Roots Biotech

Nancy Marie Brown
December 01, 2004

Two thousand years ago, a Chinese healer might have mashed the thick tuberous root of the Mongolian snake gourd, steeped it in fresh water, sieved out the grounds, and then sponged the liquor on a tumor, mixed it with other medicines to treat diabetes, or prescribed it as a potion against pregnancy.

Today, the preparation is higher tech: Glass cylinders filled with steel coils and knots, roots growing on them thick as hair, water percolating through, picking up whatever chemicals the roots produce. Then distilling, separating, concentrating . . . The result is a protein that kills the AIDS virus in human lymphocytes.

yellowish rounded mass of roots

Roots of the Chinese medicinal cucumber become a chemical factory in a biotechnological collaboration between plant physiologists and chemical engineers.

The Mongolian snake gourd—a.k.a. the Chinese medicinal cucumber or Trichosanthes kirilowii—is an excellent candidate for chemical prospecting, a term coined by Cornell biologist Thomas Eisner to describe the systematic searching of nature for "drugs, pesticides, natural food flavorings, more healthful cooking oils, even perfume components." The basic protocol is to first examine plants known to be "biologically active"; in general, any plant used in traditional medicines is fair game.

In the 1980s, Chinese chemical prospectors isolated the active ingredient in the Mongolian snake gourd. Tricosanthin, as the protein was named, was the dominant protein in the plant's storage root, accounting for some 25 percent of the extractable protein in some populations of the plant. It was already in clinical trials in China for use in abortions and to treat ectopic pregnancies when researchers in Taiwan and the United States discovered that this protein with the 2,000-year-old medicinal history could inhibit the reproduction of the HIV virus in human lymphocytes grown in cell cultures.

Soon tricosanthin, under the street name "compound Q," was being used clandestinely to treat AIDS patients in San Francisco. Hector Flores, an associate professor of plant pathology at Penn State, read about it in the New York Times. "Since Hector's focus is in root metabolism," says his graduate student Brett Savary, "the statement that this protein came from the roots caught his interest. He obtained seeds from a medicinal garden in Japan. The seeds germinated, he grew his first cultures—and at about that time, I arrived."

Savary had completed a master's degree in botany at the University of Tennessee, where he'd developed an interest in medicinal plants; he joined Flores's research group at Penn State in order to combine botany, biochemistry, and plant physiology. He would soon have to add biotechnology to that list.

"I'm not a medical researcher," says Savary. "What I've been focusing on is understanding the biosynthesis of tricosanthin in the plant. If we understand how the protein is synthesized in the plant, we can perhaps make a better protein, or produce the protein itself for use."

In the case of compound Q, Savary set out to produce it in a system Flores and his chemical engineering colleague, Wayne Curtis, had developed to transform roots into chemical factories.

To begin, explains Savary, "we take advantage of a natural genetic engineer, Agrobacterium rhizogenes." This bacterium, responsible for "hairy root" disease in plants, contains an extra piece of DNA, called a plasmid, that easily slips into a plant's own genetic code, reprogramming it to overproduce roots.

"The roots are essentially immortalized," says Savary. "They will continue to grow as roots, generation after generation," never needing shoots, leaves, or fruit. Yet, "They express the normal metabolism of root cells. They are genetically stable over time. You have normal mitoses, normal cell divisions. It's very well regulated and controlled.

"Jumping to the end of the story," Savary adds, "we found that these root cultures will produce tricosanthin"—compound Q—"but at very low levels. The synthesis of this protein, we found, is associated with the change from primary roots to secondary, or storage, roots. That's the most important result of this study.

"We don't have an experimental system established for storage roots yet. That's sort of the Holy Grail for this project, being able to induce the secondary growth pattern that causes the broadening into a storage root. First you have to define a system to take fibrous roots and turn them into storage roots, and we don't have that. It would open up an entire new route of research for this lab.

"But we're not moving too fast on that now. The interest in compound Q has started to wane." Clinical trials by AIDS researchers have uncovered disappointing side effects. "They're trying to understand it better now. It's not like taxol," Savary adds, "which was recently approved as a therapy." (Another of Flores' students, is working on that anticancer plant product.) "Taxol brings a greater return on your research investment."

But work is going ahead on the engineering side, by several of Curtis's graduate students, to transform Trichosanthes kirilowii into a 20th-century roots factory. "Three of us are working on reactors," says graduate student Divakar Ramakrishnan, "using three different designs: a bubble column, a submerged flow reactor, and a trickle flow reactor." By altering how the glass columns are packed with steel coils and knots, how air and nutrients are filtered to the roots, and how the chemicals are extracted, they are hoping to maximize production of any useful chemicals the wild Trichosanthes kirilowii (or, perhaps, a genetically engineered cousin) might excrete.

"The fibrous roots also produce another protein, a chitinase, that has antifungal properties," Savary notes. "A hypothesis I have is that this protein may interact with tricosanthin to protect the roots against soil invertebrates, say, nematodes." Savary plans to test his hypothesis on nematodes in the lab and to isolate the gene that codes for this chitinase, in hopes that he'll have a chemical worth collecting—or imitating.

For as Eisner has said of any chemical prospecting, "Chances are the chemicals are new. Chances are the chemicals are interesting. Nature has been a constant source of inspiration to the chemist." Brett Savary and Divakar Ramakrishnan, along with Fabricio Medina-Bolivar and Gurmeet Singh, won third place in the Health and Life Sciences category of the 1994 Graduate Research Exhibition. Savary and Medina-Bolivar are Ph.D. students in the intercollege graduate degree program in plant physiology, The Graduate School, University Park, PA 16802; 814-865-2956. Ramakrishnan and Singh are Ph.D. students in the department of chemical engineering, College of Engineering, 158 Fenske Lab; 865-2574. Their advisers are Hector Flores, Ph.D., associate professor of plant pathology (865-2955) and Wayne Curtis, Ph.D., assistant professor of chemical engineering (863-4805). The research reported here is funded by the National Science Foundation, the Pennsylvania Research Corporation, and a Sigma Xi Research Grant-in-Aid.

Last Updated December 01, 2004