Toward redirecting cardenolide biosynthesis in tropical milkweed: from 5-alpha to 5-beta configuration

Cardenolides, traditionally used in heart medications, are produced in plants such as milkweed, wallflower, and foxglove. Besides their biosynthetic complexity, plant cardenolide production plays a vital role in fitness and coevolution with insects. Beyond modifications on glycones and genins, the configuration on the 5-carbon of the steroid core, as seen in wallflower, is increasingly recognized as a novel way to expand the repertoire of cardenolide metabolites. However, milkweed produces only 5α-cardenolides, lacking this configuration, which limits our understanding of its importance in its interaction with insects. To address this, our research aims to reconfigure cardenolide biosynthesis in tropical milkweed by knocking out a gene (CYP87A) to reduce constitutive cardenolide production and overexpressing a wallflower gene coding for progesterone 5β-reductase (P5βR) to produce 5β-cardenolides. By infiltrating two Agrobacterium strains (EHA105 and MSU440) with two plasmids (pTrans221-Cas9-CYP97AgRNA and pEAQ-P5βR) into tropical milkweed seedlings, we harvested stem and leaf samples and conducted untargeted HPLC-MS metabolic profiling, data processing with XCMS and CAMERA software packages, and multivariate analysis in R. Our principal component analysis indicated that agroinfiltration with all treatments significantly altered the overall chemical profile and constitutive cardenolide accumulation. Compared to controls, most major 5α-cardenolide compounds showed significantly reduced abundance, especially in stem samples. Notably, we identified new genin features in both stem and leaf samples infiltrated by EHA105 or MSU440 with the pEAQ-P5βR plasmid, suggesting possible production of 5β-cardenolides. These results highlight the potential for producing novel cardenolides for medicinal testing and plant defense studies.

I am grateful to have worked in Georg Jander’s lab under the mentorship of Fumin Wang. When I entered the BTI high school internship program, I had minimal wet-lab experience, but I am leaving with a wealth of new skills. I learned essential lab techniques, from basic micropipetting to more advanced procedures like PCR, gel electrophoresis, and restriction cloning. Besides, I enhanced my knowledge of R programming, particularly in data structuring, subsetting, and ggplot2 visualization. I am especially thankful for Fumin’s guidance in untargeted metabolic profiling and principal component analysis (PCA). This internship has significantly developed my critical thinking and communication skills; I learned how to effectively organize and explain my results and their significance. Lastly, I cherished the meetings and lunches with my labmates, which fostered a friendly and positive work environment.