Corn Primed for Making Biofuel

Shoots and leaves: To facilitate the breakdown of cellulose into fermentable sugars for making ethanol, Mariam Sticklen of Michigan State University is genetically modifying corn with genes that produce cellulose-degrading enzymes in the plant’s stems and leaves. The enzymes are activated only after the corn is harvested, when the plant is ground up.
Credit: Michigan State University

In an effort to help boost the nation's supply of biofuels, researchers have created three strains of genetically modified corn to manufacture enzymes that break down the plant's cellulose into sugars that can be fermented into ethanol. Incorporating such enzymes directly into the plants could reduce the cost of converting cellulose into biofuel.

Last year, new federal regulations called for production of renewable fuels to increase to 36 billion gallons annually--nearly five times current levels--by 2022. Today, nearly all fuel ethanol in the United States is produced from corn kernels. To meet the required increase, researchers are turning to other sources, such as cellulose, a complex carbohydrate found in all plants. Corn leaves and stems, prairie grasses, and wood chips are leading candidates for supplies of cellulose. Cellulosic ethanol has many advantages over that produced from corn kernels. Cellulose is not only extremely abundant and inexpensive; studies also suggest that the production and use of ethanol from cellulose could yield fewer greenhouse gases.

However, the biggest obstacle to making cellulosic ethanol commercially feasible is the breakdown of cellulose. Enzymes that degrade cellulose, called cellulases, are typically produced by microbes grown inside large bioreactors, an expensive and energy-intensive process. "In order to make cellulosic ethanol really competitive, we really need to bring those costs down," says Michael J. Blaylock, vice president of system development at Edenspace, a crop biotechnology firm based in Manhattan, KS.

Mariam Sticklen, professor of crop and soil science at Michigan State University, in East Lansing, figured that she could eliminate the cost of manufacturing enzymes by engineering corn plants to produce the enzymes themselves. Instead of relying on the energy-intensive process of producing them in bioreactors, "the plants use the free energy of the sun to produce the enzymes," she says.

Typically, the breakdown of cellulose requires three different cellulases. Last year, Sticklen reported modifying corn with a gene for a cellulase that cuts the long cellulose chains into smaller pieces. The gene came from a microbe that lives in a hot spring. A month later, Sticklen inserted a gene derived from a soil fungus into the corn genome. That gene codes for an enzyme that breaks the smaller pieces of cellulose into pairs of glucose molecules. In this latest effort, Sticklen has modified corn to produce an enzyme that splits the glucose pairs into individual sugar molecules; the enzyme is naturally produced by a microbe that lives inside a cow's stomach. The final result: three strains of corn, each of which produces an enzyme essential to the complete breakdown of cellulose.

To avoid the possibility of transferring the genes to other crops or wild plants, the enzymes are only produced in the plant's leaves and stems, not in its seeds, roots, or pollen, says Sticklen. What's more, to prevent the corn from digesting itself, she engineered the plants so that the enzymes accumulate only in special storage compartments inside the cells, called vacuoles. The cellulases are released only after the plant is harvested, during processing. Sticklen described her modified crops last week at the American Chemical Society's national meeting in New Orleans.

Although it's possible to incorporate all three genes in a single plant, says Sticklen, using three different varieties of corn, each carrying a different gene, will allow her to control the conversion of cellulose into sugars. Preliminary studies show that the enzymes are just as efficient as commercially available enzymes when combined at a ratio of 1:4:1, she says. The results suggest that mixing the three different plants using the same ratios will provide the best outcome.

"I think the strategy of compartmentalizing the enzymes in the vacuoles is terrific," says Susan Leschine, a microbiologist at the University of Massachusetts Amherst. "The question I have is, do the enzymes work under conditions that are realistic?" For instance, different microbe species secrete their own cellulases that work synergistically to chip away at the cellulose fibers. It's unclear, Leschine says, how well an enzyme taken from a microbe that lives in a hot spring will work with an enzyme drawn from a soil fungus. "These different enzymes may not be active under the same conditions," she says.

Edenspace, which is currently developing Sticklen's technology, expects to begin field trials of her genetically modified corn within the year, with the goal of commercializing the technology within the next three years, says Blaylock. The company is not alone in pursuing this strategy: Agrivida, an agricultural biotech company based in Medford, MA, is also genetically modifying corn to simplify the production of cellulosic ethanol.

"This really is a worthwhile path to follow," says Michael Ladisch, professor of agricultural and biological engineering at Purdue University, in West Lafayette, IN. "However, at the end of the day, it's more complicated than it seems." The main obstacle is finding ways to ensure that the enzymes will survive the chemical and physical pretreatment needed to remove the lignin--the tough polymer in cell walls that provides plants with strength--from the cellulose fibers, says Ladisch, who is currently on leave from Purdue to serve as the chief technical officer at Mascoma, a biofuels company based in Brighton, MA.

One solution is to engineer the plants so that they require only a mild pretreatment. For instance, Sticklen is working on reducing the amount of lignin contained in corn, as well as modifying the molecular configuration of lignin, which would make it easier to break down. Although her work is currently focused on modifying corn, Sticklen says that the technology could eventually be transferred to other crops as well, such as switchgrass.

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