Biology subjects

Walls, M. T.

Publications and source records attributed to Walls, M. T..

5 recordsLinked to original sources

bioRxiv2026

Enhanced GPP synthesis by Erg20p-peptide fusions and biomolecular condensates boosts monoterpene production in yeast

Monoterpenes are a diverse class of natural products with broad industrial and pharmaceutical applications. While there is great interest in transitioning their production from chemical synthesis and natural source extraction to yeast bioprocesses, this approach remains limited by the dual functionality of the endogenous farnesyl diphosphate synthase Erg20p, which produces the monoterpene precursor geranyl diphosphate (GPP) but favors its subsequent conversion to farnesyl diphosphate (FPP). To address this limitation, we recruited Erg20p and monoterpene synthases into synthetic membraneless organelles, improving production. In doing so, we found that short C-terminal peptide fusions used for recruitment also significantly enhance GPP synthase activity relative to FPP synthase activity. The combined effects of metabolic spatial organization and GPP synthase activity enhancement significantly boost production of different monoterpenes, including geraniol titers exceeding 4 g/L. The strategies presented here can be readily integrated with other traditional metabolic engineering approaches to build yeast strains with high levels of monoterpene production.

bioengineering↗

bioRxiv2026

Spatial organization of enzymes and cofactors in synthetic membrane-associated condensates (sMACs)

Cells spatially organize metabolic enzymes to optimize flux through complex biochemical networks. Here, we engineered synthetic Membrane-Associated Condensates (sMACs), artificial assemblies that form on intracellular membranes through liquid-liquid phase separation, to couple metabolic compartmentalization with cofactor partitioning. Using intrinsically disordered regions (IDRs) characterized by simulations and validated by experiments, we identified an IDR sequence, Dbp1n, that selectively enrich NADH and NADPH, establishing distinct local redox microenvironments. At the endoplasmic reticulum (ER) membrane, NADPH-enriched sMACs enhanced cytochrome P450 activity by improving electron transfer and supporting in situ regeneration. At the plasma membrane, NADH-enriched sMACs provided scaffolds for a xylose uptake and assimilation pathway that exploits localized NADH/NAD recycling to improve flux. These results collectively demonstrated that condensate composition and membrane context can be engineered to enhance metabolic functions. sMACs thus provide a modular framework for creating programmable, membrane-associated microreactors, enabling new strategies for metabolic engineering and offering insights into how phase separation can be exploited in living cells.

synthetic biology↗

bioRxiv2023

A Modular Design for Synthetic Membraneless Organelles Enables Compositional and Functional Control

Living cells organize a wide variety of processes through compartmentalization into membraneless organelles, known as biomolecular condensates. Given their ubiquitous presence across a wide spectrum of different organisms and cell types, biomolecular condensates are increasingly considered to offer great potential for biotechnological applications. However, native condensates remain difficult to harness for engineering applications, both due to their intertwined mechanisms of assembly and compositional control, and potential disruptions to native cellular processes. Here, we demonstrate a modular framework for the formation of synthetic condensates designed to decouple cluster formation and protein recruitment. Synthetic condensates are built through constitutive oligomerization of intrinsically-disordered regions (IDRs), which drive the formation of condensates whose composition can be independently defined through fused interaction domains. The composition of the proteins driven to partition into the condensate can be quantitatively described using a binding equilibrium model, demonstrating predictive control of how component expression levels and interaction affinity determine the degree of protein recruitment. Finally, the engineered system is utilized to regulate protein interactions and metabolic flux by harnessing the systems compositional tunability.

synthetic biology↗

bioRxiv2023

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility

Endogenous biomolecular condensates, comprised of a multitude of proteins and RNAs, can organize into multiphasic structures, with compositionally-distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here, we utilize in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein region (IDR) results in enhanced immiscibility and multiphase formation. Interestingly, we found that oligomerization tunes the miscibility of IDRs in an asymmetric manner, with the effect being more pronounced when the IDR exhibiting stronger homotypic IDR interactions is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism which cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

biophysics↗

bioRxiv2018

Mapping local and global liquid-liquid phase behavior in living cells using light-activated multivalent seeds

Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce a biomimetic optogenetic system, \"Corelets\", and utilize its rapid and quantitative tunability to map the first full intracellular phase diagrams, which dictate whether phase separation occurs, and if so by nucleation and growth or spinodal decomposition. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

biophysics↗