Jailbreaking Lignin - Voiding Nature's Warranty On Cellulose

Lignin is nature’s plastic. It gives plants the ability to stand tall and to withstand weather, insects, and diseases. But while lignin is useful for plants, it’s an obstacle to humans who want to use cellulose—a type of sugar found in plant cells that is tightly interwoven with lignin—to make biofuels.

John Carlson and Ming Tien

John Carlson (left) and Ming Tien are pushing their research to bring technology to the marketplace to help make cellulosic ethanol a viable fuel.

“To get to cellulose to make biofuel, you have to get around the lignin barrier,” says John Carlson, professor of molecular genetics in the Department of Ecosystem Science and Management and director of the Schatz Center for Tree Molecular Genetics.

Yet breaking the bond between cellulose and lignin is no easy task. Researchers have long sought cheaper, easier, and better ways to do this. Carlson’s collaborator Ming Tien, professor of biochemistry in the Eberly College of Science, and others have researched the use of naturally occurring enzymes in fungi to degrade the lignin barrier, for example. Other researchers have tried to genetically engineer plants with less lignin, but found that the resulting plants grew more slowly or weren’t sufficiently hardy.

New Approach to an Old Problem

In 2004, Carlson and Tien hatched a plan for how to modify lignin without decreasing its content within the plant cells. They would use genetic engineering to transfer a gene encoding a cell wall protein from parsley into young hybrid poplar trees. They would do this just as the poplar trees were becoming woody through the production of lignin. The goal was to replace some of the crosslinks, or connections, between lignin and cellulose with crosslinks between lignin and the new protein.

The idea worked. The modified crosslinking produced a type of lignin that is similar in strength to regular lignin, so the trees had the strength to grow. “The important difference,” Carlson says, “is that we were able to break up this lignin using enzymes that cut proteins, which are already commercially available, rather than enzymes that attack lignin, which are not.”

According to Carlson, the introduced crosslinks are weak spots where protein-cutting enzymes, called “proteases,” can work. “Using this method, more cellulose is released from lignin and becomes available for industrial or agricultural processing,” he says.

Postdoctoral fellow Haiying Liang had experience doing this type of genetic engineering, called “transformation,” with hybrid poplar trees, the first tree for which the entire genome was sequenced. It also grows rapidly and sprouts from a cutting, all of which made it a model test subject.

From the beginning of the process, Liang had surprising success with the transformation. After growing the transformed trees in a greenhouse for about a year, to about 10 feet tall, Liang compared them with wild hybrid poplar. She found that total lignin content, plant structure, and growth rate were unchanged in the transformed trees. But most important, she also found that the amount of sugar released by proteases was higher in the transformed trees than in the wild hybrid trees, proving that lignin digestibility increased.

Through trial and error, the researchers found that the amino acid tyrosine crosslinked most readily with cellulose, so they experimented with transferring genes that were coded for various proteins that are high in tyrosine. They identified a particular gene that works exceptionally well.

The National Science Foundation refers to this type of work in which a technology is created that enables the creation of products and processes as a “platform technology.” In this case, the platform technology is the gene that aids in the breakdown of lignin in woody plants. Its use could increase the efficiency of biofuel production with woody plants, forage use by grazing animals, and pulp and paper production.

Upon learning about the team’s results, several biotechnology companies approached Carlson and Tien wanting to license the technology for further exploration. But the pair decided they wanted to try to optimize the process further before licensing it. They wanted to determine just how useful the process really was.

Lignolink Is Born

In December 2012, Penn State obtained a patent for the technology. Lignolink, Inc., the start-up company that Carlson and Tien founded, optioned the technology.
Recently, Lignolink received a Small Business Innovation Research grant from the National Science Foundation to optimize the process for corn. Carlson points out that besides the challenges of working with a new species, they and their two employees still have a lot to learn about the process, even in hybrid poplar trees. For instance, they want to discover the optimal number for the percent increase in protein-lignin linkages, and how well other proteins might work. There’s a fine balance between achieving the goal of increasing lignin digestibility and decreasing plant fitness.

Carlson and Tien hope to refine the technology within about three years so it can be licensed to biotechnology companies. Carlson estimates that the companies will need about five additional years to develop the most efficient cultivars and get through the testing and regulatory processes before there might be field trials with corn.

The timeline for poplar is less certain. The U.S. Department of Agriculture has not yet permitted large-scale field trials of genetically transformed trees because of concerns that the transgene could escape into wild trees. However, neither Carlson nor Tien thinks this would happen or would make a difference in plant health.
“We’re just taking something nature makes and putting a little more of it in the cell wall,” says Tien.

Will Cellulosic Biofuel Become Viable?

Societal concerns about genetic engineering and the difficulty of releasing cellulose from lignin are two major impediments to the potential future for biofuel made from nontraditional sources.

“Right now we’re at a crossroads in biofuels,” says Carlson. “The first generation of biofuels came from sugar cane, sugar beets, and corn, plants from which people have a long history of extracting sugars and using yeast to ferment the sugars into alcohol—usually ethanol. It is relatively easy and cost effective to make biofuel from those plants.”

In contrast, the biofuels industry has not been so successful with cellulosic ethanol. It’s made from the inedible parts of plants, including corn stalks and leaves, switchgrass, Miscanthus grass species, woodchips, and lawn and tree trimmings. But actual production of the fuel from cellulosic materials is not currently at required commercial scales.

“If cellulosic ethanol is to become a viable fuel, we need a technology such as Lignolink to make it more cost effective,” says Carlson.

Until earlier this year, the demand for cellulosic ethanol was there, although the supply wasn’t. The U.S. Environmental Protection Agency had set a standard for how much cellulosic ethanol should be in gasoline, but the fuel just wasn’t available. In January, the petroleum industry successfully fought to have the standard nullified.

Beyond Biofuel

Even if cellulosic biofuel never takes off, the methodology has other important potential uses. One is in forage crops.

A cow eating a forage plant extracts from the plant a certain amount of cellulose. Some of the plant is indigestible because of the tight interweaving of lignin and cellulose. Lignolink technology may allow the genetic engineering of forage cultivars to free more cellulose from lignin. This would let the cow draw more energy from the plant using its own naturally occurring proteases, thus increasing feeding efficiency, and therefore, the value of the forage crop.

More easily digestible forages might also help reduce digestive problems, such as excess gas, that occur when grazing animals eat crops higher in lignin. This could lessen greenhouse gas production, potentially slowing climate change. Globally, livestock are responsible for a significant share of greenhouse gas emissions.

The technology should also be useful in pulp and paper making, where pretreatment to separate cellulose from lignin is expensive and energy intensive and often uses harsh chemicals.

No matter which business sectors end up licensing the Lignolink technology, it seems likely to make an important difference in the world. For one, it might help feed the world by allowing animals to get more nutrition from plants.

Alternatively, says Tien, “Nobody believes there’s a limitless supply of fossil fuels. Plants are still the best solar-energy-capturing 
devices we know of, and we’ll always be dependent on them. Anything that can make plants’ lignin easier to break down will improve the efficiency and viability of biofuel production.”

Joy Drohan