Biology subjects

Mangan, N.

Publications and source records attributed to Mangan, N..

4 recordsLinked to original sources

bioRxiv2025

Evaluation of Bacterial Microcompartment Cofactor Recycling and Permeability with a Model Guided In Vitro Assay

Biomanufacturing is a promising strategy for sustainable chemical production. However, challenges such as cofactor competition and low pathway flux prevent competitive titers. Some bacteria address these challenges by encapsulating metabolic pathways in bacterial microcompartments (MCPs), many of which contain dedicated cofactor recycling enzymes. We sought to determine how pathway cofactor recycling and intermediate sequestration in MCPs benefit pathway performance using an in vitro assay and kinetic model of the 1,2-propanediol utilization (Pdu) system. Guided by model simulations, we performed experimental design to characterize permeability, a key and difficult-to-measure property of MCPs. Using our model and measurements of metabolite concentrations over time, we estimate MCP permeability values in the range of 10-5 cm/s. We also demonstrated that NAD+/NADH recycling in the Pdu MCP benefits increased pathway flux. This study integrates experiments and systems modeling to advance our understanding of why pathways are encapsulated and to inform bioengineering applications.

biochemistry↗

bioRxiv2024

Encapsulation of Select Violacein Pathway Enzymes in the 1,2-Propanediol Utilization Bacterial Microcompartment to Divert Pathway Flux

A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells encapsulating a metabolic pathway - for this purpose. We aim to determine to what degree this hypothesized benefit is conferred to encapsulated pathways. To this end, we used bacterial microcompartments to encapsulate select enzymes from the violacein pathway, which is composed of five enzymes that produce violacein as the main product and deoxyviolacein as a side product. Importantly, we studied the pathway in a cell-free context, allowing us to hold constant the concentration of unencapsulated and encapsulated enzymes and increase our control over reaction conditions. The VioE enzyme is a branch point in that it makes the precursor for both violacein and deoxyviolacein, the VioC enzyme is required for production of deoxyviolacein, and the VioD enzyme is required for violacein production. When we encapsulated VioE and VioC and left VioD unencapsulated, the product profile shifted toward deoxyviolacein and away from violacein compared to when VioC and VioD were both unencapsulated. This work provides the first fully quantitative evidence that microcompartment-based encapsulation can be used to divert pathway flux to the encapsulated pathway. It provides insight into why certain pathways are encapsulated natively and could be leveraged for metabolic engineering applications.

synthetic biology↗

bioRxiv2024

Chromatin endogenous cleavage provides a global view of RNA polymerase II transcription kinetics

Chromatin immunoprecipitation (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo. The occupancy of transcription factors (TFs) from ChIP-seq agrees well with an alternative method, chromatin endogenous cleavage (ChEC-seq2). However, ChIP-seq and ChEC-seq2 reveal strikingly different patterns of enrichment of yeast RNA polymerase II. We hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.

molecular biology↗

bioRxiv2017

Spatially organizing biochemistry: choosing a strategy to translate synthetic biology to the factory

Natural biochemical systems are ubiquitously organized both in space and time. Engineering the spatial organization of biochemistry has emerged as a key theme of synthetic biology, with numerous technologies promising improved biosynthetic pathway performance. One strategy, however, may produce disparate results for different biosynthetic pathways. We propose a spatially resolved kinetic model to explore this fundamental design choice in systems and synthetic biology. We predict that two example biosynthetic pathways have distinct optimal organization strategies that vary based on pathway-dependent and cell-extrinsic factors. Moreover, we outline this design space in general as a function of kinetic and biophysical properties, as well as culture conditions. Our results suggest that organizing biosynthesis has the potential to substantially improve performance, but that choosing the appropriate strategy is key. The flexible mathematical framework we propose can be adapted to diverse biosynthetic pathways, and lays a foundation to rationally choose organization strategies for biosynthesis.

synthetic biology↗