Directed Evolution Teaches an Old Enzyme New Tricks
Over the last few billion years, chemical reactions have run rampant on planet Earth, combining atoms and molecules in new and increasingly complex ways, testing the limits of universal physical laws. Through the evolutionary force of natural selection, these reactions created self-replicating nucleic acids, microbial cells, wooly mammoths, and, perhaps the pinnacle of evolution, Duck Dynasty.
It’s a fine-tuned but maddeningly slow process, at least for those of us unable to hang around for a few million years. Fortunately, Frances Arnold has found a way to accelerate evolution, expanding the limits of biological capability in the process. Arnold is a Professor of Chemical Engineering, Bioengineering, and Biochemistry at Caltech, where she has worked at the forefront of directed evolution and probed the abilities of recombined proteins to perform biochemical reactions. Arnold was awarded the National Medal of Technology and Innovation on December 21st, a recognition given to 11 inventors across the country.
Directed evolution works by re-shuffling the deck of a protein’s sequence, producing hundreds of new enzyme variants at a time, and seeing how the resulting molecular machines perform the desired reaction. Enzymes that do worse are discarded; those that do better move on to the next round of evolution – wash, rinse, repeat. It’s a high-throughput way to identify more efficient proteins. “Why on Earth would you do just one experiment at a time?” asks Arnold, summarizing the time saving benefits of directed evolution.
Of course, it would be most efficient to simply construct the perfect protein from scratch, writing the code letter by letter, but this type of “rational design” requires a level of functional understanding that is decades away. “It’s not going to happen in my lifetime,” says Arnold. “The precise interactions of 4000 atoms plus 7000 water molecules? Good luck figuring that out. We’re going to have to learn some new tricks.”
Microbes, on the other hand, “are self-replicating, self-repairing catalysts,” as Arnold puts it; you don’t really need to the intricate workings of how an enhanced enzyme works, only that it does.
As the developer of a proven method for protein enhancement and re-purposing, Arnold now has the luxury of choice in deciding which projects to work on. “I’m mostly interested in projects that are extremely high-risk, high-reward,” she says. “Things that we’ve done before, where we’re just trying to incrementally improve something that already exists, that’s not fun for any of these smart people I get to work with.”
The Arnold lab’s latest finding was a challenge worthy of the brainpower. While previous successes had optimized reactions that proteins were known to perform, the next step was to take reactions that were previously the exclusive provenance of synthetic chemistry and perform them with microbial enzymes suited for another task altogether.
Cytochrome P450 enzymes are red-colored proteins best known for their ability to add single oxygen atoms into organic molecules – lipids, hormones, or drugs, for example. Arnold and her colleagues have been working with the bacterial version of cytochrome P450 for ten years, trying to convince it to perform new tricks. They recently discovered laboratory-generated versions that form cyclopropanes (a group of three carbon atoms, joined by single bonds in a triangle-like arrangement). Cyclopropanes are key intermediates in the production of many pharmaceuticals and other industrially produced materials, and the current way they are made often uses toxic metals and solvents. After a few rounds of directed evolution and sifting through hundreds of P450 variants, the team found versions of the enzyme able form cyclopropanes efficiently, in a way that biology had never been able to do before.
And after years of modifying existing pathways successfully (isobutanol production), and not-so-successfully (methanol from methane), Arnold sees her group’s cytochrome P450 work as a watershed moment, a way to make nature do unnatural things, with the aim of solving substantial problems. “I install whole new chemistries into biology,” she says; “the idea is to go where nature doesn’t care to, and in this way we’re exploring where evolution can go, with a little coaxing from us.”