Louise Horsfall

Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer.

The use of dioxygen by HIF prolyl hydroxylase (PHD1).

The hypoxic response in animals is mediated by hydroxylation of proline residues in the alpha-subunit of hypoxia inducible factor (HIF). Hydroxylation is catalysed by prolyl-4-hydroxylases (PHD isozymes in humans) which are iron(II) and 2-oxoglutarate dependent oxygenases. Mutation of the arginine proposed to bind 2-oxoglutarate and of the 2His-1-carboxylate iron(II) binding motif in PHD1 dramatically reduces its activity. The source of the oxygen of the product alcohol is (>95%) dioxygen.

Protein Expression, Aggregation, and Triggered Release from Polymersomes as Artificial Cell‐like Structures

Bringing droplets to life: A cytoskeletal protein (red dots, see scheme) is expressed in artificial cells composed of biocompatible polymersomes, which encapsulate expression machinery and amino acid building blocks. Release of the expressed proteins can be triggered by a negative osmotic shock.

Exploring the potential of metallic nanoparticles within synthetic biology

The fields of metallic nanoparticle study and synthetic biology have a great deal to offer one another. Metallic nanoparticles as a class of material have many useful properties. Their small size allows for more points of contact than would be the case with a similar bulk compound, making nanoparticles excellent candidates for catalysts or for when increased levels of binding are required. Some nanoparticles have unique optical qualities, making them well suited as sensors, while others display para-magnetism, useful in medical imaging, especially by magnetic resonance imaging (MRI). Many of these metallic nanoparticles could be used in creating tools for synthetic biology, and conversely the use of synthetic biology could itself be utilised to create nanoparticle tools. Examples given here include the potential use of quantum dots (QDs) and gold nanoparticles as sensing mechanisms in synthetic biology, and the use of synthetic biology to create nanoparticle-sensing devices based on current methods of detecting metals and metalloids such as arsenate. There are a number of organisms which are able to produce a range of metallic nanoparticles naturally, such as species of the fungus Phoma which produces anti-microbial silver nanoparticles. The biological synthesis of nanoparticles may have many advantages over their more traditional industrial synthesis. If the proteins involved in biological nanoparticle synthesis can be put into a suitable bacterial chassis then they might be manipulated and the pathways engineered in order to produce more valuable nanoparticles.

Nickel and platinum group metal nanoparticle production by Desulfovibrio alaskensis G20.

Desulfovibrio alaskensis G20 is an anaerobic sulfate reducing bacteria. While Desulfovibrio species have previously been shown to reduce palladium and platinum to the zero-state, forming nanoparticles in the process; there have been no reports that D. alaskensis is able to form these nanoparticles. Metal nanoparticles have properties that make them ideal for use in many industrial and medical applications, such as their size and shape giving them higher catalytic activity than the bulk form of the same metal. Nanoparticles of the platinum group metals in particular are highly sought after for their catalytic ability and herein we report the formation of both palladium and platinum nanoparticles by D. alaskensis and the biotransformation of solvated nickel ions to nanoparticle form.

Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis

Sialylated oligosaccharides, present on mammalian outer‐cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host’s immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages, the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP‐Neu5Ac synthetase (CNS). Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine, and the steady‐state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction, pyrophosphate. Mutations that caused the greatest loss in activity included K142A, D211A, D209A and a series of mutations at residue Q104, highlighted from sequence‐alignment studies of related enzymes, demonstrating significant roles for these residues in the catalytic mechanism of CNS. The mutations of D211A and D209A provide strong evidence for a previously proposed metal‐binding site in the enzyme, and the results of our mutations at residue Q104 lead us to include this residue in the metal‐binding site of an intermediate complex. This suggests that, like the sugar‐activating lipopolysaccharide‐synthesizing CMP‐2‐keto‐3‐deoxy‐manno‐octonic acid synthetase enzyme KdsB, CNS recruits two Mg2+ ions during the catalytic cycle.

Microbial enzyme systems for lignin degradation and their transcriptional regulation

Lignocellulosic biomass is the most abundant renewable resource in nature and has received considerable attention as one of the most promising alternatives to oil resources for the provision of energy and certain raw materials. The phenolic polymer lignin is the second most abundant constituent of this biomass resource and has been shown to have the potential to be converted into industrially important aromatic chemicals after degradation. However, due to its chemical and structural nature, it exhibits high resistance toward mechanical, chemical, and biological degradation, and this causes a major obstacle for achieving efficient conversion of lignocellulosic biomass. In nature, lignin-degrading microorganisms have evolved unique extracellular enzyme systems to decompose lignin using radical mediated oxidative reactions. These microorganisms produce a set of different combinations of enzymes with multiple isozymes and isoforms by responding to various environmental stimuli such as nutrient availability, oxygen concentration and temperature, which are thought to enable effective decomposition of the lignin in lignocellulosic biomass. In this review, we present an overview of the microbial ligninolytic enzyme systems including general molecular aspects, structural features, and systematic differences in each microorganism. We also describe the gene expression pattern and the transcriptional regulation mechanisms of each ligninolytic enzyme with current data.

Cytoskeletal Protein Expression and its Association within the Hydrophobic Membrane of Artificial Cell Models

We demonstrate the cell-free expression of the actin-like protein MreB and observed its preference to localize and aggregate at the membrane interface of a water-in-oil-in-water droplet as happens in vivo. These monodisperse microdroplets were produced on a large scale using microfluidics. We suggest a possible use of this platform for theory validation in fundamental biological studies.

Fusion of Pyruvate Decarboxylase and Alcohol Dehydrogenase Increases Ethanol Production in Escherichia coli

Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.

Beyond Genetic Engineering: Technical Capabilities in the Application Fields of Biocatalysis and Biosensors

Synthetic biology allows the generation of complex recombinant systems using libraries of modular components. Two major near-market applications are whole-cell biosensors and biocatalysts for conversion of lignocellulosic biomass to biofuels and chemical feedstocks. Whole cell biosensors consist of cells genetically modified so that binding of a specific analyte to a receptor in the cell triggers generation of a specific output which can be detected and quantified. Since these systems are intrinsically modular in nature, with separate systems for signal detection, signal processing, and generation of the output, they are well suited to a synthetic biology approach. Likewise, effective degradation of cellulosic biomass requires a battery of different enzymes working together to degrade the matrix, expose the polysaccharide fibres, hydrolyse these to release sugars, and convert the sugars to useful products. Synthetic biology provides a useful set of tools to generate such systems. In this chapter we consider how synthetic biology has been applied to these applications, and look at possible future developments in these areas.