Synthetic biology approaches towards the recycling of metals from the environment
Capeness, M. J. & Horsfall, L. E.
28 Aug 2020, In : Biochem Soc Trans. 48(4):1367-1378.

Metals are a finite resource and their demand for use within existing and new technologies means metal scarcity is increasingly a global challenge. Conversely, there are areas containing such high levels of metal pollution that they are hazardous to life, and there is loss of material at every stage of the lifecycle of metals and their products. While traditional resource extraction methods are becoming less cost effective, due to a lowering quality of ore, industrial practices have begun turning to newer technologies to tap into metal resources currently locked up in contaminated land or lost in the extraction and manufacturing processes. One such technology uses biology for the remediation of metals, simultaneously extracting resources, decontaminating land, and reducing waste. Using biology for the identification and recovery of metals is considered a much 'greener' alternative to that of chemical methods, and this approach is about to undergo a renaissance thanks to synthetic biology. Synthetic biology couples molecular genetics with traditional engineering principles, incorporating a modular and standardised practice into the assembly of genetic parts. This has allowed the use of non-model organisms in place of the normal laboratory strains, as well as the adaption of environmentally sourced genetic material to standardised parts and practices. While synthetic biology is revolutionising the genetic capability of standard model organisms, there has been limited incursion into current practices for the biological recovery of metals from environmental sources. This mini-review will focus on some of the areas that have potential roles to play in these processes.

Understanding the role of SilE in the production of metal nanoparticles by Morganella psychrotolerans using MicroScale Thermophoresis
Pantidos, N. & Horsfall, L.
25 Mar 2020, In : N. Biotechnol. 55:1-4.

Metal nanoparticle synthesis has been observed in several species of bacteria but the underlying mechanisms of synthesis are not well understood. Morganella psychrotolerans is a Gram-negative psychrophilic bacterium that is able to tolerate relatively high concentrations of Cu and Ag ions, and it is through the associated resistance pathways that this species is able to convert metal ions to nanoparticles. The purpose of this study was to investigate the mechanism of nanoparticle synthesis, looking at the interaction of the metal binding protein SilE with metal ions using MicroScale Thermophoresis (MST). MST assays give a rapid and accurate determination of binding affinities, allowing for the testing of SilE with a range of environmentally significant metal ions. The binding affinities (Kd) of Ag+ and Cu2+ were measured as 0.17 mM and 0.13 mM respectively, consistent with the observations of strong binding reported in the literature, whereas the binding to Al3+ and Co2+ was measured as Kd values of 4.19 mM and 1.35 mM respectively.

High Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) for the characterisation of enzymatic processing of commercial lignin
Echavarri-Bravo, V., Tinzl, M., Kew, W., Cruickshank, F., Logan Mackay, C., Clarke, D. J & Horsfall, L.E.
25 Sep 2019, In : N. Biotechnol. 52:1-8 

Lignin and lignin components of woody biomass have been identified as an attractive alternative to fossil fuels. However, the complex composition of this plant polymer is one of the drawbacks that limits its exploitation. Biocatalysis of lignin to produce platform chemicals has been receiving great attention as it presents a sustainable approach for lignin valorisation. Aligned with this area of research, in the present study we have applied ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to identify the preferred lignin substrates of a ligninolytic enzyme, a laccase produced by the terrestrial fungus Trametes versicolor. A commercial lignin was incubated with the laccase and acetosyringone (a laccase mediator) for up to 168 h and direct infusion electrospray FT-ICR MS enabled the identification of thousands of molecular species present in the complex lignin sample at different incubation time points. Significant changes in the chemical composition of lignin were detected upon laccase treatment, which resulted in a decrease in the molecular mass distribution of assigned species, consistent with laccase lytic activity. This reduction was predominantly in species classified as lignin-like (based on elemental ratios) and polymeric in nature (>400 Da). Of particular note was a fall in the number of species assigned containing sulfur. Changes in the chemical composition/structure of the lignin polymer were supported by FT-IR spectroscopy. We propose the use of FT-ICR MS as a rapid and efficient technique to support the biotechnological valorisation of lignin as well as the development and optimization of laccase-mediator systems for treating complex mixtures.

Shotgun proteomic analysis of nanoparticle-synthesizing Desulfovibrio alaskensis in response to platinum and palladium

Capeness, M., Imrie, L., Mühlbauer, L., Le Bihan, T. & Horsfall, L.
30 Jul 2019, In : Microbiology. 165(2):1282-1294.

Platinum and palladium are much sought-after metals of critical global importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them and synthesize elemental nanoparticles. Despite this ability, very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also seven proteins that were specific to either platinum or palladium. Overexpression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and is informative regarding how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as a way to capture contaminant metals from the environment.

Production of Biogenic Nanoparticles for the Reduction of 4-Nitrophenol and Oxidative Laccase-Like Reactions
Capeness, M. J., Echavarri-Bravo, V. & Horsfall, L. E.
7 May 2019, In : Front Microbiol. 10: 997.

Biogenic nanoparticles present a wide range of possibilities for use in industrial applications, their production is greener, they can be manufactured using impure feedstocks, and often have different catalytic abilities compared to their chemically made analogs. Nanoparticles of Ag, Pd, Pt, and the bi-elemental PdPt were produced by Morganella psychrotolerans and Desulfovibrio alaskensis and were shown to be able to reduce 4-nitrophenol, an industrial and toxic pollutant. Nanoparticles were recovered post-reaction and then reused, thus demonstrating continued activity. Biogenic PdNPs were shown to have enhanced specificity in a wide pH activity range in the oxidation of the three common substrates used 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,6-Dimethoxyphenol and (2,6-DMP) and 3,3′,5,5′-Tetramethylbenzidine (TMB) to determine oxidase-like activity. Overall Pd in a nanoparticle form exhibited higher oxidation activity than its ionic counterpart, highlighting the potential of biogenic nanoparticles over the use of ions or chemically made elemental forms.

Synthetic Biology for the development of bio-based binders for greener construction materials

Echavarri-Bravo, V., Eggington, I. & Horsfall, L.
Jun 2019, In : MRS Comms. 9(2):474-485.

The development of more sustainable construction materials is a crucial step towards the reduction of CO2 emissions to mitigate Climate Change issues and minimise environmental impacts of the associated industries. Therefore there is growing demand for bio-based binders which are not only safer towards human and environmental health but also facilitate cleaner disposal of the construction materials and enable their compostability.

Here, we summarise the most relevant bio-based polymers and molecules with applications in the construction sector. Due to the biological nature of these materials, the existing biotechnological processes, including Synthetic Biology, for their development and production have been evaluated.

Room temperature bioproduction, isolation and anti-microbial properties of stable elemental copper nanoparticles

Pantidos, N., Edmundson, M. C. & Horsfall, L.
25 Jan 2018, In : N. Biotechnol. 40(B):275-281.

In nanoparticle production there are a number of important considerations that must be made. Producing nanoparticles of uniform size and shape is vital, but no less important is ensuring the production process is as efficient as possible in time, cost and energy. Traditional chemical and physical methods of nanoparticle production often involve high temperatures and pressures, as well as the use of toxic substrates; in contrast the bioproduction of nanoparticles is greener and requires a smaller input of energy resources. Here we outline a method for the straightforward bioproduction of stable, uniform elemental (zero-valent) copper nanoparticles at room temperature, and demonstrate how their size and shape can be modified by subsequent pH manipulation. We also highlight a potential application for these biogenic copper nanoparticles by demonstrating their potential to inhibit bacterial growth.

The contribution of microbially-produced nanoparticles to sustainable development goals

Cueva, M. & Horsfall, L.
Sep 2017, In : Microb. Biotechnol. 10(5):1212-1215.

Nanoparticles (NPs), particles having one or more dimensions below 100 nm, are currently being synthesized through chemical and physical methods on an industrial scale. However, these methods for the synthesis of NPs do not fit with sustainable development goals. NP synthesis, through chemical and physical methods, requires high temperatures and/or pressures resulting in high energy consumption and the generation of large amounts of waste. In recent years, research into the synthesis of NPs has shifted to more green and biological methods, often using microorganisms. A biological approach has many advantages over chemical and physical methods. Reactions are catalysed in aqueous solutions at standard temperature and pressure (cost effective and low energy syntheses). This method does not require solvents or harmful chemicals, making NP biosynthesis a greener and more eco-friendly method. Furthermore, NP synthesis by microbes does not require the use of pure starting materials; thus it can simultaneously be used for the bioremediation of contaminated water, land and waste, and the biosynthesis of NPs. Therefore the biosynthesis of NPs contributes to the sustainable development goals, while the alternative physical and chemical methods exclusively utilize scarce and expensive resources for NP synthesis

Characterisation of a new family of carboxyl esterases with an OsmC domain

Jensen, M. B. V., Horsfall, L. E., Wardrope, C., Togneri, P. D., Marles-Wright, J. & Rosser, S. J.
16 Nov 2016, In : PLoS ONE. 11(11).

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.

Nickel and platinum group metal nanoparticle production by Desulfovibrio alaskensis G20

Capeness, M. J., Edmundson, M. & Horsfall, L. E.
25 Dec 2015, In : New Biotechnology. 32(6):727-731.

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 ofthe 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.

Construction of a modular arsenic resistance operon in E. coli and the production of arsenic nanoparticles
Edmundson, M. C. & Horsfall, L.
20 Oct 2015, In : Front. Bioeng. Biotechnol. 3:160.

Arsenic is a widespread contaminant of both land and water around the world. Current methods of decontamination such as phytoremediation and chemical adsorbents can be resource and time intensive, and may not be suitable for some areas such as remote communities where cost and transportation are major issues. Bacterial decontamination, with strict controls preventing environmental release, may offer a cost-effective alternative or provide a financial incentive when used in combination with other remediation techniques. In this study, we have produced Escherichia coli strains containing arsenic-resistance genes from a number of sources, overexpressing them and testing their effects on arsenic resistance. While the lab E. coli strain JM109 (the “wild-type”) is resistant up to 20 mM sodium arsenate, the strain containing our plasmid pEC20 is resistant up to 80 mM. When combined with our construct pArsRBCC arsenic-containing nanoparticles were observed at the cell surface; the elements of pEC20 and pArsRBCC were therefore combined in a modular construct, pArs, in order to evaluate the roles and synergistic effects of the components of the original plasmids in arsenic resistance and nanoparticle formation. We have also investigated introducing the lac operator in order to more tightly control expression from pArs. We demonstrate that our strains are able to reduce toxic forms of arsenic into stable, insoluble metallic As(0), providing one way to remove arsenate contamination, and which may also be of benefit for other heavy metals.

Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol production in escherichia coli

Lewicka, A. J., Lyczakowski, J. J., Blackhurst, G., Pashkuleva, C., Rothschild-Mancinelli, K., Tautvaišas, D., Thornton, H., Villanueva, H., Xiao, W., Slikas, J., Horsfall, L., Elfick, A. & French, C.
19 Dec 2014, In : ACS Synth. Biol. 3(12):976-978.

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.

Exploring the potential of metallic nanoparticles within synthetic biology

Edmundson, M. C., Capeness, M. & Horsfall, L.
25 Dec 2014, In : New Biotechnology. 31(6):572-578.

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.

Biological synthesis of metallic nanoparticles by bacteria, fungi and plants
Pantidos N, & Horsfall LE.
9 Oct 2014, In : J. Nanomed. Nanotechnol. 5(5):233.

Over the past few decades interest in metallic nanoparticles and their synthesis has greatly increased. This has resulted in the development of numerous ways of producing metallic nanoparticles using chemical and physical methods. However, drawbacks such as the involvement of toxic chemicals and the high-energy requirements of production make it difficult for them to be widely implemented. An alternative way of synthesising metallic nanoparticles is by using living organisms such as bacteria, fungi and plants. This “green” method of biological nanoparticle production is a promising approach that allows synthesis in aqueous conditions, with low energy requirements and low-costs. This review gives an overview of some of these environmentally friendly methods of biological metallic nanoparticle synthesis. It also highlights the potential importance of these methods in assessing nanoparticle risk to both health and the environment.

Microbial enzyme systems for lignin degradation and their transcriptional regulation
Furukawa T, Bello FO & Horsfall L.
9 Dec 2014, In : Front Biol. 9(6):448-471. 

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.

Accessibility statement