Shaping the Future of Farming

Thirty-five years ago, when CALS bacteriologist Winston Brill and his colleagues set out to exploit science’s newfound ability to manipulate genes to confer new traits on crop plants, the technology was, literally, a shot in the dark.

Working in a facility in Middleton, just west of Madison, Brill and his team blasted plant cells using a gene gun—a device that fired microscopic gold beads laden with DNA.

The idea was to introduce foreign genes that could confer new abilities on the plants that would ultimately be grown from the altered cells. First as Cetus of Madison, Inc., later as Agracetus and still later as a research and development outpost of Monsanto Company, the Middleton lab was, by all accounts, a hub of plant biotechnology innovation.

“Agracetus was the first in the world to engineer soybean, first in the world to engineer cotton, first in the world to field-test a genetically engineered plant,” recalls Brill, who was recruited by Cetus to establish the lab in the early 1980s. “Thus, the Madison area and the UW influence led to historically important events.”

In December 2016, the $10 million,100,000-square-foot facility—a warren of labs, greenhouses and growth chambers—was donated to UW–Madison by Monsanto to become the Wisconsin Crop Innovation Center (WCIC).

The hope, according to agronomy professor Shawn Kaeppler BS’87—now WCIC’s director—is that the center will add to its string of plant biotechnology achievements as one of just a few public facilities in the country dedicated to plant transformation, where genetically modified plant cells are taken from tissue culture and regenerated into large numbers of complete fertile plants.

The advent of the WCIC “is an unprecedented opportunity to add capabilities and capacity we couldn’t afford otherwise,” says Kaeppler, an expert on corn. Its acquisition by UW–Madison, he and others note, comes at an opportune time as powerful new techniques in synthetic biology are poised to make the development of plants with new or improved traits much more than a shot in the dark with a gene gun.

WCIC will function very much like a core facility, providing cell culture, phenotyping and plant transformation services for researchers at UW– Madison and other universities. It is also coming online at a time when the need for such resources is acute.

“There is a recognized need nationally,” explains agronomy professor Heidi Kaeppler BS’87, an expert in plant transformation who is serving as WCIC’s transformation technology director. “There are just a few public facilities around the U.S. and demand is outpacing the abilities of those facilities. It is a bottleneck.”

For researchers like bacteriology and agronomy professor Jean-Michel Ané, a member of the WCIC scientific advisory board, the new center means he will be able to devote more time to exploring such things as the genetic interplay that occurs when plants and bacteria collude to draw nutrients from the air through the act of nitrogen fixation.

Nitrogen-fixing plants such as soybean, alfalfa and clover are staples of modern agriculture. They are essential to the crop rotation practices that prevent exhaustion of soil from crops such as corn. Ané and many other scientists have long dreamed of engineering the ability to fix nitrogen into plants like corn to transcend the need for expensive and environmentally harmful chemical fertilizers.

However, engineering complex traits such as nitrogen fixation in plants that don’t have that innate ability is a monumental scientific and technological undertaking. To begin with, there are two organisms—the plant and a bacterium—working cooperatively. Each has its own genome, and many different genes from each organism are in play to accommodate the act of drawing life-sustaining nutrients from the air.

To confer that trait on corn, for example, is an exercise far more complicated than tinkering with one or a few genes, notes Ané. “The goal is to create maize that has this association. However, modifying a single gene will not be sufficient,” he says. “We modify many genes at a time. There is a lot of trial and error. We need to try many combinations.”

Those combinations come about in the lab as scientists alter individual plant cells by adding or subtracting genes of interest. Today, scientists can harness new techniques such as CRISPR– Cas9—a fast, cheap and accurate genome editing tool—and potent new cloning technologies that allow scientists to easily assemble multiple DNA fragments and their assorted genes into novel sequences.

Even with potent new tools like CRISPR–Cas9, engineering plants is a big, difficult task. A gene needs to be dropped in the right place on the genome and be in association with the right “promoters,” segments of DNA that initiate gene transcription, the first step toward expressing a new gene in an organism. Once plant cells are genetically altered, they must be transformed into large numbers of actual plants for further testing in the lab and, ultimately, the field. It is essential to know, for example, that the new genetic construct is stable, that the new genes are passed from generation to generation, and what effects they may have on plant growth or yield.

The promise of WCIC, Ané believes, will be the opportunity to work through all of those steps more efficiently and cost-effectively, and carry projects from the lab to the field much faster.

“We can focus on really doing science instead of growing plants,” Ané says. “We can now make genetic constructs very quickly. Within a month we can make hundreds of constructs. The limiting aspect is plant transformation. However, the scale of transformation we can do at WCIC allows us to think seriously about applying synthetic biology to plants.”

To begin with, WCIC is providing plant transformation services for corn, soybean and sorghum, big commercially important crop species. But Shawn Kaeppler envisions WCIC playing a role, as well, with crop plants that have not yet risen to the top of commercial research agendas.

To date, commercial interest has focused primarily on just a handful of traits—insect and herbicide resistance—in a handful of widely planted crops. Uncharted territory, Kaeppler says, exists in the full range of crop plants and their many different traits.

A ready example is switchgrass, a native perennial that is under the microscope at the Great Lakes Bioenergy Research Center (GLBRC), a U.S. Department of Energy- funded multi-institutional research center headquartered on the UW–Madison campus. The grass is seen as a potential feedstock for converting its biomass to liquid fuel. However, efficient conversion of plant materials to energy remains a challenge, and plant genetics will play a big role in refining the traits that will make that possible.

“WCIC will help lead us to the next generation of crop breeding and plant genetics,” explains Kate VandenBosch, the dean of CALS, referencing, broadly, the genetic makeup of the crop plants in play. “Scientific agencies at the federal level have invested a lot in understanding genomes, but we still have a lot of work to do to understand how those genes function.”

Indeed, genetic sequencing technologies have advanced to the point where new plant genomes are sequenced with increasing regularity. The genomes of crop plants like watermelon, cucumber, potato, soybean, wheat, corn and many others have been sequenced, but as VandenBosch notes, exploring those sequences to identify the genes that govern plant traits is an unexplored frontier.

Shawn Kaeppler’s own research, for example, is a window to both the complexity and opportunity that lurk in the genomes of plants. One of his interests is the complex of genes—involving anywhere from tens to hundreds of genes—that governs the root architecture of corn. Knowing more about the combination of genes that directs the plant to send shoots into the soil, it might one day be possible to engineer a plant that can send its roots deeper into the earth, providing farmers with a hedge against drought.

“Fifty to 70 percent of all maize genes are expressed in roots,” Kaeppler says. “Some control processes in all parts of a plant, and some specifically control root development and response to environmental stimuli.”

A gene of interest for Kaeppler and his team is one that influences root angle. “Altering root angle even five to 10 degrees can dramatically increase the rate that roots get deep in the soil,” as well as how much root biomass a plant lays down at depth, he explains.

Identifying those candidate genes and mutations of those genes means they can be selected and manipulated in the laboratory to generate plants with different root structures. At WCIC, those plants can be grown in quantity, their new qualities studied and, if promising, tested in the field. The goal, of course, is to provide a practical outcome that is useful to growers.

In plant science, numbers matter. The more plants you can grow to test a new genetic combination, the better, as there are so many variables in play.

“In many aspects of science, doing things on a large scale is critical,” says biochemistry professor Rick Amasino, an expert on flowering in plants. “To have WCIC in our capability is great. Large-scale transformation opens up a lot of possibilities.”

Amasino, who is also a member of WCIC’s scientific advisory board, views the center as an important new national resource. Individual labs, he explains, do not have the same capacity.

“This has the potential to be on a scale greater than any other university’s,” Amasino says. “Individual labs can’t generate the hundreds or thousands of transgenic plants needed to fully test certain hypotheses. Labs around the country and, hopefully, around the world can now do experiments they couldn’t otherwise do. There are so many opportunities out there.”

A Facility With Deep CALS Roots

The name is new, but the Wisconsin Crop Innovation Center (WCIC) holds a prominent place in the young history of agricultural biotechnology. The facility also has long and deep ties to CALS researchers and alumni.

Originally known as Cetus of Madison, Inc., the Middleton facility—owned by
the Cetus Corporation of Emeryville, California—opened in 1981 under the direction of CALS bacteriology professor Winston Brill. The Wisconsin Alumni Research Foundation (WARF) played a key funding role in the early days of the company.

Cetus of Madison, Inc. initially focused on evaluating and testing a wide variety of natural rhizobia species to better understand their role in nitrogen fixation and nodulation in legumes, with the hope of someday enabling maize to have that capacity.

As interest in biotechnology grew in the early 1980s, the facility’s focus changed to inventing and innovating ways to introduce genes into plants. In 1984, Cetus Corp. sold half of its interest in Cetus of Madison, Inc. to the WR Grace Co.—and thus the company name “Agracetus” was born.

Great discoveries followed. An electric “gene gun” and transformation methods developed at Agracetus revolutionized the plant transformation process. Many plant species were subsequently transformed, including tobacco, peanut, sunflower, soybean, maize, cotton, cranberry, canola, poplar, wheat and rice. CALS researchers Kenneth Raffa, Brent McCown PhD’69 and Elden Stang, as well as WCIC associate director Michael Petersen BS’87 (then still an undergraduate) and Richard Heinzen MS’74, collaborated with Agracetus scientists during that period. But that wasn’t the only significant research taking place. Other studies critical to agricultural improvement focused on cotton fiber quality, transformation process improvements, polymerase chain reaction (PCR) method development, insect and disease resistance and herbicide tolerance. A number of CALS faculty, including Michael Sussman, Richard Amasino and Andrew Bent, were highly involved in consulting with Agracetus in many of these areas.

In 1990, WR Grace Co. acquired full ownership of Agracetus. During the early 1990s, Agracetus ventured into research in DNA vaccines—using an improved “gene gun”—and contracted plant transformation services to others within the industry, including, most notably, the Monsanto Company. Collaborating with biological systems engineering professor Richard Straub PhD’80 (now CALS senior associate dean) and other CALS researchers, the company also worked on producing industrial enzymes in plants.

After successfully generating plants that eventually became commercial products
for Monsanto, including Roundup Ready Soybeans and Bollgard Cotton, the facility was acquired by Monsanto in 1996.

Over the next 20 years, Monsanto used the facility as its primary site for soybean and cotton transformation. Other R&D at the site included corn, canola, wheat, rice and alfalfa transformation, gene expression, molecular testing and seed chipping/genotyping.

The site was considered a “center of excellence” for Monsanto due to its highly innovative employees, high throughput transformation capabilities and ability to consistently perform above and beyond expectations.

In July of 2016, Monsanto relocated a number of remote functions back to its St. Louis headquarters in the interest of business consolidation. In the hope that the Middleton facility would continue to work toward the betterment of agriculture, Monsanto the following December donated it to longtime collaborator the University of Wisconsin– Madison, along with University Research Park.

Not surprisingly, given the long history of CALS involvement, agronomy professor Shawn Kaeppler BS’87 was chosen to serve as facility director.

For the Birds

Slipping into a patch of woods in western Dane County, Jim Berkelman ignores the swarming mosquitoes and strains to sort through the early- morning chatter of warblers, robins and vireos and the nearby drum of a pileated woodpecker. “I’m hearing something I wouldn’t expect to hear,” says Berkelman, a lecturer in the Department of Forest and Wildlife Ecology at CALS and a volunteer contributor to the Wisconsin Breeding Bird Atlas II, a comprehensive, volunteer-powered survey of birds that nest in Wisconsin.

Experienced birders use their ears as much as their eyes to identify species, and Berkelman thinks he hears a northern parula, a small warbler that doesn’t typically nest this far south. Finding a bird, Berkelman explains, is only the start. The point of the Atlas, he notes, is to identify and map where birds in Wisconsin are courting, nesting, breeding and raising their broods.

To be sure of that, “atlasers,” as volunteer observers like Berkelman are called, must find tangible evidence that a species has actually taken up residence. A nest, of course, is the most obvious clue. But most birds are assiduously covert in their nesting and only conspicuous players like robins, herons, orioles, house wrens and bluebirds construct their nests in ways that make them easy to find and identify.

Other definitive hallmarks of breeding birds include observations of birds carrying nesting material or food for nestlings; distraction displays where birds seek to draw animals, other birds or humans away from a nest; and, of course, fledglings. Some bird species are fastidious as well and carry fecal matter away from occupied nests. Such an observation is also a telltale sign of breeding and can be used by an atlaser to confirm breeding activity and provide a new data point that science can ultimately draw on.

Following a rising wooded path to the top of a hill, Berkelman’s rounds on this warm June day encompass two different types of ecosystems: forest, and open fields and prairie. His block is designated as a “priority block,” a specified block within a six-block “quad” on a grid of more than 7,000 three-mile-by-three-mile blocks that covers Wisconsin. Within that grid are 1,175 priority blocks, each of which requires at least a year’s documentation of breeding birds within a five-year period to ensure that the state is uniformly surveyed for the new Atlas. In addition, there are 153 “specialty blocks” that have unique habitat, are of high conservation value or are of particular interest to ornithologists.

Today, Berkelman is recording his data the old-fashioned way: with pen and notebook. Later, he can plug his observations into Atlas eBird, an online checklist program that is a direct conduit to the database that is the bedrock of the Wisconsin Breeding Bird Atlas.

Data, of course, are the raw material of science. Astronomers gather it by measuring and parsing starlight. Molecular biologists get data by plumbing the sequence of the chemical base pairs that make up a gene or genome. Meteorologists numerically dissect the many variables of weather—temperature, precipitation, wind, clouds.

To be sure, most data collection is a laborious and numbing process—the antithesis of the eureka moment. Harvesting data can be very expensive, too, as the tools of modern science have become bigger, more complex and more powerful in their ability to see farther or smaller, drill deeper, or accelerate particles to higher energies. Indeed, much of what we hear about modern scientific discovery rests on the pillars of sophisticated technology. Think of the Hubble Space Telescope, the Large Hadron Collider, the IceCube Neutrino Observatory and the Human Genome Project as just a few examples.

But while technology is taking science to new heights, it’s also giving a boost to the age-old methods of data gathering like the ones Berkelman uses in his efforts to document the presence of breeding birds. The Internet and personal computing technology are being used like never before to crowd-source traditional observational data collected by a growing cadre of citizen scientists. Groups of people or individuals armed with laptops and app-laden smartphones are collectively logging everything from trash in the ocean and flying ants to cosmic rays and precipitation, giving working scientists access to oceans of new data and the revelations that come from subsequent analysis and interpretation.

In the realm of ecology, citizen science has gained a new standing as researchers have tapped into the potential of an interested public. Citizen science projects, mapping things like the presence and behaviors of bumblebees, manta rays, butterflies and bats, have fueled dozens of published studies.

It’s proven to be a powerful resource for Ben Zuckerberg, a professor of forest and wildlife ecology at CALS. North American birds and their distribution on a changing landscape are a primary focus of his research, a significant portion of which depends on data gathered by volunteer observers.

For instance, Zuckerberg and post-doctoral fellow Karine Princé drew on citizen science data to tell us that the cast of characters we see at our bird feeders in the winter is shifting, most likely due to climate change. Their study of wintering songbirds shows that some species, once rare during the Wisconsin winter, are shifting their ranges north, remaking the resident communities of birds that visit our backyard feeders.

The conclusions of the study rested on two decades of data gathered by thousands of citizen scientists through the Cornell University Laboratory of Ornithology’s Project Feederwatch.

“Birds have always been important environmental indicators,” Zuckerberg explains. Rapidly declining songbird populations in the 1950s and 1960s, he notes, were used to help ascertain the consequences of widespread use of the chemical insecticide DDT, which was subsequently banned, first in Wisconsin and then nationally.

The DDT story was famously informed by the unintended involvement of ordinary citizens who gathered baseline data in the form of bird eggs. In the 19th century, collecting bird eggs was a widespread hobby, an artifact of the Victorian obsession with the natural world. Many collections ended up in museums where, decades later, CALS ornithologist Joseph Hickey and his students used them to document the thinning of eggshells subsequent to the widespread introduction of DDT into the environment in the 1940s and ’50s.

Today the contributions of citizen scientists tend to be more directed, and the advent of personal computers and smartphones, in particular, are making participation easier, more immediate and more effective. And a prime example of that trend is the Wisconsin Breeding Bird Atlas, a collaborative project by the Wisconsin Department of Natural Resources (DNR), the Wisconsin Society for Ornithology, the Wisconsin Bird Conservation Initiative and the Western Great Lakes Bird and Bat Observatory.

This year, the group launched a second iteration of the Atlas. Zuckerberg and other scientists are working with Atlas coordinators and waiting in anticipation of a flood of new data from the project, which recruits volunteers statewide to survey thousands of designated blocks over a five-year period for evidence of breeding birds.

The first Wisconsin Breeding Bird Atlas featured data collected by nearly 1,600 volunteers between 1995 and 2000. As its name implies, the Atlas is a survey that documents the distribution and abundance of birds breeding in Wisconsin. It provides critical baseline information about bird species that live in our state and is an important benchmark in terms of assessing potential changes in bird populations over time due to things like habitat loss and climate change. It also helps document avian diversity, the state of endangered and rare bird species, and habitat needs in Wisconsin.

Such data, explains Zuckerberg, help scientists make sense of a world that involves players ranging from microbes to plants and animals, including birds. There are so many moving parts that capturing a wide snapshot of what exists where at a given point in time can give scientists insightful information about the dynamics, nuances and health of an ecosystem.

“Ecology is necessarily a messy endeavor,” Zuckerberg observes. “But at certain scales, it all becomes very clear.”

Drawing on things like Breeding Bird Atlas data, Zuckerberg and other scientists can get at the scales that matter: geography and time. As the Wisconsin Breeding Bird Atlas II effort gets under way, ecologists are laying the groundwork for analyzing the data by formulating hypotheses and ideas about what the data might show and how it will compare to data in the first iteration of the Atlas, which, according to the Wisconsin Society of Ornithology, “represented the largest coordinated field effort in the history of Wisconsin ornithology.”

Data collection for the Wisconsin Breeding Bird Atlas II began in 2015 and runs through 2019. In September the DNR released findings for the first Atlas season. Volunteers submitted nearly 24,000 checklists documenting the location and breeding activity of 229 species of birds. These early data show that wild turkeys are on the move, now populating nearly every corner of our state. And eight species of birds new to the Wisconsin breeding landscape since the last survey—including the iconic whooping crane—have cropped up in the new Atlas data.

“The stories that come out of the data are so robust,” Zuckerberg says. “We go in with our ideas of what we’re going to uncover, and some of the patterns just jump out at us.”

The major advantage of the Wisconsin Breeding Bird Atlas, according to noted ornithologist Stan Temple, a CALS emeritus professor in forest and wildlife ecology, is that it documents the relationship between birds and the places they require to successfully reproduce. “Habitat affinity is where the Atlas works best,” Temple explains.

Temple cites other long-standing citizen science efforts to document birds. The North American Breeding Bird Survey was officially launched in 1966. Conducted during the breeding season, volunteers traverse by car more than 3,700 randomly selected 24.5-mile road transects in the United States and Canada. Stopping every half-mile, volunteers document every bird seen or heard in a three-minute span before moving to the next observing station. The North American Breeding Bird Survey, Temple argues, is the gold standard for measuring population trends among birds.

A more recent citizen science effort—one that capitalizes on personal computing technology and helps inform the Wisconsin Breeding Bird Atlas—is the aforementioned eBird. Taking old-fashioned pen and paper checklists into the digital age, eBird is an online checklist linked to a central database. Used by amateur and professional birders, eBird logs millions of bird observations worldwide in any given month through a simple and intuitive web interface. The Wisconsin Breeding Bird Atlas II is the first state Atlas effort to employ it.

“We’re in the information age now,” explains Nick Anich, the Wisconsin DNR Breeding Bird Atlas coordinator. “We have eBird. We’re excited to use this new system. The developers have put an awful lot of effort into the checklist input, and they just launched the maps function. And the data update at least every 24 hours, so we can see things in real time.”

But can the information gathered by armies of citizen scientists be trusted? Can it help researchers predict the future of Wisconsin’s environment? How is it validated? Can scientists get over any qualms they might have about data collected beyond the strict parameters of controlled experiments and expert observation?

Zuckerberg, who has published on the use and value of crowd-sourced data, believes that many scientists are coming around to the idea that the data indeed represent an accurate picture of the natural world. “There has always been some skepticism about it in ecology. But studies show it is valuable data that are relatively accurate for picking up ecological patterns and processes,” Zuckerberg says.

“There are entire subfields of ecology dependent on these data. Theories in macroecology and how species respond to widespread environmental changes, such as pollution or climate change, for example,” Zuckerberg observes, referencing the study of relationships between living organisms and their environments at large spatial scales. “We wouldn’t be able to do anything like that without citizen science.”

That kind of insight is essential, Zuckerberg stresses, as broad-scale environmental change due to pollution, deforestation, reforestation and climate change will have significant and possibly lasting effects on birds in many different types of ecosystems.

According to Temple, the power of citizen science lies in the sheer numbers of observers. As a new CALS faculty member in 1976, Temple launched the Wisconsin Checklist Project. “The Wisconsin Checklist Project did in the predigital age what eBird does now,” Temple explains. “It is a rigorous way of engaging lots of bird-watchers in a very systematic way.”

For the most part, Temple says, the data are trustworthy. “Bird-watchers are used to keeping records, so you’re not asking them to do anything that already isn’t part of the culture. Mistakes in observing and recording happen, but it is safe to say those few errors become insignificant noise in comparison to the strength of the signal: the overwhelming number of accurate observations.”

For atlasers like Florence Edwards-Miller, a 31-year-old communications specialist from Madison, the chance to go into the field and gather data blends neatly with her deep-felt appreciation of the natural world.

Trekking through the prime birding habitat of Madison’s Nine Springs E-way on a rainy midsummer morning, Edwards-Miller is on a mission. An experienced birder, she knows she can confirm any number of breeding birds that use the settling ponds of Madison’s Metropolitan Sewerage District to raise their broods. And she is eager to contribute those little bits of data to the Wisconsin Breeding Bird Atlas effort.

“You can’t make good decisions unless you know what’s out there,” says Edwards-Miller. “I believe in science. I believe in the importance of the data.”

In a little more than an hour, she confirms the presence of breeding mallards, Canada geese and red-winged blackbirds—all pedestrian wetland species—by noting offspring and, in the case of the blackbirds, a cantankerous distraction display.

It takes a little longer to find the killdeer fledglings, but at the end of our circuit around the pond, there they are: little puffballs on stilts trailing behind their foraging parents. It’s a beautiful sight. And another valuable data point for the Wisconsin Breeding Bird Atlas.

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 http://go.wisc.edu/wildyeast

To watch an interview with Chris Todd Hittinger, visit http://go.wisc.edu/hittingerinterview


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.”

The Shocking Truth

It is well known that a certain kind of fish swims the world’s waters protected, as it were, by its very own stun gun.

Unknown, until now, is how electric fish evolved such a defense. A team of researchers led by CALS biochemistry professor Michael Sussman has established the genetic basis for the electric organ, an anatomical feature found only in fish. It evolved independently half a dozen times in environments ranging from the flooded forests of the Amazon to murky marine environments.

“These fish have converted a muscle to an electric organ,” says UW Biotechnology Center director Sussman, who began this research almost a decade ago. The study, recently published in the journal Science, provides evidence to support the idea that the six electric fish lineages used essentially the same genes and developmental and cellular pathways to make the electric organ, which fish use to communi- cate with mates, navigate, stun prey, and as a shock- ing defense. The jolt from an electric organ can be several times more powerful than the current from a standard household electrical outlet.

Worldwide, there are hundreds of electric fish in six broad lineages. Their taxonomic diversity is so great that Darwin himself cited electric fishes as critical examples of convergent evolution, where unrelated animals independently evolve similar traits to adapt to a particular environment or ecological niche.

The new work includes the first draft assembly of the complete genome of the South American electric eel. “A six-foot eel is a top predator in the water and is in essence a frog with a built-in five-and-a- half-foot cattle prod,” says Sussman. “Since all of the visceral organs are near the face, the remaining 90 percent of the fish is almost all electric organ.”

Electric fish have long fascinated humans. The ancient Egyptians used the torpedo, an electric marine ray, in an early form of electrotherapy to treat epilepsy. Much of what Benjamin Franklin and other pioneering scientists learned about electric- ity came from studies of electric fish. In Victorian times, parties were organized where guests would form a chain to experi- ence the shock of an electric fish.

All muscle cells have electrical potential. Simple contraction of a muscle will release a small

amount of voltage. But at least 100 million years ago some fish began to amplify that potential by evolv- ing from muscle cells another type of cell called an electrocyte—larger cells, organized in sequence and capable of generating much higher voltages than those used to make muscles work.

The “in-series alignment” of the electrocytes and the unique polarity of each cell allows for the “sum- mation of voltages, much like batteries stacked in series in a flashlight,” says Sussman.

In addition to sequencing and assembling DNA from the electric eel genome, the team produced protein sequences from the cells of the electric organs and skeletal muscles of three other electric fish lin- eages using RNA sequencing and analysis.

“I consider exotic organisms such as the electric fish to be one of nature’s wonders and an important gift to humanity,” says Sussman. “Our study dem- onstrates nature’s creative powers and its parsimony, using the same genetic and developmental tools to invent an adaptive trait time and again in widely disparate environments.”

And the findings may be useful to humans. “By learning how nature does this, we may be able to manipulate the process with muscle in other organ- isms and, in the near future, perhaps use the tools of synthetic biology to create electrocytes for generating electrical power in bionic devices within the human body or for uses we have not thought of yet,” says Sussman.

Sussman’s collaborators include Harold Zakon of the University of Texas at Austin and Manoj Samanta of the Systemix Institute in Redmond, Washington. The study was funded by the National Science Foundation, the W. M. Keck Foundation and the National Institutes of Health.

Sloths Thrive at Chocolate Source

Like many and much more nimble Neotropical fauna, sloths are running out of room to maneuver.

As forests in South America and Central America are cleared for agriculture and other human uses, populations of these arboreal leaf eaters, which depend on large trees for both food and refuge, can become isolated and at risk. But one type of sustainable agriculture, shade-grown cacao plantations, could become critical refuges and bridges between intact forests for the iconic animals.

In Costa Rica, CALS forestry and wildlife ecology professors Jonathan Pauli and Zach Peery are using a complex of intact tropical forest, pasture, and banana and pineapple plantations—all connected by a large, shade-grown cacao farm—as a field laboratory to explore the ecology of two species of sloths in a rapidly changing environment.

“We know a lot about sloth physiology,” says Pauli. “But when it comes to sloth ecology and behavior, we know almost nothing. It’s a giant black box.”

But some of that mystery is being peeled away as studies of both the brown-throated three-toed sloth and Hoffmann’s two-toed sloth, two common species, are yielding new insights into their mating habits and how the animals navigate the landscape.

The fact that sloths require forested habitat and are sedentary makes them vulnerable to deforestation, says Peery. “Once a tract of tropical forest has been cleared, sloths have relatively little capacity to seek out new habitats.”

But the shade-grown cacao plantation, with its tall trees and network of cables for moving the pods that ultimately become chocolate, seems to be a de facto refuge and transit hub.

“Because of the diverse overstory of native trees, the cacao farm appears to provide excellent habitat for both species of sloths,” explains Peery. “We want to compare sloth populations in cacao to populations in intact tropical forests to see if cacao provides habitat that is of as high a quality as their natural forests.”

Fleshing out those ecological parameters, however, requires a better basic understanding of sloth behavior, knowledge the CALS researchers are now beginning to accumulate.

For example, in a study recently published in Animal Behavior, Pauli and Peery described the mating system of Hoffmann’s two-toed sloths and showed that, unlike many other animals, the females tend to disperse from their home range and that the breeding territories of males can slightly overlap, with males tolerating competitors on the fringes but excluding them, sometimes violently, from the core. And Hoffmann’s two-toed sloths of both sexes seem to have multiple partners as well. “They’re more promiscuous than previously thought,” says Pauli. “We see a much more flexible system of multiple matings.”

That’s not so for the three-toed sloth. In another study, published in PLoS ONE in December, they found that three-toed sloths are strongly polygynous—males exclude other male competitors and mate with many females.

In addition to contributing to basic sloth knowledge, these findings should help wildlife and land managers in the Neotropics make sound decisions to better balance development and conservation.

“Understanding how shade-grown agriculture can benefit sensitive tropical animals such as sloths is highly relevant, considering the ongoing and rapid loss of biodiversity in the Neotropics,” notes Pauli. “What kinds of ecological services can these already altered landscapes provide? Can we mitigate future biodiversity loss with a greater emphasis on shade-grown agricultural systems than crops grown in monocultures? That’s the future we’re facing.”

Because of their sedentary nature and dependence on forest, sloths can be viewed as an “umbrella species,” says Peery. “Protecting sloths could indirectly protect many other animal species in tropical forests that are harder to measure and study.”