Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution.

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

Chloroplast-containing coacervate micro-droplets as a step towards photosynthetically active membrane-free protocells.

Encapsulation of structurally and functionally intact chloroplasts within coacervate micro-droplets is used to prepare membrane-free protocells capable of light-induced electron transport.

Nanoparticle-based membrane assembly and silicification in coacervate microdroplets as a route to complex colloidosomes.

The chemical construction of complex colloidosomes consisting of a molecularly crowded polyelectrolyte-enriched interior surrounded by a continuous shell of closely packed silica nanoparticles is studied using optical and fluorescence microscopy, high-resolution X-ray microcomputed tomography, and synchrotron radiation X-ray tomographic microscopy. The colloidosomes are prepared by addition of partially hydrophobic silica nanoparticles to dodecane dispersions of positively or negatively charged coacervate microdroplets consisting of aqueous mixtures of poly(diallyldimethylammonium chloride) (PDDA) and adenosine 5'-triphosphate (ATP) or PDDA and poly(acrylic acid) (PAA), respectively. Interfacial assembly of the nanoparticles produces a polydisperse population of well-defined PDDA/PAA droplets with diameters ranging from 50 to 950 µm. In contrast, reconstruction of the PDDA/ATP coacervate interior occurs on addition of the silica nanoparticles to produce a nanoparticle-stabilized oil-in-coacervate-in-oil multiphase emulsion. Transfer of the coacervate-containing colloidosomes into water and replication of their internal structure are achieved by addition of tetramethoxysilane, which serves as both a cross-linking and silicification agent to produce mineralized PDDA/PAA or PDDA/ATP microstructures with a uniform solidified texture or multichambered interior, respectively. The integration of colloidosome and coacervate technologies offers a route to a new type of multifunctional microcompartmentalized system based on the membrane-mediated incarceration of molecularly crowded chemical environments.

Pharmaceutical and personal care products in sewage treatment works.

In this study a number of analytical procedures are described to determine pharmaceuticals and personal care products (PPCPs) and their metabolites during sewage treatment. The work shows that PPCPs occur in sewage influent and are removed by various wastewater treatment processes. PPCPs include a wide range of chemicals such as prescription drugs as well as diagnostic agents, fragrances, sun-screen agents, and various other compounds commonly present in household items (e.g. detergents, cleaners, toothpastes etc.). During this study a number of PPCPs including painkillers (aspirin, ibuprofen), cholesterol control medication (clofibric acid), antibacterial agents (triclosan), musks (including galaxolide and tonalide), X-ray contrast media (diatrizoate), cancer treatment drugs (cyclophosphamide) and anti-depressant drugs (fluvoxamine) were investigated. Analysis was carried out using a number of techniques. Samples were extracted using solid phase extraction or liquid-liquid extraction and the extracts analysed using capillary gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring or liquid chromatography mass spectrometry (LC-MS) or LC-MS-MS. The results obtained show that aspirin, clolibric acid, diatrozate, fluvoxamine and cyclophosphamide were not detected in any of the crude sewage or sewage effluent samples above the limit of detection of the applied methods. Ibuprofen was detected in all crude sewage samples as well as in all effluent samples with one exception. Removal of ibuprofen by the different STWs was generally between 80-100%, with the exception of one STW where removal was poor (14.4 to 44%). Triclosan was also detected in all crude sewage samples and in all sewage effluent samples. The highest concentration of triclosan detected was 3100 ng l(-1). A high removal efficiency was observed in effluent samples taken on two occasions (average removal 95.6%). The concentrations of musks detected in the crude sewage were generally low except for galaxolide and tonalide. The results from STW effluent samples showed significant removal of galaxolide (70-83% removal) except at one STW (STW 1) where removal was low (57% and 39%). Similar removal efficiencies for tonalide were achieved at these STWs (73-96%) except at STW 1 where removal was poor (53%).