image: Dr. Meng Wang working in his labratory.
Credit: Paul Kovach
The insatiable Pac Man and Ms. Pac Man, from the 1980s arcade games, provide an ideal vision for how natural microorganisms such as bacteria and fungi could degrade toxic pollutants, a process known as biodegradation. Imagine an oil spill approaching a pristine coastline, the black sheen shimmering ominously in the sun. Enter the iconic yellow circles, chomping hydrocarbon molecules and leaving behind a trail of clear blue water.
Unfortunately, it’s not that easy. As catalysts, some enzymes in microorganisms can effectively degrade toxic contaminants, but it can be a slow process that leaves behind intermediates—materials that themselves can be toxic.
Meng Wang, assistant professor of civil and environmental engineering at the University of Pittsburgh Swanson School of Engineering, received a prestigious National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award of $550,000 to develop a nanostructure cocktail of enzymes that will more efficiently break down toxic materials without producing dangerous intermediates. His project, “Advancing Biodegradation Through Protein Nanocompartment-Based Cargo Encapsulation for Organic Contaminant Removal,” seeks to dramatically improve biodegradation while advancing biomanufacturing.
“As an undergraduate student, I began researching how fungi and other microorganisms can degrade contaminants,” said Wang. “It is a promising tool that is sustainable and can be less expensive than traditional methods of cleaning toxic sites.”
Microbes like Phanerochaete chrysosporium and Dehalococcoides contain enzymes that can help break down pollutants such as pesticides, industrial solvents, or spilled oil.
“The problem is that to effectively break down harmful substances, it takes multiple enzymes—it’s not a single reaction,” Wang said. “However, some enzymes won’t cooperate, and they slow the process. And while breaking down toxic substances, some enzymes can create harmful intermediates.”
Cages and cocktails
As a graduate student and a postdoctoral researcher at the University of California Los Angeles (UCLA), Wang began studying unique nanostructures called protein nanocompartments. “They are like cages that can form on their own, and we applied these cages to encapsulate enzymes, enhancing their stability and robustness,” said Wang.
Since coming to Pitt in 2020, Wang has been seeking to harness protein cage encapsulation to address the limitations of biodegradation and make the process more efficient and safer. “I want to use protein cages to combine and regulate multiple enzymes, creating a kind of ‘nano-reactor,’ where the intermediates can transfer more efficiently from one enzyme to the next,” said Wang. “This will help expedite the biodegradation process and reduce the buildup of potentially harmful intermediates. My aim is to find the best cocktail of enzymes and assemble it with precision, which requires developing new methods to control and fine-tune enzyme encapsulation within protein cages.”
To achieve this, Wang uses affinity tags, which are smaller proteins attached, like tails, to larger ones. By designing and testing affinity tags with varying molecular properties, Wang will investigate how these properties affect enzyme encapsulation efficiency. This knowledge will guide controlled enzyme encapsulation through tag engineering and help optimize the cocktail to create an efficient cascade breaking down parts of the contaminant.
Given that these nanostructures are imperceptible to the human eye, to test his work Wang uses dynamic light scattering (DLS), a technique that involves shooting a laser into a sample and analyzing the pattern of scattered light, and transmission electron microscopy (TEM), which involves a beam of electrons illuminating a structure. “With DLS and TEM, we can determine if we have assembled the cage,” said Wang.
He also uses Fast Protein Liquid Chromatography (FPLC), a technique that separates molecules by their size, showing large ones earlier and smaller ones later. “FPLC allows us to confirm whether enzymes are encapsulated within the cages and to separate the encapsulated enzymes from the unencapsulated ones,” he noted.
For this project, Wang will focus on developing bioagents that can more efficiently break down the compound 1,2,3-trichloropropane, which belongs to a large group of persistent contaminants found at polluted sites around the country. He will analyze how enzyme cocktails interact with this pollutant and work to fully convert it to non-toxic products.
Beyond its overarching aim of enabling more efficient biodegradation and thus improving the tools to clean dangerous polluted sites, Wang hopes this research will advance biomanufacturing by revealing new ways to precisely control enzyme encapsulation in protein cages. The research could enable scientists to adapt mixtures of enzymes for breaking down different toxic substances more easily and potentially apply the technology to biofuel production, resource recovery, and more.
“I am so grateful for this support from the National Science Foundation,” said Wang. “I am also grateful for the support from my colleagues and collaborators here at Pitt, and from my mentors and advisors at other institutions. It’s an honor to have the opportunity to advance this important research that I began exploring as an undergraduate, and I’m excited about the possibility of creating more efficient biodegradation.”