Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products

Abstract

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A 2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.

Heterologous Production of Unnatural Flavipucine Family Products Provides Insights into Flavipucines Biosynthesis

“…Membrane-bound small hypothetical proteins were proved to sequester and mold intermediates for the skeleton formation in hirsutellones biosynthesis. 24 Hence, FlvB was likely responsible for stabilizing the unstable intermediate 5a/5b for subsequent transformation instead of the catalytic function, which was also supported by detecting trace amounts of 14a and 11a in the mutant Δf lvB (Figure S9). Further heterologous expression of flvABD in S. cerevisiae led to the detection only of 10a/10b, also supporting our hypothesis (Figure S7A).…”

“…A similar significant difference could also be observed between the A. nidulans strains harboring FlvA B CDEFG and harboring FlvACDEFG (Figure and Figure S7B). Membrane-bound small hypothetical proteins were proved to sequester and mold intermediates for the skeleton formation in hirsutellones biosynthesis . Hence, FlvB was likely responsible for stabilizing the unstable intermediate 5a / 5b for subsequent transformation instead of the catalytic function, which was also supported by detecting trace amounts of 14a and 11a in the mutant Δ flvB (Figure S9).…”

Discovery and Characterization of a Terpene Biosynthetic Pathway featuring a Norbornene-forming Diels-Alderase

“…Extensive modifications of the cycloaraneosene skeleton are expected to ready the molecule for norbornene formation: 1) unlike IMDA reactions observed for polyketide chains that are conformationally flexible to position dienes and dienophiles in spatial proximity (Fig. 1b) [8][9][10][11][12][13][14][15] , the polycyclic terpene molecule is rigid, thereby requiring breaking one or more C-C bonds to afford rotational freedom; 2) desaturation of sp 3 -sp 3 C-C bond(s) in the cyclized terpenes such as cycloaraneosene must take place to generate the diene moiety; and 3) the diene and especially the dienophile require activation to lower the transition state (TS) energy barrier for IMDA. Whereas dienophiles in polyketides that undergo IMDA are typically conjugated to electron withdrawing groups as a result of polyketide synthase programing, the hydrocarbon scaffold of a terpene molecule must be strategically oxidized prior to the pericyclic reaction.…”

“…2b, Supplementary Notes, Supplementary Table 5, Supplementary Figs. 4,[14][15][16][17][18][19]. The stereochemistries of the diol 2 were determined by NOESY based on the reported stereochemistry for cycloaraneosene 19,28 .…”

Optimization of Protoplast Preparation and Establishment of Genetic Transformation System of an Arctic-Derived Fungus Eutypella sp.

“…In recent years, many secondary metabolites of filamentous fungi have been found to possess unique structure and pharmacological activities, which has attracted scientists’ extensive attention ( Guo et al, 2021 ; Ohashi et al, 2021 ; Qian et al, 2021 ). Eutypella species are filamentous fungi, belonging to Ascomycetes , Xylariales , and Diatrypaceae ( Lu et al, 2014 ).…”