The Future, Unzipped

John Ralph PhD’82 talks with the easy, garrulous rhythms of his native New Zealand, and often seems amiably close to the edge of laughter.

So he was inclined toward amusement last year when he discovered that some portion of the Internet had misunderstood his latest research. Ralph—a CALS biochemist with joint appointments in biochemistry and biological systems engineering—had just unveiled a way to tweak the lignin that helps give plants their backbone. A kind of a natural plastic or binder, lignin gets in the way of some industrial processes, and Ralph’s team had cracked a complicated puzzle of genetics and chemistry to address the problem. They call it zip-lignin, because the modified lignin comes apart—roughly—like a zipper.

One writer at an influential publication called it “self-destructing” lignin. Not a bad turn of phrase—but not exactly accurate, either. For a geeky science story the news spread far, and by the time it had spread across the Internet, a random blogger could be found complaining about the dangers of walking through forests full of detonating trees.

Turning the misunderstanding into a teachable moment, Ralph went image surfing, and his standard KeyNote talk now contains a picture of a man puzzling over the shattered remains of a tree. “Oh noooo!” the caption reads. “I’ll be peacefully walking in a national park and these dang GM trees are going to be exploding all around me!”

That’s obviously a crazy scenario. But if the technology works as Ralph predicts, the potential changes to biofuels and paper production could rewrite the economics of these industries, and in the process lead to an entirely new natural chemical sector.

“When we talk to people in the biofuels industry, what we are hearing is that creating value from lignin could be game-changing,” says Timothy Donohue, a CALS professor of bacteriology and director of the UW–Madison-based Great Lakes Bioenergy Research Center, where Ralph has a lab. “It could be catalytic.”

After cellulose, lignin is the most abundant organic compound on the planet. Lignin surrounds and shapes our entire lives. Most of us have no idea—yet we are the constant beneficiaries of its strength and binding power.

When plants are growing, it’s the stiffening of the cell wall that creates their visible architecture. Carbohydrate polymers—primarily cellulose and hemicelluloses—and a small amount of protein make up a sort of scaffolding for the construction of plant cell walls. And lignin is the glue, surrounding and encasing this fibrous matrix with a durable and water-resistant polymer—almost like plastic. Some liken lignin to the resin in fiberglass.

Without lignin, the pine cannot soar into the sky, and the woody herb soon succumbs to rot. Found primarily in land plants, a form of lignin has been identified in seaweed, suggesting deep evolutionary origins as much as a billion years ago.

“Lignin is a funny thing,” says Ralph, who was first introduced to lignin chemistry as a young student during a holiday internship at New Zealand’s Forest Research Institute. “People who get into it for a little bit end up staying there the rest of their lives.”

The fascination is born, in part, from its unique chemistry. Enzymes, proteins that catalyze reactions, orchestrate the assembly of complex cell wall carbohydrates from building blocks like xylose and glucose. The types of enzymes present in cells therefore determine the composition of the wall.

Lignin is more enigmatic, says Ralph. Although its parts (called monomers) are assembled using enzymes, the polymerization of these parts into lignin does not require enzymes but instead relies on just the chemistry of the monomers and their radical coupling reactions. “It’s combinatorial, and so you make a polymer in which no two molecules are the same, perhaps anywhere in the whole plant,” says Ralph.

This flexible construction is at the heart of lignin’s toughness, but it’s also a major obstacle for the production of paper and biofuels. Both industries need the high-value carbohydrates, especially the cellulose fraction. And both have to peel away the lignin to get to the treasure inside. A combination of heat, pressure, and caustic soda is standard procedure for liberating cellulose to make paper; bleach removes the remaining lignin. In the biofuels industry, a heat and acid or alkaline treatment is often used to crack the lignin so that it is easier to produce the required simple sugars from cellulose. Leftover lignin is typically burned.

The economic cost of these treatments alone is significant, and lignin pretreatment is at the heart of many of the more egregious environmental costs of paper. On the biofuels side, lowering treatment costs to liberate carbohydrates from lignin could change the very economics of biofuels. In these large-scale, industrial processes, saving a percentage point or two is often worthwhile, but the Holy Grail is a quantum jump.

“Because it’s made this way”—Ralph jams his hands together, crazy-wise, fingers twisted together into a dramatic representation of lignin polymerization—“there is no chemistry or biology that takes it apart in an exquisite way,” he says. “We actually stepped back and thought: How would we like to design lignin? If we could introduce easily cleavable bonds into the backbone, we could break it like a hot knife through butter. How much can you actually mess with this chemistry before the tree falls down?”

Ralph’s team had their eureka moment more than 15 years ago, and have been trying to bring it to life ever since.

With a background in forage production and ruminant nutrition, John Grabber, an agronomist at the USDA–Agricultural Research Services’ Dairy Forage Research Center in Madison, got pulled into lignin chemistry through the barn door. On his family’s dairy farm he grew up with lignin stuck to his boots, though he never knew it. But during graduate school he became interested in how plants are digested by cows. Cell walls are potentially a great source of digestible carbohydrates—most plants contain anywhere from 30 to 90 percent of their mass in their cell walls—but it is entangled with lignin. “You quickly find out that lignin is the main barrier to feed digestion,” he says.

Grabber began working on a model system to understand plant lignification—for corn in particular—in 1989. After meeting at a conference, Grabber joined Ralph and plant physiologist Ronald Hatfield at the Dairy Forage Research Center back in 1992. There were many projects ongoing, but Grabber remained interested in trying to fully understand the structural characteristics of the lignin: how it’s made and how to modify it. In his model system they could make any kind of lignin they wanted to study, and see how the changes affected utilization.

Ralph and Hatfield advocated for the work, helping to find funding and offering their expertise. “If I had worked for somebody else I probably wouldn’t be doing this work,” says Grabber. “John and Ron gave me freedom and support to do it.”

Around the same time, Fachuang Lu joined Ralph’s lab seeking a Ph.D. His journey into lignin chemistry was not, at first, his idea. A native of mainland China, he’d enjoyed a successful undergraduate career in Beijing studying chemical engineering, then found himself assigned by the college to a master’s program in lignin chemistry. Lignin is an ingredient in the slurry of chemicals used in oil drilling, and that was his specialty. In 1989 Lu left Beijing for a teaching position at Guangxi University, but three years later he decided to continue his education. Though he’d never met Ralph, he was fascinated by the chemistry and applied to study in his lab.

As Ralph, Grabber, Hatfield and Lu continued to tinker with lignin chemistry, momentum began to build in the lab. Though lignin created a snowflake universe of different molecules, there were rules of assembly. A complex chemical pathway enabled lignin construction, with a mechanism that remained constant across different families of plants, but with many potential building blocks.

Ralph and his colleagues were the first to detail what was happening to lignin as the controlling genes of the biosynthetic pathway were turned on and off, a task ably completed by a slew of outstanding collaborators worldwide with expertise in biotechnological methods—but who lacked the diagnostic structural tools to determine what the plant was doing in response.

Ralph’s team quickly learned that lignification was somewhat flexible. “We figured that we could engineer lignin well beyond the previously held bounds,” says Ralph. As various pathways and chemical possibilities danced in their heads, it struck them: What if, during lignification, they could persuade the plant to slip in a few monomers that had easily broken chemical bonds? If they did it right, lignin would retain its structural value to the plant, but be easier to deal with chemically.

“In the course of our conversation we realized that if plants could do this, it could really revolutionize how readily you could make paper,” recalls Grabber. Says Ralph: “It’s almost impossible to tell which one of us actually verbalized it first—it is one of those great outcomes of the group dynamic.”

Lu’s particular genius was synthesizing the various complex chemicals needed, particularly a novel monomer-conjugate called coniferyl ferulate. It was the key to the zip-lignin—the teeth of the zipper. “He’s got to be one of the best in terms of making molecules,” says Grabber.

They were thrilled by such a revelation, but, in retrospect, they soon realized it was sort of an obvious idea—one suggested by the underlying chemistry and biochemistry of a pathway that was becoming increasingly well understood. Yet it was a discovery of huge potential value. They dropped into stealth mode and began to work on it. They finished important research and stuck it in drawers—signature research, the kind that, when finally published, would capture journal covers. And yet they sat on it, quietly chipping away for nearly a decade.

It helped that there was a flurry of controversy in the field—what Chemical & Engineering News called “the lignin war.” “Part of the reason we could sit on it was that, at the time, making these kinds of molecules was so far-fetched,” says Grabber. “Probably if we had talked about it, people would have laughed at us.”

But as the idea for zip-lignin grew in principle, it became stronger. Lu, Hatfield and colleague Jane Marita MS’97 PhD’01 found that balsa trees and a fiber crop known as kenaf produced very small amounts of coniferyl ferulate. But even as the idea seemed more and more feasible, Hatfield and Marita couldn’t isolate the gene needed to manufacture coniferyl ferulate because of its very low expression in these plants.

And they got stuck. “At the beginning we were thinking that this is just a fantastic idea, but we really didn’t have that much confidence,” says Lu. “Maybe John [Ralph] had more confidence than me.” So they just kept at it. “Every step you think, yes, we are closer, closer, closer.”

In 2008 Ralph moved his work from the Dairy Forage Research Center into UW labs, with research projects under the recently formed Great Lakes Bioenergy Research Center (GLBRC). The center, launched with a $125 million grant from the U.S. Department of Energy that has since been renewed, was just one manifestation of the money and intellectual heft infusing biofuels research—and for zip-lignin it was a lucky move.

During the center’s first full meeting, Curtis Wilkerson, a plant biologist at GLBRC partner Michigan State University, was sitting in the audience when Ralph took his turn at the podium.

Wilkerson is a cell wall specialist. Though lignin is a third of the wall’s carbon and is essential to the way plants conduct water, he confesses he’d never given it much thought. In a room full of cell wall specialists, Ralph would “likely be the only person talking about lignin,” he says. “It just split that way a long time ago. People like myself had very little exposure to what John was thinking.”

It was this kind of academic silo that a place like GLBRC was supposed to breach. Ralph talked about putting ester bonds into lignins and his team’s long search for the elusive enzyme. Wilkerson saw a solution. Due to recent technical advances, the price of determining all of the expressed enzymes in a plant had become more refined and much less expensive. He offered to use these recent developments to try to find the missing enzyme to enable zip-lignin.

From the previous work, Wilkerson knew essentially the size and shape of the puzzle piece he was looking for. He began, quite literally with Google, trolling through the scientific literature looking for a plant that made a lot of coniferyl ferulate. The Chinese medicinal “dong quai” or Chinese angelica (Angelica sinensis) soon emerged as a candidate. Its roots contained about 2 percent coniferyl ferulate.

The team used knowledge about the likely type of enzyme they were searching for and successfully identified the gene and its enzyme that could produce coniferyl ferulate. The whole search took less than six months.

Would you believe an essential tool for the genetic engineering of poplars is a hole punch? That’s the word from Shawn Mansfield, a molecular biochemist at the University of British Columbia, who took the zip-gene from the Angelica and made it work in poplar, a popular tree in the biomass and forest products industry.

Working from Wilkerson’s gene, the first job was figuring out how to tag the new protein so that it fluoresced during imaging. While not necessary to the function of the genetically modified plant, it essentially allows the scientists to check their work: see where the protein is, how much is there, and if it is behaving as a protein should.
Mansfield’s lab also had to find a way to turn the gene on at the right time and place. It could make all the coniferyl ferulate one wanted, but if it wasn’t made at the right time and tissue, there would be no zip-lignin.

After perfecting these finer points, the gene is inserted into a special bacterium—and then the hole punch finally comes into play. Disks punched from poplar leaves are mixed with bacteria that have been inoculated with a special chemical that stimulates the bacteria to share their DNA around. Then the leaf disks are put in a special growth medium. As many as 12 shoots might emerge off of a single disk, but the lab would select and nurture only one shoot from each disk.

In the end they had about 15 successful transgenic candidates that they grew in the greenhouse and then shipped off to Wilkerson and Ralph for further study. Final selection was made based on the amount of fluorescent yellow the trees gave off, and from a newly devised analytical method developed by Lu and Ralph that was particularly diagnostic for the incorporation of the zip monomer into the lignin polymer.

The team knew that genetically modified organisms are not popular or easily talked about—never mind the exploding trees. The idea of reworking a fundamental building block of the plant world will breed resistance.

Ralph argues that this is already part of nature’s vocabulary: they’ve found their building blocks within the plant kingdom, including mutants that do similar things. And now that they know what they are looking for, Steven Karlen, a member of Ralph’s group, is continuing to find more evidence that Mother Nature is doing it herself. “We managed to persuade plants to do this,” Ralph says. “Chances are that nature has already attempted it and you could actually get there by breeding.”

It’s no surprise that Mansfield, who created the final transgenic tree, argues that there is a role for this kind of technology. “We as scientists should be wise in advocating for the proper use of it,” he cautions. “I would never force it on anybody. I would never try to sway people to think that it is the end-all or be-all for everything.”
But given the growing human population and rising CO2 levels, something like zip-lignin has a definite use in reducing the carbon footprint by reducing processing energy and chemical loads. “That means there are less environmental pollutants that need to be cleaned up afterwards,” Mansfield says.

“Our ecological footprint can be much reduced using these kinds of transgenic trees,” he argues. “The caveat is that we need to be very smart about where and how we plant them.”

Not many things in the natural world can take apart lignin, but any homeowner with a deck knows that fungi are up to the task. A recent analysis of mushroom genomes suggests that fungi evolved this ability about 300 million years ago. This is about the end of the Carboniferous era, when earth’s coal production began to slow down. Coincidence? Perhaps not. Now that wood could rot, it probably slowed the burial of organic carbon via tree trunks and other lignin-rich plants.

Could the discovery of zip-lignin signal another transition, and hasten our move away from fossil fuels laid down in the Carboniferous?

Tim Donohue likes to think so. He likens biofuels now to the early oil industry, when oil was simply being turned into liquid fuel while the by-products were burned or dumped. It took a few decades for inventors to capitalize on this now valuable stream of raw materials to build the modern chemical industry.

“Lignin is about 25 to 30 percent of carbon in the plant. So if we’re going to catalyze an industry that makes clean energy and chemicals from plant biomass, figuring out what to do with the lignin is going to be key,” Donohue says.

People in the industry used to joke that you could do a lot of things with lignin except make money from it. But that may be changing. “The economics and profitability of the industry will be very different if lignin can be turned into valuable compounds,” says Donohue.

One of the early efforts to make use of lignin was in Rothschild, Wisconsin, at a company now known as Borregaard LignoTech. When processed properly, lignin has many uses, from the manufacture of vanilla flavor to additives for concrete. There is even a small amount of it in the battery of your car that allows it to keep recharging.

Jerry Gargulak is research manager at Borregaard LignoTech, and learned about zip-lignin recently in his capacity as a scientific advisor to the GLBRC. Despite its many uses, Gargulak and his colleagues dream about a time when lignin can replace carbon black in tires and be used to build carbon fibers and structural plastics.

Zip-lignin and the ideas behind it could bring this day closer. “It gives us a technology that might yield a more interesting lignin-derived starting material,” Gargulak says. “It could potentially lead to a lot of innovation downstream in lignin technology.” But he emphasizes, “There are a lot of i’s to be dotted and t’s to be crossed.”

This story is just beginning. Zip-lignin has a patent and has excited industrial interest that could be worth significant dollars. Ralph and his colleagues continue working to further refine the process, increasing the percentage of zippable bonds in poplar and also inserting the gene into more plants, such as corn and Brachypodium, both grasses.
And in the basement of the shiny new Wisconsin Energy Institute building, where the GLBRC is based, two massive new nuclear magnetic resonance (NMR) spectrometers work 24/7, providing a level of detail into lignin that Ralph has never had before.

“We spend a lot of time looking at these Rorschach test–like figures,” Ralph says of the information generated from the NMR. “The detail in them is unbelievable. These things have been revolutionizing what we do.”

Forever Rising

To begin to understand the outsized potential and sheer weirdness of yeast, it helps to consider the genetics behind one of the world’s most successful and useful microorganisms. It also helps to consider lager.

Lager, or cold-brewed beer, is made possible by the union of two distinct species of yeast. About 500 years ago, these two species, Saccharomyces eubayanus and Saccharomyces cerevisiae, joined in a Bavarian cellar. They gave us a hybrid organism that today underpins an annual global market for lager estimated at one-quarter of a trillion dollars.

“We would not have lager if there hadn’t been a union equivalent to the marriage of humans and chickens,” notes Chris Todd Hittinger PhD’07, a CALS professor of genetics and a co-discoverer of S. eubayanus, the long-sought wild species of yeast that combined with the bread- and wine-making S. cerevisiae to form the beer. “That’s just one product brewed by one interspecies hybrid.”

Yeasts, of course, are central to many things that people depend on, and the widespread domestication in antiquity of S. cerevisiae is considered pivotal to the development of human societies. Bread and wine, in addition to beer, are the obvious fruits of taming the onecelled fungi that give us life’s basics. But various strains and species of yeasts also are partly responsible for cheese, yogurt, sausage, sauerkraut, kimchi, whiskey, cider, sake, soy sauce and a host of other fermented foods and beverages.

Baker’s yeast, according to yeast biologist Michael Culbertson, an emeritus professor and former chair of UW– Madison’s Laboratory of Genetics, ranks as “one of the most important organisms in human history. Leavened bread came from yeast 5,000 years ago.”

Beyond the table, the microbes and their power to ferment have wide-ranging applications, including in agriculture for biocontrol and remediation, as well as for animal feed and fodder. They are also widely used to make industrial biochemicals such as enzymes, flavors and pigments.

What’s more, yeasts are used to degrade chemical pollutants and are employed in various stages of drug discovery and production. Human insulin, for instance, is made with yeast. By inserting the human gene responsible for producing insulin into yeast, the human variant of the hormone is pumped out in quantity, supplanting the less effective bovine form of insulin used previously.

Transforming corn and other feedstocks, such as woody plant matter and agricultural waste, to the biofuel ethanol requires yeast. Hittinger is exploring the application of yeast to that problem through the prism of the Great Lakes Bioenergy Research Center (GLBRC), a Department of Energy-funded partnership between UW–Madison and Michigan State University. Hittinger leads a GLBRC “Yeast BiodesignTeam,” which is probing biofuel applications for interspecies hybrids as well as genome engineering approaches to refine biofuel production using yeasts.

“There are lots and lots of different kinds of yeasts,” explains Hittinger. “Yeasts and fungi have been around since Precambrian time—hundreds of millions of years, for certain. We encounter them every day. They’re all around us and even inside us. They inhabit every continent, including Antarctica. Yeasts fill scores of ecological niches.”

The wild lager beer parent, S. eubayanus, for example, was found after a worldwide search in the sugarrich environment of Patagonian beech trees—or, more specifically, in growths, called “galls,” bulging from them. (How S. eubayanus got to Bavaria hundreds of years ago and made the lager hybrid possible remains a mystery.) It is possible, notes Hittinger, to actually smell the S. eubayanus yeast at work, churning alcohol from the sugars in the galls themselves.

Though the merits of known yeast species for making food, medicines and useful biochemicals are numerous, there are likely many more valuable applications of existing and yet-to-bediscovered yeasts.

For Hittinger and the community of yeast biologists at UW–Madison and beyond, a critical use is in basic scientific discovery. The use of yeast as a research organism was pioneered by Louis Pasteur himself, and much of what we know about biochemical metabolism was first studied in yeasts.

Since the 1970s, the simple baker’s variety of yeast has served as a staple of biology. Because yeasts, like humans and other animals, are eukaryotes— organisms composed of cells with a complex inner architecture, including a nucleus—and because of the ease, speed and precision with which they can be studied and manipulated in the lab, they have contributed significantly to our understanding of the fundamentals of life. And because nature is parsimonious, conserving across organisms and time useful traits encoded as genes, the discoveries made using yeast can often be extended to higher animals, including humans.

“The model yeast, S. cerevisiae, has been instrumental in basic biology,” says Hittinger. “It has told us something aging. In terms of understanding basic processes, it’s a tough model system to beat. It’s a champion model organism for genetics and biochemistry.”

“It is widely unappreciated how thevast terrain of biology has been nourished by yeast,” argues Sean B. Carroll, a CALS professor of genetics and one of the world’s leading evolutionary thinkers. It was in Carroll’s lab a decade ago as a graduate student that Hittinger first turned his attention to yeast, coauthoring a series of high-profile papers that, among other things, used the yeast model to catch nature in the act of natural selection, the proof in the pudding of evolutionary science.

Now the model is about to shift into an even higher gear. The work of Hittinger and others is poised toenhance the yeast model, add many new species to the research mix, and begin to make sense of the evolutionary history of a spectacularly successful and ubiquitous organism. The advent of cheap and fast genomics—the ability to sequence and read the DNA base pairs that make up the genes and genomes of yeasts and all other living organisms—along with the tools of molecular biology and bioinformatics promise a fundamental new understanding and order for yeast biology.

“This is all about weaponry,” explains Carroll, noting that Hittinger, in addition to possessing “great benchtop savvy and skill,” has armed himself remarkably well to exploit yeast genetics through the mutually beneficial prisms of molecular biology, evolutionary biology and bioinformatics (which harnesses computers to help make sense of the bumper harvests of data). “He has a determination and resolve to get the answer to any important question— whatever it takes,” says Carroll.

The big questions on the table for Hittinger and others include ferreting out “the genetic factors that drive species diversification and generate biodiversity,” and weaving that granular understanding into the larger fabric of biology. Because the functional qualities of all the various yeast species differ in order for the microbe to thrive in the many different environments it inhabits, the genetic code that underpins their different physiological and metabolic features varies accordingly.

In short, it takes a diversity of talents to inhabit every major terrestrial and aquatic environment the world has to offer. Species that thrive in South American tree galls and species that eke out a living on human skin require different skill sets in order to cope with vastly different environments and utilize different resources. Each of those skills is determined by the organism’s genetic makeup, and as scientists discover and extract the lode of genomic data found in new species discovered in the wild, new and potentially useful genetic information and metabolic qualities will come to light.

These are big, basic biological questions. But their answers promise far more than simply satisfying scientific curiosity. Yeasts are big business. They are medically and industrially important. The secrets they give up will, without a doubt, amplify our ability to produce food, medicine and industrial biochemicals.

To lay the groundwork, Hittinger and an international collaboration of yeast biologists are setting out, with support from the National Science Foundation (NSF), to map the genetic basis of metabolic diversity by sequencing the genomes of the 1,000 or so known species of yeast in the subphylum that includes Saccharomyces. Three hundred times smaller than the human genome, a typical yeast genome consists of 16 linear chromosomes and, roughly, 6,000 genes and 12 million letters of DNA.

“This is the best possible time to be a yeast biologist,” avers Hittinger. “Our collections have been vastly improved, and we can sequence genomes a hundred at a time. The important thing to know is that yeast is not just one organism or one species. There are thousands of yeasts, and they each have their own evolutionary history.”

Acquiring new species from the wild and sequencing their genomes will enable Hittinger and his colleagues to construct an accurate yeast family tree.

“If we don’t understand what’s out there and how they evolved, we’re notgoing to understand how to make use of them,” Hittinger notes. “Now, we can rip ’em open, get a peek at their genomes and see what the differences are and how they’ve changed over time.”

Thus stalking new strains and species of yeast in the wild is an essential part of the program, according to Hittinger, who routinely dispatches students, including undergraduates, to seek out new yeasts in nature. Half of all the known species of yeast have been described scientifically only within the past 15 years, meaning scientists have only a limited understanding of the world’s yeast diversity.

“Until recently, most strain collections have been paltry and biased towards domesticated strains,” says Hittinger. “If we can expand our understanding of the wild relatives, we can use them as an evolutionary model.Yeasts have a much less welldeveloped history in ecology and natural history.”

A recent yeast hunting excursion in Wisconsin by one of Hittinger’s students yielded three strains of the same S. eubayanus lager yeast parent found in tree galls in South America. Discovered near Sheboygan, the yeast has been cultured in Hittinger’s lab and samples have been provided to CALS food science professor James Steele, whose group is setting up a new comprehensive program in fermentation science and, with the help of a gift from Miller-Coors, a new pilot brewery lab in Babcock Hall. (Steele is also looking to support other fermented beverages in Wisconsin—namely, wine and cider— in both production and education. See sidebar on page 20.)

“We grew up a few hundred billion cells, gave them to Jim Steele to brew beer, and we’re eagerly awaiting the results,” says Hittinger, who explains that another focus of his lab is making interspecies hybrids, such as the lager hybrid. “Now that we’ve identified the wild species, we can make crosses in the lab to make hybrids that produce flavors people are interested in.”

In the food science realm, says Steele, yeast research is focused on the functional characteristics—fermentation qualities, sugar utilization, flavors—of a particular strain of yeast. “How does microbial physiology link to flavor in fermented beverages?” he asks.

Saccharomyces strains are the workhorse and best-known yeasts, including many of the most medically and biotechnologically important. With the $2 million award from NSF,
Hittinger and his colleagues will use the genomes to develop a robust taxonomy of important yeasts and look for the genetic footprints that give rise to yeast biodiversity, an  evolutionary history of their metabolic, ecological and pathogenic qualities. Such an understanding will elevate yeast to a new plane as a model and will undoubtedly serve as the basis of valuable new technologies.

Hittinger cautions, however, that sequencing yeast genomes is only a start: “We can very easily read gene sequences, but we don’t yet know how to interpret them fully. We will need to read those bases and make functional predictions” to extend both the knowledge of yeast biology and their potential use in industry.

“But if it weren’t for that natural diversity, we wouldn’t be able to enjoy Belgian beers,” says Hittinger, referencing the gifts conferred by different yeasts and their varied genetic underpinnings, resulting in the different flavors of ales, lagers and Belgians.

One of the central metabolic qualities of the familiar yeasts, of course, is their ability to ferment. Put simply, fermentation is a process by which cells partially oxidize or burn sugar. Among yeasts, the propensity to ferment in the presence of oxygen has evolved only in Saccharomyces species and a few others.

“To make a living using this process, you have to be a glucose hog,” says Hittinger. “But you don’t burn it all the way. You leave some energy on the table. Ethanol burns because it is unoxidized fuel.”

Different kinds of cells can perform fermentation if they become oxygenstarved Human cells, for example, ferment when starved of oxygen, causing painful muscle cramps. Given enough sugar, cancer cells can ferment, and do so to survive in oxygen-poor environments.

Indeed, Hittinger’s research on the cellular resemblance between Saccharomyces yeasts and cancer cells (for which he recently was named a Pew Biomedical Scholar) focuses on identifying which steps in yeast evolution were key to making the transition from respiratory to fermentative metabolic activity, as well as the sequence of those evolutionary events.

“Armed with that information, we should be able to shed some light on how cancer cells make that same transition over an individual’s lifetime,” says Hittinger.

Genes, Hittinger knows, hold the secrets to the functional qualities of yeast. Those microbial secrets, in turn, promise us food, fuel, pharmaceuticals— and, of course, beer. Like bread and wine, the gift of lager is no small thing. Who knows what other gifts, large and small, may lurk in the genes of these microorganisms?

Headed into the wild? If so, you could help Chris Todd Hittinger’s team identify new yeast species and strains. To learn more, visit

To watch an interview with Chris Todd Hittinger, visit

Brewing Beer-6005

Food science professor James Steele (left) and students are creating a red lager to be brewed by the Wisconsin Brewing Company. Steele and colleagues are launching a fermented foods and beverages program to take research and teaching to the next level.

“Farm to Glass” and More: Fermenting a Growth Industry

We all know Wisconsin as the land of beer and cheese. But in the not too distant future, Wisconsin may also become famous for other fermented products, notably wine and cider, thanks to growing public taste for those products and a blossoming wine- and cider-making culture in the Badger State.

Wisconsin now has about 110 wineries—up from 13 in 2000—and has been adding around a dozen new ones each year in recent years. Many of these operations could use some help, which is on the way in the form of a newly appointed CALS-based outreach specialist whose job is to support the state’s wine and hard apple cider industry.

Leaders of the Wisconsin Grape Growers Association, the Wisconsin Vintners Association and the Wisconsin Winery Association worked with CALS faculty in food science and horticulture to apply for a Specialty Crop Block Grant to support the position through the Wisconsin Department of Agriculture, Trade and Consumer Protection, with the associations providing matching funds. The specialist is scheduled to start working in early 2015.

The position is part of a larger effort to boost fermentation in Wisconsin. CALS food science professor James Steele and his colleagues are laying the groundwork for a comprehensive fermented foods and beverages program through the Department of Food Science—a program that will take to the next level much of the research and teaching the department has been building on for decades.

Already the program is bearing fruit—or, one might more literally say, “bearing beer.” Over the spring 2015 semester, students participating in Steele’s Fermented Foods & Beverages Laboratory will create and develop a new red lager recipe to be brewed by the Wisconsin Brewing Company and sold at the Memorial Union.

A central goal of the program, Steele explains, is to help improve the quality of fermented food and beverage products. As such, the functional roles played by yeast to influence such characteristics as flavor, color and other attributes will be very much in the spotlight.

“Yeast is a key player, beyond the shadow of a doubt,” says Steele. “It is extremely important, but from a food science perspective, it hasn’t gotten a lot of attention.”

With the help of yeast researchers such as Chris Todd Hittinger and his genetics colleague Audrey Gasch, Steele hopes to create an environment where the food science nuances of fermentation are teased out to the benefit of both growers and the producers of fermented foods and beverages.

The basic fermentation characteristics of various yeast strains are of interest, according to Steele: “For example, how does microbial physiology link to flavor in fermented beverages? These collaborations give us opportunities to look for new strains or develop new strains that could allow for the production of beverages with different flavors. And what we learn in one industry, we can apply to another.”

Connecting Our Ways of Knowing

In any other classroom, mention of planting “Three Sisters” might cause confusion. But in Becky Nutt’s science class at Oneida Nation High School, located on a tribal reservation in northern Wisconsin, most students know that the Three Sisters are corn, beans and squash, crops that in Native American tradition are planted together in a single mound.

Guided by Nutt, their questions focus on photosynthesis, the process by which plants like the Three Sisters convert sunlight into the energy they need to grow and produce oxygen. The lesson culminates with each student pretending to be an atom of a particular element in that process— oxygen, carbon or hydrogen—and “form bonds” by holding hands or throwing an arm around a classmate’s shoulders. It’s a fun lesson that resonates, judging by both the enthusiastic participation and the thoughtful entries each student writes afterward in a logbook.

The students know the lesson as part of a “pilot curriculum from UW–Madison,” as Nutt tells them—perhaps the easiest way to explain POSOH (poh-SOH), which is both the Menominee word for “hello” and an acronym for “Place-based Opportunities for Sustainable Outcomes and High Hopes.” The program is being developed in partner- ship with both Oneida and Menominee communities.

But what POSOH really represents is a new way of teaching science. Funded by a $4.7 million grant awarded by the U.S. Department of Agriculture in 2011, the program has the mission of helping prepare Native American students for bioenergy and sustainability-related studies and careers. POSOH aims to achieve that by offering science education that is both place-based and culturally relevant, attributes that have been shown to improve learning.

“We’re hoping to help make science relevant to young people,” says CALS biochemistry professor and POSOH project director Rick Amasino. “Bioenergy and sustainability offer an entrée into broader science education.”

For Native American students, sustainability is an obvious fit for science discussion, Amasino notes. The Native American concept of thinking in “seven generations”—how the natural resource management decisions we make today could affect people far into the future—has sustainability at its foundation, and most Native American traditions reflect that value. The Three Sisters, for example, offer a way to discuss not only photosynthesis but also indigenous contributions to our knowledge of agronomy, including how mixed crops support long-term soil health and animal habitat.

An innovative program like POSOH is needed because current teaching methods are not proving effective with Native American students. Native American students score lower in reading and math than their white counter- parts in elementary and high school, and only a low percentage have ACT scores that indicate college readiness, according to “The State of Education for Native Students,” a 2013 report by The Education Trust. Other studies show higher dropout rates and unemployment among Native Americans—and, specifically, that Native Americans are vastly underrepresented in STEM fields as students, teachers and professionals.

Verna Fowler, president of the College of Menominee Nation, sees POSOH as offering a crucial connection. Her tribal community college, along with CESA 8, the state public education authority that includes the Menominee Indian School District, has been a key partner in developing and piloting POSOH. Other leading partners include Michigan State University and, within UW–Madison, the Great Lakes Bioenergy Research Center.

“POSOH takes you into science in the natural world and helps you relate your concepts and understanding so that you understand science is all around you,” says Fowler. “Sometimes that’s what we miss in our classrooms. A lot of students are afraid of science classes. They don’t realize what a scientific world they’re living in.”

In developing POSOH materials, Amasino serves as the go-to guy for verifying the science. “The main thing I do is work with everyone to keep the science accurate,” he says.

Curriculum development and other POSOH activities are led by CALS researcher and POSOH co-director Hedi Baxter Lauffer, who has a rich background in K–12 science education. In a previous project she worked with California state universities in developing a multiyear math and science education program with diverse ethnic communities in the Los Angeles Unified School District. Alongside her work with POSOH, Lauffer directs the Wisconsin Fast Plants Program, which operates worldwide.

From the start Lauffer saw POSOH as a trailblazing effort. “We wanted to create a model for how a culturally responsive science curriculum can emerge from the community it is serving,” she says. “There’s nothing else like it.”

Lauffer knew her group was on to something during early curriculum design sessions with local educators, Native American community elders and students, particularly when she participated in a talking circle with seventh- and eighth-graders from the Menominee Indian School District. The kids were asked a simple question: “How do you take care of the forest—and how does the forest take care of you?”

“They had all kinds of stories about the plants and animals that live there,” says Lauffer. “They were saying things like, ‘I take my nephew into the forest and teach him to pick up his trash. He needs to know that it’s a beautiful place to play.’ It was clear that their connection to nature was strong—and that’s an opportunity for making science learning relevant and valuable.”

Initial steps for curriculum development included building key institutional partnerships and forming teams for curriculum design that brought in a wide range of Native American voices. Team members include scientists, assessment professionals, and teachers of science, education and Native American culture, some of whom are field-testing the materials.

The group is creating curricula for grades seven through nine. Seventh grade is complete, comprised of a fat lesson book and accompanying DVD with graphics and other enrichment materials. The other grades will be completed by the end of 2015, the project’s final year.

Other POSOH activities include after-school science clubs facilitated by undergraduate interns who also serve as informal mentors. This work is conducted in partnership with the Sustainable Development Institute at the College of Menominee Nation under the direction of Kate Flick BS’06, who studied community and environmental sociology at CALS and now serves as POSOH’s education coordinator.

Thumbing through the seventh- grade lesson book, it is immediately clear that cultural relevance is placed front and center. A typical textbook might pay tribute to cultural relevance with sidebars while the main text carries on with “science as usual.” With POSOH materials, cultural relevance is embedded in the meat of the text.

The seventh-grade curriculum, for example, is called “Netaenawemakanak” —Menominee for “All My Relatives”— and its six units focus on various scientific aspects of the Menominee Forest, such as organisms, microhabitats and ecological interactions. Students learn how such terms as evidence, protocol and conceptual models are used in science and, as a final lesson, how to formulate their own stewardship action plan based on what they’ve learned.

And it’s not just what the students learn, but how they learn it. POSOH incorporates forms of teaching and learning that are rooted in Native American culture, such as:

• Storytelling—Scientific concepts are imparted through stories involving the everyday lives of young Native American protagonists as well as figures from Native American legends and folktales.

• Perspective-taking—Students are invited to look at ecosystems from the viewpoint of animals, plants and other natural resources.

• “Careful noticing”—Students use all their senses when getting to know an environment, paying close attention to what is and is not present. In an exercise in the forest, for example, students are asked not only what they see, smell and hear, but also, “How do the woods make you feel?”

“These are age-old practices in indigenous pedagogy, but they aren’t widely seen as such. They’re so fundamental that I think they’re often overlooked,” says Linda Orie, an enrolled member of the Oneida tribe who taught middle-school science at the Menominee Tribal School. She now works on the POSOH curriculum team.

Orie considers POSOH a huge eye- opener for students. “It’s probably one of the first times they’ve seen anything in science class that has anything to do with Native Americans or Native American contributions to science and forestry,” she says. “Especially for a Menominee, that’s really important because most of them live on the reservation and a lot of their parents are employed through the lumber mill.”

“So they live and breathe the forest, but they don’t often get that instruction in the classroom,” Orie continues. “It was a huge gaping hole in the curriculum when I started teaching at the tribal school.”

By drawing upon indigenous ways of teaching and learning, POSOH helps bridge a gap between how students experience nature and how knowledge about it is imparted in the classroom. POSOH team member Robin Kimmerer, for example, says that as a professor of forest biology and as a Native American, she’s had to work hard to reconcile two distinct perspectives.

“In science we are asked to objectify the world, to view it in a strictly material, intellectual way,” says Kimmerer, who earned her doctorate in botany at UW–Madison and now teaches at the State University of New York. “In indigenous ways of knowing, we’re reminded that we can understand the world intellectually, physically, emotionally and spiritually—and that we can’t really claim to understand something unless we engage all four elements,” she says. POSOH team member Justin Gauthier, an enrolled Menominee who as a teenager attended a Native American boarding school, has come to think of science as another language for indigenous ways of knowing nature. In science, he says, “They’re using numbers, they’re using experimentation. It’s just different language.”

That recognition helped science feel more approachable to him.

“I used to perceive science as being outside of my experience. It was meant for scientists to do in a lab in a white coat. When I started thinking about how it tied into the ways that I was thinking, I felt that it had always been a part of my life and I had just never given it much credence,” he says.

Gauthier, a returning adult student, is earning his bachelor’s degree in English at UW–Madison and plans to teach in a tribal college after earn- ing an MFA in creative writing. He serves POSOH as a curriculum writer. Gauthier suggested naming the seventh- grade curriculum Netaenawemakanak (“All My Relatives”) because it is often uttered as a kind of one-word prayer when entering and leaving the sweat lodge. To him, among other things, the word expresses Native American regard for nature.

POSOH is not only helping fill a gap in science education. Project intern McKaylee Duquain, a junior majoring in forest science, notes that POSOH is filling a gap in cultural knowledge among young Native Americans as well. As an enrolled Menominee who attended tribal schools, Duquain confesses to not knowing what the Three Sisters were until late in high school—and she learned about it on her own.

“It wasn’t even offered when I was a student,” she says. “I’m not the most traditional person out there—I try to practice the traditional ways, but you can only do so much in this day and age. I feel like having that knowledge incorporated into your everyday learning life in school would definitely cement it in more.”

The program’s most enthusiastic ambassadors are the teach- ers and students who have been using it. So far the POSOH curriculum has been taught in 25 Wisconsin classrooms with the participation of some 135 students. Another 140 students have worked with POSOH materials in other settings, such as outreach programs conducted by undergraduate interns and the project’s high school club, called the Sustainability Leadership Cohort.

“I love that the POSOH curriculum brings science to a local level,” says Dan Albrent, a science teacher at De Pere’s Ashwaubenon High School, where he’s been piloting POSOH materials for the past two years. “Students a lot of times wonder why we are even learning all these complex things in science and just want a reason. POSOH does a nice job of bringing in real-life situations and issues that are literally close to home. And never in the curriculum are students sitting and listening to a lecture. They are actively talking and working with real data and real situations to solve problems.”

To him, POSOH represents the future of science education. “I truly believe this is how science should be taught,” Albrent says. “At the moment there is no better alternative for helping our kids realize the importance of learning science for our communities.”

Becky Nutt, of Oneida Nation High School, is just as convinced. She appreciates the program’s emphasis on reading and writing, which is not a given in science class—but important, she notes, in both communicating science and demonstrating understanding.

“Most important from my view is the integration of Native American culture into the materials,” says Nutt. “If, through these materials, we can foster better relationships between our Native students and their communities and other individuals and their communities, then we are on the right track.”

POSOH team member Linda Orie is taking a break from the classroom while earning her master’s degree in curriculum and instruction at UW– Madison—but she plans to return
to teaching in tribal schools and sees POSOH as a life-changing tool to bring with her.

“My career goal is to transform Indian education because it is stuck in this terrible rut,” Orie says. “Working in the tribal school I saw a lot of opportunity for growth. It was heartbreaking to see so much potential and not have colleagues that saw the same. And not seeing as many Native American teachers as there could be or should be in the schools. The kids need the best curriculum and the best teachers, and they’re not getting that right now. I want to be part of the change.”

That Orie, as an Oneida, backs the program so strongly speaks to perhaps the program’s greatest indicator of success—the acceptance it has earned in Native communities.

“We’ve been presenting POSOH to different schools, to different areas, to our boards of education and so on, and they’re very enthused about it— extremely enthused, I must say,” says College of Menominee Nation president Verna Fowler.

That enthusiasm is no accident, but the result of the program being developed within and in partnership with Native communities. Patty Loew, who is a professor of life sciences communication at CALS and an enrolled member of the Bad River Band of Lake Superior Ojibwe, just happened to be on hand during a POSOH presentation on the Menominee Reservation and was heartened by what she saw.

“I’ve been in a lot of situations where UW people try to engage with community members and it’s like pulling teeth for reasons that vary, but often come down to a basic mistrust of researchers,” Loew says. In those encounters, she says, “People are either being polite or they’ll have their arms folded and are just quietly listening or maybe hiding their resentment.”

“That was not the case on this day,” Loew says. “People were really engaged, they were discussing, they had ideas, it was emotional. It was clear to me that the community’s handprints were all over this project. They not only were hosting the research, they had shaped it, they were contributing to it, they were using the materials in their classrooms, they had a lot of pride in it. And I was really impressed.”

POSOH team member Justin Gauthier also knew about the mistrust firsthand—and saw it melt away.

“Historically in Indian Country there’s been this sort of stigma toward outside groups coming into the community, studying groups of people, taking data out of that community—and nary shall the two meet again,” Gauthier says. “But I really like and respect the way that the POSOH process is set up because, while the leadership team
is made up of people from within and without that community, the ideas—the voices at the table—are respected and integrated into the process. I feel like when we finish the project the curriculum and the relationships we’ve built are going to remain strong.”

“And that could be the big takeaway for me from this project,” Gauthier says. “Communities have the right to be wary of people coming in and studying them. But when you have a project like this, where the end result is meant to be a gift for that community, then you really see the beauty of cultures blossom and open up.”

That could be the big takeaway for Amasino and Lauffer as well. They and their team conceived of POSOH as an experiment in developing culturally integrated science curricula in a way that could be applied in various settings around the country.

“Our overarching mission is to build a transformational model for place- based collaborations dedicated to preparing all learners, especially those who are underrepresented in science and science education,” says Lauffer. “These community-based processes are what the project will share more broadly as it draws to a close. We plan to pass on lessons from POSOH to many other communities who can then build on our work and continue improving science teaching and learning.”

To learn more about POSOH, visit You can also watch the following video: