In synthetic biology, microbes are often used to produce proteins based on natural models. So far, however, its use has been limited to small proteins. Researchers have now succeeded in overcoming this hurdle and microbially producing the very large muscle protein titin. Spun fibers of this molecule result in a particularly stable, tear-resistant and flexible material that can also be sustainably produced and biodegraded.
Some of the most extraordinary and high-performance materials can be found in nature – from extremely stable spider threads to underwater shell silk to the super-rubber resilin, which is found in the ankle bones of fleas, for example. Previous attempts to technically replicate such materials did not come close to the original materials and also lagged behind the natural models in terms of environmental friendliness: “Artificially produced high-performance polymers are usually not biodegradable and are obtained from crude oil through energy-intensive processes with toxic solvents and by-products “, Writes a research team led by Christopher Bowen from Washington University in St. Louis.
Microbes as protein producers
Bowen and his colleagues have instead taken a different approach to produce a new high-performance material: They have found a method for genetically modified microbes to produce very large proteins true to the original. “Many small molecules are already produced sustainably using microbes,” the researchers write. “A direct microbial synthesis of high-performance polymers, on the other hand, is a major challenge.”
This is because most of nature’s super materials are made up of very large proteins. “However, these are extremely difficult to produce in microbes because the efficiency of such large molecules is low and the appropriately modified microbes are genetically unstable,” the researchers explain. To get around this problem, they decided on a trick. They only provided the microbes with small sections of the large total protein and added places where the fragments can be joined together. In this way they succeeded in producing the muscle protein titin – the largest known human protein.
Outstanding resilience
“In muscle tissue, titin gives the muscle a combination of strength, cushioning ability and rapid mechanical recovery,” the researchers explain. In order to achieve similar properties with the microbially produced titin, they stretch the proteins into fibers. To do this, they first destroyed the three-dimensional structure of the proteins with a denaturing solution. The proteins unfolded in this way were pressed into water through a fine-meshed needle so that the individual titin molecules linked to form fibers while they were being refolded. “In fact, uniform cylindrical fibers formed,” the researchers say.
With a diameter of ten micrometers, these fibers are ten times thinner than a human hair. According to the researchers, around 250 meters of titin fibers can be obtained from a one-liter microbial culture. Next, the researchers tested whether the fibers made with microbes actually had the desired properties of natural muscle fibers. The result: “Both the strength and the resilience of these fibers far exceed the values measured for muscle fibers and individual myofibrils,” the researchers say. “In addition, these resilience values even exceed those of many of the toughest synthetic and natural materials.” The microbially produced titin is even more stable than Kevlar.
Versatile application possibilities
Molecular analyzes confirmed that this extraordinary resilience is actually due to the structure of particularly large proteins. “This underscores the value of the new strategy for making large polymers,” write Bowen and his colleagues. According to the researchers, the new technology can also be used to produce other large proteins. “The nice thing about this system is that it really is a platform that can be used anywhere,” says Bowen’s colleague Cameron Sargent. “We can take proteins from different natural contexts, then put them in this platform for polymerization and make larger, longer proteins for different material applications with greater sustainability.”
From the researchers’ point of view, the artificial titin has numerous potential applications. Similar to Kevlar, it could be used for bulletproof vests, for example, but also for biomedical purposes. Since it is almost identical to the proteins found in muscle tissue, it is likely to be biocompatible and could therefore be used for surgical sutures, for example.
Source: Christopher Bowen (Washington University in St. Louis, USA) et al., Nature Comminications, doi: 10.1038 / s41467-021-25360-6