The tropical flowering plant Madagascar periwinkle produces the anti-cancer drug vinblastine. More than 35 enzymes are dedicated to the synthesis of this compound.
How does a plant synthesize an anti-cancer drug in 35 steps? And why?
Vinblastine does not represent an anomaly: many of the ca. 400,000 species of plants found on earth produce molecules of comparable complexity using lengthy metabolic pathways consisting of enzymes, regulatory factors and transporters. Elucidation of these pathways has historically been challenging, but over the last 15 years we have developed tools and approaches to streamline plant pathway elucidation and engineering. Our research group has led efforts to elucidate complex plant natural product pathways, and use this knowledge to understand the mechanism, evolution and function of these pathways.
Discovery of new plant biosynthetic genes
Many of the metabolic pathways that produce natural products in plants have not been characterized at the gene level. We have elucidated many natural product pathways, with a particular focus on the monoterpene indole alkaloids and iridoid natural products. To the left is the biosynthetic pathway of strychnine, which we elucidated in 2022.
To use our self organizing map developed by Marc Jones in Payne et al. (2017) Nature Plants:
Along with a variety of biochemical approaches to rapidly identify new biosynthetic genes in plants, we use state-of-the-art sequencing and bioinformatics techniques, often in collaboration with the Buell group at the University of Georgia.
We have recently collaborated with the Buell Lab to apply single cell RNA-seq technologies to elucidation of plant pathways. The figure to the right shows the co-expression profiles of vinblastine biosynthetic enzymes using standard RNA-seq data, compared with co-expression profiles using single cell RNA-seq data.
The advances that have been made in sequencing technologies over the last 10 years has revolutionized what we are able to do in plant metabolism.
Mechanism and evolution of biosynthetic pathways
Our group has a strong interest in enzyme mechanism. We use mutagenesis, X-ray crystallography, and a variety of other biochemical and biophysical techniques to understand how enzymes work and how these enzymes can be used in metabolic engineering and as biocatalysts. We are also interested in the evolution of enzymes, and we have used approaches such as ancestral sequence reconstruction to try to understand how unusual enzyme activity may have evolved. To the the right is a model of an enzyme active site of a medium chain alcohol dehydrogenase. We can mutate the residues in red to switch the stereoselectivity of the reduction, along with the stereochemistry of the resulting alkaloid product.
We study examples where the biosynthesis of a natural product evolved independently, so that we can see how nature found different chemical solutions to synthesize the same molecule. There are many beautiful examples of natural product convergent evolution in plants. Additionally, sometimes plants and insects make the same natural product! For example, we are comparing and contrasting the biosynthetic pathway of the natural prodcuct nepetalactone in the plant catnip (where nepetalactone is the active ingredient of catnip that makes cats go wild) and female aphids (where nepetalactone is a sex pheromone), as shown in the figure on the left. We are looking at the biosynthetic pathways of other terpenes in a variety of insects to understand how natural product biosynthesis evolved differently in plants and animals.
Reconstitution of plant pathways
We are exploring ways to reconstitute individual branches of plant pathways in tractable host organisms, such as Nicotiana benthamiana and yeast. We use this approach to better understand the individual steps of the pathway, as well as to try to generate proof of concept reconstitution systems for the production of high value molecules. We also use transient expression of Nicotiana benthamiana capable to produce “new-to-nature” products. This reprogramming of biosynthetic pathways to produce unnatural products may lead to the development of molecules with new biological activities.
We also use in planta protein expression to answer a variety of biophysical questions. For example, we can explore interactions among biosynthetic proteins by co-expressing labeled versions of these biosynthetic enzymes in Nicotiana benthamiana and then monitoring the resulting interactions by fluorescence or luminescence.