Building New Metabolic Pathways

Building New Metabolic Pathways

By Kevin Coss

Microbes are nothing if not industrious. The metabolic pathways (linked series of chemical reactions) in these tiny organisms lead them to crank out a wide variety of molecules for all sorts of purposes — and that’s what has Mike Freeman’s attention.

“Some of these pathways are for chemical communication, some are for warfare, and others are completely unknown to us,” says Freeman, Ph.D., member of the University of Minnesota BioTechnology Institute and assistant professor of biochemistry, molecular biology, and biophysics. “A large part of what our lab does is looking at those genes and those pathways in unique organisms and studying them for the purpose of both basic science and biotechnology.”

Freeman’s research involves understanding how each piece of a biological system works and then combining and mixing those parts to “recircuit” pathways. This field, synthetic biology, aims to redirect microbes’ natural industriousness to produce a different, more desirable product. The intended purpose can span a wide range of applications, from producing compounds that help address bacteria’s growing resistance to antibiotics to developing molecules for use in the fragrance industry.

“You could use a microbe to do something it doesn’t already do in nature, for example, or engineer it to do it much better,” Freeman says. “We’re trying to put things together in unique ways to do what humans want, not what nature wants.”

Understanding the full scope of how a given pathway works, what it produces, and what larger purpose it serves requires a wealth of expertise. Fortunately, Freeman is part of the Synthetic Biology Research Cluster, which brings together faculty, postdoctoral fellows, staff, and students who study in the field to intermingle and learn from one another. This arrangement helps to spark collaborations that inspire new lines of research and enables the group to apply for larger grants with multiple principal investigators.

Freeman’s current research focus is on modifications that give molecules called peptides (smaller versions of proteins, with fewer pieces acting as “links” in their chain) more desirable properties. Peptide-based medications, such as the insulin used to treat diabetes, generally can’t be taken orally because stomach acids destroy the active compounds before they can provide any benefit. Modifying these peptides to remain stable through the digestive tract would open the possibility for peptide-based drugs to be taken as a pill — an option that makes it easier and more comfortable for patients to adhere to their medication schedule.

As another example, Freeman and his team are studying anti-cancer compounds derived from a specific type of fungus. Two promising varieties of peptides had previously been isolated from it, but the team went further in depth to study and isolate other “flavors” of these peptides with unique and useful characteristics. By optimizing the growing conditions in the lab for the fungi’s mycelia — the long, stringlike filaments that spread through the soil — they have been able to isolate a number of compounds from this fungus.

Apart from the medical field, a stronger understanding of synthetic biology could also help solve problems in manufacturing, agriculture, and other industries. While these solutions are appealing, what inspires Freeman the most about his research is the basic science — specifically, how much of it remains undiscovered.

In many other areas of science, researchers build upon a strong foundation of existing knowledge. Synthetic biology is different. It’s only recently that advances in genomic technology made it possible to study the workings and purposes of genes, proteins, and pathways at this level of detail.

“In some cases, the enzymes that we find, the pathways, the molecules have never been seen before in science,” he says. “Being able to explore these unknowns and add to the general scientific knowledge for humanity is what’s really satisfying to me.”

Kate Adamala

Kate Adamala

KATE ADAMALA

Assistant Professor
Genetics, Cell Biology, and Development

kadamala@umn.edu
protobiology.org

Research Interests

Synthetic cells, Cell-free protein expression, Engineering genetic pathways

Bio

Kate Adamala is a biochemist whose research focuses around building synthetic cells. Her lab studies the origin and early evolution of life, explores possibilities of using synthetic biology to colonize space, and aims to shape the future of biotechnology and medicine. The lab’s research utilizes synthetic cell technologies to make tools for metabolic engineering, drug development, and biosensing.

Tapping the Talents of Enzymes

Tapping the Talents of Enzymes

BTI researchers are working to discover, understand, and improve our ability to enlist the help of molecules that catalyze life.

by Mary Hoff

Enzymes are the movers and shakers of the biomolecular world. A class of proteins found in every cell of every living thing, they bring together molecules that would otherwise be reluctant to interact so they can combine, exchange parts, or otherwise alter each other.

In nature, enzymes facilitate thousands of processes from photosynthesis to decomposition. Enzymes speed up chemical reactions thousands of times faster than the reactions would occur on their own. Over the millennia, humans have found ways apply this superpower to accomplish a variety of useful goals from making beer and cheese, to making our laundry whiter and brighter, to cranking out millions of copies of a sample of DNA to solve crimes or find long-lost relatives.

“And that’s just the beginning,” says Romas Kazlauskas, professor in the department of Biochemistry, Molecular Biology, and Biophysics. “Enzymes can also learn how to do different things. We’re trying to figure out which parts are important and why they’re important, then design something different to do a reaction that doesn’t occur naturally.” By identifying enzymes, improving understanding of how they work, and applying them to new tasks, Kazlauskas and BTI colleagues are taking advantage of the billions of years of evolution that produced these molecules to make medicines, degrade pollutants, speed industrial processes, and more.

 

Trash to Treasure Kazlauskas is working with an enzyme found in nature that degrades PET plastic. First synthesized in the 1940s, PET (a form of polyethylene) is widely used for beverage and food containers with some 70 million tons generated every year—and much of that ending up in landfills and the ocean. Current efforts to recycle the material can only produce lower-quality plastics.

“One idea is to break down to component parts and resynthesize fresh plastic that would be just as good as what you started with,” he says.

Sound like a job for an enzyme? Kazlauskas thought so, too. But the likelihood of finding a suitable one in nature was slim because PET didn’t exist for most of evolutionary history. Nevertheless, in a compost heap in Japan, researchers recently found an enzyme that bacteria use to break down the waxy part of plants that could also break down PET.

Kazlauskas, along with graduate student Colin Pierce and colleagues, is working on modifying that enzyme known as cutinase, to improve its ability to degrade PET. The goal, ultimately, is to be able to develop a commercially viable way to turn old PET into new plastics without experiencing the loss of quality. This would not only reduce the load of plastic waste, it would also reduce pressure to produce more plastic, often from fossil fuels.

Kazlauskas sees huge opportunities beyond PET for efforts to extend nature’s ability to degrade compounds. “There are a lot of cases where we start making stuff and putting it out in nature, and people say it doesn’t degrade,” he says. “We’re finding bugs that can degrade these forever chemicals.” he says. By advancing the ability to modify such natural enzymes to make them do the job even better, the hope is to avoid the buildup of undesirable, manufactured compounds in the environment and reduce the need to make more from raw materials.

 

Life on the Edge

Turning somersaults in a sulfurous hot spring or clinging tenaciously to life on a glacier, the organisms studied by McKnight Presidential Fellow Trinity Hamilton, associate professor in the department of Plant and Microbial Biology, are the stars in microscopic world’s version of “Survivor.” Having evolved within living things, most enzymes work best under fairly mild conditions with respect to things like temperature and acidity. But not all. In the hot springs of Yellowstone National Park, Hamilton is studying how microbes—and the enzymes they use to carry out the activities of daily life—can survive regular temperature fluctuations of 40 degrees Celsius (104 degrees Fahrenheit) or more. And, on the other end of the spectrum, she’s also looking at how other microorganisms that live on ice maintain their function at low temperatures in the absence of liquid water.

Hamilton studies, among other things, enzymes that facilitate photosynthesis and function well up to 72 degrees Celsius, and then suddenly quit. “We have no idea why,” she says. “It’s really hard to perform autopsies on microbes.”

The ability of bacterial enzymes to function at various temperatures is behind the process that makes it possible to sequence DNA using what is called the polymerase chain reaction (PCR) technique. The enzyme used for this was isolated from bacteria in a hot spring in Yellowstone National Park. A better understanding of how bacteria keep these proteins active under extreme conditions could open the door to advancing our ability to solve other human challenges like preserving food or thriving in the face of climate change.

Molecular Assembly Line 

For Michael Smanski, a McKnight land grant professor in the department of Biochemistry, Molecular Biology, and Biophysics, the focus is on bringing together enzymes from different sources to create an efficient work team that can produce a specific molecule.

Most recently he’s been focusing on serofendic acid, a molecule found in the blood of fetal calves. In the early 2000s, researchers in Japan discovered that serofendic acid could prevent the untimely death of neurons and potentially serve as a therapy for minimizing damage from Parkinson’s disease, strokes, and other neurological trauma. The problem? It occurs in such small quantities that it would take 1,000 calves to make a minute 3 milligrams of active substance—providing dismal prospects for practical use.

Biosynthesis and enzymes to the rescue. Using databases describing enzymes from different organisms and the type of work each does, Smanski and colleagues designed a molecular assembly line that could make serofendic acid from readily available feedstock chemicals. Then they introduced the corresponding genes into a bacterium, enabling it to make the desired product.

As any industrial engineer would know, populating an assembly line with ready workers is only part of the solution, however. For efficiency’s sake, it’s also important to have the right capacity at every step of an assembly process to ensure that partially made products don’t pile up, or a slow step doesn’t drag down everything else. To optimize production of serofendic acid, Smanski has been tweaking the activity levels of the various enzymes relative to each other.

Even as he’s working on a strategy to improve outcomes for individuals with brain disorders, Smanski is also discovering principles that can be applied more broadly to optimizing artificial biosynthetic processes.

Optimizing a process means testing multiple levels of expression for the specific gene that’s coding for each enzyme along the way. If the assembly line consists of 15 enzymes, and researchers want to test five levels of expression for each, that would require 5 15 power—more than 30 billion—individual trials. Using combinatorial mathematics, Smanski is devising a strategy for selecting from those millions of possibilities, a mere 100 or so that are most likely to succeed, making the optimization process far more manageable.

“As improvements in the technical aspects of genetic engineering make it easier to rewrite the genetic blueprints of living organisms, learning how to efficiently sift through all of the possibilities of what-to-write is becoming more important,” Smanski says. “We think that our work at the interface of combinatorial mathematics and genetic engineering will benefit any application that requires more than one or a few genes working together.”

 

Self-Assembling Scaffold

Bringing multiple enzymes together in an assembly line is far more easily said than done. The molecules involved in sequential steps must be physically located close enough to each other to be able to pass a product-in-the-making down the line. They also must be positioned correctly at their stations so they can accomplish their task.

Distinguished McKnight University Professor Claudia Schmidt-Dannert is on it. Rather than identifying enzymes to string together to accomplish a molecule-making task, she is working on the assembly line itself.

Her goal is to not only design a stable assembly line of enzymes able to make a useful product but to genetically modify a microorganism—say, the bacterium E. coli—so that it can produce it on its own. Such a self-assembling scaffold, she believes, could dramatically improve the function of molecular assembly lines, reducing the cost of producing pharmaceuticals and other useful products.

“If we stabilize enzymes, we can make reactions more efficient,” she says.

Schmidt-Dannert and colleagues recently received a patent for a self-assembling, modular scaffold that co-locates enzymes in a way that enhances their ability to efficiently produce desired molecules. Her current focus is on developing scaffolds as materials that can correctly self-assemble into 3-dimensional architectures to organize and support enzyme functions. This would allow the manufacture of a broad range of chemicals including pharmaceutically active molecules—a process that can be extremely difficult using conventional chemical synthesis methods. 

She’s also applying her strategy to making molecular assembly lines more economical by including enzymes that recycle required co-factors, which are molecules or metals that assist enzymes in assembling a molecular product.

“You need to recycle the [co-factors]. Otherwise, the process is too expensive,” she says. “A scaffold helps make that possible.”

 

The National Microbiome Data Collaborative

The National Microbiome Data Collaborative (NMDC) team is building an integrated data science ecosystem that leverages existing data standards, data resources, and infrastructure in the microbiome research space. The NMDC is launching the NMDC Ambassador program to provide training and support for early career researchers who are motivated to engage with their respective research communities to lower barriers to adoption of metadata standards.

We invite applications from early career leaders who are:

  • Familiar with the challenges of discovering, accessing, and reusing microbiome data

  • Committed to working with the NMDC to make microbiome data findable, accessible, interoperable, and reusable (FAIR)

  • Motivated to engage with researchers in their community

  • Committed to inclusion, diversity, equity, and accountability (IDEA)

Please encourage qualified early career researchers to apply by May 21, 2021. For more details about the program, visit: microbiomedata.org/community/ambassadors or email us at support@microbiomedata.org.

Understanding a Toxic Necessity

Understanding a Toxic Necessity

Jannell Bazurto, assistant professor of Plant and Microbial Biology at the University of Minnesota, is pursuing a better understanding of formaldehyde, a chemical that is carcinogenic, toxic, and produced by all living things.

By Reed Grumann

If you dissected a pig in high school biology, you might remember a sharp, acrid smell permeating the classroom and the teacher’s warning about a carcinogenic chemical called formaldehyde. Though often labeled a killer chemical, every organism on Earth, including humans, produces small amounts of formaldehyde. In very small quantities, it’s manageable. Produce or consume too much, and formaldehyde will kill otherwise healthy cells by attacking critical proteins and DNA.

While most organisms can neutralize small amounts of formaldehyde, researchers are just beginning to understand the mechanisms involved in formaldehyde regulation. Jannell Bazurto, a member of the BioTechnology Institute and an assistant professor of Plant and Microbial Biology, looks for clues in Methylobacterium, a type of bacteria that produces and neutralizes formaldehyde levels that would kill most other microbes.

As their name implies, methylobacteria have a unique ability to metabolize or breakdown, single-carbon molecules like methane and methanol. The process helps the cell maintain enough energy to survive, but also generates formaldehyde as an intermediate step. Fortunately, the bacteria are also uniquely equipped to handle sudden changes in concentrations of the toxin.

A key mechanism of Methylobacterium is an enzyme that converts formaldehyde into less harmful chemicals. While the enzyme is sufficient at normal levels of formaldehyde concentration, it’s not enough to handle exceptionally higher formaldehyde levels. To identify the function of two additional proteins suspected of playing a role in regulating formaldehyde levels, a group of researchers at the University of Idaho removed them from bacterial cells. And just like a cake without sugar and eggs, something was off. “If you don’t have [the two proteins] … you can see [the cells] accidentally overproduce formaldehyde, and they end up secreting it in the growth medium,” says Bazurto.

It’s still unclear exactly how these two proteins keep formaldehyde levels low in methylobacterium, but they aren’t alone in their efforts. Dozens of genes express proteins as formaldehyde levels change—a strong indicator of their importance in regulating the toxin. The challenge for Bazurto is knowing which of these genes, and the proteins they encode, actually play a role in formaldehyde metabolism. By manipulating each gene and looking at the results, Bazurto hopes to crack the code and establish which genes impact formaldehyde metabolism and their role in the process.

Once understood, these metabolic pathways could be hardwired into other microbes (like E. coli) through genetic engineering. Modified E. coli could consume methanol, neutralize formaldehyde, or produce marketable chemicals like biofuels and organic acids. In some facilities, formaldehyde is produced in large quantities as a building block for other chemicals. Wastewater remediation at these facilities would greatly benefit from bacteria genetically modified to directly consume formaldehyde and withstand toxic concentrations.

Throughout its industrial lifecycle, formaldehyde has the potential to creep into our air and water, putting humans at risk of exposure. The relationship between excessive formaldehyde exposure and human health issues—cancer, respiratory issues, and skin irritation—has been well established, yet we still know very little about how humans (and other organisms) sense and handle exposure to formaldehyde. As the search for practical applications in biotechnology, medicine, and environmental remediation continues, Bazurto remains fascinated by the basic science and “scenario where we actually know how to resolve formaldehyde toxicity itself.”

Reed Grumann is a writing intern in the Science Communication Lab, majoring in microbiology and political science. He can be reached at gruma017@umn.edu.

Image courtesy of Janelle Buzurto. Timecourse of Methylobacterium chemotaxing toward a capillary tube that has a formaldehyde plug in it. (Zero and five minutes). Cells seen faintly in the background at zero minutes begin to move toward the plug by the five minute point, forming a halo around the end of the tube.