Qing-Li He

The medicinal fungus Wulingshen, comprising multiple Xylaria species, inhabits deserted termite mounds as sclerotia. To explore the molecular basis of its niche adaptation, we employed a synthetic biology-driven approach. Metagenomic and transcriptomic mining of wild specimens identified a conserved biosynthetic gene cluster. Its heterologous reconstruction in the fungal host Aspergillus oryzae enabled the characterization of a family of α-pyrone metabolites and, crucially, the elucidation of a spatially separated aldehyde-alcohol cycle. In this self-protection system, an extracellular oxidase (WlsA) converts an alcohol precursor to a reactive aldehyde, while an intracellular reductase (WlsE) catalyzes the reverse reaction. The aldehyde product exhibits potent toxicity against termites in vitro, suggesting a potential role in ecological interactions. This work establishes a functional genomics platform that decodes cryptic ecological metabolism by integrating multiomics with heterologous pathway expression, providing a generalizable strategy for discovering and mechanistically understanding niche-specific natural products.

Supplementary Figure S1-S4 Supplementary Table S1

(a) Western blot analysis of glycoprotein patterns in MDA-MB-231 cells after treatment with the indicated concentrations of penfluridol for 24 hours. Red arrowhead, immature glycoproteins. (b) Western blot analysis of protein expression pattern of ACE2 in HEK293T cells after treatment with the indicated concentrations of penfluridol for 24 hours. (c) In HEK293T cells, FLAG-tagged ACE2 was treated for 24 hours, collected by immunoprecipitation, subjected to SDS-PAGE, and analyzed by western blot. (d) Pearson correlation coefficient of three independent groups of (c) through N-glycan profiling.

(a) The stability of proteins was determined by cellular thermal shift at different concentrations of penfluridol.

(a) Western blot analysis of protein expression pattern of PD-L1 in HEK293T cells after the exposure to the indicated concentrations of penfluridol for 24 hours. (b) Protein expression pattern of PD-L1 in B16-F10 cells after treatment with the indicated concentrations of penfluridol for 12 hours as determined by western blot. (c) The toxicity effect of penfluridol on B16-F10 (top) and B16-OVA (bottom) cells. (d) Western blot analysis of protein expression of MAN1A1 in B16-OVA cells treated with the indicated siRNA.

(a) qPCR analysis of the GRP78 and CHOP expression in MDA-MB-231 cells after the treatment with the indicated concentrations for 6 hours. (b) Western blot analysis of Integrin β1 expression pattern in MDA-MB-231 cells after indicated drugs treatment for 24 hours. (c) Western blot analysis of protein expression pattern of Integrin β1 in MDA-MB-231 cells treated with the indicated siRNA.

Fraxinus chinensis Roxb. (Qinpi), a traditional Chinese medicinal plant, accumulates abundant coumarins that contribute to its anti-inflammatory and other bioactivities. However, the enzymatic basis for coumarin structural diversification in this species remains largely unexplored. Here, through transcriptome-wide identification of the 2-oxoglutarate-dependent dioxygenase (2OGD) family in F. chinensis, followed by phylogenetic analysis, heterologous expression, and in vitro enzyme assays, we identified FcDOH2, a member of the DOXC31 subfamily, which exhibits substrate-dependent bifunctionality, catalyzing the C6-O-demethylation of scopoletin (3) to esculetin (2) and the C8-hydroxylation of umbelliferone (1) to daphnetin (6). Using AlphaFold3-based structural modeling, molecular docking, and alanine scanning mutagenesis, we revealed that residues R155 and R221 are essential for both activities through stabilizing hydrogen bonds, whereas residue F312 acts as a functional switch, being critical for demethylation but negatively regulating hydroxylation. These findings uncover a rare bifunctional 2OGD with substrate-dependent catalytic plasticity, providing mechanistic insights into coumarin diversification in medicinal plants and a structural basis for future enzyme engineering.

Chemoproteomic profiling provides a complementary approach for linking natural products to their corresponding enzymes. A previously unrecognized polyphenol oxidase, SmPPOA, was identified from Salvia miltiorrhiza using a 3D carbene-immobilized salvianolic acid B. SmPPOA catalyzes oxidative lactonization of salvianolic acid B to generate salvianolic acid O via a cascade of oxidation and intramolecular Michael addition using the carboxylate anion as the Michael donor. This strategy can facilitate discovery of biosynthetic enzymes for plant natural products.

CsCYP21A, a steroid 21-hydroxylase from Bufo bufo gargarizans, exhibits unprecedented sequential oxidations. Optimizing Pichia pastoris biotransformation conditions enhanced C21-hydroxylation selectivity, converting 14 substrates to 21-hydroxylated products, with 10 conversions of >80% and 4 yields of >80%. Hydrocortisone production reached 1.5 g L^-1 day^-1 with 100 g/L wet biomass. CsCYP21A's versatility enables integration into the synthesis of over 10 steroidal drugs, offering a sustainable biocatalytic platform for green pharmaceutical manufacturing.