Biology subjects

Tepper, K.

Publications and source records attributed to Tepper, K..

7 recordsLinked to original sources

bioRxiv2025

Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals

TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster, to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli, and NsfI from Enterobacter cloacae.

synthetic biology↗

bioRxiv2025

Distinct Membrane Binding Properties of the Two Non-visual Arrestins

Membrane interactions play a crucial role in regulating arrestin activation and its binding to phosphorylated G protein-coupled receptors (GPCRs). Here, we utilize in vitro biophysical approaches and cell-based fluorescence intensity fluctuation analysis to systematically compare the membrane-binding properties of the two highly conserved arrestin subtypes, arrestin-2 and arrestin-3, under basal and stimulated conditions. Our findings reveal that arrestin-2 selectively engages the PI(4,5)P2-containing nanodiscs via its C-edge, whereas arrestin-3 primarily utilizes its finger loop to interact with negatively charged lipids. Notably, while the lipid bilayer alone does not activate arrestin, it synergistically enhances arrestin-2/3 activation in conjunction with a phosphorylated GPCR C-tail. Additionally, the spacing between receptor phosphorylation sites and the lipid bilayer modulates arrestin-membrane assembly. Live cell tracking further demonstrates that arrestin-2 and arrestin-3 exhibit distinct plasma membrane dissociation dynamics. These findings provide novel insights into the mechanisms governing arrestin activation and its functional interplay with membranes.

biochemistry↗

bioRxiv2024

Biomanufacturing of a functional microbial phytase in an insect host.

BackgroundInsects, such as Black Soldier Flies (Hermetia illucens), are increasingly used as sustainable animal feed ingredients that can be reared on plentiful organic substrates such as agricultural residues and pre-consumer food waste. Genetically engineering insects to heterologously express feed additive enzymes has the potential to generate more value from organic waste, while improving livestock health and productivity. Phytases are widely used feed additive enzymes that hydrolyse the phosphate groups from the myo-inositol backbone of phytic acid, a phosphate rich antinutrient compound that monogastric animals cannot efficiently digest. Dietary phytase supplementation improves absorption of phosphorous, proteins, and cationic nutrients, while mitigating the negative environmental effects of phytic acid rich excreta. ResultsWe evaluated the potential of using insects to biomanufacture microbial feed additive enzymes by engineering the model insect, Drosophila melanogaster, to express phytases. One histidine acid phytase, three beta propellor phytases, three purple acid phosphatases, and one PTP-like phytase were selected for screening in D. melanogaster. Transgenic flies expressing the AppA histidine acid phytase from E. coli had 27.82 FTU/g of phytase activity, which exceeds the 0.5-1.0 FTU/g required in animal feed. Maximum activity from AppA phytase expressed by D. melanogaster was observed at pH 5 and 55 {degrees}C, however, more than 50% of phytase activity was present at 25 {degrees}C and pH 2. Here we demonstrate that insects may be suitable hosts for the heterologous expression of a microbial phytase enzyme with applications for improving animal feed nutrition and organic waste valorisation.

synthetic biology↗

bioRxiv2024

Rapid generation and screening of transgenic black soldier fly (Hermetia illucens)

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

synthetic biology↗

bioRxiv2023

Methylmercury demethylation and volatilization by animals expressing microbial enzymes.

Methylmercury is a highly toxic pollutant that accumulates in food webs where it is inaccessible to current remediation technologies. We demonstrate that animals can be engineered to express the microbial enzymes, organomercurial lyase (MerB) and mercuric reductase (MerA), to bioremediate methylmercury. MerA and MerB from Escherichia coli were functional in invertebrate (Drosophila melanogaster) and vertebrate (Danio rerio) model systems and converted methylmercury into volatile Hg0. The engineered animals tolerated higher exposures to methylmercury and accumulated less than half as much mercury relative to their wild-type counterparts. The outcomes of this research could be applied to reduce mercury contamination in farmed and recreationally caught fish, for species conservation, and to restore value to organic wastes contaminated with mercury.

synthetic biology↗

bioRxiv2021

Bioremediation of industrial pollutants by insects expressing a fungal laccase

Inadequate management of household and industrial wastes pose major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often require highly pre-processed inputs and sophisticated infrastructure, is not feasible for many waste streams. Concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder made from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.

synthetic biology↗

bioRxiv2020

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD, with pro-aggregant mutation, e.g., {Delta}K280 or P301L, [~]13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, [~]28 kD). Since the diameter of the reporter GFP ([~]3 nm) is [~]6.5 times larger than the {beta}-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N- or C-terminally) TauRD and TauFL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD{Delta}K-GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from TauRD{Delta}K). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of TauRD{Delta}K fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-{beta} structure. By contrast, the elongated particles formed by TauRD{Delta}K-GFP have MPL values around [~]2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.

neuroscience↗