Utilizing Enzymes to Help Treat Tuberculosis

Tuberculosis, or TB, is a contagious bacterial infection that primarily infects the lungs. While curative treatments exist for TB, more than 80 percent of cases occur in low- and middle-income nations where drug-resistant strains of the disease are on the rise and the price of medications is a burden. A next-generation class of antibiotics has shown good promise in targeting multidrug- and extensively drug-resistant TB, but their current manufacturing cost is a barrier to access in poorer countries that need them the most.

Now, researchers working with Nobel Laureate Frances Arnold, Caltech's Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry, have developed a method that could lower those production costs and increase access to better treatments.

"In our lab, we use enzymes—nature's catalytic machinery—to make valuable molecules in a more efficient and sustainable way," says Ziqi Li, a postdoctoral scholar research associate in the Arnold group. "We wanted to find a more accessible route to making effective antibiotics against TB."

Li is lead author of a paper published September 23 in Nature that describes the team's findings.

The researchers targeted a type of organic compound called 5-(S)-aminomethyl oxazolidinone used for many pharmaceutical applications. Those applications include the synthesis of a new group of antibiotics that can target bacterial cellular growth, making them promising candidates against antibiotic resistance.

Many drug molecules can exist in two mirror-image forms, much like a left and a right hand. Both forms contain the same atoms, but at one key point in the molecule, called a stereogenic center, those atoms are arranged differently in three-dimensional space. That arrangement controls the molecule's overall shape, which determines how it interacts with proteins and other biomolecules in the body. For oxazolidinone antibiotics, only one of the two forms, known as the (S) form, kills bacteria.

Oxazolidinones are often made as a mixture of the two mirror-image forms. Because only the (S) form is effective against TB, half of each batch can't be used and goes to waste. To increase yields, and potentially lower costs, Li and the team set out to build the stereogenic center from scratch using enzymes, producing only the (S) form.

"We have more than 5,000 enzymes in our freezer, so we went digging into those libraries, screened a few hundred, and found one that gave us a starting point," Li says. "It wasn't necessarily a very good starting point, but we iteratively improved this enzyme to ultimately get to a final point where it's high yield and high selectivity."

The process involved using directed evolution, a bioengineering method that earned Arnold the Nobel Prize in Chemistry in 2018. The process, developed in the early 1990s, is used to make new and improved enzymes in the laboratory using the principles of evolution. Scientists begin by inducing mutations to the DNA, or gene, that encodes a particular enzyme. An array of thousands of mutated enzymes is produced and then tested for a desired trait. The top-performing enzymes are selected, and the process is repeated to further enhance the enzymes' performances.

"The vast and wonderful chemistry of life is a result of billions of years of enzyme evolution, which is still ongoing," says Arnold, who is also director of the Donna and Benjamin M. Rosen Bioengineering Center at Caltech. "But with these tools, we can make enzymes that do so much more efficiently and with little waste, reducing the cost of producing what we need for our daily lives."

The team's new method provides a strategy that enables efficient synthesis of 5-(S)-aminomethyl oxazolidinone antibiotics—including clinically relevant and discovery-stage versions—while improving cost efficiency and reducing waste generation. Li hopes that pharmaceutical companies will take their findings and continue to perfect the method.

"We solved the first step to prove that the enzymes can make these molecules," Li says. "But there's still a very long way to go before we know whether it is possible to produce it in mass quantities. However, this proof-of-concept work is an important step forward in making medications that are both effective and more affordable."

The Nature paper is titled "Chiral oxazolidinones via biocatalytic aziridination of unactivated alkenes." Additional Caltech coauthors are graduate students Deirdre Hanley and Chi Zhang, former undergraduate researcher Sophia J. Wu (BS '25), former graduate students Zi-Yang Qin (PhD '26) and Francesca-Zhoufan Li (PhD '25), former NIH Postdoctoral Fellow Edwin Alfonzo, and lab manager Sabine Brinkmann-Chen. Yu Zhang, Pei-Pei Xie, and Peng Liu from the University of Pittsburgh are also coauthors. The work was supported by the Gates Foundation, the Jacobs Institute for Molecular Engineering for Medicine at Caltech, the National Institutes of Health, and the National Science Foundation.