The Genetics of Ethanol Production

Two recently released genome studies are expected to provide answers to how to bolster the production of ethanol from corn and biomass.
By Jessica Ebert | May 09, 2008
Like the blueprint of a home showing all its support beams, plumbing and wiring, the genetic information carried in the cells of all living things provide information about the growth, development and persistence of those organisms. Boiled down to its basic building blocks, the deoxyribonucleic acid (DNA) of every human, plant, animal or microbe consists of four chemical components called nucleotides, which are denoted A, T, C and G. These molecules are grouped into segments called genes, which are the basic units of heredity. Genes direct the production of proteins and other molecules that ultimately define things such as how an organism looks, how it moves and how it responds to changes in its environment. To have access to the complete sequence of an organism is like being given an instruction manual for the construction of that form of life. Scientists mine these genetic sequences to learn such things as how bacteria cause disease because unlocking the genetic cues that lead to sickness can also reveal the key to prevention.

Scientists hope the same will hold true for the production of biofuels; that by unraveling the information trapped in the DNA of various bioenergy feedstocks or the organisms associated with them, researchers will uncover the means to improve or optimize these feedstocks or to identify better enzymes for degrading cellulose and hemicellulose or for converting biomass into fuels. The genomes of several organisms linked to biofuels production have already been sequenced including the poplar tree and the microbes found in the termite gut. Now, researchers report the results of two sequencing efforts—one to sequence the corn genome and one to sequence a fungus that plays a pivotal role in the growth and productivity of the poplar tree.

Corn's Genetic Blueprint
The effort to sequence the corn genome was initiated in the late 1990s. At this time, the National Corn Growers Association worked with Sen. Kit Bond, R-Mo., to advocate for the establishment of the National Plant Genome Initiative. This research program was ultimately established in 1998 with the long-term aim of exploring the structure and function of the DNA of certain plants, including corn. However, the sequencing technology available in the late 20th century was not advanced enough to deal with the corn genome. "The corn genome is as big as the human genome but more complex," explains Patrick Schnable, a geneticist at Iowa State University who has been studying the maize genome since the early 1980s. "It was the corn geneticists who suggested that we first develop the techniques and reagents that we would need to sequence the genome. A lot of energy went into building these tools." For example, because it's not feasible to sequence an intact genome of billions of bases, an organism's DNA is chopped into small fragments and sequenced. Those sequences are then pieced together and assigned to their respective chromosomes to provide a complete genome sequence. This latter step takes a significant amount of supercomputing power, which requires advanced software technology.

ISU researchers led by Srinivas Aluru have developed algorithms and software that allow for more information to be extracted from genome sequences more quickly. By 2005, the time was right for the National Science Foundation, under the auspices of the NPGI, the USDA and the U.S. DOE to fund a three-year, $32 million project to sequence the corn genome. The effort is led by Richard Wilson who directs the Genome Sequencing Center at the Washington University School of Medicine in St. Louis, Mo. The team includes researchers from the University of Arizona in Tucson, Cold Spring Harbor Laboratory in New York and ISU.

The completion of the first draft of the sequence was announced at the 50th Annual Maize Genetics Conference in Washington, D.C., on Feb. 28. "The first draft of the genome sequence is exciting because it's the first comprehensive glimpse at the blueprint for the corn plant," Wilson says. "Scientists now will be able to accurately and efficiently probe the corn genome to find ways to improve breeding and subsequently increase crop yields and resistance to drought and disease." This draft covers 95 percent of the 2.5 billion nucleotides or A, C, G, T bases that make up the corn genome. "Although it's still missing a few bits, the draft genome sequence is empowering," Wilson says. "Virtually all the information is there, and while we may make some small modifications to the genetic sequence, we don't expect major changes."

The variety of corn used in the project was developed by ISU and USDA researchers. Dubbed B73, this line is known for conferring high-yield properties to hybrids and is widely used to produce many commercial hybrids. Although scientists will spend the remaining year of the grant refining and finalizing the sequence, the genetic data is available to the public at www.maizesequence.org. Researchers are already studying the genetic data to find ways to make the corn plant more nutritious, more drought-resistant or more efficient for ethanol production among other things.

Schnable's team was one of several that, for instance, recognized that the corn genome contains twice as many genes as the human genome. Although it's not intuitively obvious why this would be, he explains that the human genome directs the development of elements like the nervous system, which allows for thinking and responding to the environment. Plants don't have this ability to reason. "Plants, in a sense, are more hardwired for solutions," Schnable says. "They need more of that than we do." Hence, they need more genes.

In Schnable's lab, they're trying to mine the 50,000 to 60,000 genes of corn to find those associated with a phenomenon called hybrid vigor, or the likelihood that when you cross two varieties, the resulting hybrid plant will exceed the parents in height, yield and biomass—simply their vigor. "It's like pulling the lever on a slot machine, sometimes it works, sometimes it doesn't," Schnable explains. "We'd like to figure out what's going on inside the slot machine so that when we pull the lever we know it's going to work." In other words, the goal is to be able to predict which varieties should be crossed to make the best hybrid for properties like yield, starch content for ethanol production or lignocellulose for cellulosic ethanol.

In addition, Schnable is trying to identify the genes that determine the plants' cell wall composition. Whereas most researchers are trying to engineer lignocellulose crops with cell walls that are more easily digestible, Schnable is going the other way. "If we grow crops with cell walls that don't break down easily, soil organic matter will increase and move carbon dioxide will be removed from the atmosphere," he explains. It's another form of carbon sequestration. "Having a genome sequence opens the door to all kinds of improvements," Schnable adds.

A Fungal Genome
In a second genome study, researchers associated with the U.S. DOE Joint Genome Institute recently reported in the journal Nature, the sequence of the fungus Laccaria bicolor. The mission of the JGI is to sequence and conduct data analysis on genomes selected by the DOE's new bioenergy research centers. The genetic information generated by JGI will aid in the centers' mandate to develop biobased alternatives to fossil fuels.

L. bicolor is a target organism for sequencing because it forms beneficial partnerships with plants. "Laccaria is a symbiotic associate of poplar," says Jerry Tuskan, a plant geneticist and program lead for JGI's Laboratory Science Program. "It's a fungus that infects the root systems of poplar trees but the infection is actually beneficial to both organisms." L. bicolor produces long, branching filaments that form a sheath around the root. These filaments also extend into the surrounding soil where they take in elements like nitrogen, phosphorus and potassium and transfer these nutrients to the plant. In return, the plant provides the fungus with sugars and phosphates, which the microbe needs to grow. "There's a mutualistic exchange of responsibilities in a way," Tuskan says.

But how does this relate to bioenergy? Plants intended for biofuel production such as poplar, willow, switchgrass or miscanthus have to compete with land destined for raising food crops. So the lands that are economically available for biomass crops tend to be more marginal areas—lands where you would have to add nutrients or water to maximize productivity. "By modifying the relationship between poplar and Laccaria, we can plant or deploy poplar onto marginal lands without irrigating or fertilizing," Tuskan explains. "The symbiotic relationship allows poplar to obtain more nutrients and tolerate drought better because the root system is enhanced by Laccaria." This allows producers to capitalize on lands that may not have been available otherwise.

Optimizing the relationship between poplar and L. bicolor for various soil conditions requires an understanding of the genes involved in the interactions between these two organisms. "The genome is a predictive catalog of genes contained in an organism," Tuskan says. "Without the catalog we can do informative studies. We can make crosses and measure progeny and predict a genetic gain, but we do that without understanding the genes behind that gain. When you have the genome available it allows you to target and predict candidates."

Tuskan led the effort to sequence poplar in 2006, the first tree genome to be sequenced. He is also a member of the team of scientists who wrote the proposal to have JGI sequence the L. bicolor genome. "Now we have both genomes," he explains. "We can maximize the benefits of that mutualistic relationship by studying how they interact and what they do to benefit each other."

This is the research direction that Tuskan is currently pursuing. One of the major insights into the Laccaria-poplar interaction provided by the fungal genome is that L. bicolor harbors a wealth of genes for the production of proteins that get excreted from the organism's cells. "It turns out that these proteins are signals," Tuskan explains. "When the protein comes in contact with poplar it is recognized by poplar." This chemically mediated familiarity allows the fungus to intercalate between the cells of poplar and infect the root system. In addition, a second class of proteins was unexpectedly found in the Laccaria genome that allows the fungus to interact with plant cell walls, which favors the growth of the microbe.

The Laccaria team is now trying to bring out the subtleties of these interactions by inoculating poplar with Laccaria and studying gene expression in both organisms all the way through maturation. "We're trying to understand how the genetic networks between the two organisms communicate with each other." Then, the researchers will place the plant-fungus system under various constraints such as drought stress, nitrogen stress, cold and heat stress, and identify the genes involved in the organism's ability to interact and collaborate to deal with those exposures. "Hopefully we'll learn from that and be able to regulate how poplar will be able to tolerate drought or nitrogen limits in the field," Tuskan explains. "Natural selection optimized the relationship for survival in the wild," he says. "But what we can do now is optimize this community for bioenergy production."

Jessica Ebert is an Ethanol Producer Magazine staff writer.