Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from Pestalotiopsis fici and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from Saccharomyces cerevisiae. In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (1–28) revealed the corresponding aldehyde formation in 14 cases (1–14). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, 4) and vanillic acid (10). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.

Epidithiodiketopiperazines (ETPs) are a class of fungal secondary metabolites (SMs) featuring a transannular disulfide bridge at the diketopiperazine (DKP) core. The complex structures and biological activities have attracted widespread attention from biologists and chemists. In this study, we identified five novel ETP derivatives 4–7 and 4′ from three gene deletion mutants of Trichoderma hypoxylon CGMCC 3.17906, including ΔtdaP, ΔtdaQ, and ΔtdaQΔtdaI. Their structures were characterized through NMR and HR-ESI-MS data analysis. Compounds 4 and 4′ have unique heteroatom substitutions at the α and α′ positions, compound 5 possesses a unique α, β′-disulfide bridge, and compounds 6 and 7 contain a C3′-(thio)carbonyl group. Based on structural elucidation and biosynthetic pathway of α, β′-disulfide bridged ETPs, we also proposed the formation of 4–7 and 4′. This study not only expands the chemical diversity of ETPs, but also offers new mechanistic insights into the biosynthetic pathways of fungal ETPs.