Tricarbocyclic core formation of tyrosine-decahydrofluorenes implies a three-enzyme cascade with XenF-mediated sigmatropic rearrangement as a prerequisite

Tyrosine-decahydrofluorene derivatives feature a fused[6.5.6]tricarbocyclic core and a 13-membered para-cyclophane ether.Herein,we identified new xenoacremones A,B,and C(1-3)from the fungal strain Xenoacremonium sinensis ML-31 and elucidated their biosynthetic pathway using gene deletion in the native strain and heterologous expression in Aspergillus nidulans.The hybrid polyketide synthaseenonribosomal peptide synthetase(PKS-NRPS)XenE together with enoyl reductase XenG were confirmed to be responsible for the formation of the tyrosine-nonaketide skeleton.This skeleton was subsequently dehydrated by XenA to afford a pyrrolidinone moiety.XenF catalyzed a novel sigmatropic rearrangement to yield a key cyclohexane intermediate as a prerequisite for the formation of the multi-ring system.Subsequent oxidation catalyzed by XenD supplied the substrate for XenC to link the para-cyclophane ether,which underwent subsequent spontaneous Diels-Alder reaction to give the end products.Thus,the results indicated that three novel enzymes XenF,XenD,and XenC coordinate to assemble the[6.5.6]tricarbocyclic ring and para-cyclophane ether during biosynthesis of complex tyrosine-decahydrofluorene derivatives.

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new“non-natural”analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities.To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,de novo synthesis of cyathane diterpenes in a geranylgeranyl pyrophosphate engineered Saccharomyces cerevisiae is investigated.Aided by homologous analyses,one new unclustered FAD-dependent oxidase EriM accounting for the formation of allyl aldehyde and three new NADP(H)-dependent reductases in the biosynthesis of cyathanes are identified and elucidated.By combinatorial biosynthetic strategy,S.cerevisiae strains generating twenty-two cyathane-type diterpenes,including seven"unnatural"cyathane xylosides(12,13,14a,14b,19,20,and 22)are established.Compounds 12-14,19,and 20 show significant neurotrophic effects on PC 12 cells in the dose of 6.3-25.0μmol/L.These studies provide new insights into the divergent biosynthesis of mushroom-originated cyathanes and a straightforward approach to produce bioactive cyathane-type diterpenes.