How Phase Separation Affects Biomolecular Functions

Enhancing Enzyme Stability and Metabolic Yield in Nicotiana benthamiana Using Synthetic Biomolecular Condensates Recent work published in Plant Biotechnology Journal by Lindström Battle et al. (2025) demonstrates that synthetic biomolecular condensates can be used to improve the performance of engineered metabolic pathways in plants.   𝐁𝐚𝐜𝐤𝐠𝐫𝐨𝐮𝐧𝐝: 𝐂𝐡𝐚𝐥𝐥𝐞𝐧𝐠𝐞𝐬 𝐢𝐧 𝐏𝐥𝐚𝐧𝐭-𝐁𝐚𝐬𝐞𝐝 𝐌𝐞𝐭𝐚𝐛𝐨𝐥𝐢𝐜 𝐄𝐧𝐠𝐢𝐧𝐞𝐞𝐫𝐢𝐧𝐠 N. benthamiana is widely used for transient expression of recombinant proteins and metabolites. However, two common limitations affect its utility as a biofactory: - Proteolytic degradation of heterologous enzymes, reducing their accumulation+activity. - Toxicity or instability of metabolic intermediates and final products within the host cytoplasm. These issues reduce yield and limit the feasibility of complex or multi-step pathways.   𝐖𝐡𝐚𝐭 𝐚𝐫𝐞 𝐁𝐢𝐨𝐦𝐨𝐥𝐞𝐜𝐮𝐥𝐚𝐫 𝐂𝐨𝐧𝐝𝐞𝐧𝐬𝐚𝐭𝐞𝐬 ? Cells can organize proteins and metabolites into biomolecular condensates, which are non-membrane-bound compartments formed by phase separation. These structures concentrate specific molecules and have been shown to influence biochemical activity and stability. A useful analogy: Similar to how oil droplets form in water, biomolecular condensates emerge when certain proteins or RNAs cluster into a dense phase, surrounded by a more dilute background. This organization is driven by multivalent, low-affinity interactions. Synthetic condensates have previously been applied in yeast and mammalian systems, but their utility in plant hosts has not been extensively studied.   𝐎𝐯𝐞𝐫𝐯𝐢𝐞𝐰 𝐚𝐧𝐝 𝐊𝐞𝐲 𝐅𝐢𝐧𝐝𝐢𝐧𝐠𝐬 Synthetic condensates were engineered in N. benthamiana leaves using a scaffold built from RGG domains known to promote phase separation. These were paired with SYNZIP domains to recruit enzymes into the condensates. They tested two metabolic pathways: - Citramalate biosynthesis (single enzyme: MdCMS) - Poly-3-hydroxybutyrate (PHB) biosynthesis (three enzymes) Main results: Enzyme recruitment to condensates was successful and did not impair enzymatic activity. Enzyme accumulation increased up to 7.4-fold when targeted to condensates, compared to untargeted controls. PHB granules were found to localize within condensates, suggesting a sequestration effect that may reduce cytotoxicity or degradation. These results indicate that condensates may enhance metabolic output through a combination of enzyme stabilization, increased local concentration, and potential metabolite channeling or protection.   Synthetic condensates offer a promising alternative to membrane-bound organelles for plant metabolic engineering, with advantages such as: - Easier targeting - Compatibility with cytosolic pathways - Reduced vulnerability to degradation #SyntheticBiology #MetabolicEngineering #PlantBiotechnology #BiomolecularCondensates #NicotianaBenthamiana   Read here: https://lnkd.in/e3hcrC5n

GOLDILOCK UBIQUITINATION HOLDS THE KEY TO OPTIMAL REGENERATIVE CAPACITY In a lecture I am delivering next month, one topic I will be discussing is protein ubiquitination. Ubiquitination is a tightly regulated, highly specific, and ATP-dependent biological process carried out by a complex cascade of enzymes. Ubiquitination is an essential player in protein homeostasis, serving to remove unwanted or damaged proteins rapidly. This process and heat and cold shock proteins significantly affect our health.  This article describes research exploring how protein quality control systems regulate the formation of condensates within cells. Condensates are liquid droplets that form through phase separation, allowing the cell to quickly assemble specific components in response to stressors. The researchers established a "Goldilocks" framework showing an optimal spacing between ubiquitin units in polyubiquitin chains that maximizes condensate formation with the protein UBQLN2. Too much spacing or too little impairs condensate formation. This is significant for regenerative medicine because proper protein quality control and condensate dynamics are essential for healthy cellular function and stress response. Stem and progenitor cells needed for regenerative therapies rely extensively on precisely regulating proteins and condensates. Dysfunction of these systems can lead to neurodegenerative diseases like ALS. By elucidating the molecular details of how condensate assembly/disassembly is modulated, this research provides principles to optimize stem cell cultures and improve cell survival after transplantation.  The "Goldilocks" concept that an optimal intermediate state maximizes condensate formation likely applies to phase separation regulation in many contexts. Manipulating phase separation could allow precise control over the localization of specific factors that need to be concentrated together for differentiation or other stem cell behaviors. This study advances our understanding of an essential cellular process and regulatory mechanism influencing approaches to enhance regenerative therapies. This modulatory ability means ubiquitination allows precise control over protein phase separation dynamics and allows cells to regulate the assembly of liquid compartments and condensates tightly. The central new insight is that polyubiquitin chain length, spacing, linkage type, and abundance can critically influence phase separation - highlighting new regulatory roles for ubiquitination in controlling protein condensates via phase separation. JP https://lnkd.in/enMCBEmA

Phase separation and autophagy A research team has now discovered the conditions necessary for autophagy to start. They were also able to artificially create these conditions and thus trigger the degradation of otherwise non-degradable molecules in yeast cells. Targeting autophagy in this way is a promising approach for promoting the degradation of aggregates that can otherwise form plaques in neurodegenerative diseases such as Alzheimer‘s, as well as to improve the efficacy of cancer treatments. In order for the degradation of cellular components through autophagy to occur, they must first be recognised as waste. This is done by receptor and other adapter molecules. However, it was previously unknown how exactly these molecules trigger the subsequent steps. “We have now been able to show that the receptors must bind weakly to the material to be disposed of for autophagy to start,” explains the author. “If they bind too strongly, the process is not triggered.” What initially sounds counterintuitive could be explained by the researchers with the help of computer simulations and experiments on living yeast cells and human cells in cell culture: the weak binding causes the receptors to remain mobile and form random clusters. “When the point of critical concentration has been reached, phase separation occurs: the adapter molecules come together and form a droplet, similar to oil in water,” explains another author. “Such a liquid accumulation has different physical properties than the individual molecules serving as a flexible platform for all other molecules involved in autophagy.” To test their hypothesis, the researchers introduced virus particles into yeast cells that the cells are normally unable to break down. By modifying the virus particles so that autophagy receptors could weakly bind to them, the researchers were able to trigger the degradation of the viral protein. However, if they modified the surface so that the receptors bound strongly to it, no degradation took place. #ScienceMission #ScienceMission https://lnkd.in/gzV8s7n2