Scientists are becoming increasingly aware of how the human microbiome, or the collection of microbes the live on and inside of us, has a major connection to health and human physiology. Microbial engineering, which changes the structure of the microbiome through methods such as probiotics, antibiotics, and microbe transplants, has been found to be a useful strategy for improving human health, but the mechanisms underlying this improvement are still unclear and difficult to test. However, a team of researchers hopes that their new study, published in Microbiology Spectrum, will provide a platform that will make mechanistic studies on the microbiome more feasible.

The study was conducted by the labs of Yong-Su Jin (BSD/MME/CABBI), a professor of bioengineering, and Michael Miller (MME co-leader/IGOH), a professor of food microbiology, based at University of Illinois Urbana-Champaign, along with first author Jungyeon Kim, a former postdoctoral researcher in Jin’s lab and now assistant professor at Seoul National University in South Korea. The researchers utilize a genetically engineered strain of Saccharomyces boulardii, a species of yeast, as their delivery vehicle, or ‘chassis,’ to deliver bioactive proteins into the gut. The yeast is commonly used as a probiotic, and the researchers say that it’s not only easy to genetically engineer, it also moves quickly through the gut, unlike other options for vehicles.

“When we first started this study, many people were using a probiotic E. coli strain as their chassis,” said Kim. “The problem with E. coli is that it’s great at colonizing the gut, and can stay in the system for several months. Even after you have recovered from a disease, the E. coli could still be in the gut producing recombinant proteins, which can trigger immune responses and inflammation. S. boulardii on the other hand leaves the system in just 1-3 days. This feature of yeast makes it easy to control the supply of recombinant proteins.”

The goal of the study was to genetically engineer the yeast to produce lysozyme, an antimicrobial protein that mammals naturally produce in milk, tears, saliva, and more. The researchers used CRISPR/Cas9 genome editing to integrate the human gene for lysozyme directly into the yeast genome. The engineered yeast was then fed to mice for 2 weeks, and the microbiome of these mice was compared to mice fed either wild-type unengineered yeast or a saline solution.

First, the researchers measured the presence of lysozyme in the gut and fecal matter of the mice fed the engineered yeast, to verify that the yeast was producing and delivering lysozyme into the gut. After verifying this, they measured the gut microbiome and fecal metabolome (the collection of metabolites microbes produce) of the mice across all three treatment groups.

While the diversity of the microbiome increased across all groups, the mice fed either type of yeast saw significant increases in the diversity of microbes present in the gut, including increases in the ratio of gram-positive to gram-negative bacteria. The researchers say this is to be expected, due to the probiotic nature of the yeast.

When they specifically examined the mice fed the lysozyme-secreting yeast, they found the structure of their gut microbiome and diversity of their fecal metabolome was significantly altered compared to mice fed saline or wild-type yeast. The researchers concluded that the lysozyme secretions by the yeast had indeed impacted the gut microbial community.

“We found a dramatic increase in firmicutes, or gram-positive bacteria, compared to gram-negative bacteria in the mice fed S. boulardii,” said Kim. “We investigated whether there were changes to specific strains, and found that probiotic bacteria increased after administration of lysozymes. We also found increases in diversity of microbiome, and decreases in sugar found in blood in the mice given yeast. So, we’re thinking administration of this engineered yeast could be helpful for maintaining a healthy microbiome or preventing growth of pathogens.”

“What’s cool is that we show that our engineered S. boulardii is able to produce proteins in the gut that significantly affect the microbiome,” said Miller. “But I think the bigger picture is that lysozyme is just a starting point. We can engineer the yeast to make any bioactive protein that we want, and have them deliver that cargo functionally to the gut.”

The researchers say that the activity of the yeast could be improved, as they may not have had enough sugar or nutrients to proliferate fully within the gut. However, for a follow-up study the researchers added a new genetic pathway to the yeast that will allow them to utilize lactose, the sugar found in milk. The lactose can then be fed to the mice alongside the yeast to provide a new fuel source for the newly engineered yeast. The researchers have already found that doing so dramatically increases lysozyme production in the gut.

In the future, the researchers are hoping to figure out how to deliver specific quantities of a target protein into the gut to be used in therapeutics. Jin says the ultimate goal of their research would be to utilize engineered yeast in “in-food fermentation,” such that the yeast that’s already in foods people enjoy, like baked goods, milk, and alcohol, would produce additional proteins that help maintain healthy gut microbiomes.

“My vision is to use this engineered yeast in food,” said Jin. “We already use yeast for making bread, wine, beer and such. But if we create these fermented foods using engineered microorganisms designed to be helpful for the gut microbiome, we can enjoy the benefits of the engineered microorganism simply through the consumption of food.”

The study was funded by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries, the Ministry of Agriculture, Food, and Rural Affairs, and the USDA. The paper can be found at https://doi.org/10.1128/spectrum.00780-23.