We often look to the smallest life-forms for help solving the biggest problems: Microbes can make foods and beverages, cure diseases, treat waste, and even clean up pollution. Yeast and bacteria can also convert plant sugars into biofuels and chemicals traditionally derived from fossil fuels, which are a key component of most plans to slow climate change.

Now, researchers at CALS and the Great Lakes Bioenergy Research Center (GLBRC) have engineered bacteria that can produce two products — at the same time — from underutilized plant fiber. And unlike humans, these multitasking microbes can do both things equally well. “To my knowledge, it’s one of the first times you can make two valuable products simultaneously in one microbe,” says Tim Donohue, GLBRC director and a professor of bacteriology at CALS.

The discovery, detailed in an article in the December 2023 issue of the journal Applied and Environmental Microbiology, could help make those biofuels more sustainable and commercially viable by tapping into multiple chemical markets.

“In principle, the strategy lowers the net greenhouse gas emissions and improves the economics,” Donohue says. “The amount of energy and greenhouse gas that you need to make two products in one pot is going to be less than running two pots to make one product in each pot.”

Every Molecule Counts

The quest to replace fossil fuels with sustainable alternatives hinges on extracting the most value possible from renewable biomass. As with petrochemicals, every molecule counts: Low-volume, high-value products help keep the fuel more affordable.

One of the biggest barriers is a part of the plant cell wall called lignin, which binds together the walls’ main molecular components — carbohydrates called cellulose and hemicellulose — to make plants sturdy.

Lignin is the world’s second largest source of renewable carbon, but its complex and irregular structure make it notoriously difficult to break apart into useful components. There’s even a saying in the paper industry: You can make anything from lignin, except money.

That’s why scientists with GLBRC have studied a microbe called Novosphingobium aromaticivorans (or simply Novo for short). Discovered in sediment contaminated with petroleum products, Novo can survive by feeding on ringed carbon and hydrogen molecules such as toluene, naphthalene, and xylene, which are also found in lignin.

While other bacteria can digest some of the aromatics in lignin, Novo eats nearly all, funneling them into smaller compounds through a series of chemical reactions it uses to capture energy.

Some of those intermediates can be substituted for petroleum-based chemicals used in common plastic products, such as soda bottles. It’s just a matter of rewiring the microbe to stop the digestive assembly line at the desired chemical.

Fortunately, Novo also happens to be amenable to genetic modification. In 2019, a team of GLBRC researchers engineered a strain of Novo that produces a chemical known as PDC (2-pyrone-4,6-dicarboxylic acid), which is used to make products such as nylon and polyurethane.

More recently, a team in Donohue’s lab discovered other modifications that allow Novo to make a different plastic precursor, muconic acid (cic,cis-muconic acid, or ccMA) from a mix of aromatic compounds in poplar tree lignin that had been chemically treated.

But they didn’t stop there.

“We’re not going to solve our carbon emissions problem by only producing two products,” says Ben Hall MS’21, PhD’23, a recent doctoral graduate in genetics and coauthor of the paper.

Donohue’s team used genomic modeling to come up with a list of potential products that could be made from bio- mass aromatics. To make it on the list, the products needed to be valuable and able to be produced in large quantities with fewer than five genetic changes. Near the top of the list was zeaxanthin, one of a group of organic pigments known as carotenoids.

Carotenoids — which give distinctive hues to carrots, pumpkins, salmon, and even flamingos (via the birds’ steady diet of algae and shrimp) — are used as animal feed, nutritional supplements, pharmaceuticals, and cosmetics. They have a cumulative market value worth tens of billions of dollars per year.

Researchers already knew that Novo had the genes to produce another carotenoid with little market value. Based on the bacteria’s genome sequence, they suspected zeaxanthin is a stepping stone to that less valuable carotenoid in the process that cells use to make complex molecules. They just needed to alter the right genes to stop the digestive assembly line at the more valuable product.

By deleting or adding selected genes, the research team engineered Novo strains that produced zeaxanthin as well as other valuable carotenoids, such as beta-carotene, lycopene, and astaxanthin, when grown on an aromatic compound commonly found in lignin.

Next, the team showed that the engineered Novo bacteria could produce the same carotenoids from a liquor made from ground and treated sorghum stems, a solution that contains a mixture of aromatics that many industrial bacteria can’t digest.

One Pot, Two Products

Hall then wondered what would happen if he combined the genetic changes needed to make PDC and a carotenoid in the same microbe. The resulting strains produced both PDC and the target carotenoid with no discernable loss to either yield. Even better, the bacteria accumulate carotenoids within their cells, which must be separated from the solution that contains the PDC, which they excrete.

“We’re already separating the cells from the media,” Hall says. “Now we would have a product coming out of both.”

The next steps include testing whether engineered strains can co-produce carotenoids and ccMA, which Donohue thinks is possible, and to engineer strains to improve yields in industrial conditions.

While there are lucrative markets for each of these products, Donohue and Hall say the real value of the discovery is the ability to add multiple functions to this biological platform.

“To me, it’s both the strategy and the products,” Donohue says. “Now that we’ve done this, I think it opens the door to see if we can create other microbial chassis that make two products.”

What Is the GLBRC?
The Great Lakes Bioenergy Research Center (GLBRC) is a federally funded interdisciplinary science hub housed at the Wisconsin Energy Institute on the UW campus and operated in partner- ship with Michigan State University.

The center aims to develop sustainable alternatives to gasoline, diesel, and other hydrocarbon fuels and products that are currently made from petroleum. GLBRC researchers — including faculty and staff from CALS and elsewhere at UW — are also working to increase plant productivity and develop cost-effective processes to convert as much non-food plant material as possible into chemicals used to make products such as polyester, nylon, lubricants, and plastics. One of the ultimate goals is to lower net greenhouse gas emissions from the production and use of fuels and chemicals.

The GLBRC was established in 2007 by the Biological and Environmental Re-search program in the U.S. Department of Energy (DOE) Office of Science. In 2023, DOE renewed the center’s grant for another five years, authorizing up to $147.5 million in additional funding.
Learn more at glbrc.org.