Biology subjects

Polizzi, K. M.

Publications and source records attributed to Polizzi, K. M..

9 recordsLinked to original sources

bioRxiv2026

Iterative Bump-and-hole engineering creates a bioorthogonal reporter for N-acetylglucosaminyltransferase I

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

biochemistry↗

bioRxiv2022

Resource-aware construct design in mammalian cells

Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report quantification of the resource load imposed by different mammalian genetic components and identify construct designs with increased performance and reduced resource footprint. We use these to generate improved synthetic circuits and optimise the co-expression of co-transfected cassettes, shedding light on how this can be useful for bioproduction and biotherapeutic applications. This work provides the scientific community with a framework to take resource demand into consideration when designing mammalian constructs to achieve robust and optimised gene expression.

synthetic biology↗

bioRxiv2022

Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: a safe format for engineered living materials

In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.

bioengineering↗

bioRxiv2022

Immobilised enzyme cascade for targeted glycosylation

Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro. This novel proof-of-concept system is enabled by immobilised enzymes produced with a "one-step immobilisation/purification" method to express, biotinylate in vivo and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered Pichia pastoris, yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised {beta}-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 - 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures for functional and clinical evaluation. The use of immobilised enzymes enables the economical modification of cell-based material supporting applications at a large industrial scale.

bioengineering↗

bioRxiv2021

A curcumin direct protein (DiPro) biosensor for cell-free prototyping

In synthetic biology, biosensors are routinely coupled to a gene expression cascade for detecting small molecules and physical signals. We posit that an alternative direct protein (DiPro) biosensor mechanism, could provide a new opportunity for rapid detection of specific chemicals. Herein, we reveal a fluorescent curcumin DiPro biosensor, based on the Escherichia coli double bond reductase (EcCurA) as a detection system. We characterise the EcCurA DiPro biosensor and propose enhanced curcumin fluorescence is generated through {pi}-{pi} stacking between protein and ligand. Using a cell-free synthetic biology approach, we use the EcCurA DiPro biosensor to fine tune 10 reaction parameters (cofactor, substrate, and enzyme levels) for cell-free biosynthesis, assisted through acoustic liquid handling robotics. Overall, we increase EcCurA-curcumin fluorescence by 80-fold. We speculate that a generic DiPro biosensor fluorescence mechanism can be further exploited for a wider range of chemicals that share intrinsic fluorescence and have a suitable binding protein.

synthetic biology↗

bioRxiv2021

CHOmpact: a reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability

Metabolic modelling has emerged as a key tool for the characterisation of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimise the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are, therefore, difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy multi-objective optimisation and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and, with further development, will serve as a platform to develop dynamic metabolic models for the control and optimisation of biopharmaceutical cell culture processes.

systems biology↗

bioRxiv2017

Tools for engineering coordinated system behaviour in synthetic microbial consortia

Advancing synthetic biology to the multicellular level requires the development of multiple orthogonal cell-to-cell communication channels to propagate information with minimal signal interference. The development of quorum sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on reducing signal leakage between systems of cognate AHL/transcription factor pairs. However, the use of non-cognate signals as a design feature has received limited attention so far. Here, we demonstrate the largest library of AHL-receiver devices constructed to date with all cognate and non-cognate chemical signal interactions quantified and we develop a software tool that allows automated selection of orthogonal chemical channels. We use this approach to identify up to four orthogonal channels in silico and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels will facilitate the design of synthetic microbial consortia for novel applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation, and spatial organisation.

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↗

bioRxiv2017

Computational Re-Design of Synthetic Genetic Oscillators for Independent Amplitude and Frequency Modulation

Engineering robust and tuneable genetic clocks is a topic of current interest in Systems and Synthetic Biology with wide applications in biotechnology. Synthetic genetic oscillators share a common structure based on a negative feedback loop with a time delay, and generally display only limited tuneability. Recently, the dual-feedback oscillator was demonstrated to be robust and tuneable, to some extent, by the use of chemical inducers. Yet no engineered genetic oscillator currently allows for the independent modulation of amplitude and period. In this work, we demonstrate computationally how recent advances in tuneable synthetic degradation can be used to decouple the frequency and amplitude modulation in synthetic genetic oscillators. We show how the range of tuneability can be increased by connecting additional input dials, e.g. orthogonal transcription factors that respond to chemical, temperature or even light signals. Modelling and numerical simulations predict that our proposed re-designs enable amplitude tuning without period modulation, coupled modulation of both period and amplitude, or period adjustment with near-constant amplitude. We illustrate our work through computational re-designs of both the dual-feedback oscillator and the repressilator, and show that the repressilator is more flexible and can allow for independent amplitude and near-independent period modulation.

synthetic biology↗