Synthetic biology is a relatively new, but rapidly growing industry, gaining traction in many business sectors across the ever-growing bioeconomy. An even newer arm of the SynBio field is engineering living therapeutics, or synthetic biotics. It’s not a new concept to use microbial species as tiny cellular factories to produce materials in a more sustainable way than harvesting them from nature. But what is new is the idea of engineering therapeutic properties or therapeutic production into probiotics. These “living therapeutics” can act as a trojan horse for delivering therapeutic payloads by synthesizing them in the GI tract or elsewhere. This exciting therapeutic delivery system could help revolutionize how we treat some diseases, including genetic disorders and cancers.

Microbes in Human Health

Microbes play several key roles in human health. Since some microbes are pathogenic, a critical research focus is studying mechanisms of antimicrobial resistance as well as discovering novel antimicrobial agents. Additionally, many therapeutics such as proteins or small molecules are made in living cells. Though mammalian cells are better at expressing some therapeutic proteins, engineered microbes are an excellent alternative because they are less expensive to culture, easier to control genetically, and can produce higher titers of therapeutic compounds. Additionally, microbes can be ingested as probiotics and living therapeutics.

Probiotics and living therapeutics

Humans have been developing a mutually beneficial relationship with microbes for thousands of years. Probiotics are microbes that have beneficial health effects. A great example of this is probiotics for gut flora, which has a symbiotic relationship with our own gut cells. Probiotics in our gut can aid in digestion and combat pathogens. During an invasion of pathogenic bacteria, probiotics can outcompete and reduce the pathogenic population. Some probiotic bacteria achieve this by acidifying the intestinal lumen. This creates a harsh environment that pathogenic bacteria cannot tolerate and reduces infection.

Using synthetic biology, probiotic strains can be engineered to have additional beneficial effects like producing and delivering drugs directly at the site where they are needed. A variety of labs across the world are genetically engineering probiotics for other purposes, like breaking down indigestible inflammatory compounds, producing nucleic acid vaccines, and breaking down alcohol biproducts to abate a hangover. The most profound use of E. coli Nissle1917 is its ability to target and combat colon cancer, which several labs are developing using various approaches. Though the field of living therapeutics is still in the early stages, it could become the next generation of microbial therapeutics production.

A popular probiotic strain that has been used for living therapeutics research is E. coli Nissle1917. The Nissle1917 strain was first identified as a beneficial probiotic in 1917 during WWI and is commercialized as Mutflor® in many countries today. However, in order to be able to use this microbe as a living therapeutic for human health applications we need to develop a better understanding of its genetics and improved tools that would allow us to engineer beneficial properties into the strain.

Building the first genome-wide knockout library in E. coli Nissle1917

With this goal in mind, Inscripta scientists have built the first Genome-Wide Knock Out (GWKO) cell library in E. coli Nissle1917 using the Onyx engineering platform. GWKO libraries are a popular tool to study and engineer microbial strains, but this has never been done in E. coli Nissle1917, due to it being a more difficult strain to engineer than other lab-optimized strains. This library contains a mixture of edited cells, each with a single gene knocked out, resulting in 3,254 gene KOs in total. Each gene KO is more or less evenly distributed throughout the library, allowing for observing about 83% of the gene knockout variants in 1 mL of cells. This first-of-its-kind library was created in less than a month, thanks to rapidly accelerated design and building of the libraries on the Onyx platform.

Rapidly engineering an improved phenotype into a probiotic strain

The important outcome of this project is that the E. coli Nissle1917 GWKO library can be used to study and discover genomic changes that have beneficial effects under specific conditions or designer strains with enhanced specific functionality. One such function that could enhance E. coli Nissle1917’s utility as a probiotic and living therapeutic is a strain with better survivability at low pH. The human gut has varying acidity, from 2.5 to 7.5, so an optimized probiotic strain would be able to survive a wide range of pH conditions. To understand which knockouts could confer a benefit under low pH, we grew the E. coli Nissle1917 cell library in normal (pH 7.0) and acidic (pH 5.0) media. We then sequenced the Onyx barcodes to see which variants were enriched in the low pH conditions. Approximately one hundred designs were enriched in pH 5, suggesting potential targets for enhancing cell survival in acidic conditions.

The future of synthetic biotics in the clinic

The field of living therapeutics is still new, but many scientists are working on engineering probiotic microbes to have beneficial effects. The company Synlogic has an ongoing clinical trial for the rare disease phenylketonuria (PKU). People living with this condition lack the enzyme needed to break down the amino acid phenylalanine, leading to harmful buildup. PKU patients who continue to ingest phenylalanine, which is found in most foods containing protein, will likely suffer from severe, irreversible brain damage. In this therapeutic application, people were administered an engineered Nissle1917 SYNB1934 strain that was shown to lower the levels of phenylalanine in the gut. The SYNB1934 strain showed promising results in Phase 2 trials recently and will be advancing to Phase 3, giving hope to people with PKU to have a less restrictive diet.

This study demonstrates the feasibility of using living cells as both factories and delivery systems for therapeutics, producing and delivering therapeutic enzymes within the patient at the precise location where they are needed. This foundational research opens up possibilities for other diseases characterized by the lack of an enzyme or metabolite, where that molecule can be produced by a therapeutic probiotic. Synlogic is working toward developing Nissle1917-based living therapeutics for other metabolic disorders, like Inflammatory Bowl Disorder, metabolic disorders including Homocystinuria, Hyperoxaluria and Maple Syrup Urine Disorder, and cancer. Improving our ability to engineer and study microbial systems would help us design more effective probiotics or living medicines for treatment of human diseases in the future. Instead of ingesting a therapeutic compound in a pill, future diseases and conditions may come from ingesting a microbe engineered to contain genes encoding the therapeutic.