Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.     

Universality in ant behaviour

Prediction for social systems is a major challenge. Universality at the social level has inspired a unified theory for urban living but individual variation makes predicting relationships within societies difficult. Here, we show that in ant societies individual average speed is higher when event duration is longer. Expressed as a single scaling function, this relationship is universal because for any event duration an ant, on average, moves at the corresponding average speed except for a short acceleration and deceleration at the beginning and end. This establishes cause and effect within a social system and may inform engineering and control of artificial ones.     

Current status and future trends for disposable technology in the biopharmaceutical industry

Single use technology (SUT) is in widespread use throughout the biopharmaceutical industry where it is replacing traditional stainless steel plant particularly for the manufacture of small volume, high value products. The key drivers for SUT uptake include reduction of regulatory risk, lower capital and energy costs and increased facility flexibility. Disposable technology is now well established for cell culture, liquid handling and storage but its application to some downstream operations is proving more intractable. Single‐use chromatography resins are generally uneconomic at process scale and there are limitations on large‐scale tangential flow filtration capacities. The development of disposable sensor technology is lagging behind that of bioreactors and capacities for microbial culture are presently limited to 2000 litres. Concerns regarding compatibility of equipment from different suppliers and extractable and leachable testing are being addressed but continue to influence uptake of SUT by traditional manufacturers. Complete ‘plug and play’ disposable process trains, however, are becoming increasingly available and are being embraced by Contract Manufacturing Organisations (CMOs) with multiproduct facilities. © 2013 Society of Chemical Industry     

Predicting Blast Waves from the Axial Direction of a Cylindrical Charge

Bare, cylindrical, explosive charges produce secondary shock waves in the direction of least presented area. Whilst the source of these shock waves was explored in the 1940’s, no attempt was made to predict them. This paper describes the detonation of bare, cylindrical charges of PE4 (RDX binder 88/12 %), mass 0.2 to 0.46 kg and with a length to diameter ratio of 4 to 1. High speed camera footage showed (i) the formation of the separate, primary, shock waves from the sides and ends of the charge, (ii) Mach reflection of these separate shock waves, giving rise to reflected, secondary shock waves, and (iii) the secondary shock waves catching and merging with the primary shock wave. In the axial direction, the secondary shock wave’s peak overpressure and impulse exceeded that of the primary shock wave for scaled distances, Z=R/M^1/3 ≥3.9 m kg^−1/3, where M is the mass in kg and R the distance from the charge in m. It was found possible to predict the primary peak overpressure, P, at all distances in the axial direction, for a constant length to diameter ratio, using P=3075 Z⁻³−1732 Z⁻²+305 Z⁻¹. Close in the primary peak overpressure is proportional to M/R³ in the axial direction. It was not possible to predict the secondary peak overpressure with the data obtained. The total impulse from both shock waves, I, in the axial direction can be predicted using I=746(M^2/3/R)³−708(M^2/3/R)²+306(M^2/3/R).     

Biocatalysis with Thermostable Enzymes: Structure and Properties of a Thermophilic ‘ene’‐Reductase related to Old Yellow Enzyme

We report the crystal structure of a thermophilic “ene” reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T_m>70 °C) and shows increased resistance to denaturation in water‐miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene‐reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water‐miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.     

Combustion of oil palm stone in a pilot-scale fluidised bed reactor

Biomass is being generated in vast amounts from oil palm plantations particularly in developing countries such as Malaysia, Thailand and Indonesia. Oil palm stone (OPS) is currently considered a waste material and has not previously been considered for energy purposes. The main objective of this study was to investigate the thermochemical conversion of OPS in a pilot-scale fluidised bed combustor. The net heating value of OPS was 24.93 MJ/kg. The effect of primary air flowrate and initial bed temperature were the main parameters investigated. The bed and bed's surface temperature were found to decrease as the primary air flowrate increased. In all tests CO emissions were less than 0.2%. The emissions of SO₂ and HCl ranged from 0.02 ppm to 0.05 ppm, significantly below the permitted levels set by legislation. Stable combustion was observed at a bed temperature of 950 °C. The most abundant elements found in the ash were Al, Ca, Fe, K, Mg, Mn, P, S and Si. However, due to the temperature regime used in the study fouling would not be an issue.     

Hierarchical virtual screening for the discovery of new molecular scaffolds in antibacterial hit identification

One of the initial steps of modern drug discovery is the identification of small organic molecules able to inhibit a target macromolecule of therapeutic interest. A small proportion of these hits are further developed into lead compounds, which in turn may ultimately lead to a marketed drug. A commonly used screening protocol used for this task is high-throughput screening (HTS). However, the performance of HTS against antibacterial targets has generally been unsatisfactory, with high costs and low rates of hit identification. Here, we present a novel computational methodology that is able to identify a high proportion of structurally diverse inhibitors by searching unusually large molecular databases in a time-, cost- and resource-efficient manner. This virtual screening methodology was tested prospectively on two versions of an antibacterial target (type II dehydroquinase from Mycobacterium tuberculosis and Streptomyces coelicolor), for which HTS has not provided satisfactory results and consequently practically all known inhibitors are derivatives of the same core scaffold. Overall, our protocols identified 100 new inhibitors, with calculated K_i ranging from 4 to 250 μM (confirmed hit rates are 60% and 62% against each version of the target). Most importantly, over 50 new active molecular scaffolds were discovered that underscore the benefits that a wide application of prospectively validated in silico screening tools is likely to bring to antibacterial hit identification.     

A survey of Irish fruit and vegetable waste and by-products as a source of polyphenolic antioxidants

In this study, fruit and vegetable by-product and waste sources in Ireland were tested for their antioxidant activity and polyphenol content. The highest levels of antioxidants measured by both ferric reducing antioxidant power (FRAP) and diphenyl-picrylhydrazyl (DPPH) assays, were detected in whole kiwifruit. Of the vegetable by-products, broccoli stems showed the best antioxidant potential. The level of polyphenols as assessed by the Folin–Ciocalteu Reagent (FCR) was significantly correlated with the level of polyphenols by HPLC–DAD (r = 0.93). The level of polyphenols assessed by HPLC–DAD was not significantly correlated with the antioxidant assays. Apple pomace and vegetable by-products were found to be good sources of both polyphenols and antioxidants and due to their abundance may be exploitable resources to use as food ingredients.