How We Engineered a Probiotic to Break Down Acetaldehyde

• Genetic engineering: the process of modifying an organism’s DNA to give it new traits or abilities
• Acetaldehyde: an unwanted byproduct of alcohol metabolism
• Enzyme: a protein that speeds up chemical reactions in living organisms
• Chromosome: the structure of DNA that carries genes and determines inherited traits
• Locus: the specific position of a gene on the chromosome, which helps determine where and how much the gene is used
• Expression: the process by which a gene’s information is used to make proteins or other molecules
• Bacterial endospore (spore): a tough, dormant form of bacteria that can survive extreme conditions until the environment is favorable again
• Germination: the process by which a bacterial spore "wakes up" and returns to active growth when conditions improve

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Introduction

Our microbiologists at ZBiotics engineered a probiotic strain of Bacillus subtilis with a specific purpose: to help break down acetaldehyde in the gut. This novel probiotic works by converting acetaldehyde, a toxic byproduct of alcohol metabolism, into acetate—a harmless substance that the body can easily process. We recently published a peer-reviewed article in the scientific journal PLOS ONE — “Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase” — where we go into the details of how we built and validated our genetically engineered probiotic. This blog aims to provide you with a high-level overview of our publication, including the experiments and results that demonstrate the function of this innovative probiotic.

Understanding the problem: acetaldehyde

When alcohol is consumed, most of it is quickly absorbed into the bloodstream and subsequently processed in the liver through a two-step process. First, an enzyme called alcohol dehydrogenase (ADH) converts alcohol into acetaldehyde. Then, acetaldehyde is further broken down into acetate by another enzyme, acetaldehyde dehydrogenase (ALDH). However, a small amount of alcohol is not absorbed and is instead processed in the gut by ADH-producing bacteria, where it is converted into acetaldehyde (Salspuro, 1996;Jokelainen et al., 1994). Without enough ALDH-producing bacteria to break it down further, acetaldehyde can build up and cause some of the undesirable next-day effects of drinking. With this knowledge, we focused on developing a solution to break down acetaldehyde levels in the gut using a genetically engineered probiotic.

Developing the solution: an engineered probiotic

We chose Bacillus subtilis as the foundation for genetic engineering due to its long history of safe use, ability to form resilient spores that survive harsh environments (including your hot porch in the summer, and your very acidic stomach), and well-characterized genetics and genetic tractability (Su et al., 2020). These traits allow it to effectively deliver beneficial enzymes—such as ALDH—to the gut, where they can function in real-world conditions. Additionally, B. subtilis does not take up permanent residence in the gut, making it an attractive candidate to deliver a function without altering the gut microbiome.

In this study, we set out to create a probiotic that can make enough ALDH to rapidly break down acetaldehyde. We introduced into the B. subtilis genome the gene that produces ALDH from another soil bacteria, Cupriavidus necator, which frequently co-mingles with B. subtilis in the environment. This enzyme works the same way as the one produced in the liver for efficient acetaldehyde metabolism. However, having the gene alone isn’t enough. The location of the gene within the genome affects when and how much enzyme is produced by the bacteria. To increase production, we inserted the gene into a region of the genome responsible for producing motility proteins, known as the hag locus. This region is stable and produces proteins in high quantities, ensuring that ALDH is expressed at the desired levels (Kearns & Losick, 2005).

These engineering decisions were important, as they enabled the probiotic to be effective shortly after consumption. But since the probiotic would be consumed, efficacy was not the only factor considered during its design. We also prioritized safety by minimizing edits to the genome and limiting the use of foreign DNA traditionally used to regulate gene expression. By using tools native to B. subtilis, we took a conservative approach without compromising the probiotic’s effectiveness. To learn more about our commitment to GEM safety, take a look at some of our recent collaborations with regulatory officials and industry experts!

Key results: testing the probiotic

Ensuring effective enzyme production

To confirm that the engineered probiotic could produce an enzyme reliably, continuously, and effectively, we performed tests at the hag locus using a reporter protein called LacZ. When LacZ is expressed, it causes a visible color change that can be easily quantified. This allowed us to verify that the hag locus supports strong and stable expression of ALDH in relevant conditions.

Our engineered probiotic works in simulated gut conditions

Our previous experiment allowed us to quantify the activity of the engineered probiotic in laboratory conditions, using a nutrient-rich growth medium called Luria-Bertani medium (LB). While these conditions support bacterial growth for research purposes, we can use a simulated intestinal fluid (SIF) to measure activity in conditions more closely resembling those found in the human body. We also wanted to look directly at the ability of the bacteria to express our specific enzyme of interest, ALDH. So we used our strain engineered to express ALDH in the same hag locus that we validated in the previous experiment, and assessed ALDH activity directly in both LB and SIF. As seen below, while ALDH activity is present in both LB and SIF, the numbers are slightly lower in gut-simulated conditions. These results provide us with more realistic numbers to use when deciding how much bacteria are needed for optimal acetaldehyde breakdown in the gut.

Our engineered probiotic effectively removes acetaldehyde

Our next set of experiments confirmed that the probiotic strain could drastically reduce acetaldehyde levels. We observed over 99% of acetaldehyde removal when the engineered probiotic was present, significantly outperforming the unmodified strain, which only reduced acetaldehyde by about 16.5%. This demonstrated that the engineered probiotic expressing ALDH could effectively and rapidly reduce acetaldehyde.

In addition to the results detailed above, we also conducted baseline studies to ensure that the genetic modifications we made did not interfere with the bacteria’s normal growth, spore formation, or germination. Separately, we also performed extensive testing to demonstrate the safety of the strain, which we published in another peer-reviewed paper in the Journal of Toxicology.

Engineered probiotics offer targeted solutions to real-world problems

Our research shows that Bacillus subtilis can be engineered to provide targeted benefits to consumers. By carefully selecting where we can guide the bacteria to edit their genetic material, we can increase desired protein production while minimizing the amount of foreign DNA introduced into the system. In addition, spore-forming bacteria have practical advantages as probiotics—they’re easy to store, have a long shelf life, and activate in the gut where they’re needed. We applied this to a real world problem, the accumulation of acetaldehyde in the gut after drinking. By engineering B. subtilis to produce ALDH, we are able to target the cause of many unpleasant next-day effects of alcohol consumption.

Genetic engineering can shape the future

This research demonstrates how genetic engineering can be used to make the next generation of probiotics. By modifying Bacillus subtilis—a familiar and safe bacteria—to produce ALDH, we created a probiotic with a clear and defined benefit for the user. The success of this approach suggests that genetic engineering could play a more prominent role in the probiotic industry, allowing for the development of products that tackle other common challenges. Our “hag locus” engineering method can be adapted to deliver a variety of beneficial enzymes, opening the door to new applications of probiotics in the future. This research just scratches the surface of what genetically engineered probiotics can offer, solving modern problems with modern solutions.