Controlling Polymersome Size through Microfluidic-Assisted Self-Assembly: Enabling 'Ready to Use' formulations for biological applications

The self-assembly of poly(ethylene glycol)-block-poly(trimethylene carbonate) PEG-b-PTMC copolymers into vesicles, also referred as polymersomes, was evaluated by solvent displacement using microfluidic systems. Two microfluidic chips with different flow regimes (micromixer and Herringbone) were used and the impact of process conditions on vesicle formation was evaluated. As polymersomes are sensitive to osmotic variations, their preparation under conditions allowing their direct use in biological medium is of major importance. We therefore developed a solvent exchange approach from DMSO (Dimethylsulfoxide) to aqueous media with an osmolarity of 300 mOsm.L-1, allowing their direct use for biological evaluation. We evidenced that the organic/aqueous solvent ratio does not impact vesicle size, but the total flow rate and copolymer concentration have been observed to influence the size of polymersomes. Finally, nanoparticles with diameters ranging from 76 nm to 224 nm were confirmed to be vesicles through the use of multi-angle light scattering in combination with cryo-TEM (Cryo-Transmission Electron Microscopy) characterization

Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli

BACKGROUND: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. RESULTS: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (d)-xylose isomerase, (d)-xylulose-1-kinase, (d)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (d)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. CONCLUSIONS: We have demonstrated the feasibility to produce EG from (d)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0312-7) contains supplementary material, which is available to authorized users

A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts

International audienceA reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of -(1,3) and -(1,6)-glucan in the total -glucan of the cell wall. In the first step, chitin and -(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo--(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo--(1,6)-glucanase from Trichoderma harzianum with -glucosidase from Aspergillus niger to hydrolyse the remaining -glucan. This latter component corresponded to a highly branched -(1,6)-glucan that contained about 75-80% of linear -(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of -(1,3), -(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 degrees C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of -(1,3)-glucan accompanied by an increase of -(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition

Membrane properties of giant polymer and lipid vesicles obtained by electroformation and PVA gel-assisted hydration methods

Giant Unilamellar Vesicles (GUV) are the most commonly used biomimetic membrane model. Their production in physiological conditions through the well-known electroformation process is complex and other gel-assisted hydration procedures have recently emerged to circumvent some limitations of the process. In this work, the membrane physical properties of Giant Polymer Unilamellar Vesicles (Polymersomes) and lipid vesicles (Liposomes) obtained from the gel-assisted hydration procedure and electroformation, have been evaluated thoroughly by different techniques: micropipettes aspiration (MPA), Fluorescence Recovery After Photo Bleaching (FRAP) and Laurdan Generalized Polarization (GP). The study was performed on GUVs made of either phospholipids (POPC and DPPC) or amphiphilic copolymers based on Poly(dimethylsiloxane) and poly(ethyleneoxide). A significant deviation of stretching modulus, lateral diffusion coefficient and membrane packing was observed for GUVs formed by the PVA gel-assisted hydration method compared to the classical electroformation process. We were able to show that this deviation was due to the presence of PVA chains in the membrane and in the suspending medium. Globally our findings reveal that particular attention has to be paid on the gel assisted hydration process as the vesicles obtained present altered mechanical behaviour.Cinétique de Translocation de Particules à travers de Bicouches Auto-AssembléesErasmus Mundus - International Doctoral School in Functional Material

Deep Chemical and Physico-Chemical Characterization of Antifungal Industrial Chitosans—Biocontrol Applications

International audienceFive different chitosan samples (CHI-1 to CHI-5) from crustacean shells with high deacetylation degrees (>93%) have been deeply characterized from a chemical and physicochemical point of view in order to better understand the impact of some parameters on the bioactivity against two pathogens frequently encountered in vineyards, Plasmopara viticola and Botrytis cinerea. All the samples were analyzed by SEC-MALS, 1 H-NMR, elemental analysis, XPS, FTIR, mass spectrometry, pyrolysis, and TGA and their antioxidant activities were measured (DPPH method). Molecular weights were in the order: CHI-4 and CHI-5 (MW >50 kDa) > CHI-3 > CHI-2 and CHI-1 (MW < 20 kDa). CHI-1, CHI-2 and CHI-3 are under their hydrochloride form, CHI-4 and CHI-5 are under their NH 2 form, and CHI-3 contains a high amount of a chitosan calcium complex. CHI-2 and CHI-3 showed higher scavenging activity than others. The bioactivity against B. cinerea was molecular weight dependent with an IC50 for CHI-1 = CHI-2 (13 mg/L) ≤ CHI-3 (17 mg/L) < CHI-4 (75 mg/L) < CHI-5 (152 mg/L). The bioactivity on P. viticola zoospores was important, even at a very low concentration for all chitosans (no moving spores between 1 and 0.01 g/L). These results show that even at low concentrations and under hydrochloride form, chitosan could be a good alternative to pesticides

Coupling of RAFT polymerization and chemoselective post-modifications of elastin-like polypeptides for the synthesis of gene delivery hybrid vectors

Hybrids of synthetic polymers and biopolymers are known to be macromolecular systems that merge the properties of each component and overcome some of their intrinsic limitations. Elastin-like polypeptides (ELPs) are a class of biopolymers known for their genetically-encoded synthesis, monodispersity, biocompatibility and absence of toxicity, which are very attractive features for biological applications. However, the presence of numerous amino acid residues presenting electrically charged side chains within the ELP sequence makes the purification of these ELPs by Inverse Transition Cycling(ITC) difficult to achieve. Positively charged ELPs are of great interest for the design of polyelectrolyte complexes dedicated to the transport and delivery of genetic material. In this work, an ELP containing periodically spaced methionine residues was recombinantly expressed, and these residues were chemoselectively modified at the thioether side chains to introduce alkyne groups. In parallel, four cationic oligomers with different chain lengths were synthesized by Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization using a chain transfer agent containing an azido group. Hybrid cationic ELP were finally obtained by covalent coupling of the cationic oligomers onto the ELP by Huisgen azide-alkyne cycloaddition reaction. The different hybrid cationic ELPs were characterized by 1H NMR, ζ-potential and SEC analyses to assess their purity and determine their degree of functionalization, overall charge and molar mass. Then, electrostatic complexation was achieved between these hybrid cationic ELPs and plasmid DNA, allowing the determination of the optimal conditions for obtaining stable nanoparticles having a controlled size and surface potentials at different N+/P- charge ratios. Preliminary biological tests showed the reliability of such hybrid cationic ELPs to internalize efficiently genetic material into living cell

Additional file 1. of Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli

Log2 transformed expression levels of candidate glycolaldehyde reductase

Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H+-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H+-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism