Oak Ridge Lab synthetic biology expert targets plastics

From our bottled water, toothbrush and comb to our car and cellphone, it’s hard to get through the day without touching and using plastic products. Unfortunately, while this lightweight, durable material made from petroleum and natural gas is a boon for our economy, it is also an environmental threat.

In the United States, less than 10% of waste plastic is recyclable, 20% of it is burned, producing climate-altering carbon dioxide, and significant amounts end up in the ocean, choking marine life.

Adam Guss, a genetic and metabolic engineer at Oak Ridge National Laboratory, told the Friends of ORNL group that he and his colleagues are creating microbes with a special ability to deal with the problem of mixed plastic waste from milk jugs, disposable coffee cups and single-use water bottles as part of a two-step chemical and biological process.

These genetically altered bacteria from soil, called Pseudomonas putida, “eat” chemically broken-down products of modern plastics, converting them to a compound called beta-ketoadipate, which can be used to make high-performance nylon polymers.

According to an ORNL news release, the new chemical and biological process, “described in the journal Science, would replace a system that now requires painstaking, costly sorting of materials, which has resulted in only about 5% of plastics being recycled in the United States. The project is led by the National Renewable Energy Laboratory (NREL) and also brings together scientists from the Massachusetts Institute of Technology, the University of Wisconsin-Madison and ORNL under the Bio-Optimized Technologies to keep Thermoplastics out of Landfills and the Environment, or BOTTLE, Consortium.

“Different plastics contain different polymers, each with unique chemical building blocks. The BOTTLE researchers developed a process to convert mixed plastics to a single chemical product, working toward a solution that would allow recyclers to skip sorting.”

Plastic water bottles are made mostly of polyethylene terephthalate (PET). PET is a repeating polymer of terephthalate and ethylene glycol. The Guss lab has engineered the P. putida bacteria to grow on terephthalate, and ORNL’s collaborators at NREL engineered it to grow on ethylene glycol and to secrete the enzyme PETase, which breaks down the PET compound by chemical reaction with water.

NREL used chemical catalysis to further break down plastic bottles into BHET molecules that Guss and colleagues at ORNL and NREL converted into beta-ketoadipate. After separation, this compound can be used to react with other chemicals to make biodegradable polymers such as “a superior nylon product that is more water- and heat-resistant, ideal for applications such as automotive parts,” according to the release.

“We can also make these biodegradable polymers from lignocellulose and sugars,” he said.

The Guss lab also uses engineered microbes to eat another cheap feedstock, the lignin and lignocellulose in green plant biomass and agricultural residues, converting it to compounds such as itaconic acid.

The Department of Energy has stated that itaconic acid is a value-added building block that could be used to form commercially valuable polymers that are biodegradable when discarded – that is, they can be decomposed by bacteria when disposed of in landfills.

In 2020, according to an Oct. 13 ORNL news release, Guss led a team that announced it had engineered the P. putida microbe to simultaneously digest five of the most abundant and difficult-to-break-down sugar and aromatic compounds of lignocellulosic biomass.

“We like the P. putida bacterium because our engineered version is good at eating aromatic materials and is tolerant of different toxic chemicals like aldehydes and solvents,” he stated. These genetically altered bacterial cells are replicated by exposing them for a short time to nitrogen, which is essential to their growth, he explained, adding that the cells are then denied nitrogen.

When the cells run out of nitrogen, this deficiency is detected by a biosensor developed by Guss’ group, which triggers a change in gene expression that leads to the production of itaconic acid while carbon is present. By selecting the mutant cells that expressed the most polymerase enzyme, the rate of the compound’s total production sped up, dropping from 30 hours to 12 hours.

“We achieved close to the maximum theoretical yield with our engineered bacteria,” Guss asserted.

Besides addressing the plastics problem, Guss and colleagues are helping DOE’s Center for Bioenergy Innovation, where he works, to find the most economical way to make jet and ship fuel from plant biomass, such as poplar trees and switchgrass, instead of fossil fuels. Unlike today’s aviation and marine fuel, biomass fuel is carbon neutral because it has already absorbed the carbon dioxide it will later release when burned.

Jets and ships, he added, can’t be electrified like cars, so they must operate in the future on carbon-neutral liquid fuels made of plant biomass and agricultural residues. The goal, he said, is to make plant-based fuel at a cost that is competitive with petroleum – around 25 cents a pound, or $2 a gallon.

The Guss lab uses synthetic biology methods to engineer microorganisms. These methods include the ability to extract and link together DNA sequences that give microorganisms a certain trait and then to insert this foreign DNA into a different microbe being engineered to yield a specific product. The DNA is transcribed into RNA, which instructs the cells to synthesize a particular protein. They have collected various DNA sequences that enable them to, for example, increase the gene expression in target bacteria to produce a higher amount of a desired enzyme, a type of protein that stimulates, or catalyzes, chemical reactions.

One DNA insertion method for manipulating millions of microbial cells, he added, is “to use an electric field to punch holes in each cell’s membrane.” The inserted DNA contains a gene that inactivates antibiotics. The way to eliminate the cells that fail to accept the introduced DNA is to expose all the cells to antibiotics, leaving only the engineered microbes.

They also created new methods for genetic engineering in bacteria, including developing molecular tools to engineer new types of bacteria and to rapidly insert new DNA into a bacterium’s chromosome.

“Genetic tools are critical for the understanding of how microbes work and for manipulation of the microbes for different applications,” Guss told FORNL. “These tools are helping enable the conversion of plastic waste and lignocellulose compounds in plant biomass to renewable fuels and chemicals.”

“We work with about 10 microorganisms for metabolic engineering and 20 to 30 microorganisms for developing genetic tools to help our partners in academia and industry do their genetic engineering faster and better,” Guss said. “We are always looking for collaborations and new interesting organisms.”

Although none of the processes have been proven to work at an industrial scale yet, Guss expressed excitement about their collaborations with multiple companies for the biological production of solvents, polymer building blocks and biodegradable super-absorbent, renewable biopolymers to replace petroleum-derived polyacrylate in disposable baby diapers.

“They are trying to scale up these processes,” Guss said. “They hope to have commercial processes soon.”

This article originally appeared on Oakridger: Oak Ridge Lab synthetic biology expert targets plastics