Structural Determination of an Orphan Natural Product Using Microcrystal Electron Diffraction and Genome Mining

More than 60% of pharmaceuticals are related to natural products (NPs), chemicals produced by living organisms. Hence, new methods that accelerate natural product discovery are poised to profoundly impact human health. Of the many challenges that remain in natural product discovery, none are as pervasive as structural elucidation, as determination of the molecular structure of a newly discovered natural product can take months, years, or in some cases be altogether unachievable. This challenge can be fueled by lack of sufficient material for spectroscopic analysis, or difficulties in sourcing the producing organism. Even in cases where the analyte is abundant, its physical properties, including molecular structure, can prevent unambiguous structural determination. Here we report the use of microcrystal electron diffraction (MicroED), an emerging cryogenic electron microscopy (CryoEM) technique, in combination with genome mining, to address these challenges. As proof-of-principle, we apply these techniques to fischerin ( 1 ), an orphan NP isolated more than 30 years ago, with potent cytotoxicity but ambiguous structural assignment. We utilize genome mining methods to reconstruct its biosynthetic pathway and highlight the sensitivity of MicroED through the precise determination of the solid-state structure of 1 from sub-micron thick crystals. This structural solution serves as a powerful demonstration of the synergy of MicroED and synthetic biology in NP discovery, technologies that when taken together will ultimately accelerate the rate at which new drugs are discovered.