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

Abstract

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 CO 2 -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 acidone of the best-studied lichen metabolites, with considerable and renewed interest for pharmaceutical and lifestyle applications.Spraying an aqueous solution of [U-13 C 6 ]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.

The Lichens’ Microbiota, Still a Mystery?

“…One method to study metabolic pathways and fluxes of lichens in vivo is based on the utilization of stable isotope labeling. They are based on the supply of the labeled tracer to the growth medium (Kuhn et al, 2019). The positional labeling patterns can reveal the biosynthetic history of the studied products (Kinoshita et al, 2015).…”

“…An additional study reveals that cyano-and tripartite lichens have a strong respiratory response to glucose by conducting carbon dioxide flux measurements and phospholipid acid analysis with experimental application of 13 C 6 (Campbell et al, 2013). Recently, Kuhn et al (2019) performed in vivo labeling experiments with Usnea dasypoga using 13 CO 2 or [U-13 CO 6 ]glucose and reconstructed the biosynthetic pathway of usnic acid.…”

Characterization of Sunflower Oil Extracts from the Lichen Usnea barbata

“…For a rapid identification and quantification of UA in crude extracts of U. barbata , we used 1 H-NMR spectroscopy of the mixture that relied on the detection of specific and well separated signals for UA in the concurrent presence of abundant lipid signals from the extractant ( Figure 2 D). The NMR signals of UA were unequivocally assigned by two-dimensional NMR experiments, as reported earlier [ 48 ]. It turned out that the downfield shifted 1 H singlet at 13.33 ppm due to the OH group at C-8 of UA (for carbon numbering, see Figure 1 ) was most useful for quantifying the polyketide in the complex mixture (cf.…”

Towards a Systems Biology Approach to Understanding the Lichen Symbiosis: Opportunities and Challenges of Implementing Network Modelling

“…The literature on lichen metabolism has been largely focused on understanding the exchange of key nutrients between symbionts ( Lines et al, 1989 ; Kono et al, 2020 ; ten Veldhuis et al, 2020 ) and identifying lichen secondary metabolites and their biosynthetic pathways (i.e., metabolite profiling) ( Fahselt, 1994 ; Aubert et al, 2007 ; Elix and Stocker-Worgotter, 2008 ; Mittermeier et al, 2015 ; Bertrand et al, 2018b ; Brakni et al, 2018 ; Calcott et al, 2018 ; Kuhn et al, 2019 ; Goga et al, 2020 ; Figure 4 ). In the 1960s, observations of carbohydrate storage and translocation between the symbionts of Peltigera polydactyla ( Smith and Drew, 1965 ; Drew and Smith, 1967a , b ) together with a series of similar studies on other lichens ( Smith et al, 1969 ) established the foundations for studying the metabolic interplay in lichens.…”