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Innovative Method Boosts Protein Production in Bacteria

Biomedical engineers from Duke University have pioneered a novel synthetic method that significantly enhances the ability of bacteria to produce specific proteins, including those that might otherwise be lethal to them, such as antibiotics.

The process involves guiding bacteria to synthesize synthetic disordered proteins that aggregate to create structures known as biological condensates. These condensates serve as compartments that can efficiently recruit mRNA—the molecule that transmits gene instructions—along with the cellular machinery required for protein synthesis, thereby accelerating protein production rates.

This advancement holds considerable promise for industries relying on bacterial systems to generate various products including pharmaceuticals, industrial chemicals, and biofuels.

The findings were published online on February 10 in the journal Nature Chemistry.

Biological condensates function as essential components in nature, utilized by all living cells to coordinate or segregate biochemical activities. Since their discovery in 2009, there has been intense research focused on their roles and potential applications in cellular processes.

“Condensates enable cells to quickly adjust gene expression in response to environmental changes. This rapid modulation allows cells to alter protein production in a matter of minutes rather than the longer timescales associated with DNA-level changes,” explained Daniel Shapiro, a PhD student in Ashutosh Chilkoti’s laboratory. Chilkoti is the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke. “However, naturally occurring condensates are incredibly complex and difficult to engineer. Our lab is one of the few that is successfully directing cells to create synthetic versions customized for specific tasks.”

The Chilkoti Laboratory specializes in the study of elastin-like polypeptides (ELPs), which are long, disordered proteins able to cluster or disperse based on various factors, including temperature and acidity.

In 2023, the laboratory was the first to demonstrate that bacteria could be engineered to produce these synthetic proteins and form condensates that had a notable impact on biological processes.

“Our previous research showed that we could design new molecular components from scratch, instruct cells to produce them, and assemble these elements inside the cell to create novel biological machines,” Chilkoti stated. “This research is at the forefront of an emerging field that is paving the way for innovative ways to reprogram biological systems.”

While earlier studies illustrated that synthetic biological condensates could enhance the activity of biomolecular machinery, they did not specifically dictate to the cells which processes to optimize or which proteins to generate.

This latest work builds upon that foundation by specifically instructing bacterial cells to synthesize ELPs that not only form condensates but also bind to particular RNA sequences, which are essential for transporting the genetic templates needed for protein synthesis. The researchers believe that by gathering these RNA molecules into a concentrated condensate, they enhance their accessibility to the cellular machinery responsible for translating them into proteins.

“Instead of sequestering RNA from the cell’s machinery, we concentrate it within a sort of reaction vessel that promotes a higher rate of protein production,” Shapiro noted. “While creating more RNA can increase protein levels, the challenge has been enhancing the translation process once RNA is formed. This is where our approach has made a significant impact.”

The research team is keen to expand on their platform. Preliminary results suggest that increasing the viscosity of these condensates can lead to a reduction in protein output. Understanding and manipulating such variables will provide the researchers with tools to better control production rates. Additionally, Shapiro is investigating how the mRNA structure influences its translation efficiency.

This innovative research holds potential applications in at least two key sectors. Many biological therapeutics, including antibodies, vaccines, and immune modulators, are typically produced in mammalian cells due to the need for specific biochemical machinery not found in bacteria. The introduction of synthetic condensates may allow for the assembly of necessary components to enable bacteria to produce these therapeutics more effectively. Furthermore, condensates could theoretically help contain the potentially harmful proteins produced, an issue often encountered in the efficient production of antibiotics and antimicrobial peptides.

This project received funding from the Air Force Office of Scientific Research (FA9550-20-1-0241) and the National Institutes of Health (MIRA R35GM127042).

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