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Abstract Background Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield. Results To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5’ untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps. Conclusions The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.

Abstract Industrial biotechnology has not developed as fast as expected due to some challenges including the emergences of alternative energy sources, especially shale gas, natural gas hydrate (or gas hydrate) and sand oil et al. The weaknesses of microbial or enzymatic processes compared with the chemical processing also make industrial biotech products less competitive with the chemical ones. However, many opportunities are still there if industrial biotech processes can be as similar as the chemical ones. Taking advantages of the molecular biology and synthetic biology methods as well as changing process patterns, we can develop bioprocesses as competitive as chemical ones, these including the minimized cells, open and continuous fermentation processes et al.

Abstract Background Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). Results Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. Conclusions Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.

Abstract Background Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. Results The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and −16.4°C, one melting temperature (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. Conclusions It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.

Abstract Background Halophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far. Results The draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment. Conclusions The accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.

Atherosclerosis is a chronic inflammatory disease that can cause acute cardiovascular events. Activation of the NOD‐like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome enhances atherogenesis, which links lipid metabolism to sterile inflammation. This study examines the impact of an endogenous metabolite, namely ketone body 3‐hydroxybutyrate (3‐HB), on a mouse model of atherosclerosis. It is found that daily oral administration of 3‐HB can significantly ameliorate atherosclerosis. Mechanistically, 3‐HB is found to reduce the M1 macrophage proportion and promote cholesterol efflux by acting on macrophages through its receptor G‐protein‐coupled receptor 109a (Gpr109a). 3‐HB–Gpr109a signaling promotes extracellular calcium (Ca2+) influx. The elevation of intracellular Ca2+ level reduces the release of Ca2+ from the endothelium reticulum (ER) to mitochondria, thus inhibits ER stress triggered by ER Ca2+ store depletion. As NLRP3 inflammasome can be activated by ER stress, 3‐HB can inhibit the activation of NLRP3 inflammasome, which triggers the increase of M1 macrophage proportion and the inhibition of cholesterol efflux. It is concluded that daily nutritional supplementation of 3‐HB attenuates atherosclerosis in mice. [ABSTRACT FROM AUTHOR]

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high‐salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self‐flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self‐flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self‐flocculating H. campaniensis was developed for growth and the production of poly‐3‐hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self‐flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L −1·hr −1 for the batch process compared to 0.82 and 0.33 g·L −1·hr −1 for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time. The wastewaterless open and continuous batch process is conducted by stopping agitation and aeration after 48‐hour growth, most cells flocculated and sedimented to the bottom of the bioreactor within 1 minute, followed by removing the sedimented cells and restarting the cell growth using the supernatant containing residual cells. The interval time between two batches was less than 10 minutes. The collected sedimented CDW reached 300 g/L. No wastewater was generated throughout the whole process. [ABSTRACT FROM AUTHOR]

High-cell-density cultivation is an effective way to improve the productivity of microbial fermentations and in turn reduce the cost of the final products, especially in the case of intracellular products. Halomonas bluephagenesis TD01 is a halophilic platform bacterium for the next generation of industrial biotechnology with a native PHA synthetic pathway, able to grow under non-sterile continuous fermentation conditions. A selection strategy for mutant strains that can grow to a high cell density was developed. Based on an error-prone DNA polymerase III ε subunit, a genome-wide random mutagenesis system was established and used in conjunction with an artificial high cell density culture environment during the selection process. A high-cell-density H. bluephagenesis TDHCD-R3 obtained after 3 rounds of selection showed an obvious enhancement of resistance to toxic metabolites including acetate, formate, lactate and ethanol compared to wild-type. H. bluephagenesis TDHCD-R3-8-3 constructed from H. bluephagenesis TDHCD-R3 by overexpressing an optimized phaCAB operon was able to grow to 15 g/L cell dry weight (CDW) containing 94% PHA in shake flask studies. H. bluephagenesis TDHCD-R3-8-3 was grown to more than 90 g/L CDW containing 79% PHA compared with only 81 g/L with 70% PHA by the wild type when incubated in a 7-L fermentor under the same conditions. [ABSTRACT FROM AUTHOR]

Different forms of biopolyester PHBVHHx microspheres were prepared so as to compare the mammalian cell behaviors in suspension cultivation system. Based on a microbial terpolyester PHBVHHx consisting of 3-hydroxybutyrate (HB), 3-hydroxyvalerate (HV), and 3-hydroxyhexanoate (HHx), solid microspheres (SMSs), hollow microspheres (HMSs), and porous microspheres (PMS) were successfully prepared by a modified solvent evaporation method involving gas-in-oil-in-water (G1/O/W2) double emulsion, water-in-oil-in-water (W1/O/W2) double emulsion and oil-in-water (O/W) single emulsion, respectively. Generally, PMSs have diameters ranging from 330 to 400 μm with pore sizes of 10 to 60 μm. The pores inside the PMSs were found well interconnected compared with PHBVHHx prepared by the traditional solvent evaporation method, resulting in the highest water uptake ratio. When inoculated with human osteoblast-like cells lasting 6 days, PMS showed much better cell attachment and proliferation compared with other less porous microspheres due to its large inner space as a 3 D carrier. Cell migration towards surface and other interconnected inner pores was clearly observable. Dead or apoptotic cells were found more common among less porous SMSs or HMSs compared with highly porous PMSs. It is therefore concluded that porous PHBVHHx microspheres with larger surface open pores and interconnected inner pores can serve as a carrier or scaffold supporting more and better cell growth for either injectable purposes or simply supporting cell growth. [ABSTRACT FROM AUTHOR]

The concept of “morphology engineering” is proposed here. There are many genes involved in maintaining the bacterial shapes. The manipulations of these genes allow us to change the bacterial shapes from rods to fibers or to small spheres or large spheres. The advantages of morphology engineered bacteria for bio-production including accelerated growth, high cell density, simplification of downstream separation, enlarged space for more inclusion body accumulation and reduction on the cost of bio-production, have recently started to be exploited. So far only a few shape related genes have been manipulated for bioprocess benefits, many more genes are to be exploited for various cell morphologies. The limits of bacterial lengths and diameters may depend on how we manipulate relevant genes. Over time, these limits can be broken to enhance bioprocess competitiveness including improvements on the effectiveness of up- and downstream bioprocessing. Morphology engineering is just starting to show its promises. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are a family of diverse biopolyesters synthesized by bacteria. PHA diversity, as reflected by its monomers, homopolymers, random and block copolymers, as well as functional polymers, can now be generated by engineering the three basic synthesis pathways including the acetoacetyl-CoA pathway, in situ fatty acid synthesis, and/or β-oxidation cycles, as well as PHA synthase specificity. It is now possible to tailor the PHA structures via genome editing or process engineering. The increasing PHA diversity and maturing PHA production technology should lead to more focused research into their low-cost and/or high-value applications. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHAs) are a family of polyesters synthesized by bacteria. Similarly to the genome, transcriptome, and proteome (the entire array of nucleic acids and proteins present in a cell or population of cells at a given time), the PHA spectrum exhibits diverse and dynamic modifications – the ‘PHAome’ – reflecting not only by the diversity of monomers, homopolymers, random and block copolymers, functional and graft polymers, molecular weights, and combinations of the above, but also the ranges of PHAs with various molecular weights and monomer ratios that are present at a particular timepoint in a bacterial cell. Echoing the Materials Genome Initiative (MGI) launched in 2011 to develop an infrastructure to accelerate advanced materials discovery and deployment, understanding the PHAome and ensuring an ample supply of PHAs based on it will promote the discovery of new properties and applications of this family of advanced materials. [ABSTRACT FROM AUTHOR]

This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 282-288, 2015. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are a family of polyesters synthesized by microorganisms under unbalanced growth conditions. PHA including poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly-3-hydroxyoctanoate (PHO) are now available in sufficient quantity for tissue engineering medical application studies due to their reasonable biocompatibility, adjustable mechanical properties, and controllable biodegradability. This paper reviews many achievements based on PHA for medical devices development, tissue repair, artificial organ construction, drug delivery, and nutritional/therapeutic uses. Combined with the recent FDA approval for P4HB clinical application, one can expect a good prospect for PHA application in the medical fields. [ABSTRACT FROM AUTHOR]

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase ( fadD) of Escherichia coli MG1655 in P. putida KT2442. FadLPp, which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadDEc, which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid β-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L−1 extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ Pst, fadD Ec and fadL Pp achieved 2.32 g L−1 extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L−1 extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL Pp and fadD Ec genes combined with the polyhydroxyalkanoates depolymerase gene phaZ. [ABSTRACT FROM AUTHOR]

Abstract: Terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx) abbreviated as P(3HB-co-4HB-co-3HHx), was produced by recombinant Aeromonas hydrophila 4AK4 harboring polyhydroxyalkanoate (PHA) synthesis genes phaPCJ. The monomer content of 4HB in the terpolyesters was dependent on the concentration of 1,4-butanediol, ranging from 4.3 to 8.5mol% in the presence of 5–25g/l 1,4-butanediol, respectively, accompanying by a decreasing 3HHx content from 22 to 17.6mol% and a constant 3HB content of 74mol% during growth in lauric acid containing mineral medium. Number average molecular weights of the terpolyesters were 440,000 to 550,000, much higher than 220,000 of the copolyester consisting of 3HB and 12mol%3HHx commonly produced by wild type A. hydrophila 4AK4. Thermal and mechanical properties of the above terpolyesters were better than those of homopolyester poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB-co-7mol%4HB), P(3HB-co-12mol%3HHx). Moreover, physical properties of the terpolyesters changed with variations of monomer composition. The terpolyesters were characterized using nuclear magnetic resonance (NMR), gas chromatography (GC), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurements. The introduction of 4HB and 3HHx monomers into PHB enhanced the thermal stability, changed its crystallinity and flexibility compared with homopolyester PHB and their two monomer containing copolyesters. [Copyright &y& Elsevier]

Abstract: Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB (phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB-co-3HV-co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43gl−1 to 3.29gl−1, 20.7% to 35.6%, 2.3mol% to 7.1mol%, respectively. Number average molecular (M n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB-co-5mol%3HV) or P(3HB-co-12mol%3HHx). In addition, the terpolyester had lower melting (T m) temperatures and enthalpy of fusions (ΔH m) than PHB did. [Copyright &y& Elsevier]

Samples of polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 4–20% (mol/mol) 3-hydroxyhexanoate (3HHx) were characterized as potential components of blood-contact biomaterials. In an erythrocyte contact hemolysis assay, all tested PHBHHx films had substantially reduced reactivity, typically displaying about 2-fold less hemolytic activity compared with that of PHBV. Both 12% and 20% containing PHBHHx also bound less platelets than other films. After a 120-min exposure to platelet-rich plasma (PRP), few platelets adhered to the 12% and 20% containing PHBHHx films, while numerous platelets were seen on PHBV. Surface properties investigation suggested along with increasing 3HHx content, PHBHHx co-polymer films became smoother and smoother, which may contribute to lower platelet adhesion of PHBHHx containing high HHx content in a short-term contact to platelet-rich plasma. In a long-term contact to PRP, the difference in crystallization of PHBVand PHBHHx can be a critical parameter for platelet adhesion. Human umbilical vein endothelial cells (HUVECs) grew well on PHBHHx containing high content of 3HHx, indicating that both had good biocompatibility with HUVECs. While gelatin-coated or lipase-treated polyesters improved HUVECs proliferation compared with that on uncoated films, platelet adhesion was also decreased on gelatin-coated polyester. The hemocompatibility and biocompatibility of PHBHHx film were markedly improved. Thus, PHBHHx, particularly the surface-modified PHBHHx film, is promising for blood-contact materials. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaPAh from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P212121, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å . [ABSTRACT FROM AUTHOR]

Abstract: As a prerequisite for tissue engineering applications, researchers must understand the effect on local cell types of the degradation products of biodegradable polymers. Polyhydroxybutyrate (PHB) has received special interest as an implant material, because it degrades to release a normal component of blood and tissue, D,L-β-hydroxybutyrate (HB). We report that HB (0.02g/l) promoted cell proliferation in cultured L929 cells plated at high cell density (1×105 cells/well) but not lower cell densities. While HB did not affect cell cycle progression, it significantly inhibited cell death. HB treatment prevented necrosis, reducing cell membrane permeability 4h following serum withdrawal from the medium, and for all subsequent time points. In summary, HB promotes proliferation of L929 cells in high-density cultures by preventing apoptotic and necrotic cell death. This property makes biodegradable polymers containing HB, such as PHBHHx, attractive candidates for tissue engineering applications, especially those requiring the regeneration of large numbers of cells. [Copyright &y& Elsevier]

Abstract: Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future. [Copyright &y& Elsevier]

Abstract: An inactive (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase (PhaGPm) was cloned from a newly isolated Proteobacteria Pseudomonas mendocina LZ. It is the first characterized native inactive PhaG protein. Sequence analysis indicated that there were only two sites where the amino acid sequence differed between this inactive protein and the functional PhaGPp from P. putida. The differences were located at position 78 and in the region 109–113 in the amino acid sequence. Mutagenesis was carried out to investigate these two sites. A recombinant strain harboring a S78C PhaGPp mutant accumulated polyhydroxyalkanoates (PHA) at 11.9% of the cellular dry weight, as compared to the 21.6% PHA produced by the recombinant harboring the wild-type PhaGPp. On the other hand, the changes in the amino acid region 109–113 of PhaGPp to its corresponding region of PhaGPm resulted in negligible PHA accumulation. This demonstrated that region 109–113 in PhaG is relatively important for transacylase activity, while position 78 just plays a supporting role for the enzyme. Furthermore, 3-D structural models of PhaGPp and PhaGPm developed by computational prediction revealed that the variation in amino acids at 109–113 leads to the destruction of the PhaG catalytic center, resulting in the loss of enzyme activity. [Copyright &y& Elsevier]

Abstract: The gene phaPAh, encoding the protein phasin that is associated with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) granule of Aeromonas hydrophila 4AK4, was cloned and characterized. Recombinant strains harboring additional copies of the phasin gene (phaPAh) and the polyhydroxyalkanoate (PHA) synthase gene (phaCAh) accumulated PHBHHx copolyesters consisting of 21 mol% 3-hydroxyhexanoate (3HHx) as compared to 14 mol% 3HHx produced by wild type strain. The molecular weight of PHBHHx produced by the above recombinants was lower than that obtained from the wild type strain grown under similar conditions. Over-expression of phaPAh led to the production of more PHA granules but with reduced sizes. SDS–PAGE showed that PhaPAh was the predominant protein present in the PHBHHx granules. The RT-PCR results suggested that phasin PhaPAh, regulated phaCAh gene at the transcription level. Gene PhaPWe from Wautersia eutropha (formerly Ralstonia eutropha; encoding a 20kDa protein with low amino acid homology to the A. hydrophila 13kDa protein) cloned into A. hydrophila 4AK4 exhibited similar effects on PHBHHx production and PHBHHx composition. These data suggest that the phasins could represent a protein family possessing similar functions but different structures. [Copyright &y& Elsevier]