Researchers have spent decades—and billions of dollars—sequencing animal and crop genomes, but fungi have historically been the forgotten middle child of genomics, only noticed when they're ruining bread or colonizing toes.
"This neglect is kind of remarkable considering how fungi have shaped modern medicine," says chemical and biomolecular engineer Xue "Sherry" Gao . "From the serendipitous discovery of penicillin to cholesterol-lowering statins, we owe many recent breakthroughs in longevity to fungal chemistry. But despite this, the vast majority of the fungal kingdom remains a black box."
A main driver for this mystery is that when grown in sterile lab conditions, fungi turn off the drug-producing gene pathways they synthesize in the wild to fight off bacteria.
"To turn those silent pathways back on, we needed a powerful way to precisely manipulate fungal genome, such as editing their master regulatory genes, but traditional tools weren't up to the task," Gao says.
Now, Gao and her team at the School of Engineering and Applied Science have developed a novel genome editing tool, called fPE7max, to navigate the complex genetic architecture of thread-like molds known as filamentous fungi—think Aspergillus, or the Penicillium that gave the world penicillin—and finally unlock the secrets of this overlooked kingdom.
Their findings are published in Nature Biotechnology .
"We isolated 18 distinct complex molecules, eight of which possessed chemical structures entirely new to science," says first author Chunxiao Sun, a postdoctoral researcher in the Gao Lab. "Of these uncovered molecules, three exhibited promising anti-cancer properties. These molecules can serve as lead compounds for disease treatment, providing a vital new pipeline for drug discovery."
Sun says that one novel molecule showed selective toxicity against human breast, hepatic, and leukemia cancer cells.
Rewriting the genomics textbook for fungi
Over the last decade, CRISPR-Cas9 has been the headline-grabbing gene-splicing tool. But Gao explains that in filamentous fungi, which are rich sources of antibacterial compounds, it can be a blunt instrument, resulting in unintended mutations.
A newer technology called prime editing avoids double-strand breaks entirely, allowing for precise control over DNA sequences. But adapting prime editing for the fungal kingdom was a challenge.
First, the team had to ensure their genetic instructions actually survived the trip through the cell. Prime editing relies on a guide RNA—a molecular instruction manual that tells the tool where to go and what new code to write. But when researchers try to make massive edits, these instruction manuals can get unreasonably long, making them fragile and prone to degrading before the editing job is done.
Their workaround was integrating a special protein—fLa—into their tool. fLa acts as a sturdy, protective binder that shields the fragile RNA instructions, allowing fPE7max to handle the massive DNA insertions and deletions that cause other tools to break down.
Second, the team had to stop the fungal cells from spotting the researchers' new edits, flagging them as errors, and reverting the DNA back to its original sequence. To outsmart that, the team incorporated a specialized protein that mutes the fungus's natural repair system just long enough for the new genetic code to permanently take hold.
Ancient organisms, new science
The resulting platform, fPE7max, achieves editing efficiency approaching 90%. And by using fPE7max to flip the switch on these silent fungal gene clusters, the team uncovered previously unknown compounds.
To test their new tool, the researchers targeted the regulatory sequences of a master gene called laeA, which controls a vast network of biosynthetic pathways. By using fPE7max to precisely edit out the molecular roadblocks that naturally keep this gene's translation repressed, they successfully awakened silent gene clusters across several different fungal species, finding molecules with promising anti-cancer properties.
"It's a compelling proof-of-concept demonstrating that the next generation of life-saving therapeutics might already exist in nature," Gao adds.
Looking ahead, the team plans to deploy fPE7max across a much wider array of fungal species to continue hunting for novel natural products. The researchers hope to move away from the treasure-hunt approach of searching for wild fungi that might produce useful drugs and into an era of systematic optimization.
Xue "Sherry" Gao is the Presidential Penn Compact Associate Professor in the Department of Chemical and Biomolecular Engineering , the Department of Bioengineering , and the Center for Precision Engineering for Health at the University of Pennsylvania.
Chunxiao Sun is a postdoctoral researcher in the Gao Lab at Penn Engineering.
Other authors include Chris Keum, Qiuyue Nie, Yihui Shen, and Naomi Straub of Penn Engineering.
This research was supported by the National Institutes of Health (NIH grant R35GM138207) and startup funds provided by the University of Pennsylvania.
