and γ-butyrolactone under open non-sterile conditions. Gene orfZ encoding 4HB-CoA transferase of Clostridium kluyveri was integrated into the genome to achieve P(3HB-co-4HB) accumulation comparable to that of strains encoding orfZ on plasmids. Fed-batch cultivations conducted in 1-L and 7-L fermentors, respectively, resulted in over 70g/L cell dry weight (CDW) containing 63% P(3HB-co-12mol% 4HB) after 48h under non-sterile conditions. The processes were further scaled up in a 1000-L pilot fermentor to reach 83g/L CDW containing 61% P(3HB-co-16mol% 4HB) in 48h, with a productivity of 1.04g/L/h, again, under non-sterile conditions. The elastic P(3HB-co-16mol% 4HB) shows an elongation at break of 1022±43%. Results demonstrate that the engineered Halomonas bluephagenesis TD01 is a suitable industrial strain for large scale production under open non-sterile conditions.

[Progress on polyhydroxyalkanoates (PHA)].

Microbial polyhydroxyalkanoates (PHA) has developed with more diversity and more advanced manufacturing technology. Diversity has now been reflected by diverse monomers, diverse structures and diverse polymerization modes, giving the concept of "PHAome". In addition, the application of synthetic biology and the development of seawater-based biotechnology reduce the production cost of PHA, making PHA more economically competitive. Some examples of commercialized PHA products are described here. Besides, PHA with high value added applications has been exploited.

[Preparation and characterization of polyhydroxyalkanoate bioplastics with antibacterial activity].

Polyhydroxyalkanoates (PHAs), as a novel class of biopolymer, are attracting more attention due to their diverse material properties and environment-independent biodegradability. Here we report the preparation of PHA exhibiting efficient antibacterial activity by embedding Nisin, a food additive generally recognized as safe, into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a type of PHA with high biocompatibility. We first prepared Nisin-containing PHBHHx films using solvent casting method. Confocal laser scanning microscopy analysis showed that a well-mixed integrated structure of the films with an even distribution of the Nisin particles in the PHBHHx matrices. Then the antimicrobial activity of PHBHHx/Nisin films against Micrococcus luteus was quantified on agar plate by measuring the size of inhibition zone. Cultivation in liquid media further confirmed the releasing of Nisin from the films and the long-time antibacterial activity. Results showed that the threshold of Nisin concentration for long-time and effective inhibition against bacteria growth is 25 µg/g. These results altogether establish a technological foundation for the application of PHA in biomedicine and food industry.