Expanding Uses for Bioengineered Bacterial Spores

(Tufts University Newswise) A remarkable quality of bioengineering is the fact that scientists can take biological processes honed by millions of years of evolution and use them to efficiently create drugs, chemicals, and other products to improve our lives. Now Tufts researchers have found new ways to expand the potential for using bacterial spores as catalysts for chemical reactions, biofuel production, or breaking down pollutants.

When some species of bacteria find themselves in an environmentally stressful situation like extreme heat, cold, aridity, loss of nutrients, or even exposure to disinfectants, they can hunker down and form spores—hardened protein-coated spheres protecting a DNA-filled core. The spores remain stable and dormant for years—even centuries—waiting patiently until the right conditions allow them to resurrect into active bacteria once again.

This extraordinary stability has made bacterial spores great candidates for bioengineering. Researchers are designing spores to express drugs, industrial enzymes, and catalysts, and be used as biological sensors, as useful molecules are fused to the spore’s outer coat of proteins. The products fused to the proteins can be stored and distributed without the need for refrigeration, or can be used in applications under extreme conditions, such as high heat or exposure to harsh chemicals.

While promising, the technology has encountered hurdles, including the fact that only 12 of the nearly 50 proteins that coat spores have been explored as potential objects to fuse with new substances.

Now Nik Nair, associate professor of chemical and biological engineering, and his team have expanded the list of fusion candidates to as many as 33 of the proteins that coat bacterial spores, suggesting an approach that might lead to a much wider range of bioengineered products. They describe the work in a paper in the journal JACS Au.

“Spore engineering is still an emerging technology,” said Nair. “Most products are in the development stage and are not ready for widespread commercial application. We are hopeful that expanding the target list for fusion can speed up this process.”

The types of bacterial spore products could include oral vaccine delivery, for example, where spores with antigens on their surface pass through the gastrointestinal tract to stimulate a mucosal immune response. This makes them highly attractive for distribution to remote locations without needing refrigeration and for needle-free vaccination.

The spores can also be engineered to glow due to fluorescence in the presence of specific chemical compounds, making them great candidates for detecting toxins in harsh environments.

By displaying enzymes on their surface, engineered spores can also function as catalysts for chemical reactions, biofuel production, or breaking down pollutants.

As a proof of concept, Nair and his research team fused the outer spore proteins with enzymes that can degrade polyethylene terephthalate (PET), a hard plastic used in many products like water bottles and automotive parts.

To do this, they surveyed their expanded list of spore proteins to find the most stable and effective fusion product. For the PET-dissolving enzyme, the small spore coat assembly protein A (SscA) was the best of 33 proteins they tested for fusion. It yielded fourfold higher activity than any other fusion, breaking down the monomers of PET. On actual PET solid plastic, the enzyme fused to the outer coat protein Y (CotY) yielded higher activity, consistent with the fact that it is more accessible on the surface of the spore’s outer coat.

The researchers also suggest that combining fusion products in spores might be a way to create a multi-step process of breaking down solid plastics and then metabolizing the released chemicals further into environmentally safe forms.

As bioengineered spores make their way toward commercial applications, a key question is whether the spores can be prevented from reactivating as bacteria when released in the environment.

“We have a good understanding of what activates spores to become replicating bacteria again,” said Nair. “If we delete five specific genes, they’ll never germinate and always remain spores. Product safety will be a critical part of introducing spores to widespread applications.”

Nair suggested that SscA, CotY, and other spore proteins could be candidates for more bioengineered products. Continuing development of this technology is being carried out by a new startup company emerging from this research, called Caravel Bio, led by Trevor Nicks, EG23, a former graduate student in Nair’s lab and co-author of the study. Todd Chappell, former postdoctoral researcher in the Nair lab was the first author of this study. READ MORE