Researchers of the Li Lab, led by Prof. Sijin Li, chemical and biomolecular engineering, recently developed a new yeast-based screening method to identify key enzymes that synthesize medicinal compounds in plants.
Many commercially available drugs are derived from natural compounds made in plants, including aspirin, morphine, and some types of chemotherapy. Identifying the biosynthetic pathways to obtain these compounds is of great pharmaceutical significance, but existing methods have limited efficiency, according to Li.
Currently, scientists analyze plant transcriptomes, the gene products in a cell, to identify hundreds of genes that could potentially code for proteins of medicinal interest. They then must biochemically determine the function of each gene with specific substrates and reaction conditions.
At Cornell, the Li Lab has developed a new technique, which expands upon existing methods, that uses genetically-engineered yeast to predict the biosynthetic pathways at the protein level by examining protein-protein interactions.
“We chose yeast for several reasons,” said co-first author Chang Liu, a grad student in the Li lab. “For one, yeast has a quick turnaround time — we can grow it in one day and use it for experiments. Another reason is that our lab has a lot of tools to engineer yeast.”
After using transcriptomics to identify proteins of interest, the researchers engineered the genes of interest into the yeast to see which ones produce proteins that interact with one another. This genetic engineering strategy allows many copies of the genes to be made as the yeast replicates, enabling the researchers to track gene products. Experiments were then conducted to confirm these protein interactions.
In these experiments, proteins were labeled with a special kind of light called fluorescence and viewed under a microscope to see which ones associate with one another. Overall, this method significantly reduced the number of genes that need to be biochemically characterized and can save time and financial resources, according to Liu.
The researchers demonstrated the technique using kratom leaves. Kratom is a Southeast Asian tree that produces a compound called mitragynine, a morphine alternative with understudied pharmaceutical potential, according to Li.
“[Kratom] has less potential to induce dangerous respiratory depression, which is the reason why morphine and opioid derivatives can be lethal,” Li said. “Our goal is to use our technology to produce [mitragynine] and further engineer it so that it can ultimately create a next-generation painkiller.”
To identify the biochemical pathways of mitragynine, the researchers screened 20 enzyme candidates in kratom with their yeast-based technique. They predicted that six of these enzymes would produce mitragynine and other targeted chemicals. Additional biochemical testing confirmed four of the six enzymes are functional, which makes them relevant for further study.
The timeline of the study began in early 2021, when Yinan Wu, postdoc in the Li lab and co-first author of the study, conducted transcriptomic analysis to predict kratom enzyme candidates. The group spent half a year analyzing the kratom plant before moving into yeast-based screening, which was led by Liu. Wu was then able to characterize protein activity, and the lab identified the chemicals of interest in 2022, wrapping up and publishing the study in 2023.
Ultimately, Li is optimistic about the broad applications of this technology in future drug discovery and clinical research.
“There is an interest in clinical trials with kratom-derived medical products, but it is difficult to isolate the pure chemical, and when the chemical is not pure, it can still lead to side effects,” Li said. “Our technology opens the gates to large-scale production of pure compounds to advance clinical trials.”
The Li Lab will continue to pursue this trajectory of work, integrating new techniques, such as a cell characterization technology called flow cytometry, to further streamline their biochemical characterization method for the identification and extraction of medicinal plant properties. Li noted that they are also collaborating with other researchers at Cornell to study the biological activity of the chemicals the lab produces.
Kaitlin Chung can be reached at [email protected].