Biomedical engineers at Duke University have demonstrated a new synthetic approach that turbocharges bacteria into producing more of a specific protein, even proteins that would normally destroy them, such as antibiotics.
The technique directs bacteria to produce synthetic disordered proteins that bunch together to form compartments called biological condensates. When these compartments trap mRNA carrying instructions for specific proteins together with the machinery needed to implement them, they can greatly enhance the rate of protein production.
The technique could be a boon to industries that use bacteria to produce a wide range of products such as pharmaceuticals, industrial chemicals and biofuels.
The results appeared online February 10 in the journal Nature Chemistry.
Biological condensates are useful tools that are already abundant in nature. All cells use condensates to trap together or separate biomolecular machinery to dial its activity up and down. Since the phenomenon was discovered in 2009, their uses and functions have been a topic of vigorous study.
"Condensates are useful for cells to temporarily control gene expression in response to new conditions or challenges because, by quickly controlling gene expression at the protein production level, rather than at the DNA level, they can make changes in which proteins are produced in minutes instead hours or even days." said Daniel Shapiro, a PhD student working in the laboratory of Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke. "But naturally occurring condensates are extremely complicated and difficult to engineer. Our lab is one of the few directing cells to make synthetic versions that can be specifically tailored to fit our purposes."
The Chilkoti Laboratory specializes in elastin-like polypeptides, or ELPs for short. These long, disordered proteins are like globs of noodles that can be tailored to clump together or dissolve apart based on a wide range of variables such as temperature or acidity.
In 2023, the laboratory was the first to show that bacteria could be programmed to make these synthetic disordered proteins and have them form condensates that affect biological machinery.
"That work showed that we, as biomedical engineers, could design new molecular parts from the ground up, convince cells to make them, and assemble these parts inside the cell to make a new machine," Chilkoti said. "It was the beginnings of an emerging field that is now allowing us to reprogram life in new and exciting ways."
That research, among other results, showed that synthetic biological condensates could trap together biomolecular machinery to speed up their work. But it did not seek to specifically instruct the cell which processes to speed up or which proteins to produce.
This new work builds on that base to do exactly that. The researchers instructed bacteria cells to produce ELPs that form condensates and also bind to specific RNA sequences, which copy and carry the blueprints for building proteins from DNA to the rest of the cell. By drawing these RNA sequences together into a dense condensate, the researchers believe it made them more readily available for the cell's protein-making machinery to find and use them.
"Rather than hiding the RNA from the cell's machinery, it seems to bring it all together at a higher concentration into a sort of reaction crucible that increases the rate of protein production," Shapiro said. "You can make a cell express more RNA and make more of its protein, but once the RNA is made, there are very few ways of enhancing the rate at which proteins are translated from it. That's what we did here, which is very exciting."
Moving forward, the researchers are continuing to build out their platform. For example, experiments indicate that if these condensates are designed to be more viscous, they produce fewer proteins. Effects such as this gives the team handles to use to control the rate of production. Shapiro is also working to see how the structure of the mRNA being targeted affects its rate of production.
The research could be useful in at least two large classes of industry. Many biological therapeutics, like antibodies, vaccines and immune proteins, are made in mammalian cells because they require chemical machinery that doesn't exist in bacteria. By using these synthetic condensates, Shapiro sees a route to trapping together the requisite pieces of the puzzle to help bacteria efficiently create these therapeutics. The other possibility is using condensates to sequester the proteins being produced so that they can't harm the host bacterium, which is a common roadblock in efficiently producing antibiotics and other antimicrobial proteins.
This research was supported by the Air Force Office of Scientific Research (FA9550-20-1-0241) and the National Institutes of Health (MIRA R35GM127042).
"Synthetic biomolecular condensates enhance translation from a target mRNA in living cells." Daniel Mark Shapiro, Sonal Deshpande, Seyed Ali Eghtesadi, Miranda Zhong, Cassio Mendes Fontes, David Fiflis, Dahlia Rohm, Junseon Min, Taranpreet Kaur, Joanna Peng, Max Ney, Jonathan Su, Yifan Dai, Aravind Asokan, Charles Gersbach, and Ashutosh Chilkoti. Nature Chemistry, 2025. DOI: 10.1038/s41557-024-01706-7
