Researchers from ETH Zurich have succeeded in introducing large quantities of unnatural amino acids into bacteria, enabling the creation of innovative and highly efficient designer proteins. These can be used as more efficient catalysts or more effective drugs.
In brief
Researchers from ETH Zurich have modified a bacterial transport system so that it can efficiently introduce large quantities of unnatural amino acids into cells, disguised as a kind of Trojan horse.
All organisms manufacture their proteins from the same 20 amino acids. Additionally, unnatural amino acids can be used to produce designer proteins with new functions.
The new system allows the efficient biotechnological mass production of these designer proteins. Applications range from precise therapeutics and more efficient catalysts to improved imaging techniques.
Life uses 20 amino acid building blocks, from which proteins are assembled. Although limiting the number to 20 building blocks allows for a wide variety of protein structures and functions, it also sets clear chemical boundaries. In the lab, however, chemists can theoretically synthesise thousands of artificial amino acids, many of which have completely new properties.
These artificial building blocks can be specifically incorporated into proteins of living cells using biotechnological methods. "Proteins with specifically inserted unnatural amino acids open up many new possibilities both for medical and industrial applications as well as for scientific research," explains Kathrin Lang, Professor of Chemical Biology at ETH Zurich.
Novel therapeutics and powerful enzymes
Artificial amino acids open up new possibilities in all areas of protein-based applications. Thanks to additional chemical groups, therapeutic proteins can function more effectively and in a more targeted manner. Fluorescent components or molecules with atoms such as chlorine or fluorine, which don't normally occur in proteins, can improve imaging techniques in medicine and research. Enzymes with novel catalytic capabilities are becoming possible, and specific cross-linking amino acids can be used to manufacture proteins that work efficiently even in extreme external conditions such as heat or pressure. Moreover, special coupling groups allow attachment of active substances to carrier proteins for reliable transport of drugs to diseased tissue.
Hijacking a bacterial transport system
Until now, the targeted insertion of synthetic amino acids into proteins has been significantly less efficient than producing proteins comprising only the 20 natural amino acids. Applications have therefore generally been limited to small-scale research projects. A significant bottleneck is that often only very small quantities of unnatural amino acids enter the bacteria used for biotechnological production.
Now, Lang's group has developed a solution that allows artificial amino acids to be introduced into bacteria efficiently. This means the "amino acid toolbox" can be feasibly expanded for widespread use in medicine and the biotech industry. To that end, the researchers have hijacked a natural transport system of the bacterium E. coli. This normally serves to transport short protein fragments, known as peptides, into the cell from the surrounding area.
The transport system is made up of two units: a channel in the cell membrane and a shuttle component. The shuttle unit recognises peptides with a length of three or four amino acids and delivers them to the channel, which then funnels them into the cell's interior. Once there, the peptides are broken down into their individual amino acid building blocks. These are then available to the cellular machinery in order to synthesise new proteins. As the system has to work for all natural amino acid combinations, it's not particularly picky. Peptides containing artificial amino acids are also funnelled through - albeit often only in small quantities, if at all.
An evolutionary approach to specific binding
The biochemists from ETH used two tricks to allow the transport system to also import unnatural amino acids in large quantities. Firstly, they packed these amino acids into short, synthetic peptides, in which they were surrounded by natural building blocks. The transporter readily allows this "cargo" to pass - like a molecular Trojan horse.
Secondly, the researchers made targeted modifications to the shuttle component. To do this, they determined the molecular structure of the binding site for the peptides in the shuttle. They then progressively and systematically modified this area in experiments until the binding site was tailored to a specific peptide with artificial amino acids.
To tailor the site in this way, the researchers used methods that emulate biological evolution in high speed. This approach can now be used to tailor the transport system to a wide range of peptides with unnatural amino acids. For example, the team was even able to introduce bulky or negatively charged amino acids that couldn't previously be imported into cells at all.
"The unnatural amino acids are now available in large quantities inside E. coli cells, the most common bacteria used in biotechnology. This allows to efficiently incorporate various artificial building blocks into proteins using genetic code expansion methods," says Tarun Iype, a doctoral student in Lang's group and one of the lead authors of the study.
"In many cases, it's therefore possible to produce designer proteins containing unnatural amino acids just as efficiently as their natural counterparts," adds Maximilian Fottner, Senior Scientist in Lang's group, who was also a lead author of the study. ETH Zurich has applied for a patent on the new method.
Expanding the system to other synthetic molecules
This method currently works in E. coli bacteria. "We're also working to design a comparable system in human cells," says Lang. "This could be used to produce human-like proteins with properties that make them better suited to a wide range of therapeutic applications."
However, the ETH biochemists' plans also extend beyond amino acids, as Lang explains: "We want to develop the system further so that it can also import other molecules that previously couldn't enter cells." These imported molecules could then serve as the source materials for the efficient biotechnological production of complex chemical compounds whose synthesis is currently inefficient or impossible.
Reference
Iype T, Fottner M, Böhm P, Piedrafita C, Möller Y, Groll M, Lang K: Hijacking a bacterial ABC transporter for genetic code expansion. Nature 2025, 647: 1045, doi: external page 10.1038/s41586-025-09576-w
