Exploring the Potential of Antibody Mimetics for Detecting Environmental Contaminants

Over the past 15-20 years, there has been growing interest and concern from the scientific and regulatory communities over the potential risks of emerging environmental contaminants (ECs). State-of-the-art techniques used for monitoring ECs do not provide the high spatial and temporal resolution measurements required to better understand and mitigate the risks. Immunoassays, which use antibodies to detect a target compound with high affinity, specificity and selectivity, partly address these limitations. However, the use of antibodies for the detection of small-sized, non-immunogenic environmental contaminants, presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies, but with reduced batch-to-batch variability, increased stability, and in vitro selection to ensure rapid discovery of binders against a wide range of targets. This study explores the potential of Affimers, a recent example of antibody mimetics, as suitable bioreceptors for the detection of small organic molecules. Methylene blue (MB), a redox-active molecule used as a fabric dye and diclofenac (DCF), an important environmental contaminant, were selected as the target compounds and Affimers against MB and DCF were developed by the BioScreening Technology Group, University of Leeds. The objectives of this project were to a) demonstrate that the developed Affimers can bind to the selected targets with very high affinity, b) assess their performance in the complexity of environmental water samples (selectivity), and c) investigate the potential of an Affimer-based assay for small molecule detection. Target immobilisation for Affimer characterisation was achieved using long-chained alkanethiol linkers coupled with oligoethylene glycol (LCAT–OEG) and the immobilisation approach was evaluated through electrochemical measurements and infrared spectroscopy. Subsequently, binding between the immobilised targets and target-specific Affimers was quantified using quartz crystal microbalance with dissipation monitoring (QCM-D). Affimer affinity studies revealed Affimer dissociation constants (KD=13.7 nM and 73 nm for MB and DCF Affimers respectively) was comparable to that of high affinity antibodies. Furthermore, the high selectivity of MB-Affimers was demonstrated using limnetic water samples. Finally, an Affimer-based competition ELISA was demonstrated (LOD=75 nM), illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, non-immunogenic compounds. These findings are very promising, encouraging further research into Affimer-based assays and biosensors in order to introduce a novel, alternative path for rapid, on-site monitoring of contaminants in the environment

Antibody mimetics for the detection of small organic compounds using a quartz crystal microbalance

Conventional immunoassays rely on antibodies that provide high affinity, specificity and selectivity against a target analyte. However, the use of antibodies for the detection of small-sized, non-immunogenic targets, such as pharmaceuticals and environmental contaminants presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies but with reduced batch-to- batch variability, high stability and in vitro selection to ensure rapid discovery of binders against a wide range of targets. In this work we explore the potential of Affimers, a recent example of antibody mimetics, as suitable bio-receptors for the detection of small organic target compounds, here methylene blue. Target immobilisation for Affimer characterisation was achieved using long-chained alkanethiol linkers coupled with oligoethyleneglycol (LCAT-OEG). Using quartz crystal microbalance with dissipation monitoring (QCM-D), we determine the affinity constant, KD , of the methylene blue Affimer to be comparable to that of antibodies. Further, we demonstrate the high selectivity of Affimers for its target in complex matrices, here a limnetic sample. Finally, we demonstrate an Affimer-based competition assay, illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, non-immunogenic compounds

Glycosylation Fosters Interactions between Model Sea Urchin Spicule Matrix Proteins. Implications for Embryonic Spiculogenesis and Biomineralization

The formation of embryonic mineralized skeletal elements (spicules) in the sea urchin requires the participation of proteins, many of which may interact with one another and assist in the creation of an extracellular matrix wherein mineral formation takes place. To probe this, we created a sea urchin spicule recombinant model protein pair system wherein we tested the interactions between two major spicule proteins, SpSM50 and the glycoprotein, SpSM30B/C. Both proteins are strong hydrogelators that manipulate early and later events in mineral formation. We discovered that the anionic glycan moieties of SpSM30B/C are required for interaction with the SpSM50 protein and that these interactions are Ca(II)- independent. In addition, when these proteins form a complex, they create hybrid hydrogel particles that are physically distinct from their individual counterparts. Thus, glycan-mediated interactions play an important role in in vitro spicule protein assembly and most likely within the spicule itself

Organic cultivation and deficit irrigation practices to improve chemical and biological activity of mentha spicata plants

Intensive crop production and irrational use of fertilizers and agrochemicals have ques-tionable effects on the quality of products and the sustainable use of water for agricultural purposes. Organic cultivation and/or deficit irrigation are, among others, well appreciated practices for a sustainable crop production system. In the present study, spearmint plants (Mentha spicata L.) were grown in different cultivation schemes (conventional versus organic cultivation, full versus deficit irrigation), and effects on the plant physiological and biochemical attributes were examined in two harvesting periods. Deficit irrigation decreased plant growth, but increased total phenolics, flavonoids, and antioxidant capacity of the plants at the second harvest. Spearmint nutrient accumulation was affected by the examined cultivation practices; nitrogen was decreased in organic cultivation, potassium and sodium were elevated at full-irrigated plants, while magnesium, phosphorus, and copper levels were higher at the deficit-irrigated plants. However, conventional/full-irrigated plants had increased height and fresh biomass at the first harvest. Essential oil content decreased at the second harvest in organic and/or deficit treated plants. Additionally, deficit irrigation affected plant growth and delayed the formation of carvone from limonene. The essential oils were further evalu-ated with regard to their bioactivity on a major vineyard pest Lobesia botrana. Volatile compounds from all essential oils elicited strong electroantennographic responses on female insects antennae, highlighting the role of carvone, which is the major constituent (~70%) in all the tested essential oils. M. spicata essential oils also exhibited larvicidal activity on L. botrana, suggesting the potential of their incorporation in integrated pest management systems

Selective Synergism Created by Interactive Nacre Framework-Associated Proteins Possessing EGF and vWA Motifs: Implications for Mollusk Shell Formation

In the nacre layer of the <i>Pinctada fucata</i> oyster shell there exists a multimember proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally defined combinatorial model consisting of two recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain (vWA)) and r-n16.3 (containing an EGF-like domain), whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca²⁺-mediated protein–protein interactions (∼4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca²⁺ there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp/Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization

Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation

Production of moth sex pheromones for pest control by yeast fermentation

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations. Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits. Here we demonstrate a biotechnological method for the production of (Z)-hexadec-11-en-1-ol and (Z)-tetradec-9-en-1-ol, using engineered yeast cell factories. These unsaturated fatty alcohols are pheromone components or the immediate precursors of pheromone components of several economically important moth pests. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting yeast-derived pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a conventionally produced synthetic pheromone mixture. We further demonstrated the production of (Z)-tetradec-9-en-1-yl acetate, the main pheromone component of the fall armyworm Spodoptera frugiperda. Taken together our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control via yeast fermentation