Mining bacterial blueprints yields novel process for creation of fuel and chemical compounds


Tim Donohue, UW-Madison bacteriology professor and director of the Great Lakes Bioenergy Research Center, supervised the lab that identified the makeup of 19Fu-FA — a compound with promising potential applications. Photo: Great Lakes Bioenergy Research Center

A team of researchers at the University of Wisconsin-Madison has identified the genes and enzymes that create a promising compound — the 19 carbon furan-containing fatty acid (19Fu-FA). The compound has a variety of potential uses as a biological alternative for compounds currently derived from fossil fuels.

Researchers from the Great Lakes Bioenergy Research Center (GLBRC), which is headquartered at UW-Madison and funded by the U.S. Department of Energy, discovered the cellular genomes that direct 19Fu-FA’s synthesis and published the new findings Aug. 4 in the journal Proceedings of the National Academy of Sciences.

“We’ve identified previously uncharacterized genes in a bacterium that are also present in the genomes of many other bacteria,” says Tim Donohue, GLBRC director and UW-Madison bacteriology professor. “So, we are now in the exciting position to mine these other bacterial genomes to produce large quantities of fatty acids for further testing and eventual use in many industries, including the chemical and fuel industries.”

The novel 19Fu-FAs were initially discovered as “unknown” products that accumulated in mutant strains of Rhodobacter sphaeroides, an organism being studied by the GLBRC because of its ability to overproduce hydrophobic, or water-insoluble, compounds. These types of compounds have value to the chemical and fuel industries as biological replacements for plasticizers, solvents, lubricants or fuel additives that are currently derived from fossil fuels. The team also provides additional evidence that these fatty acids are able to scavenge toxic reactive oxygen species, showing that they could be potent antioxidants in both the chemical industry and cells.

Cellular genomes are the genetic blueprints that define a cell’s features or characteristics with DNA. Since the first genome sequences became available, researchers have known that many cells encode proteins with unknown functions according to the instructions specified by the cell’s DNA. But without known or obvious activity, the products derived from these blueprints remained a mystery.

As time has gone on, however, researchers have realized that significant pieces of these genetic blueprints are directing the production of enzymes — proteins that allow cells to build or take apart molecules in order to survive. These enzymes, it turned out, create new and useful compounds for society.

“I see this work as a prime example of the power of genomics,” Donohue says. “It is not often that one identifies genes for a new or previously unknown compound in cells. It is an added benefit that each of these compounds has several potential uses as chemicals, fuels or even cellular antioxidants.”

A cross-disciplinary, collaborative effort between GLBRC chemists, biochemists and bacteriologists in departments at UW-Madison and Michigan State University yielded the chemical identity of the fatty acid compounds and identified the specific genes that direct their synthesis in bacteria.

“I don’t think this discovery would have been possible,” says Rachelle Lemke, the paper’s lead author and a research specialist in Donohue’s lab, “without the analytical and intellectual expertise of the members from this center.”

Two CALS teams among winners of Wisconsin Energy and Sustainability Challenge

Two CALS-based teams took silver in the 2014 Wisconsin Energy and Sustainability Challenge.

Second place in The Dvorak Energy Innovation Prize contest – and $1,500 – went to Team Drsti.

Vitamin A deficiency is a health concern throughout the developing world that affects more than 200 million children every year. Team Drsti, made of members Chris Johnson and Mary Pitassi, both masters students in Bacteriology, have described their product as “a sustainable solution to vitamin A deficiency.” Project Drsti will develop a probiotic bacterium engineered to make a precursor of vitamin A. The strain will be inexpensive to produce, store and transport, and can be easily added to yogurt or other fermented dairy products.

Second Place in The Global Stewards Sustainability Prize – and $1,500 – went to a team called Sustainable Fertilizer Recovery from Wastewater.

The Sustainable Fertilizer Recovery from Wastewater team is made of Tyler Anderson, a soil science graduate research assistant, Rania Bashar, PhD in biological systems engineering, Cody Calkins, soil science undergraduate, Grant Herrman, biological systems engineering undergraduate, and Logan Voellinger, biological systems engineering. Their concept is to develop and incorporate an electrodialysis cell at existing wastewater treatment facilities to capture and concentrate nitrogen, which can be used in fertilizers.

Richard Gourse elected to American Academy of Arts and Sciences

University of Wisconsin-Madison bacteriologist Richard L. Gourse is among leaders from academia, business, public affairs and the arts and humanities elected to membership in the American Academy of Arts and Sciences, it was announced today.

Richard L. Gourse

Richard L. Gourse

The Ira L. Baldwin Professor of Bacteriology, Gourse joins an eminent class of inductees that includes Nobel laureates, winners of the Wolf and Pulitzer Prizes as well as Grammy, Emmy, Oscar and Tony award winners.

Gourse joined the UW-Madison faculty in 1988 and is currently chair of the bacteriology department.

Working primarily with the model organism Escherichia coli, Gourse is well known for his studies of how genes are expressed in cells, primarily transcription initiation and the control of ribosome synthesis. Previous honors include election as a fellow for the American Academy of Microbiology and the American Association for the Advancement of Science, both in 2003. In 2007, he received the National Institutes of Health Merit Award.

The American Academy of Arts and Sciences was established in 1780 and each year elects “thinkers and doers” as fellows, among them George Washington, Benjamin Franklin, Daniel Webster, Ralph Waldo Emerson, Margaret Meade and Martin Luther King. Its current membership includes 250 Nobel laureates and more than 60 Pulitzer Prize winners.

Wisconsin team to search for new antibiotics from untapped microbes

Facing an imminent global public health crisis, a University of Wisconsin-Madison research team has been awarded up to $16 million from the National Institutes of Health to find new sources of antibiotics to combat the rising number of deadly antibiotic-resistant infections.

“The number of antibiotic-resistant strains has increased while the discovery of new antibiotics has slowed to a crawl. In fact, there are no new antibiotics,” said Dr. David Andes, professor of medicine and division chief of infectious diseases at the UW School of Medicine and Public Health.

In the 1980s, pharmaceutical companies were seeking Food and Drug Administration (FDA) approval of 10 to 20 antibiotics a year. Andes said there has been more than an 80 percent decrease in development of antibiotics since that time.

“The inability to mine novel natural resources for antimicrobials is a major bottleneck for attacking the drug-resistance crisis,” he said.

“Our team has developed a completely new paradigm for anti-infective drug discovery,” said Andes. “We have developed novel ways of finding new antibiotics and testing them rapidly. It’s a fresh approach catalyzed by complementary input from basic and physician scientists, microbiologists, chemists and pharmacologists who are thinking about the same problem.”

New sources for symbiotic organisms

Andes is a co-principal investigator for a National Institutes of Health (NIH) Center of Excellence for Translational Research (CETR). The other is Cameron Currie from the department of bacteriology in the UW College of Agricultural and Life Sciences. Other members of the team are Michael Hoffman, Dr. Bruce Klein, Dr. Rod Welch and Harvard researcher Jon Clardy. The project grant runs for five years.

Traditionally, soil has been mined for antimicrobials that are used to develop antibiotics. But the UW-Madison team has been studying other natural products from animals, insects, plants and marine life. Andes said the study of soil has become a dead end because the same microbes are turning up over and over again.

Andes said the scientists are looking in new places for symbiotic organisms, those that have an interdependent relationship, that are highly likely to have a biological effect.

The scientists are traveling around the globe to harvest insects, plants and marine life. Once specimens are collected, Currie sequences the genome of each product and then decides if it is promising enough to merit further testing.

Tim Bugni, an assistant professor of pharmaceutical sciences, then uses a rapid and accurate method to determine if the microbes are making something never found before. A third part of the research attempts to coax an organism to make compounds by mimicking its environment.

“We’ve also seen that the compounds that microbes are making are evolutionarily selected to be safe because they protect the animal from the environment, from infection threats,” said Andes.

The team is looking at two groups of relevant microbes: fungi associated with infections in immunocompromised patients like cancer and transplant patients, and bacteria responsible for the majority of U.S. hospital infections. The U.S. Centers for Disease Control and Prevention says more than two million drug-resistant infections a year are reported.

“There are patients in almost every hospital with infections that have absolutely no treatment options,” said Andes.

 Building on previous research

The research builds on the work of the UW Antimicrobial Drug Discovery and Development Center, established in 2007. The Wisconsin Partnership Program and various NIH Challenge grants funded the research.

“We’ve been finding large numbers of new compounds at a rate greater than what the pharmaceutical industry ever did,” said Andes.

The goal of the Center of Excellence for Translational Research is to find one drug lead in each of the next five years.

The project is funded by NIH grant number U19AI109673.

This article was originally published on the UW-Madison School of Medicine and Public Health News & Events webpage.

Saving the honeybee

This video features CALS entomology professor Christelle Guedot and bacteriology professor Cameron Currie discussing the issue of Colony Collapse Disorder and scientific efforts at CALS to understand this devastating disease of honeybees. It aired twice in early 2014 during the Wisconsin edition of the Big Ten Network show “Live Big.”

Decades on, bacterium’s discovery feted as paragon of basic science

Over time, the esoteric and sometimes downright strange quests of science have proven easy targets for politicians and others looking for perceived examples of waste in government — and a cheap headline.

In 1966, microbiologist Tom Brock‘s National Science Foundation-supported treks to Yellowstone National Park to study life in the park’s thermal springs might easily have been singled out as yet another feckless science project, deserving of a “Golden Fleece Award.”

Photo: Tom and Kathie Brock

Tom and Kathie Brock enjoy the 140 acres of restored prairie, oak savannah and woodlands that they established as the Pleasant Valley Conservancy in Black Earth. Photo: Jeff Miller

But Brock, then a professor at Indiana University, and Hudson Freeze, an undergraduate student working with Brock at Mushroom Spring in the Lower Geyser Basin of Yellowstone, found something that by any measure validates investments in basic science, the undirected search for new knowledge.

At the time, Brock was simply interested in studying the microbial ecology of the thermal springs that make Yellowstone famous. It was a frontier of knowledge as scientific dogma held that life, or at least photosynthetic life, could not occur at temperatures above 160 degrees Fahrenheit.

But to their astonishment, Brock and Freeze found a bacterium, later dubbed Thermus aquaticus, that made its entire living in the scalding waters of Mushroom Spring. The discovery set the stage for the branch of science that studies life in extreme environments and, most tangibly, yielded an enzyme, Taq polymerase, that is central to the technology for amplifying DNA — revolutionizing everything from medical diagnostics to criminal forensics.

“We were looking for a simple system where one could do basic research in microbial ecology. Everything fell out of that,” recalls Brock, now an emeritus professor of bacteriology at UW-Madison. “We didn’t know there were these organisms that could live in boiling water.”

Now, almost 40 years after discovering and describing the organism, Brock and Freeze are being recognized with the Golden Goose Award, an award aimed at celebrating “seemingly obscure studies that have led to major breakthroughs and resulted in significant societal impact.”

Conferred by a gaggle of organizations — the American Association for the Advancement of Science, the Association of American Universities, the Association of Public and Land-grant Universities, the Breakthrough Institute, the Progressive Policy Institute, the Science Coalition, the Task Force on American Innovation, and United for Medical Research — the Golden Goose Award is given for federally-funded research that has led to “demonstrable, significant human and economic benefits.”

The enzyme derived from the Thermus aquaticus bacterium powers polymerase chain reaction, or PCR. It is a technique used routinely to take trace amounts of DNA and amplify it for identification or study. The Taq polymerase is useful because it is stable at the high temperatures PCR requires to denature DNA and exponentially replicate target strands. The technology — for which its inventor, Kary Mullis, was awarded the Nobel Prize — is widely used in molecular biology, criminal forensics and medical diagnostics.

The Golden Goose Award was originally conceived by Congressman Jim Cooper of Tennessee to help educate members of Congress and the public about the value of basic scientific research. The name is a play on the Golden Fleece Awards, given by the late Wisconsin Sen. William Proxmire between 1975 and 1988, targeting specific federally funded research grants as examples of government waste.

“We’ve all read stories about the study with the wacky title, the research project from left field,” Cooper says. “But off-the-wall science yields medical miracles. We can’t abandon research funding only because we can’t predict how the next miracle will happen.”

Taq polymerase has gone on to be a workhorse molecule of science in good measure because Brock and Freeze deposited samples of the organism in the American Type Culture Collection, a repository where others can freely obtain cultures of the organism for little or no cost. It was there that Mullis and his collaborators found Thermus aquaticus and its enzyme when they were looking to replace an E. coli enzyme that did not survive the high temperatures of PCR.

Although Brock began his career at the Upjohn Co. looking for new antibiotics, after a few years he turned to the professor business and basic research: “Everything I ever did was basic,” says Brock. “It’s great that this discovery happened, but I wouldn’t have predicted it.”

Brock and Freeze will receive their award at a Capitol Hill ceremony on Sept. 19.

Protecting our pollinators: CALS researchers seek answers, solutions to bee die-off

People and bees have a long shared history. Honeybees, natives of Europe, were carried to the United States by early settlers to provide honey and wax for candles. As agriculture spread, bees became increasingly important to farmers as pollinators, inadvertently fertilizing plants by moving pollen from male to female plant parts as they collected nectar and pollen for food. Today, more than two-thirds of the world’s crop plants—including many nuts, fruits and vegetables—depend on animal pollination, with bees carrying the bulk of that load.

It’s no surprise that beekeeping has become a big business in the farm-rich Midwest. Wisconsin is one of the top honey-producing states in the country, with more than 60,000 commercial hives. The 2012 state honey crop was valued at $8.87 million, a 31 percent increase over the previous year, likely due in part to the mild winter of 2011–2012.

But other numbers are more troubling. Nationwide, honeybee populations have dropped precipitously in the past decade even as demand for pollination-dependent crops has risen. The unexplained deaths have been attributed to colony collapse disorder (CCD), a mysterious condition in which bees abandon their hives and simply disappear, leaving behind queens, broods and untouched stores of honey and pollen. Annual overwintering losses now average around 30 percent of managed colonies, hitting 31.1 percent this past winter; a decade ago losses were around 15 percent. Native bee species are more challenging to document, but there is some evidence that they are declining as well.

Despite extensive research, CCD has not been linked to any specific trigger. Parasitic mites, fungal infections and other diseases, poor nutrition, pesticide exposure and even climate change all have been implicated, but attempts to elucidate the roles of individual factors have failed to yield conclusive or satisfying answers. Even less is known about native bees and the factors that influence their health.

Poised at the interface of ecology and economy, bees highlight the complexity of human interactions with natural systems. As reports of disappearing pollinators fill the news, researchers at CALS are investigating the many factors at play—biological, environmental, social—to figure out what is happening to our bees, the impacts of our choices as farmers and consumers, and where we can go from here.

Please visit Grow magazine to read the whole story.

Researchers unearth bioenergy potential in leaf-cutter ant communities

As spring warms up Wisconsin, humans aren’t the only ones tending their gardens.

At the University of Wisconsin-Madison Department of Bacteriology, colonies of leaf-cutter ants cultivate thriving communities of fungi and bacteria using freshly cut plant material.

While these fungus gardens are a source of food and shelter for the ants, for researchers, they are potential models for better biofuel production.

“We are interested in the whole fungus garden community, because a lot of plant biomass goes in and is converted to energy for the ants,” says Frank Aylward, a bacteriology graduate student and researcher with the Great Lakes Bioenergy Research Center.

Illustration: Leaf-cutter ants

Illustration of the leaf-cutter ant Atta cephalotes in its fungus garden habitat. The variation in texture between the top and bottom strata represents the different stages of biomass degradation occurring in each layer of the fungus garden. Image credit: Cara Gibson

Aylward is the lead author of a study identifying new fungal enzymes that could help break down cellulosic-or non-food-biomass for processing to fuel. His work appears on the cover of the June 15 issue of the journal Applied and Environmental Microbiology.

“All the enzymes that we found are similar to known enzymes, but they are completely new; no one had identified or characterized them until now, ” Aylward says.

Building on Aylward’s previous study of these gardens, the researchers relied on genome sequencing provided by the U.S. Department of Energy Joint Genome Institute (JGI) and support from Roche Applied Science’s 10 Gigabase Grant Program to understand the unique roles of fungi and bacteria. In addition to sequencing the genome of Leucoagaricus gongylophorous, the fungus cultivated by leaf-cutting ants, the researchers looked at the genomes of entire, living garden communities.

“We really tried as thoroughly as possible to characterize the biomass degrading enzymes produced,” Aylward says. “Identifying all these new enzymes really opens the door to technological applications, because we could potentially mix and match them with others that we already know about to achieve even better biomass degradation.”

In a symbiotic relationship, L. gongylophorous provides food for the leaf-cutter ant Atta cephalotes by developing fruiting bodies rich in fats, amino acids and other nutrients. To fuel production of these fruiting bodies, the fungus needs sugar, which comes in the form of long cellulose molecules packed inside the leaf clippings the ants deliver. To get at the sugars, the fungus produces enzymes that break the cellulose apart into glucose subunits.

After sequencing the L. gongylophorous genome, the researchers noticed that the fungus seemed to be doing the lion’s share of cellulose degradation with its specialized enzymes. However, they also realized that it was by no means working alone: in fact, the gardens are also home to a diversity of bacteria that may help boost the fungus’s productivity.

“We think there could potentially be a division of labor between the fungus and bacteria,” says Garret Suen, co-author of the study and a UW-Madison assistant professor of bacteriology and Wisconsin Energy Institute researcher.

Photo: Garret Suen

Garret Suen

The researchers have a few leads in their investigation of the mysterious role of bacteria in leaf-cutter ant communities, which they are pursuing in collaboration with JGI. In addition to providing nitrogen and key vitamins, the bacteria appear to help the fungus access energy-rich cellulose by breaking apart other plant polymers that encase it, such as hemicellulose.

Accessing and deconstructing cellulose is also the goal of GLBRC researchers, who want to ferment the stored sugars to ethanol and other advanced biofuels. Enzymes such as those of the leaf-cutting ants’ fungus specialize in breaking down leaves, but understanding how they work in the context of the ant community could help researchers create similar methods for processing cellulosic biofuel feedstocks, such as corn stalks and grasses.

The researchers are discovering, however, that both the beauty and the challenge of the leaf-cutter ant garden lie in its complexity. A peek into UW-Madison’s resident colony in the Microbial Sciences Building reveals a metropolis of brown insects bustling around the pale, pitted surface of the fungus garden, many with leaf sections held aloft. The strong resemblance to a small city drives home the point that energy production in such a meticulously coordinated system would be difficult to replicate in a lab or a bio-refinery.

“In an industrial setting, you need a system that’s reproducible, sustainable, controlled — and that produces a consistent level of ethanol,” Suen says.

A potential alternative to re-creating these natural processes is to extract, replicate and purify biomass-degrading enzymes synthetically. New enzymes could be added to known combinations and tested for their ability to break down biofuel feedstocks. However, this process can be time-consuming and costly.

To put their findings in perspective, the researchers plan to study other insects in addition to ants, including certain species of termites and beetles, which also act as gardeners in fungal communities. They hope that a better understanding of these complex systems will help them share their biomass-degrading secrets with bioenergy researchers.

“It’s difficult to think that we can actually find a process that improves on nature,” says Aylward, “so it probably makes sense to learn from it.”

Beetle-dwelling yeast holds promise for biofuel production

Tom Jeffries is a scientist in the truest sense of the word. His passion for studying biofuel is evident as he describes is 40-year career in the field, yet he emphasizes that he is careful not to favor one technology over another.

Instead, Jeffries relies on pragmatism to guide his research.

“Yeast are very resilient, there is a long history of their commercialization, and we understand the technology,” he says of yeast’s role in converting plant biomass to ethanol.

“Making it work economically is what’s hard.”

Jeffries, a Great Lakes Bioenergy Research Center scientist and University of Wisconsin-Madison bacteriology professor, has recently published a paper describing a new type of yeast that can ferment plant sugars to ethanol much more efficiently than other species. He thinks this microscopic critter could have a big impact on economical commercial biofuel production.

“Yeast are probably the most practicable way to get renewable fuels today,” Jeffries says.

Though yeast may not be the first thing to spring to mind when the subject of renewable energy is raised, these single-celled organisms are metabolic powerhouses. For biofuel researchers, Saccharomyces cerevisiae is nothing short of a household name thanks to its appetite for sugars from cellulosic—or non-food—plant material, and its ability to ferment them to ethanol.

But Jeffries’ interest has been captured by Spathaspora passalidarum, a highly specialized yeast species that grows in the bellies of wood-boring beetles. Because it plays a key role in the beetles’ digestion, S. passalidarum is naturally adapted to fermenting sugars found in wood that S. cerevisiae can’t process. While S. cerevisiae is able to ferment simple sugars like glucose and sucrose, S. passalidarum is also able to convert xylose, cellobiose, arabinose, and galactose to ethanol.

Finding an organism that can ferment xylose is a particularly important breakthrough for biofuel researchers, because xylose is an essential building block of hemicellulose—a polymer that makes up about 20 percent of most plants.

“People tend to think that yeasts are all the same, but they’re not,” says Jeffries. [S. passalidarum] can use cellulosic and hemicellulosic sugars efficiently, whereas S. cerevisiae cannot unless it has been genetically engineered.”

Jeffries hypothesizes that these two yeast taxa diverged as long ago as 180 million years, around the time that flowering plants began to evolve. It is possible that while S. cerevisiae evolved in association with insects that consume sugars from flowering plants, S. passalidarum instead took up residence in the mid-guts of passalid beetles—large insects of an inch or more in size that inhabit rotting logs. Their unique habitat requires S. passalidarum to ferment sugars found in the wood pulp ingested by their beetle hosts.

“[S. passalidarum] has all these metabolic capacities that we must to engineer into S. cerevisiae…they have them already because they’ve evolved in that environment,” explains Jeffries.

The biofuel benefits of the new yeast, which was first discovered in 2006 by biologist Meredith Blackwell of Louisiana State University, are more than just theoretical. Jeffries, who founded biotechnology startup Xylome in 2007, is already looking for ways to produce the yeast on a commercial scale and integrate them into the existing bioenergy infrastructure.

Jeffries is interested in providing technologies to independent corn grain ethanol producers that will help them shift their focus to cellulosic feedstocks. For example, with S. passalidarum’s unique fermentation abilities, these companies could produce ethanol from the leaves and stalks of corn plants (corn stover) rather than the kernels. Jeffries predicts that this approach could help ethanol producers ameliorate ‘food versus fuel’ issues, as well as increase output by up to 50 percent.

“There are 122 independent ethanol production plants, each with an average capacity of about 50 million gallons per year, that produce 40 percent of all the ethanol in the U.S. from corn grain,” Jeffries says.

“They have the infrastructure, they have the distillation facilities, they have the fermentation facilities…so you don’t have to make a lot of changes to these producers to be able to add corn stover as a feedstock.”

Jeffries has also been in conversations with GLBRC collaborator MBI, a Michigan company specializing in the de-risking and scale-up of bio-based technologies. MBI is working to produce commercial quantities of corn stover that has been pretreated to make it more easily processed to ethanol. The pretreatment process, known as AFEXTM* was developed by GLBRC researcher Bruce Dale at Michigan State University.

A unique advantage of the AFEXTM process that it turns loose, dry cellulosic biomass into dense pellets—pellets that resemble corn grain in size and shape.

“If we could use AFEXTM pellets as a fermentation feedstock, that would provide an abundant, cost-effective sugar source for microbes to convert into biofuels like ethanol, as well as bio-based chemicals,” says MBI President and CEO Bobby Bringi.

Much of the rhetoric surrounding sustainable energy involves references to new technological frontiers and sweeping innovations. But Jeffries says that an industrial shift to cellulosic feedstocks—aided by yeast that can naturally process them to ethanol—is promising precisely because it would require only slight changes to the way grain ethanol producers currently operate.

“A lot of people think about biomass and bioenergy as major changes to the existing technology, and that’s not really the way technology evolves—technology evolves at the margins,” he says.

“When you are going commercial with [a technology] it has to work…it can’t work 90 percent of the time, or even 95 percent of the time. It has to work. So industry tends to look for incremental, rather than earth-shaking, changes.”

*AFEXTM  is a registered trademark of MBI.