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GOLDEN OPPORTUNITY: Joan Combie's small company finds microbes in Yellowstone National Park. A small yet growing opportunity exists for biochemists and engineers interested in turning "extremozymes" into industrial catalysts. In nature, these peculiar enzymes fuel microbes that live in scalding sea vents, hot springs, and other adverse locales. Such hardiness means extremozymes might function in hotter, more high-pressure manufacturing conditions than can today's industrial enzymes. As a result, extremozymes could speed up or maximize reactions used to make food, detergents, and drugs; remediate toxic waste; and drill for oil. LOOKING DOWNSTREAM: Jay Short sees opportunities for chemists in the engineering phase. "Take the food industry," offers Jay Short, chief technology officer at Recombinant BioCatalysis Inc., a private company in Sharon Hill, Pa., that isolates and sells extremozymes, among other enzymes. "[Manufacturing] processes need to be sterile. If you can run things above 60C, it is automatically sterile. The problem is, most of your enzymes are inactivated. Now, [with extremozymes], you can keep it sterile and not worry about it." According to Short and others, the worldwide enzyme market is estimated at $2 billion. If researchers learn to reproduce and engineer extremozymes in the lab -- a potentially tricky task -- the hardy chemicals could be a hot industrial product. Short compares coming job opportunities in extremozyme research to those spawned by human genome sequencing efforts. "My view is that the biggest opportunities are going to be downstream, in the engineering phase of enzyme development," he says, suggesting that well-rounded chemists comfortable with enzyme biochemistry will be in demand. "That's where many jobs will be." In the hot springs of Yellowstone National Park alone, researchers have discovered more than 30 extremophile species, half of which have known biotech potential for processing starch, bleaching paper, or producing ethanol. More than 20 companies and universities have gotten permits to search Yellowstone for microbes, including Genencor International Inc. of Rochester, N.Y.; Stratagene Corp. of La Jolla, Calif.; and New England BioLabs Inc. of Beverly, Mass. Microbe genome discoveries are catapulting extremozymes into the news. In August, a research team led by Carol J. Bult, a scientist at The Institute for Genomic Research (TIGR) in Rockville, Md., and molecular evolutionist Carl Woese of the University of Illinois in Urbana-Champaign announced that they had sequenced the full genome of a microbe called Methanococcus jannaschii, found in the Pacific Ocean's thermal vents (C.J. Bult et al., Science, 273:1058-73, 1996). The project was funded by the Department of Energy (DOE)-sponsored Microbial Genome Initiative, which will sequence four more extreme microbes. Each organism may offer novel enzymes as well as insight into the evolution of life on earth. CHALLENGES: Glenn Nedwin notes potential problems ahead. Despite excitement over Methanococcus, observers caution that it will take a lot of research to turn extremozymes into industrial products. "Just because something grows at a high temperature doesn't mean the enzyme will work in a commercial process [at that temperature]," remarks Glenn Nedwin, president of Davis, Calif.-based Novo Nordisk Biotech Inc., the United States enzyme research arm of Novo Nordisk A/S of Denmark. Novo Nordisk sells more than 700 enzyme-based products. "I think people tend to overestimate [extremozymes'] immediate use and underestimate problems with the economics," Nedwin says. For industrial use, he adds, an enzyme must be reproduced on a large scale and used in a complex environment with other chemicals. It's this kind of challenge that offers enterprising scientists new opportunities, observes Robert Kelly, a chemical engineer at North Carolina State University. "The [extremozyme] field so far has been made up of individuals that happened to get interested in it," Kelly says. "There has been no systematic plan to bring a discipline into the area. So to bring [an extremozyme] up from a heated pool to application, a researcher has needed to be a microbiologist, biochemist, biophysicist, and engineer-one person has worn all those hats at the same time. The field now needs people who are experts in these particular areas." Slow Beginnings Extremozymes and their parent microbes are not new. In 1965, for example, researchers sampling life at Yellowstone identified an unusual, heat-loving bacterium named Thermus aquaticus. But this and other exotic microbes often ended up on lab shelves while researchers made other discoveries. "Back in the 1980s, there were really very few hyperthermophilic [heat-loving] microbes that were isolated, and we didn't know much about them," recalls John Baross, an oceanographer at the University of Washington in Seattle. "In 1980, there were only two microbial species [in the Archaea domain] that we knew could grow above 80°C." The Archaea represent one domain of life; the other two are Eucarya -- which include humans, plants, and animals -- and Bacteria. To complicate matters, studying extreme microbes in the lab hasn't been easy. Scientists have had to maintain the odd chemical diets and living conditions that these organisms prefer. For example, a microbe may thrive with plenty of sulfur, lots of heat, and no oxygen. To mimic the heat of a sea vent, researchers sometimes leave the microbes in fermenters with the steam turned on far longer than usual. And some of the organisms even produce highly corrosive gases. "Let's just say all the conventional techniques for growing these organisms are out the window," says Kelly with a laugh. "Because a lot of things are unconventional, there aren't a lot of ready-made systems to handle these enzymes. So researchers have had to adapt existing systems or buy new ones." Over time, Kelly points out, scientists have come to grips with these challenges and characterized many extremozymes. In the 1980s, researchers used T. aquaticus to get the enzyme Taq polymerase, which subsequently became the star of high-heat polymerase chain reaction (PCR) technology. PCR allows scientists to rapidly multiply DNA from just a few strands. The patents for Taq polymerase, the enzyme that makes PCR possible, have been the subject of a lengthy dispute between Swiss drug giant Hoffmann-LaRoche and Madison, Wis.-based Promega Corp. Hoffmann-LaRoche acquired the Taq patents from the now-defunct Cetus Corp. in 1991. It is possible that the complex dispute, which is now being played out in federal district court in San Francisco, will conclude with one or more of Hoffmann-LaRoche's patents being invalidated. However, this outcome is far from certain, and any resolution likely is years away. AMPLIFIED ROLE: Tom White cites extremozymes' importance in PCR. Enzymes useful in PCR still win a fair share of extremozymes' headlines. "The primary focus is to increase the fidelity of PCR amplification or to allow [PCR cycles] to run longer," explains Tom White, vice president of research and development at Alameda, Calif.-based Roche Molecular Systems Inc., a member of the Roche group that includes Hoffmann-LaRoche. Up to 10 scientists at Roche Molecular Systems work on extremozymes. "Within our group," White says, "extremozymes play a big role." "PCR probably brought more interest to extremozymes than any other single event," adds Joan Combie, one half of a two-person company, J.K. Research in Bozeman, Mont. The company finds and sells Yellowstone microbes to biotech companies. Trained as a toxicologist, Combie has been hunting Yellowstone microbes for more than 10 years. J.K. Research recently received a grant from the U.S. Navy to explore an extremozyme that can blister paint off old aircraft-an environmentally friendly alternative to chemical bleaching. "Until recently, these microbes and enzymes were just a lab curiosity," Combie remarks. "Now, people are calling us and saying, 'I need a microbe that does this.'" As extremozymes expand beyond PCR applications into other industries, the field must scale up its expectations, Kelly states. That means mass production. "A few grams of a DNA polymerase might take care of the world for a year," he says. "But for commercial applications like gas drilling or detergent use, you're going to need literally tons of enzymes." To get that much material,researchers are turning to genetics. Rather than roam the wild for easy-to-use extremozymes, some scientists want to engineer desirable extremozyme properties into more common enzymes. BIOTECH BOOM: Frank Landesberger notes the industry's role. This strategy, notes Nedwin, may be the best way to make extremozymes work. Genetic engineering already provides some of the common proteases used in making detergent. "Extremozymes are another tool in the arsenal," he says. "Maybe by studying their structure and domains, we can engineer useful enzymes." "The extremozyme field is standing on the shoulders of the biotech industry," adds Frank Landsberger, director of the Office of Science and Technology Development at Mount Sinai School of Medicine in New York. "Biotech has learned how to fish out genes of interest and grow them in other organisms." Biotech also comes into play with genome sequencing. At Recombinant BioCatalysis, some 40 molecular biologists, microbiologists, chemists, and biochemists work with robotic programs that sequence and analyze genes from extremophiles found in the wild. Researchers then sort the genes according to enzymatic activity. Typical of the emerging extremozyme field, Recombinant BioCatalysis contracts with academic scientists who collect microbes from hot springs and seas. The two-year-old company currently works with about 10 university researchers. GOVERNMENT INTEREST: D. Jay Grimes sees DOE funding expanding. For academic scientists, basic extremozyme research is promising, according to D. Jay Grimes of DOE's office of energy research. "DOE's research dollars [for studies of extremozymes] are definitely expanding," says Grimes. "I can't say just how much DOE money there is because it comes in pieces, in several different programs. But we are definitely interested in novel enzymes." In October, for example, DOE will announce a new bioremediation program that encourages researchers to genetically modify toxin-eating organisms, including extremozymes' parent microbes. "Some of the Methanococcus proteins appear to be metal-binding proteins, which means they might be useful in removing metals from waste streams," notes Grimes. "The enzymes might also offer genes for engineering into other organisms for bioremediation." CALLING ALL EXPERTS: Robert Kelly notes need for experience. Baross adds that because so little is known about extremozymes and their parent microbes, researchers have much to study. "There is a lot more work to be done on the physiology and biochemistry of these organisms," he says. "Since we just tapped into this new domain, we're going to see a massive blooming of research on enzyme diversity that corresponds to [extreme] organisms." Adding to the mystery and opportunity, researchers estimate that they have studied less than 2 percent of all environmental microbes. "Right now, we're studying just a small fraction of what's out there," Kelly says. And that means many more extremozymes have yet to be discoverered. Kathryn S. Brown is a freelance science writer based in Columbia, Mo.

Scientists Find Jobs Turning 'Extremozymes' Into Industrial Catalysts

Sep 29, 1996

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