Addition by subtraction. By removing a specific gene, researchers reduced the amount of lignin (stained red) by 36% in cells in a plant’s stem, making it easier to recover sugar-rich parts of the plant that can be converted to automotive

Lisa Sundin; (inset) Ruben Vanholme

Fuel From Waste?

In the biofuels business, the idea of converting nonfood plants to fuels is like a mirage: enticing but seemingly just out of reach. To do it, the industry must add a cocktail of expensive enzymes to break down the plants into sugars that microbes can easily transform into chemical precursors for gasoline, diesel, and jet fuel. Now researchers have used genetic engineering to overcome these problems. If the technique can be improved further, nonagricultural plants could be used to make all of the most common transportation fuels.

Most biofuel makers today use microbes to synthesize ethanol from either cornstarch or sugar produced from sugarcane. That has raised widespread concerns over food security because corn and sugarcane grown for ethanol compete with valuable agricultural land needed to feed the planet's 7 billion people. Biofuel brewers would prefer to convert either agricultural waste or other nonfood plants, such as trees and grasses, to fuels. Sugar and cornstarch are easy to ferment and convert to ethanol. But nonfood plants, or "cellulosic biomass," are more complex.

Cellulosic biomass contains a mixture of chainlike sugars called cellulose and hemicellulose that are embedded in a woody material called lignin. To convert cellulose and hemicellulose to fuels, researchers must first break them down into individual sugars that microbes can eat. That's normally done by adding a cocktail of six enzymes to a solution containing the plants. But brewing up those enzymes and purifying them is expensive. It typically costs about 25 cents per liter of the final fuel.

To get around this problem, researchers have inserted genes for these enzymes into biofuel-making bugs. In 2009, for example, researchers at Mascoma, a biofuels company in Lebanon, New Hampshire, inserted several such enzymes into two different microbes already capable of converting simple sugars to ethanol. The added genes allowed the bugs to live on cellulose, breaking it down and converting it to ethanol, a technology the company is now commercializing. But in most countries, ethanol is not the primary transportation fuel.

So a team led by Jay Keasling, a bioengineer at the Joint BioEnergy Institute in Emeryville, California, worked to extend the strategy to make more commonly used fuels. They used Escherichia coli, a bacterium into which it's relatively easy to insert new genes. They started by creating two strains of E. coli, inserting genes for breaking down cellulose in one and genes for breaking down hemicellulose in the other. They then split each of these two strains into three groups and to each group added genes for one of three different metabolic pathways that allow the microbes to make chemical precursors for either gasoline, diesel, or jet fuel.

After treating a common cellulosic biofuels plant called switchgrass with a compound known as an ionic liquid (IL) to break apart the plant fibers and reduce the lignin, the researchers added their engineered E. coli. And in an upcoming issue of the Proceedings of the National Academy of Sciences, the team reported that their E. coli could grow on IL-treated switchgrass and generate all three fuels.

"It's an important proof of concept," says Bruce Dale, a chemical engineer at Michigan State University in East Lansing. However, he hastens to add that the work is "a long way from being any sort of commercial process." For now, the bugs still produce only about one-tenth of the enzymes they need to break cellulose and hemicellulose, and the amount of fuel they generate is low. Keasling says that his team will try to improve both of those issues by engineering similar genes into yeast, a more industrially useful microbe. If it works, the true promise of biofuels may finally come within reach.