Evolution Gets an Assist

Frances Arnold ’79 was awarded the 2018 Nobel Prize in Chemistry for her work on the directed evolution of enzymes, sharing the prize with George P. Smith and Gregory P. Winter. The following story about her pioneering work was featured in PAW’s Oct. 22, 2014, issue.

It took nature tens of millions of years to develop the cytochrome P450 protein. It took Frances Arnold ’79 only a few weeks to make it better. A pioneer of a form of bioengineering known as “directed evolution,” Arnold and her team of researchers at the California Institute of Technology generate new versions of P450 that do things nature never designed them to do, including mapping chemical signals in the brain and testing the toxicity of drugs.

Cytochrome P450 is an enzyme that helps the body metabolize everything from food to medicine, but with just a few genetic tweaks, Arnold can get it to perform entirely new functions. Where P450s naturally insert oxygen atoms in the drugs we ingest, for example, modified P450s can insert carbon and nitrogen instead. That excites biologists because it shows how quickly molecules can adapt to new opportunities, and it excites chemists because they can use these biological catalysts to make complex drug molecules or pesticides that are not deadly to bees and animals. Arnold herself has made compounds that could replace more toxic pesticides. “Many are derivatives of natural pesticides like what is found in chrysanthemum flowers,” she says. 

Genetically altered P450s also might be useful as contrast agents for better MRI scans, providing clues about how chemical signals in the brain are connected to specific feelings or behaviors. Functional MRIs today can provide only an indirect picture of what is going on inside the brain, Arnold says: They can see oxygen molecules binding to hemoglobin in the blood, but that is an imperfect proxy for the activities of neurotransmitters such as dopamine and serotonin.

Because P450 is similar to hemoglobin in that it, too, binds to oxygen, Arnold thought it might be possible to evolve a P450 that could bind to dopamine instead. Which mutations in P450 would enable it to do that was a mystery, so her team randomly mutated the P450 gene and inserted the mutations into E. coli bacteria (a type of bacteria that reproduces quickly and is easy to work with), which produced P450 proteins with those changes. She and her researchers then used algorithms to comb through the mutated P450 proteins for any that showed signs of binding to dopamine, extracted their DNA, and inserted them into a new generation of bacteria. Repeating the process for several generations, within a few weeks the scientists had bred the kind of protein they wanted. Think of a botanist breeding a more colorful orchid, only much faster. 

Directed evolution — taking natural proteins and rewriting their DNA by accumulating mutations over multiple generations — allows scientists “to rewrite the code of life” to solve human problems, Arnold says. The technique is increasingly common in biochemistry, and Arnold, more than anyone, is the person who invented it. Researchers at MIT have taken her mutated P450 protein and found that it does indeed enable them to trace dopamine activity in MRI scans of rat brains. Though still not perfected, the technique shows promise for use in scans of human brains as well, which could enable neuroscientists to get a much clearer picture of what happens in the brain when we feel joy or pain. 

Arnold’s techniques have enabled pharmaceutical companies to manufacture drugs without using toxic chemicals and energy companies to produce biofuels that replace fossil fuels. Her processes have helped her found three companies and secure more than 40 patents, earning millions of dollars for Caltech. She is one of a handful of living scientists to be elected to all three of the nation’s most prestigious scientific societies — the National Academy of Engineering, the Institute of Medicine, and the National Academy of Sciences — and has been awarded the National Medal of Technology and Innovation. In May, she was inducted into the National Inventors Hall of Fame, joining such luminaries as Alexander Graham Bell, Henry Ford, and Eli Whitney. 

A layman might be forgiven for seeing a mad-scientist quality in Arnold’s tweaking of nature. Others might say that directed evolution gives bioengineers the power to play God. Leave it to a fellow scientist, though, to sum up her work more accurately, if a little piquantly. Asked what it is that Arnold does, David MacMillan, Princeton’s James S. McDonnell Distinguished University Professor of Chemistry, replies: “She hijacks biological enzymes and makes them do her bidding.” 

Caltech’s campus occupies 124 acres in sunny Pasadena. The buildings tend toward office-park utilitarian, and at first glance, the suite where Frances Arnold works has little to draw the eye. Only the photographs that show her meeting President Obama and Queen Elizabeth II hint that something very unusual is going on there. 

Arnold is a professor of chemical engineering, bioengineering, and biochemistry, but she acknowledges that her professional evolution has seen its own share of random mutations. The granddaughter of a three-star general and daughter of William Howard Arnold *55, a nuclear physicist and president of the first private uranium-enrichment facility in the United States, Frances Arnold grew up outside Pittsburgh. If science was in her blood, military conformity was not; she hitchhiked to Washington, D.C., at 15 to join an anti-Vietnam War protest. She moved out of her parents’ house while still in high school, supporting herself by driving a cab and lying about her age so she could work as a waitress in a jazz bar.

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Joe Lertola, Bryan Christie Design

A self-described “worse than mediocre” student, she nevertheless was accepted to Princeton and chose mechanical and aerospace engineering because, she says, the department had the fewest academic requirements for an engineering degree. Arnold used that extra time to study economics and foreign languages, joking with her characteristic throaty laugh that she also majored in “Italian postdocs.” After her sophomore year, she took a year off, went to Italy, and got a job in a factory that made parts for nuclear reactors, but the point was not to further her academic career. It was an adventure, and that love of adventure never has waned: Her hobbies include scuba diving, skiing, dirt-bike riding, and hiking in the California mountains while staying in a cabin that lacked plumbing and electricity. For her sabbatical in 2004, she took her family on a yearlong, round-the-world tour with long stops in Australia, Egypt, Namibia, Madagascar, South Africa, and Wales.

Upon returning to Princeton from Italy for her junior year, she spent time studying at the Center for Energy and Environmental Studies with a young group of engineers and scientists who were trying to solve the riddle of sustainable energy. Robert Socolow, now professor emeritus but then the center’s director, describes the group in its early days as “bold” and “driven” in its determination to work out complicated problems, qualities he says Arnold has exhibited throughout her career. Arnold remembers the researchers as being “activists who really cared about where our energy was going to come from and at what cost.” She became interested in that, too.

After graduation, she worked as an engineer in Brazil and South Korea and at the Solar Energy Research Institute in Colorado before going to graduate school at the University of California, Berkeley. She developed a love of biochemistry while pursuing her Ph.D. in chemical engineering, which she earned in 1985. She went to Caltech as a postdoc the following year and has been there ever since. Nevertheless, Arnold has said that she was not sure she wanted to become a scientist or engineer until she actually became one. She had always imagined being a diplomat or a CEO.

Genetic engineering already had begun by the time Arnold entered the field, as scientists experimented with ways to manipulate DNA and insert it into organisms so they could read it. Arnold first began trying to design new proteins “rationally,” starting from scratch, but grew discouraged that no one in the field seemed to be able to make useful ones. Rather than trying to reinvent the genetic wheel, Arnold hit upon the idea of using evolution by artificial selection, in effect making the natural evolutionary process work for her.

“Mother Nature has been the best bioengineer in history,” she explained in an article for Engineering and Science two years ago. “Why not harness the evolutionary process to design proteins?” 

There are now many ways of doing directed evolution, but the processes Arnold developed, and still uses, are deceptively simple. Starting with the DNA in a particular protein, Arnold makes mutations, usually by introducing a copying enzyme that inserts random mistakes in the genetic code. The scientists also can recombine DNA from different species in a kind of molecular sex. Although Arnold can do this in her own lab, it is often easier to have another DNA lab make them to her specifications and mail her a billion or so mutated genes in a test tube. 

However she gets them, Arnold inserts these mutated genes into bacteria, which then read their altered DNA and produce what the DNA tells them to produce. Arnold sifts through the new generation of proteins to see which ones exhibit the properties (such as binding with dopamine) she wants them to have. When she finds proteins with the desired qualities, she extracts their DNA and repeats the process, breeding generation after generation of proteins until she gets the proteins that will do what she wants. 

As the National Inventors Hall of Fame described Arnold’s work on its website, directed evolution “mimics Darwinian evolution in the lab under an extremely accelerated timescale. With directed evolution, however, the human experimenter decides the selection of the ‘fittest.’ Thus directed evolution is like breeding, but at the molecular level.”

The evolutionary process that turned ancient primates into humans or pterodactyls into eagles involved small genetic mutations over countless generations and tens of millions of years. A bacterial generation, fortunately, only lasts for about 20 minutes, so Arnold can evolve a new protein in the lab in anywhere from a few days to a few weeks. And if only one new protein in a million mutants pans out, she is happy to use that one and throw the 999,999 failures away.

Over the last 20 years, directed evolution has been widely adopted and applied across academia and industry to make everything from pharmaceuticals to laundry detergents. Arnold’s research at Caltech is funded by the departments of Energy and Defense and the National Science Foundation, among others, but some of it is done to order for private industry. Although Arnold has started her own companies and serves as an adviser to several others, she says she prefers research and teaching to running a business. 

She has often been one of a small group of women in a male-dominated field, something she says has not bothered her. Although she recalls being treated with a certain amount of condescension by male colleagues early in her career, she says she was lucky to have come along at a time when there was a demand for women engineers. Besides, she adds, “in academics, it’s getting your voice out that’s important. It’s getting somebody to listen to you. I had no problem with that. People were always curious about what I had to say.”

She has worked in the same three-room laboratory, just a floor above her office, for the last 28 years. It is full of test tubes, petri dishes, microscopes, and refrigerators. Trays of God-only-knows-what strain of bacteria grow on the counter. On any day, her team of 22 graduate students and postdocs has several projects going on, with Arnold supervising and directing the work. 

Much of her research has been directed toward what she calls “green chemistry,” enabling industry to use biological processes instead of toxic chemicals. Directed evolution has enabled the pharmaceutical company Merck, in collaboration with Codexis, a company Arnold advises, to manufacture its diabetes drug Januvia with genetically engineered enzymes rather than the heavy metal rhodium, a process that is cheaper and produces less waste. New companies are looking to use biodegradable biological enzymes to replace chemical pesticides.

Arnold with postdoc Christopher Prier *14 in her lab.

Steve Anderson Photography

Inspired by her undergraduate work at Princeton, Arnold has devoted much of her career to finding alternative sources of energy. She has, for example, developed an enzyme that can thrive in airless environments, enabling Gevo Inc., a company she co-founded, to produce biofuels without air-circulating equipment. Gevo also uses fermented plant materials to produce fuels that one day might replace petroleum-based gasoline and jet fuel. Directed evolution also has contributed to a new field called “bioremediation,” which uses mutated organisms to eat oil spills or toxic waste at Superfund sites (Arnold has not worked in this area herself). 

Still another promising application is in a field called optogenetics: using light to control nerve cells. Shining light on certain microbial proteins found in the ocean, for example, can open channels in their cell membranes. Arnold’s lab is taking these ocean-microbe proteins and breeding them into versions that can be activated inside the human brain, which could lead to the development of new treatments for Parkinson’s disease, schizophrenia, autism, and other neurological disorders.

Early in her career, Arnold recalls, her methodology was viewed skeptically by “gentleman scientists” who thought that proteins should be designed rationally rather than built scattershot and looked askance at her focus on manipulating the outcomes before she fully understood the processes that drove them. But, she told herself, “I’m not a gentleman and I’m not a scientist” — Arnold defines herself first and foremost as an engineer — “so I didn’t mind.”

Saying she is not a scientist is debatable, as directed evolution draws upon chemistry, molecular biology, chemical engineering, and physics. Says Princeton chemistry professor Michael Hecht, a longtime friend, “She has combined the kind of thinking that an evolutionary biologist would do with real engineering thinking and real chemistry thinking. That’s unusual. She does basic science but thinks like an engineer, focused on real-world applications.”

In 2011, Arnold shared the $500,000 Charles Stark Draper Prize, which is given by the National Academy of Engineering and has been called engineering’s Nobel Prize. Later that year, President Obama awarded her the National Medal of Technology and Innovation for “pioneering research” on biofuels and green chemicals. She has spoken at conferences around the world, including the World Economic Forum in Davos, Switzerland. 

On a quiet day in early June, two of Arnold’s postdoctoral students were busy taking DNA from three different parent proteins to make something that would not have been possible in nature, where the breeding rules are pretty rigid: Organisms reproduce only with the same kind of organisms, and there are never more than two parents. As Arnold likes to put it, a big part of her job is to “force molecules to have sex,” and in the new world of biotechnology, anything goes. She can, for example, cross a cat’s DNA with a dog’s DNA (or an elephant’s or an apple’s, for that matter), take the resulting proteins, and use them for some other function. 

“We’ll take one form of life and turn it into another form of life that is useful or beautiful to us,” she explains. 

If it all seems like playing God in the laboratory, Arnold points out that genetic engineering is nothing new. Dopamine-binding cytochromes may not exist in nature, but neither do labradoodles, thoroughbred racehorses, Big Boy tomatoes, or nectarines. Directed evolution simply allows scientists to crossbreed at the genetic level. (Genetically modified foods, which have been the subject of some controversy, are created by a different method, and Arnold does not work on them.) As for the risk of inadvertently breeding some mutant bacterium that eats Pasadena, Arnold says that she never works with infectious agents and that none of her specimens could survive for long outside the lab. She wants to make sure bioengineering is communicated accurately, and even works as an adviser to the Science and Entertainment Exchange, a program established by the National Academy of Sciences to assist Hollywood screenwriters who write about science.

Someone could write a good screenplay about directed evolution, but it is no longer a new story. More than four decades after genetic engineering began, the science of splicing genes is relatively familiar. Arnold sees two principal challenges ahead.

One is a simple question of capacity. Researchers may be able to run a million genetic experiments in a few days, but reviewing their results, interpreting them, and using them to devise the next million mutations takes time.

The bigger problem is conceptual. If directed evolution lets nature handle the evolution, humans still must do the directing, and that requires imagination. Which new sequence of DNA will make a viable biofuel? Which will make a better laundry detergent? Which will form a P450 protein that binds to dopamine?

“We only have the sequence nature gives us to start with,” Arnold explains. “Instead of studying what biology has already made, we have to imagine what biology could make. You can say, ‘Oh, I want a cure for cancer,’ but that doesn’t tell you what evolutionary pathway will take you from here to there. What are the intermediate steps?”

One could call this trying to improve on nature, but Arnold believes the relationship works both ways. “I see a future in which nature gives us a helping hand,” she said in a recent video for the BBC. “Instead of destroying the natural world, why can’t we use it to solve the kinds of problems that we are facing?”  

Mark F. Bernstein ’83 is PAW’s senior writer.