Evolution On Fast Forward

Letting nature do the heavy lifting, gene scientists are creating super new materials

Some 140 years after Charles Darwin penned The Origin of Species, educators, parents, and theologians are still challenging his theories of evolution. But amid all this, a new kind of evolution is on the loose. It's called directed evolution, and to hear its advocates, it is the most important development in biotechnology since the advent of genetic engineering 25 years ago. "Evolution isn't just a grand idea anymore," says Andrew D. Ellington, a biochemist at the University of Texas at Austin. "It's big business, something that makes money and products."

Directed evolution is essentially sex in a test tube. It involves subjecting a naturally occurring compound to a rapid evolutionary process, with scientists imposing some controls from the sidelines. The compound might be a protein with anticancer properties--promising, but too toxic to administer in large doses. By rapidly shuffling and mutating the genes that make this protein, scientists create superior versions in a matter of weeks rather than eons.

The power to derive new medicines and materials has captured the attention of Dow Chemical, DuPont/Pioneer Hi-Bred, Glaxo Wellcome, and Eli Lilly, among others. After just 10 years of experimentation, directed evolution has already borne commercial fruit: a laundry detergent enzyme from the Danish firm Novo Nordisk and an anti-tumor drug in early human trials from the San Diego-based biotech company Ixsys Inc. Within the next two years, dozens of other products are expected to follow, from industrial enzymes to new and improved vaccines made by companies such as Maxygen, Phylos, and Diversa.

Directed evolution differs from conventional gene splicing and so-called rational drug design, in which scientists use mathematical algorithms to customize molecules so they will perform as drugs. These techniques have produced few blockbuster drugs so far. Indeed, fast evolution doesn't depend on the recent decades of gene research and giant databases of proteins. The beauty of directed evolution is that engineers can leave the heavy lifting to nature, which functions as an extremely potent algorithm. "This technology teaches you to be humble," says Russell J. Howard, president and CEO of biotech startup Maxygen Inc. in Redwood City, Calif. "Nature has a way of providing rare solutions that could never in a million years have been predicted or designed from first principles," he adds.

If Charles Darwin were alive today, he would nod in agreement. As he first elucidated, nature pressures species to diversify through a built-in process of adaptation, which we now link with random mutation. Most mutations will hinder an individual organism's survival, but a relatively small number of mutations are "adaptive," yielding individual plants or animals that are fitter than their cousins. Their offspring and subsequent generations supplant the earlier species. After millions of years of this natural selection, a completely new species emerges.

Farmers have been intervening in evolution for centuries, selecting breeds of higher-yielding corn or beefier cattle. "Directed evolution is just a high-tech version of this process," says Frances H. Arnold, a California Institute of Technology biochemist and a pioneer in the field. "It's breeding at the molecular level. Instead of doing it with whole organisms, we are doing it with particular genes or proteins."

DESIRABLE TRAITS. To orchestrate this process at the molecular level, scientists first need to choose a target gene to be the "parent." Using chemicals or other methods, they make millions of offspring genes--variants that differ slightly from the parent in their genetic makeup. The offspring genes are introduced into bacteria or yeast. Researchers then observe the genes and pluck out those variants that display a desirable trait--say, the ones that work better at a higher temperature. Bioengineers can then take these higher-performing molecules and breed them to one another, reorganizing the genetic information to create offspring that remain active at even higher temperatures.

When breeding cattle or new strains of corn, farmers are limited to using the genetic information of just two parents. With genes, however, it's possible to mate 50 or a 100 in a single step. This way, the researchers can integrate the desirable traits from many parent genes into a single offspring gene. They can even create genes with whole new functions since the process results in combinations of DNA never before seen. By repeating the mutation and breeding steps, it's possible to strengthen the desired trait in a matter of weeks or months. Often, just one or two rounds of evolution is all that is needed to increase a compound's performance 100 times. Ixsys researchers, in one round of evolution, created an antitumor drug called Vitaxin that is 2,000 times as potent as the starting anticancer compound.

INDUSTRIAL STRENGTH. Enzymes also present compelling targets for directed evolution. Arnold's lab is focused on breeding them for use in chemical synthesis. She wants to make clean, highly selective catalysts that will save the industry money and trouble by eliminating energy-intensive procedures and toxic byproducts.

Researchers at Diversa, a biotech company in San Diego, are also using directed evolution to improve enzymes, starting with exotic microorganisms isolated from extreme environments such as the hot springs of Yellowstone National Park. They claim to have improved enzymatic activity as much as 39,000 times. The company is currently collaborating with ZymeQuest Inc. of Beverly, Mass., to evolve enzymes that strip blood-type factors from the surface of red blood cells. According to Jay M. Short, Diversa's president and chief executive, the goal is to turn A and B blood types into type O, the universal donor blood.

At Maxygen Inc., the company that originated the molecular-breeding concept, cell wizards take a different approach. Instead of starting with a single parent gene and mutating it, they begin with a family of genes that are similar in overall structure but different in tiny yet specific ways. Skipping the mutation step altogether, they then breed this family of molecules--a process that amounts to chopping the different DNA sequences into pieces and splicing them back together in all possible combinations. The result of this so-called family shuffling is a highly diverse pool of new molecules.

In collaboration with Novo Nordisk, Maxygen has used this approach to improve the performance of one of the most thoroughly studied industrial enzymes of all time, subtilisin. Businesses spend about $500 million annually to purchase subtilisin for applications that include food and leather processing and as a biological additive to laundry detergents. So important is subtilisin that researchers have mapped and patented every single one of its components. Chemists have spent decades trying to improve its performance under stressful conditions, such as at high temperatures or in alkaline solutions. At best, they have seen a twofold improvement.

Maxygen researchers claim much more dramatic improvements. They have shuffled the DNA sequences of 26 subtilisins, each from a different bacterial species, and recombined them. It took them just six weeks to screen through 654 offspring and identify new versions that were three to four times more robust, not just at higher temperatures but also in alkaline and other destabilizing solvents.

Maxygen researchers didn't just improve enzymatic activity. In lab experiments, they were also able to imbue molecules with entirely new functions. The investigators recombined the sequences of 20 human genes that code for interferon-alpha, a protein that defends the body against viral infections. They then selected combinations that bind, not to human cells, but to mouse cells. After two rounds of shuffling, the researchers generated a new form of the protein that could bind to mouse cells 300,000 times more tightly than the original version (and several times stronger than the native version). "This was a shoot-for-the-moon experiment," says Maxygen's Howard.

As with any powerful and innovative technology, directed evolution comes with its own risks. In theory, terrorists could use the technique to create a germ perhaps 30,000 times as virulent as any that exists today.

But proponents of directed evolution say the benefits outweigh the risks. Arnold foresees a future in which the technology is used to create new drugs with fewer side effects, crop varieties that improve nutrition, and chemical processes that help the environment. "It's staggering," she says. "Directed evolution is going to be the design paradigm in all areas of biotechnology."

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