enzymes

Genetically engineered bacteria break down industrial contaminants

Once that was done, the researchers started looking through the genomes of species that have been identified as breaking down industrial contaminants. The breakdown of complex molecules typically involves more than one enzyme, and the genes for these enzymes tend to end up clustered together so they can be produced as a single, large RNA that encodes all the proteins needed. This simplifies regulating their production, making it easy to ensure the bacteria only make the proteins if the molecule they break down is actually present. In this case, the clusters ranged from just three genes all the way up to 11.

Once nine of these gene clusters were identified, the DNA that would encode them was ordered and assembled into a single DNA molecule in yeast. The researchers took some time while ordering this DNA to better optimize the genes to be active and produce proteins in Vibrio natriegens, as opposed to whatever species the genes were normally used by.

From yeast, each of these individual gene clusters was inserted into Vibrio natriegens, creating different strains that could digest one of the following: benzene, toluene, phenol, naphthalene, biphenyl, DBF29, and dibenzothiophene (DBT). (Some of the nine clusters target the same contaminant.) Each of these bacterial strains was then put in a solution with the chemical they were engineered to digest. Five of the nine worked, giving researchers strains that could digest biphenyl, phenol, napthalene, DBF, and toluene.

Good, but limited

From there, the researchers developed a system that would enable them to iteratively insert a new gene cluster at the tail end of a previously inserted gene cluster. This allowed them to build up a cluster of clusters, eventually including all five of the ones that had shown activity in the earlier tests. Given two days, this single strain could remove about a quarter of the phenol, a third of the biphenyl, 30 percent of the DBF, all of the naphthalene, and nearly all of the toluene.

AI used to design a multi-step enzyme that can digest some plastics

AI, biochemistry, Biology, catalysts, chemistry, Computer science, enzymes, Science / Kris Guyer / February 15, 2025

And it worked. Repeating the same process with an added PLACER screening step boosted the number of enzymes with catalytic activity by over three-fold.

Unfortunately, all of these enzymes stalled after a single reaction. It turns out they were much better at cleaving the ester, but they left one part of it chemically bonded to the enzyme. In other words, the enzymes acted like part of the reaction, not a catalyst. So the researchers started using PLACER to screen for structures that could adopt a key intermediate state of the reaction. This produced a much higher rate of reactive enzymes (18 percent of them cleaved the ester bond), and two—named “super” and “win”—could actually cycle through multiple rounds of reactions. The team had finally made an enzyme.

By adding additional rounds alternating between structure suggestions using RFDiffusion and screening using PLACER, the team saw the frequency of functional enzymes increase and eventually designed one that had an activity similar to some produced by actual living things. They also showed they could use the same process to design an esterase capable of digesting the bonds in PET, a common plastic.

If that sounds like a lot of work, it clearly was—designing enzymes, especially ones where we know of similar enzymes in living things, will remain a serious challenge. But at least much of it can be done on computers rather than requiring someone to order up the DNA that encodes the enzyme, getting bacteria to make it, and screening for activity. And despite the process involving references to known enzymes, the designed ones didn’t share a lot of sequences in common with them. That suggests there should be added flexibility if we want to design one that will react with esters that living things have never come across.

I’m curious about what might happen if we design an enzyme that is essential for survival, put it in bacteria, and then allow it to evolve for a while. I suspect life could find ways of improving on even our best designs.

Science, 2024. DOI: 10.1126/science.adu2454 (About DOIs).

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