Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes

‘Ene’-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The b,b-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the a,b-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56% and poor enantioselectivity (16% ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of ‘ene’-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds

Efficient bio-production of citramalate using an engineered Escherichia coli strain

Citramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l-1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l-1 in total) and 1.4 g l-1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l-1, with a productivity of 1.85 g l-1 h-1 and a conversion efficiency of 0.48 gcitramalate g-1 glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d -xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway

Oxidation of cadaverine by putrescine oxidase from Rhodococcus erythropolis

BACKGROUNDPutrescine oxidase (EC 1.4.3.10) is of interest for the microbial production of unsubstituted platform nitrogen (N-)heterocycles, because it only requires inexpensive oxygen as co-substrate. Putrescine oxidase from Rhodococcus erythropolis (Re-PuO) was shown previously to catalyze the oxidation of cadaverine; however, there is little information in the literature about the robustness of this enzyme for biotechnological applications. The aim of this study was to investigate the suitability of Re-PuO for the bioproduction of 1-piperideine from cadaverine under different reaction conditions.RESULTSThe formation of 1-piperideine catalyzed by Re-PuO was demonstrated using o-aminobenzaldehyde as a reagent to trap the cyclic imine and shift the equilibrium for cyclization. A direct assay of Re-PuO activity for cadaverine oxidation was then implemented, by monitoring oxygen consumption. Characterization of the reaction mixture by 1H NMR and mass spectrometry confirmed the presence of piperideine dimers and trimers, yet the quantification of the reaction products could not be achieved. The optimum temperature and pH conditions for enzyme activity were determined as 55 °C and 8.5, respectively. At pH 7.5, the enzyme retained its activity after 65 h incubation at 25 °C, but lost 75% of its activity after 1 h incubation at 55 °C. The enzyme showed no substrate inhibition at concentrations as high as 100 mmol L–1 cadaverine. Complete biotransformation of cadaverine was observed in whole cells at physiological conditions.CONCLUSIONSThese results successfully demonstrate the potential of putrescine oxidase for the bioproduction of N-heterocycles from cadaverine. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI)

The putative mevalonate diphosphate decarboxylase from Picrophilus torridus is in reality a mevalonate-3-kinase with high potential for bioproduction of isobutene

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway.MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme’s potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min-1 g cells-1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min-1 mg protein-1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway

Counting defects with the two-point correlator

We study how topological defects manifest themselves in the equal-time two-point field correlator. We consider a scalar field with Z_2 symmetry in 1, 2 and 3 spatial dimensions, allowing for kinks, domain lines and domain walls, respectively. Using numerical lattice simulations, we find that in any number of dimensions, the correlator in momentum space is to a very good approximation the product of two factors, one describing the spatial distribution of the defects and the other describing the defect shape. When the defects are produced by the Kibble mechanism, the former has a universal form as a function of k/n, which we determine numerically. This signature makes it possible to determine the kink density from the field correlator without having to resort to the Gaussian approximation. This is essential when studying field dynamics with methods relying only on correlators (Schwinger-Dyson, 2PI).Comment: 11 pages, 7 figures

A novel causal mechanism for grey squirrel bark stripping: The Calcium Hypothesis

AbstractGrey squirrels, Sciurus carolinensis, damage trees in the UK by stripping bark and eating the underlying phloem; squirrel motivation for damage is, however, unknown. Damage can result in deterioration of timber quality and a significant economic toll on the forestry industry. Prediction of severe damage followed by targeted killing of squirrels is the current recommended management option. However, the use of warfarin (an anticoagulant poison) is now restricted in the UK and other more humane methods of killing are labour-intensive, so an alternative solution is needed. A better understanding of what motivates grey squirrels to strip bark may enable a preventive approach to be developed. Whilst the bark stripping literature has explored predictive factors affecting the likelihood of damage, causal understanding is lacking. The aim of this review is to introduce the Calcium Hypothesis as a possible explanation for bark stripping, with a view to informing the prevention of damage. The Calcium Hypothesis states that grey squirrels damage trees to ameliorate a calcium deficiency. The main predictive factors of bark stripping behaviour each inform and lend support to the Calcium Hypothesis. Calcium is stored in tree phloem, and damage increases with phloem width, providing squirrels with more calcium per unit area ingested. Calcium levels increase in trees as active growth resumes after winter dormancy, this occurs immediately prior to the main bark stripping season of May–July, and trees growing most vigorously are at increased risk of damage. It is likely grey squirrels also have a requirement for calcium during the bark stripping season. Adult females will be under post-parturition pressures such as lactation, and juveniles will be going through their main period of bone growth, both of which likely represent a requirement for calcium – which supports an observed positive correlation between juvenile abundance and bark stripping. A high autumnal seed crop increases juvenile recruitment the following spring, and could also induce a requirement for calcium to a population due to the high phosphorus to calcium ratio of seeds. To further investigate the hypothesis, the extent to which grey squirrels can utilise calcium oxalate, as calcium occurs in bark, should be determined, and also the extent to which grey squirrels undergo seasonal periods of calcium deficiency. Increasing our causal understanding of bark stripping could inform the future development of preventive measures to aid forest management

Formation of topological defects in gauge field theories

When a symmetry gets spontaneously broken in a phase transition, topological defects are typically formed. The theoretical picture of how this happens in a breakdown of a global symmetry, the Kibble-Zurek mechanism, is well established and has been tested in various condensed matter experiments. However, from the viewpoint of particle physics and cosmology, gauge field theories are more relevant than global theories. In recent years, there have been significant advances in the theory of defect formation in gauge field theories, which make precise predictions possible, and in experimental techniques that can be used to test these predictions in superconductor experiments. This opens up the possibility of carrying out relatively simple and controlled experiments, in which the non-equilibrium phase transition dynamics of gauge field theories can be studied. This will have a significant impact on our understanding of phase transitions in the early universe and in heavy ion collider experiments. In this paper, I review the current status of the theory and the experiments in which it can be tested.Comment: Review article, 43 pages, 7 figures. Minor changes, some references added. Final version to appear in IJMP

The Evolving Professional Identity of the Clinical Research Nurse - A Qualitative Exploration

BACKGROUND: Clinical research nurses (CRN) make a significant contribution to healthcare research within the UK and internationally. However, lack of clarity about their role, and scope of practice renders their contribution within the profession and in the minds of the wider public invisible. This has implications in terms of promoting the role nurses play not only in terms of recruitment, retention, and care of research participants but also as research leaders of the future. AIM: To examine the perspectives of CRNs in the UK on their professional role identity, in order to inform the professional practice of Clinical Research Nursing. METHODS: Exploratory qualitative design using thematic analysis conducted within a realist paradigm. FINDINGS: Participants viewed the positive aspects of their identity 'as agents of change' who were fundamental to the clinical research process. Resourcefulness and the ability to guide members of the research team were valued as key to job satisfaction. Successful navigation through the complexity of advice, support, management and leadership tasks related to their role in caring for research patients were role affirming and generated a sense of pride. However, lack of recognition, clarity of the role and career development opportunities within an identified structure undermined the CRN identity and optimism about progression in the future. Participants reported feeling invisible to colleagues within the clinical community, isolated and excluded from wider nursing groups. IMPLICATIONS: The study describes UK CRN practice, highlighting the positive benefits and challenges associated with the role, including the need to support professional development to maximise their research contribution. Drawing on international comparators the study makes recommendations to establish well-defined educational, career and promotional pathways that include opportunities for research leadership