Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP⁺-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB _Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB _Av ). In comparison with the wild type strain expressing pPHB _Av , the specific accumulation of PHB (g_PHB/g_DCW) in MG1655 Δack-pta/pTrcgapN/pPHB _Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.     

Synthesis of xylan-graft-poly(l-lactide) copolymers via click chemistry and their thermal properties

Di-O-(6-azidohexanoyl)-xylan-graft-poly(l-lactide)s (XylC6N₃-g-PLLAs) were prepared by grafting propargyl-terminated poly(l-lactide) onto di-O-(6-azidohexanoyl)-xylan (XylC6N₃) via click chemistry. Di-O-(6-azidohexanoyl)-xylan (XylC6N₃) was prepared via two steps from xylan extracted from eucalyptus kraft pulp with aqueous sodium hydroxide solution. Propargyl-terminated poly(l-lactide)s (PLLA) with three different molecular weights were synthesized via ring-opening polymerization of l-lactide using propargyl alcohol as initiator and tin (II) octanoate (Sn(Oct)₂) as catalyst. XylC6N₃ and propargyl-terminated PLLAs were treated with N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) and copper(I) bromide, and the graft copolymers XylC6N₃-g-PLLAs were obtained. DSC measurements revealed that the glass transition temperatures (T_g) of the copolymers decreased compared to that of XylC6N₃, suggesting that the grafted PLLA side-chains act as an internal plasticizer for xylan. TGA measurements revealed that XylC6N₃-g-PLLAs had higher decomposition temperatures than those of XylC6N₃ or PLLA, and that the decomposition temperatures of the copolymers increased with decrease in the number of PLLA side-chains grafted to the xylan main-chain.     

Enzymatic grafting of natural phenols to flax fibres: Development of antimicrobial properties

Unbleached flax fibres for paper production were treated with laccase from Pycnoporus cinnabarinus and low molecular weight phenols (syringaldehyde - SA, acetosyringone - AS and p-coumaric acid - PCA) to evaluate the potential of this treatment to biomodify high cellulose content fibres. After the enzymatic treatment with the phenols, an increase in kappa number was found, probably due to a covalent binding of the phenoxy radicals on fibres. Grafting was more evident in pulps treated with PCA (an increase of 4 kappa number points with respect to the laccase control was achieved). Paper handsheets from treated pulps showed antimicrobial activity against the bacteria tested: Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. An important reduction on microbial count was obtained after incubation of liquid cultures of the bacteria with grafted handsheets. AS and PCA grafted fibres showed a high antibacterial activity on K. pneumoniae, getting a nearly total growth inhibition. AS fibres also caused a high reduction in bacterial population of P. aeruginosa (97% reduction). Optical properties of handsheets from treated pulps were also determined, showing a brightness decrease and increase in coloration, evaluated by CIE L*a*b* system, caused by the laccase induced grafting of the phenols. The results suggest that these low molecular weight phenols, covalently bound to the flax fibres by the laccase treatment, can act as antimicrobial agents and produce handsheets with antimicrobial activity.     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

Elevated levels of laccase synthesis by Pleurotus pulmonarius BPSM10 and its potential as a dye decolorizing agent

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases that have industrial value. In the present study, forty-five isolates of wild mushrooms were screened for laccase production. Eight of the isolates exhibited exploitable levels of substrate oxidation capacity. Isolate BPSM10 exhibited the highest laccase activity of 103.50?U/ml. Internal Transcribed Spacer (ITS) rRNA gene sequencing was used to identify BPSM10 as Pleurotus pulmonarius. The response of BPSM10 to two nutritional media supplemented with various inducers was characterized and the results indicated that Malt Extract Broth (MEB) supplemented with Xylidine increased laccase production by 2.8× (349.5?U/ml) relative to the control (122?U/ml), while Potato Dextrose Broth (PDB) supplemented with xylidine increased laccase production by 1.9× (286?U/ml). BPSM10 has the ability to decolorize seven synthetic dyes in a liquid medium. Maximum decolorization was observed of malachite green (MG); exhibiting 68.6% decolorization at 100?mg/L. Fourier-transform infrared spectroscopy (FTIR) was employed to confirm the decolorization capacity. The present study indicates that P. pulmonarius BPSM10 has the potential to be used as a potent alternative biosorbent for the removal of synthetic dyes from aqueous solutions, especially in the detoxification of polluted water.     

Microwave initiated synthesis and application of polyacrylic acid grafted carboxymethyl cellulose

An environmentally benign and efficient route of synthesis of polyacrylic acid grafted carboxymethyl cellulose (CMC-g-PAA) is developed using microwave radiation alone to initiate the grafting reaction. The synthesis is optimized in terms of percentage grafting and intrinsic viscosity, by varying the microwave irradiation time and monomer (acrylic acid) concentration. The grafted product has been characterized by various physicochemical characterization techniques (intrinsic viscosity measurement, FTIR spectroscopy, SEM morphology study and elemental analysis). FTIR spectroscopy confirmed that free radicals are formed on polysaccharide backbone by cleavage of 1°-OH bond, indicating microwave effect and not thermal decomposition as the cause of free radical generation. The application of the grafted product as flocculant for river water clarification, towards augmentation of drinking water supply has been investigated.     

<Emphasis Type="Italic">UGP</Emphasis> gene expression and UDP-glucose pyrophosphorylase enzymatic activity in grafting annonaceous plants

Grafting is commonly used to propagate commercial fruit species to ensure that the genetic characteristics of selected clones are maintained. However, the biochemical and molecular mechanisms involved in the graft incompatibility of woody trees are not well understood. We investigated the effect of grafting in vegetative growth, UDP-glucose pyrophosphorylase expression and activity of Annonaceous grafted plants: atemoya (Annona cherimola Mill. x Annona squamosa L.) ‘Thompson’ grafted onto different rootstocks, araticum-de-terra-fria (Annona emarginata Schltdl. H. Rainer “var. terra-fria”), araticum-mirim (Annona emarginata Schltdl. H. Rainer “var. mirim”) and biribá (Annona mucosa Schltdl. H. Rainer) at different post-grafting times. The growth of atemoya grafted onto araticum-mirim was lower than that of the rootstocks araticum-de terra-fria and biribá. The results also indicated that grafting alters UGPase gene expression; showing the combination atemoya grafted onto araticum-de-terra-fria (a compatible union) the higher levels of gene expression during the early stages of grafting development. However, no significant differences were detected in UGPase enzyme activity between the graft combinations. In addition, SDS-PAGE and MALDI-TOF analyses detected similar UGPase amino acid sequences in ungrafted atemoya samples to cherimoya (Annona cherimola Mill.), a female parent of the atemoya hybrid. These findings suggest that expression of the UGPase protein is related to graft compatibility in grafted Annona plants.     

Inhibition de l'adhérence de Porphyromonas gingivalis sur la surface de titane greffé de poly(styrène sulfonate de sodium)

Dental implant-associated infections as peri-implantitis represent one of the major causes of osteointegration failures of oral implants. Adhesion of Porphyromonas gingivalis, one of the bacterial strains mainly involved in such infections, is tightly dependent on the topographical and/or physico-chemical properties of the implant surfaces. As a matter of fact, we showed that the grafting of one bioactive polymer such as poly(sodium styrene sulfonate) onto titanium implant surfaces allowed a sensitive decrease of Staphylococcus aureus adhesion (> 40%). The aim of the study consists in evaluating the adhesion of P. gingivalis onto titanium surfaces grafted with poly(sodium stryrene sulfonate) in order to elaborate implants exhibiting appropriate inhibiting properties towards the adhesion of periodontal pathogens. The grafting of poly(sodium stryrene sulfonate) onto titanium surfaces is carried out in two steps: chemical oxydation of titanium to initiate radical species then grafting of poly(sodium stryrene sulfonate) by radical polymerization. Chemical characterization of the surfaces is achieved by Fourier transformed infrared spectroscopy (FTIR). Bacterial adhesion was studied on grafted and non grafted (control) titanium surfaces, preadsorbed or not by plasmatic proteins. Protein adsorption as well as bacteria adhesion is followed by fluorescence spectroscopy by using proteins or bacteria previously labelled with fluorescence probes; the quantification of adsorption and bacteria adhesion are performed by image analysis. Results showed that protein adsorption is more important (~3 times) and that P. gingivalis adhesion is strongly inhibited (~73%) onto poly(sodium styrene sulfonate) grafted surfaces when compared to titanium control. Moreover, the inhibition of bacterial adhesion on grafted surfaces preadsorbed with plasma proteins is comparable to that observed on grafted surfaces preadsorbed with fibronectin. In conclusion, the obtained results evidenced that the grafting of titanium surface by poly(sodium styrene sulfonate) led to significant inhibition of P. gingivalis adhesion and that this inhibitory activity involved adsorbed proteins. Poly(sodium styrene sulfonate) grafted titanium surfaces present a high interest for the elaboration of oral implants in various clinical dental applications.     

Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris

A modified laccase gene, CcLCC6, from Coprinopsis cinerea was chemically synthesized according to the yeast codon bias and expressed in Pichia pastoris. The main properties of laccase, effects of ions and inhibitors, and optimal condition for decolouring malachite green (MG) were investigated in this study. The optimal pH level and temperature of laccase are 3.0 and 40 °C, respectively. The metal ions Mn²⁺, Zn²⁺, Fe³⁺ and Al³⁺ could inhibit laccase activity, as well as 1 mM of sodium dodecyl sulphate and sodium thiosulphate. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as a mediator, was necessary in decolorizing MG. The optimal pH and temperature for MG decolorization were 3.0 and 50 °C, respectively. Approximately 0.02 μM recombinant laccase could effectively decolour 0.05 mM of MG in 1 h. CcLCC6I could inhibit the toxicity of MG to P. pastoris. This is the first report on the successful expression in P. pastoris of CcLCC6I and its enzymatic property. Laccase can also be considered as a candidate for treating industrial effluent containing MG.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Corynebacterium glutamicum using propionate as a precursor

Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Enzyme-mediated coupling of a bi-functional phenolic compound onto wool to enhance its physical,mechanical and functional properties

Wool fibres have been modified with nordihydroguaiaretic acid (NDGA) to improve their performance at use. This water insoluble bi-functional phenolic compound has been grafted on wool through a laccase enzyme catalyzed reaction in an aqueous–ethanol mixture. The capacity of laccase to oxidise NDGA in this aqueous–organic medium has been studied electrochemically. The increase of CH₂, CH₃ and aromatic groups signal in the DRIFT spectra, together with SEM images of the enzymatically modified fabrics confirmed the covalent grafting of NDGA on wool. This one step enzymatic process for grafting of NDGA improved the physical and mechanical properties of wool fabrics such as shrink resistance, crease recovery and tensile strength. Furthermore, the NDGA imparted to the textile material strong antioxidant activity and UV protection.     

Transport of mRNA molecules coding NAC domain protein in grafted pear and transgenic tobacco

Grafting is an important cultivation method and recent research on the mechanism of interactions between rootstock nad scion is focused on the long-distance transport of mRNA and small RNAs in the phloem. Among these transportable molecules, NACP gene coding NAM, ATAF1/2, CUC2 (NAC) domain protein might be involved in apical meristem development. Here, we report the transport of NACP mRNA between Chinese pear (Pyrus bretschneideri) cv. Yali (scion) and the wild Pyrus betulaefolia Bunge (rootstock). Our results indicated that NACP mRNA can be transported in both directions from the 3^rd to 10^th day after micro-grafting. It can also be transported to the shoot apex 30 to 70 cm away from graft-union in 2-year-old grafted trees. For further investigation, transgenic tobaccos with 35S: P. betulaefolia-NACP construct were grafted on wild-type tobaccos (Nicotiana tabacum L. cv. Samsun). The sustainable transport of Pyrus-NACP mRNA through the graft-union occurred from the 15^th day after grafting.     

Grafting Chitosan with Polyethylenimine in an Ionic Liquid for Efficient Gene Delivery

Modifying chitosan (CS) with polyethylenimine (PEI) grafts is an effective way to improve its gene transfection performance. However, it is still a challenge to conduct the grafting with fine control and high efficiency, particularly for the modification of water-insoluble CS. Herein, a novel method to graft CS with PEI (1.8 kDa, PEI-1.8) was developed by using ionic liquid 1-butyl-3-methyl imidazolium acetate ([BMIM]Ac) as a reaction solvent, water-insoluble CS as a reaction substrate and 1,1-carbonyldiimidazole (CDI) as a linking agent. The grafting reaction was greatly accelerated and the reaction time was largely shortened to 4 h by taking advantages of the good solubility of CS, the enhanced nucleophilicity of amino groups and the preferential stability of the activated complexes in the ionic liquid. The chitosan-graft-polyethylenimine (CS-g-PEI) products were characterized by ¹H NMR, FTIR and GPC. PEI-1.8 was quantitatively grafted to CS through urea linkages, and the grafting degree (GD) was conveniently tuned by varying the molar ratios of PEI-1.8 to D-glucosamine units of CS in the range of 9.0 × 10^-3 to 9.0 × 10^-2. Compared with CS, the synthesized CS-g-PEI copolymers showed higher pDNA-binding affinity, which increased with the GD as shown in Agarose gel electrophoresis. The dynamic light scattering (DLS) experiment demonstrated that the CS-g-PEI/pDNA polyplexes had suitable particle sizes and proper ζ-potentials for cell transfection. The CS-g-PEI copolymer with a medium GD of 4.5% conferred the best gene transfection, with the efficiency 44 times of CS and 38 times of PEI-1.8 in HEp-2 cells. The cytotoxicity of CS-g-PEI was tested and found nearly as low as that of CS and much lower than that of PEI.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

Comparison of acellular and cellular bioactivity of poly 3-hydroxybutyrate/hydroxyapatite nanocomposite and poly 3-hydroxybutyrate scaffolds

Nanocomposites have recently been identified as a useful scaffolding material in tissue engineering applications. Poly (3-hydroxybutyrate)/hydroxyapatite nanoparticles (P3HB)/(nHA) porous scaffolds were successfully fabricated through a solvent casting and particulate leaching technique. P3HB/nHA and P3HB scaffolds were prepared by the same technique for comparison. The structure of the nanocomposite and P3HB scaffolds was observed by SEM. The Energy Disperssive X-ray Analysis (EDXA, map of Ca) results indicated that HA nanoparticles were homogeneously dispersed in the P3HB matrix. X-ray diffraction (XRD) analysis showed that P3HB and HA coexist in the nanocomposite. Transmission electron microscopy (TEM) images also showed that the particle size of HA was 30 ～ 40 nm. The porosity of the scaffolds was 84%, and macropores and micropores coexisted and interconnected throughout the scaffolds. Acellular bioactivity experiments showed that more HA crystals formed on the surface of the nanocomposite scaffold than on the P3HB scaffold after 4 weeks immersion in Simulated Body Fluid (SBF). Cell culture experiments demonstrated that the P3HB/nHA nanocomposite scaffold had a better tendency of proliferation and Alkaline Phosphatase (ALP) activity to MG 63 cells than the pure P3HB scaffold. It was found that nHA addition can improve acellular and cellular bioactivity of the P3HB scaffold.     

On the reactivity of the Melanocarpus albomyces laccase and formation of coniferyl alcohol dehydropolymer (DHP) in the presence of ionic liquid 1-allyl-3-methylimidazolium chloride

Some ionic liquids are able to dissolve wood, including lignin and lignocellulose, and thus they provide an efficient reaction media for modification of globally abundant wood-based polymers. Lignin can be modified with laccases (EC 1.10.3.2), multicopper oxidases, which selectively catalyze the oxidation of phenolic hydroxyl to the phenoxy radical in lignin by using oxygen as the co-substrate and an electron acceptor. Many enzymes, including laccases, retain their catalytic activity in the presence of ionic liquids. However, the enzyme activity is usually decreased in the presence of ionic liquids, and the most deactivating ionic liquids have been observed to be those dissolving wood most efficiently. In the present study the activity, pH optimum and catalyzed oxidation of coniferyl alcohol by the laccase from the ascomycete Melanocarpus albomyces was investigated in the ionic liquid 1-allyl-3-methyl-imidazolium chloride ([Amim]Cl), known to dissolve wood and expected to affect the laccase activity. Indeed, with an increasing concentration of [Amim]Cl, the activity of M. albomyces laccase decreased, and the pH range of the enzyme activity was narrowed. The pH optimum, using 2,6-dimethoxyphenol as the substrate, was shifted from 6.5 to 6.0 when the amount of [Amim]Cl was increased to 60% (m-%). It was also found that the inhibition of laccase with NaN₃ was not as severe in the ionic liquid as in water. The insoluble fraction of the dehydropolymer (DHP) formed in the presence of [Amim]Cl had clearly higher molecular weight compared to the one formed in water. DHPs formed in the absence and presence of [Amim]Cl both contained β-5, β–β, β-O-4, α-CO/β-O-4 and α-O-4/β-O-4 structures. However, in the presence of [Amim]Cl, less β-O-4, slightly less β-5 and more β–β structures were formed.     

A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).     

An ionic liquid as reaction media for radiation-induced grafting of thermosensitive poly (N-isopropylacrylamide) onto microcrystalline cellulose

This paper reports a homogeneous modification of microcrystalline cellulose (MCC) in ionic liquids via radiation-induced grafting. Thermosensitive poly (N-isopropylacrylamide) (PNIPAAm) was successfully grafted onto MCC in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) ionic liquid using γ-ray irradiation technique at room temperature. The grafting yield (GY) increased with dose up to 40 kGy, while decreased slightly with dose rate from 22 to 102 Gy/min. The results of TGA indicated that cellulose grafted PNIPAAm (cellulose-g-PNIPAAm) had higher thermal stability than that of ungrafted regenerated cellulose (reg-cellulose). The crystalline structure of original MCC was largely destroyed during the dissolution process according to the XRD profiles, and grafting PNIPAAm onto cellulose further decreased the intensity of crystallinity. SEM showed that reg-cellulose and cellulose-g-PNIPAAm films displayed dense and homogeneous morphology. Moreover, the resulting cellulose-g-PNIPAAm exhibited obvious thermal sensitivity with a lower critical solution temperature around 35 °C, which was observed from the swelling behavior in water at different temperatures.