A facile in vivo procedure to analyze metabolic pathways in intact lichens

Lichen secondary metabolites show important biological activities as well as pharmaceutical and chemotaxonomic potential. In order to utilize such substances of interest, detailed knowledge of their biosynthetic pathways is essential. ¹³ CO₂ -pulse/chase experiments using intact thalli of the lichen Usnea dasopoga resulted in multiple ¹³ C-labeled isotopologs in amino acids, but not in the dibenzofuran derivative usnic acid - one of the best-studied lichen metabolites, with considerable and renewed interest for pharmaceutical and lifestyle applications. Spraying an aqueous solution of [U-¹³ C₆ ]glucose onto the thalli of U. dasopoga afforded a specific mixture of multiple ¹³ C-labeled isotopologs in usnic acid. One- and two-dimensional NMR analysis of the crude lichen extract corroborated the polyketide biosynthetic pathway via methylphloroacetophenone but not via phloroacetophenone. With usnic acid as an exemplar, we provide proof-of-principle experiments that can be used in general to study metabolic pathways and fluxes in intact lichens.