Biology subjects

Freemont, P. S.

Publications and source records attributed to Freemont, P. S..

8 recordsLinked to original sources

bioRxiv2025

Hollow-fibre biomanufacturing and cell-free engineering of HEK293 extracellular vesicles

Extracellular vesicles (EVs) are lipid-delineated nanoparticles that are produced by most cell types. EVs contain complex molecular cargoes that can have useful therapeutic or vaccine immunological effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro, that can co-localise and integrate with exogenously added EVs. To advance this type of cell-free EV engineering we manufactured, isolated and characterised HEK293 cell EVs. These EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. In our most optimal conditions, up to 4.83 x1011 /ml of HEK293 EVs were successfully cell-free engineered with a fusion membrane protein incorporating CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3) and monomeric green lantern (mGL). Finally, we also demonstrated that nano flow cytometry is a powerful tool for assessing cell-free EV engineering efficiency. In the future cell-free EV engineering could help accelerate future EV discoveries and the development of EV translational applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/677537v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@2f6b33org.highwire.dtl.DTLVardef@1270337org.highwire.dtl.DTLVardef@442c2aorg.highwire.dtl.DTLVardef@1afabcc_HPS_FORMAT_FIGEXP M_FIG C_FIG Technology readinessCell-free systems are a well-established part of the engineering biology toolkit. Indeed, several applications are already at TRL9 stage, including commercially available cell-free protein synthesis kits, drug screening assays, field-tested medical biosensors, as well as therapeutic antibody manufacturing that has now reached cell-free reaction volume scales of up to 4500 L. However, cell-free extracellular vesicle (EV) engineering is currently at TRL3 / 4 stage. Whilst our study helps to further advance cell-free EV engineering, key challenges remain. To accelerate technology readiness, improvements in scalable EV isolation methods, increased eukaryotic cell-free protein synthesis yields, and assay automation will likely speed up cell-free EV engineering workflows. Furthermore, artificial intelligence-guided design workflows could be used to rapidly create community accessible libraries of EV membrane protein scaffolds, therapeutic, cell targeting and other modular elements for use in EV engineering studies. Finally, the development of EV potency assays for functional assessment of cell-free engineered EVs are needed to progress prototyped designs towards clinical translation. If these challenges are addressed, we envision that cell-free EV engineering will become an important approach in EV biological research and clinical applications. HighlightsO_LICell-free extracellular vesicle (EV) engineering could be utilised to rapidly prototype and test novel biotechnological, vaccine, or therapeutic EVs for foundational or translational applications. C_LIO_LIOur data highlight some of the impacts that hollow fibre-based cell culture, EV isolation methods and different cell-free engineering approaches have on cell-free EV engineering workflows. C_LIO_LIWe demonstrate several characterisation assays and technologies, including nanoflow cytometry, that can be used to assess cell-free EV engineering efficiency. C_LI

synthetic biology↗

bioRxiv2023

On-grid purification of electron microscopy samples via a 3D-printed flow-cell

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

biophysics↗

bioRxiv2022

Simple, low-cost production of DNA MS2 virus-like particles as molecular diagnostic controls

Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA or in vitro transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce MS2 virus-like particles, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced virus-like particles were packaged with Hepatitis B Virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory.

synthetic biology↗

bioRxiv2021

Complex structure of the Bacillus subtilis CdaA c-di-AMP cyclase domain and the phosphoglucomutase GlmM

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signalling molecule, which plays a key role in osmotic regulation in bacteria. Cellular c-di-AMP levels are tightly regulated, as both high and low levels have a negative impact on bacterial growth. Here, we investigated how the activity of the main Bacillus subtilis c-di-AMP cyclase CdaA is regulated by the phosphoglucomutase GlmM. c-di-AMP is produced from two molecules of ATP by proteins containing a deadenylate cyclase (DAC) domain. CdaA is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. Here we show, using the soluble catalytic B. subtilis CdaACD domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM proteins, both of which form dimers in the structures, and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus providing molecular details on how GlmM acts as cyclase inhibitor. The function of a key amino acid residue in CdaACD in complex formation was confirmed by mutagenesis analysis. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed inhibition mechanism of CdaA by GlmM is conserved among Firmicutes.

microbiology↗

bioRxiv2020

Bacillus subtilis YngB is a UTP-glucose-1-phosphate uridylyltransferase contributing to wall teichoic acid modification and glycolipid formation during anaerobic growth

UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When wild-type and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen limited growth condition.

microbiology↗

bioRxiv2020

AL-PHA beads: bioplastic-based protease biosensors for global health applications

Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads - a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads - resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.

synthetic biology↗

bioRxiv2017

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

The polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3M (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising polyhydroxyalkanoates production.

synthetic biology↗

bioRxiv2017

A semi-synthetic strategy for derivatization of the violacein natural product scaffold

Natural products and their analogues are often challenging to synthesise due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, 6 halogenated analogues of violacein or deoxyviolacein were generated, demonstrating promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via Suzuki-Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein, we demonstrate a flexible and rapid approach to access diverse chemical space that can be applied to a wide range of natural product scaffolds.

synthetic biology↗